EP4587568A2 - Compositions et méthodes de traitement de maladies neurologiques - Google Patents

Compositions et méthodes de traitement de maladies neurologiques

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Publication number
EP4587568A2
EP4587568A2 EP23773208.6A EP23773208A EP4587568A2 EP 4587568 A2 EP4587568 A2 EP 4587568A2 EP 23773208 A EP23773208 A EP 23773208A EP 4587568 A2 EP4587568 A2 EP 4587568A2
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EP
European Patent Office
Prior art keywords
nucleic acid
atxn2
acid sequence
seq
inhibitory nucleic
Prior art date
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EP23773208.6A
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German (de)
English (en)
Inventor
Christopher Shaw
Youn Bok Lee
Romain JOUBERT
Do Young Lee
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Kings College London
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Kings College London
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Publication of EP4587568A2 publication Critical patent/EP4587568A2/fr
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia

Definitions

  • Ataxin-2 is a protein involved in several functions, such as formation of stress granules, P- bodies, and regulation of mRNA translation.
  • Ataxin-2 protein is encoded by the ATXN2 gene and is associated with diseases such as spinocerebellar ataxia-2 (SCA2), as CAG trinucleotide repeat mutations in the ATXN2 gene can give rise to neuronal degeneration.
  • SCA2 spinocerebellar ataxia-2
  • Exemplary viral vectors described herein that encode inhibitory nucleic acid constructs are adeno-associated viral (AAV) vectors, such as pseudotyped AAV2/8 and AAV2/9 vectors.
  • AAV adeno-associated viral
  • a patient diagnosed as having a disease associated with wild-type or mutant ATXN2 can be administered an inhibitory nucleic acid, such as an interfering RNA construct, or a vector encoding the same, so as to reduce the expression of wild-type or mutant mRNA transcripts.
  • an inhibitory nucleic acid such as an interfering RNA construct, or a vector encoding the same, so as to reduce the expression of wild-type or mutant mRNA transcripts.
  • compositions and methods described herein can be used to treat patients having SCA2, as such patients may be administered an inhibitory nucleic acid construct or a viral vector, such as an AAV vector, encoding such a construct, thereby reducing the expression of mRNA transcripts encoding wild-type or mutated Ataxin-2 protein.
  • Wild-type Ataxin-2 gene typically contains from about 13 to about 31 CAG trinucleotide repeats, with 22 repeats being the most common.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 15, 16, 17, 18, 19, 20, 21, or more contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 17 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 19 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 20 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand has at least 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% complementarity to a segment of 21 contiguous nucleotides within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises at least 10, at least 11, at least 12, at least 13, at least 14, at least 15, at least 16, at least 17, at least 18, at least 19, at least 20, or at least 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises at least 10 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises at least 11 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises at least 12 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises at least 17 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises at least 18 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the guide strand comprises from 18 to 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153. In some embodiments, the guide strand comprises 19, 20, or 21 contiguous nucleotides that are fully complementary to a contiguous polynucleotide of equal length within the region of the ATXN2 mRNA transcript having the nucleic acid sequence of any one of SEQ ID NOs: 103-153.
  • the inhibitory nucleic acid comprises a hairpin having a nucleic acid sequence that is at least 85% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52- 102 (e.g., at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical to the nucleic acid sequence of any one of SEQ ID NOs: 52-102).
  • the AAV comprises a capsid disclosed, e.g., in WO 2017/218842, the disclosure of which is incorporated herein by reference.
  • the AAV comprises a capsid protein disclosed in Lin et al. Mol Brain 13:138 (2020), the disclosure of which is incorporated herein by reference.
  • the AAV comprises an AAV2-retro or an AAV9-retro capsid protein.
  • the synthetic virus is chimeric virus, mosaic virus, or pseudotyped virus, and/or comprises a foreign protein, synthetic polymer, nanoparticle, or small molecule.
  • the neurological disease is SCA2, and the subject may be one that has a plurality of CAG trinucleotide repeat mutations in the ATXN2 locus, e.g., at least 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, or 100 CAG repeats.
  • the neurological disease is ALS, and the subject may be one that has a plurality of CAG trinucleotide repeat mutations in the ATXN2 locus, e.g., at least 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, or 40 CAG repeats.
  • the neurological disease is Huntington’s disease
  • the subject may be one that has upregulated ATXN2.
  • the inhibitory nucleic acid, viral vector, or pharmaceutical composition is administered to the subject by a route selected from intrathalamic, intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intracerebroventricular, intraocular (e.g., intravitreal), intraventricular, intralumbar, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, percutaneous, intratracheal, intraarterial, intravascular, and oral administration, inhalation, perfusion, lavage, or any combination thereof.
  • FIG.1 abbreviations: ATXN2, Ataxin-2; A2, Ataxin-2; HEK, Human embryonic kidney.
  • FIG.2A is a bar graph showing the knockdown efficacy of several ATXN2-specifc miRNA constructs from FIG.1.
  • FIG.2B abbreviations: ATXN2, Ataxin-2; HEK, Human embryonic kidney; GAPDH, Glyceraldehyde-3-phosphate dehydeogenase; qPCR, quantitative polymerase chain reaction.
  • FIG.2C is a bar graph showing the knockdown efficacy of several ATXN2-specific miRNA constructs from FIG.1.
  • FIG.2C is a bar graph showing the knockdown efficacy of several ATXN2-specific miRNA constructs from FIG.1. Data were obtained from a qPCR assay performed in HEK293 cells after viral transduction and are presented as a ratio of ATXN2 to GAPDH mRNA signal.
  • PBS group represents no silencing of the endogenous ATXN2 mRNA and serves as a baseline.
  • the experimental groups received AAV injections with doses of 1e8 viral genomes/hemisphere (vg/hem) or 1e9 vg/hem.
  • the left and right panels show expression of mouse Atxn2 mRNA in the mouse thalamus and sub-cortex, respectively.
  • FIG.4B abbreviations: Atxn2, Ataxin-2; PBS, Phosphate-buffered saline.
  • the term "anneal” refers to the formation of a stable duplex of nucleic acids by way of hybridization mediated by inter-strand hydrogen bonding, for example, according to Watson- Crick base pairing.
  • the nucleic acids of the duplex may be, for example, at least 50% complementary to one another (e.g., about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, or 100% complementary to one another.
  • the "stable duplex" formed upon the annealing of one nucleic acid to another is a duplex structure that is not denatured by a stringent wash.
  • exemplary stringent wash conditions include temperatures of about 5° C less than the melting temperature of an individual strand of the duplex and low concentrations of monovalent salts, such as monovalent salt concentrations (e.g., NaCl concentrations) of less than 0.2 M (e.g., 0.2 M, 0.19 M, 0.18 M, 0.17 M, 0.16 M, 0.15 M, 0.14 M, 0.13 M, 0.12 M, 0.11 M, 0.1 M, 0.09 M, 0.08 M, 0.07 M, 0.06 M, 0.05 M, 0.04 M, 0.03 M, 0.02 M, 0.01 M, or less).
  • monovalent salt concentrations e.g., NaCl concentrations
  • the “length” of a nucleic acid refers to the linear size of the nucleic acid as assessed by measuring the quantity of nucleotides from the 5’ to the 3’ end of the nucleic acid. Exemplary molecular biology techniques that may be used to determine the length of a nucleic acid of interest are known in the art.
  • the term “operably linked” refers to a first molecule (e.g., a first nucleic acid) joined to a second molecule (e.g., a second nucleic acid), wherein the molecules are so arranged that the first molecule affects the function of the second molecule.
  • the two molecules may or may not be part of a single contiguous molecule and may or may not be adjacent to one another.
  • a promoter is operably linked to a transcribable polynucleotide molecule if the promoter modulates transcription of the transcribable polynucleotide molecule of interest in a cell.
  • two portions of a transcription regulatory element are operably linked to one another if they are joined such that the transcription-activating functionality of one portion is not adversely affected by the presence of the other portion.
  • Two transcription regulatory elements may be operably linked to one another by way of a linker nucleic acid (e.g., an intervening non-coding nucleic acid) or may be operably linked to one another with no intervening nucleotides present.
  • one segment of a nucleic acid molecule is considered to “overlap with” another segment of the same nucleic acid molecule if the two segments share one or more constituent nucleotides.
  • two segments of the same nucleic acid molecule are considered to “overlap with” one another if the two segments share 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 100, or more, constituent nucleotides.
  • the two segments are not considered to “overlap with” one another if the two segments have zero constituent nucleotides in common.
  • Percent (%) sequence complementarity with respect to a reference polynucleotide sequence is defined as the percentage of nucleic acids in a candidate sequence that are complementary to the nucleic acids in the reference polynucleotide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence complementarity.
  • a given nucleotide is considered to be “complementary” to a reference nucleotide as described herein if the two nucleotides form canonical Watson-Crick base pairs.
  • Watson-Crick base pairs in the context of the present disclosure include adenine-thymine, adenine-uracil, and cytosine-guanine base pairs.
  • a proper Watson-Crick base pair is referred to in this context as a “match,” while each unpaired nucleotide, and each incorrectly paired nucleotide, is referred to as a “mismatch.”
  • Alignment for purposes of determining percent nucleic acid sequence complementarity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software. Those skilled in the art can determine appropriate parameters for aligning sequences, including any algorithms needed to achieve maximal complementarity over the full length of the sequences being compared.
  • the percent sequence complementarity of a given nucleic acid sequence, A, to a given nucleic acid sequence, B, is calculated as follows: 100 multiplied by (the fraction X/Y) where X is the number of complementary base pairs in an alignment (e.g., as executed by computer software, such as BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B.
  • nucleic acid sequence A is not equal to the length of nucleic acid sequence B
  • percent sequence complementarity of A to B will not equal the percent sequence complementarity of B to A.
  • a query nucleic acid sequence is considered to be “completely complementary” to a reference nucleic acid sequence if the query nucleic acid sequence has 100% sequence complementarity to the reference nucleic acid sequence.
  • Percent (%) sequence identity with respect to a reference polynucleotide or polypeptide sequence is defined as the percentage of nucleic acids or amino acids in a candidate sequence that are identical to the nucleic acids or amino acids in the reference polynucleotide or polypeptide sequence, after aligning the sequences and introducing gaps, if necessary, to achieve the maximum percent sequence identity. Alignment for purposes of determining percent nucleic acid or amino acid sequence identity can be achieved in various ways that are within the capabilities of one of skill in the art, for example, using publicly available computer software such as BLAST, BLAST-2, or Megalign software.
  • percent sequence identity values may be generated using the sequence comparison computer program BLAST.
  • percent sequence identity of a given nucleic acid or amino acid sequence, A, to, with, or against a given nucleic acid or amino acid sequence, B, (which can alternatively be phrased as a given nucleic acid or amino acid sequence, A that has a certain percent sequence identity to, with, or against a given nucleic acid or amino acid sequence, B) is calculated as follows: 100 multiplied by (the fraction X/Y) where X is the number of nucleotides or amino acids scored as identical matches by a sequence alignment program (e.g., BLAST) in that program’s alignment of A and B, and where Y is the total number of nucleic acids in B.
  • sequence alignment program e.g., BLAST
  • the term “pharmaceutically acceptable” refers to those compounds, materials, compositions, and/or dosage forms, which are suitable for contact with the tissues of a subject, such as a mammal (e.g., a human) without excessive toxicity, irritation, allergic response and other problem complications commensurate with a reasonable benefit/risk ratio.
  • a mammal e.g., a human
  • wild-type or non-mutant form of a gene refers to a nucleic acid that encodes a protein associated with normal or non-pathogenic activity (e.g., a protein lacking a mutation, such as a repeat region expansion that results in higher risk of developing, onset, or progression of a neurodegenerative disease).
  • mutation refers to any change in the structure of a gene, e.g., gene sequence, resulting in an altered form of the gene, which may be passed onto subsequent generations (hereditary mutation) or not (somatic mutation).
  • Gene mutations include the substitution, insertion, or deletion of a single base in DNA or the substitution, insertion, deletion, or rearrangement of multiple bases or larger sections of genes or chromosomes, including repeat expansions.
  • Ataxin 2 or “Ataxin-2” or “ATXN2” refers to a protein encoded by the ATXN2 gene, which contains a polyglutamine (polyQ, CAG repeat) tract.
  • ATXN2 gene or transcript may refer to normal alleles of ATXN2, which usually have 22 or 23 repeats, or mutated alleles having intermediate ( ⁇ 24-32 repeats) or longer repeat expansions ( ⁇ 33 to >100 repeats).
  • ATXN2 refers to mammalian ATXN2, including human ATXN2.
  • Exemplary ATXN2 proteins that may be targeted using the compositions and methods of the disclosure include a protein having the amino acid sequence represented by NCBI ID NP_001297050.1, as well as naturally occurring variants thereof.
  • an exemplary ATXN2 gene has the nucleic acid sequence of NCBI ID NC_000012.12:c111599673-111452214, or a naturally occurring variant thereof.
  • an inhibitory nucleic acid is a single stranded or double stranded molecule.
  • An inhibitory nucleic acid may further comprise a passenger strand sequence on a separate strand (e.g., double stranded duplex) or in the same strand (e.g., single stranded, self-annealing duplex structure).
  • an inhibitory nucleic acid is an interfering RNA molecule, such as short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or double-stranded RNA (dsRNA).
  • the term “interfering RNA” refers to an RNA, such as an siRNA, miRNA, or shRNA that suppresses the expression of a target RNA transcript by way of (i) annealing to the target RNA transcript, thereby forming a nucleic acid duplex; and (ii) promoting the nuclease-mediated degradation of the RNA transcript and/or (iii) slowing, inhibiting, or preventing the translation of the RNA transcript, such as by sterically precluding the formation of a functional ribosome-RNA transcript complex or otherwise attenuating formation of a functional protein product from the target RNA transcript.
  • an RNA such as an siRNA, miRNA, or shRNA that suppresses the expression of a target RNA transcript by way of (i) annealing to the target RNA transcript, thereby forming a nucleic acid duplex; and (ii) promoting the nuclease-mediated degradation of the RNA transcript and/or (iii) slowing, inhibit
  • RNA platforms are described, for example, in Lam et al., Molecular Therapy – Nucleic Acids 4:e252 (2015); Rao et al., Advanced Drug Delivery Reviews 61:746-769 (2009); and Borel et al., Molecular Therapy 22:692-701 (2014), the disclosures of each of which are incorporated herein by reference in their entirety.
  • a “microRNA” or “miRNA” refers to a small non-coding RNA molecule capable of mediating silencing of a target gene by cleavage of the target mRNA, translational repression of the target mRNA, target mRNA degradation, or a combination thereof.
  • miRNA is transcribed as a hairpin or stem-loop (e.g., having a self-complementary, single-stranded backbone) duplex structure, referred to as a primary miRNA (pri-miRNA), which is enzymatically processed (e.g., by Drosha, DGCR8, Pasha, etc.) into a pre-miRNA.
  • pri-miRNA primary miRNA
  • Pre-miRNA is exported into the cytoplasm, where it is enzymatically processed by Dicer to produce a miRNA duplex with the passenger strand and then a single- stranded mature miRNA molecule, which is subsequently loaded into the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • Reference to a miRNA may include synthetic or artificial miRNAs.
  • a “synthetic miRNA” or “artificial miRNA” or “amiRNA” refers to an endogenous, modified, or synthetic pri-miRNA or pre-miRNA (e.g., miRNA backbone or scaffold) in which the endogenous miRNA guide sequence and passenger sequence within the stem sequence have been replaced with a miRNA guide sequence and a miRNA passenger sequence that direct highly efficient RNA silencing of the targeted gene (see, e.g., Eamens et al. (2014), Methods Mol. Biol.1062:211-224).
  • the term “antisense strand sequence” or “guide strand sequence” of an inhibitory nucleic acid refers to a sequence that is substantially complementary (e.g., at least 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% complementary) to a region of about 10-50 nucleotides (e.g., about 15-30, 16-25, 18-23, or 19-22 nucleotides) of the mRNA of the gene targeted for silencing.
  • the antisense sequence is sufficiently complementary to the target mRNA sequence to direct target-specific silencing, e.g., to trigger the destruction of the target mRNA by the RNAi machinery or process.
  • the antisense sequence or guide strand sequence refers to the mature sequence remaining following cleavage by Dicer.
  • the term “sense sequence” or “passenger strand sequence” of an inhibitory nucleic acid refers to a sequence that is homologous to the target mRNA and partially or completely complementary to the antisense strand sequence or guide strand sequence of an inhibitory nucleic acid.
  • the antisense strand sequence and sense strand sequence of an inhibitory nucleic acid are hybridized to form a duplex structure (e.g., forming a double-stranded duplex or single-stranded self- annealing duplex structure).
  • expression construct refers to any type of genetic construct containing a nucleic acid (e.g., transgene) in which part or all the nucleic acid encoding sequence is capable of being transcribed.
  • expression includes transcription of the nucleic acid, for example, to generate a biologically active polypeptide product or inhibitory RNA (e.g., siRNA, shRNA, miRNA) from a transcribed gene.
  • inhibitory RNA e.g., siRNA, shRNA, miRNA
  • the transgene is operably linked to expression control sequences.
  • the term “transgene” refers to an exogenous nucleic acid that has been transferred naturally or by genetic engineering means into another cell and is capable of being transcribed, and optionally translated.
  • the term “gene expression” refers to the process by which a nucleic acid is transcribed from a nucleic acid molecule, and often, translated into a peptide or protein. The process can include transcription, post-transcriptional control, post-transcriptional modification, translation, post-translational control, post translational modification, or any combination thereof. Reference to a measurement of “gene expression” may refer to measurement of the product of transcription (e.g., RNA or mRNA), the product of translation (e.g., peptides or proteins). As used herein, the term “inhibit expression of a gene” means to reduce, down-regulate, suppress, block, lower, or stop expression of the gene.
  • the expression product of a gene can be an RNA molecule transcribed from the gene (e.g., an mRNA) or a polypeptide translated from an mRNA transcribed from the gene. Typically, a reduction in the level of an mRNA results in a reduction in the level of a polypeptide translated therefrom.
  • the level of expression may be determined using standard techniques for measuring mRNA or protein.
  • “neurodegenerative disease” or “neurodegenerative disorder” refers to diseases or disorders that exhibit neural cell death as a pathological state.
  • a neurodegenerative disease may exhibit chronic neurodegeneration, e.g., slow, progressive neural cell death over a period of several years, or acute neurodegeneration, e.g., sudden onset or neural cell death.
  • Chronic neurodegenerative diseases include Alzheimer’s disease, Parkinson’s disease, Huntington’s disease, spinocerebellar ataxia type 2 (SCA2), frontotemporal lobar dementia (FTLD), and amyotrophic lateral schlerosis (ALS).
  • Chronic neurodegenerative diseases include diseases that feature TDP-43 proteinopathy, which is characterized by nucleus to cytoplasmic mislocalization, deposition of ubiquitinated and hyper-phosphorylated TDP-43 into inclusion bodies, protein truncation leading to formation of toxic C-terminal TDP-43 fragments, and protein aggregation.
  • TDP-43 proteinopathy diseases include ALS, FTLD, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), dementia Parkinsonism ALS complex of guam (G-PDC), Pick’s disease, hippocampal sclerosis, Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease.
  • Acute neurodegeneration may be caused by ischemia (e.g., stroke, traumatic brain injury), axonal transection by demyelination or trauma (e.g., spinal cord injury or multiple sclerosis).
  • a neurodegenerative disease may exhibit death of mainly one type of neuron or of multiple types of neurons.
  • the term “repeat region” refers to segments within a gene of interest or an RNA transcript thereof containing nucleic acid repeats, such as the poly CAG sequence in the ATXN2 gene.
  • a repeat region is considered to be an “expanded repeat region,” a “repeat expansion,” or the like, if the number of nucleotide repeats in the repeat region exceeds the quantity of repeats ordinarily found in the repeat region of a wild-type form of the gene or RNA transcript thereof.
  • the wild-type human ATXN2 genes typically contain from 13 to 31 CAG repeats.
  • “Expanded repeat regions” and “repeat expansions” in the context of the ATXN2 gene or an RNA transcript thereof thus include repeat regions containing greater than 31 repeats, among others.
  • the term “sample” refers to a specimen (e.g., blood, blood component (e.g., serum or plasma), urine, saliva, amniotic fluid, cerebrospinal fluid, tissue (e.g., placental or dermal), pancreatic fluid, chorionic villus sample, or cells) isolated from a subject.
  • the subject may be, for example, a patient suffering from a disease described herein, such as a disease associated with expression of wild-type or mutant ATXN2 (e.g., SCA2, ALS, Huntington’s disease, Parkinson’s disease.).
  • a disease described herein such as a disease associated with expression of wild-type or mutant ATXN2 (e.g., SCA2, ALS, Huntington’s disease, Parkinson’s disease.).
  • the phrases “specifically binds” and “binds” refer to a binding reaction which is determinative of the presence of a particular molecule, such as an RNA transcript, in a heterogeneous population of ions, salts, small molecules, and/or proteins that is recognized, e.g., a mutant ATXN2 RNA transcript.
  • a ligand that specifically binds to a species (e.g., an RNA transcript) may bind to the species, e.g., with a KD of less than 1 mM.
  • a ligand that specifically binds to a species may bind to the species with a KD of up to 100 ⁇ M (e.g., between 1 pM and 100 ⁇ M).
  • a ligand that does not exhibit specific binding to another molecule may exhibit a K D of greater than 1 mM (e.g., 1 ⁇ M, 100 ⁇ M, 500 ⁇ M, 1 mM, or greater) for that particular molecule or ion.
  • a variety of assay formats may be used to determine the affinity of a ligand for a specific protein.
  • solid-phase ELISA assays are routinely used to identify ligands that specifically bind a target protein. See, e.g., Harlow & Lane, Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1988) and Harlow & Lane, Using Antibodies, A Laboratory Manual, Cold Spring Harbor Press, New York (1999), for a description of assay formats and conditions that can be used to determine specific protein binding.
  • the terms “subject” and “patient” refer to an organism that receives treatment for a particular disease or condition as described herein (such as a disease associated with expression of an ATXN2 mutant, e.g., SCA2). Examples of subjects and patients include mammals, such as humans, receiving treatment for a disease or condition described herein.
  • the term “transcription regulatory element” refers to a nucleic acid that controls, at least in part, the transcription of a gene of interest. Transcription regulatory elements may include promoters, enhancers, and other nucleic acids (e.g., polyadenylation signals) that control or help to control gene transcription.
  • transcription regulatory elements are described, for example, in Goeddel, Gene Expression Technology: Methods in Enzymology 185 (Academic Press, San Diego, CA, 1990).
  • treatment refers to therapeutic treatment, in which the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the progression of a disease associated with wild-type or mutant ATXN2, for example, SCA2, ALS, Huntington’s disease, and Parkinson’s disease.
  • beneficial or desired clinical results that are indicative of successful treatment include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable.
  • Treatment of a patient having SCA2 may manifest in one or more detectable changes, such as a decrease in the expression of mutant ATXN2 RNA transcripts (e.g., a decrease in the expression of ATXN2 RNA transcripts that contain expanded CAG trinucleotide repeat regions).
  • the term “vector” refers to a nucleic acid, e.g., DNA or RNA, that may function as a vehicle for the delivery of a gene of interest into a cell (e.g., a mammalian cell, such as a human cell), tissue, organ, or organism, such as a patient undergoing treatment for a disease or condition described herein, for purposes of expressing an encoded transgene.
  • a cell e.g., a mammalian cell, such as a human cell
  • tissue e.g., a mammalian cell, such as a human cell
  • exemplary vectors useful in conjunction with the compositions and methods described herein are plasmids, DNA vectors, RNA vectors, virions, or other suitable replicon (e.g., viral vector).
  • vectors have been developed for the delivery of polynucleotides encoding exogenous proteins into a prokaryotic or eukaryotic cell. Examples of such expression vectors are disclosed in, e.g., WO 1994/11026, the disclosure of which is incorporated herein by reference.
  • Expression vectors described herein contain a polynucleotide sequence as well as, e.g., additional sequence elements used for the expression of proteins and/or the integration of these polynucleotide sequences into the genome of a mammalian cell.
  • Certain vectors that can be used for the expression of transgenes described herein include plasmids that contain regulatory sequences, such as promoter and enhancer regions, which direct gene transcription.
  • kits for expression of transgenes contain polynucleotide sequences that enhance the rate of translation of these genes or improve the stability or nuclear export of the mRNA that results from gene transcription. These sequence elements include, e.g., 5’ and 3’ untranslated regions, an internal ribosomal entry site (IRES), and polyadenylation signal site in order to direct efficient transcription of the gene carried on the expression vector.
  • the expression vectors described herein may also contain a polynucleotide encoding a marker for selection of cells that contain such a vector. Examples of a suitable marker include genes that encode resistance to antibiotics, such as ampicillin, chloramphenicol, kanamycin, or nourseothricin.
  • compositions and methods described herein are useful for treating disorders associated with expression of wild-type or mutant Ataxin-2 (ATXN2), such as spinocerebellar ataxia type 2 or spinocerebellar ataxia-2 (SCA2), amyotrophic lateral sclerosis (ALS), Huntington’s disease, FTD (Frontotemporal dementia), TDP-43 (TAR DNA binding protein 43) proteinopathies, and others.
  • Ataxin-2 Ataxin-2
  • ATXN2 wild-type or mutant Ataxin-2
  • SCA2 spinocerebellar ataxia type 2 or spinocerebellar ataxia-2
  • ALS amyotrophic lateral sclerosis
  • FTD Ferontotemporal dementia
  • TDP-43 TAR DNA binding protein 43 proteinopathies
  • compositions described herein include inhibitory nucleic acid constructs such as interfering RNA constructs, for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA) that suppress the expression of wild-type or mutant mRNA transcripts transcribed from wild- type or mutant genes.
  • interfering RNA constructs for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA) that suppress the expression of wild-type or mutant mRNA transcripts transcribed from wild- type or mutant genes.
  • shRNA short hairpin RNA
  • miRNA microRNA
  • Ataxin-2 and Disorders Associated with Ataxin-2 ATXN2 protein is a cytoplasmic protein that is a component of stress granules. Stress granules are transient, subcellular compartments induced by arrest of protein translation and include a number of proteins known to be mutated in subjects with neurodegenerative disease (Brown and Al- Chalabi, N Engl J Med (2017) 377:162-172).
  • ATXN2 contains a sequence of glutamine residues, known as a polyglutamine repeat (polyQ), that in normal individuals is about 22 amino acids in length. Expansions of this polyglutamine repeat to a length of 34 or longer is found in individuals with neurodegenerative diseases, including spinocerebellar ataxia type 2 (SCA2). This disease is characterized by progressive death of Purkinje neurons in the cerebellum and other neuronal cell types. Patients with SCA2 develop ataxia, sensory problems, and other clinical features, which worsen over time.
  • polyQ polyglutamine repeat
  • SCA2 spinocerebellar ataxia type 2
  • Ataxin-2 polyglutamine repeat e.g., between 27 and 40 glutamine residues
  • ALS amyotrophic lateral sclerosis
  • the pathogenic functions of polyQ disease proteins that occur with polyQ expansion may be attributed to the gain of toxicity associated with the development of intranuclear inclusion bodies or with soluble toxic oligomers (Lajoie et al., PLoS One, 2011, 5: e15245). While SCA2 patient brains are characterized by loss of Purkinje cells, SCA2 Purkinje cells lack inclusion bodies indicating polyQ- expanded ataxin-2 may cause toxicity that is unrelated to inclusion body formation (Huynh et al., Ann. Neurol., 1999, 45: 232–241). Functions gained in polyQ-expanded ataxin-2 may include anomalous accumulation in Golgi bodies (Huynh et al., Hum. Mol.
  • Ataxin-2 is present in stress granules and P-bodies suggesting functions in sequestering mRNAs and protein translation regulation during stress (Nonhoff et al., Mol. Biol. Cell, 2007, 18: 1385–1396). Ataxin-2 overexpression interfered with the P-body assembly, while underexpression interfered with stress granule assembly (Nonhoff et al., Mol. Biol. Cell, 2007, 18: 1385–1396). Interactions with polyA-binding protein 1, the RNA splicing factor A2BP1/Fox1 and polyribosomes further support roles for ataxin-2 in RNA metabolism (Shibata et al., Hum. Mol.
  • Ataxin-2 also interacts with the ALS-related protein TDP-43 in an RNA-dependent manner and familial and sporadic ALS associates with the occurrence of long normal CAG repeat expansion ATXN2 (Elden et al., Nature, 2010, 466: 1069–1075; Van Damme et al., Neurology, 2011, 76: 2066–2072).
  • SCA2 is an autosomal dominant neurodegenerative disease characterized by progressive functional and cell loss of neurons in the cerebellum, brain stem and spinal cord.
  • the cause of SCA2 is CAG expansion in the ATXN2 gene resulting in polyglutamine (polyQ) expansion in the ataxin-2 protein.
  • SCA2 Patients with SCA2 are characterized by progressive cerebellar ataxia, slow saccadic eye movements and other neurologic features such as neuropathy (Pulst, S.M. (ed.), Genetics of Movement Disorders. Elsevier, Inc., Amsterdam, 2003, pp.19–34.). Moderate CAG expansion in the ATXN2 gene is also associated with parkinsonism or ALS indistinguishable from the idiopathic forms of these diseases (Kim et al., Arch. Neurol., 2007, 64: 1510–1518; Ross et al., Hum. Mol. Genet., 2011, 20: 3207–3212; Corrado et al., Hum.
  • ATXN2 is a causative agent (e.g., SCA2)
  • reducing ATXN2 levels may be useful for treating neurodegenerative diseases where ATXN2 is a causative agent (e.g., SCA2)
  • ATXN2 is not the causative agent but modifies TDP-43 pathological aggregation.
  • Aspects of the disclosure relate to inhibitory nucleic acids, such as interfering RNA molecules (e.g., short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), micro RNAs (miRNAs), including artificial miRNAs), that when administered to a subject reduce the expression or activity of Ataxin-2 in the subject.
  • interfering RNA molecules e.g., short interfering RNAs (siRNAs), short hairpin RNAs (shRNAs), micro RNAs (miRNAs), including artificial miRNAs
  • This advantageous feature is based, in part, on the surprising discovery that inhibitory nucleic acid constructs that anneal to wild-type or repeat-expanded RNA targets can be used to suppress the expression of these RNA transcripts.
  • the compositions and methods described herein can thus attenuate the expression of wild-type or pathological RNA transcripts.
  • the sections that follow provide a description of exemplary inhibitory nucleic acid constructs, such as interfering RNA constructs, that may be used in conjunction with the compositions and methods described herein, as well as a description of vectors encoding such constructs and procedures that may be used to treat diseases associated with expression of wild-type or mutant ATXN2.
  • a patient having a disease characterized by expression of wild-type or mutant ATXN2 may be administered an interfering RNA molecule, a composition containing the same, or a vector encoding the same, so as to suppress the expression of an RNA transcript.
  • interfering RNA molecules that may be used in conjunction with the compositions and methods described herein for the treatment of diseases associated with expression of wild-type or mutant ATXN2, such as SCA2, ALS, Huntington’s disease, FTD, TDP-43 proteinopathies, Parkinson’s disease and others, are siRNA molecules, miRNA molecules, and shRNA molecules, among others.
  • the siRNA may be single stranded or double stranded.
  • miRNA molecules in contrast, are single-stranded molecules that form a hairpin, thereby adopting a hydrogen-bonded structure reminiscent of a nucleic acid duplex.
  • the interfering RNA may contain an antisense or “guide” strand that anneals (e.g., by way of complementarity) to the repeat-expanded mutant RNA target.
  • the interfering RNA may also contain a “passenger” strand that is complementary to the guide strand and, thus, may have the same nucleic acid sequence as the RNA target.
  • Exemplary interfering RNA molecules that anneal to ATXN2 RNA may be used in conjunction with the compositions and methods described herein for the treatment of diseases associated with expression of wild-type or mutant ATXN2 shown in Table 2, below. Table 2.
  • Exemplary inhibitory nucleic acids useful for suppressing ATXN2 expression Methods of Treating Diseases Characterized by Expression of Wild-type or Mutant ATXN2 Using the compositions and methods described herein, a patient experiencing and/or having a disease associated with expression of wild-type or mutant ATXN2, such as SCA2, ALS, Huntington’s disease, FTD, TDP-43 proteinopathies, Parkinson’s disease, among others, can be administered an inhibitory nucleic acid construct, such as an interfering RNA construct, for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA), or a vector encoding the same, so as to reduce the expression of wild-type or mutant RNA transcripts.
  • siRNA short inter
  • the present disclosure provides methods for inhibiting the expression or activity of ATXN2 in a cell, comprising administering a composition of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) to a cell, thereby inhibiting the expression or activity of ATXN2 in the cell.
  • a composition of the present disclosure e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition
  • the cell is a CNS cell.
  • the cell is a non-neuronal cell or neuronal cell of the CNS.
  • the non-neuronal cell of the CNS is a glial cell, astrocyte, or microglial cell.
  • the cell is in vitro.
  • the cell is from a subject having one or more symptoms of a neurodegenerative disease or suspected of having a neurodegenerative disease.
  • the cell expresses an ATXN2 having at least 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, or more CAG trinucleotide (polyglutamine) repeats.
  • the cell expresses an ATXN2 having about 22 or 23 repeats, 24-32 repeats, or 33-100 or more repeats.
  • the present disclosure provides methods for inhibiting the expression or activity of ATXN2 in the central nervous system of a subject, comprising administering a composition of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) to the subject, thereby inhibiting the expression or activity of ATXN2 in the subject.
  • a composition of the present disclosure e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition
  • the present disclosure provides methods for treating a subject having or suspected of having a neurodegenerative disease, comprising administering a composition of the present disclosure (e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition) to the subject, thereby treating the subject.
  • a composition of the present disclosure e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition
  • the term "treat” refers to preventing or delaying onset of neurodegenerative disease (e.g., SCA2, ALS/FTLD, Huntington’s disease, Alzheimer's disease, Parkinson's disease, etc.); reducing severity of neurodegenerative disease; reducing or preventing development of symptoms characteristic of neurodegenerative disease; preventing worsening of symptoms characteristic of neurodegenerative disease, or any combination thereof.
  • neurodegenerative disease e.g., SCA2, ALS/FTLD, Huntington’s disease, Alzheimer's disease, Parkinson's disease, etc.
  • reducing severity of neurodegenerative disease reducing or preventing development of symptoms characteristic of neurodegenerative disease
  • preventing worsening of symptoms characteristic of neurodegenerative disease or any combination thereof.
  • Neurodegenerative diseases that may be treated in a subject using the compositions of the present disclosure include neurodegenerative diseases where ATXN2 is a causative agent (e.g., SCA2), as well as neurodegenerative diseases where ATXN2 is not the causative agent (e.g., directly causative) but modifies TDP-43 pathological aggregation.
  • ATXN2 is a causative agent (e.g., SCA2)
  • ATXN2 is not the causative agent (e.g., directly causative) but modifies TDP-43 pathological aggregation.
  • Neurodegenerative diseases associated with TDP-43 proteinopathy include ALS, FTLD, primary lateral sclerosis, progressive muscular atrophy, limbic-predominant age-related TDP-43 encephalopathy, chronic traumatic encephalopathy, dementia with Lewy bodies, corticobasal degeneration, progressive supranuclear palsy (PSP), dementia Parkinsonism ALS complex of guam (G-PDC), Pick’s disease, Perry syndrome, cerebral age-related TDP-43 with sclerosis (CARTS), hippocampal sclerosis, Huntington’s disease, Parkinson’s disease, and Alzheimer’s disease.
  • the neurodegenerative disease is SCA2.
  • the subject with SCA2 may be one that has a plurality of CAG trinucleotide repeat mutations in the ATXN2 locus, e.g., at least 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, or 100 CAG repeats.
  • ATXN2 is a validated target for treating SCA2 as disclosed in Giunti et al. Brain 121, 459-467 (1998).
  • the neurodegenerative disease is ALS.
  • the subject is characterized as having an ATXN2 allele having at least 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 50, 60, 70, 80, 90, 100, or more CAG trinucleotide (polyglutamine) repeats.
  • the subject is characterized as having an ATXN2 allele having about 22 or 23 repeats, 24-32 repeats, or 33-100 or more repeats.
  • the methods for treatment of the present disclosure reduces, prevents, or slows development or progression of one or more symptom characteristic of a neurodegenerative disease.
  • the one or additional therapies that may be used in combination with the inhibitory nucleic acids of the present disclosure include: inhibitory nucleic acids or antisense oligonucleotides that target neurodegenerative disease related genes or transcripts, gene editing agents (e.g., CRISPR, TALEN, ZFN based systems) that target neurodegenerative related genes, agents that reduce oxidative stress, such as free radical scavengers (e.g., Radicava (edaravone), bromocriptine); antiglutamate agents (e.g., Riluzole, Topiramate, Lamotrigine, Dextromethorphan, Gabapentin and AMPA receptor antagonist (e.g., Talampanel)); Anti-apoptosis agents (e.g., Minocycline, Sodium phenylbutyrate and Arimoclomol); Anti-inflammatory agents (e.g., ganglioside, Celecoxib, Cyclosporine, Nimesulide, Azathi
  • a subject treated in any of the methods described herein is a mammal (e.g., mouse, rat), preferably a primate (e.g., monkey, chimpanzee), or human.
  • a composition of the present disclosure e.g., inhibitory nucleic acid, isolated nucleic acid comprising an expression construct encoding an inhibitory nucleic acid, vector, rAAV particle, pharmaceutical composition
  • a composition of the present disclosure is directly injected into the CNS of the subject.
  • direct injection into the CNS is intrathalamic injection, intracerebral injection, intraparenchymal injection, intrathecal injection, intrastriatal injection, subpial injection, or any combination thereof.
  • direct injection into the CNS is direct injection into the cerebrospinal fluid (CSF) of the subject, optionally wherein the direct injection is intracisternal injection, intraventricular injection, intralumbar injection, or any combination thereof.
  • CSF cerebrospinal fluid
  • viral vectors examples include AAV, retrovirus, adenovirus (e.g., Ad5, Ad26, Ad34, Ad35, and Ad48), parvovirus (e.g., adeno- associated viruses), coronavirus, negative strand RNA viruses such as orthomyxovirus (e.g., influenza virus), rhabdovirus (e.g., rabies and vesicular stomatitis virus), paramyxovirus (e.g.
  • rAAV vectors useful in the conjunction with the compositions and methods described herein include recombinant nucleic acid constructs that contain (1) a transgene encoding an inhibitory nucleic acid construct, such as an interfering RNA construct described herein (such as an siRNA, shRNA, or miRNA described herein), and (2) one or more nucleic acids that facilitate and expression of the heterologous genes.
  • the viral nucleic acids may include those sequences of AAV that are required in cis for replication and packaging (e.g., functional ITRs) of the DNA into a virion.
  • Such rAAV vectors may also contain marker or reporter genes.
  • the AAV comprises a capsid disclosed, e.g., in WO 2017/218842, the disclosure of which is incorporated herein by reference.
  • the AAV comprises a capsid protein disclosed in Lin et al. Mol Brain 13:138 (2020), the disclosure of which is incorporated herein by reference.
  • the AAV comprises an AAV2-retro or an AAV9-retro capsid protein.
  • AAV virions that have mutations within the virion capsid may be used to infect particular cell types more effectively than non-mutated capsid virions.
  • suitable AAV mutants may have ligand insertion mutations for the facilitation of targeting AAV to specific cell types.
  • the construction and characterization of AAV capsid mutants including insertion mutants, alanine screening mutants, and epitope tag mutants is described in Wu et al., J. Virol.74:8635-45 (2000).
  • Other rAAV virions that can be used in methods of the invention include those capsid hybrids that are generated by molecular breeding of viruses as well as by exon shuffling. See, e.g., Soong et al., Nat.
  • a transgene such as a transgene encoding an inhibitory nucleic acid described herein
  • a target cell e.g., a target cell from or within a human patient suffering from RNA dominance
  • electroporation can be used to permeabilize mammalian cells (e.g., human target cells) by the application of an electrostatic potential to the cell of interest.
  • Mammalian cells such as human cells, subjected to an external electric field in this manner are subsequently predisposed to the uptake of exogenous nucleic acids. Electroporation of mammalian cells is described in detail, e.g., in Chu et al., Nucleic Acids Research 15:1311 (1987), the disclosure of which is incorporated herein by reference. A similar technique, Nucleofection TM , utilizes an applied electric field in order to stimulate the uptake of exogenous polynucleotides into the nucleus of a eukaryotic cell.
  • Lipofection represents another technique useful for transfection of target cells. This method involves the loading of nucleic acids into a liposome, which often presents cationic functional groups, such as quaternary or protonated amines, towards the liposome exterior. This promotes electrostatic interactions between the liposome and a cell due to the anionic nature of the cell membrane, which ultimately leads to uptake of the exogenous nucleic acids, for example, by direct fusion of the liposome with the cell membrane or by endocytosis of the complex.
  • cationic functional groups such as quaternary or protonated amines
  • Magnetic beads are another tool that can be used to transfect target cells in a mild and efficient manner, as this methodology utilizes an applied magnetic field in order to direct the uptake of nucleic acids.
  • This technology is described in detail, for example, in US 2010/0227406, the disclosure of which is incorporated herein by reference.
  • Another useful tool for inducing the uptake of exogenous nucleic acids by target cells is laserfection, a technique that involves exposing a cell to electromagnetic radiation of a particular wavelength in order to gently permeabilize the cells and allow polynucleotides to penetrate the cell membrane. This technique is described in detail, e.g., in Rhodes et al., Methods in Cell Biology 82:309 (2007), the disclosure of which is incorporated herein by reference.
  • vesicles also referred to as Gesicles
  • Gesicles for the genetic modification of eukaryotic cells is described in detail, e.g., in Quinn et al., Genetic Modification of Target Cells by Direct Delivery of Active Protein [abstract].
  • Methylation changes in early embryonic genes in cancer [abstract] in: Proceedings of the 18th Annual Meeting of the American Society of Gene and Cell Therapy; 2015 May 13, Abstract No.122.
  • a transgene such as a transgene encoding an inhibitory nucleic acid construct, such as an interfering RNA construct (for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) described herein), into a target cell, and particularly into a human cell.
  • an interfering RNA construct for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), microRNA (miRNA) described herein
  • siRNA short interfering RNA
  • shRNA short hairpin RNA
  • miRNA microRNA
  • Transposons are polynucleotides that encode transposase enzymes and contain a polynucleotide sequence or gene of interest flanked by 5’ and 3’ excision sites. Once a transposon has been delivered into a cell, expression of the transposase gene commences and results in active enzymes that cleave the gene of interest from the transposon. This activity is mediated by the site-specific recognition of transposon excision sites by the transposase. In some instances, these excision sites may be terminal repeats or inverted terminal repeats.
  • the transgene of interest can be integrated into the genome of a mammalian cell by transposase-catalyzed cleavage of similar excision sites that exist within the nuclear genome of the cell. This allows the transgene of interest to be inserted into the cleaved nuclear DNA at the complementary excision sites, and subsequent covalent ligation of the phosphodiester bonds that join the gene of interest to the DNA of the mammalian cell genome completes the incorporation process.
  • the transposon may be a retrotransposon, such that the gene encoding the target gene is first transcribed to an RNA product and then reverse- transcribed to DNA before incorporation in the mammalian cell genome.
  • transposon systems are the piggybac transposon (described in detail in, e.g., WO 2010/085699) and the sleeping beauty transposon (described in detail in, e.g., US 2005/0112764), the disclosures of each of which are incorporated herein by reference as they pertain to transposons for use in gene delivery to a cell of interest.
  • Another tool for the integration of target transgenes into the genome of a target cell is the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, a system that originally evolved as an adaptive defense mechanism in bacteria and archaea against viral infection.
  • CRISPR clustered regularly interspaced short palindromic repeats
  • the CRISPR/Cas system includes palindromic repeat sequences within plasmid DNA and an associated Cas9 nuclease. This ensemble of DNA and protein directs site specific DNA cleavage of a target sequence by first incorporating foreign DNA into CRISPR loci. Polynucleotides containing these foreign sequences and the repeat-spacer elements of the CRISPR locus are in turn transcribed in a host cell to create a guide RNA, which can subsequently anneal to a target sequence and localize the Cas9 nuclease to this site.
  • RNA transcript expression level of a wild-type or pathological RNA transcript can be ascertained, for example, by a variety of nucleic acid detection techniques. Additionally, or alternatively, RNA transcript expression can be inferred by evaluating the concentration or relative abundance of an encoded protein produced by translation of the RNA transcript. Protein concentrations can also be assessed, for example, using functional assays. Using these techniques, a reduction in the concentration of wild-type or pathological RNA transcripts in response to the compositions and methods described herein can be observed, while monitoring the expression of the encoded protein.
  • RNA transcript expression can be evaluated by a number of methodologies known in the art, including, but not limited to, nucleic acid sequencing, microarray analysis, proteomics, in-situ hybridization (e.g., fluorescence in-situ hybridization (FISH)), amplification-based assays, in situ hybridization, fluorescence activated cell sorting (FACS), northern analysis and/or PCR analysis of RNAs.
  • FISH fluorescence in-situ hybridization
  • FACS fluorescence activated cell sorting
  • Nucleic Acid-based methods for detection of RNA transcript expression include imaging- based techniques (e.g., Northern blotting or Southern blotting), which may be used in conjunction with cells obtained from a patient following administration of, for example, a vector encoding an inhibitory nucleic acid construct (such as an interfering RNA, for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA) described herein) or a composition containing such an inhibitory nucleic acid construct.
  • an inhibitory nucleic acid construct such as an interfering RNA, for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA) described herein
  • a composition containing such an inhibitory nucleic acid construct such as an interfering RNA, for example, short interfering RNA (siRNA), short hairpin RNA (shRNA), or microRNA (miRNA) described herein
  • RNA expression levels may be determined using microarray-based platforms (e.g., single- nucleotide polymorphism arrays), as microarray technology offers high resolution. Details of various microarray methods can be found in the literature. See, for example, U.S. Pat. No.6,232,068 and Pollack et al., Nat. Genet.23:41-46 (1999), the disclosures of each of which are incorporated herein by reference in their entirety.
  • nucleic acid microarrays mRNA samples are reverse transcribed and labeled to generate cDNA. The probes can then hybridize to one or more complementary nucleic acids arrayed and immobilized on a solid support.
  • Protein expression assays suitable for use with the compositions and methods described herein include proteomics approaches, immunohistochemical and/or western blot analysis, immunoprecipitation, molecular binding assays, ELISA, enzyme-linked immunofiltration assay (ELIFA), mass spectrometry, mass spectrometric immunoassay, and biochemical enzymatic activity assays.
  • proteomics methods can be used to generate large-scale protein expression datasets in multiplex.
  • the peptide microarray may include a plurality of binders, including, but not limited to, monoclonal antibodies, polyclonal antibodies, phage display binders, yeast two-hybrid binders, aptamers, which can specifically detect the binding of specific oligonucleotides, peptides, or proteins.
  • binders including, but not limited to, monoclonal antibodies, polyclonal antibodies, phage display binders, yeast two-hybrid binders, aptamers, which can specifically detect the binding of specific oligonucleotides, peptides, or proteins.
  • Examples of peptide arrays may be found in U.S. Patent Nos. 6,268,210, 5,766,960, and 5,143,854, the disclosures of each of which are incorporated herein by reference in their entirety.
  • Mass spectrometry may be used in conjunction with the methods described herein to identify and characterize transgene expression in a cell from a patient (e.g., a human patient) following delivery of the transgene. Any method of MS known in the art may be used to determine, detect, and/or measure a protein or peptide fragment of interest, e.g., LC-MS, ESI-MS, ESI-MS/MS, MALDI-TOF-MS, MALDI-TOF/TOF-MS, tandem MS, and the like.
  • Mass spectrometers generally contain an ion source and optics, mass analyzer, and data processing electronics.
  • Mass analyzers include scanning and ion-beam mass spectrometers, such as time-of-flight (TOF) and quadruple (Q), and trapping mass spectrometers, such as ion trap (IT), Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), may be used in the methods described herein. Details of various MS methods can be found in the literature. See, for example, Yates et al., Annu. Rev. Biomed. Eng.11:49-79, 2009, the disclosure of which is incorporated herein by reference in its entirety.
  • TOF time-of-flight
  • Q quadruple
  • trapping mass spectrometers such as ion trap (IT), Orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR)
  • proteins in a sample obtained from the patient can be first digested into smaller peptides by chemical (e.g., via cyanogen bromide cleavage) or enzymatic (e.g., trypsin) digestion.
  • Complex peptide samples also benefit from the use of front-end separation techniques, e.g., 2D-PAGE, HPLC, RPLC, and affinity chromatography.
  • the digested, and optionally separated, sample is then ionized using an ion source to create charged molecules for further analysis.
  • Ionization of the sample may be performed, e.g., by electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), photoionization, electron ionization, fast atom bombardment (FAB)/liquid secondary ionization (LSIMS), matrix assisted laser desorption/ionization (MALDI), field ionization, field desorption, thermospray/plasmaspray ionization, and particle beam ionization. Additional information relating to the choice of ionization method is known to those of skill in the art. After ionization, digested peptides may then be fragmented to generate signature MS/MS spectra.
  • ESI electrospray ionization
  • APCI atmospheric pressure chemical ionization
  • FAB fast atom bombardment
  • LIMS liquid secondary ionization
  • MALDI matrix assisted laser desorption/ionization
  • field ionization field desorption
  • the pharmaceutical forms suitable for injectable use include sterile aqueous solutions or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions (described in US 5,466,468, the disclosure of which is incorporated herein by reference).
  • the formulation may be sterile and may be fluid to the extent that easy syringability exists.
  • Formulations may be stable under the conditions of manufacture and storage and may be preserved against the contaminating action of microorganisms, such as bacteria and fungi.
  • isotonic agents for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by the use in the compositions of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • a solution containing a pharmaceutical composition described herein may be suitably buffered, if necessary, and the liquid diluent first rendered isotonic with sufficient saline or glucose.
  • aqueous solutions are especially suitable for administration by a route selected from intrathalamic, intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intracerebral, intracerebroventricular, intraocular (e.g., intravitreal), intraventricular, intralumbar, intravenous, intramuscular, subcutaneous, intraperitoneal, intradermal, transdermal, parenteral, intranasal, percutaneous, intratracheal, intraarterial, intravascular, and oral administration, inhalation, perfusion, lavage, or any combination thereof.
  • sterile aqueous media that can be employed will be known to those of skill in the art in light of the present disclosure.
  • one dosage may be dissolved in 1 ml of isotonic NaCl solution and either added to 1000 ml of hypodermoclysis fluid or injected at the proposed site of infusion. Some variation in dosage will necessarily occur depending on the condition of the subject being treated. The person responsible for administration will, in any event, determine the appropriate dose for the individual subject. Moreover, for human administration, preparations may meet sterility, pyrogenicity, general safety, and purity standards as required by FDA Office of Biologics standards.
  • a pharmaceutical composition containing, for example, an inhibitory nucleic acid described herein, typically includes a pharmaceutically acceptable diluent or carrier.
  • prodrugs include one or more conjugate group attached to a nucleic acid molecule, wherein the conjugate group is cleaved by endogenous nucleases within the body.
  • Lipid moieties have been used in nucleic acid therapies in a variety of methods.
  • the nucleic acid is introduced into preformed liposomes or lipoplexes made of mixtures of cationic lipids and neutral lipids.
  • DNA complexes with mono- or poly-cationic lipids are formed without the presence of a neutral lipid.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to a particular cell or tissue.
  • a lipid moiety is selected to increase distribution of a pharmaceutical agent to fat tissue. In certain embodiments, a lipid moiety is selected to increase distribution of a pharmaceutical agent to muscle tissue.
  • pharmaceutical compositions include a delivery system. Examples of delivery systems include, but are not limited to, liposomes and emulsions. Certain delivery systems are useful for preparing certain pharmaceutical compositions including those including hydrophobic compounds. In certain embodiments, certain organic solvents such as dimethylsulfoxide are used.
  • pharmaceutical compositions include one or more tissue-specific delivery molecules designed to deliver the one or more pharmaceutical agents of the present invention to specific tissues or cell types. For example, in certain embodiments, pharmaceutical compositions include liposomes coated with a tissue-specific antibody.
  • compositions include a co-solvent system.
  • co-solvent systems include, for example, benzyl alcohol, a nonpolar surfactant, a water-miscible organic polymer, and an aqueous phase.
  • co-solvent systems are used for hydrophobic compounds.
  • a non-limiting example of such a co-solvent system is the VPD co- solvent system, which is a solution of absolute ethanol including 3% w/v benzyl alcohol, 8% w/v of the nonpolar surfactant Polysorbate 80TM and 65% w/v polyethylene glycol 300.
  • the proportions of such co-solvent systems may be varied considerably without significantly altering their solubility and toxicity characteristics.
  • Certain solvents suitable for use in pharmaceutical compositions for injection include, but are not limited to, lipophilic solvents and fatty oils, such as sesame oil, synthetic fatty acid esters, such as ethyl oleate or triglycerides, and liposomes.
  • Routes of Administration and Dosing Viral vectors, such as AAV vectors and others described herein, containing a transgene encoding an inhibitory nucleic acid of the disclosure may be administered to a patient (e.g., a human patient) by a variety of routes of administration.
  • Example 1 Determining the knockdown efficacy of ATXN2-specific miRNA constructs in HEK293 cells via dual luciferase reporter assay Objective The objective of this study was to evaluate the knockdown efficacy of several ATXN2 miRNA candidates in HEK293 cells via the dual luciferase reporter assay. This study was designed to determine the efficiency of the miRNA constructs in silencing ATXN2 mRNA.
  • a dual luciferase reporter assay was then performed to measure the efficacy of the knockdown achieved by each miRNA construct in comparison to a negative control, which effectuates no significant silencing.
  • the construct numbering in this example is the same as Example 1. Results In result, we observed that the miRNA constructs chosen for this study were able to silence ATXN2. Percentage of luciferase activity relative to negative control ranged from 20% to 54% (FIG. 2A), with miRNA construct 1 showing the best knockdown efficacy of those tested. Knockdown efficacy is inverse to the percentage luciferase activity relative to negative control, therefore, the lower the percentage value of the percentage luciferase activity relative to negative control, the higher the knockdown efficacy.
  • mice In these mice, a 169 kb human BAC (RP11-798L5) containing the entire 150 kb human ATXN2 locus including regulatory regions was engineered to replace the endogenous ATXN2 exon-1 CAG22 with CAG72 repeats.
  • the thalamus and sub-cortex from one hemisphere were harvested 6 weeks post-intrathalamic administration and analysed for mouse and human ATXN2 mRNA expression using qPCR. Following tissue harvesting, a probe-based qPCR assay was performed using homogenized mouse tissue to measure the efficacy of the knockdown achieved by each ATXN2-specific miRNA construct in comparison with a negative control.
  • Vector expression was confirmed by measuring the quantity of viral genomes (vg) using qPCR in the thalamus (data not shown).
  • the experiments in this study were performed twice, starting with a small-scale pilot study with construct 2 followed by a large-scale study including multiple ATXN2-specific miRNA constructs.
  • Both mouse and human ATXN2 mRNA were measured in the mouse thalamus and sub-cortex in the small-scale pilot study whereas only human ATXN2 mRNA was measured in the large-scale study.
  • the experimental groups received AAV injections with doses of 1e8 viral genomes/hemisphere (vg/hem) or 1e9 vg/hem in the pilot study whereas in the large-scale study only AAV doses of 1e9 vg/hem were injected.

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Abstract

L'invention concerne des compositions et des méthodes pour le traitement de troubles associés à l'expression de transcrits d'ARN d'ataxine-2 (ATXN2) de type sauvage ou mutants, comprenant des troubles caractérisés par des gènes contenant des régions de répétition étendues de manière aberrante qui peuvent conduire à un phénotype pathologique. L'invention concerne des constructions d'ARN inhibitrices qui suppriment l'expression d'ATXN2, ainsi que des vecteurs viraux, tels que des vecteurs viraux adéno-associés, codant pour de telles molécules d'ARN inhibitrices.
EP23773208.6A 2022-09-16 2023-09-15 Compositions et méthodes de traitement de maladies neurologiques Pending EP4587568A2 (fr)

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