antisense oligomers for the treatment of conditions and diseases

专利摘要:
  Alternative splicing events in the SCN1A gene can lead to non-productive mRNA transcripts that, in turn, can lead to aberrant protein expression, and therapeutic agents that can target alternative splicing events in the SCN1A gene can modulate the level of expression of functional proteins in patients with Dravet's syndrome and / or inhibit aberrant protein expression. These therapeutic agents can be used to treat a condition caused by a deficiency of the SCN1A, SCN8A or SCN5A protein.
公开号:BR112020003591A2
申请号:R112020003591-2
申请日:2018-08-24
公开日:2020-09-01
发明作者:Isabel Aznarez；Zhou Han
申请人:Stoke Therapeutics, Inc.；
IPC主号:

专利说明:

[001] [001] This request claims the benefit of Provisional US Order No. 62 / 550,462, filed on August 25, 2017 Provisional US Application No. 62 / 575,901, filed on October 23, 2017, Provisional US Order No. 62 / 667,356, filed on May 4, 2018, Provisional US Order No. 62 / 671,745, filed on May 15, 2018, each of which is incorporated into this specification by reference in its entirety. BACKGROUND OF THE INVENTION
[002] [002] Nervous system disorders are often associated with channelopathies, characterized by the altered function of ion channels that mediate neuronal excitability, neuronal interactions and brain functions in general. Mutations in the SCNIA gene, which is part of the SCN1A-SCN2A-SCN3A gene cluster encoding pore-forming subunits of the sodium channel controlled by neuronal voltage, are associated with the development of several diseases and conditions, for example, Dravet syndrome (DS) (Miller et al, 1,993-
[003] [003] In this specification, a method of modulating the expression of SCNI1A protein in a cell that has an mRNA containing an RNA decay-inducing exon mediated by nonsense mutations is revealed in certain modalities
[004] [004] This specification discloses, in certain modalities, a method of treating a disease or condition in an individual in need by modulating the expression of SCNIA protein in an individual's cell, which comprises: the contact of the individual's cell with a therapeutic agent that modulates the splicing of an exon inducing mRNA mediated by nonsense mutations (NMD exon) by an mRNA in the cell that contains the NMD exon and encodes SCN1A, thereby modulating the level of processed mRNA that encodes the SCNIA protein and modulating SCNIA protein expression in the individual's cell.
[005] [005] All publications, patents and patent applications mentioned in this specification are incorporated into this specification by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference. BRIEF DESCRIPTION OF THE DRAWINGS
[006] [006] The novel characteristics of the invention are presented with particularity in the appended claims. A better understanding of the characteristics and advantages of the present invention will be obtained by reference to the following detailed description which presents illustrative modalities, in which the principles of the invention are used and in the accompanying drawings, in which:
[007] [007] FIG. 1 depicts a schematic representation of a target mRNA that contains a nonsense mutation-mediated RNA decay exon (NMD exon mRNA) and therapeutic agent-mediated exclusion of nonsense-mediated mRNA decay to increase expression of the full-length target protein or functional RNA. FIG. 1A shows a cell divided into nuclear and cytoplasmic compartments. In the nucleus, the pre-mRNA transcript of a target gene is spliced to generate mRNA and that mRNA is exported to the cytoplasm and translated into the target protein. For this target gene, some fraction of the mRNA contains a nonsense-mediated decay-inducing mRNA exon (NMD exon mRNA) that is degraded in the cytoplasm thus leading to the absence of target protein production. FIG. 1B shows an example of the same cell divided into nuclear and cytoplasmic compartments. Treatment with a therapeutic agent, for example, an antisense oligomer (ASO), promotes the exclusion of nonsense-mediated decay-inducing mRNA exon and results in an increase in mRNA, which, in turn, translates into levels higher target protein. FIG. 1C is a schematic representation of therapeutic ASO-mediated exclusion of a nonsense-mediated decay-inducing mRNA exon, which transforms a non-productive mRNA into a productive mMRNA and increases the expression of the full-length target protein by the productive mRNA.
[008] [008] FIG. 2 depicts the identification of an exemplary nonsense-mediated mRNA decay (NMD) exon in the SCNIA gene. The identification of the NMD-inducing exon in the SCNIA gene is shown using comparative genomics, visualized in the UCSC genome browser. The bottom panel shows a graphical representation of the SCNIA gene at scale. The conservation level across 100 vertebrate species is shown as peaks. The largest peaks correspond to exons
[009] [009] FIG. 3A depicts confirmation of NMD-inducing exon by treatment with cycloheximide. RT-PCR analysis using Neuro 2A cytoplasmic RNA (mouse neural progenitor cells) treated with DMSO (CHX-) or treated with cycloheximide (CHX +) and primers in exon 21 and an exon downstream confirmed the presence of a band that corresponds to the NMD-inducing exon (21x). The identity of the product was confirmed by sequencing. Densitometry analysis of the bands was performed to calculate the percentage of inclusion of 21x exon of total SCNIA transcript. The treatment of Neuro 2A with cycloheximide (CHX +) to inhibit NMD led to a 2-fold increase in the product corresponding to the NMD-inducing 21x exon in the cytoplasmic fraction (cf. light gray bar, CHX-, for dark gray bar, CHX +) .
[010] [010] FIG. 3B depicts confirmation of NMD-inducing exon by treatment with cycloheximide. Analysis by RT-PCR using cytoplasmic RNA from RenCell VM (human neural progenitor cells) treated with DMSO (CHX-) or treated with cycloheximide (CHX +) and primers in exon 20 and exon 23 confirmed the presence of a band that corresponds to the NMD-inducing exon (20x). The identity of the product was confirmed by sequencing. Densitometry analysis of the bands was performed to calculate the percentage of inclusion of the 20x exon of total SCNIA transcript. The treatment of RenCell VM with cycloheximide (CHX +) to inhibit NMD led to a 2-fold increase in the product corresponding to the NMD-inducing exon 20x in the cytoplasmic fraction (cf. light gray bar, CHX-, for dark gray bar, CHX +) .
[011] [011] FIG. 4 depicts an ASO walk from the exemplary SCNIA 20x exon region. A graphic representation of an ASO walk performed for sequences that target the SCNIA 20x exon region upstream of the splice site 3 ", through the splice site 3 ', exon 20x, through the splice site 5' and a site of splice 5 'downstream splice using 2'-MOE ASOs, PS framework, is shown, ASOS were designed to cover these regions by changing 5 nucleotides at once.
[012] [012] FIG. 5A depicts ASO walk from the 20x exon region of SCNIA evaluated by RT-PCR. A representative PAGE shows SYBR-Safe stained RT-PCR products from SCN1A-treated (Simulated), treated with SMN control ASO (SMN) or treated with a 2'-MOE ASO that targets the exon 20x as described in that specification in the Examples and in the description of FIG. 4, at a concentration of 20 µM in RenCell VM cells by gimnite uptake. Two products corresponding to the inclusion of exon 20x (upper band) and total length (exclusion of exon 20x, lower band) were quantified.
[013] [013] FIG. 5B depicts a graphical plot of the percentage of exon 20x inclusion by the data in FIG. 5A.
[014] [014] FIG. 5C depicts a graph of the normalized full-length products for internal control of RPL32 and the rate of increase in relation to the Simulated is plotted. The black line indicates a ratio of 1 and no change with respect to the Simulated.
[015] [015] FIG. 6 depicts an ASO walk from the 20x exon region of exemplary SCNIA evaluated by RT-qPCR. Amplification results of SCNIA by RT-qgPCR with SYBR Green normalized to RPL32, obtained using the same ASO capture experiment that were evaluated by RT-PCR with SYBR-Safe as shown in FIG. 5, are plotted as an increase ratio in relation to the Simulated, confirming the results of RT-PCR with SYBR-Safe. The black line indicates a ratio of 1 (no change from the Simulated).
[016] [016] FIG. 7A depicts a table with members of the voltage-controlled sodium channel alpha subunit members. Arrows correspond to the colors of the bar in FIG. 7B. X means no expression detected.
[017] [017] FIG. 7B depicts selected ASOs evaluated by Tagman-qPCR from SCNIA, SCN2A, SCN3A, SCN8A and SCN9A to assess target selectivity. Amplification results by Tagman-qPCR normalized to RPL32, obtained using ASOs Ex20x + 1, IVS20x + 18 and IVS20x + 33, are plotted as an increase ratio in relation to the Simulated. The black line indicates a ratio of 1 (no change from the Simulated).
[018] [018] FIG. 8A depicts exemplary dose-dependent effect of selected ASOs in cells treated with CXH. A representative PAGE showing simulated-treated RT-PCR stained with SYBR-Safe mouse Scnla
[019] [019] FIG. 8B depicts a graphical plot of the percentage of exon 20x inclusion by the data in FIG. T7A. The black line indicates no change with respect to the Simulated.
[020] [020] FIG. 8C depicts an exemplary graph of the normalized full-length products for internal control of Hprt and the rate of increase in relation to the Simulated are plotted. The black line indicates a ratio of 1 and no change with respect to the Simulated.
[021] [021] FIG. 9A depicts exemplary results of intravitreal injection (IVT) of selected ASOs in C57BL6J mice (male, 3 months old). PAGE gels from RT-PCR products stained with SYN-Safe from Scnla mouse from left eye injected with PBS (1 ul) (-) or right eye injected with ASO 2'-MOE IVS20x-21, Ex2l1x + 1, IVS21x + 18, IVS21x + 33 or Cep290 (negative control ASO; Gerard et al, Mol. Ther. Nuc. Ac., 2015) (1 nl) (+) at a concentration of 10 mM, are shown. Ex2l1x + 1, IVS21x + 18 and IVS21x + 33 (mouse nomenclature) and Ex20x + 1, IVS20x + 18 and IVS20x + 33 (human nomenclature) are identical. Two products that correspond to the inclusion of exon 21x (upper band) and total length (exclusion of exon 21x, lower band) were quantified.
[022] [022] FIG. 9B depicts a graphical plot of the 21x exon inclusion percentage by the data in FIG. 9A. White bars correspond to eyes injected with ASO and gray bars correspond to eyes injected with PBS, n = 5 in each group.
[023] [023] FIG. 9C depicts a graph of the full-length products that have been normalized for internal Gapdh control and the ratio of increased eye injected with ASO to eye injected with PBS is plotted. The black line indicates a proportion of 1 and no change with respect to PBS, n = 5 in each group.
[024] [024] FIG. 10A depicts exemplary results of intracerebroventricular injection (ICV) of selected ASOs in C57BL6J mice (male, 3 months old). PAGE gels from RT-PCR products stained with SYN-Safe from Scnla mouse from non-injected brains (-, control without ASO), or brains injected with 300 µg of ASO from 2'-MOE Cep290 (negative control ASO; Gerard and cols., Mol. Ther. Nuc. AcC., 2015), Ex21x + 1, IVS21x + 18, IVS21x + 33 are shown. Ex21x + 1, IVS21x + 18 and IVS21x + 33 (mouse nomenclature) and Ex20x + 1, IVS20x + 18 and IVS20x + 33 (human nomenclature) are identical. Two products that correspond to the inclusion of exon 21x (upper band) and total length (exclusion of exon 21x, lower band) were quantified.
[025] [025] FIG. 10B depicts a graphical plot of the 21x exon inclusion percentage by the data in FIG. 10A, n = 6 (each targeting ASO), n = 5 (ASO Cep290), n = 1 (not injected, control without ASO).
[026] [026] FIG. 10C depicts a graph of results from a Taqgman-qPCR assay performed using two different probes that span the junction of exons 21 and 22. The products were normalized for internal Gapdh control and the ratio of increased brains injected with ASO to brains injected with Cep290 is plotted. The black line indicates a ratio of 1 and no change with respect to Cep290, n = 6 (each targeting ASO), n = 5 (ASO Cep290), n = 1 (not injected, control without ASO).
[027] [027] FIG. 11A depicts exemplary results of intracerebroventricular injection (ICV) of selected ASOs in C57BL6J mice (male, 3 months old). PAGE gels from RT-PCR products stained with SYBR-Safe from Scenla mouse brains injected with 300 µg of Cep290 (negative control ASO; Gerard et al., Mol. Ther. Nuc. AC., 2015), or injected brains with 33 µg, 100 µg and 300 µg of 2 'MOE Ex21x + 1. Ex21x + l (mouse nomenclature) and Ex20x + 1, (human nomenclature) are identical. Two products that correspond to the inclusion of exon 21x (upper band) and total length (exclusion of exon 21x, lower band) were quantified.
[028] [028] FIG. 11B depicts a graphical plot of the 21x exon inclusion percentage by the data in FIG. 11A, n = 5 (each group).
[029] [029] FIG. 11C depicts a graph of the results of a Taqgman-qPCR assay performed using two different probes that span the junction of exons 21 and 22. The products were normalized for internal Gapdh control and the ratio of increased brains injected with ASO to brains injected with Cep290 is plotted. The black line indicates a ratio of 1 and unchanged with respect to Cep290, n = 5 (each group).
[030] [030] FIG. 12A depicts exemplary results of intracerebroventricular injection (ICV) of a selected ASO in C57BL6J mice (postnatal day 2). PAGE gels from RT-PCR products stained with SYN-Safe from Scnla mouse from non-injected brains (-, control without ASO), or from brains injected with 20 µg ASO of 2'-MOE Ex2l1x + 1 are shown. Two products that correspond to the inclusion of exon 21x (upper band) and total length (exclusion of exon 21x, lower band) were quantified. Ex21x + 1 (mouse nomenclature) and Ex20x + 1 (human nomenclature) are identical.
[031] [031] FIG. 12B depicts a graphical plot of the 21x exon inclusion percentage by the data in FIG. 12A, n = 4 (each group).
[032] [032] FIG. 12C depicts a graph of results from a Taqman-qPCR assay performed using two different probes that span the junction of exons 21 and 22. The products were normalized for internal Gapdh control and the ratio of increased brains injected with ASO to brains control without ASO is plotted. The black line indicates a proportion of 1 and without change in relation to the control without ASO, n = 4 (each group).
[033] [033] FIG 13A depicts a graphical plot of the percentage of exon 21x inclusion in the indicated mouse CNS samples.
[034] [034] FIG 13B depicts a graphical plot of the percentage of exon 20x inclusion in the indicated human CNS samples.
[035] [035] FIG. 14A depicts a graphical plot of the percentage decrease in the inclusion of 21x exon in the doses indicated.
[036] [036] FIG. 14B depicts a graphical plot of the percentage increase in Scnla mRNA at the indicated doses.
[037] [037] FIG. 14C depicts a graphical plot of the percent increase in Nav 1.1 protein levels at the indicated doses.
[038] [038] FIG. 15A depicts a graphical plot of the percentage decrease in the inclusion of 21x exon in the doses indicated.
[039] [039] FIG 15B depicts a graphical plot of the percentage increase in Scnla mRNA at the indicated doses.
[040] [040] FIG. 16 depicts a selected ASO targeting Scnla administered at a dose of 10 µg by means of ICV injection in mice on day 2 postnatally assessed on day 5 post-injection by Tagman-qPCR from SCNIA, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCNI0A and SCNI1A to assess target selectivity. Amplification results by Taqgman-qPCR normalized for Gapdh, obtained using ASO Ex20x + l1, are plotted as an increase ratio in relation to mice injected with PBS.
[041] [041] FIG. 17 depicts exemplary results of intracerebroventricular injection (ICV) on postnatal day 2 of an ASO selected at the dose indicated in wild type Fl (WT) mice or heterozygous Dravet (HET) mice from 129S-Scnlatwkea x C5S7BL / 6J crosses in 3 days post-injection.
[042] [042] FIG. 17A depicts a graph of the results of a Taqgman-qPCR assay performed using a probe that transposes exons 21 and 22. The products were normalized for internal Gapdh control and the ratio of increase in brains injected with ASO in relation to brains injected with PBS com is plotted.
[043] [043] FIG. 17B depicts a results plot of a western blot performed using an anti-Navl.l antibody. The products were normalized to bands stained with Ponceau and the ratio of increase in brains injected with ASO in relation to brains injected with PBS with is plotted.
[044] [044] FIG. 18 depicts exemplary results of an ASO microwalk from the 20x exon region of SCNIA in RenCells through free uptake. ASOs were designed to cover regions around three targeting ASOs previously identified in FIG. 6 (marked by stars) by changing 1 nucleotide at once (6-41) or by decreasing the length of ASO 17 (1-5). The graph depicts the percentage of exon 20x inclusion as measured by qPCR with SYBR Green. The black line indicates no change with respect to the one without ASO (=).
[045] [045] FIG. 19 is a graphical plot of the increase in the level of Scnla mRNA in coronal slides of mouse brains over time post-injection of an ASO targeting SCNIA. As pictured, the increase in the Scnla mRNA level was maintained for at least 80 days post-injection.
[046] [046] FIG. 20 is an exemplary survival curve that demonstrates the 100% survival benefit provided by an ASO targeting SCNIA in a Dravet mouse model. + / + Tapes for the WT genotype and +/- tapes for the 129S-scnlatmkea heterozygous genotype (Dravet mouse model); A tapes for treatment with PBS and B tapes for treatment with ASO. As pictured, mice in the A +/- group (Dravet mice receiving treatment with PBS) began to die around the 16th postnatal day, while all mice in the other three groups, including group B +/- (Dravet mice that receiving treatment with ASO), survived until at least the 35th postnatal day.
[047] [047] Intervening sequences or introns are removed by a large, highly dynamic RNA-protein complex called the spliceosome, which orchestrates complex interactions between “primary, small transcripts — nuclear RNAs (SNnRNAs) and a large number of splice -ome proteins are assembled ad hoc at each intron in an orderly manner, starting with recognition of the 5 'splice site (5's) by Ul snRNA or the 3' splice site (3's) via the U2 path, which involves the connection of the auxiliary factor U2 ( U2AF) to the 3'ss region to facilitate the connection of U2 to the branch point sequence (BPS). U2AF is a stable heterodimer composed of a 65 kD subunit encoded by U2AF2 (U2AF65), which binds to the polypyrimidine trace (PPT) and a 35 kD subunit encoded by U2AFl (U2AF35), which interacts with highly conserved AG dinucleotides in 3'ss and stabilizes the binding of UZ2AF65. In addition to the BPS / PPT unit and 3's / 5's, precise splicing requires sequences or auxiliary structures that activate or repress splice site recognition, known as intronic or exonic splicing promoters or silencers. These elements allow genuine splice sites to be recognized among a large excess of cryptic or pseudo-sites in the genome of higher eukaryotes, which have the same sequences,
[048] [048] The decision as to whether or not the splice should be done can typically be modeled as a stochastic and non-deterministic process, so that even the most well-defined splicing signals can sometimes splice incorrectly. However, under normal conditions, pre-mRNA splicing proceeds at surprisingly high fidelity. This is attributed, in part, to the activity of adjacent exonic and intronic auxiliary splicing regulatory elements (ESRs or ISRs). Typically, these functional elements are classified as exonic or intronic promoters (ESEss or ISEs) or silencers (ESSs or ISSs) based on their ability to stimulate or inhibit splicing, respectively. Although there is now evidence that some cis-acting auxiliary elements may act to influence the kinetics of the spliceosome assembly, for example, the arrangement of the complex between Ul snRNP and the 5'ss, it seems very likely that many elements will work in line with proteins of binding to trans-acting RNA (RBPs). For example, the serine-rich and arginine-rich family of RBPs (SR proteins) is a conserved family of proteins that play a crucial role in defining exons. SR proteins promote exon recognition by recruiting components of the pre-spliceosome to adjacent splice sites or by antagonizing the effects of ESSs in the surroundings. The repressive effects of ESSs can be mediated by members of the heterogeneous nuclear ribonucleoprotein (hnRNP) family and can alter the recruitment of central splicing factors to adjacent splice sites. In addition to their roles in the regulation of splicing, it is suggested that silencing elements have a role in the repression of pseudoexons, sets of intronic trap splice sites with the typical spacing of an exon, but without a functional open reading frame. ESEs and ESSs, in cooperation with their cognate trans-acting RBPs represent important components in a set of splicing controls that specify how, where and when mMRNASs are assembled from their precursors.
[049] [049] The sequences that mark exon-intron boundaries are degenerate signs of variable forces that can occur at high frequency within human genes. In multi-exon genes, different pairs of splice sites can be linked together in many different combinations, creating a diverse array of transcripts from a single gene. This is commonly referred to as alternative pre-mMRNA splicing. Although most mRNA isoforms produced by alternative splicing can be exported by the nucleus and translated into functional polypeptides, different mRNA isoforms from a single gene can vary markedly in their translation efficiency. In those mRNA isoforms with premature termination codons (PTCs) it is likely that at least 50 bp upstream of an exon junction complex will be targeted for degradation by the nonsense-mediated mRNA (NMD) decay pathway. Mutations in traditional splicing (BPS / PPT / 3'ss / 5'ss) and auxiliary motifs can cause aberrant splicing, for example, exon-skipping or inclusion of cryptic (or pseudo-) exon or splice site activation and contribute significantly for human morbidity and mortality. Both aberrant and alternative splicing patterns can be influenced by natural DNA variants in exons and introns.
[050] [050] Considering that the exon-intron limits can occur in any of the three positions of a codon, it is evident that only a subset of alternative splicing events can maintain the canonical open reading frame. For example, only exons that are evenly divisible by 3 can be skipped or included in the mRNA without any change in the reading frame. Splicing events that do not have compatible phases will induce a frame shift. Unless reversed by downstream events, frame-shifts can certainly lead to one or more PTCs, probably resulting in subsequent NMD degradation. NMD is a translation-coupled mechanism that eliminates mRNAS that contain PTCs. NMD can act as a surveillance route that exists in all eukaryotes. NMD can reduce errors in gene expression by eliminating mRNA transcripts that contain premature stop codons. The translation of these aberrant mRNASsS could, in some cases, lead to deleterious gain-of-function or dominant negative activity of the resulting proteins. NMD targets not only transcripts with PTCs, but also a wide array of mRNA isoforms expressed by many endogenous genes, suggesting that NMD is an important regulator that directs both fine and gross adjustments to the steady state RNA levels in the cell.
[051] [051] An NMD-inducing exon (NIE) is an exon or a pseudo-exon, which is a region within an intron and can activate the NMD pathway if included in a mature RNA transcript. In constitutive splicing events, the intron that contains an NIE is normally removed, but the intron or a portion of it (for example, NIE) can be retained during alternative or aberrant splicing events. Mature mRNA transcripts that contain such an NIE may be non-productive due to frame-shifts that induce the NMD pathway. The inclusion of an NIE in mature RNA transcripts may impair gene expression. MRNA transcripts that contain an NIE can be referred to as "mNRNA that contains NIE" or "NMD exon mRNA" in the present disclosure.
[052] [052] Cryptic splice sites (or splice pseudo-sites) have the same splicing recognition sequences as genuine splice sites, but are not used in splicing reactions. These numerically outperform genuine splice sites in the human genome by an order of magnitude and are usually repressed by very little understood molecular mechanisms. Cryptic 5 'splice sites have the NNN / GUNNNN or NNN / GCNNNN consensus, where N is any nucleotide and / is the exon-intron boundary. Cryptic 3 'splice sites have NAG / N consensus. Their activation is positively influenced by surrounding nucleotides that make them more similar to the optimal consensus of authentic splice sites, specifically MAG / GURAGU and YAG / G, respectively, in which Mé C or A, Ré GouAeY is CouU.
[053] [053] Splice sites and their regulatory sequences can be readily identified by those skilled in the art using appropriate publicly available algorithms listed, for example, in Kralovicova, JJ. and
[054] [054] Cryptic splice sites or regulatory splicing sequences can compete for RNA-binding proteins, such as U2AF, with an NIE splice site. In one embodiment, an agent can bind to the cryptic splice site or splicing regulatory sequences to prevent binding of RNA-binding proteins and, thus, favor the use of NIE splice sites.
[055] [055] In one embodiment, the cryptic splice site may not comprise the 5 'or 3' splice site of the NIE. The cryptic splice site can be at least 10 nucleotides upstream of the 5 "NIE splice site. The cryptic splice site can be at least 20 nucleotides upstream of the 5" NIE splice site. The cryptic splice site can be at least 50 nucleotides upstream of the 5 ”NIE splice site. The cryptic splice site can be at least 100 nucleotides upstream of the 5 "NIE splice site. The cryptic splice site can be at least 200 nucleotides upstream of the 5" NIE splice site.
[056] [056] The cryptic splice site can be at least nucleotides downstream of the NIE 3 "splice site. The cryptic splice site can be at least 20 nucleotides downstream of the NIE 3" splice site. The cryptic splice site can be at least 50 nucleotides downstream of the NIE 3 "splice site. The cryptic splice site can be at least 100 nucleotides downstream of the NIE 3" splice site. The cryptic splice site there may be at least 200 nucleotides downstream of the 3 'NIE splice site.
[057] [057] In some embodiments, the methods of the present disclosure explore the presence of NIE in the pre-mRNA transcribed by the SCNIA gene. Splicing of the SCNIA NIE pre-mRNA species identified to produce functional mature SCNIA mRNA can be induced with the use of a therapeutic agent such as, for example, an ASO that stimulates exon skipping of an NIE. The induction of exon-skipping can result in inhibition of an NMD pathway. The resulting mature SCNIA mRNA can be translated normally without activation of the NMD pathway, thereby increasing the amount of SCNI1A protein in the patient's cells and alleviating symptoms of a condition associated with SCNIA deficiency, for example, Dravet syndrome (DS ); epilepsy, generalized, with febrile seizures plus, type 2; febrile, familial seizures, 3A; autism; epileptic encephalopathy, early childhood, 13; sinus node disease 1; Alzheimer's disease; or SUDEP.
[058] [058] In various embodiments, the present disclosure provides a therapeutic agent that can target SCNIA mMRNA transcripts to modulate, for example, increase or inhibit, splicing or protein expression level. The therapeutic agent can be a small molecule, polynucleotide or polypeptide. In some embodiments, the therapeutic agent is an ASO. Several regions or sequences in the pre-
[059] [059] In some embodiments, a therapeutic agent described in that specification modulates the binding of a factor involved in splicing the NMD exon mRNA.
[060] [060] In some embodiments, a therapeutic agent described in that specification interferes with the binding of a factor involved in splicing the NMD exon mRNA.
[061] [061] In some embodiments, a therapeutic agent described in that specification prevents the binding of a factor involved in splicing the NMD exon mRNA.
[062] [062] In some embodiments, a therapeutic agent targets a target portion located in an intronic region between two canonical exon regions of the NMD exon mRNA encoding SCNIA and in which the intronic region contains the NMD exon.
[063] [063] In some embodiments, a therapeutic agent targets a targeted portion that overlaps at least partially with the NMD exon.
[064] [064] In some embodiments, a therapeutic agent targets a targeted portion that at least partially overlaps an intron upstream of the NMD exon.
[065] [065] In some embodiments, a therapeutic agent targets a targeted portion within the NMD exon.
[066] [066] In some embodiments, a therapeutic agent targets a target portion comprising at least about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive NMD exon nucleotides. In some embodiments, a therapeutic agent targets a targeted portion comprising at most about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20 , 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive NMD exon nucleotides. In some embodiments, a therapeutic agent targets a target portion comprising about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22 , 23, 24, 25, 26, 27, 28, 29,
[067] [067] In some embodiments, a therapeutic agent targets a targeted portion proximal to the NMD exon.
[068] [068] In some embodiments, ASO targets a sequence of about 4 to about 300 nucleotides upstream (or 5 ') from the 5' end of the NIE. In some embodiments, ASO targets a sequence of about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about 200 nucleotides, about 200 to about 250 nucleotides about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides, about 550 to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides, about 950 to about 1,000 nucleotides, about 1,050 to about
[069] [069] In some embodiments, ASO targets a sequence of about 4 to about 300 nucleotides upstream (or 5 ') from the 5' end of the NIE. In some embodiments, the ASO targets a sequence of at least about 1 nucleotide, at least about 10 nucleotides, at least about 20 nucleotides, at least about 50 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides , at least about 90 nucleotides, at least about 95 nucleotides, at least about 96 nucleotides, at least about 97 nucleotides, at least about 98 nucleotides, at least about 99 nucleotides, at least about 100 nucleotides, at least about 101 nucleotides, at least about 102 nucleotides, at least about 103 nucleotides, at least about 104 nucleotides, at least about 105 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least at least about 150 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about that of 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, or at least about 1,000 nucleotides upstream (or 5 ') from the 5' end of the NIE region.
[070] [070] In some embodiments, the ASO targets a sequence of about 4 to about 300 nucleotides upstream (or 5 ') from the 5' end of the NIE. In some embodiments, ASO targets a sequence at most about 10 nucleotides, at most about 20 nucleotides, at most about 50 nucleotides, at most about 80 nucleotides, at most about 85 nucleotides, at most about 90 nucleotides , at most about 95 nucleotides, at most about 96 nucleotides, at most about 97 nucleotides, at most about 98 nucleotides, at most about 99 nucleotides, at most about 100 nucleotides, at most about 101 nucleotides, at most about 102 nucleotides, at most about 103 nucleotides, at most about 104 nucleotides, at most about 105 nucleotides, at most about 110 nucleotides, at most about 120 nucleotides, at most about 150 nucleotides, maximum about 200 nucleotides, maximum about 300 nucleotides, maximum about 400 nucleotides, maximum about 500 nucleotides, maximum about 600 nucleotides, maximum about 700 nucleotides, at most about 800 nucleotides, at most about 900 nucleotides, at most about 1,000 nucleotides, at most about 1,100 nucleotides, at most about 1,200 nucleotides, at most about 1,300 nucleotides, at most about 1,400 nucleotides or, at most about
[071] [071] In some embodiments, the NIE as described in this specification is located between GRCh37 / hg1l9: chr2: 166,863,740 and GRCh37 / hg1l9: chr2: 166,863,803, as depicted in FIG. 2. In some embodiments, the 5 "end of the NIE is located at GRCh37 / hgl9: chr2: 166,863,803. In some embodiments, the 3 '" end of the NIE is located at GRCh37 / hgl9: chr2: 166,863,740.
[072] [072] In some embodiments, ASO targets a sequence of about 4 to about 300 nucleotides upstream (or 5 ') of the GRCh37 / hgl19: chr2: 166,863,803 genomic site. In some embodiments, ASO targets a sequence of about 1 to about 20 nucleotides, about 20 to about 50 nucleotides, about 50 to about 100 nucleotides, about 100 to about 150 nucleotides, about 150 to about about 200 nucleotides, about 200 to about 250 nucleotides, about 250 to about 300, about 250 to about 300 nucleotides, about 350 to about 400 nucleotides, about 450 to about 500 nucleotides about 550 up to about 600 nucleotides, about 650 to about 700 nucleotides, about 750 to about 800 nucleotides, about 850 to about 900 nucleotides about 950 to about 1,000 nucleotides, about 1,050 to about 1,100 nucleotides, about 1,150 to about
[073] [073] In some embodiments, ASO targets a sequence of about 4 to about 300 nucleotides upstream (or 5 ") of the GRCh37 / hgl9: chr2: 166,863,803 genomic site. In some embodiments, ASO targets at least one sequence about 1 nucleotide, at least about 10 nucleotides, at least about 20 nucleotides, at least about 50 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides, at least about 90 nucleotides, at least about 95 nucleotides, at least about 96 nucleotides, at least about 97 nucleotides, at least about 98 nucleotides, at least about 99 nucleotides, at least about 100 nucleotides, at least about 101 nucleotides, at least about 102 nucleotides, at least about 103 nucleotides, at least about 104 nucleotides, at least about 105 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least about 150 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least at least about 900 nucleotides, or at least about 1,000 nucleotides upstream (or 5 ') of the GRCh37 / hg1l9: chr2: 166,863,803 genomic site. In some embodiments, ASO targets a sequence of about 4 to about 300 nucleotides downstream (or 3 ') of GRCh37 / hg19: chr2: 166,863,740. In some embodiments, the ASO targets a sequence of at least about 1 nucleotide, at least about 10 nucleotides, at least about 20 nucleotides, at least about 50 nucleotides, at least about 80 nucleotides, at least about 85 nucleotides , at least about 90 nucleotides, at least about 95 nucleotides, at least about 96 nucleotides, at least about 97 nucleotides, at least about 98 nucleotides, at least about 99 nucleotides, at least about 100 nucleotides, at least about 101 nucleotides, at least about 102 nucleotides, at least about 103 nucleotides, at least about 104 nucleotides, at least about 105 nucleotides, at least about 110 nucleotides, at least about 120 nucleotides, at least at least about 150 nucleotides, at least about 200 nucleotides, at least about 300 nucleotides, at least about 400 nucleotides, at least about 500 nucleotides, at least about that of 600 nucleotides, at least about 700 nucleotides, at least about 800 nucleotides, at least about 900 nucleotides, or at least about 1,000 nucleotides downstream of GRCh37 / hgl9: chr2: 166,863,740. In some embodiments, ASO targets a sequence of more than 300 nucleotides downstream of GRCh37 / hgl9: chr2: 166,863,740.
[074] [074] In some modalities, ASO targets a sequence of about 4 to about 300 nucleotides upstream (or 5 ") of the genomic site GRCh37 / hgl9: chr2: 166,863,803. In some modalities, ASO targets a sequence at most about 10 nucleotides, maximum about 20 nucleotides, maximum about 50 nucleotides, maximum about 80 nucleotides, maximum about 85 nucleotides, maximum about 90 nucleotides, maximum about 95 nucleotides, maximum about 96 nucleotides, at most about 97 nucleotides, at most about 98 nucleotides, at most about 99 nucleotides, at most about 100 nucleotides, at most about 101 nucleotides, at most about 102 nucleotides, at most about 103 nucleotides, maximum about 104 nucleotides, maximum about 105 nucleotides, maximum about 110 nucleotides, maximum about 120 nucleotides, maximum about 150 nucleotides, maximum about 200 nucleotides, maximum about 300 nucleotides, at most about 400 nucleotides, at most about 500 nucleotides, at most about 600 nucleotides, at most about 700 nucleotides, at most about 800 nucleotides, at most about 900 nucleotides, at most about 1,000 nucleotides, at most about 1,100 nucleotides, at most about 1,200 nucleotides, at most about 1,300 nucleotides, at most about 1,400 nucleotides or at most about 1,500 nucleotides upstream (or 5) of the GRCh37 / hg19 genomic site : chr2: 166,863,803. In some embodiments, ASO targets a sequence of about 4 to about 300 nucleotides downstream (or 3 ") of GRCh37 / hgl9: chr2: 166,863,740. In some embodiments, ASO targets a sequence at most about 10 nucleotides, at most about 20 nucleotides, at most about 50 nucleotides, at most about 80 nucleotides, at most about 85 nucleotides, at most about 90 nucleotides, at most about 95 nucleotides, at most about 96 nucleotides, no maximum about 97 nucleotides, maximum about 98 nucleotides, maximum about 99 nucleotides, maximum about 100 nucleotides, maximum about 101 nucleotides, maximum about 102 nucleotides, maximum about 103 nucleotides, maximum about 104 nucleotides, at most about 105 nucleotides, at most about 110 nucleotides, at most about 120 nucleotides, at most about 150 nucleotides, at most about 200 nucleotides, at most about 300 nucleotides, at maximum about 400 nucleotides, maximum about 500 nucleotides, maximum about 600 nucleotides, maximum about 700 nucleotides, maximum about 800 nucleotides, maximum about 900 nucleotides or, maximum, about 1,000 nucleotides, at most about 1,100 nucleotides, at most about 1,200 nucleotides, at most about 1,300 nucleotides, at most about 1,400 nucleotides or at most about 1,500 nucleotides downstream of GRCh37 / hg19: chr2: 166,863,740. In some embodiments, ASO targets a sequence of more than 300 nucleotides downstream of GRCh37 / hgl19: chr2: 166,863,740.
[075] [075] As described in that specification in the Examples, the SCNIA gene (SEQ ID. No. 1) was analyzed for NIE and inclusion of a portion of intron 20 (SEQ ID. No. 4) (that portion is called exon 20x throughout the present disclosure) was observed. In some embodiments, the ASOs revealed in this specification report target a pre-mRNA that contains NIE (SEQ ID. No. 2) transcribed by a SCNIA genomic sequence. In some embodiments, the ASO targets a pre-mRNA transcript that contains NIE from a SCNIA genomic sequence that comprises a portion of the intron 20. In some embodiments, the ASO targets a pre-mRNA transcript that contains NIE from a genomic sequence of SCNIA comprising exon 20x (SEQ ID. No. 6). In some embodiments, the ASO targets a pre-mRNA transcript that contains the ID's NIE. SEQ. No. 2 or 12. In some embodiments, the ASO targets a pre-mRNA transcript that contains NIE's ID. SEQ. No. 2 or 12 comprising an NIE. In some embodiments, the ASO targets a pre-mRNA transcript that contains the ID's NIE. SEQ. No. 2 comprising exon 20x (SEQ ID. No. 10). In some embodiments, the ASOs disclosed in this specification report target a SCNIA pre-mRNA sequence (SEQ ID. No. 2 or 12). In some embodiments, the ASO targets a SCNIA pre-mRNA sequence that comprises an NIE (ID. DE
[076] [076] In some embodiments, the SCNIA pre-mRNA transcript containing NIE is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity for the ID. SEQ. No.: 1 or 11. In some embodiments, the SCNIA NIE pre-mRNA transcript comprises a sequence of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99 % or 100% sequence identity for any of the IDS. SEQ. Nº: 2-10 and 12-20.
[077] [077] In some embodiments, the SCNIA pre-mRNA transcript containing NIE (or NMD exon mMRNA) comprises a sequence of at least about 80%, 85%, 90%, 95% 97% or 100% string identity for any of the IDS. SEQ. Nº: 2, 7-10, 12 and 17-20. In some embodiments, the SCNIA pre-mRNA transcript that contains NIE (or NMD exon mMRNA) is encoded by a sequence of at least about 80%, 85%, 90%, 95%, 97% or 100% of string identity for IDS. SEQ. No.: 1, 3-6, 11 and 13-16. In some embodiments, the target portion of the NMD exon mRNA comprises a sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity for a region comprising at least 8 nucleic acids contiguous to the IDS. SEQ. Nº: 2, 7-10, 12 and 17-20.
[078] [078] In some embodiments, ASO targets exon 20 of a pre-mRNA that contains SCNIA NIE that comprises 20x exon of NIE. In some embodiments, ASO targets a sequence of exon 21 downstream (or 3 ") of exon 20x of NIE. In some embodiments, ASO targets a sequence of about 4 to about 300 nucleotides upstream (or 5 ') of the 5 'end of the 20x exon. In some embodiments, the ASO targets a sequence of about 4 to about 300 nucleotides downstream (or 3') of the 3 "end of the 20x exon. In some modalities, the ASO has a sequence according to any of the IDS. SEQ. No. 21-67. In some modalities, the ASO has a sequence according to any of the IDS. SEQ. No. *: 210-256.
[079] [079] In some embodiments, the ASO targets a sequence upstream of the 5 ”end of an NIE. For example, ASOsS that target a sequence upstream of the 5 'end of an NIE (for example, 20x exon in human SCNIA, or 21x exon in mouse SCNIA) can comprise a sequence with at least 80%, 85%, 90% , 95%, 97% or 100% sequence identity for any of the IDS. SEQ. No. *: 21-38. As another example, ASOs that target a sequence upstream of the 5 'end of an NIE (e.g., 20x exon in human SCNIA, or 21x exon in mouse SCNI1A) may comprise a sequence of at least 80%, 85%, 90 %, 95%, 97% or 100% sequence identity for any of the IDS. SEQ.
[080] [080] In some embodiments, ASO targets 20x exon in a SCNIA pre-mRNA that contains NIE that comprises 20x exon. In some embodiments, the ASO targets a sequence of the 20x exon downstream (or 3 ') of the 5' end of the 20x exon of a SCNIA pre-mRNA. In some embodiments, the ASO targets a 20x exon sequence upstream (or 5 ') from the 3 "end of the 20x exon of a SCNIA pre-mRNA.
[081] [081] In some embodiments, the target portion of the SCNIA pre-mRNA that contains NIE is in intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 (intron numbering that corresponds to the mRNA sequence in NM 006920). In some embodiments, hybridization of an ASO to the target portion of the pre-mRNA NIE results in exon-skipping of at least one of the NIE within intron 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 and subsequently increases the production of SCNI1A protein. In some embodiments, hybridization of an ASO to the targeted portion of the pre-mRNA NIE inhibits or blocks exon-skipping of at least one of the NIE within intron 1, 2, 3, 4, 5, 6, 7, 8, 9 , 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 or 25 and subsequently decreases the production of SCNIA protein. In some embodiments, the target portion of the SCNIA pre-mRNA that contains NIE is at intron 20. Those skilled in the art can determine the number of the corresponding intron in any isoform based on an intron sequence provided in that specification or using of the number provided in reference to the mRNA sequence in NM 006920, NM 001202435, NM 001165964 or NM 001165963. Those skilled in the art can also determine flanking exon sequences in any isoform of SCNIA for targeting using the methods of the invention, based on the methods in an intron sequence provided in that specification or using the intron number provided in reference to the mRNA sequence in NM 006920, NM 001202435, NM 001165964 or NM 001165963.
[082] [082] In some embodiments, the methods and compositions of the present disclosure are used to modulate, for example, increase or decrease, the expression of SCNIA by inducing or inhibiting exon-skipping of a pseudoexon of a pre-mRNA containing SCN1A NIE. In some embodiments, a pseudoexon is a sequence within any of introns 1-25. In some modalities, the pseudoexon is a sequence within any of the introns 2, 4, 6, 13, 14, 15, 16, 17, 18, 20, 21, 22, 23, 24 and 25. In some modalities, the pseudoexon is a sequence within any of introns 15, 18 and 19. In some embodiments, the pseudoexon can be any SCNIA intron or a portion thereof. In some embodiments, the pseudo-exon is within the intron 20. The numbering of the SCNIA intron used in this specification corresponds to the mRNA sequence in NM 006920. It is understood that the intron numbering may change in reference to a SCNIA isoform sequence different.
[083] [083] The SCNIA gene can encode the SCNIA protein (voltage-controlled sodium channel, type I, alpha subunit), which can also be called the voltage-controlled sodium channel alpha subunit Na ', l.1. Also described above, SCN1A mutations in DS are spread throughout the protein. More than 100 new mutations have been identified across the gene with the most debilitating ones emerging again. These comprise mutations by truncations (47%), of sense change (missense) (43%), deletions (3%) and splice site (7%). The percentage of individuals who carry SCNIA mutations varies between 33 and 100%. Most of the mutations are formed by unprecedented changes (88%).
[084] [084] In some embodiments, the methods described in this specification are used to modulate, for example, increase or decrease, the production of a functional SCNI1A protein. As used in this specification, the term "functional" refers to the amount of activity or function of an SCNIA protein that is required to eliminate any one or more symptoms of a treated condition, for example, Dravet's syndrome; epilepsy, generalized, with febrile seizures plus, type 2; febrile, familial seizures, 3A; autism; epileptic encephalopathy, early childhood, 13; sinus node disease 1; Alzheimer's disease; or SUDEP. In some embodiments, the methods are used to increase the production of a partially functional SCNIA protein. As used in this specification, the term “partially functional” refers to any amount of activity or function of the SCNIA protein that is less than the amount of activity or function that is required to eliminate or prevent any or more symptoms of a disease or condition. In some embodiments, a partially functional protein or RNA will have at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, or at least 95% less activity compared to the fully functional protein or RNA.
[085] [085] In some embodiments, the method is a method of increasing the expression of the SCNIA protein by an individual's cells that have a pre-mRNA that contains NIE encoding the SCNIA protein, in which the individual has Dravet's syndrome caused by a deficient amount of SCNIA protein activity and where the deficient amount of SCNIA protein is caused by haploinsufficiency of the SCNI1A protein. In this modality, the individual has a first allele that encodes a functional SCNIA protein and a second allele from which the SCNIA protein is not produced. In another such modality, the individual has a first allele that encodes a functional SCNIA protein and a second allele that encodes a non-functional SCNIA protein. In another such modality, the individual has a first allele that encodes a functional SCNIA protein and a second allele that encodes a partially functional SCNIA protein. In any of these modalities, the antisense oligomer binds to a targeted portion of the pre-mRNA that contains NIE transcribed by the second allele, thereby inducing exon-skipping of the pseudoexon by the pre-mRNA and causing an increase in the level of mMRNA mature encoding functional SCNIA protein and an increase in SCNIA protein expression in the individual's cells.
[086] [086] In related modalities, the method is a method of using an ASO to increase the expression of a functional protein or RNA. In some embodiments, an ASO is used to increase the expression of SCNI1A protein in an individual's cells that have a pre-mRNA that contains NIE encoding SCNIA protein, in which the individual has the deficiency, for example, Dravet syndrome (DS ) (also known as SMEI); childhood severe myoclonic epilepsy (SMEI) -orderline (SMEB); febrile seizure (FS); epilepsy, generalized, with febrile seizures plus (GEFS +); epileptic encephalopathy, early childhood, 13; generalized cryptogenic epilepsy; focal cryptogenic epilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; West syndrome; “idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); pacemaker syndrome on the left 1; early childhood SCNIA encephalopathy; early childhood epileptic encephalopathy (EEEE); or autism, in the amount or function of an SCNIA protein. In some embodiments, an ASO is used to increase the expression of SCNI1A protein in an individual's cells, in which the individual has a deficiency, for example, early childhood epileptic encephalopathy, 13; in the amount or function of the SCN8A protein. In some embodiments, an ASO is used to increase the expression of SCN1A protein in an individual's cells, in which the individual has a deficiency, for example, sinus node disease 1; in the amount or function of a SCNS5A protein.
[087] [087] In some embodiments, the pre-mRNA transcript that contains NIE that encodes the protein that causes the disease or condition is targeted by the ASOs described in this specification. In some embodiments, a pre-mRNA transcript that contains NIE that encodes a protein that does not cause the disease is targeted by ASOs. For example, a disease that is the result of a mutation or deficiency of a first protein in a particular pathway can be improved by targeting a pre-mRNA that contains NIE that encodes a second protein, thereby increasing the production of the second protein . In some embodiments, the function of the second protein is able to compensate for the mutation or deficiency of the first protein (which causes the disease or condition).
[088] [088] In some modalities, the individual has: (a) a first mutant allele of which: (i) the SCNIA protein is produced at a reduced level, compared to production by a wild type allele, (ii) the protein SCNIA is produced in a form that has reduced function, compared to an equivalent wild-type protein, or (iii) the SCNIA protein or functional RNA is not produced; and (b) a second mutant allele of which: (i) the SCNIA protein is produced at a reduced level, compared to production by a wild type allele, (ii) the SCNIA protein is produced in a form that has reduced function , compared to an equivalent wild-type protein, or (iii) the SCNIA protein is not produced, and in which the pre-mRNA containing NIE is transcribed by the first and / or the second allele. In these modalities, the ASO binds to a target portion of the pre-mRNA that contains NIE transcribed by the first or second allele, thereby inducing exon-skipping of the pseudoexon by the pre-mRNA that contains NIE and causing an increase in the level of mMRNA encoding SCNIA protein and an increase in expression of the target protein or functional RNA in the individual's cells. In these modalities, the target protein or functional RNA that has an increase in the level of expression resulting from the exon skipping of the pseudoexon by the pre-mRNA that contains NIE is in a form that has reduced function, compared to the equivalent wild-type protein ( partially functional), or that has full function, compared to the equivalent wild-type protein (fully functional).
[089] [089] In some embodiments, the level of mRNA encoding SCNI1A protein is increased 1.1 to 10 times when compared to the amount of mRNA encoding SCN1A protein that is produced in a control cell, for example, one that does not it is treated with the antisense oligomer or one that is treated with an antisense oligomer that does not bind to the target portion of the SCNIA pre-mRNA that contains NIE.
[090] [090] In some embodiments, an individual treated using the methods of the present disclosure expresses a partially functional SCN1A protein by an allele, wherein the partially functional SCN1A protein is caused by a frame-shift mutation, a nonsense mutation, an exchange mutation of sense, or a partial gene deletion. In some embodiments, an individual treated using the methods of the invention expresses a non-functional SCNIA protein by an allele, in which the non-functional SCNI1A protein is caused by a frame-shift mutation, a nonsense mutation, a sense exchange mutation, a partial gene deletion, in an allele. In some embodiments, an individual treated using the methods of the invention has a deletion of the entire SCNIA gene, in an allele.
[091] [091] In some embodiments, the method is a method of decreasing the expression of the SCNIA protein by cells in an individual that have a pre-mRNA that contains NIE that encodes the SCNIA protein and in which the individual has a gain-de -function in Navl.1. In this modality, the individual has an allele from which the SCNIA protein is produced in a high amount or an allele that encodes a mutant SCN1A that induces increased Navl.1 activity in the cell. In some modalities, the increased activity of Navl.1 is characterized by a prolonged or almost persistent sodium current mediated by the mutant Navl.l channel, a slowing of rapid inactivation, a positive change in inactivation in the steady state, increased channel availability during repetitive stimulation, persistent increased non-inactivated sodium currents induced by depolarization, delayed entry into inactivation, accelerated recovery from rapid inactivation, and / or rescue of folding defects by incubation at a lower temperature or co-expression of interacting proteins. In any of these modalities, the antisense oligomer binds to a targeted portion of the pre-mRNA that contains NIE transcribed by the second allele, thereby inhibiting or blocking the exon-skipping of the pseudoexon by the pre-mRNA and causing a decrease in level of mature mRNA encoding functional SCNI1A protein and a decrease in SCNI1A protein expression in the individual's cells.
[092] [092] In related modalities, the method is a method of using an ASO to decrease the expression of a functional protein or RNA. In some embodiments, an ASO is used to decrease the expression of SCNI1A protein in an individual's cells that have a pre-mRNA that contains NIE that encodes SCN1A protein. In some modalities, the individual has a gain-of-function mutation in Na, vl.1, for example, migraine. In some modalities, an ASO is used to decrease the expression of SCNIA protein in an individual's cells, the individual has a gain-of-function mutation in Navl.1, for example, migraine, family hemiplegic, 3.
[093] [093] In some embodiments, the level of mRNA that encodes SCNIA protein is decreased 1.1 to 10 times when compared to the amount of mRNA that encodes SCN1A protein that is produced in a control cell, for example, one that does not it is treated with the antisense oligomer or one that is treated with an antisense oligomer that does not bind to the target portion of the SCNIA pre-mRNA that contains NIE.
[094] [094] In some embodiments, an individual treated using the methods of the present disclosure expresses a mutant SCN1A protein by an allele, in which the mutant SCNIA protein is caused by a frame-shift mutation, a nonsense mutation, a sense exchange mutation , or a partial gene deletion and in which the mutant SCNIA protein causes a high level of Na activity, l.1. In some embodiments, an individual treated using the methods of the present disclosure expresses a high amount of SCNIA protein by an allele as a result of a frame-shift mutation, a nonsense mutation, a sense exchange mutation or a partial gene deletion.
[095] [095] In embodiments of the present invention, an individual may have a mutation in SCNIA. Mutations in SCNI1A may be spread throughout that gene. The SCNIA protein can consist of four domains. Said SCNI1A domains can have transmembrane segments. Mutations in said SCNIA protein can arise throughout that protein. Said SCNIA protein can consist of at least two isoforms. Mutations in SCNI1A can be composed of R931C, R946C, M9341I, R1648C or R1648H. In some cases, mutations in a C-terminus of an SCNI1A protein can be observed. Mutations in an SCNIA protein can also be found in loops between segments 5 and 6 of the first three domains of that SCNIA protein. In some cases, mutations in an N-terminus of an SCNI1A protein can be observed. Exemplary mutations within SCNIA include, without limitation, R222X, R712X, 1227S, R1892X, W952X, R1245X, R1407X, W1434R, c.4338 + 1G> A, S1516X, L1670fsX1678 or KI846fsX1856. Mutations that can be targeted with the present invention can also encode a pore of an ion channel.
[096] [096] In some embodiments, the methods and compositions described in this specification can be used to treat DS. In other modalities, the methods and compositions described in this specification can be used to treat severe childhood myoclonic epilepsy (SMEI). In other modalities, the methods and compositions described in this specification can be used to treat borderline Dravet syndrome; epilepsy, generalized, with febrile seizures plus, type 2; febrile, familial seizures, 3A; migraine, family hemiplegic, 3; autism;
[097] [097] In some embodiments, an individual who has any SCNIA mutation known in the art and described in the literature cited above (for example, by Hamdan et al., 2009, Mulley et al., 2005) can be treated using the methods and compositions described in this specification. In some embodiments, the mutation is within any SCNIA intron or exon.
[098] [098] As used in this specification, a “pre-mRNA that contains NIE” is a pre-mRNA transcript that contains at least one pseudoexon. Alternative or aberrant splicing may result in the inclusion of (at least one) pseudoexon in the mature mRNA transcripts. The terms "mature mRNA" and "fully-spliced mRNA" are used interchangeably in this specification to describe a fully processed mRNA. The inclusion of (at least one) pseudoexon can be non-productive mRNA and lead to mature mMRNA NMD. Mature mRNA that contains NIE can sometimes lead to aberrant protein expression.
[099] [099] In some embodiments, the included pseudo-exon is the most abundant pseudo-exon in a population of pre-mRNAs that contain NIE transcribed by the gene that encodes the target protein in a cell. In some embodiments, the included pseudo-exon is the most abundant pseudo-exon in a population of pre-mRNAs that contain NIE transcribed by the gene encoding the target protein in a cell, where the population of pre-mRNAs that contain NIE comprises two or more pseudo-exons included. In some embodiments, an antisense oligomer targeting the most abundant pseudoexon in the population of pre-mRNAs that contain NIE encoding the target protein induces exon-skipping of one or two or more pseudoexons in the population, including the pseudoexon to which the oligomer antisense is targeted or binds. In modalities, the target region is in a pseudo-exon which is the most abundant pseudo-exon in a pre-mRNA that contains NIE that encodes the SCNI1A protein.
[100] [100] The degree of exon inclusion can be expressed as a percentage exon inclusion, for example, the percentage of transcripts in which a certain pseudoexon is included. In summary, the percentage exon inclusion can be calculated as the percentage of the amount of RNA transcripts with the inclusion of exon, over the sum of the average amount of RNA transcripts with inclusion of exon plus the average amount of RNA transcripts excluding exon.
[101] [101] In some embodiments, an included pseudoexon is an exon that is identified as an included pseudoexon based on a determination of at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, or at least about 50% inclusion. In embodiments, an included pseudoexon is an exon that is identified as an included pseudoexon based on a determination of about 5% to about 100%, about 5% to about 95%, about 5% to about 90 %, about 5% to about 85%, about 5% to about 80%, about 5% to about 75%, about 5% to about 70%, about 5% to about 65 %, about 5% to about 60%, about 5% to about 55%, about 5% to about 50%, about 5% to about 45%, about 5% to about 40 %, about 5% to about 35%, about 5% to about 30%, about 5% to about 25%, about 5% to about 20%, about 5% to about 15 %, about 10% to about 100%, about 10% to about 95%, about 10% to about 90%, about 10% to about 85%, about 10% to about 80 %, about 10% to about 75%, about 10% to about 70%, about 10% to about 65%, about 10% to about 60%, about 10% to about 55 %, about 10% until about about 50%, about 10% to about 45%, about 10% to about 40%, about 10% to about 35%, about 10% to about 30%, about 10% to about 25%, about 10% to about 20%, about 15% to about 100%, about 15% to about 95%, about 15% to about 90%, about 15% to about 85%, about 15% to about 80%, about 15% to about 75%, about 15% to about 70%, about 15% to about 65%, about 15% to about 60%, about 15% to about 55%, about 15% to about 50%, about 15% to about 45%, about 15% to about 40%, about 15% to about 35%, about 15% to about 30%, about 15% to about 25%, about 20% to about 100%, about 20% to about 95%, about 20% to about 90%, about 20% to about 85%, about 20% to about 80%, about 20% to about 75%, about 20% to about 70%, about 20% to about 65%, about 20% to about 60%, about 20% to about 55%, about 20% to about 50%, about 20% to about 45%, about 20% to about 40%, about 20% to about 35%, about 20% to about 30%, about 25% to about 100%, about 25% to about 95%, about 25% to about 90%, about 25% to about 85%, about 25% to about 80%, about 25% to about 75%, about 25% to about 70%, about 25% to about 65%, about 25% to about 60%, about 25% to about 55%, about 25% to about 50%, about 25% to about 45%, about 25% to about 40%, or about 25% to about 35% of inclusion. ENCODE data (described, for example, by Tilgner et al., 2012, “Deep Sequencing of Subcellular RNA Fractions Shows Splicing to be Predominantly Co-Transcriptional in the Human Genome but Inefficient for lncRNAS”, Genome Research 22 (9): 1,616 -25) can be used to assist in identifying exon inclusion.
[102] [102] In some embodiments, contact of cells with an ASO that is complementary to a targeted portion of a SCNIA pre-mRNA transcript results in an increase in the amount of SCNIA protein produced by at least 10, 20, 30, 40 , 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500 or 1,000%, compared to the amount of protein produced by a cell in the absence of ASO / no treatment.
[103] [103] In some embodiments, contact of cells with an ASO that is complementary to a targeted portion of a SCNIA pre-mRNA transcript results in a decrease in the amount of SCNIA protein produced by at least 10, 20, 30, 40 , 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500 or 1,000%, compared to the amount of protein produced by a cell in the absence of ASO / no treatment.
[104] [104] In some embodiments, contact of cells with an ASO that is complementary to a targeted portion of a SCNIA pre-mRNA transcript results in an increase in the amount of mRNA encoding SCNIA, including the mature mRNA encoding the protein -target. In some embodiments, the amount of mRNA encoding SCNIA protein, or the mature mMRNA encoding SCNIA protein, is increased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500 or 1,000%, compared to the amount of protein produced by a cell in the absence of ASO / no treatment. In some embodiments, the total amount of mMRNA encoding SCNIA protein, or mature mRNA encoding SCNIA protein produced in the cell with which the antisense oligomer is brought into contact is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 time, at least c about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times compared to the amount of mature RNA produced in an untreated cell, for example, an untreated cell or a cell treated with a control compound. A control compound can be, for example, an oligonucleotide that is not complementary to a target portion of the SCNI1A pre-mRNA that contains NIE.
[105] [105] In some embodiments, contact of cells with an ASO that is complementary to a targeted portion of a SCNIA pre-mRNA transcript results in a decrease in the amount of mRNA that encodes SCNIA, including the mature mRNA that encodes the protein -target. In some embodiments, the amount of mRNA encoding SCNIA protein, or the mature mMRNA encoding SCNIA protein, is decreased by at least 10, 20, 30, 40, 50, 60, 80, 100, 150, 200, 250, 300, 350, 400, 450, 500 or 1,000%, compared to the amount of protein produced by a cell in the absence of ASO / no treatment. In some embodiments, the total amount of mMRNA encoding SCNIA protein, or mature mRNA encoding SCNIA protein produced in the cell with which the antisense oligomer is brought into contact is decreased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times,
[106] [106] The NIE can be of any length. In some embodiments, the NIE comprises a complete sequence of an intron, when it may then be referred to as intron retention. In some embodiments, the NIE can be a portion of the intron. In some embodiments, the NIE may be a portion of the 5 "end of an intron including a 5's sequence. In some embodiments, the NIE may be a portion of the 3" end of an intron including a 3's sequence. In some modalities, the NIE may be a portion within an intron without including a 5's sequence In some modalities, the NIE may be a portion within an intron without including a 3's sequence In some modalities, the NIE it can be a portion within an intron without including a 5's sequence or a 3's sequence. In some embodiments, the NIE can be 5 nucleotides to 10 nucleotides in length, 10 nucleotides to 15 nucleotides in length, 15 nucleotides to 20 nucleotides in length, from 20 nucleotides to 25 nucleotides in length, from 25 nucleotides to 30 nucleotides in length, from 30 nucleotides to 35 nucleotides in length, from 35 nucleotides to 40 nucleotides in length, from 40 nucleotides to 45 nucleotides in length otids in length, from 45 nucleotides to 50 nucleotides in length, from 50 nucleotides to 55 nucleotides in length, from 55 nucleotides to 6 € 60 nucleotides in length, from 60 nucleotides to 65 nucleotides in length, from 65 nucleotides to 70 nucleotides in length , from 70 nucleotides to 75 nucleotides in length, from 75 nucleotides to 80 nucleotides in length, from 80 nucleotides to 85 nucleotides in length, from 85 nucleotides to 90 nucleotides in length, from 90 nucleotides to 95 nucleotides in length or from 95 nucleotides to 100 nucleotides in length.
[107] [107] The inclusion of a pseudoexon can lead to a frame shift and the introduction of a premature termination codon (PIC) in the mature mRNA transcript making the transcript a NMD target. The transcript of mature mRNA that contains NIE can be transcribed from non-productive mRNA that does not lead to protein expression. The PIC can be present in any position downstream of an NIE. In some modalities, the PIC can be present in any exon downstream of an NIE. In some modalities, the PIC may be present within the NIE. For example, the inclusion of exon 20x in an mRNA transcript encoded by the SCNIA gene can induce a PIC in the mRNA transcript, for example, a PIC in exon 21 of the mMRNA transcript.
[108] [108] In various embodiments of the present disclosure, compositions and methods comprising a therapeutic agent are provided to modulate the level of SCNIA protein expression. In some embodiments, compositions and methods for modulating alternative SCNAl pre-mRNA splicing are provided in this specification. In some embodiments, compositions and methods for inducing exon-skipping in SCNIA pre-mRNA splicing, for example, for inducing skipping of a pseudoexon during SCNIA pre-mRNA splicing, are provided in this specification. In other embodiments, therapeutic agents can be used to induce the inclusion of an exon in order to decrease the level of protein expression.
[109] [109] In some embodiments, a therapeutic agent disclosed in this specification is a small molecule, a polypeptide, or a polynucleic acid polymer. In some cases, the therapeutic agent is a small molecule. In some cases, the therapeutic agent is a polypeptide. In some cases, the therapeutic agent is a polynucleic acid polymer. In some cases, the therapeutic agent is a repressive agent. In additional cases, the therapeutic agent is a promoting agent.
[110] [110] A therapeutic agent disclosed in this specification may be an NIE-suppressing agent. A therapeutic agent can comprise a polynucleic acid polymer.
[111] [111] In accordance with one aspect of the present disclosure, in that specification is provided a method of treating or preventing a condition associated with a functional SCNIA protein deficiency, which comprises administering an NIE-repressing agent to an individual to increase levels of functional SCNIA protein, where the agent binds to a region of the pre-mRNA transcript to decrease the inclusion of NIE in the mature transcript. For example, in that specification a method of treating or preventing a condition associated with a deficiency of functional SCNIA protein is provided, which comprises administering an NIE-repressing agent to an individual to increase levels of functional SCNIA protein, where the agent binds to an intron region that contains an NIE (for example, intron 20 in the human SCNI1A gene) of the pre-mRNA transcript or to a regulatory sequence for NIE activation in the same intron.
[112] [112] When reference is made to reducing the inclusion of NIE in the mature mRNA, the reduction may be complete, for example, 100%, or it may be partial. The reduction can be clinically significant. The reduction / correction can be in relation to the level of inclusion of NIE in the individual without treatment, or in relation to the amount of inclusion of NIE in a population of similar individuals. The reduction / correction can be at least 10% less inclusion of NIE in relation to the average individual, or to the individual before treatment. The reduction can be at least 20% less inclusion of NIE in relation to an average individual, or to the individual before treatment. The reduction can be at least 40% less inclusion of NIE in relation to an average individual, or to the individual before treatment. The reduction can be at least 50% less inclusion of NIE in relation to an average individual, or to the individual before treatment. The reduction can be at least 60% less inclusion of NIE in relation to an average individual, or to the individual before treatment. The reduction can be at least 80% less inclusion of NIE in relation to an average individual, or to the individual before treatment. The reduction can be at least 90% less inclusion of NIE in relation to an average individual, or to the individual before treatment.
[113] [113] When reference is made to increasing levels of active SCNIA protein, the increase can be clinically significant. The increase may be in relation to the level of active SCNIA protein in the untreated individual, or in relation to the amount of active SCNI1A protein in a population of similar individuals. The increase can be at least 10% more active SCNIA protein compared to the average individual,
[114] [114] In embodiments in which the NIE-repressing agent comprises a polynucleic acid polymer, the polynucleic acid polymer can be about 50 nucleotides in length. The polynucleic acid polymer can be about 45 nucleotides in length. The polynucleic acid polymer can be about 40 nucleotides in length. The polynucleic acid polymer can be about 35 nucleotides in length. The polynucleic acid polymer can be about 30 nucleotides in length. The polynucleic acid polymer can be about 24 nucleotides in length. The polynucleic acid polymer can be about 25 nucleotides in length. The polynucleic acid polymer can be about 20 nucleotides in length.
[115] [115] The polynucleic acid polymer sequence can be at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85% 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 99.5% complementary to a target sequence of an mRNA transcript, for example, a partially processed mRNA transcript. The sequence of the polynucleic acid polymer can be 100% complementary to a target sequence of a pre-mRNA transcript.
[116] [116] The polynucleic acid polymer sequence can have 4 or fewer pairing errors for a target sequence of the pre-mRNA transcript. The polynucleic acid polymer sequence can have 3 or less pairing errors for a target sequence of the pre-mRNA transcript. The polynucleic acid polymer sequence can have 2 or less pairing errors for a target sequence of the pre-mRNA transcript. The sequence of the polynucleic acid polymer may have 11 or fewer pairing errors for a pre-mRNA transcript target sequence. The sequence of the polynucleic acid polymer may have no mismatch for a target sequence of the pre-mRNA transcript.
[117] [117] The polynucleic acid polymer can hybridize specifically to a target sequence of the pre-mRNA transcript. For example, the polynucleic acid polymer can have 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5% or
[118] [118] The polynucleic acid polymer can have a sequence of at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93% , 94%, 95%, 96%, 97%, 98%, 99% or 99.5% sequence identity for a sequence selected from the group consisting of IDS. SEQ. Nº: 21-
[119] [119] When reference is made to a polynucleic acid polymer sequence, those skilled in the art will understand that one or more substitutions can be tolerated, optionally two substitutions can be tolerated in the sequence, so that it maintains the ability to hybridize to the target sequence; or, when the substitution is in a target sequence, the ability to be recognized as the target sequence. References to sequence identity can be determined by aligning BLAST sequences using parameters-
[120] [120] A composition comprising an antisense oligomer that induces exon-skipping by binding to a target portion of a SCNIA pre-mRNA containing NIE is provided in this specification. As used in this specification, the terms “ASO” and “antisense oligomer” are used interchangeably and refer to an oligomer such as a Ppolinucleotide, which comprises nucleobases that hybridize to a target nucleic acid sequence (for example, a SCNIA pre-mRNA that contains NIE) by Watson-Crick base pairing or oscillation base pairing (GU). The ASO can have the exact sequence complementary to the target sequence or almost complementarity (for example, sufficient complementarity to bind to the target sequence and increase splicing at a splice site). ASOs are designed so that they bind (hybridize) to a target nucleic acid (for example, a targeted portion of a pre-mRNA transcript) and remain hybridized under physiological conditions. Typically, if they hybridize to a site other than the desired (target) nucleic acid sequence, they hybridize to a limited number of sequences that are not a target nucleic acid (for a few different sites than a target nucleic acid). The design of an ASO can take into account the occurrence of the nucleic acid sequence of the targeted portion of the pre-mRNA transcript or a sufficiently similar nucleic acid sequence at other locations in the genome or cellular pre-mRNA or transcriptome, so that the The likelihood that the ASO will link to other sites and cause “off-target” effects is limited. Any antisense oligomers known in the art, for example, in PCT Application No. PCT / US2014 / 054151, published as WO 2015/035091, entitled “Reducing Nonsense-Mediated mRNA Decay”, incorporated by reference in this specification, can be used to the practice of the methods described in this specification.
[121] [121] In some embodiments, ASOS "specifically hybridize" to or are "specific" to a target nucleic acid or a targeted portion of a pre-mRNA that contains NIE. Typically this hybridization occurs at a Tr substantially greater than 37 ° C, preferably at least 50 ° C, and typically between 60 ° C to approximately 90 ° C. Such hybridization preferably corresponds to stringent hybridization conditions. At a certain ionic strength and pH, Tn is the temperature at which 50% of a target sequence hybridizes to a complementary oligonucleotide.
[122] [122] Oligomers, for example, oligonucleotides, are "complementary" to each other when hybridization occurs in an antiparallel configuration between two single-stranded polynucleotides. A double-stranded polynucleotide can be "complementary" to another polynucleotide if hybridization can occur between one strand of the first and the second polynucleotide. Complementarity (the degree to which one polynucleotide is complementary to another) is quantifiable in terms of the proportion (for example, the percentage) of bases on opposite strands that are supposed to form hydrogen bonds between them, according to base pairing rules generally accepted. The sequence of an antisense oligomer (ASO) need not be 100% complementary to that of its target nucleic acid to hybridize. In certain embodiments, ASOs may comprise at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98 % or at least 99% sequence complementarity for a target region within the target sequence of nucleic acids to which they target. For example, an ASO in which 18 out of 20 nucleobases of the oligomeric compound are complementary to a target region and therefore would hybridize specifically would represent 90 percent complementarity. In this example, the remaining non-complementary nucleobases would be grouped together or spaced with complementary nucleobases and need not be contiguous with each other or with complementary nucleobases. The percentage of complementarity of an ASO with a region of a target nucleic acid can be determined routinely using BLAST programs (basic local alignment research tools) and PowerBLAST programs known in the art (Altschul et al., J. Mol. Biol. , 1990, 215, 403-410; Zhang and Madden, Genome Res., 1997, 7, 649-656).
[123] [123] An ASO does not need to hybridize for all nucleobases in a target sequence and the nucleobases to which it hybridizes can be contiguous or non-contiguous. ASOs can hybridize over one or more segments of a pre-mRNA transcript, so that intervening or adjacent segments are not involved in the hybridization event (for example, a loop structure or hairpin structure can be formed). In certain embodiments, an ASO hybridizes to non-contiguous nucleobases in a target pre-mRNA transcript. For example, an ASO can hybridize to nucleobases in a pre-mRNA transcript that are separated by one or more nucleobases to which the ASO does not hybridize.
[124] [124] The ASOs described in this specification include nucleobases that are complementary to nucleobases present in a targeted portion of a pre-mRNA that contains NIE. The term "ASO" encompasses oligonucleotides and any other oligomeric molecule that comprises nucleobases capable of hybridization to a complementary nucleobase in a target mRNA, but does not comprise a portion of sugar, for example, a peptide nucleic acid (PNA). ASOsS can comprise naturally occurring nucleotides, nucleotide analogs, modified nucleotides, or any combination of two or three of the foregoing. The term "naturally occurring nucleotides" includes deoxyribonucleotides and ribonucleotides. The term "modified nucleotides" includes nucleotides with modified or substituted sugar groups and / or which have a modified framework. In some embodiments, all ASO nucleotides are modified nucleotides. Chemical modifications of ASOs or components of ASOs that are compatible with the methods and compositions described in this specification will be evident to those skilled in the art and can be found, for example,
[125] [125] One or more nucleobases from an ASO can be any naturally occurring, unmodified nucleobase, for example, adenine, guanine, cytosine, thymine and uracil, or any synthetic or modified nucleobase that is sufficiently similar to an unmodified nucleobase from so that it is capable of hydrogen bonding to a nucleobase present in a target pre-mRNA. Examples of modified nucleobases include, without limitation, hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, S5-methylcytosine and 5-hydroxymethylcytosine.
[126] [126] The ASOs described in this specification also comprise a framework structure that connects the components of an oligomer. The terms "frame structure" and "oligomer bonds" can be used interchangeably and refer to the connection between ASO monomers. In naturally occurring oligonucleotides, the framework comprises a 3'-5 'phosphodiester bond that connects sugar portions of the oligomer. The framework structure or oligomer bonds of the ASOs described in this specification may include (without limitation) phosphorothioate, phosphorodithioate, phosphoroselenoate, phosphorodiselenoate, phosphoranilothioate, phosphoraniladate, phosphoramidate and the like. See, for example, LaPlanche et al., Nucleic Acids Res. 14: 9,081 (1986); Stec et al, JJ. Am. Chem. Soc. 106: 6,077 (1984), Stein et al., Nucleic Acids Res. 16: 3,209 (1988), Zon et al., Anti-Cancer Drug
[127] [127] In modalities, the stereochemistry in each of the phosphorus internucleotide bonds in the ASO framework is random. In modalities, the stereochemistry in each of the phosphorus internucleotide bonds in the ASO framework is controlled and is not random. For example, US Patent Application Publication No. 2014/0194610, “Methods for the Synthesis of Functionalized Nucleic Acids”, incorporated in this specification by reference, describes methods for independently selecting chirality dexterity in each phosphor atom in an oligomer nucleic acid. In embodiments, an ASO used in the methods of the invention including, without limitation, any of the ASOs presented in that specification in Tables 5 and 6, comprises an ASO that has non-random internucleotide bonds. In embodiments, a composition used in the methods of the invention comprises a pure diastereomeric ASO. In embodiments, a composition used in the methods of the invention comprises an ASO having a diastereomeric purity of at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94% at least about 95%, at least about 96%, at least about 97%, at least about 98%, at least about 99%, about 100%, about 90% to about 100 %, about 91% to about 100%, about 92% to about 100%, about 93% to about 100%, about 94% to about 100% about 95% to about 100% , about 96% to about 100%, about 97% to about 100%, about 98% to about 100%, or about 99% to about 100%.
[128] [128] In modalities, ASO has a non-random mix of Rp and Sp configurations in its phosphorus internucleotide bonds. For example, it has been suggested that a mixture of Rp and Sp is needed in antisense oligonucleotides to achieve a balance between good activity and nuclease stability (Wan et al., 2014, “Synthesis, Biophysical Properties and Biologic Activity of Second Generation Antisense Oligonucleotides Containing Chiral Phosphorothioate Linkages ”, Nucleic Acids Research 42 (22):
[129] [129] In modalities, an ASO used in the methods of the invention including, without limitation, any of the ASOs presented in that specification in the IDS. SEQ. No. 21-114, comprises about 5-100% Sp, at least about 5% Sp, at least about 10% Sp, at least about 15% Sp, at least about 20% Sp Sp, at least about 25% Sp, at least about 30% Sp, at least about 35% Sp, at least about 40% Sp, at least about 45% Sp, at least about 50% Sp, at least about 55% Sp, at least about 60% Sp, at least about 65% Sp, at least about 70% Sp, at least about 75% Sp , at least about 80% Sp, at least about 85% Sp, at least about 90% Sp, or at least about 95% Sp, with the remainder Rp, or about 100% Sp In modalities, an ASO used in the methods of the invention including, without limitation, any of the ASOs presented in this specification in the IDS. SEQ. No.: 21-114, comprises about 10% to about 100% Sp, about 15% to about 100% Sp, about 20% to about 100% Sp, about 25% to about 100% Sp, about 30% to about 100% Sp, about 35% to about 100% Sp, about 40% to about 100% Sp, about 45% to about 100% Sp, about 50% to about 100% Sp, about 55% to about 100% Sp, about 60% to about 100% Sp, about 65% to about 100% Sp , about 70% to about 100% Sp, about 75% to about 100% Sp, about 80% to about 100% Sp, about 85% to about 100% Sp, about from 90% to about 100% Sp, or about 95% to about 100% Sp, about 20% to about 80% Sp, about 25% to about 75% Sp, about 30% to about 70% Sp, about 40% to about 60% Sp, or about 45% to about 55% Sp, with the rest Rp.
[130] [130] Any of the ASOs described in that specification may contain a sugar portion comprising ribose or deoxyribose, as present in naturally occurring nucleotides, or a modified sugar or sugar analog portion, including a morpholino ring.
[131] [131] In some embodiments, each ASO monomer is modified in the same way, for example, each ASO framework bond comprises a phosphorothioate bond or each ribose sugar moiety comprises a 2'-O-methyl modification. These modifications that are present in each of the monomeric components of an ASO are called "uniform modifications". In some examples, a combination of different modifications may be desired, for example, an ASO may comprise a combination of phosphorodiamidate bonds and sugar moieties that comprise morpholino (morpholino) rings. Combinations of different modifications for an ASO are called "mixed modifications" or "mixed chemicals".
[132] [132] In some modalities, the ASO comprises one or more modifications of the framework. In some embodiments, the ASO comprises one or more modifications of the sugar portion. In some embodiments, the ASO comprises one or more modifications to the framework and one or more modifications to the sugar portion. In some embodiments, the ASO comprises a 2'MOE modification and a phosphorothioate framework. In some embodiments, ASO comprises a morpholino phosphorodiamidate (PMO). In some embodiments, ASO comprises a peptide nucleic acid (PNA). Any of the ASOs or any component of an ASO (for example, a nucleobase, sugar portion, framework) described in this specification can be modified to obtain desired ASO properties or activities or to reduce unwanted ASO properties or activities. For example, an ASO or one or more components of any ASO can be modified to increase the binding affinity for a target sequence in a pre-mRNA transcript; reduce binding to any non-target sequence; reduce degradation by cellular nucleases (ie RNase H); increase the uptake of ASO in a cell and / or in the nucleus of a cell; change the pharmacokinetics or pharmacodynamics of ASO; and / or modulate the ASO half-life.
[133] [133] In some embodiments, ASOs are composed of 2'-O- (2-methoxyethyl) (MOE) phosphorothioate-modified nucleotides. ASOsS composed of these nucleotides are especially well suited to the methods revealed in this specification; it was demonstrated that oligomers that have these modifications have significantly increased resistance to degradation by nuclease and increased bioavailability, which makes them suitable, for example, for oral release in some modalities described in this specification. See, for example, Geary et al, J. Pharmacol. Exp. Ther. 2001; 296 (3): 890-7; Geary et al, J. Pharmacol. Exp. Ther. 2001; 296 (3) 898-904.
[134] [134] ASO synthesis methods will be known to those skilled in the art. Alternatively or in addition, ASOs can be obtained from a commercial source.
[135] [135] Unless otherwise specified, the left end of the single-stranded nucleic acid sequences (eg, pre-mRNA transcript, oligonucleotide, ASO etc.) is the 5th end and the left direction of nucleic acid sequences single or double stranded is called the 5 "direction. Similarly, the right end or direction of a nucleic acid sequence (single or double stranded) is the 3 'end or direction. Generally, a region or sequence that is 5 'for a reference point in a nucleic acid is referred to as "upstream" and a region or sequence that is 3 "for a reference point in a nucleic acid is referred to as" downstream. "Generally, the direction or end 5 'of an mRNA is where the initiation or start codon is located, while the end or direction 3' "is where the termination codon is located. In some ways, nucleotides that are upstream of a reference point in an acid nucleic can be designated by a negative number, while nucleotides that are downstream of a reference point can be designated by a positive number. For example, a reference point (for example, an exon-exon junction in mRNA) can be designated as the “zero” site and a nucleotide that is directly adjacent and upstream of the reference point is designated “minus one”, for example, “-l1”, while a nucleotide that is directly adjacent and downstream from the reference point is designated “plus one”, for example, “+1”.
[136] [136] In some embodiments, ASOs are complementary to (and bind to) a targeted portion of a SCNIA pre-mRNA that contains NIE that is downstream (in the 3 "direction) of the 5" splice site (or end 3 ”of the NIE) of the exon included in a SCNIA pre-mRNA that contains NIE (for example, the direction designated by positive numbers in relation to the 5 'splice site). In some embodiments, the ASOs are complementary to a targeted portion of the SCNIA pre-mRNA that contains NIE that is within the region approximately +1 to about +500 with respect to the splice site 5 '(or 3' end) of the included exon. In some embodiments, ASOsS may be complementary to a targeted portion of a SCNIA pre-mRNA that contains NIE that is within the region between +6 and +496 nucleotides with respect to the 5 "splice site (or 3" end) of the included exon.
[137] [137] In some embodiments, ASOs are complementary to (and bind to) a targeted portion of a SCNIA pre-mRNA that contains NIE that is upstream (in the 5 'direction) of the 5 "splice site (or end 3 ') of the exon included in a SCNIA pre-mMRNA that contains NIE (for example, the direction designated by negative numbers in relation to the 5' splice site). In some embodiments, ASOs are complementary to a targeted portion of the pre -SCNIA mRNA containing NIE that is within the region approximately -—- 4 to about -270 with respect to the 5 'splice site (or 3' end) of the included exon. In some embodiments, ASOs may be complementary to a targeted portion of a SCNIA pre-mRNA that contains NIE that is within the region between nucleotides -1 and -264 with respect to the 5 "splice site (or 3" end) of the included exon. In some ways, ASOs are complementary to a target portion that is within the region approximately -1 to about -270, about -1 to about -260, about -1 to about -250, about -1 to about -240, about -1 to about -230, about -1 to about -220, about -1 to about -210, about -1 to about -200, about -1 to about -190, about -1 to about -180, about -1 to about -170, about -1 to about -160, about -1 to about -150, about -1 to about -140, about -1 to about -130, about -1 to about -120, about -1 to about -110, about -1 to about -100, about -1 to about -90, about -1 to about -80, about -1 to about -70, about -1 to about -60, about -1 to about -50, about -1 to about -40, about -1 to about -30, or about -1 to about -20 with respect to splice site 5 '(or 3 "end) of the included exon. In some respects, ASOs are complementary to a target portion that is within the region of about -1 to about -50, from about -50 to about -100, from about -100 to about -150, from about - 150 to about -200, or from about -200 to about -250 with respect to the 5 'splice site (or 3' end) of the included exon.
[138] [138] In some embodiments, ASOs are complementary to a targeted region of a SCNIA pre-mRNA that contains NIE that is upstream (in the 5 'direction) of the splice site 3' (or 5 'end) of the included exon in a SCNIA pre-mRNA that contains NIE (for example, in the direction designated by negative numbers). In some embodiments, the ASOS are complementary to a targeted portion of the SCNIA pre-mRNA that contains NIE that is within the region approximately -1 to about -500 with respect to the splice site 3 '(or 5th end) of the included exon . In some embodiments, the ASOsS are complementary to a targeted portion of the SCNIA pre-mRNA that contains NIE that is within the -1l to -496 region with respect to the 3 'splice site of the included exon. In some respects, ASOs are complementary to a target portion that is within the region of approximately -1l1 to about -500, about -1 to about -490, about -l to about -
[139] [139] In some embodiments, ASOs are complementary to a target region of a SCNIA pre-mRNA that contains NIE that is downstream (in the 3 'direction) of the 3' splice site (5 'end) of the exon included in a SCNIA pre-mRNA that contains NIE (for example, in the direction designated by positive numbers). In some embodiments, the ASOS are complementary to a targeted portion of the SCNIA pre-mRNA that contains NIE which is within the region of about +1 to about +100 with respect to the 3 'splice site of the included exon. In some respects, ASOs are complementary to a target portion that is within the region approximately +1 to about +90, about +1 to about +80, about +1 to about +70, about +1 up to about +60, about +1 to about +50, about +1 to about +40, about +1 to about +30, about +1 to about +20, or about + 1 to about +10 with respect to the 3 'splice site of the included exon.
[140] [140] In some embodiments, the target portion of the SCNIA pre-mRNA that contains NIE is within the +100 region with respect to the 5 'splice site (3' end) of the included exon to -100 with respect to the splice site 3 '(5' end) of the included exon. In some embodiments, the target portion of the SCNIA pre-mRNA that contains NIE is within the NIE. In some embodiments, the target portion of the SCNIA pre-mRNA that contains NIE comprises a pseudoexon and intron boundary.
[141] [141] ASOs can be of any length suitable for specific binding and effective splicing enhancement. In some embodiments, ASOs consist of 8 to 50 nucleobases. For example, the ASO can have 8, 9, 10, 11, 12, 13, 14, 15,
[142] [142] In some embodiments, two or more ASOsS with different but complementary chemicals are used for the same target portion of the pre-mRNA that contains NIE. In some embodiments, two or more ASOs are used that are complementary to target portions other than the pre-mRNA that contains NIE.
[143] [143] In embodiments, the antisense oligonucleotides of the invention are chemically linked to one or more moieties or conjugates, for example, a targeting moiety or other conjugate that increases the oligonucleotide activity or cell uptake. Such portions include, without limitation, a lipid moiety, for example, a cholesterol moiety, a cholesteryl moiety, an aliphatic chain, for example, dodecanediol or undecyl residues, a polyamine or a polyethylene glycol chain, or acetic acid adamantane. Oligonucleotides comprising lipophilic moieties and methods of preparation have been described in the published literature. In embodiments, the antisense oligonucleotide is conjugated to a moiety that includes, without limitation, an abasic nucleotide, a polyether, a polyamine, a polyamide, peptides, a carbohydrate, for example, N-acetylgalactosamine (GalNAc), N-Ac -Glucosamine (GluNAc), or mannose (for example, mannose-6-phosphate), a lipid, or a polyhydrocarbon compound. Conjugates can be attached to one or more of any nucleotides that comprise the antisense oligonucleotide at any of several positions in the sugar, base or phosphate group, as implied in the art and described in the literature, for example, using a linker. Linkers can include a divalent or trivalent branched linker. In modalities, the conjugate is attached to the 3 ”end of the antisense oligonucleotide. Methods of preparing oligonucleotide conjugates are described, for example, in U.S. Patent No. 8,450,467, "Carbohydrate Conjugates as Delivery Agents for Oligonucleotides", incorporated by reference in that specification.
[144] [144] In some embodiments, the nucleic acid to be targeted by an ASO is a SCNIA pre-mRNA that contains NIE expressed in a cell, for example, a eukaryotic cell. In some embodiments, the term "cell" can refer to a population of cells. In some embodiments, the cell is in an individual. In some embodiments, the cell is isolated from an individual. In some embodiments, the cell is ex vivo. In some embodiments, the cell is a cell or cell line relevant to a condition or disease. In some embodiments, the cell is in vitro (for example, in cell culture).
[145] [145] Pharmaceutical compositions or formulations comprising the agent, for example, antisense oligonucleotide, of the described compositions and for use in any of the described methods can be prepared according to conventional techniques well known in the pharmaceutical industry and described in the published literature . In embodiments, a pharmaceutical composition or formulation for the treatment of an individual comprises an effective amount of any antisense oligomer as described in that specification, or a pharmaceutically acceptable salt, solvate, hydrate or ester thereof. The pharmaceutical formulation comprising an antisense oligomer can further comprise a pharmaceutically acceptable excipient, diluent or carrier.
[146] [146] Pharmaceutically acceptable salts are suitable for use in contact with the tissues of humans and lower animals without unnecessary toxicity, irritation, allergic response, etc. and are compatible with a reasonable risk / benefit ratio (see, for example, SM Berge et al, J. Pharmaceutical Sciences, 66: 1-19 (1977), incorporated in this specification by reference for that purpose. Salts may be prepared in situ during the final isolation and purification of the compounds, or separately by reaction of the free base function with a suitable organic acid Examples of pharmaceutically acceptable non-toxic acid addition salts are salts of an amino group formed with inorganic acids such as example, hydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid and perchloric acid or with organic acids such as acetic acid, oxalic acid, maleic acid, tartaric acid, citric acid, succinic acid or malonic acid or by using other methodologies documented as, for example, ion exchange. Other pharmaceutically acceptable salts include adipate, alginate, ascorbate, aspartate, benzenesulfonate, ben zoate, bisulfate, borate, butyrate, camphorate, camphorsulfonate, citrate, cyclopentanopropionate, digluconate, dodecyl sulphate, ethanesulfonate, format, fumarate, glucoheptonate, glycerophosphate, gluconate, hemisulfate, heptanoate, lactate, hydroxide, hydroxide, hydroiodide laurate, lauryl sulfate, malate, maleate, malonate, methanesulfonate, 2-
[147] [147] In embodiments, the compositions are formulated in any of several possible dosage forms, such as, without limitation, tablets, capsules, gel capsules, liquid syrups, soft gels, suppositories and enemas. In embodiments, the compositions are formulated as suspensions in aqueous, non-aqueous or mixed media Aqueous suspensions may also contain substances that increase the viscosity of the suspension including, for example, sodium carboxymethylcellulose, sorbitol and / or dextran. The suspension may also contain stabilizers. In embodiments, a pharmaceutical formulation or composition of the present invention includes, without limitation, a solution, emulsion, microemulsion, foam or formulation containing liposome (e.g., cationic or non-cationic liposomes).
[148] [148] The pharmaceutical composition or formulation described in this specification may comprise one or more penetration enhancers, carriers, excipients or other active or inactive ingredients, as appropriate and well known to those skilled in the art or described in the published literature. In embodiments, liposomes also include sterically stabilized liposomes, for example, liposomes that comprise one or more specialized lipids. These specialized lipids result in liposomes with an increased circulation half-life. In embodiments, a sterically stabilized liposome comprises one or more glycolipids or is derivatized with one or more hydrophilic polymers, for example, a portion of polyethylene glycol (PEG). In embodiments, a surfactant is included in the pharmaceutical formulation or compositions. The use of surfactants in pharmaceutical products, formulations and emulsions is well known in the art. In embodiments, the present invention employs a penetration promoter to effect the efficient release of the antisense oligonucleotide, for example, to assist diffusion across cell membranes and / or to increase the permeability of a lipophilic drug. In modalities, penetration promoters are a surfactant, fatty acid, bile salt, chelating agent or non-chelating non-surfactant.
[149] [149] In modalities, the pharmaceutical formulation comprises “multiple antisense oligonucleotides. In embodiments, the antisense oligonucleotide is administered in combination with another drug or therapeutic agent.
[150] [150] In some embodiments, the ASOs disclosed in the present disclosure can be used in combination with one or more additional therapeutic agents. In some embodiments, the (one or more) additional therapeutic agents may comprise a small molecule. For example, the (one or more) additional therapeutic agents may comprise a small molecule described in WO 2016128343 A1, WO 2017053982 A1, WO 2016196386 A1, WO 201428459 A1, WO 201524876 A2, WO 2013119916 A2 and WO 2014209841 A2, which are incorporated by reference in that specification in its entirety. In some embodiments, the (one or more) additional therapeutic agents comprise an ASO that can be used to correct intron retention. In some embodiments, (one or more) other agents are selected from the ASOs listed in Table la or Table 1b.
[151] [151] Any of the compositions provided in this specification can be administered to an individual. The term "person" can be used interchangeably with "individual" or "patient". An individual can be a mammal, for example, a human or animal such as, for example, a non-human primate, a rodent, a rabbit, a mouse, a mouse, a horse, a monkey, a goat, a cat, a dog , a cow, a pig or a sheep. In modalities, the individual is a human. In modalities, the individual is a fetus, an embryo or a child. In other embodiments, the individual may be another eukaryotic organism, for example, a plant. In some embodiments, the compositions provided in that specification are administered and a cell ex vivo.
[152] [152] In some embodiments, the compositions provided in that specification are administered to an individual as a method of treating a disease or disorder. In some modalities, the individual has a genetic disease, for example, any of the diseases described in this specification. In some modalities, the individual is at risk of having a disease, for example, any of the diseases described in this specification. In some embodiments, the individual is at increased risk of having a disease or disorder caused by insufficient protein or insufficient protein activity. If an individual is "at an increased risk" of having a disease or disorder caused by insufficient protein or insufficient protein activity, the method involves preventive or prophylactic treatment. For example, an individual may be at an increased risk of having this disease or disorder because of the family's history of the disease. Typically, individuals at an increased risk of having this disease or disorder benefit from prophylactic treatment (for example, by preventing or delaying the onset or progression of the disease or disorder). In embodiments, a fetus is treated in the womb, for example, by administering the ASO composition to the fetus directly or indirectly (for example, through the mother).
[153] [153] Pathways suitable for administering ASOs of the present invention may vary depending on the type of cell to which ASO release is desired. Multiple tissues and organs are affected by Dravet's syndrome; epilepsy, generalized, with febrile seizures plus, type 2; febrile, familial seizures, 3A; migraine, family hemiplegic, 3; autism; epileptic encephalopathy, early childhood, 13; sinus node disease 1; Alzheimer's disease or SUDEP, with the brain being the tissue most significantly affected. The ASOs of the present invention can be administered to patients parenterally, for example, by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal injection or intravenous injection.
[154] [154] In some embodiments, the disease or condition is induced by a mutation in Na, l.1 (a protein encoded by the SCNIA gene). In some cases, the mutation is a loss-of-function mutation in Na, l.1. In some cases, the loss-of-function mutation in Na, l.1 comprises one or more mutations that decrease or impair the function of Navl.l1 (for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) in relation to the function of a Na, l.1 wild type. In some cases, the loss-of-function mutation in Navl.l comprises one or more mutations that result in a disease phenotype. Exemplary loss-of-function mutations include, without limitation, R859C, T875M, V1353L, I1656M, R1657C, Al685V, M1841T and R1916G.
[155] [155] In other cases, the mutation is a gain-of-function mutation in Na, vl.1. In such cases, the gain-of-function mutation comprises one or more mutations that prolong the activation of Navl.l1 (for example, by 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 95%, or more) in relation to the function of a wild type Na, l.1. In such cases, the gain-of-function mutation in Na, l.1 comprises one or more mutations that result in a disease phenotype. Exemplary gain-of-function mutations include, without limitation, DI188V, WI1204R, R1648H and DI866Y.
[156] [156] In some embodiments, the disease or condition is an encephalopathy. In some cases, encephalopathy is induced by a loss-of-function mutation in Navl.1.
[157] [157] In some embodiments, encephalopathy is epileptic encephalopathy. Exemplary epileptic encephalopathies include, without limitation, Dravet's syndrome (DS) (also known as severe childhood myoclonic epilepsy or SMEI); childhood severe myoclonic epilepsy (SMEI) -orderline (SMEB); febrile seizure (FS); epilepsy, generalized, with febrile seizures plus (GEFS +); epileptic encephalopathy, early childhood, 13; generalized cryptogenic epilepsy; focal cryptogenic epilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; West syndrome; “idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); early childhood encephalopathy due to SCN1A; early childhood epileptic encephalopathy (EEEE); or pacemaker syndrome on the left 1. In some modalities, the disease or condition is epileptic encephalopathy, optionally selected from Dravet syndrome (DS) (also known as severe childhood myoclonic epilepsy or SMEI); childhood severe myoclonic epilepsy (SMEI) -orderline (SMEB); febrile seizure (FS); epilepsy, generalized, with febrile seizures plus (GEFS +); epileptic encephalopathy, early childhood, 13; generalized cryptogenic epilepsy; focal cryptogenic epilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); and pacemaker syndrome on the left 1.
[158] [158] In some cases, GEFS + is epilepsy, generalized, with febrile plus seizures, type 2.
[159] [159] In some cases, the febrile seizure consists of febrile, familial seizures, 3A.
[160] [160] In some cases, SMEB is SMEB without generalized waveform (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB without more than one SMEI characteristic (SMEB-
[161] [161] In some embodiments, diseases or conditions induced by a loss-of-function mutation in Na, v.1 include, without limitation, Dravet's syndrome (DS) (also known as SMEI); childhood severe myoclonic epilepsy (SMEI) -orderline (SMEB); febrile seizure (FS); epilepsy, generalized, with febrile seizures plus (GEFS +); epileptic encephalopathy, early childhood, 13; generalized cryptogenic epilepsy; focal cryptogenic epilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; West syndrome; “idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); pacemaker syndrome on the left 1; early childhood SCNIA encephalopathy; early childhood epileptic encephalopathy (EEEE); autism; or partial malignant migrant convulsions from childhood.
[162] [162] In some embodiments, the disease or condition is induced by a gain-of-function mutation in Na, l.l. Exemplary diseases or conditions associated with a gain-of-function mutation in Navl.l include, without limitation, migraine. In some cases, the disease or condition induced by a gain-of-function mutation in Navl.1 is migraine.
[163] [163] In some cases, migraine is migraine, family hemiplegic, 3.
[164] [164] In some embodiments, the disease or condition is a genetic epilepsy of Na ', l.1. Genetic epilepsy of Navl.1 may include a loss-of-function mutation in Na'vl.1 or a gain-of-function mutation in Navl.l.
[165] [165] In some embodiments, the disease or condition is associated with a haploinsufficiency of the SCNIA gene. Exemplary diseases or conditions associated with a haploinsufficiency of the SCNIA gene include, without limitation, Dravet's syndrome (DS) (also known as SMEI); childhood severe myoclonic epilepsy (SMEI) -orderline (SMEB); febrile seizure (FS); epilepsy, generalized, with febrile seizures plus (GEFS +); epileptic encephalopathy, early childhood, 13; generalized cryptogenic epilepsy; focal cryptogenic epilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); pacemaker syndrome on the left 1; early childhood SCNIA encephalopathy; early childhood epileptic encephalopathy (EEEE); or partial malignant migrant convulsions from childhood. In some cases, the disease or condition is Dravet's syndrome (DS (also known as SMEI); severe myoclonic epilepsy of childhood (SMEI) -borderline (SMEB); febrile seizure (FS); epilepsy, generalized, with febrile plus seizures ( GEFS +); epileptic encephalopathy, early childhood, 13; cryptogenic generalized epilepsy; cryptogenic focal epilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome;
[166] [166] In some cases, the disease or condition is Dravet's syndrome (DS).
[167] [167] Dravet's syndrome (DS), also known as severe childhood myoclonic epilepsy (SMEI), is an epileptic encephalopathy that presents in the first year of life. Dravet's syndrome is an increasingly recognized epileptic encephalopathy in which the clinical diagnosis is supported by the finding of mutations in the sodium channel gene in approximately 70-80% of patients. Ion channel gene mutations play an important role in the pathogenesis of a range of epilepsy syndromes, causing some epilepsies to be considered channelopathies. Voltage-controlled sodium channels (VGSCs) play an essential role in neuronal excitability; therefore, it is not surprising that many mutations associated with DS have been identified in the gene encoding a VGSC subunit. The disease is described, for example, by Mulley et al, 2005 and the description of the disease in OMIM + * + 607208 (“Online Mendelian Inheritance” in “Man, Johns Hopkins University” 1966-2015), both incorporated by reference in this descriptive report.
[168] [168] Between 70% and 80% of patients carry abnormalities in the sodium channel al subunit gene (SCNIA) and truncation mutations are responsible for about 40% and have a significant correlation with an earlier age of onset of seizures . Sequencing mutations are found in about 70% of cases and comprise truncation (40%) and sense change (40%) mutations, with the remainder being splice site changes. Most mutations are new, but familial mutations occur in 5-10% of cases and are usually meaning-changing. The remaining SCNIA mutations comprise splice site and sense change mutations, most of which fall within the pore-forming region of the sodium channel. At the moment, more than 500 mutations have been associated with DS and are randomly distributed throughout the gene (Mulley et al., Neurol. 2006, 67, 1.094-1.095).
[169] [169] The SCNIA gene is located in the sodium channel gene pool on human chromosome 2924 and encodes the pore-forming subunits a known as neuronal voltage controlled sodium channel Navl.1. The SCNIA gene transposes approximately 100 kb of genomic DNA and comprises 26 exons. The SCNIA protein consists of four domains, each with six transmembrane segments. Two splice variants have been identified that result in a long and short isoform that differ in the presence or absence of 11 amino acids in the cytoplasmic loop between domains 1 and 2, in exon 11 (Miller et al., 19993-2.015 and Mulley et al. , 2005, 25, 535-542, incorporated in this specification by reference).
[170] [170] Alternative splicing events in the SCNIA gene can lead to non-productive mRNA transcripts which, in turn, can lead to aberrant protein expression and therapeutic agents that can target alternative splicing events in the SCNI1A gene can modulate the level of expression of functional proteins in patients with DS and / or inhibit aberrant protein expression. These therapeutic agents can be used to treat a condition caused by a deficiency of the SCNI1A protein.
[171] [171] One of the alternative splicing events that can lead to non-productive mRNA transcripts is the inclusion of an extra exon in the mRNA transcript that can induce nonsense-mediated mRNA decay. The present disclosure provides compositions and methods for modulating alternative SCNIA splicing to increase production of mature mRNA encoding protein and, thus, translated functional SCNIA protein. These compositions and methods include antisense oligomers (ASOs) that can cause exon-skipping and promote constitutive splicing of SCNIA pre-mRNA. In various embodiments, functional SCNIA protein can be increased using the methods of the disclosure to treat a condition caused by deficiency of the SCNI1A protein.
[172] [172] In some cases, the disease or condition is SMEB.
[173] [173] In some cases, the disease or condition is GEFS +.
[174] [174] In some cases, the disease or condition is a febrile seizure (eg, febrile, family, 3A).
[175] [175] In some cases, the disease or condition is autism (also known as autism spectrum disorder or ASD).
[176] [176] In some cases, the disease or condition is migraine (for example, migraine, familial hemiplegic, 3).
[177] [177] In some cases, the disease or condition is Alzheimer's disease.
[178] [178] In some embodiments, the disease or condition is SCN2A encephalopathy.
[179] [179] In some embodiments, the disease or condition is SCN8A encephalopathy.
[180] [180] In some embodiments, the disease or condition is SCNSA arrhythmia.
[181] [181] In modalities, the antisense oligonucleotide is administered with one or more agents capable of promoting the penetration of the antisense oligonucleotide in question through the blood-brain barrier by any method known in the art. For example, the release of agents by administering an adenovirus vector to motor neurons in muscle tissue is described in US Patent No. 6,632,427, “Adenoviral-Vector-Mediated Gene Transfer into Medullary Motor Neurons”, incorporated in this specification by reference . The release of vectors directly to the brain, for example, to the striatum, to the thalamus, to the hippocampus or to the substantia nigra, is described, for example, in US Patent No. 6,756,523, “Adenovirus Vectors for the Transfer of Foreign Genes into Cells of the Central Nervous System Particularly in Brain ”, incorporated in this specification by reference.
[182] [182] In embodiments, the antisense oligonucleotides are linked or conjugated with agents that provide desirable pharmaceutical or pharmacodynamic properties. In modalities, the antisense oligonucleotide is coupled to a substance, known in the art to promote penetration or transport across the blood-brain barrier, for example, an antibody to the transferrin receptor. In modalities, the antisense oligonucleotide is linked to a viral vector, for example, to make the antisense compound more effective or to increase transport across the blood-brain barrier. In modalities, the osmotic rupture of the blood-brain barrier is aided by an infusion of sugars, for example, meso erythritol, xylitol, D (+) galactose, D (+) lactose, D (+) xylose, myulinositol dulcitol, L (-) fructose , D (-) mannitol, D (+) glucose, D (+) arabinose, D (-) arabinose, cellobiose, D (+) maltose, D (+) raffinose, L (+) rhamnose, D (+) melibiosis , D (-) ribose, adonitol, D (+) arabitol, L (-) arabitol, D (+) fucose, L (-) fucose, D (-) lixose, L (+) lixose and L (-) lixose , or amino acids, for example, glutamine, lysine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glycine, histidine, leucine, methionine, phenylalanine, proline, serine, threonine, tyrosine, valine and taurine. Methods and materials for increasing penetration of the blood-brain barrier are described, for example, in US Patent No. 9,193,969, “Compositions and Methods for Selective Delivery of Oligonucleotide Molecules to Specific Neuron Types”, US Patent No. 4,866,042, “Method for the Delivery of Genetic Material Across the Blood-Brain Barrier ”, US Patent No. 6,294,520,“ Material for Passage Through the Blood-Brain Barrier ”and US Patent No. 6,936,589,“ Parenteral Delivery Systems ”, each incorporated into that descriptive report by reference.
[183] [183] In embodiments, an ASO of the invention is coupled to a dopamine reuptake inhibitor (DRI), a selective serotonin reuptake inhibitor (SSRI), a norepinephrine reuptake inhibitor (NRI), a norepinephrine reuptake inhibitor -dopamine (NDRI) and a serotonin-norepinephrine-dopamine reuptake inhibitor (SNDRI), using methods described, for example, in US Patent No. 9,193,969 incorporated in this specification by reference.
[184] [184] In modalities, individuals treated using the methods and compositions are evaluated for improvements in condition using any methods known and described in the art.
[185] [185] Also within the scope of this disclosure are methods for identifying or determining ASOs that induce exon-skipping of a SCNIA pre-mRNA that contains NIE. For example, a method may comprise the identification or determination of ASOs that induce pseudo-exon-skipping of a SCNIA pre-mRNA that contains NIE. ASOs that hybridize specifically to different nucleotides within the pre-mRNA target region can be evaluated to identify or determine ASOs that increase the rate and / or extent of splicing of the target intron. In some embodiments, the ASO may block or interfere with the site (or sites) of a repressor (or repressors) / splicing silencer connection. Any method known in the art can be used to identify (determine) an ASO that, when hybridized to the target exon region, results in the desired effect (for example, pseudo-exon-skipping, protein production or functional RNA). These methods can also be used for the identification of ASOsS that induce exon-skipping of the included exon by binding to a target region on an intron that flanks the included exon, or on an non-included exon. An example of a method that can be used is provided below.
[186] [186] An evaluation round, referred to as an ASO "walk" can be performed using ASOsS that have been designed to hybridize to a pre-mRNA target region. For example, ASOs used in the ASO walk can be covered every 5 nucleotides from approximately 100 nucleotides upstream from the included exon 3 'splice site (for example, an exon sequence portion located upstream from the target exon / included) up to approximately 100 nucleotides from a 3 'splice site downstream of the target exon / included and / or from approximately 100 nucleotides upstream from the included exon 5' splice site to approximately 100 nucleotides from a target exon 5 'splice downstream of the target / included exon (e.g., a portion of the exon sequence located downstream of the target / included exon). For example, a first ASO of 15 nucleotides in length can be designed to hybridize specifically to nucleotides +6 to +20 with respect to the 3 'splice site of the target / included exon. A second ASO can be designed to hybridize specifically to nucleotides +11 to +25 with respect to the splice site 3 'of the target / included exon. ASOS are designed to span the target region of the pre-mRNA. In modalities, ASOs can be covered more closely, for example, every 1, 2, 3 or 4 nucleotides. In addition, ASOs can be covered from 100 nucleotides to a 5 'splice site downstream, up to 100 nucleotides upstream of the 3' splice site. In some modalities, ASOs can be covered from around
[187] [187] One or more ASOs, or a control ASO (an ASO with a scrambled sequence, a sequence that is not expected to hybridize to the target region) are released, for example, by transfection, into a cell line relevant to disease that expresses the target pre-mRNA (for example, a pre-mRNA that contains NIE described in that specification). The exon-skipping effects of each of the ASOs can be assessed by any method known in the art, for example, by (RT) -PCR with reverse transcriptase using primers that span the splice junction, as described in Example 4 A reduction or absence of a longer RT-PCR product produced using primers that span the region containing the included exon (for example, including NIE flanking exons) in cells treated with ASO, when compared to cells treated with control ASO, indicates that the splicing of the target NIE was increased. In some embodiments, the exon-skipping efficiency (or splicing efficiency for the intron splice that contains the NIE), the ratio of pre-mRNA spliced to unspliced, the rate of splicing or the extent of splicing can be increased using the ASOs described in this specification. The amount of protein or functional RNA that is encoded by the target pre-mRNA can also be assessed to determine whether each ASO has achieved the desired effect (for example,
[188] [188] A second round of evaluation, called a "micro-walk" ASO, can be performed using ASOs that have been designed to hybridize to a pre-mRNA target region. ASOs used in the micro-walk ASO are covered at each 1 nucleotide to further refine the nucleotide acid sequence of the pre-mRNA which, when hybridized to an ASO, results in exon-skipping (or increased NIE splicing).
[189] [189] ASO-defined regions that promote splicing of the target intron are explored in more detail through a "micro-walk" ASO, which involves ASOs spaced in l-nt steps, as well as longer, typically 18 ASOs -25 nt.
[190] [190] As described for the ASO walk above, the ASO micro-walk is performed by releasing one or more ASOs, or a control ASO (an ASO with a scrambled sequence, a sequence that is not expected to hybridize to the region- target), for example, by transfection, into a disease-relevant cell line that expresses the target pre-mRNA. The splicing-inducing effects of each of the ASOs can be evaluated by any method known in the art, for example, by (RT) -PCR with reverse transcriptase using primers that transpose the NIE, as described in this specification (see , for example, Example 4). A reduction or absence of a longer RT-PCR product produced using primers that transpose the NIE in ASO treated cells, when compared to control ASO treated cells,
[191] [191] ASOsS which, when hybridized to a region of a pre-mRNA, result in exon-skipping (or increased splicing of the intron containing an NIE) and increased protein production, can be tested in vivo using animal models, for example, models in transgenic mice in which the full-length human gene was knocked-in or in disease models in humanized mice. Appropriate pathways for administering ASOs may vary depending on the disease and / or the types of cells to which the release of ASOs is desired. ASOs can be administered, for example, by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal injection or intravenous injection. After administration, the cells, tissues and / or organs of the animals in the model can be evaluated to determine the effect of treatment with ASO, for example, by splicing evaluation
[192] [192] As described in this specification in several examples, exon 20x in the human SCNIA gene is equivalent to exon 21x in the mouse SCNIA gene.
[193] [193] Also within the scope of the present disclosure is a method for identifying or validating an NMD-inducing exon in the presence of an NMD inhibitor, for example, cycloheximide. An exemplary method is provided in FIG. 3 and Example 2. SPECIFIC MODALITIES
[194] [194] Modality 1. A method of modulating SCNIA protein expression in a cell that has an mRNA that contains a nonsense mutation-mediated RNA decay exon (NMD exon mRNA) and encodes SCNIA protein, the method comprising the contact of a therapeutic agent with the cell, whereby the therapeutic agent modulates the splicing of the NMD exon by the NMD exon mRNA encoding SCN1A protein, thereby modulating the level of processed mRNA encoding SCNIA protein and modulating expression of SCN1A protein in the cell.
[195] [195] Modality 2. A method of treating a disease or condition in an individual in need by modulating the expression of SCNIA protein in an individual's cell, comprising: contact of the individual's cell with a therapeutic agent that modulates the splicing of an exon inducing mRNA mediated by nonsense mutations (NMD exon) by an mRNA in the cell that contains the NMD exon and encodes
[196] [196] Mode 3. The method of mode 1 or 2, wherein the therapeutic agent (a) binds to a targeted portion of the NMD exon mMRNA encoding SCN1A; (b) modulates the binding of a factor involved in splicing the NMD exon mRNA; or (c) the combination of (a) and (b).
[197] [197] Mode 4. The method of mode 3, in which the therapeutic agent interferes with the binding of the factor involved in splicing the NMD exon of a region of the target portion.
[198] [198] Mode 5. The method of mode 3 or 4, in which the target portion is proximal to the NMD exon.
[199] [199] Modality 6. The method of any of modalities 3 to 5, in which the target portion has, at most, about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides , about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream 5 from the NMD exon.
[200] [200] Modality 7. The method of any of modalities 3 to 6, in which the target portion has at least about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 40 nucleotides, about of 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide upstream of the 5 'end of the NMD exon.
[201] [201] Mode 8. The method of any of the modalities 3 to 5, in which the target portion has a maximum of about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides , about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of the end 3 from the NMD exon.
[202] [202] Mode 9. The method of any of the modalities 3 to 5 or 8, in which the target portion has at least about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide downstream of the 3 'end of the NMD exon.
[203] [203] Mode 10. The method of any of modalities 3 to 9, in which the target portion is located in an intronic region between two canonical exon regions of the NMD exon mMRNA encoding SCNIA and in which the intronic region contains the NMD exon.
[204] [204] Mode 11. The method of any of modes 3 to 10, in which the target portion overlaps at least partially with the NMD exon.
[205] [205] Mode 12. The method of any of modes 3 to 11, in which the target portion overlaps at least partially with an intron upstream of the NMD exon.
[206] [206] Mode 13. The method of any of modes 3 to 12, wherein the target portion comprises 5 "NMD-intron junction or NMD 3-junction exon.
[207] [207] Mode 14. The method of any of modes 3 to 13, wherein the target portion is within the NMD exon.
[208] [208] Mode 15. The method of any of modes 3 to 14, wherein the target portion comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 , 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive nucleotides of the NMD exon.
[209] [209] Mode 16. The method of either mode 1 to 15, wherein the NMD exon mRNA encoding SCN1A comprises a sequence of at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for any of the IDS. SEQ. No.: 2 or 7-10.
[210] [210] Mode 17. The method of any of modalities 1 to 16, in which the NMD exon mRNA encoding SCN1A is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95% , 97% or 100% sequence identity for IDS. SEQ. No: 1 or 3-6.
[211] [211] Modality 18. The method of any of modalities 3 to 17, in which the target portion has, at most, about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides , about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of the genomic site GRCh37 / hgl9: chr2: 166,863,803.
[212] [212] Mode 19. The method of any of modalities 3 to 18, in which the target portion has about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 30 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide upstream of the GRCh37 / hgl9: chr2: 166,863,803 genomic site.
[213] [213] Mode 20. The method of any of modes 3 to 17, in which the target portion has, at most,
[214] [214] Method 21. The method of any of modalities 3 to 17 or 20, in which the target portion has about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide downstream of the genomic site GRCh37 / hg19: chr2: 166,863,740.
[215] [215] Mode 22. The method of any of modes 3 to 21, wherein the target portion of the NMD exon mRNA encoding SCNIA comprises a sequence of at least 80%, 85%, 90%, 95%, 97 % or 100% sequence identity for a region comprising at least 8 contiguous nucleic acids of the IDS. SEQ. No.: 2 or 7-10.
[216] [216] Mode 23. The method of mode 22, in which the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95 %, 97% or 100% identity for any of the IDS. SEQ. No.: 21-67, 210-256, or 304-379.
[217] [217] Mode 24. The method of either mode 3 to 21, wherein the target portion of the NMD exon mMRNA encoding SCNIA is within the RNA decay inducing exon mediated by 20x nonsense SCNI1A mutations.
[218] [218] Mode 25. The method of mode 24, in which the therapeutic agent is an antisense oligomer (ASO) and in which ASO comprises a sequence that has at least about 80%, 85%, 90%, 95 %, 97% or 100% identity for any of the IDS. SEQ. No. 42-50, or 231-239.
[219] [219] Modality 26. The method of any of modalities 3 to 21, in which the target portion of the NMD exon mRNA encoding SCNIA is upstream or downstream of the RNA decay inducing exon mediated by 20x nonsense mutations of SCNIA.
[220] [220] Mode 27. The method of mode 26, in which the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95 %, 97% or 100% identity for any of the IDS. SEQ. No.: 21-38, 53-67, 210-227, or 242-256.
[221] [221] Mode 28. The method of any of modes 3 to 21, wherein the targeted portion of the NMD exon mRNA comprises an exon-intron junction of the SCNIA exon 20x.
[222] [222] Mode 29. The method of mode 28, in which the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95 %, 97% or 100% identity for any of the IDS. SEQ. No.: 39-41, 51, 52, 228-230, 240 or 241.
[223] [223] Mode 30. The method of any of modalities 1 to 29, in which the therapeutic agent promotes the exclusion of NMD exon from the processed mRNA encoding SCNI1A protein.
[224] [224] Mode 31. The method of mode 30, in which exclusion of the NMD exon from the processed mRNA encoding SCNIA protein in the cell placed in contact with the therapeutic agent is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1, 1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times at least about 1.1 times, at least about 1.5 times at least s about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times compared to excluding the NMD exon from the processed mMRNA encoding SCNI1A protein in a control cell.
[225] [225] Mode 32. The method of mode 30 or 31, in which the therapeutic agent increases the level of the processed mRNA encoding SCNIA protein in the cell.
[226] [226] Mode 33. The method of any of modes 30 to 32, in which an amount of the processed mMRNA encoding SCNIA protein in the cell placed in contact with the therapeutic agent is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times , about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 time, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times , or at least about 10 times, compared to a total amount of the processed mRNA encoding SCN1A protein in a control cell.
[227] [227] Mode 34. The method of any of modes 30 to 33, wherein the therapeutic agent increases the expression of SCNIA protein in the cell.
[228] [228] Mode 35. The method of any of modes 30 to 34, in which an amount of SCNI1A produced in the cell brought into contact with the therapeutic agent is increased by about 1.1 to about 10 times, about 1, 5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 up to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 about 3 times to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about d and 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about times, compared to the total amount of SCNIA produced in a control cell.
[229] [229] Mode 36. The method of any of modes 2 to 35, in which the disease or condition is induced by a loss-of-function mutation in Navl.1.
[230] [230] Modality 37. The method of any of modalities 2 to 36, in which the disease or condition is associated with haploinsufficiency of the SCNIA gene and in which the individual has a first allele encoding a functional SCNIA and a second allele by which SCNI1A is not produced or is produced at a reduced level, or a second allele that encodes a non-functional SCNIA or a partially functional SCNIA.
[231] [231] Mode 38. The method of any of modes 2 to 37, where the disease or condition is encephalopathy.
[232] [232] Mode 39. The method of mode 38, in which the encephalopathy is epileptic encephalopathy.
[233] [233] Mode 40. The method of any of modes 2 to 37, where the disease or condition is Dravet's syndrome (DS); childhood severe myoclonic epilepsy (SMEI) -orderline (SMEB); febrile seizure (FS); epilepsy, generalized, with febrile seizures plus (GEFS +); epileptic encephalopathy, early childhood, 13; generalized cryptogenic epilepsy; focal cryptogenic epilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; West syndrome; “idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); pacemaker syndrome on the left 1; autism; or partial malignant migrant convulsions from childhood.
[234] [234] Mode 41. The method of mode 40, in which GEFS + is epilepsy, generalized, with febrile seizures plus, type 2.
[235] [235] Mode 42. The method of mode 40, in which the febrile seizure consists of febrile, familial seizures, 3A.
[236] [236] Mode 43. The method of mode 40, where SMEB is SMEB without generalized wave-tip (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB without more than one SMEI characteristic (SMEB-O ), or intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC).
[237] [237] Mode 44. The method of any one of modalities 1 to 43, in which the therapeutic agent promotes the exclusion of NMD exon from the processed mRNA encoding SCNIA protein and increases SCNIA expression in the cell.
[238] [238] Mode 45. The method of any one of modalities 1 to 44, in which the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85% , 90%, 95%, 97% or 100% complementary to any of the IDS. SEQ. No. 22-24, 26, 27, 29-35, 37-62, 64-67, or 304-379.
[239] [239] Mode 46. The method of any one of modalities 1 to 29, in which the therapeutic agent inhibits the exclusion of NMD exon from the processed mRNA encoding SCNI1A protein.
[240] [240] Mode 47. The method of mode 46, in which exclusion of the NMD exon from the processed mRNA encoding SCNIA protein in the cell placed in contact with the therapeutic agent is decreased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1, 1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about from 2 to about 6 times, about 2 to about 7 times about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times at least about 1.1 times, at least about 1.5 times at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times compared to excluding the NMD exon from the processed mMRNA encoding SCNI1A protein in a control cell.
[241] [241] Mode 48. The method of mode 46 or 47, in which the therapeutic agent decreases the level of the processed mMRNA that encodes SCNI1A protein in the cell.
[242] [242] Mode 49. The method of any of modes 46 to 48, in which a quantity of the processed mRNA encoding SCNIA protein in the cell brought into contact with the therapeutic agent is decreased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times , about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times about 4 to about 8 times, about 4 to about 9 times, at least at least about 1.1 times, at least about 1, 5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times, compared to a total amount of the processed mRNA encoding SCN1A protein in a control cell.
[243] [243] Mode 50. The method of any of modes 46 to 49, in which the therapeutic agent decreases the expression of SCNIA protein in the cell.
[244] [244] Mode 51. The method of any of modes 46 to 50, in which an amount of SCNIA produced in the cell brought into contact with the therapeutic agent is decreased by about 1.1 to about 10 times, about 1, 5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 up to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about d and 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about times, compared to the total amount of SCNIA produced in a control cell.
[245] [245] Mode 52. The method of any of modes 2 to 29 or 46 to 49, in which the disease or condition is induced by a gain-of-function mutation in Navl.1.
[246] [246] Mode 53. The method of mode 52, in which the individual has an allele by which SCNIA is produced at an increased level, or an allele that encodes a mutant SCN1A that induces increased Navl.1 activity in the cell.
[247] [247] Mode 54. The method of mode 52 or 53, where the disease or condition is migraine.
[248] [248] Mode 55. The method of mode 54, in which the migraine is migraine, family hemiplegic, 3.
[249] [249] Mode 56. The method of any of the modes 2 to 49, in which the disease or condition is a genetic epilepsy of Navl.1.
[250] [250] Mode 57. The method of any of modes 46 to 56, wherein the therapeutic agent inhibits the exclusion of NMD exon from the processed mRNA encoding SCNIA protein and decreases the expression of SCNIA in the cell.
[251] [251] Method 58. The method of any of the modalities 46 to 57, in which the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85% , 90%, 95%, 97% or 100% complementary to any of the IDS. SEQ. Nº: 21, 25, 28, 36 or 63.
[252] [252] Mode 59. The method of any of the previous modalities, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a modification of the framework comprising a phosphorothioate bond or a bond phosphorodiamidate.
[253] [253] Mode 60. The method of any of the previous modalities, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a morpholino phosphorodiamidate, a blocked nucleic acid, a nucleic acid peptide, a 2'-O-methyl moiety, a 2'-Fluorine moiety or a 2'-O-methoxyethyl moiety.
[254] [254] Modality 61. The method of any of the previous modalities, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises at least a modified portion of sugar.
[255] [255] Mode 62. The method of mode 61, where each portion of sugar is a portion of modified sugar.
[256] [256] Mode 63. The method of any of the previous modalities, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, l1 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to nucleobases or 12 to 15 nucleobases.
[257] [257] Mode 64. The method of any of modalities 3 to 63, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99% or 100%, complementary to the target portion of the NMD exon mRNA encoding the protein.
[258] [258] Modality 65. The method of any of the previous modalities, in which the method still comprises evaluation of the expression of SCNI1A mRNA or protein.
[259] [259] Modality 66. The method of any of the modalities 2 to 65, in which the individual is a human.
[260] [260] Mode 67. The method of any of modes 2 to 65, in which the individual is a non-human animal.
[261] [261] Mode 68. The method of any of modes 2 to 65, in which the individual is a fetus, an embryo or a child.
[262] [262] Mode 69. The method of any of the previous modalities, in which the cells are ex vivo.
[263] [263] Mode 70. The method of any of modes 2 to 69, in which the therapeutic agent is administered by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal or intravenous injection to the individual.
[264] [264] Mode 71. The method of any of modes 2 to 65, wherein the method still comprises administering a second therapeutic agent to the individual.
[265] [265] Mode 72. The method of mode 71, in which the second therapeutic agent is a small molecule.
[266] [266] Mode 73. The method of mode 71, in which the second therapeutic agent is an ASO.
[267] [267] Mode 74. The method of mode 73, in which the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95%, 97% or 100% complementary to any of the IDS. SEQ. No.: 115-161.
[268] [268] Mode 75. The method of mode 71, in which the second therapeutic agent corrects intron retention.
[269] [269] Mode 76. The method of any of modes 2 to 65, where the disease or condition is Alzheimer's disease, SCN2A encephalopathy, SCN8A encephalopathy or SCN5A arrhythmia.
[270] [270] Mode 77. The method of mode 30, 32 or 34, where the disease or condition is Alzheimer's disease, SCN2A encephalopathy, SCN8A encephalopathy or SCN5A arrhythmia.
[271] [271] Mode 78. A method of treating Dravet's syndrome (DS); epilepsy, generalized, with febrile seizures plus, type 2; febrile, familial seizures, 3A; migraine, family hemiplegic, 3; autism; epileptic encephalopathy, early childhood, 13; sinus node disease 1; Alzheimer's disease or unexpected sudden death in epilepsy (SUDEP) in a needy individual, by increased expression of a target protein or functional RNA by an individual's cell, where the cell has an mRNA that contains a decay-inducing exon RNA mediated by nonsense mutations (NMD exon mMRNA) and where the NMD exon mRNA encodes the target protein or functional RNA, the method comprising contacting the individual's cell with a therapeutic agent that binds to a targeted portion of the NMD exon mRNA encoding the target protein or functional RNA, whereby the RNA decay-inducing exon mediated by nonsense mutations is excluded by the NMD exon mRNA encoding the protein-
[272] [272] Mode 79. The method of mode 78, where the target protein is SCNIA.
[273] [273] Mode 80. A method of increasing SCNIA protein expression by a cell that has an mRNA that contains a nonsense mutation-mediated RNA decay exon (NMD exon mRNA) and encodes SCNIA protein, the method comprising contact of the cell with an agent that binds to a targeted portion of the NMD exon mRNA encoding SCNIA protein, whereby the RNA decay inducing exon mediated by nonsense mutations is excluded by the NMD exon mRNA encoding SCNIA protein thereby increasing the level of processed mRNA encoding SCNIA protein and increasing the expression of SCN1IA protein in the cell.
[274] [274] Mode 81. A method of treating a disease or condition in an individual in need by increasing the expression of SCNI1A protein in an individual's cell, comprising: contact of the individual's cell with a therapeutic agent that binds to a target portion of an RNA decay-inducing exon mediated by nonsense mRNA mutations encoding the SCNIA protein or functional SCNIA RNA, whereby the exon-inducing RNA decay mediated by nonsense mutations is excluded by the NMD exon mRNA encoding the SCNIA protein or functional SCNIA RNA thereby increasing the level of processed mRNA encoding the functional SCNIA protein or SCNIA RNA and increasing the expression of the functional SCNIA protein or SCNIA RNA in the individual's cell; wherein the disease or condition is associated with a mutation of a gene other than a SCNIA gene, aberrant expression of a protein encoded by a gene other than a SCNIA gene or aberrant expression of an RNA encoded by a gene other than a SCNIA gene.
[275] [275] Mode 82. The method of mode 81, in which a symptom of the disease or condition is reduced by about 2 times, 3 times, 4 times, 5 times, 6 times, 7 times, 8 times, 9 times, 10 times or more.
[276] [276] Mode 83. The method of mode 81 or 82, in which a symptom of the disease or condition is reduced by about 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% , 95% or 100% with an increase in SCNI1A protein expression.
[277] [277] Mode 84. The method of any of modes 81 to 83, in which the progression of the disease or condition is reduced by about 2 times, 3 times, 4 times, 6 times, 7 times, 8 times, 9 times, 10 times, or more with an increase in SCNI1A protein expression.
[278] [278] Mode 85. The method of any of the modes 81 to 84, in which the progression of the disease or condition is reduced by about 20%, 30%, 40%, 50%, 60% 70%, 80%, 90%, 95% or 100% with an increase in SCNI1A protein expression.
[279] [279] Mode 86. The method of any of modes 81 to 85, in which increased expression of the SCNIA protein or functional SCNIA RNA compensates for the mutation of a gene other than a SCNIA gene, the aberrant expression of a coded protein by a gene other than a SCN1A gene or the aberrant expression of an RNA encoded by a gene other than a SCNIA gene.
[280] [280] Mode 87. The method of any of the modes 81 to 86, in which the disease or condition is early childhood epileptic encephalopathy, 13.
[281] [281] Mode 88. The method of any of modes 81 to 87, in which the individual has a mutation in the SCN8A gene.
[282] [282] Mode 89. The method of any of modalities 81 to 86, in which the disease or condition is syndrome of the pacemaker on the left 1.
[283] [283] Mode 90. The method of any of modalities 81 to 86 or 88, in which the individual has a mutation in the SCN5A gene.
[284] [284] Mode 91. The method of any of modes 81 to 86, where the disease or condition is Alzheimer's disease.
[285] [285] Mode 92. A method of treating a disease or condition in a needy individual, which comprises administering to the individual a composition comprising an antisense oligomer, the antisense oligomer comprising a sequence of at least 8 nucleotides contiguous which is at least 80%, 85%, 90%, 95%, 97% or 100% complementary to SCNIA intron 20.
[286] [286] Mode 93. A method of treating a disease or condition in a needy individual, which comprises administering to the individual a composition comprising an antisense oligomer, the antisense oligomer comprising a sequence of at least 8 nucleotides contiguous which is at least 80%, 85%, 90%, 95%, 97% or 100% complementary to any of the IDS. SEQ. No.
[287] [287] Mode 94. The method of any of the modalities 78 to 93, in which the RNA decay-inducing exon mediated by nonsense mutations is spliced by the NMD exon mRNA encoding the target protein or functional RNA.
[288] [288] Mode 95. The method of any of the modalities 78 to 94, in which the target protein does not comprise an amino acid sequence encoded by the exon inducing RNA mediated by nonsense mutations.
[289] [289] Method 96. The method of any of the modalities 78 to 95, wherein the target protein is a full-length target protein.
[290] [290] Mode 97. The method of any of the modes 78 to 96, in which the agent is an antisense oligomer (ASO) complementary to the target portion of the NMD exon mMRNA.
[291] [291] Mode 98. The method of any of the modalities 78 to 97, in which the mRNA is pre-mRNA.
[292] [292] Mode 99. The method of any of the modalities 78 to 98, in which the contact comprises the contact of the therapeutic agent with the mMRNA, in which the mRNA is in a cell nucleus.
[293] [293] Mode 100. The method of any of the modes 78 to 99, wherein the target protein or functional RNA corrects a deficiency in the target protein or functional RNA in the individual.
[294] [294] Mode 101. The method of any of modes 78 to 100, wherein the cells are in or are from an individual with a condition caused by a deficient amount or SCNI1A protein activity.
[295] [295] Mode 102. The method of any of the modalities 78 to 101, in which the deficient amount of the target protein is caused by haploinsufficiency of the target protein, in which the individual has a first allele that encodes a functional target protein and a second allele from which the target protein is not produced or produced at a reduced level, or a second allele that encodes a non-functional or partially functional target protein and in which the antisense oligomer binds to a targeted portion of an NMD exon mRNA transcribed by the first allele.
[296] [296] Mode 103. The method of any of the modalities 78 to 101, in which the individual has a condition caused by a disorder that results from a deficiency in the quantity or function of the target protein, in which the individual has: (a ) a first mutant allele of which: (i) the target protein is produced at a reduced level, compared to production by a wild type allele, (ii) the target protein is produced in a form that has reduced function, compared to an equivalent wild-type protein, or (iii) the target protein is not produced, and (b) a second mutant allele of which: (1) the target protein is produced at a reduced level, compared to production by a wild-type allele, (ii) the target protein is produced in a form that has reduced function, compared to an equivalent wild-type protein, or (iii) the target protein is not produced, and where when the individual has a first mutant allele (a) (iii), the second mutant allele is (b) (i) or (b) (ii) and where, when the individual has a second mutant allele (b) (iii), the first mutant allele is (a) (i) or (a) (ii) and where the NMD exon mMRNA is transcribed by the first mutant allele which is (a) (i) or (a) (ii), and / or by the second allele which is (b) (i) or (b) (ii).
[297] [297] Mode 104. The method of mode 103, in which the target protein is produced in a form that has reduced function, compared to the equivalent wild-type protein.
[298] [298] Mode 105. The method of mode 103, in which the target protein is produced in a form that is fully functional, compared to the equivalent wild-type protein.
[299] [299] Mode 106. The method of any of the modalities 78 to 105, wherein the target portion of the NMD exon mRNA is within the RNA decay inducing exon mediated by nonsense mutations.
[300] [300] Mode 107. The method of any of the modalities 78 to 105, in which the target portion of the NMD exon mRNA is upstream or downstream of the RNA decay-inducing exon mediated by nonsense mutations.
[301] [301] Mode 108. The method of any of the modalities 78 to 107, wherein the NMD exon mRNA comprises a sequence with at least about 80%, 85%, 90%, 95% 97% or 100% string identity for any of the IDS. SEQ. No.: 2, 7-10, 12 and 17-20.
[302] [302] Mode 109. The method of any of the modalities 78 to 107, in which the NMD exon mRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for IDS. SEQ. No.: 1, 3-6, 11 and 13-16.
[303] [303] Mode 110. The method of any of the modalities 78 to 107, wherein the target portion of the NMD exon mRNA comprises a sequence of at least 80%, 85%, 90%, 95%, 97% or 100 % sequence identity for a region comprising at least 8 contiguous IDS nucleic acids. SEQ. Nos: 2, 7-10, 12 and 17-20.
[304] [304] Mode 111. The method of any of the modalities 78 to 110, in which the agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95%, 97% or 100% identity for any of the IDS. SEQ. No. 21-114.
[305] [305] Mode 112. The method of any of the modalities 78 to 105, wherein the targeted portion of the NMD exon mRNA is within the RNA decay inducing exon mediated by SCNIA 20x nonsense mutations.
[306] [306] Mode 113. The method of mode 112, where the agent is an antisense oligomer (ASO) and where ASO comprises a sequence that has at least about 80%, 85%, 90%, 95% , 97% or 100% identity for any of the IDS. SEQ. No. 42-50, or 231-239.
[307] [307] Mode 114. The method of any of the modalities 78 to 105, wherein the target portion of the NMD exon mRNA is upstream or downstream of the RNA decay-inducing exon mediated by 20x nonsense SCNIA mutations.
[308] [308] Mode 115. The method of mode 114, in which the agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95% , 97% or 100% identity for any of the IDS. SEQ. No.: 21-38, 53-67, 210-227, or 242-256.
[309] [309] Mode 116. The method of either mode 78 to 105, wherein the target portion of the NMD exon mRNA comprises an exon-intron junction of the SCNIA exon 20x.
[310] [310] Mode 117. The method of mode 116, in which the agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95% , 97% or 100% identity for any of the IDS. SEQ. No.: 39-41, 51, 52, 228-230, 240 or 241.
[311] [311] Mode 118. The method of any of the modalities 78 to 105, wherein the target portion of the NMD exon mRNA is within the 21x RNA decay inducer mediated by Scnla nonsense mutations.
[312] [312] Mode 119. The method of mode 118, in which the agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95% , 97% or 100% identity for any of the IDS. SEQ. No.: 89-97.
[313] [313] Mode 120. The method of any of the modalities 78 to 105, in which the target portion of the NMD exon mRNA is upstream or downstream of the exon 21x exon inducing RNA mediated by Scnla nonsense mutations.
[314] [314] Mode 121. The method of mode 120, where the agent is an antisense oligomer (ASO) and where ASO comprises a sequence that has at least about 80%, 85%, 90%, 95% , 97% or 100% identity for any of the IDS. SEQ. No. 68-85 and 100-114.
[315] [315] Mode 122. The method of either mode 78 to 105, wherein the target portion of the NMD exon mRNA comprises an exon-intron junction of Secnla's 21x exon.
[316] [316] Modality 123. The method of modality 122, in which the agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95% , 97% or 100% identity for any of the IDS. SEQ. Nº: 86-88 and 98-99.
[317] [317] Method 124. The method of any of the modalities 78 to 123, wherein the target protein produced is full-length protein, or wild-type protein.
[318] [318] Mode 125. The method of any of the modes 78 to 124, in which the total amount of processed mRNA encoding the target protein or functional RNA produced in the cell placed in contact with the antisense oligomer is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1 , 1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least c about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times, compared to the total amount of processed mRNA encoding the target protein or functional RNA produced in a control cell.
[319] [319] Mode 126. The method of any of the modes 78 to 124, in which the total amount of target protein produced by the cell brought into contact with the antisense oligomer is increased by about 1.1 to about times, about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 up to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times at least about 1.1 times, at least about 1.5 times, eg it at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times compared to the total amount of target protein produced by a control cell.
[320] [320] Mode 127. The method of any of the modalities 78 to 126, in which the agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a modification of the framework comprising a phosphorothioate bond or a phosphorodiamidate bond.
[321] [321] Mode 128. The method of any of the modes 78 to 127, in which the agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a morpholino phosphorodiamidate, a blocked nucleic acid, an acid peptide nucleic, a 2'-O-methyl moiety, a 2 "-Fluorine moiety or a 2'-O-methoxyethyl moiety.
[322] [322] Mode 129. The method of any of the modes 78 to 128, in which the agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises at least a modified portion of sugar.
[323] [323] Mode 130. The method of mode 129, where each portion of sugar is a portion of modified sugar.
[324] [324] Mode 131. The method of any of the modalities 78 to 130, in which the agent is an antisense oligomer (ASO) and in which the antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases , 8 to 35 nucleobases 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 35 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 40 to nucleobases, 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 20 nucleobases, 10 to 20 nucleobases, 10 to nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, l12 to nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases or 12 to 15 nucleobases.
[325] [325] Mode 132. The method of any of the modalities 78 to 131, in which the agent is an antisense oligomer (ASO) and in which the antisense oligomer is at least 80%, at least 85%, at least at least 90%, at least 95%, at least 98%, at least 99% or 100%, complementary to the target portion of the NMD exon mRNA encoding the protein.
[326] [326] Modality 133. The method of any of the modalities 78 to 132, in which the method still comprises evaluation of the expression of SCNI1A mRNA or protein.
[327] [327] Mode 134. The method of any of modalities 1 to 133, in which Dravet's syndrome; epilepsy, generalized, with febrile seizures plus, type 2; febrile, familial seizures, 3A; migraine, family hemiplegic, 3; autism; epileptic encephalopathy, early childhood, 13; sinus node disease 1; Alzheimer's disease or sudden unexpected death in epilepsy (SUDEP) is treated and in which the antisense oligomer binds to a target portion of an NMD exon SCNIA mRNA, where the target portion is within a selected sequence of IDS. SEQ. Nº: 7-10 e 17-
[328] [328] Modality 135. The method of any of the modalities 78 to 134, in which the individual is a human.
[329] [329] Modality 136. The method of any of the modalities 78 to 135, in which the individual is a non-human animal.
[330] [330] Method 137. The method of any of the modalities 78 to 136, in which the individual is a fetus, an embryo or a child.
[331] [331] Mode 138. The method of any of the modes 78 to 137, in which the cells are ex vivo.
[332] [332] Method 139. The method of any of the modalities 78 to 138, in which the therapeutic agent is administered by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal injection or intravenous injection to the individual.
[333] [333] Mode 140. The method of any of the modalities 78 to 139, in which the method still comprises the administration of a second therapeutic agent to the individual.
[334] [334] Mode 141. The method of mode 140, where the second therapeutic agent is a small molecule.
[335] [335] Mode 142. The method of mode 140, where the second therapeutic agent is an ASO.
[336] [336] Mode 143. The method of mode 142, in which the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95%, 97% or 100% identity for any of the IDS. SEQ. No. 1155-161.
[337] [337] Mode 144. The method of any of the modalities 140 to 142, in which the second therapeutic agent corrects intron retention.
[338] [338] Mode 145. An antisense oligomer as used in a method of any of modalities 78 to 144.
[339] [339] Mode 146. An antisense oligomer comprising a sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for any of the IDS. SEQ. No. 21-114.
[340] [340] Mode 147. A pharmaceutical composition comprising the 145 or 146 antisense oligomer and an excipient.
[341] [341] Mode 148. A method of treatment of a needy individual, which comprises administering the pharmaceutical composition of modality 147 to the individual, where administration is by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, injection intravitreal or intravenous injection.
[342] [342] Modality 149. A composition comprising a therapeutic agent for use in a method of increasing the expression of a target protein or functional RNA by cells to treat a disease or condition associated with a deficient protein or deficient functional RNA in a needy individual, where the deficient protein or deficient functional RNA is deficient in quantity or activity in the individual, where the target protein is: (a) the deficient protein; or (b) a compensating protein that functionally increases or replaces the deficient protein or in the individual; and where the functional RNA is: (c) the defective RNA; or (d) a compensating functional RNA that functionally increases or replaces the defective functional RNA in the individual; wherein the therapeutic agent increases the exclusion of the RNA decay inducing exon mediated by nonsense mutations by the NMD exon mRNA encoding the target protein or functional RNA, thereby increasing the production or activity of the target protein or RNA functional in the individual.
[343] [343] Modality 150. A composition comprising a therapeutic agent for use in a method of treating a disease or condition in a needy individual, the method comprising the step of modulating SCN1A protein expression by cells of the individual, wherein the cells have an mMRNA that contains an exon inducing RNA decay mediated by nonsense mutations (NMD exon mRNA) and encodes SCNIA protein, the method comprising contacting cells with the therapeutic agent, whereby exclusion of decay inducing exon of RNA mediated by nonsense mutations by the NMD exon mRNA encoding SCNIA protein is modulated, thereby modulating the level of processed mRNA encoding SCNIA protein and modulating the expression of SCNIA protein in the individual's cells.
[344] [344] Modality 151. The composition of modality 150, in which the disease or condition is selected from the group consisting of: Dravet's syndrome (DS); childhood severe myoclonic epilepsy (SMEI) -orderline (SMEB); febrile seizure (FS); epilepsy, generalized, with febrile seizures plus (GEFS +); epileptic encephalopathy, early childhood, 13; generalized cryptogenic epilepsy; focal cryptogenic epilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); pacemaker syndrome on the left 1; autism; or migraine, family hemiplegic, 3; and Alzheimer's disease.
[345] [345] Mode 152. The composition of any of modalities 150 to 151, wherein the SCNIA protein and NMD exon mRNA are encoded by the SCNIA gene.
[346] [346] Mode 153. The composition of any of modes 149 to 152, in which the RNA decay-inducing exon mediated by nonsense mutations is amended by the NMD exon mRNA encoding the SCNI1A protein.
[347] [347] Mode 154. The composition of any of the modalities 149 to 153, in which the SCNI1A protein does not comprise an amino acid sequence encoded by the RNA decay-inducing exon mediated by nonsense mutations.
[348] [348] Mode 155. The composition of any of modes 149 to 154, wherein the SCNI1A protein is a full-length SCN1A protein.
[349] [349] Mode 156. The composition of any of modes 149 to 155, wherein the therapeutic agent is an antisense oligomer (ASO) complementary to the targeted portion of the NMD exon mMRNA.
[350] [350] Mode 157. The composition of any of modalities 149 to 156, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer targets a portion of the NMD exon mMRNA that is within the exon inducing RNA decay mediated by nonsense mutations.
[351] [351] Mode 158. The composition of any of modalities 149 to 156, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer targets a portion of the NMD exon mMRNA that is upstream or downstream of the exon inducing RNA decay mediated by nonsense mutations.
[352] [352] Mode 159. The composition of any of modalities 149 to 158, where the target protein is SCNIA.
[353] [353] Mode 160. The 159 mode composition, wherein the NMD exon mMRNA comprises a sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for any of the IDS. SEQ. Nº: 2, 7-10, 12 and 17-20.
[354] [354] Mode 161. The composition of mode 159, wherein the NMD exon mMRNA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% identity string for the ID. SEQ. Nº: 1, 3- 6, 11 and 13-16.
[355] [355] Mode 162. The 159 mode composition, wherein the target NMR exon mRNA portion comprises a sequence with at least 80%, 85%, 90%, 95%, 97% or 100% sequence identity for a region comprising at least 8 contiguous ID nucleic acids. SEQ. No.: 2, 7-10, 12 and 17-20.
[356] [356] Mode 163. The composition of any of modalities 159 to 162, wherein the target portion of the NMD exon mRNA (i) is within the RNA decay inducing exon mediated by 20x nonsense mutations, (ii) upstream or downstream of the RNA decay inducing exon mediated by 20x nonsense mutations, or (iii) comprises an exon-intron junction of the RNA decay inducing exon mediated by 20x nonsense mutations.
[357] [357] Mode 164. The composition of any of the modalities 159 to 163, in which the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85% , 90%, 95%, 97% or 100% identity for any of the IDS. SEQ. No. 21-114.
[358] [358] Mode 165. The composition of any of modes 149 to 164, in which the disease or condition is induced by a loss-of-function mutation in Navl.1.
[359] [359] Modality 166. The composition of any of the modalities 149 to 165, in which the disease or condition is associated with haploinsufficiency of the SCNIA gene and in which the individual has a first allele encoding a functional SCNIA and a second allele by which SCNI1A is not produced or is produced at a reduced level, or a second allele that encodes a non-functional SCNIA or a partially functional SCNIA.
[360] [360] Modality 167. The composition of any of modalities 149 to 166, in which the disease or condition is encephalopathy, optionally induced by a loss-of-function mutation in Navl.1.
[361] [361] Modality 168. The composition of modality 167, in which the encephalopathy is epileptic encephalopathy.
[362] [362] Modality 169. The composition of modality 165 or 166, in which the disease or condition is Dravet's syndrome (DS); childhood severe myoclonic epilepsy (SMEI) -orderline (SMEB); febrile seizure (FS); epilepsy, generalized, with febrile seizures plus (GEFS +); epileptic encephalopathy, early childhood, 13; generalized cryptogenic epilepsy; focal cryptogenic epilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; West syndrome; idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); pacemaker syndrome on the left 1; autism; or partial malignant migrant convulsions from childhood.
[363] [363] Modality 170. The composition of modality 168, in which GEFS + is epilepsy, generalized, with febrile seizures plus, type 2.
[364] [364] Mode 171. The composition of mode 168, in which the febrile seizure consists of febrile, familial seizures, 3A.
[365] [365] Modality 172. The composition of modality 168, in which SMEB is SMEB without generalized wave-tip (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB without more than one SMEI characteristic (SMEB-O ), or intractable childhood epilepsy with “generalized tonic-clonic seizures (ICEGTC).
[366] [366] Mode 173. The composition of any of the modalities 165 to 172, wherein the therapeutic agent promotes the exclusion of NMD exon from the processed mRNA encoding SCNIA protein and increases the expression of SCN1A in the cell.
[367] [367] Mode 174. The composition of any of the modalities 165 to 173, in which the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85% , 90%, 95%, 97% or 100% complementary to any of the IDS. SEQ. No. 22-24, 26, 27, 29-35, 37-62, or 64-67.
[368] [368] Modality 175. The composition of any of the modalities 149 to 164, in which the disease or condition is induced by a gain-of-function mutation in Navl.1.
[369] [369] Mode 176. The composition of any of modes 149 to 164 or 175, in which the individual has an allele by which SCNIA is produced at an increased level, or an allele that encodes a mutant SCNIA that induces increased Navl activity .1 in the cell.
[370] [370] Mode 177. The composition of any of the modalities 149 to 164, 175, or 176, in which the disease or condition is migraine.
[371] [371] Modality 178. The composition of modality 177, in which migraine is migraine, family hemiplegic, 3.
[372] [372] Mode 179. The composition of any of the modalities 149 to 164, 175, or 176, in which the disease or condition is a genetic epilepsy of Navl.1.
[373] [373] Mode 180. The composition of any of the modalities 149 to 164, or 175 to 179, in which the therapeutic agent inhibits the exclusion of NMD exon from the processed mRNA encoding SCNI1A protein and decreases the expression of SCNIA in the cell.
[374] [374] Mode 181. The composition of any of the modalities 149 to 164, or 175 to 180, in which the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95%, 97% or 100% complementary to any of the IDS. SEQ. Nº: 21, 25, 28, 36 or 63.
[375] [375] Mode 182. The composition of any of modalities 149 to 181, wherein the processed mRNA encoding the target protein or functional RNA is a full-length mature mRNA, or a mature wild-type mRNA.
[376] [376] Mode 183. The composition of any of the modes 149 to 182, wherein the target protein produced is full-length protein, or wild-type protein.
[377] [377] Mode 184. The composition of any of the modes 149 to 183, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a modification of the framework comprising a phosphorothioate bond or a phosphorodiamidate bond.
[378] [378] Mode 185. The composition of any of modes 149 to 184 in which the therapeutic agent is an antisense oligomer (ASO) and in which said antisense oligomer is an antisense oligonucleotide.
[379] [379] Mode 186. The composition of any of modes 149 to 185, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a morpholino phosphorodiamidate, a blocked nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2'-Fluorine moiety or a 2'-O-methoxyethyl moiety.
[380] [380] Mode 187. The composition of any of modes 149 to 186, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises at least a modified sugar portion.
[381] [381] Modality 188. The composition of modality 187, in which each portion of sugar is a portion of modified sugar.
[382] [382] Mode 189. The composition of any of modalities 149 to 188, in which the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases , l1 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases , 12 to 25 nucleobases, 12 to nucleobases or 12 to 15 nucleobases.
[383] [383] Mode 190. A composition comprising an antisense oligomer, the antisense oligomer comprising a sequence of at least 8 contiguous nucleotides that is at least 80%, 85%, 90%, 95%, 97% or 100 % complementary to intron 20 of SCNIA.
[384] [384] Mode 191. A composition comprising an antisense oligomer, the antisense oligomer comprising a sequence of at least 8 contiguous nucleotides that is at least 80%, 85%, 90%, 95%, 97% or 100 % complementary to any of the IDS. SEQ. No. 7-10.
[385] [385] Mode 192. A pharmaceutical composition comprising the therapeutic agent of any of the compositions of modalities 149 to 191 and an excipient.
[386] [386] Mode 193. A method of treating an individual in need, which comprises administering the pharmaceutical composition of modality 192 to the individual, where administration is by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, injection intravitreal or intravenous injection.
[387] [387] Mode 194. A pharmaceutical composition comprising: an antisense oligomer that hybridizes to a target sequence of a SCNIA mRNA transcript, wherein the SCNIA mRNA transcript comprises an RNA decay-mediating exon mediated by nonsense mutations, in which the antisense oligomer induces exclusion of the RNA decay-inducing exon mediated by nonsense mutations in the transcribed SCNI1A mRNA; and an acceptable pharmaceutical excipient.
[388] [388] Mode 195. The pharmaceutical composition of mode 194, wherein the SCNIA mRNA transcript is a NMD exon mRNA transcript from SCNI1A.
[389] [389] Mode 196. The pharmaceutical composition of mode 194 or 195, wherein the target portion of the SCNIA NMD exon mRNA transcript (i) is within the RNA decay inducing exon mediated by 20x nonsense mutations, (ii ) is upstream or downstream of the RNA decay inducing exon mediated by 20x nonsense mutations, or (iii) comprises an exon-intron junction of the RNA decay inducing exon mediated by 20x nonsense mutations.
[390] [390] Mode 197. The pharmaceutical composition of mode 194 or 196, wherein the NMR exon mRNA transcript from SCNIA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity for any of the IDS. SEQ. Nº: 1, 3-6, 11 and 13-16.
[391] [391] Mode 198. The pharmaceutical composition of mode 194 or 196, wherein the NMR exon mRNA transcript of SCNI1A comprises a sequence of at least about 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity for any of the IDS. SEQ. No.: 2, 7-10, 12 and 17-20.
[392] [392] Mode 199. The pharmaceutical composition of mode 194, wherein the antisense oligomer comprises a modification of the framework comprising a phosphorothioate bond or a phosphorodiamidate bond.
[393] [393] Mode 200. The pharmaceutical composition of mode 194, in which the antisense oligomer is an antisense oligonucleotide.
[394] [394] Mode 201. The pharmaceutical composition of mode 194, wherein the antisense oligomer comprises a morpholino phosphorodiamidate, a blocked nucleic acid, a peptide nucleic acid, a 2'-O-methyl moiety, a 2 "-Fluorine moiety or a 2'-O-methoxyethyl moiety.
[395] [395] Mode 202. The pharmaceutical composition of mode 194, in which the antisense oligomer comprises at least a portion of modified sugar.
[396] [396] Mode 203. The pharmaceutical composition of mode 194, wherein the antisense oligomer comprises 8 to 50 nucleobases, 8 to 40 nucleobases, 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases, 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, to 40 nucleobases , 10 to 35 nucleobases, 10 to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to nucleobases, 11 to 50 nucleobases, 11 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to nucleobases, 12 to 25 nucleobases, 12 to 20 nucleobases or 12 to 15 nucleobases.
[397] [397] Mode 204. The pharmaceutical composition of mode 194 or 195, in which the antisense oligomer is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or is 100% complementary to a targeted portion of the NMD exon mRNA transcript of SCN1A.
[398] [398] Mode 205. The pharmaceutical composition of mode 194 or 195, wherein the target portion of the SCNIA NMD exon mRNA transcript is within a selected sequence of the IDS. SEQ. Nº: 2, 7-10, 12 and 17-20.
[399] [399] Mode 206. The pharmaceutical composition of mode 194, in which the antisense oligomer comprises a nucleotide sequence that has at least about 80%, 85%, 90%, 91%, 92%, 93%, 94 %, 95%, 96%, 97%, 98% or 99% sequence identity for any of the IDS. SEQ. No. 21-114.
[400] [400] Modality 207. The pharmaceutical composition of modality 194, in which the antisense oligomer comprises a nucleotide sequence selected from the IDS. SEQ. No.: 21-114.
[401] [401] Mode 208. The pharmaceutical composition of any of the modalities 194 to 207, in which the pharmaceutical composition is formulated for intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal injection or intravenous injection.
[402] [402] Mode 209. A method of inducing processing of a deficient SCNIA mRNA transcript to facilitate the removal of an exon inducing RNA decay mediated by nonsense mutations to produce a fully processed SCNIA mRNA transcript encoding a form functional of a SCNIA protein, the method comprising: (a) contacting an antisense oligomer with an individual target cell; (b) hybridization of the antisense oligomer to the defective SCNIA mRNA transcript, wherein the defective SCNIA mRNA transcript is capable of encoding the functional form of a SCNIA protein and comprises at least one RNA decay inducing exon mediated by nonsense mutations; (c) removing the (at least one) RNA decay-inducing exon mediated by nonsense mutations from the deficient SCNIA mRNA transcript to produce the fully processed SCNIA mMRNA transcript encoding the functional form of SCNIA protein; and (d) the translation of the functional form of SCNIA protein by the fully processed SCNIA mRNA transcript.
[403] [403] Modality 210. A method of treating an individual who has a condition caused by a deficient amount or activity of SCNIA protein, which comprises administering to the individual an antisense oligomer comprising a nucleotide sequence of at least about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity for any of the IDS. SEQ. No. 24-114.
[404] [404] Mode 211. An evaluation method for an agent that enhances the gene expression of a target protein or functional RNA by a cell, in which the cell has an mMRNA that contains an exon inducing RNA decay mediated by nonsense mutations (NMD exon mRNA) and wherein the NMD exon mRNA encodes the target protein or functional RNA, the method comprising: (a) contacting a test agent targeting the NMD exon mRNA with a first cell; (b) the contact of a control agent with a second cell; (c) determining a first level in the first cell, where the first level is a level of (i) an RNA transcript encoded by the NMD exon mRNA that does not comprise the RNA decay inducing exon, or (ii) a protein encoded by the NMD exon mRNA that does not comprise an amino acid sequence encoded by the RNA decay-inducing exon; (d) determining a second level in the second cell, where the second level is a level of (i) an RNA transcript encoded by the NMD exon mRNA that does not comprise the RNA decay inducing exon, or (ii) a protein encoded by the NMD exon mRNA that does not comprise an amino acid sequence encoded by the RNA decay-inducing exon; where the first level is greater than the second level; and (e) selecting the test agent.
[405] [405] Mode 212. An evaluation method for an agent that increases the gene expression of a target protein or functional RNA by a cell, in which the cell has an mMRNA that contains an exon inducing RNA decay mediated by nonsense mutations (NMD exon mRNA) and wherein the NMD exon mRNA encodes the target protein or functional RNA, the method comprising: (a) contacting a test agent targeting the NMD exon mRNA with a first cell; (b) the contact of a control agent with a second cell; (c) determining a first level in the first cell, where the first level is a level of (i) an RNA transcript encoded by the NMD exon mRNA comprising the RNA decay inducing exon, or (ii) an protein encoded by the NMD exon mRNA which comprises an amino acid sequence encoded by the RNA decay inducing exon; (d) determining a second level in the second cell, where the second level is a level of (i) an RNA transcript encoded by the NMD exon mRNA comprising the RNA decay inducing exon, or (ii) a protein encoded by the NMD exon mRNA which comprises an amino acid sequence encoded by the RNA decay inducing exon; where the first level is less than the second level; and (e) selecting the test agent.
[406] [406] Mode 213. The method of mode 211 or 212, wherein the method comprises contacting a protein synthesis inhibitor with the first cell and the second cell; wherein the first level is a level of an RNA transcript encoded by the NMD exon mRNA which comprises the RNA decay inducing exon; and wherein the second level is a level of an RNA transcript encoded by the NMD exon mRNA comprising the RNA decay inducing exon.
[407] [407] Mode 214. A method of treatment of Dravet's syndrome (DS), epilepsy, generalized, with febrile seizures plus, type 27 febrile seizures, family, 3A; migraine, family hemiplegic, 3; autism; epileptic encephalopathy, early childhood, 13; sinus node disease 1; Alzheimer's disease or SUDEP (sudden unexpected death in epilepsy) in a needy individual, by increased expression of a target protein or functional RNA by an individual's cell, in which the cell has an mRNA that contains a decay-inducing exon RNA mediated by nonsense mutations (NMD exon mMRNA) and where the NMD exon mRNA encodes the target protein or functional RNA, the method comprising contacting the individual's cell with a therapeutic agent that modulates the splicing of the mMRNA of the NMD exon encoding the target protein or functional RNA, whereby the RNA decay-inducing exon mediated by nonsense mutations is excluded by the NMD exon mRNA encoding the target protein or functional RNA, thereby increasing the level of processed mRNA encoding the target protein or functional RNA and increasing the expression of the target protein or functional RNA in the individual's cell.
[408] [408] Mode 215. A method of increasing the expression of SCNIA protein by a cell that has an mRNA that contains an exon inducing RNA decay mediated by nonsense mutations (NMD exon mRNA) and encodes SCNIA protein, the method comprising the contact of the cell with an agent that modulates the splicing of the NMD exon mRNA encoding SCNIA protein, whereby the exon inducing RNA decay mediated by nonsense mutations is excluded by the NMD exon mRNA encoding SCNIA protein, thereby increasing way, the level of processed mRNA encoding SCNIA protein and increasing the expression of SCN1A protein in the cell.
[409] [409] Mode 216. The method of mode 214 or 215, wherein the agent: (a) binds to a targeted portion of the NMD exon mRNA that encodes the target protein or functional RNA; (b) binds to one or more components of a spliceosome; or
[410] [410] Although preferred embodiments of the present invention have been “shown and described in this specification, it will be obvious to those skilled in the art that these modalities are provided by way of example only. Numerous variations, changes and substitutions will now occur to those skilled in the art without departing from the invention. It should be noted that several alternatives to the modalities of the invention described in this specification can be employed in the practice of the invention. It is desired that the following claims define the scope of the invention and that the methods and structures within the scope of those claims and their equivalents are covered by it. EXAMPLES
[411] [411] The present invention will be more specifically illustrated by the following Examples. However, it should be understood that the present invention is not limited by these examples in any way.
[412] [412] Shotgun sequencing of the entire transcriptome was performed using new generation sequencing to reveal a snapshot of transcripts produced by the SCN1A gene to identify NIE inclusion events. For this purpose, pulleyA + RNA from nuclear and cytoplasmic fractions from HCN (human cortical neurons) was isolated and cDNA libraries constructed using the Illumina's TruSeaga Stranded mRNA Library Prep Kit. The libraries were sequenced pair-end resulting in readings of 100 nucleotides that were mapped to the human genome (February 2009, assembly GRCh37 / hg19). The results of the sequencing for SCNIA are shown in Fig. 2. Briefly, Fig. 2 shows the mapped readings visualized using the UCSC genome browser (operated by the “UCSC Genome Informatics Group” (“Center for Biomolecular Science & Engineering”, University of California, Santa Cruz, 1156 High Street, Santa Cruz, CA 95064) and described, for example, by Rosenbloom et al., 2015, “The UCSC Genome Browser Database: 2015 Update”, Nucleic Acids Research 43 “Database Issue”, doi: 10.1093 / nar / gkull77) and the coverage and number of readings can be inferred from the peak signals. The height of the peaks indicates the level of expression given by the density of the readings in a particular region. The bottom panel shows a graphical representation of the SCNIA gene at scale. The conservation level across 100 vertebrate species is shown as peaks. The largest peaks correspond to exons (black boxes), while no peaks are observed for most introns (lines with arrowheads). Conservation peaks were identified at intron 20 (NM 006920), shown in the middle panel. Inspection of the conserved sequences identified an exon-like sequence of 64 bp (bottom panel, sequence highlighted in gray) flanked by splice sites 3 'and 5' (underlined sequence). The inclusion of this exon leads to a frame-shift and the introduction of a premature termination codon in exon 21 making the transcript an NMD target.
[413] [413] Exemplary sequences of the SCNIA gene, pre-mRNA, exon and intron are summarized in Table 2. The sequence for each exon or intron is summarized in Table 3.
[414] [414] RT-PCR analysis using cytoplasmic RNA from Neuro 2A mouse cells treated with DMSO (CHX-) or treated with cycloheximide (CHX +) (FIG. 3A) and RenCell VM (human neuroprogenitor cells) (FIG. 3B) and primers in exon 20 and exon 23 confirmed the presence of a band that corresponds to the NMD inducing exon (20x). The identity of the product was confirmed by sequencing. Densitometry analysis of the bands was performed to calculate the percentage of inclusion of the 20x exon of total SCNIA transcript. The treatment of RenCell VM with cycloheximide (CHX +) to inhibit NMD led to a 2-fold increase in the product corresponding to the NMD-inducing exon 20x in the cytoplasmic fraction (cf. light gray bar, CHX-, for dark gray bar, CHX +) . Example 3: ASO Walk from the Exon 20x region of SCNIA
[415] [415] A graphical representation of the ASO walk performed for sequences that target the SCNIA exon 20x region immediately upstream of the splice site 3 ', through the splice site 3', exon 20x, through the splice site 5 'and a 5 ”splice site downstream using 2'-MOE ASOs, PS framework, is shown in FIG. 4. ASOs were designed to cover these regions by changing 5 nucleotides at once.
[416] [416] ASO walk sequences can be evaluated, for example, by RT-PCR. In FIG. 5A, a representative PAGE shows SYBR- stained RT-PCR products from simulated-SCNIA-treated (Simulated), treated with SMN control ASO (SMN), or treated with a 2'-MOE ASO that targets exon 20x region as described in that specification in Example 3 and in the description of FIG. 4, at a concentration of 20 µM in RenCell VM cells by gimnite uptake. Two products that correspond to the inclusion of exon 20x (upper band) and total length (exclusion of exon 20x, lower band) were quantified and the percentage of inclusion of exon 20x is plotted on the bar graph (FIG. 5B). The black line indicates no change with respect to the Simulated. The full-length products have also been normalized for the internal control of RPL32 and the ratio of increase to the Simulated is plotted on the bar graph (FIG. 5C). The black line indicates a ratio of 1 and no change with respect to the Simulated.
[417] [417] SCNIA amplification results by RT-qPCR with SYBR Green normalized to RPL32, obtained using the same ASO capture experiment that were evaluated by RT-PCR with SYBR-Safe as shown in FIG. 6, are plotted as an increase ratio in relation to the Simulated, confirming the results of RT-PCR with SYBR-Safe. The black line indicates a ratio of 1 (no change from the Simulated).
[418] [418] In FIG. 8A, a representative PAGE shows RT-PCR products stained with SYBR-Safe from Scnla mice treated in a simulated manner (Simulated, RNAiMAX exclusively), or treated with ASO of 2'-MOE Ex2l1x + 1 that targets 21x exon (nomenclature of mouse, corresponds to human exon 20x), at concentrations of 30 nM, 80 nM and 200 nM in Neuro 2A cells (mouse neuroblastoma) by RNAiMAX transfection. Ex2l1x + l (mouse nomenclature) and Ex20x + 1 (human nomenclature) are identical. Two products corresponding to the inclusion of exon 21x (upper band) and total length (exclusion of exon 21x, lower band) were quantified and the percentage of inclusion of exon 21x is plotted on the bar graph (FIG. 8B). Full-length products have also been standardized for internal HPRT control and the ratio of increase to Simulated is plotted on the bar graph (FIG. 8C). The black line indicates a ratio of 1 and no change with respect to the Simulated.
[419] [419] FIG. 9A shows PAGES of RT-PCR products stained with SYBR-Safe from Scnla mouse from left eye injected with PBS (1 ul) (-) or right eye injected with 2'-MOE ASO IVS20x-21, Ex2l1x + 1, IVS21x +18, IVS21x + 33 or Cep290 (negative control ASO; Gerard et al., Mol. Ther. Nuc. Ac., 2015) (11l) (+) at a concentration of 10 mM. Ex2l1x + 1, IVS21x + 18 and IVS21xX + 33 (mouse nomenclature) and Ex20x + 1, IVS20x + 18 and IVS20x + 33 (human nomenclature) are identical. Two products that correspond to the inclusion of exon 21x (upper band) and total length (exclusion of exon 21x, lower band) were quantified and the percentage of inclusion of exon 21x is plotted in FIG. 9B. White bars correspond to eyes injected with ASO and gray bars correspond to eyes injected with PBS, n = 5 in each group. The full-length products were normalized for GAPDH internal control and the ratio of increased eye injected with ASO to eye injected with PBS is plotted in FIG. 9C. The black line indicates a proportion of 1 and no change with respect to PBS, n = 5 in each group.
[420] [420] FIG. 10A shows PAGEs of RT-PCR products stained with SYN-Safe from Scnla mouse from non-injected brains (-, control without ASO), or brains injected with 300 µg of ASO from 2'-MOE Cep290 (ASO from negative control; Gerard et al., Mol. Ther. Nuc. Ac., 2015), Ex2l1x + 1, IVS21x + 18, IVS21x + 33. Ex21x + 1, IVS21x + 18 and IVS21x + 33 (mouse nomenclature) and Ex20x + 1, IVS20x + 18 and
[421] [421] FIG. 11 depicts the exemplary dose-dependent response to ICV injection of selected ASOs in CS7BL6J mice (males, 3 months old). FIG. 11A shows PAGE gels of RT-PCR products stained with SYBR-Safe from Scnla mouse brains injected with 300 µg of Cep290 (negative control ASO; Gerard et al., Mol. Ther. Nuc. AC., 2015), or brains injected with 33 µg, 100 µg and 300 µg of 2 'MOE Ex2lx + l. Ex21l1x + 1 (mouse nomenclature) and Ex20x + 1, (human nomenclature) are identical. Two products that correspond to the inclusion of exon 21x (upper band) and total length (exclusion of exon 21x, lower band) were quantified. FIG. 11B depicts a graphical plot of the 21x exon inclusion percentage by the data in FIG. 11A, n = 5 (each group). FIG. 11C depicts a graph of the results of a Taqgman-qPCR assay performed using two different probes that span the junction of exons 21 and 22. The products were normalized for internal Gapdh control and the ratio of increased brains injected with ASO to brains injected with Cep290 is plotted. The black line indicates a ratio of 1 and unchanged with respect to Cep290, n = 5 (each group).
[422] [422] FIG. 12 depicts exemplary results of ICV injection of a selected ASO in C57BL6J mice (postnatal day 2). FIG. 12A shows PAGE gels of RT-PCR products stained with SYN-Safe from Scnla mouse from non-injected brains (-, control without ASO), or from brains injected with 20 ug ASO of 2'-MOE Ex21x + 1 are shown. Two products that correspond to the inclusion of exon 21x (upper band) and total length (exclusion of exon 21x, lower band) were quantified. Ex2l1x + l (mouse nomenclature) and Ex20x + 1l (human nomenclature) are identical. FIG. 12B depicts a graphical plot of the 21x exon inclusion percentage by the data in FIG. 12A, n = 4 (each group). FIG. 12C depicts a graph of results from a Taqgman-qPCR assay performed using two different probes that span the junction of exons 21 and 22. The products were normalized for internal Gapdh control and the ratio of increased brains injected with ASO to brains control without ASO is plotted. The black line indicates a proportion of 1 and without change in relation to the control without ASO, n = 4 (each group).
[423] [423] Dravet's syndrome (DS) is a devastating childhood genetic disease characterized by severe seizures,
[424] [424] FIG. 14A depicts a graphical plot of the percentage decrease in the inclusion of 21x exon in the doses indicated (n = 3-6 per group). FIG. 14B depicts a graphical plot of the percentage increase in Scnla mRNA at the indicated doses (n = 3-6 per group). FIG. 14C depicts a graphical plot of the percent increase in Nav 1.1 protein levels at the indicated doses (n = 2 per group).
[425] [425] FIG. 15A depicts a graphical plot of the percentage decrease in the inclusion of 21x exon in the doses indicated (n = 4 per group). FIG 15B depicts a graphical plot of the percentage increase in Scnla mRNA at the indicated doses (n = 4 per group).
[426] [426] FIG. 16 depicts an ASO targeting selected Scnla administered at a dose of 10 µg by means of ICV injection in mice on day 2 postnatally assessed on day 5 post-injection by Tagman-qPCR from SCNIA, SCN2A, SCN3A, SCN4A, SCN5A, SCN7A, SCN8A, SCN9A, SCNIO0A and SCNI1A to assess target selectivity. Results of amplification by Taqgman-qPCR normalized to Gapdh, obtained using ASO Ex20x + 1l, are plotted as an increase ratio in relation to mice injected with PBS (n = 3-6 per group).
[427] [427] FIG. 17 depicts exemplary results of intracerebroventricular injection (ICV) on postnatal day 2 of an ASO selected at the dose indicated in wild type Fl (WT) mice or heterozygous Dravet (HET) mice from 129S-ScnlatmiXea x C57BL / 6J crosses in 3 days post-injection (n = 9-14 per group). FIG. 17A depicts a graph of results from a Taqman-qPCR assay performed using a probe that transposes exons 21 and 22. The products were normalized for internal Gapdh control and the ratio of increased brains injected with ASO to brains injected with PBS com is plotted. FIG. 17B depicts a results plot of a western blot performed using an anti-Navl.1 antibody. The products were normalized to bands stained with Ponceau and the ratio of increase in brains injected with ASO in relation to brains injected with PBS with is plotted.
[428] [428] FIG. 19 is a graphical plot of the increase in the level of Scnla mRNA in coronal slides of mice brains along the time post ICV injection of an ASO targeting SCNIA. As pictured, the increase in the Scnla mRNA level, as quantified by Tagman-qPCR, was maintained for at least 80 days post-injection (n = 3-9 per group).
[429] [429] FIG. 20 is an exemplary survival curve that demonstrates the 100% survival benefit provided by an ASO targeting SCNIA in a Dravet mouse model. Dravet WT and heterozygous (+/-) mice, F1 offspring from 129S-scnlatmiKkea x CSTBL / 6J crosses, received an injection
[430] [430] FIG. 18 depicts exemplary results of an ASO microwalk from the 20x exon region of SCNIA in RenCells through free uptake. ASOs were designed to cover regions around three targeting ASOs previously identified in FIG. 6 (marked by stars) by changing 1 nucleotide at once (641) or by decreasing the length of ASO 17 (1-5). The graph depicts the percentage of exon 20x inclusion as measured by qPCR with SYBR Green. The black line indicates no change with respect to the one without ASO (=).
[431] [431] The ASO sequences are summarized in Table 7a and Table 7b.
[432] [432] Although preferred embodiments of the present invention have been “shown and described in this specification, it will be obvious to those skilled in the art that these modalities are provided by way of example only.
Numerous variations, changes and substitutions will now occur to those skilled in the art without departing from the invention.
It should be noted that several alternatives to the modalities of the invention described in this specification can be employed in the practice of the invention.
It is desired that the following claims define the scope of the invention and that the methods and structures within the scope of those claims and their equivalents are covered by it.

权利要求:
Claims (77)
[1]
1. Method of modulating SCNIA protein expression in a cell that has an mRNA that contains a nonsense RNA decay inducing exon (NMD exon mRNA) and encodes SCNIA protein, the method characterized by the fact that it comprises the contact of a therapeutic agent with the cell, whereby the therapeutic agent modulates the splicing of the NMD exon by the NMD exon mRNA encoding SCN1A protein, thereby modulating the level of processed mRNA encoding SCNIA protein, and modulating expression of SCN1A protein in the cell.
[2]
2. Method of treating a disease or condition in an individual in need by modulating the expression of SCN1A protein in an individual's cell, characterized by the fact that it comprises: the contact of the individual's cell with a therapeutic agent that modulates the splicing of a exon inducing mRNA in the nonsense sense (NMD exon) by an mRNA in the cell that contains the NMD exon and encodes SCNI1A thereby modulating the level of processed mRNA encoding the SCNIA protein, and modulating protein expression SCNIA in the individual's cell.
[3]
Method according to claim 1 or 2, characterized in that the therapeutic agent: (a) binds to a targeted portion of the NMD exon mRNA encoding SCNI1A; (b) modulates the binding of a factor involved in splicing the NMD exon mRNA; or (c) a combination of (a) and (b).
[4]
4, Method according to claim 3, characterized by the fact that the therapeutic agent interferes with the binding of the factor involved in splicing the NMD exon of a region of the target portion.
[5]
5. Method according to claim 3, characterized by the fact that the target portion is proximal to the NMD exon.
[6]
6. Method, according to claim 5, characterized by the fact that the target portion has, at most, about
1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of the 5 'end of the NMD exon.
[7]
7. Method according to claim 5, characterized by the fact that the target portion has at least about
1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide upstream of the 5 'end of the NMD exon.
[8]
8. Method, according to claim 5, characterized by the fact that the target portion has, at most, about
1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of the 3 'end of the NMD exon.
[9]
9. Method, according to claim 5, characterized by the fact that the target portion has at least about
1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide downstream of the 3 "end of the NMD exon.
[10]
10. Method according to claim 3, characterized by the fact that the target portion is located in an intronic region between two canonical exonic regions of the NMD exon mRNA encoding SCNI1A, and in which the intronic region contains the exon of NMD.
[11]
11. Method according to claim 3, characterized in that the target portion at least partially overlaps the NMD exon.
[12]
12. Method according to claim 3, characterized in that the target portion at least partially overlaps with an intron upstream of the NMD exon.
[13]
13. Method according to claim 3, characterized in that the target portion comprises 5 "NMD exon-intron junction or 3" NMD exon-intron junction.
[14]
14. Method according to claim 3, characterized in that the target portion is within the NMD exon.
[15]
15. Method according to claim 3, characterized in that the target portion comprises about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, or more consecutive NMD exon nucleotides.
[16]
16. Method according to claim 1 or 2, characterized in that the NMD exon mMRNA encoding SCNIA comprises a sequence of at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for any of the IDS. SEQ. Nº: 2 or 7-10.
[17]
17. Method according to claim 1 or 2, characterized by the fact that the NMD exon mMRNA encoding SCNIA is encoded by a genetic sequence with at least about 80%, 85%, 90%, 95%, 97% or 100% sequence identity for IDS. SEQ. Nº: 1 or 3-6.
[18]
18. Method according to claim 5, characterized by the fact that the target portion has a maximum of about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides upstream of the GRCh37 / genomic site hgl9: chr2: 166,863,803.
[19]
19. Method, according to claim 5, characterized by the fact that the target portion has about
1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide upstream of the GRCh37 / hgl9: chr2 genomic site: 166,863,803.
[20]
20. Method according to claim 5, characterized by the fact that the target portion is, at most, about 1,500 nucleotides, about 1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 70 nucleotides, about 60 nucleotides, about 50 nucleotides downstream of the GRCh37 / genomic site hglº:
chr2: 166,863,740.
[21]
21. Method according to claim 5, characterized by the fact that the target portion has about
1,000 nucleotides, about 800 nucleotides, about 700 nucleotides, about 600 nucleotides, about 500 nucleotides, about 400 nucleotides, about 300 nucleotides, about 200 nucleotides, about 100 nucleotides, about 80 nucleotides, about 170 nucleotides, about 60 nucleotides, about 50 nucleotides, about 40 nucleotides, about 30 nucleotides, about 20 nucleotides, about 10 nucleotides, about 5 nucleotides, about 4 nucleotides, about 2 nucleotides, about 1 nucleotide downstream of the GRCh37 / hgl9: chr2 genomic site: 166,863,740.
[22]
22. Method according to claim 3, characterized in that the target portion of the NMD exon mRNA encoding SCNIA comprises a sequence of at least 80%, 85%, 90%, 95%, 97% or 100 % sequence identity for a region comprising at least 8 contiguous IDS nucleic acids. SEQ. Nº: 2 or 7-10.
[23]
23. Method according to claim 1 or 2, characterized by the fact that the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95%, 97% or 100% identity for any of the IDS. SEQ. Nos: 21-67, 210-256, or 304-379.
[24]
24. Method according to claim 3, characterized in that the target portion of the NMD exon mRNA encoding SCNIA is within the RNA decay inducing exon in the 20x nonsense sense of SCNIA.
[25]
25. Method according to claim 24, characterized by the fact that the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90% , 95%, 97% or 100% identity for any of the IDS. SEQ. Nos: 42-50, or 231-239.
[26]
26. Method according to claim 3, characterized in that the target portion of the NMD exon mRNA encoding SCNIA is upstream or downstream of the RNA decay-inducing exon in the 20x nonsense sense of SCNI1A.
[27]
27. Method according to claim 26, characterized by the fact that the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90% , 95%, 97% or 100% identity for any of the IDS. SEQ. Nos: 21-38, 53-67, 210-227, or 242-256.
[28]
28. Method according to claim 3, characterized in that the target portion of the NMD exon mRNA comprises an exon-intron junction of the 20x exon of SCNI1A.
[29]
29. Method according to claim 28, characterized by the fact that the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90% , 95%, 97% or 100% identity for any of the IDS. SEQ. No. :; 39-41, 51, 52, 228-230, 240, or 241.
[30]
30. Method according to claim 1 or 2, characterized in that the therapeutic agent promotes the exclusion of NMD exon from the processed mRNA encoding SCNI1A protein.
[31]
31. Method according to claim 30, characterized in that the exclusion of the NMD exon from the processed mMRNA encoding SCNIA protein in the cell placed in contact with the therapeutic agent is increased by about 1.1 to about 10 times , about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least the about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about times, compared to excluding the NMD exon from the processed mRNA encoding SCNIA protein in a control cell.
[32]
32. Method according to claim 30, characterized in that the therapeutic agent increases the level of the processed mMRNA that encodes SCNIA protein in the cell.
[33]
33. The method of claim 30,
characterized by the fact that an amount of the processed mRNA encoding SCNI1A protein in the cell placed in contact with the therapeutic agent is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 up to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1, 1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 up to about 7 times, about 2 to about 8 times about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1 , 5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times compared to a total amount of the processed mRNA encoding SCNIA protein in a control cell.
[34]
34. Method according to claim 30, characterized in that the therapeutic agent increases the expression of SCNIA protein in the cell.
[35]
35. Method according to claim 30, characterized in that an amount of SCNIA produced in the cell placed in contact with the therapeutic agent is increased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times at least about 1.1 times at least about 1.5 times at least about 2 times times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times, compared with a total amount of SCNIA produced in a control cell.
[36]
36. Method according to claim 2, characterized by the fact that the disease or condition is induced by a loss-of-function mutation in Na, vl.1.
[37]
37. Method according to claim 36, characterized by the fact that the disease or condition is associated with haploinsufficiency of the SCNIA gene, and in which the individual has a first allele encoding a functional SCNIA, and a second allele by which SCNIA it is not produced or is produced at a reduced level, or a second allele encoding a non-functional SCNIA or a partially functional SCNIA.
[38]
38. Method according to claim 36, characterized by the fact that the disease or condition is encephalopathy.
[39]
39. Method according to claim 38, characterized by the fact that encephalopathy is epileptic encephalopathy.
[40]
40. Method, according to claim 36, characterized by the fact that the disease or condition is Dravet's syndrome (DS); childhood severe myoclonic epilepsy (SMEI) -frontier (SMEB); febrile seizure (FS); epilepsy, generalized, with febrile seizures plus (GEFS +); epileptic encephalopathy, early childhood, 13; generalized cryptogenic epilepsy; focal cryptogenic epilepsy; myoclonic-astatic epilepsy; Lennox-Gastaut syndrome; West syndrome; “idiopathic spasms; early myoclonic encephalopathy; progressive myoclonic epilepsy; alternating hemiplegia of childhood; unclassified epileptic encephalopathy; sudden unexpected death in epilepsy (SUDEP); pacemaker syndrome on the left 1; autism; or partial malignant migrant convulsions from childhood.
[41]
41. Method, according to claim 40, characterized by the fact that GEFS + is epilepsy, generalized, with febrile seizures plus, type 2.
[42]
42. Method according to claim 40, characterized by the fact that the febrile seizure is febrile, familiar, 3A seizures.
[43]
43. Method according to claim 40, characterized by the fact that SMEB is SMEB without generalized wave-tip (SMEB-SW), SMEB without myoclonic seizures (SMEB-M), SMEB without more than one SMEI characteristic (SMEB - O), or intractable childhood epilepsy with generalized tonic-clonic seizures (ICEGTC).
[44]
44, Method according to claim 36, characterized by the fact that the therapeutic agent promotes the exclusion of NMD exon from the processed mRNA encoding SCNIA protein and increases SCNIA expression in the cell.
[45]
45. Method according to claim 36, characterized by the fact that the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90% , 95%, 97% or 100% complementary to any of the IDS. SEQ. No. 22-24, 26, 27, 29-35, 37-62, 64-67, or 304-379.
[46]
46. Method according to claim 1 or 2, characterized in that the therapeutic agent inhibits the exclusion of NMD exon from the processed mRNA encoding SCNI1A protein.
[47]
47. Method according to claim 46, characterized by the fact that the exclusion of the NMD exon from the processed mMRNA encoding SCNIA protein in the cell placed in contact with the therapeutic agent is decreased by about 1.1 to about 10 times , about 1.5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times,
about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about times, compared to the exclusion of NMD exon from the processed mRNA encoding SCNIA protein in a control cell .
[48]
48. Method according to claim 46, characterized by the fact that the therapeutic agent decreases the level of the processed mMRNA encoding SCNIA protein in the cell.
[49]
49. Method according to claim 46, characterized in that an amount of the processed mRNA encoding SCNI1A protein in the cell brought into contact with the therapeutic agent is decreased by about 1.1 to about 10 times, about 1 , 5 to about 10 times, about 2 to about 10 times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1, 1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times, at least about 1.1 times, at least about 1.5 times, at least about 2 times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times , or at least about 10 times compared to a total amount of the processed mRNA encoding SCNIA protein in a control cell.
[50]
50. Method according to claim 46, characterized in that the therapeutic agent decreases the expression of SCNI1A protein in the cell.
[51]
51. Method according to claim 46, characterized in that an amount of SCNIA produced in the cell brought into contact with the therapeutic agent is decreased by about 1.1 to about 10 times, about 1.5 to about 10 times, about 2 to about times, about 3 to about 10 times, about 4 to about 10 times, about 1.1 to about 5 times, about 1.1 to about 6 times, about 1.1 to about 7 times, about 1.1 to about 8 times, about 1.1 to about 9 times, about 2 to about 5 times, about 2 to about 6 times, about 2 to about 7 times, about 2 to about 8 times, about 2 to about 9 times, about 3 to about 6 times, about 3 to about 7 times, about 3 to about 8 times, about 3 to about 9 times, about 4 to about 7 times, about 4 to about 8 times, about 4 to about 9 times at least about 1.1 times at least about 1.5 times, at least about 2 times times, at least about 2.5 times, at least about 3 times, at least about 3.5 times, at least about 4 times, at least about 5 times, or at least about 10 times, compared with a total amount of SCNIA produced in a control cell.
[52]
52. Method according to claim 2, characterized by the fact that the disease or condition is induced by a gain-of-function mutation in Navl.1.
[53]
53. Method according to claim 52, characterized by the fact that the individual has an allele by which SCNIA is produced at an increased level, or an allele that encodes a mutant SCNIA that induces increased Na, l.1 activity in cell.
[54]
54. Method according to claim 52, characterized by the fact that the disease or condition is migraine.
[55]
55. Method according to claim 54, characterized by the fact that migraine is migraine, family hemiplegic, 3.
[56]
56. Method according to claim 2, characterized by the fact that the disease or condition is a genetic epilepsy of Na, vl.1.
[57]
57. Method according to claim 52, characterized in that the therapeutic agent inhibits the exclusion of NMD exon from the processed mRNA encoding SCNIA protein and decreases the expression of SCNI1A in the cell.
[58]
58. Method according to claim 52, characterized by the fact that the therapeutic agent is an antisense oligomer (ASO) and in which the ASO comprises a sequence that has at least about 80%, 85%, 90% , 95%, 97% or 100% complementary to any of the IDS. SEQ. Nº: 21, 25, 28, 36 or 63.
[59]
59. Method according to claim 1 or 2, characterized in that the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a modification of the framework comprising a phosphorothioate bond or a phosphorodiamidate bond.
[60]
60. Method according to claim 1 or 2, characterized in that the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises a morpholino phosphorodiamidate, a blocked nucleic acid, an acid peptide nucleic, a 2'-O-methyl moiety, a 2'-Fluorine moiety or a 2'-O-methoxyethyl moiety.
[61]
61. Method according to claim 1 or 2, characterized by the fact that the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer comprises at least a modified sugar portion.
[62]
62. Method according to claim 61, characterized in that each sugar portion is a modified sugar portion.
[63]
63. Method according to claim 1 or 2, characterized by the fact that the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer consists of 8 to 50 nucleobases, 8 to 40 nucleobases , 8 to 35 nucleobases, 8 to 30 nucleobases, 8 to 25 nucleobases, 8 to 20 nucleobases, 8 to 15 nucleobases, 9 to 50 nucleobases, 9 to 40 nucleobases, 9 to 35 nucleobases, 9 to 30 nucleobases, 9 to 25 nucleobases , 9 to 20 nucleobases, 9 to 15 nucleobases, 10 to 50 nucleobases, 10 to 40 nucleobases, 10 to 35 nucleobases, to 30 nucleobases, 10 to 25 nucleobases, 10 to 20 nucleobases, 10 to 15 nucleobases, 11 to 50 nucleobases, l1 to 40 nucleobases, 11 to 35 nucleobases, 11 to 30 nucleobases, 11 to 25 nucleobases, 11 to 20 nucleobases, 11 to 15 nucleobases, 12 to 50 nucleobases, 12 to 40 nucleobases, 12 to 35 nucleobases, 12 to 30 nucleobases, 12 to 25 nucleobases, 12 to nucleobases or 12 to 15 nucleobases.
[64]
64. Method according to claim 3, characterized by the fact that the therapeutic agent is an antisense oligomer (ASO) and in which the antisense oligomer is at least 80%, at least 85%, at least 90 %, at least 95%, at least 98%, at least 99% or 100%, complementary to the target portion of the NMD exon mRNA encoding the protein.
[65]
65. Method, according to claim 1, characterized by the fact that the method still comprises evaluation of the expression of SCNIA mRNA or protein.
[66]
66. Method, according to claim 2, characterized by the fact that the individual is a human.
[67]
67. Method, according to claim 2, characterized by the fact that the individual is a non-human animal.
[68]
68. Method according to claim 2, characterized by the fact that the individual is a fetus, an embryo or a child.
[69]
69. Method according to claim 1 or 2, characterized by the fact that the cells are ex vivo.
[70]
70. Method, according to claim 2, characterized by the fact that the therapeutic agent is administered by intrathecal injection, intracerebroventricular injection, intraperitoneal injection, intramuscular injection, subcutaneous injection, intravitreal or intravenous injection of the individual.
[71]
71. Method according to claim 2, characterized by the fact that the method still comprises administering a second therapeutic agent to the individual.
[72]
72. The method of claim 71,
characterized by the fact that the second therapeutic agent is a small molecule.
[73]
73. The method of claim 71, characterized by the fact that the second therapeutic agent is an ASO.
[74]
74. Method according to claim 73, characterized by the fact that the ASO comprises a sequence that has at least about 80%, 85%, 90%, 95%, 97% or 100% complementary to any of the IDS . SEQ. No. 115-
161.
[75]
75. Method according to claim 71, characterized in that the second therapeutic agent corrects intron retention.
[76]
76. Method according to claim 2, characterized by the fact that the disease or condition is Alzheimer's disease, SCN2A encephalopathy, SCN8A encephalopathy or SCNS5A arrhythmia.
[77]
77. Method according to claim 30, 32 or 34 characterized by the fact that the disease or condition is Alzheimer's disease, SCN2A encephalopathy, SCN8A encephalopathy or SCN5A arrhythmia.

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法律状态:
2020-11-17| B07D| Technical examination (opinion) related to article 229 of industrial property law [chapter 7.4 patent gazette]|Free format text: DE ACORDO COM O ARTIGO 229-C DA LEI NO 10196/2001, QUE MODIFICOU A LEI NO 9279/96, A CONCESSAO DA PATENTE ESTA CONDICIONADA A ANUENCIA PREVIA DA ANVISA. CONSIDERANDO A APROVACAO DOS TERMOS DO PARECER NO 337/PGF/EA/2010, BEM COMO A PORTARIA INTERMINISTERIAL NO 1065 DE 24/05/2012, ENCAMINHA-SE O PRESENTE PEDIDO PARA AS PROVIDENCIAS CABIVEIS. |

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2020-12-15| B07E| Notification of approval relating to section 229 industrial property law [chapter 7.5 patent gazette]|

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2021-05-04| B07A| Application suspended after technical examination (opinion) [chapter 7.1 patent gazette]|

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2021-08-24| B09B| Patent application refused [chapter 9.2 patent gazette]|

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2021-11-03| B12B| Appeal against refusal [chapter 12.2 patent gazette]|

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2021-11-23| B350| Update of information on the portal [chapter 15.35 patent gazette]|

优先权:

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申请号 | 申请日 | 专利标题

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US201762550462P| true| 2017-08-25|2017-08-25|

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US62/550,462|2017-08-25|

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US201762575901P| true| 2017-10-23|2017-10-23|

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US62/575,901|2017-10-23|

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US201862667356P| true| 2018-05-04|2018-05-04|

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US62/667,356|2018-05-04|

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US201862671745P| true| 2018-05-15|2018-05-15|

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US62/671,745|2018-05-15|

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PCT/US2018/048031|WO2019040923A1|2017-08-25|2018-08-24|Antisense oligomers for treatment of conditions and diseases|

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