viral vectors

Abstract

The present disclosure relates to an antagonist that directly or indirectly targets serine / arginine-rich splicing factor 1 (SRSF1); a viral vector comprising a nucleic acid sequence encoding the SRSF1 antagonist. Also disclosed are uses of the vector in gene therapy for the treatment of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or sporadic amyotrophic lateral sclerosis not caused by a pathological C9ORF72 hexanucleotide repeat expansion. Methods for the use of the vector in gene therapy for the treatment of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or sporadic amyotrophic lateral sclerosis (ALS), are also disclosed.

Description

[Technical Field]

[0001] Field of the Disclosure The present disclosure relates to an antagonist that directly or indirectly targets serine / arginine-rich splicing factor 1 (SRSF1); a viral vector comprising a nucleic acid sequence encoding the SRSF1 antagonist. Also disclosed are uses of the vector in gene therapy for the treatment of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or sporadic amyotrophic lateral sclerosis not caused by a pathological C9ORF72 hexanucleotide repeat expansion. Methods for the use of the vector in gene therapy for the treatment of neurodegenerative diseases, such as amyotrophic lateral sclerosis (ALS) or sporadic amyotrophic lateral sclerosis (ALS), are also disclosed. [Background technology]

[0002] Background of the Disclosure Gene therapy aims to treat diseases long-term by introducing genetic material that changes cell function. There are several gene therapy approaches, such as the delivery of functional genes to replace defective genes, the inactivation of toxic genes via gene silencing or antisense, the introduction or overexpression of genes that are not present in the host, and gene editing approaches. Genetic material is most commonly delivered using virus-based vectors, such as adenovirus (Ad), adeno-associated virus (AAV), self-complementary AAV, and retroviruses, i.e., lentiviruses.

[0003] The safety of gene therapy vectors requires particular attention to their long-term persistence in the patient's body, and gene therapy vectors must be designed to reduce genotoxic effects, immune responses, or prevent the activation of adjacent genes near the integration site. The backbone of a viral vector typically contains a protein capsid for packaging the nucleic acid to be expressed, genetic information describing the nucleic acid to be expressed located between inverted terminal repeats, and elements that enable efficient expression in the host, such as promoter elements. When delivering small-sized genetic material, such as short hairpin RNA (shRNA) or antisense oligonucleotides, a non-expressible "stuffer" nucleotide sequence is often required to increase the efficiency of shRNA or oligonucleotide nucleic acid targeting and expression and achieve optimal packaging capacity.

[0004] Neurodegenerative diseases are typically caused by neuronal dysfunction or loss and affect millions of people worldwide. Neurodegenerative diseases are more prevalent in the elderly population and include, but are not limited to, amyotrophic lateral sclerosis (ALS), multiple sclerosis, Parkinson's disease, Alzheimer's disease, motor neuron disease, and Huntington's disease. ALS and frontotemporal dementia (FTD) are adult-onset neurodegenerative diseases without effective treatments. ALS is the most common form of motor neuron disease (MND), a collective term for a group of neurological disorders characterized by the degeneration and loss of motor neurons. ALS is characterized by selective degeneration of upper and lower motor neurons, leading to muscle atrophy and premature death, usually due to respiratory failure and paralysis. Approximately 90% of ALS cases are classified as sporadic, while approximately 10% show a genetic component and familial inheritance. FTD is the second most common form of early-onset dementia, characterized by a progressive loss of nerve cells in the frontal and temporal lobes, leading to changes in cognitive function and personality.

[0005] The most common genetic cause of ALS and FTD is a hexanucleotide repeat expansion of GGGGCC in the first intron of the chromosome 9 open reading frame 72 (C9orf72) gene, termed C9ALS / FTD.

[0006] Antisense oligonucleotide therapy targeting C9ORF72 is in clinical trials, aiming to reduce the expression of the repeat expansion and therefore reduce RNA and DPR toxicity without affecting the normal expression of C9ORF72. U.S. Patent No. 10,801,027 demonstrates that depletion of the nuclear export adaptor serine / arginine-rich splicing factor 1 (SRSF1) inhibits the nuclear export of pathological C9ORF72 repeat transcripts carrying a hexanucleotide repeat expansion, and is incorporated herein by reference.

[0007] However, while SRSF1 depletion is functional in patients with ALS caused by a hexanucleotide repeat expansion, the present disclosure identifies that SRSF1 depletion also confers neuroprotection in sporadic ALS cases that are not caused by the pathological C9ORF72 hexanucleotide repeat expansion. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] U.S. Patent No. 10,801,027 [Patent Document 2] PCT / US2013 / 031644 [Patent Document 3] International Publication No. 2017207979 A1 [Non-patent literature]

[0009] [Non-Patent Document 1] Feng et al. (1997) Nature Biotechnology 15:866-870 [Non-patent document 2] Pennisi, E. (1996) Science 274:342-343 [Non-patent document 3] Russell, S.J. (1994) Eur.J.of Cancer 30A(8):1165-1171 [Non-patent document 4] Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA [Non-patent document 5] Hautbergue et al., Nature Communications 2017;8:16063 [Non-patent document 6] Meyer K et al., Proc. Natl. Acad. Sci. USA2014;111:829~832 [Non-Patent Document 7] Boudreau RL et al., Nucleic Acids Res.2013;41(1):e9 [Non-patent document 8] Castelli et al., bioRxiv 2021.05.23.445325v2 Summary of the Invention

[0010] Description of the invention According to an aspect of the present invention, there is provided a viral vector comprising a transcription cassette for expression of a nucleic acid molecule in a mammalian host cell, wherein the nucleic acid molecule is operably linked to a promoter adapted to express the nucleic acid molecule in the mammalian host cell, and wherein the vector comprises a non-expressible nucleotide sequence, and the nucleic acid molecule encodes an antagonist agent targeting serine / arginine-rich splicing factor 1 (SRSF1) or an SRSF1 peptide sequence.

[0011] Non-expressible nucleotide sequences are typically referred to as "stuffer" sequences. Stuffer nucleotide sequences are known in the art and are non-expressible nucleotide sequences that provide optimal viral packaging of virus-based vectors. Stuffer sequences are disclosed in PCT / US2013 / 031644, the entire contents of which are incorporated herein by reference. Stuffer nucleotide sequences can be placed between the viral inverted terminal repeats on either side of the transgene of interest, or two stuffer sequences could be added to each side of the transgene of interest.

[0012] In a preferred embodiment of the invention, the antagonist agent is a polypeptide or peptide.

[0013] In a preferred embodiment of the invention, the antagonist agent is a nucleic acid-based agent.

[0014] In a preferred embodiment of the invention, the nucleic acid-based agent is an antisense nucleic acid, an inhibitory RNA, or an shRNA or miRNA molecule that is complementary to a nucleic acid encoding serine / arginine-rich splicing factor 1 (SRSF1) and inhibits its expression.

[0015] Preferably, said SRSF1 comprises or consists of the sequence shown in SEQ ID NO:67.

[0016] Alternatively, said SRSF1 comprises or consists of the sequence shown in SEQ ID NO:76.

[0017] Nucleic acid-based agents are designed with reference to the sequence shown in SEQ ID NO:67, or alternatively with reference to the sequence shown in SEQ ID NO:76.

[0018] In a preferred embodiment of the invention, the nucleic acid-based agent is an inhibitory RNA.

[0019] In a preferred embodiment of the invention, the nucleic acid-based agent is an antisense RNA.

[0020] In a further preferred embodiment of the invention, said inhibitory RNA is an shRNA or miRNA molecule.

[0021] A technique for specifically ablation of gene function involves the introduction of double-stranded RNA, also known as small inhibitory or interfering RNA (siRNA, shRNA, and miRNA), into cells, which results in the destruction of mRNA complementary to the sequence contained in the siRNA molecule. siRNA molecules contain two complementary strands of RNA (sense and antisense strands) that anneal to each other to form a double-stranded RNA molecule. siRNA molecules are typically derived from exons of the gene to be ablated. The mechanism of RNA interference is still being elucidated. Many organisms respond to the presence of double-stranded RNA by activating a cascade that leads to the formation of siRNAs. The presence of double-stranded RNA activates a protein complex containing RNase III, which processes the double-stranded RNA into smaller fragments (siRNAs, approximately 21-29 nucleotides long) that become part of a ribonucleoprotein complex. The siRNA acts as a guide for the RNase complex to cleave the mRNA complementary to the antisense strand of the siRNA, thereby resulting in its destruction.

[0022] In a preferred embodiment of the invention, the inhibitory RNA molecule is 19 nucleotides (nt) to 29 nt in length. Even more preferably, the inhibitory RNA molecule is 21 nt to 27 nt in length. Preferably, the inhibitory RNA molecule is approximately 21 nt in length.

[0023] In a preferred embodiment of the invention, the inhibitory RNA comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58.

[0024] In a preferred embodiment of the invention, said shRNA comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11.

[0025] In a preferred embodiment of the invention, said shRNA comprises or consists of a nucleotide sequence as set forth in SEQ ID NO:7.

[0026] In a preferred embodiment of the invention, said shRNA comprises or consists of a nucleotide sequence as set forth in SEQ ID NO:10.

[0027] In a preferred embodiment of the invention, said shRNA comprises or consists of a nucleotide sequence as set forth in SEQ ID NO:11.

[0028] In a preferred embodiment of the invention, the peptide comprises an amino acid sequence that is at least 10 amino acids in length and comprises all or part of the amino acid sequence shown in SEQ ID NO:59.

[0029] In a preferred embodiment of the invention, the peptide comprises an amino acid sequence that is at least 32 amino acids in length and comprises the amino acid sequence shown in SEQ ID NO:59.

[0030] In preferred embodiments of the invention, the peptide is at least 10, 11, 12, 13, 14, 15, 16, 17, 18, 29, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or at least 100 amino acids in length, but shorter than the full-length amino acid sequence shown in SEQ ID NO: 60 or 61.

[0031] In a preferred embodiment of the invention, the peptide consists of the amino acid sequence shown in SEQ ID NO:59.

[0032] In an alternative embodiment of the invention, the peptide is a dominant-negative protein comprising an alteration of the amino acid sequence shown in SEQ ID NO: 60 or 61.

[0033] In a preferred embodiment of the present invention, the dominant negative protein comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 60 or 61, and the amino acid sequence is modified by the addition, deletion or substitution of one or more amino acid residues.

[0034] In a preferred embodiment of the invention, said modified protein comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 62 or 63.

[0035] In a preferred embodiment of the invention, said nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide or peptide is set forth in SEQ ID NO:89 or a sequence which is 90% identical to the sequence set forth in SEQ ID NO:89.

[0036] In a further preferred embodiment of the invention, the nucleic acid sequence is at least 36 nucleic acids in length.

[0037] In a preferred embodiment of the invention, the peptide is at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or 42 amino acids in length and comprises the amino acid sequence shown in SEQ ID NO:90.

[0038] In a preferred embodiment of the invention, the peptide comprises the amino acid sequence shown in SEQ ID NO: 75 (GSWQDLKDHMREA).

[0039] In a preferred embodiment of the invention, the viral vector comprises an RNA Pol III terminator.

[0040] Preferably, the terminator comprises the nucleic acid sequence 5' TTTTTT 3'.

[0041] In a preferred embodiment of the invention, the vector comprises an inverted terminal repeat nucleotide sequence.

[0042] Inverted terminal repeats (ITRs) are typically located upstream and downstream of the transcription cassette. Alternatively, the ITRs are upstream and downstream of the transcription cassette, the non-expressed nucleotide sequence, and any optional regulatory elements.

[0043] In a preferred embodiment of the invention, the ITR sequence is shown in SEQ ID NO:64.

[0044] In a preferred embodiment of the invention, the ITR sequence is shown in SEQ ID NO:88.

[0045] In a preferred embodiment of the invention, said promoter is selected from the group consisting of an H1 polymerase III promoter, a U6 promoter, a U7 promoter or a mammalian 7SK promoter.

[0046] In a further preferred embodiment of the invention, the promoter is an H1 polymerase III promoter.

[0047] In a preferred embodiment, the H1 polymerase III promoter is set forth in SEQ ID NO:65.

[0048] Viruses are commonly used as vectors for the delivery of exogenous genes. Commonly used vectors include recombinantly engineered enveloped or non-enveloped DNA and RNA viruses, such as baculoviridae, parvoviridae, picornoviridiae, herpesveridiae, poxviridae, adenoviridae, picornaviridae, or retroviridae, such as lentiviruses. Chimeric vectors that utilize advantageous elements of each parent vector's characteristics can also be used (see, for example, Feng et al. (1997) Nature Biotechnology 15:866-870). Such viral vectors can be wild-type or can be engineered by recombinant DNA technology to be replication-deficient, conditionally replicating, or replication-competent. Conditionally replicating viral vectors are used to achieve selective expression in specific cell types while avoiding harmful broad-spectrum infection. Conditionally replicating vectors are described in Pennisi, E. (1996) Science 274:342-343; Russell, S.J. (1994) Eur.J.of Cancer 30A(8):1165-1171.

[0049] Preferred viral vectors are derived from the adenovirus, adeno-associated virus, or retrovirus genomes.

[0050] In a preferred embodiment of the invention, the viral-based vector is an adeno-associated virus [AAV].

[0051] In a preferred embodiment of the invention, the adeno-associated virus is a self-complementary adeno-associated virus (scAAV).

[0052] In a preferred embodiment, the viral-based vector is selected from the group consisting of AAV2, AAV3, AAV6, AAV13; AAV1, AAV4, AAV5, AAV6, AAV9 and AAVrhlO.

[0053] In a preferred embodiment, the scAAV is selected from the group consisting of scAAV2, scAAV3, scAAV6, scAAV13; scAAV1, scAAV4, scAAV5, scAAV6, scAAV9 and scAAVrhlO.

[0054] In a preferred embodiment of the invention, the viral-based vector is scAAV9 or scAAVrhlO.

[0055] In an alternative preferred embodiment of the invention, said viral-based vector is a lentiviral vector.

[0056] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising a viral vector of the present invention and an excipient or carrier.

[0057] The viral vector compositions of the present invention are administered in pharmaceutically acceptable preparations. Such preparations may routinely contain pharmaceutically acceptable concentrations of salts, buffers, preservatives, compatible carriers, and adjunctive therapeutic agents. The expression vector compositions of the present invention can be administered by any conventional route, including injection or by gradual infusion over time, particularly intrathecal (e.g., lumbar puncture) and / or intracerebral routes.

[0058] The viral vector compositions of the present invention are administered in an effective amount. An "effective amount" is that amount of expression vector that, alone or together with further doses, produces the desired response. When treating a disease, the desired response is to inhibit the progression of the disease. This can involve only slowing the progression of the disease temporarily, but more preferably, permanently halting the progression of the disease. This can be monitored by conventional methods. Such amounts will, of course, depend on the particular condition being treated, the severity of the condition, individual patient parameters including age, health, size, and weight, the duration of treatment, the nature of concomitant therapy (if any), the specific route of administration, and similar factors within the knowledge and expertise of the healthcare professional. These factors are well known to those of skill in the art and can be addressed without more than routine experimentation. It is generally preferred that maximum doses of the individual components or combinations thereof be used, i.e., the highest safe dose according to sound medical judgment. However, it will be understood by those skilled in the art that a patient may require a lower or tolerated dose for medical reasons, psychological reasons, or virtually any other reason.

[0059] The viral vector composition used in the above-described method is preferably sterile and contains an effective amount of an expression vector according to the present invention to produce the desired response, in a mass or volume suitable for administration to a patient. The dose of the vector administered to a subject can be selected according to various parameters, particularly the mode of administration used and the subject's condition. Other factors include the desired duration of treatment. If the subject's response is insufficient with the initial dose applied, a higher dose (or an effective higher dose via a different, more localized delivery route) can be used, to the extent that patient tolerance allows. Other protocols for administering vector compositions, differing from those described above in terms of dose, injection schedule, injection site, mode of administration, etc., will be known to those skilled in the art. Administration of the composition to mammals other than humans (e.g., for testing or veterinary therapeutic purposes) is carried out under substantially the same conditions as described above. As used herein, a subject is a mammal, preferably a human, and also includes non-human primates, cows, horses, pigs, sheep, goats, dogs, cats, or rodents.

[0060] When administered, the viral vector compositions of the present invention are applied in pharmaceutically acceptable amounts and in pharmaceutically acceptable compositions. The term "pharmaceutically acceptable" refers to a non-toxic substance that does not interfere with the effectiveness of the biological activity of the active agent. Such preparations may conventionally contain salts, buffering agents, preservatives, compatible carriers, and optionally other therapeutic agents (e.g., those typically used in the treatment of specific disease indications). When used in medicines, salts must be pharmaceutically acceptable; however, non-pharmaceutically acceptable salts may be used conveniently to prepare pharmaceutically acceptable salts thereof and are not excluded from the scope of the present invention. Such pharmacologically and pharmaceutically acceptable salts include, but are not limited to, those prepared from the following acids: hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, maleic acid, acetic acid, salicylic acid, citric acid, formic acid, malonic acid, succinic acid, etc. Pharmaceutically acceptable salts can also be prepared as alkali metal or alkaline earth salts, e.g., sodium, potassium, or calcium salts.

[0061] Pharmaceutical compositions containing the viral vectors of the present invention may contain suitable buffers, including acetic acid as a salt, citric acid as a salt, boric acid as a salt, and phosphoric acid as a salt. The pharmaceutical compositions may also optionally contain suitable preservatives, such as benzalkonium chloride, chlorobutanol, parabens, and thimerosal.

[0062] Viral vector compositions can be conveniently presented in unit dosage form and prepared by any of the methods well known in the art of pharmacy. All methods include the step of bringing the active agent into association with the vector, which constitutes one or more accessory ingredients. Preparations can be formulated according to known methods using suitable dispersing or wetting agents and suspending agents. Sterile injectable preparations can also be sterile injectable solutions or suspensions in non-toxic parenterally acceptable diluents or solvents, for example, solutions in 1,3-butanediol. Acceptable solvents that can be employed include water, Ringer's solution, and isotonic sodium chloride solution. In addition, sterile fixed oils are conveniently employed as solvents or dispersion media. For this purpose, any bland fixed oil can be used, including synthetic mono- or diglycerides. In addition, fatty acids, such as oleic acid, can be used in the preparation of injectables. Suitable carrier formulations for oral, subcutaneous, intravenous, intramuscular, and the like administration can be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA.

[0063] According to a further aspect of the present invention there is provided a viral vector of the present invention for use as a medicament.

[0064] According to a further aspect of the present invention there is provided a viral vector of the present invention for use in the treatment of a neurodegenerative disease.

[0065] In a preferred embodiment of the invention, said neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), sporadic amyotrophic lateral sclerosis, familial ALS caused by mutations other than a pathogenic C9ORF72 repeat expansion, frontotemporal dementia (FTD), motor neuron disease, frontotemporal lobe dementia (FTLD), Huntington's-like disorder, and Fragile X-associated tremor / ataxia syndrome (FXTAS).

[0066] In a preferred embodiment of the invention, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

[0067] In a preferred embodiment of the invention, said neurodegenerative disease is sporadic and / or familial amyotrophic lateral sclerosis.

[0068] In a preferred embodiment of the invention, said neurodegenerative disease is ALS that is not caused by a pathological C9ORF72 repeat expansion.

[0069] In a preferred embodiment of the invention, said neurodegenerative disease is sporadic frontotemporal dementia (FTD).

[0070] In a preferred embodiment of the invention, said neurodegenerative disease is Fragile X-associated Tremor / Ataxia Syndrome (FXTAS).

[0071] According to a further aspect of the present invention there is provided a cell transfected with a viral vector of the present invention.

[0072] In a preferred embodiment of the invention, said cells are neurons and / or astrocytes.

[0073] In a preferred embodiment of the invention, said neurons are motor neurons and / or astrocytes.

[0074] According to a further aspect of the present invention, there is provided a method for treating or preventing a neurodegenerative disease, comprising the step of administering a therapeutically effective amount of the viral vector of the present invention to prevent and / or treat the neurodegenerative disease.

[0075] In a preferred method of the invention, said neurodegenerative disease is sporadic amyotrophic lateral sclerosis and familial amyotrophic lateral sclerosis.

[0076] In a preferred method of the invention, said neurodegenerative disease is amyotrophic lateral sclerosis.

[0077] In a preferred embodiment of the invention, said neurodegenerative disease is ALS that is not caused by a pathological C9ORF72 repeat expansion.

[0078] In a preferred method of the invention, said neurodegenerative disease is sporadic frontotemporal dementia (FTD).

[0079] In a preferred method of the invention, said neurodegenerative disease is Fragile X-associated Tremor / Ataxia Syndrome (FXTAS).

[0080] According to a further aspect of the present invention, there is provided an isolated nucleic acid molecule encoding an shRNA molecule comprising or consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11.

[0081] The present invention includes sequence variants corresponding to the specified SEQ ID NOs. Sequence variants differ from the reference sequence by 1, 2, 3, 4 or 5 nucleotide base changes.

[0082] In a preferred embodiment of the invention, the nucleic acid molecule comprises or consists of the nucleotide sequence shown in SEQ ID NO:7.

[0083] In a preferred embodiment of the invention, the nucleic acid molecule comprises or consists of the nucleotide sequence shown in SEQ ID NO:10.

[0084] In a preferred embodiment of the invention, the nucleic acid molecule comprises or consists of the nucleotide sequence shown in SEQ ID NO:11.

[0085] According to a further aspect of the present invention, there is provided an shRNA molecule comprising a nucleotide sequence selected from the group consisting of the following sequences, or a variant thereof: [ka]

[0086] In a preferred embodiment of the invention, said shRNA molecule comprises or consists of the nucleotide sequence shown in SEQ ID NO: 96 or a variant thereof.

[0087] In a preferred embodiment of the invention, said shRNA molecule comprises or consists of the nucleotide sequence shown in SEQ ID NO: 99 or a variant thereof.

[0088] In a preferred embodiment of the invention, said shRNA molecule comprises or consists of the nucleotide sequence shown in SEQ ID NO: 100 or a variant thereof.

[0089] According to an aspect of the present invention, there is provided an isolated nucleic acid molecule or shRNA of the present invention for use as a medicament.

[0090] According to a further aspect of the invention there is provided an isolated nucleic acid molecule or shRNA of the invention for use in the treatment of a neurodegenerative disease.

[0091] In a preferred embodiment of the invention, said neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), sporadic amyotrophic lateral sclerosis, familial ALS caused by mutations other than a pathogenic C9ORF72 repeat expansion, frontotemporal dementia (FTD), motor neuron disease, frontotemporal lobe dementia (FTLD), Huntington's-like disorder, and Fragile X-associated tremor / ataxia syndrome (FXTAS).

[0092] In a preferred embodiment of the invention, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

[0093] In a preferred embodiment of the invention, said neurodegenerative disease is sporadic and / or familial amyotrophic lateral sclerosis.

[0094] In a preferred embodiment of the invention, said neurodegenerative disease is ALS that is not caused by a pathological C9ORF72 repeat expansion.

[0095] In a preferred embodiment of the invention, said neurodegenerative disease is sporadic frontotemporal dementia (FTD).

[0096] In a preferred embodiment of the invention, said neurodegenerative disease is Fragile X-associated Tremor / Ataxia Syndrome (FXTAS).

[0097] According to an embodiment of the present invention, there are provided siRNA molecules comprising or consisting of nucleic acid sequences designed with reference to the shRNAs shown in SEQ ID NOs: 77 to 86.

[0098] According to an embodiment of the present invention, there is provided an siRNA molecule of the present invention for use as a pharmaceutical.

[0099] According to a further aspect of the present invention, there is provided an siRNA molecule of the present invention for use in the treatment of a neurodegenerative disease.

[0100] In a preferred embodiment of the invention, said neurodegenerative disease is selected from the group consisting of amyotrophic lateral sclerosis (ALS), sporadic amyotrophic lateral sclerosis, familial ALS caused by mutations other than a pathogenic C9ORF72 repeat expansion, frontotemporal dementia (FTD), motor neuron disease, frontotemporal lobe dementia (FTLD), Huntington's-like disorder, and Fragile X-associated tremor / ataxia syndrome (FXTAS).

[0101] In a preferred embodiment of the invention, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

[0102] In a preferred embodiment of the invention, said neurodegenerative disease is sporadic and / or familial amyotrophic lateral sclerosis.

[0103] In a preferred embodiment of the invention, said neurodegenerative disease is ALS that is not caused by a pathological C9ORF72 repeat expansion.

[0104] In a preferred embodiment of the invention, said neurodegenerative disease is sporadic frontotemporal dementia (FTD).

[0105] In a preferred embodiment of the invention, said neurodegenerative disease is Fragile X-associated Tremor / Ataxia Syndrome (FXTAS).

[0106] According to an embodiment of the present invention, there is provided a cell-penetrating polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:90.

[0107] In a preferred embodiment of the invention, the polypeptide is 12 to 42, or preferably 13 to 42 amino acids in length.

[0108] In a further preferred embodiment of the invention, said polypeptide comprises or consists of the amino acid sequence shown in SEQ ID NO:75.

[0109] According to an aspect of the present invention, there is provided a polypeptide of the present invention for use as a pharmaceutical.

[0110] According to a further aspect of the present invention there is provided a polypeptide of the present invention for use in the treatment of a neurodegenerative disease.

[0111] In a preferred embodiment of the invention, said neurodegenerative disease is selected from the group consisting of sporadic amyotrophic lateral sclerosis (ALS), amyotrophic lateral sclerosis, familial ALS caused by mutations other than a pathogenic C9ORF72 repeat expansion, frontotemporal dementia (FTD), motor neuron disease, frontotemporal lobe dementia (FTLD), Huntington's-like disorder, and Fragile X-associated tremor / ataxia syndrome (FXTAS).

[0112] In a preferred embodiment of the invention, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

[0113] In a preferred embodiment of the invention, said neurodegenerative disease is sporadic and / or familial amyotrophic lateral sclerosis.

[0114] In a preferred embodiment of the invention, said neurodegenerative disease is ALS that is not caused by a pathological C9ORF72 repeat expansion.

[0115] In a preferred embodiment of the invention, said neurodegenerative disease is sporadic frontotemporal dementia (FTD).

[0116] In a preferred embodiment of the invention, said neurodegenerative disease is Fragile X-associated Tremor / Ataxia Syndrome (FXTAS).

[0117] According to a further aspect of the present invention, there is provided an antagonist agent comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence designed with reference to human serine / arginine-rich splicing factor 1 (SRSF1), and the nucleic acid molecule inhibits expression of SRSF1.

[0118] In a preferred embodiment of the present invention, the nucleic acid molecule is a double-stranded nucleic acid molecule comprising a sense strand and an antisense strand comprising a nucleotide sequence, and the antisense nucleotide strand is adapted to anneal to a nucleic acid molecule encoding human SRSF1 by complementary base pairing.

[0119] In a preferred embodiment of the present invention, the double-stranded nucleic acid molecule is RNA. Preferably, the RNA is siRNA or miRNA.

[0120] In an alternative embodiment of the invention, the nucleic acid molecule is a single-stranded nucleotide sequence comprising an antisense nucleotide sequence, wherein the antisense nucleotide sequence is adapted to anneal to a nucleic acid molecule encoding SRSF1 by complementary base pairing.

[0121] In a preferred embodiment of the invention, the single-stranded nucleic acid is DNA.

[0122] In a further preferred embodiment of the invention, the single-stranded nucleic acid is DNA and / or RNA.

[0123] Preferably, the DNA and / or RNA is a therapeutic antisense oligonucleotide, such as an antisense oligonucleotide, a splice-switching oligonucleotide, a gapmer, or the like.

[0124] Preferably, the DNA is an antisense oligonucleotide.

[0125] In a preferred embodiment of the present invention, the nucleic acid molecule encoding human SRSF1 is set forth in SEQ ID NO:67.

[0126] In a preferred embodiment of the invention, the antagonist agent comprises a nucleic acid molecule that is at least 15 nucleotides in length.

[0127] In a preferred embodiment of the present invention, the antagonist agent comprises a nucleic acid molecule comprising the nucleotide sequence shown in SEQ ID NO: 67, wherein the nucleic acid molecule is a double-stranded inhibitory RNA and is 19 to 23 nucleotides in length.

[0128] In a preferred embodiment of the invention, the antagonist agent comprises a nucleic acid molecule comprising modified nucleotides.

[0129] In a preferred embodiment of the present invention, said double-stranded nucleic acid molecules, including sense and antisense nucleic acid molecules, comprise modified nucleotides.

[0130] In a preferred embodiment of the invention, the modified nucleotide / sugar is a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl ... nucleotide), 2'-methoxyethyl modified nucleotide, 2'-O-alkyl modified nucleotide, morpholino nucleotide, phosphoramidate, non-natural base containing nucleotide, tetrahydropyran modified nucleotide, 1,5-anhydrohexitol modified nucleotide, cyclohexenyl modified nucleotide, nucleotide containing a phosphorothioate group, nucleotide containing a phosphorodithioate (PS2), nucleotide containing a methyl phosphonate group, nucleotide containing a 5'-phosphate, and 5'-phosphate mimics, e.g., nucleotide containing a 5'-vinyl phosphate, nucleotide containing 2'-deoxy-2'-fluro, and 2' methyl sugar base.

[0131] In a preferred embodiment of the present invention, said double-stranded nucleic acid molecules, including sense and antisense nucleic acid molecules, comprise modified sugars.

[0132] In a preferred embodiment of the invention, the modified sugar comprises a modified ribosyl moiety, e.g., an -O-modified RNA, such as a 2'-O-alkyl or 2'-O-(substituted) alkyl, e.g., 2'-O-methyl, T-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, -O-propargyl, 2'-O-allyl, 2'-O-(2-amino)propyl, 2'-O-(2-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2 -(dimethylamino)ethyl; 2'-deoxy (DNA); 2'-O-(haloalkoxy)methyl, such as 2'-O-(2-chloroethoxy)methyl (MCEM), -O-(2,2-dichloroethoxy)methyl (DCEM); 2'-<3-alkoxycarbonyl, such as T-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), T-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2'-halo, such as 2'-F, FANA (2'-F arabinosyl nucleic acid); carba- and aza-sugar modifications; 3'-O-alkyl, such as 3'-O-methyl, 3'-O-butyryl, VO-propargyl, and derivatives thereof.

[0133] In a preferred embodiment of the invention, said antagonist agent comprises or consists of a nucleotide sequence designed with reference to a target nucleic acid sequence selected from the group consisting of: [ka]

[0134] In a preferred embodiment of the present invention, the antagonist agent comprises a nucleic acid molecule comprising a nucleotide sequence, wherein the nucleic acid molecule is a double-stranded inhibitory RNA and is 19 to 23 nucleotides in length.

[0135] According to a further aspect of the present invention there is provided a pharmaceutical composition comprising an antagonist agent of the present invention and an excipient or carrier.

[0136] According to a further aspect of the present invention there is provided an antagonist agent of the present invention for use as a pharmaceutical.

[0137] According to a further aspect of the present invention there is provided an antagonist agent to the compounds of the present invention for use in the treatment of neurodegenerative diseases.

[0138] In a preferred embodiment of the invention, the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

[0139] In a preferred embodiment of the invention, said neurodegenerative disease is sporadic and / or familial amyotrophic lateral sclerosis.

[0140] In a preferred embodiment of the invention, said neurodegenerative disease is ALS that is not caused by a pathological C9ORF72 repeat expansion.

[0141] In an alternative preferred embodiment of the invention, said neurodegenerative disease is sporadic frontotemporal dementia (FTD).

[0142] In an alternative preferred embodiment of the invention, said neurodegenerative disease is Fragile X-associated Tremor / Ataxia Syndrome (FXTAS).

[0143] Throughout the description and claims of this specification, the words "comprise" and "contain," as well as variations of these words, such as "comprising" and "comprises," mean "including but not limited to," and are not intended to (and do not) exclude other moieties, additives, ingredients, integers, or steps. "Consisting essentially of" means having the requisite integers, but including integers that do not materially affect the function of the requisite integers.

[0144] Throughout the description and claims of this specification, the singular encompasses the plural unless the context requires otherwise. In particular, where the indefinite article is used, the specification is to be understood as contemplating plural as well as singular, unless the context requires otherwise.

[0145] It is to be understood that any feature, integer, property, compound, chemical moiety, or group described in connection with any aspect, embodiment, or example of the invention is applicable to any other aspect, embodiment, or example described herein, to the extent not incompatible therewith.

[0146] Embodiments of the present invention will now be described, by way of example only, with reference to the following drawings: [Brief explanation of the drawings]

[0147] [Figure 1] FIG. 1 shows the timeline for differentiation and co-culture of motor neurons and astrocytes from healthy controls and sporadic ALS (sALS) patients. [Figure 2] The images show that MNs treated with lentivirus expressing SRSF1-miRNA retain neuronal process / axonal properties compared to MNs treated with LV_Ctrl-miRNA, which degenerate and die. [Figure 3] Bar graphs show MN survival (%) expressed as the ratio of MN quantified on counting day 3 to day 1. Two-way ANOVA with Tukey's multiple comparison test; NS: not significant; **: p<0.01; ***: p<0.001; ****: p<0.0001. [Figure 4] Western immunoblotting shows that all three shRNAs result in efficient depletion of SRSF1 and inhibition of RAN translation of V5-tagged DPR. [Figure 5] Bar graphs represent mean ± sem (two-way ANOVA with Tukey's multiple comparison test; NS: non-significant; ****: p<0.0001; n=3 biological replicates). Quantification in three independent triplicate experiments. [Figure 6] C9ORF72-ALS / FTD mice were injected via the cisterna magna on postnatal day 1 (P1) with either 8x10 scAAV9_Ctrl-shRNA_GFP vector genomes (vg) or 6x10 scAAV9_SRSF1-shRNA10_GFP vg. Animals were sacrificed 1 and 3 months after injection. Western blots show that the scAAV9_SRSF1-shRNA10_GFP virus results in specific depletion of SRSF1 in C9ORF72-ALS / FTD mice as well as in wild-type C57BL / 6 mice (not shown), whereas Ctrl-shRNA has no effect. GAPDH was used as a loading control. [Figure 7] FIG. 1 shows a map of scAAV_SRSF1 132-144 CPP_GFP (SEQ ID NOs: 1 and 101). [Figure 8] FIG. 1 shows a map of scAAV_SRSF1 89-120 CPP_GFP (SEQ ID NOs: 74 and 102). [Figure 9] (A) Western blots show depletion of SRSF1 and inhibition of RNA translation of sense DPR upon cotransfection with scAAV SRSF1-shRNA10_GFP, H1-CPP1_GFP, and H1-CPP2_GFP, but not when CPP transcription is driven by the RNAPII promoter. SRSF1 and DPR expression levels are quantified in triplicate biological experiments in panels B and C, respectively. (D) MTT cell proliferation assays in biological triplicates showing that scAAV SRSF1-shRNA10_GFP, H1-CPP1_GFP, and H1-CPP2_GFP alleviate cytotoxicity mediated by DPR expression, but not when CPP transcription is driven by the RNAPII promoter. [Figure 10](A) Western blots show depletion of SRSF1 and inhibition of RNA translation of antisense DPRs upon cotransfection with scAAV SRSF1-shRNA10_GFP, H1-CPP1_GFP, and H1-CPP2_GFP, but not when CPP transcription is driven by the RNAPII promoter. SRSF1 and DPR expression levels are quantified in triplicate biological experiments in panels B and C, respectively. (D) MTT cell proliferation assays in biological triplicates showing that scAAV SRSF1-shRNA10_GFP, H1-CPP1_GFP, and H1-CPP2_GFP attenuate cytotoxicity mediated by DPR expression, but not when CPP transcription is driven by the RNAPII promoter. [Figure 11] FIG. 1 shows a map of scAAV_SRSF1-shRNA10_GFP (SEQ ID NOs: 66 and 103). [Figure 12] Figure 1 shows DPR quantification in mouse brain. C9ORF72-ALS / FTD (C9-Tg) mice were intrathecally injected (via the cisterna magna) with 6 × 10 vector genomes (vg) of scAVV9_Ctrl-shRNA_GFP or two doses of scAAV9_SRSF1-shRNA10_GFP (4 × 10 and 8 × 10 vg) on postnatal days 1–2 (P1–2). Nontransgenic (NTg) mice served as controls. One month (A) and three months (B) after injection, animals were sacrificed, followed by MSD-ELISA quantification of polyGP DPR in the cerebellum / brainstem (mean ± SEM; one-way ANOVA with Tukey's correction for multiple comparisons; NS: not significant; **: p < 0.01; ****: p < 0.0001; n = 4–6 mice per group). PolyGP DPR was quantified against a standard curve established with GPx7 peptide and levels were normalized to 100% for untreated C9-Tg mice. [Figure 13]Figure 1. Viral transduction in mouse brain. Immunohistological analysis of C9ORF72-ALS / FTD mice intrathecally injected (via the cisterna magna) with 5 × 10 vector genomes (vg) of scAAV9_H1-SRSF1-CPP1_GFP or scAAV9_H1-CPP2_GFP on postnatal day 1-2 (P1-2). Animals were sacrificed one month after injection, followed by anti-GFP immunofluorescence microscopy of the brain. Representative images of midbrain sections are shown. GFP coexpression is displayed in the green channel. DAPI (blue channel) and NeuN (red channel) stain nuclei and mature neurons, respectively. Side panels: Enlarged immunofluorescence images showing scAAV9-mediated transduction and GFP expression in both neuronal and microglial cells. Scale bar represents 500 μm. [Figure 14] Figure 1 shows viral biodistribution and DPR quantification in mouse brain. C9ORF72-ALS / FTD (C9-Tg) mice were intrathecally injected (via the cisterna magna) with 5 × 10 vector genomes (vg) of scAAV9_H1-SRSF1-CPP1_GFP or scAAV9_H1-CPP2_GFP on postnatal day 1-2 (P1-2). Nontransgenic (NTg) mice served as controls. Animals were sacrificed one month after injection. (A) qPCR quantification of viral DNA extracted from the brain (cerebellum) and spinal cord (n = 3), demonstrating efficient transduction. (B) MSD-ELISA quantification of polyGP DPR in the cerebellum / brainstem (mean ± SEM; one-way ANOVA with Tukey's correction for multiple comparisons; **: p < 0.01; ****: p < 0.0001; N = 3 mice / group). PolyGP DPR was quantified against a standard curve established with GPx7 peptide and levels were normalized to 100% for untreated C9-Tg mice. DETAILED DESCRIPTION OF THE INVENTION

[0148] Materials and Methods Part 1: SRSF1 depletion promotes survival of sALS patient-derived motor neurons co-cultured with astrocytes 1 / Timeline for differentiation and co-culture of motor neurons and astrocytes from healthy controls and sporadic ALS (sALS) patients: Summary: Both iMotor Neurons (iMNs) and iAstrocytes were treated with lentivirus (LV) expressing either Ctrl-miRNA or double-stranded miRNA targeting SRSF1 (constructs described in Hautbergue et al., Nature Communications 2017;8:16063 and our patent WO2017207979A1) at a multiplicity of infection (MOI) of 5 on days 18 and 3 of differentiation, respectively, followed by establishment of cocultures from days 20 (iMNs) and 5 (iA). High-content automated live imaging quantified iMN viability at days 22, 23, and 24. In contrast to the lentiviruses used here in this system, scAAV9 does not efficiently transduce cells in vitro.

[0149] Detailed Protocol: Co-culture of Patient-Derived Astrocytes and Motor Neurons iMotor Neuron (iMN) Differentiation. Human patient- and control-derived neurons (iNeurons) were differentiated from induced neural progenitor cells (iNPCs) using a modified protocol (Meyer K et al., Proc. Natl. Acad. Sci. USA 2014;111:829-832) as previously described (Hautbergue GM et al., Nature Communications 2017;8:16063). Briefly, 100,000 iNPCs were plated in fibronectin-coated 6-well plates (Millipore) and expanded to 70-80% confluence. Once confluence was reached, iNPC medium was replaced with neuronal differentiation medium (Glutamax-containing DMEM / F-12 (Gibco) supplemented with 1% N2, 2% B27) containing 2.5 μM DAPT (Tocris) on day 1 to determine differentiation into neural lineages. On day 3, neuronal differentiation medium was supplemented with 1 μM retinoic acid (Sigma), 0.5 μM smoothened agonist (SAG) (Millipore), and 2.5 μM forskolin (Sigma) for 7 days until day 10. This protocol results in a typical yield of 70% β-III tubulin (Tuj1)-positive cells. iMotor To obtain iMNs, approximately 5,000 iNeurons per well were replated onto fibronectin-coated 96-well plates and maintained in iNeuron differentiation medium (containing retinoic acid, SAG, and forskolin) supplemented with BDNF, CNTF, and GDNF (all at 20 ng / mL) for the final 14 days of differentiation.

[0150] iAstrocyte differentiation. Human patient-derived astrocytes (iAstrocytes) were differentiated from iNPCs as previously described (Meyer K et al., Proc. Natl. Acad. Sci. USA 2014;111:829-832; Hautbergue GM et al., Nature Communications 2017;8:16063) and cultured in DMEM glutamax (Gibco) containing 10% FBS (Sigma) and 0.02% N2 (Invitrogen) for 5 days. Cells were maintained in a 37°C incubator containing 5% CO2.

[0151] Coculture of patient-derived iMNs and iAstrocytes. iAstrocytes were harvested on day 5 of differentiation, and approximately 5,000 iAstrocytes were replated on iMNs on day 20 of differentiation. Cocultured iMNs and iAstrocytes were maintained in neuronal differentiation medium containing BDNF, GDNF, and CTNF (all at 20 ng / mL) for 4 days. 12 hours after the initiation of coculture (day 21), 1 or 10 μM CPP was added to the medium, and iMNs / iAstrocytes were imaged for 72 hours on days 22, 23, and 24. For SRSF1 knockdown, iMNs and iAstrocytes were transduced separately at an MOI of 5 on day 18 of iMN differentiation and day 3 of iAstrocyte differentiation with GFP-coexpressing control or SRSF1-RNAi-expressing lentivirus (LV) (Hautbergue GM et al., Nature Communications 2017;8:16063) 48 hours prior to coculture.

[0152] Part 2: scAAV9-driven expression of SRSF1-shRNA Preclinical vector design: scAAV_SRSF1-shRNA_GFP 1 / The SRSF1-shRNA cassette targeting mouse, rat, non-human primate, and human SRSF1 carries the region 448-750 of human SRSF1 (3' end of the open reading frame), which is highly conserved with mouse SRSF1.

[0153] Human SRSF1 (NM_006924.4) SEQ ID NO: 67 [ka]

[0154] used to target human and mouse SRSF1 in lentiviral constructs (Hautbergue et al., Nature Communications 2017;8:16063 and our patent WO2017207979A1), [ka]

[0155] Design shRNA using the following website: Block-iT RNAi Designer tool: http: / / rnaidesiqner.lifetechnologies.com / rnaiexpress /

[0156] [Table 1]

[0157] [Table 2]

[0158] 2 / Align human (NM_006924.4; SEQ ID NO: 104) and mouse (NM_173374.4; SEQ ID NO: 105) SRSF1 The sequences corresponding to shRNAs 7, 10, and 11 (predicted to be most efficient with relatively small predicted off-target effects) are highlighted on the aligned human and mouse SRSF1 open reading frames.

[0159] [ka] [ka]

[0160] [Table 3]

[0161] 3 / Cloning of SRSF1-targeting shRNA into scAAV_GFP vector Next, we designed and custom synthesized the following oligonucleotides (SEQ ID NOs: 68-73) for cloning shRNAs 7, 10, and 11 into our scAAV_H1promoter_GFP vector:

[0162] BamHI cleavage / HindIII cleavage Red sequence corresponds to SRSF1 targeting region Blue sequence corresponds to antisense / mature shRNA Black sequence corresponds to hairpin loop

[0163] [ka]

[0164] 5 / Complete sequence of a preclinical scAAV vector co-expressing an SRSF1-shRNA cassette (under the constitutively expressed RNAPIII H1 promoter) and eGFP (under the weak RNAPII eF-1α core promoter to avoid potential GFP-induced toxicity)

[0165] scAAV_SRSF1-shRNA10_GFP circular sequence (5,648 bp) SEQ ID NO: 66 [ka] [ka]

[0166] 6 / Functionality of scAAV9-driven expression of SRSF1-shRNA10 in the mouse brain C9ORF72-ALS / FTD mice received 8 × 10 10 scAAV9_Ctrl-shRNA_GFP vector genome (vg) or 6 × 10 10 Either scAAV9_SRSF1-shRNA10_GFP or scAAV9_SRSF1-shRNA10_GFP vg was injected via the cisterna magna on postnatal day 1 (P1). Animals were sacrificed 1 and 3 months after injection. Western blots show that scAAV9_SRSF1-shRNA10_GFP virus leads to specific depletion of SRSF1 in C9ORF72-ALS / FTD mice and wild-type C57B16 mice (not shown), whereas Ctrl-shRNA has no effect. GAPDH was used as a loading control.

[0167] 4 / ITR sequence SEQ ID NO:64: [ka]

[0168] SEQ ID NO:88: [ka]

[0169] Cell-penetrating peptide sequence SRSF1 132-144 CPP nucleotide sequence: [ka]

[0170] SRSF1 132-144 CPP sequence corresponding to SRSF1 amino acids 132-144, V5 tag and protein transduction domain TAT amino acids 47-57: Nt-GSWQDLKDHMREAGGGKPIPNPLLGLDSTGGYGRKKRRQRRR-Ct (SEQ ID NO: 90)

[0171] 5 / scAAV_SRSF1 89-120 CPP_GFP circular sequence (5,692 bp) (SEQ ID NO: 74) [ka] [ka] [ka]

[0172] 6 / 5 / scAAV_SRSF1 132-144 CPP_GFP circular sequence (5,651 bp) (SEQ ID NO: 1) [ka] [ka] [ka]

[0173] Functionality of scAAV9-driven expression of SRSFl-shRNA10 in the mouse brain C9ORF72-ALS / FTD mice received 8 × 10 10 scAAV9_Ctrl-shRNA_GFP vector genome (vg) or 6 × 10 10 Either scAAV9_SRSF1-shRNA10_GFP or scAAV9_SRSF1-shRNA10_GFP vg was injected via the cisterna magna on postnatal day 1 (P1). Animals were sacrificed 1 and 3 months after injection. Western blots show that the scAAV9_SRSF1-shRNA10_GFP virus specifically depletes SRSF1 in C9ORF72-ALS / FTD mice and wild-type C57B16 mice (not shown), whereas Ctrl-shRNA has no effect. GAPDH was used as a loading control. [Example]

[0174] 2 / Example of iMN imaging on the 24th day High-content automated imaging (Opera Phenix) was used to quantify surviving MNs on imaging days 1, 2, and 3. Images (Figure 2) show that MNs treated with lentivirus expressing SRSF1-miRNA retain neuronal process / axonal properties compared to MNs treated with LV_Ctrl-RNAi, which generate and die.

[0175] 3 / iMN quantification Coculture of healthy control and sALS patient-derived MNs with astrocytes demonstrates that LV_SRSF1-RNAi specifically promotes sALS MN survival at a level comparable to that of SRSF1 depletion in C9ORF72-ALD patient-derived MNs (Hautbergue GM et al., Nature Communications 2017;8:16063; Castelli et al., bioRxiv 2021.05.23.445325v2). Bar graphs show MN survival (%) expressed as the ratio of MNs quantified on day 3 of counting to those on day 1. Two-way ANOVA with Tukey's multiple comparison test; NS: not significant; **: p<0.01; ***: p<0.001; ****: p<0.0001 (Figure 3). [Example]

[0176] 4 / Testing the functionality of SRSF1-shRNA in human cells and mouse brain An scAAV plasmid co-expressing GFP and SRSF1 shRNA6, 9, or 10 was co-transfected with either a sense or antisense C9ORF72 repeat reporter construct expressing a V5-tagged sense or antisense dipeptide repeat protein (DPR) in full frame in a repeat-associated non-AUG (RAN) translation mode. Western immunoblotting shows that all three shRNAs efficiently deplete SRSF1 and inhibit RAN translation of the V5-tagged DPR. SRSF1-shRNA10 was selected for virus generation and further experiments in mice because it had the lowest POTS score and predicted genome-wide off-target effects in both mice and humans. [Example]

[0177] scAAV SRSF1-shRNA, CPP1, and CPP2 inhibit sense DPR production and rescue DPR-associated cytotoxicity in a human cell model of C9ORF72-ALS / FTD. Human HEK293T cells were cotransfected with a sense G4C2x45 C9ORF72 repeat plasmid expressing a sense V5-tagged dipeptide repeat protein (DPR) in the RNA translational mode and scAAV plasmids expressing SRSF1-shRNA10 or two different cell-penetrating peptides (CPP1: SRSF1 aa89-120 CPP (SEQ ID NO: 59) and CPP2: SRSF1 aa132-144 CPP (SEQ ID NO: 75)). We tested potential expression under the mammalian ubiquitous RNA polymerase II (CBh) or RNA polymerase III (H1) promoter. As shown in Figure 9(A), Western blots demonstrate the depletion of SRSF1 and the inhibition of RNA translation of sense DPR upon cotransfection with scAAV SRSF1-shRNA10_GFP, H1-CPP1_GFP, and H1-CPP2_GFP, but not when CPP transcription is driven by the RNAPII promoter. SRSF1 and DPR expression levels are quantified in triplicate biological experiments in panels B and C, respectively (bar graphs show mean ± SEM; one-way ANOVA; NS: not significant, ****: p<0.0001). (D) MTT cell proliferation assays in biological triplicates demonstrate that scAAV SRSF1-shRNA10_GFP, H1-CPP1_GFP, and H1-CPP2_GFP attenuate cytotoxicity mediated by DPR expression, but not when CPP transcription is driven by the RNAPII promoter. Bar graphs show mean±SEM; one-way ANOVA; NS: not significant, ****: p<0.0001. [Example]

[0178] scAAV SRSF1-shRNA, CPP1, and CPP2 inhibit the production of antisense DPRs and rescue DPR-associated cytotoxicity in a human cell model of C9ORF72-ALS / FTD. Human HEK293T cells were cotransfected with an antisense G2C4x43 C9ORF72 repeat plasmid expressing an antisense V5-tagged dipeptide repeat protein (DPR) in the RNA translational manner and scAAV plasmids expressing SRSF1-shRNA10 or two different cell-penetrating peptides (CPP1: SRSF1 aa89-120 CPP (SEQ ID NO: 59) and CPP2: SRSF1 aa132-144 CPP (SEQ ID NO: 75)). We tested potential expression under the mammalian ubiquitous RNA polymerase II (CBh) or RNA polymerase III (H1) promoter. Figure 10(A) Western blot shows depletion of SRSF1 and inhibition of antisense DPR RNA translation upon cotransfection with scAAV SRSF1-shRNA10_GFP, H1-CPP1_GFP, and H1-CPP2_GFP, but not when CPP transcription is driven by the RNAPII promoter. SRSF1 and DPR expression levels are quantified in triplicate biological experiments in panels B and C, respectively (bar graphs show mean ± SEM; one-way ANOVA; NS: not significant, ****: p<0.0001). Note the absence of CPP1 and CPP2 expression from the protein-coding CBh promoter. (D) MTT cell proliferation assay in biological triplicates showing that scAAV SRSF1-shRNA10_GFP, H1-CPP1_GFP, and H1-CPP2_GFP attenuate cytotoxicity mediated by expression of DPRs, but not when CPP transcription is driven by the RNAPII promoter. Bar graphs show mean ± SEM; one-way ANOVA; NS: not significant, ****: p<0.0001. [Example]

[0179] The data on SRSF1-shRNA (FIG. 12) complements the data showing that the svAAV9-SRSF1-shRNA10 virus results in inhibition of DPR in the mouse brain and results in SRSF1 depletion in the mouse brain. [Example]

[0180] The data show that scAAV9 viruses expressing CPP1 or CPP2 and co-expressing GFP efficiently transduced neurons and glial cells in the mouse brain (Figure 13) and resulted in DPR inhibition (Figure 14).

Claims

1. A viral vector comprising a transcription cassette for expression of a nucleic acid molecule in a mammalian host cell, wherein the nucleic acid molecule is operably linked to a promoter adapted to express the nucleic acid molecule in the mammalian host cell, and wherein the vector comprises a non-expressible nucleotide sequence, and the nucleic acid molecule encodes an antagonist agent that targets serine / arginine-rich splicing factor 1 (SRSF1).

2. The viral vector of claim 1, wherein the SRSF1 comprises or consists of the sequence shown in SEQ ID NO:

67.

3. The viral vector of claim 1 or 2, wherein the antagonist agent is a nucleic acid molecule comprising a nucleic acid sequence encoding a polypeptide or peptide.

4. The viral vector of claim 1 or 2, wherein the antagonist agent is a nucleic acid-based agent.

5. A viral vector described in any one of claims 1 to 2 or 4, wherein the nucleic acid-based drug is an antisense nucleic acid, inhibitory RNA, shRNA or miRNA molecule that is complementary to a nucleic acid encoding a serine / arginine-rich splicing factor (SRSF1) and inhibits its expression.

6. 6. The viral vector of claim 5, wherein the inhibitory RNA comprises or consists of a nucleotide sequence as set forth in SEQ ID NO: 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 or 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57 or 58.

7. The viral vector of claim 5, wherein the shRNA comprises or consists of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11.

8. The viral vector of claim 7, wherein the shRNA comprises or consists of the nucleotide sequence shown in SEQ ID NO:

7.

9. The viral vector of claim 7, wherein the shRNA comprises or consists of the nucleotide sequence shown in SEQ ID NO:

10.

10. The viral vector of claim 7, wherein the shRNA comprises or consists of the nucleotide sequence shown in SEQ ID NO:

11.

11. The viral vector of claim 3, wherein the peptide comprises an amino acid sequence that is at least 32 amino acids in length and comprises the amino acid sequence set forth in SEQ ID NO:

59.

12. The viral vector of claim 3, wherein the peptide is a dominant-negative protein comprising a modified amino acid sequence shown in SEQ ID NO: 60 or 61.

13. 4. The viral vector of claim 3, wherein the modified protein comprises or consists of an amino acid sequence as set forth in SEQ ID NO: 62 or 63.

14. The viral vector of claim 3, wherein the peptide is at least 10, 11, 12, 13, 14, 15, 20, 25, 30, 35, 40 or 42 amino acids in length and comprises the amino acid sequence set forth in SEQ ID NO:

90.

15. The viral vector of claim 3 or 14, wherein the peptide comprises the amino acid sequence (GSWQDLKDHMREA) shown in SEQ ID NO:

75.

16. 16. The viral vector of any one of claims 1 to 3 and 11 to 15, wherein the viral vector comprises an RNA Pol III terminator.

17. 17. The viral vector of any one of claims 1 to 16, wherein the vector comprises an inverted terminal repeat nucleotide sequence, and optionally the ITR sequence is set forth in SEQ ID NO: 64 or SEQ ID NO:

88.

18. 18. The viral vector of claim 1, wherein the promoter is selected from the group consisting of an H1 polymerase III promoter, a U6 promoter, a U7 promoter, or a mammalian 7SK promoter.

19. 19. The viral vector of claim 18, wherein the vector is an H1 polymerase III promoter, optionally set forth in SEQ ID NO:

65.

20. 20. The viral vector of any one of claims 1 to 19, wherein the viral-based vector is an adeno-associated virus [AAV], optionally a self-complementary adeno-associated virus (scAAV).

21. The viral vector of claim 20, wherein the viral-based vector is scAAV9 or scAAVrh10.

22. 22. A pharmaceutical composition comprising a viral vector according to any one of claims 1 to 21 and an excipient or carrier.

23. 22. A viral vector according to any one of claims 1 to 21 for use as a medicament.

24. 22. A viral vector according to any one of claims 1 to 21 for use in the treatment of a neurodegenerative disease.

25. 25. The viral vector for use according to claim 24, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

26. 25. The viral vector for use according to claim 24, wherein the neurodegenerative disease is sporadic and / or familial amyotrophic lateral sclerosis.

27. 25. The viral vector for use according to claim 24, wherein the neurodegenerative disease is ALS that is not caused by a pathological C9ORF72 repeat expansion.

28. 25. The viral vector for use according to claim 24, wherein the neurodegenerative disease is sporadic frontotemporal dementia (FTD).

29. 25. The viral vector for use according to claim 24, wherein the neurodegenerative disease is Fragile X-associated Tremor / Ataxia Syndrome (FXTAS).

30. A cell transfected with a viral vector according to the invention.

31. 31. The cell of claim 30, wherein the cell is a neuron and / or an astrocyte, and optionally the neuron is a motor neuron and / or an astrocyte.

32. An isolated nucleic acid molecule encoding an shRNA molecule comprising or consisting of a nucleotide sequence selected from the group consisting of SEQ ID NOs: 2, 3, 4, 5, 6, 7, 8, 9, 10 and 11.

33. 33. The isolated nucleic acid molecule of claim 32, wherein the nucleic acid molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO:

7.

34. 33. The isolated nucleic acid molecule of claim 32, wherein the nucleic acid molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO:

10.

35. 33. The isolated nucleic acid molecule of claim 32, wherein the nucleic acid molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO:

11.

36. A cell-penetrating polypeptide comprising or consisting of the amino acid sequence set forth in SEQ ID NO:

90.

37. 37. The cell-penetrating peptide of claim 36, wherein the polypeptide is 13 to 42 amino acids in length.

38. 38. The cell-penetrating peptide of claim 37, wherein the polypeptide comprises or consists of the amino acid sequence set forth in SEQ ID NO:

75.

39. An antagonist agent comprising a nucleic acid molecule, wherein the nucleic acid molecule comprises a nucleotide sequence designed with reference to human serine / arginine-rich splicing factor 1 (SRSF1), and the nucleic acid molecule inhibits the expression of SRSF1.

40. The agent of claim 39, wherein the nucleic acid molecule is a double-stranded nucleic acid molecule comprising a sense strand and an antisense strand comprising a nucleotide sequence, and the antisense nucleotide strand is adapted to anneal to a nucleic acid molecule encoding human SRSF1 by complementary base pairing.

41. The agent according to claim 40, wherein the double-stranded nucleic acid molecule is RNA.

42. The agent according to claim 41, wherein the RNA is siRNA or miRNA.

43. The agent of claim 39, wherein the nucleic acid molecule is a single-stranded nucleotide sequence comprising an antisense nucleotide sequence, and the antisense nucleotide sequence is adapted to anneal to a nucleic acid molecule encoding SRSF1 by complementary base pairing.

44. 44. The agent of claim 43, wherein the single-stranded nucleic acid is DNA and / or RNA, and optionally the DNA and / or RNA is a therapeutic antisense oligonucleotide, such as an antisense oligonucleotide, a splice-switching oligonucleotide, a gapmer, etc.

45. The agent according to claim 44, wherein the DNA is an antisense oligonucleotide.

46. The agent according to any one of claims 39 to 45, wherein the nucleic acid molecule encoding human SRSF1 is set forth in SEQ ID NO:

67.

47. 47. The agent of claim 46, wherein the antagonist agent comprises a nucleic acid molecule that is at least 15 nucleotides in length.

48. 48. The agent of claim 47, wherein the antagonist agent comprises a nucleic acid molecule comprising the nucleotide sequence set forth in SEQ ID NO: 67, wherein the nucleic acid molecule is a double-stranded inhibitory RNA and is 19 to 23 nucleotides in length.

49. 49. The agent of any one of claims 39 to 48, wherein the antagonist agent comprises a nucleic acid molecule comprising modified nucleotides and / or modified sugars.

50. 50. The method of claim 49, wherein the double-stranded nucleic acid molecules, including sense and antisense nucleic acid molecules, comprise modified nucleotides.

51. The modified nucleotide may be a 3'-terminal deoxy-thymine (dT) nucleotide, a 2'-O-methyl modified nucleotide, a 2'-fluoro modified nucleotide, a 2'-deoxy modified nucleotide, a locked nucleotide, an unlocked nucleotide, a conformationally restricted nucleotide, a constrained ethyl nucleotide, an abasic nucleotide, a 2'-amino modified nucleotide, a 2'-O-allyl modified nucleotide, a 2'-C-alkyl modified nucleotide, a 2'-hydroxyl ...

51. The agent of claim 50, wherein the nucleotide is selected from the group consisting of nucleotides, 2'-methoxyethyl modified nucleotides, 2'-O-alkyl modified nucleotides, morpholino nucleotides, phosphoramidates, non-natural base containing nucleotides, tetrahydropyran modified nucleotides, 1,5-anhydrohexitol modified nucleotides, cyclohexenyl modified nucleotides, nucleotides containing phosphorothioate groups, nucleotides containing phosphorodithioate (PS2), nucleotides containing methylphosphonate groups, nucleotides containing 5'-phosphates, and 5'-phosphate mimics, such as nucleotides containing 5'-vinyl phosphate, nucleotides containing 2'-deoxy-2'-fluro, and 2' methyl sugar bases.

52. 52. The agent of any one of claims 49 to 51, wherein the double-stranded nucleic acid molecules comprising sense and antisense nucleic acid molecules comprise modified sugars.

53. The modified sugar may be a modified ribosyl moiety, e.g., an -O-modified RNA, such as a 2'-O-alkyl or 2'-O-(substituted) alkyl, e.g., 2'-O-methyl, T-O-(2-cyanoethyl), 2'-O-(2-methoxy)ethyl (2'-MOE), 2'-O-(2-thiomethyl)ethyl, 2'-O-butyryl, -O-propargyl, 2'-O-allyl, 2'-O-(2-amino)propyl, 2'-O-(2-(dimethylamino)propyl), 2'-O-(2-amino)ethyl, 2'-O-(2-(dimethylamino)ethyl); 2'-deoxy(DNA); 2'-O-(haloalkoxy)methyl, e.g., 2'-O-( 53. The agent of claim 52, wherein the agent is selected from the group consisting of 2-chloroethoxy)methyl (MCEM), -O-(2,2-dichloroethoxy)methyl (DCEM); 2'-<3-alkoxycarbonyl, such as T-O-[2-(methoxycarbonyl)ethyl] (MOCE), 2'-O-[2-(N-methylcarbamoyl)ethyl] (MCE), T-O-[2-(N,N-dimethylcarbamoyl)ethyl] (DCME); 2'-halo, such as 2'-F, FANA (2'-F arabinosyl nucleic acid); carba- and aza-sugar modifications; 3'-O-alkyl, such as 3'-O-methyl, 3'-O-butyryl, VO-propargyl, and derivatives thereof.

54. The antagonist agent is selected from the group consisting of: 【Chemical 1】 54. The agent of any one of claims 49 to 53, comprising a nucleotide sequence designed with reference to a target nucleic acid sequence selected from:

55. 55. A pharmaceutical composition comprising an antagonist agent according to any one of claims 39 to 54, and including an excipient or carrier.

56. 55. An antagonist agent according to any one of claims 39 to 54 for use as a medicament.

57. 55. An antagonist agent according to any one of claims 39 to 54 for use in the treatment of a neurodegenerative disease.

58. 58. The antagonist agent of claim 57, wherein the neurodegenerative disease is amyotrophic lateral sclerosis (ALS).

59. 59. The antagonist agent of claim 58, wherein the neurodegenerative disease is sporadic and / or familial amyotrophic lateral sclerosis.

60. 60. The antagonist agent of claim 58 or 59, wherein the neurodegenerative disease is ALS that is not caused by a pathological C9ORF72 repeat expansion.

61. 58. The antagonist agent of claim 57, wherein the neurodegenerative disease is sporadic frontotemporal dementia (FTD).

62. 58. The antagonist agent of claim 57, wherein the neurodegenerative disease is Fragile X-associated tremor / ataxia syndrome (FXTAS).

63. A group consisting of: 【Chemistry 2】 or a variant thereof.

64. 64. The shRNA molecule of claim 63, wherein the shRNA molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 96 or a variant thereof.

65. 64. The shRNA molecule of Claim 63, wherein the shRNA molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 99 or a variant thereof.

66. 64. The shRNA molecule of Claim 63, wherein the shRNA molecule comprises or consists of the nucleotide sequence set forth in SEQ ID NO: 100 or a variant thereof.

Images