EP4288555A1 - Vecteurs comprenant des séquences polynucléotidiques de remplissage - Google Patents

Vecteurs comprenant des séquences polynucléotidiques de remplissage

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Publication number
EP4288555A1
EP4288555A1 EP22705501.9A EP22705501A EP4288555A1 EP 4288555 A1 EP4288555 A1 EP 4288555A1 EP 22705501 A EP22705501 A EP 22705501A EP 4288555 A1 EP4288555 A1 EP 4288555A1
Authority
EP
European Patent Office
Prior art keywords
seq
nucleotides
sequence
vector
mirna
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
EP22705501.9A
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German (de)
English (en)
Inventor
Carleton Proctor Goold
Robert R. Graham
Peter JANKI
Ronald Chen
Eric Green
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Maze Therapeutics Inc
Original Assignee
Maze Therapeutics Inc
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from PCT/US2021/016939 external-priority patent/WO2021159008A2/fr
Application filed by Maze Therapeutics Inc filed Critical Maze Therapeutics Inc
Publication of EP4288555A1 publication Critical patent/EP4288555A1/fr
Pending legal-status Critical Current

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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
    • 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
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/14—Type of nucleic acid interfering N.A.
    • C12N2310/141—MicroRNAs, miRNAs
    • 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
    • C12N2320/00—Applications; Uses
    • C12N2320/30—Special therapeutic applications
    • C12N2320/32—Special delivery means, e.g. tissue-specific
    • 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
    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors
    • 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
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/38—Vector systems having a special element relevant for transcription being a stuffer
    • 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
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/50—Vector systems having a special element relevant for transcription regulating RNA stability, not being an intron, e.g. poly A signal

Definitions

  • AAV adeno-associated virus
  • the cis plasmid has a ‘backbone’ sequence – such as typically encoding the elements of bacterial origins of resistance and antibiotic resistance, needed to propagate the plasmid in bacteria – that is close in size to the intended AAV vector genome, the amount of unintended ‘reverse packaged’ sequence can increase (Hauck et al., Molecular Therapy (2009) 17:144-152). For these reasons, it may be beneficial to include ‘stuffer sequences’ that on their own do not confer unfavorable properties to packaged AAV, as material in the plasmid backbone, and for use in situations where the intended AAV payload is considerably shorter than the native AAV packaging size.
  • FIG.1 Tracks selected on UCSC genome browser to identify expressed regions, known or predicted regulatory elements and repetitive elements (including retroviral and transposable elements) within the base sequence.
  • FIGS.2A-2B show the liver enzyme function tests (FIG.2A) aspartate transaminase (AST; U/L) and (FIG.2B) alanine transaminase (ALT; U/L) measured 13- days post-dosing with processed serum of mice dosed intraveniously with vehicle or scAAV9 vectors.
  • AST aspartate transaminase
  • ALT alanine transaminase
  • FIGS.3A-3H show Fragment Analyzer traces of column purified scAAV9 vector DNA.
  • FIG.3A shows the standard high-molecular weight double peak, which upon enzymatic cleavage of the mutITR (double peak retained; FIG.3B), or wtITRs (now single peak; FIG.3C) demonstrates that the double- peak represents the full-length population.
  • FIG.3D labels the 3 components of the Fragment Analyzer trace of purified scAAV9 vector DNA: full-length vector as represented by the two peaks at the highest molecular weight, the next peak, or miR- centered truncations, and lastly truncations occurring within the stuffer sequence.
  • FIG.3E are the traces for scAAV9 H1 MCS constructs PSG11_V1 and PSG11_V2 in the top traceand bottom trace, respectively.
  • PSG11_V2 has a higher concentration of truncations compared to PSG11_V1.
  • FIG.3G are Fragment Analyzer traces of PSG11_V1 vector (top) and PSG11_V2 vector (bottom) embedded with the articifial miRNA miR-1-1 XD-14792 (XD-14792 is also referred to herein as 1784).
  • FIG.3H shows overlap of the traces in FIG.3G.
  • FIGS.4A-4B show the predicted secondary structure of 200 nucleotides adjacent to the artificial miRNA and terminator of PSG11_V1 (FIG.4A) and PSG11_V2 (FIG.4B) using Mfold web server.
  • PSG11_V1 compared to PSG11_V2 has a more desirable predicted DNA secondary structure adjacent to artificial miRNA.
  • FIGS.5A-5B show liver enzyme values (FIG.5A – AST; FIG.5B – ALT) of the vector architecture study.
  • FIG.6 shows stranded RNAseq traces from representative vectors with H1 long promoter, amiRNA 3330 in miR-100 backbone and PSG11_V5 stuffer (left) and H1 short promoter, amRNA 1784 (also referred to herein 14792) in miR-1-1 backbone and PSG11_V5 stuffer (right).
  • FIG.7 Shows the titers of the 24 constructs tested in the vector architecture study.
  • FIGS.8A-8C show miR and stuffer truncations for the scAAV9 vectors with AMELY_V3 and PSG11_V5 stuffer sequences as measured by Fragment Analyzer parallel capillary electrophoresis system.
  • miR100 has fewer miR truncations compared to miR1-1 in both AMELY_V3 and PSG11_V5 vectors.
  • the vector with H1 long promoter, both with and without an SV40 polyadenylation sequence has fewer stuffer truncations compared to vectors with H1 native promoter and H1 short promoter.
  • the vector with H1 long promoter both with and without an SV40 polyadenylation sequence, has fewer truncations in the combined miR/stuffer truncations compared to vector with H1 native promoter and vector with H1 short promoter.
  • DETAILED DESCRIPTION Prior to setting forth this disclosure in more detail, it may be helpful to an understanding thereof to provide definitions of certain terms to be used herein. Additional definitions are set forth throughout this disclosure. In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated.
  • any number range recited herein relating to any physical feature, such as polymer subunits, size or thickness, are to be understood to include any integer within the recited range, unless otherwise indicated.
  • the term “about” means ⁇ 20% of the indicated range, value, or structure, unless otherwise indicated.
  • the terms “a” and “an” as used herein refer to “one or more” of the enumerated components.
  • the use of the alternative should be understood to mean either one, both, or any combination thereof of the alternatives.
  • the terms “include,” “have” and “comprise” are used synonymously, which terms and variants thereof are intended to be construed as non-limiting.
  • nucleic acid or “polynucleotide” refer to any nucleic acid polymer composed of covalently linked nucleotide subunits, such as polydeoxyribonucleotides or polyribonucleotides.
  • nucleic acids include RNA and DNA.
  • RNA refers to a molecule comprising one or more ribonucleotides and includes double-stranded RNA, single-stranded RNA, isolated RNA, synthetic RNA, recombinant RNA, as well as modified RNA that differs from naturally-occurring RNA by the addition, deletion, substitution, and/or alternation of one or more nucleotides.
  • Nucleotides of RNA molecules may comprise standard nucleotides or non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides.
  • “DNA” refers to a molecule comprising one or more deoxyribonucleotides and includes double-stranded DNA, single-stranded DNA, isolated DNA, synthetic DNA, recombinant DNA, as well as modified DNA that differs from naturally-occurring DNA by the addition, deletion, substitution, and/or alteration of one or more nucleotides.
  • Nucleotides of DNA molecules may comprise standard nucleotides or non-standard nucleotides, such as non-naturally occurring nucleotides or chemically synthesized nucleotides.
  • isolated refers to a substance that has been isolated from its natural environment or artificially produced. As used herein with respect to a cell, “isolated” refers to a cell that has been isolated from its natural environment (e.g., from a subject, organ, tissue, or bodily fluid).
  • isolated refers to a nucleic acid that has been isolated or purified from its natural environment (e.g., from a cell, cell organelle, or cytoplasm), recombinantly produced, amplified, or synthesized.
  • an isolated nucleic acid includes a nucleic acid contained within a vector.
  • 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.
  • inhibitory nucleic acid refers to a nucleic acid that comprises a guide strand sequence that hybridizes to at least a portion of a target nucleic acid, e.g., neurodenerative disease target RNA, mRNA, pre-mRNA, or mature mRNA, and inhibits its expression or activity.
  • a target nucleic acid e.g., neurodenerative disease target RNA, mRNA, pre-mRNA, or mature mRNA
  • An inhibitory nucleic acid may target a protein coding region (e.g., exon) or non-coding region (e.g., 5’UTR, 3’UTR, intron, etc.) of a target nucleic acid.
  • 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 RNA molecule, such as a siRNA, shRNA, miRNA, or dsRNA.
  • 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., Methods Mol. Biol. (2014) 1062:211-224).
  • the nature of the complementarity of the guide and passenger sequences can be similar or different from the nature of complementarity of the guide and passenger sequences in the endogenous miRNA backbone upon which the synthetic miRNA is constructed.
  • miRNA backbone e.g., number of bases, position of mismatches, types of bulges, etc.
  • the term “microRNA backbone,” “miR backbone,” “microRNA scaffold,” or “miR scaffold” refers to a pri-miRNA or pre-miRNA scaffold, with the stem sequence replaced by a miRNA of interest, and is capable of producing a functional, mature miRNA that directs RNA silencing at the gene targeted by the miRNA of interest.
  • a miR backbone comprises a 5’ flanking region (also referred to 5’ miR context, > 9 nucleotides), a stem region comprising the miRNA duplex (guide strand sequence and passenger strand sequence) and basal stem (5’ and 3’, each about 4-13 nucleotides), at least one loop motif region including the terminal loop (>10 nucleotides for terminal loop), a 3’ flanking region (also referred to 3’ miR context, > 9 nucleotides), and optionally one or more bulges in the stem.
  • a miR backbone may be derived completely or partially from a wild type miRNA scaffold or be a completely artificial sequence.
  • 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).
  • the sense sequence or passenger strand sequence refers to the mature sequence remaining following cleavage by Dicer.
  • a “duplex,” when used in reference to an inhibitory nucleic acid refers to two nucleic acid strands (e.g., a guide strand and passenger strand) hybridizing together to form a duplex structure.
  • a duplex may be formed by two separate nucleic acid strands or by a single nucleic acid strand having a region of self-complementarity (e.g., hairpin or stem-loop).
  • the term “complementary” refers to the ability of polynucleotides to form base pairs with each other.
  • Base pairs are typically formed by hydrogen bonds between nucleotide subunits in antiparallel polynucleotide strands or a single, self-annealing polynucleotide strand.
  • Complementary polynucleotide strands can form base pairs in the Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes.
  • uracil rather than thymine is the base that is considered to be complementary to adenosine.
  • a “U” is denoted in the context of the present invention, the ability to substitute a “T” is understood, unless otherwise stated.
  • Complementarity also encompasses Watson-Crick base pairing between non-modified and modified nucleobases (e.g., 5-methyl cytosine substituted for cytosine).
  • Full complementarity, perfect complementarity or 100% complementarity between two polynucleotide strands is where each nucleotide of one polynucleotide strand can form hydrogen bond with a nucleotide unit of a second polynucleotide strand.
  • % complementarity refers to the number of nucleotides of a contiguous nucleotide sequence in a nucleic acid molecule that are complementary to an aligned reference sequence (e.g., a target mRNA, passenger strand), divided by the total number of nucleotides and multiplying by 100. In such an alignment, a nucleobase/nucleotide which does not form a base pair is called a mismatch. Insertions and deletions are not permitted in calculating % complementarity of a contiguous nucleotide sequence.
  • % identity Insertions and deletions are not permitted in the calculation of % identity of a contiguous nucleotide sequence. It is understood by skilled persons in the art that in calculating identity, chemical modifications to nucleobases are not considered as long as the Watson-Crick base pairing capacity of the nucleobase is retained (e.g., 5-methyl cytosine is considered the same as cytosine for the purpose of calculating % identity).
  • the term “hybridizing” or “hybridizes” refers to two nucleic acids strands forming hydrogen bonds between base pairs on antiparallel strands, thereby forming a duplex.
  • T m melting temperature
  • expression construct refers to any type of genetic construct containing a nucleic acid (e.g., transgene) in which part or all of 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.
  • 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).
  • 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 a 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.
  • vector refers to a genetic construct that is capable of transporting a nucleic acid molecule (e.g., transgene encoding inhibitory nucleic acid) between cells and effecting expression of the nucleic acid molecule when operably- linked to suitable expression control sequences.
  • Expression control sequences may include transcription initiation sequence, termination sequence (also referred to herein as terminator sequence), promoter sequence and enhancer sequence; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (i.e., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
  • the vector may be a plasmid, phage particle, transposon, cosmid, phagemid, chromosome, artificial chromosome, virus, virion, etc.
  • the vector Once transformed into a suitable host cell, the vector may replicate and function independently of the host genome, or may, in some instances, integrate into the genome itself.
  • host cell refers to any cell that contains, or is capable of containing a composition of interest, e.g., an inhibitory nucleic acid.
  • a host cell is a mammalian cell, such as a rodent cell, (mouse or rat) or primate cell (monkey, chimpanzee, or human).
  • a host cell may be in vitro or in vivo.
  • a host cell may be from an established cell line or primary cells.
  • a host cell is a cell of the CNS, such as a neuron, glial cell, astrocyte, and microglial cell.
  • 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 dementia (FTD), 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, FTD, 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.
  • subject e.g., human, non-human mammals, such as primates (monkey, chimpanzee), cows, horses, sheep, dogs, cats, rats, mice, guinea pigs, pigs, and transgenic species thereof.
  • Stuffer Sequences and Expression Constructs AAV preferentially packages a full-length genome, i.e., one that is approximately the same size as the native genome, and is not too big or too small.
  • expression cassettes encoding inhibitory nucleic acid sequences are substantially smaller than AAV full-length genome.
  • a stuffer sequence may be linked to an expression construct comprising a heterologous nucleic acid sequence and flanked by the 5’ ITR and 3’ ITR to expand the packagable genome, resulted in a genome whose size was near-normal in length between the ITRs.
  • the packaging capacity of AAV is about 4.7 kb between the 5’ ITR and 3’ ITR.
  • the packaging capacity is about 2.4 kb between the 5’ ITR and 3’ ITR.
  • the starting sequence for obtaining a vector stuffer sequence is of mammalian origin, such as human origin.
  • the length of the stuffer sequence may be adjusted such that the vector genome is at or close to the (natural) packaging limit of AAV capsid.
  • a vector stuffer sequence can be designed to minimize adverse effects in the context of in vivo gene therapy. For example, regions of the human genome may be identified as a source for vector stuffer sequences by identifying sequences with minimal impact if integration in the genome occurs and minimal risk of initiating unexpected transcription.
  • regions of the genome may be examined in which i) deletions and duplications were common in the population and not associated with disease-relevant phenotypes (no evidence of evolutionary pressure) and/or ii) RNA expression across human tissues was low or undetectable (lack strong intrinsic enhancers/promoter elements).
  • vector stuffer sequences can be designed to have reduced, minimized, removed, or to lack one or more elements to make the vector sequence inert or safe.
  • the vector stuffer sequence is modified to: reduce or remove expressed regions (e.g., exons + 10 bp on either side, human ESTs); reduce or remove regulatory elements (e.g., promoter sequences, enhancer sequences, repressor sequences, splicing donors or acceptors, or other cis-acting elements found in the human genome that could potentially affect transcription of the transgene); reduce or remove repetitive elements (e.g., microsatellite repeats, dinucleotides repeats, trinucleotide repeats); reduce, remove, or modify ATG codons to reduce or eliminate the possibility of peptides being generated from the filler or stuffer sequence due to latent start codons; reduce or remove CpG dinucleotides to lower likelihood of unmethylated CpG dinucleotides (from cis- plasmids generated in bacteria) inducing an innate immune response; or any combination thereof.
  • expressed regions e.g., exons + 10 bp
  • the present disclosure provides vector stuffer sequences possessing one or more of the aforementioned features, with further advantages of one or more of: high titer; low toxicity; and reduced truncations in miRNA and/or stuffer sequence.
  • the present disclosure provides a vector stuffer sequence comprising a nucleic acid of about 1300 to about 2300 nucleotides in length and having at least 75% identity to: nucleotides 489-2185 of any one of SEQ ID NOS:13-16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ
  • the vector stuffer sequence comprises a nucleic acid of about 1500-2000 nucleotides in length and having at least 75% identity to: nucleotides 489-2185 of any one of SEQ ID NOS:13-16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides 252-2132 of SEQ ID NO:26; nucleotides 252-2132 of SEQ ID NO:27; nucleotides 252-2132 of SEQ ID NO:27;
  • the vector stuffer sequence comprises a nucleic acid of about 1600 to 1900 nucleotides in length and having at least 75% identity to: nucleotides 489-2185 of any one of SEQ ID NOS:13-16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides 252-2132 of SEQ ID NO:26; nucleotides 252-2132 of SEQ ID NO:27; nucleotides 252-2132 of SEQ ID NO:27;
  • the vector stuffer sequence comprises a nucleic acid having at least 80% identity to: nucleotides 489-2185 of any one of SEQ ID NOS:13- 16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides 252-2132 of SEQ ID NO:26; nucleotides 252-2132 of SEQ ID NO:27; nucleotides 252-2132 of SEQ ID NO:28; nucleotides 357-22
  • the vector stuffer sequence comprises a nucleic acid of about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 in length. In some embodiments, the vector stuffer sequence comprises a nucleic acid having at least 85% identity to: nucleotides 489-2185 of any one of SEQ ID NOS:13- 16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides
  • the vector stuffer sequence comprises a nucleic acid of about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 in length. In some embodiments, the vector stuffer sequence comprises a nucleic acid having at least 90% identity to: nucleotides 489-2185 of any one of SEQ ID NOS:13- 16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucle
  • the vector stuffer sequence comprises a nucleic acid of about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 in length. In some embodiments, the vector stuffer sequence comprises a nucleic acid having at least 95% identity to: nucleotides 489-2185 of any one of SEQ ID NOS:13- 16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides
  • the vector stuffer sequence comprises a nucleic acid of about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 in length.
  • the vector stuffer sequence comprises a nucleic acid having at least 97% identity to: nucleotides 489-2185 of any one of SEQ ID NOS:13-16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucle
  • the vector stuffer sequence comprises a nucleic acid of about 1,300-2,300 nucleotides in length, about 1,500-2,000 nucleotides in length, or about 1,600-1,900 in length.
  • the vector stuffer sequence comprises or consists of: nucleotides 489-2185 of any one of SEQ ID NOS:13-16; nucleotides 342-2222 of SEQ ID NO:17; nucleotides 488-2177 of SEQ ID NO:18; nucleotides 489-2177 of SEQ ID NO:19; nucleotides 711-2187 of SEQ ID NO:20; nucleotides 711-2187 of SEQ ID NO:21; nucleotides 488-2177 of SEQ ID NO:22; nucleotides 711-2187 of SEQ ID NO:23; nucleotides 711-2187 of SEQ ID NO:24; nucleotides 252-2132 of SEQ ID NO:25; nucleotides 252-2132 of SEQ ID NO:25; nucle
  • the vector stuffer sequence comprises or consists of SEQ ID NO:48 or nucleotides 489-2185 of any one of SEQ ID NOS:13-16.
  • the vector is an adeno-associated virus (AAV) vector, optionally wherein the AAV vector is self-complementary.
  • the vector stuffer sequence is positioned adjacent to (e.g., 5’ or 3’) an expression construct comprising a heterologous nucleic acid sequence.
  • the heterologous nucleic acid sequence encodes a therapeutic agent.
  • the therapeutic agent comprises a nucleic acid encoding a therapeutic protein or an inhibitory nucleic acid.
  • the inhibitory nucleic acid comprises a siRNA, miRNA, shRNA, or dsRNA. In some embodiments, the inhibitory nucleic acid comprises a siRNA, miRNA, shRNA, of dsRNA targeting a neurodegenerative disease related gene.
  • the neurodegenerative disease is spinocerebellar ataxia-2, amyotrophic lateral sclerosis, frontotemporal dementia, 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, or Alzheimer’s disease.
  • the neurodegenerative disease is a polyglutamine repeat disease.
  • the inhibitory nucleic acid targets ATXN2.
  • 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 typically 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 ATNX2, including human ATXN2.
  • the expression construct comprises a heterologous nucleic acid encoding an artificial miRNA targeting ATXN2.
  • heterologous nucleic acids encoding guide sequences and passenger sequences and artificial miRNAs targeting ATXN2 are provided in Table 5.
  • RNA formats of the encoded guide, passenger, and artificial miRNA sequences are provided, as well as the DNA formats for insertion into cis plasmids and rAAV vectors.
  • a heterologous nucleic acid encodes an artificial miRNA targeting ATXN2 and comprises a guide sequence selected from SEQ ID NOS:1-4 and 71.
  • a heterologous nucleic acid encodes an artificial miRNA comprising a guide sequence provided by SEQ ID NO:1 and a passenger sequence provided by SEQ ID NO:5.
  • a heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO:2 and a passenger sequence provided by SEQ ID NO:6. In some embodiments, a heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO:3 and a passenger sequence provided by SEQ ID NO:7. In some embodiments, a heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO:4 and a passenger sequence provided by SEQ ID NO:8. In some embodiments, a heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO:71 and a passenger sequence provided by SEQ ID NO:72.
  • the heterologous nucleic acid comprises a guide sequence and passenger sequence embedded in a miRNA backbone (or scaffold).
  • the miRNA backbone is miR-100 or miR-1-1.
  • the heterologous nucleic acid encodes an artificial miRNA sequence provided by any one of SEQ ID NOs:9-12 and 73. Additional examples of ATXN2 targeting guide sequences, passenger sequences, miR backbones, and artificial miRNA sequences are provided in PCT Publication WO2021/159008, which is incorporated by reference in its entirety.
  • the expression construct further comprises one or more expression control sequences (regulatory sequences) operably linked with the transgene (e.g., nucleic acid encoding an artificial miRNA).
  • “Operably linked” sequences include expression control seuqences that are contiguous with the transgene or act in trans or at a distance from the transgene to control its expression. Examples of expression control sequences include transcription initiation sequences, termination sequences, promoter sequences, enhancer sequences, repressor sequences, splice site sequences, polyadenylation (polyA) signal sequences, or any combination thereof.
  • a promoter is an endogenous promoter, synthetic promoter, constitutive promoter, inducible promoter, tissue-specific promoter (e.g., CNS-specific), or cell-specific promoter (neurons, glial cells, or astrocytes).
  • tissue-specific promoter e.g., CNS-specific
  • cell-specific promoter e.g., glial cells, or astrocytes.
  • constitutive promoters include, Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), SV40 promoter, and dihydrofolate reductase promoter.
  • inducible promoters examples include zinc-inducible sheep metallothionine (MT) promoter, dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, T7 polymerase promoter system, the ecdysone insect promoter, tetracycline-repressible system, tetracycline-inducible system, RU486-inducible system, and the rapamycin- inducible system.
  • MT sheep metallothionine
  • Dex dexamethasone
  • MMTV mouse mammary tumor virus
  • T7 polymerase promoter system examples include the ecdysone insect promoter, tetracycline-repressible system, tetracycline-inducible system, RU486-inducible system, and the rapamycin- inducible system.
  • promoters include, for example, chicken beta-actin promoter (CBA promoter), a CAG promoter, a H1 promoter, a CD68 promoter, a JeT promoter, synapsin promoter, RNA pol II promoter, or a RNA pol III promoter (e.g., U6, H1, etc.).
  • the promoter is a tissue-specific RNA pol II promoter.
  • the tissue-specific RNA pol II promoter is derived from a gene that exhibits neuron-specific expression.
  • the neuron-specific promoter is a synapsin 1 promoter or synapsin 2 promoter.
  • the promoter is a H1 promoter.
  • the H1 promoter is promoter referred to herein as a “native” H1 promoter, such as a promoter comprising or consisting of the sequence set forth in SEQ ID NO:53.
  • the promoter is a promoter referred to herein as a “short” H1 promoter, such as a promoter comprising or consisting of the sequence set forth in SEQ ID NO:54 or nucleotides 113-203 of any one of SEQ ID NOS:17, 25-28, and 37-44.
  • the promoter is a promoter referred to herein as a “long” H1 promoter, such as a promoter comprising or consisting of the sequence set forth in SEQ ID NO:52 or nucleotides 113-343 of any one of SEQ ID NOS:13-16 and 18-24.
  • the termination sequence is a SV40 termination sequence.
  • SV40 termination sequence examples include SEQ ID NO:77; nucleotides 489-710 of any one of SEQ ID NOS:20, 21, 23, and 24; nucleotides 358- 579 of any one of SEQ ID NOS:31, 32, 35, and 36; nucleotides 349-570 of any one of SEQ ID NOS:40, 41, and 44.
  • the sequence encoding the inhibitory nucleic acid of the present disclosure is positioned in an untranslated region of an expression construct.
  • the sequence encoding the inhibitory nucleic acid of the present disclosure is positioned in an intron, a 5' untranslated region (5 'UTR), or a 3' untranslated region (3'UTR) of the expression construct. In some embodiments, the sequence encoding the inhibitory nucleic acid of the present disclosure is positioned in an intron downstream of the promoter and upstream of an expressed gene.
  • vectors and Host Cells In another aspect, provided are vectors comprising vector stuffer sequences of the present disclosure.
  • a vector can be a plasmid, cosmid, phagemid, bacterial artificial chromosome (BAC) or viral vector.
  • viral vectors examples include herpesvirus (HSV) vectors, retroviral vectors, adenoviral vectors, adeno-associated viral (AAV) vectors, lentiviral vectors, baculoviral vectors, and the like.
  • a retroviral vector is a mouse stem cell virus, murine leukemia virus (e.g., Moloney murine leukemia virus vector), feline leukemia virus, feline sarcoma virus, or avian reticuloendotheliosis virus vector.
  • a lentiviral vector is a HIV (human immunodeficiency virus, including HIV type 1 and HIV type 2, equine infectious anemia virus, feline immunodeficiency virus (FIV), bovine immune deficiency virus (BIV), and simian immunodeficiency virus (SIV), equine infectious anemia virus, or Maedi-Visna viral vector.
  • the vector is an adeno-associated virus (AAV) vector, such as a recombinant AAV (rAAV) vector, which is produced by recombinant methods.
  • AAV adeno-associated virus
  • the rAAV vector comprises the vector stuffer sequence of the present disclosure and an expression construct comprising a heterologous nucleic acid sequence (e.g., encoding an inhibitory nucleic acid).
  • the vector stuffer sequence may be positioned adjacent to, either 5’ or 3’, the expression construct comprising the heterologous nucleic acid.
  • AAV is a single-stranded, non-enveloped DNA virus having a genome that encodes proteins for replication (rep) and the capsid (Cap), flanked by two ITRs, which serve as the origin of replication of the viral genome.
  • AAV also contains a packaging sequence, allowing packaging of the viral genome into an AAV capsid.
  • a recombinant AAV vector (also referred to as rAAV vector genome, or rAAV genome) may be obtained from the wild type genome of AAV by using molecular methods to remove the all or part of the wild type genome (e.g., Rep, Cap) from the AAV, and replacing it with a non-native nucleic acid, such as a heterologous nucleic acid sequence (e.g., a nucleic acid molecule encoding an inhibitory nucleic acid).
  • a heterologous nucleic acid sequence e.g., a nucleic acid molecule encoding an inhibitory nucleic acid.
  • ITR inverted terminal repeat
  • the rAAV vector comprises a 5’ inverted terminal repeat (ITR) and a 3’ ITR flanking the expression construct and vector stuffer sequence.
  • Functional ITR sequences are necessary for the rescue, replication and packaging of the AAV viral particle.
  • a rAAV vector is defined herein to include at least those sequences required in cis for replication and packaging (e.g., functional ITRs) of the virus. All other viral sequences may be supplied in trans.
  • the rAAV only retains the 5’ ITR and 3’ ITR from the AAV genome in order to maximize the size of the transgene that can be efficiently packaged by the vector.
  • each AAV ITR is a full length ITR (e.g., approximately 145 bp in length, and containing a functional Rep binding site (RBS) and a terminal resolution site (trs)).
  • RBS Rep binding site
  • trs terminal resolution site
  • one or both of the ITRs is is modified, e.g., by insertion, deletion, or substitution, provided that the ITRs provide for functional rescue, replication, and packaging.
  • a modified ITR lacks a functional terminal resolution site (trs) and is used for production of self-complementary AAV vectors (scAAV vectors).
  • the rAAV vector is a self-complementary AAV vector comprising a mutant ITR (lacking a terminal resolution site) on the 5’ side and a wild-type AAV ITR on the 3’ side.
  • a mutant 5’ ITR lacking a terminal resolution site is set forth in SEQ ID NO:57 or nucleotides 1-106 of any one of SEQ ID NOS:13-24, 29-44, and 78-82.
  • a modified ITR is a truncated version of AAV2 ITR referred to as AITR (D-sequence and TRS are deleted).
  • the rAAV vector comprises a 5’ ITR comprising or consisting of SEQ ID NO:57 or nucleotides 1-106 of any one of SEQ ID NOS:13-24, 29-44, and 78-82.
  • the rAAV vector comprises a 3’ ITR comprising or consisting of SEQ ID NO:58, nucleotides 2192-2358 of any one of SEQ ID NOS:13-16, nucleotides 2214-2358 of any one of SEQ ID NOS:13-16, nucleotides 2229-2395 of SEQ ID NO:17, nucleotides 2251-2395 of SEQ ID NO:17, nucleotides 2184-2350 of SEQ ID NO:18, nucleotides 2206-2350 of SEQ ID NO:18; nucleotides 2206-2350 of SEQ ID NO:19; nucleotides 2216-2360 of SEQ ID NO:20; nucleotides 2216-2360 of SEQ ID NO:20; nucleo
  • the rAAV vector comprises: a 5’ ITR comprising or consisting of SEQ ID NO:57 and a 3’ ITR comprising or consisting of SEQ ID NO:58; a 5’ ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO:13 and a 3’ ITR comprising or consisting of nucleotides 2192-2358 of SEQ ID NO:13; a 5’ ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO:14 and a 3’ ITR comprising or consisting of nucleotides 2192-2358 of SEQ ID NO:14; a 5’ ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO:15 and a 3’ ITR comprising or consisting of 2192-2358 of SEQ ID NO:15; a 5’ ITR comprising or consisting of nucleotides 1-106 of SEQ ID NO:16 and a 3’ ITR comprising or consisting or consisting or
  • the rAAV vector is a mammalian serotype AAV vector (e.g., AAV genome and ITRs derived from mammalian serotype AAV), including a primate serotype AAV vector or human serotype AAV vector.
  • the rAAV vector is a chimeric AAV vector.
  • the ITRs are selected from AAV serotypes of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV 12, AAVrh8, AAVrh10, AAV-DJ8, AAV-DJ, AAV- PHP.A, AAV-PHP.B, AAVPHP.B2, AAVPHP.B3, AAVPHP.N/PHP.B-DGT, AAVPHP.B- EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B- SNP(3), AAVPHP.B-SNP,
  • the heterologous nucleic acid sequence encodes a therapeutic agent.
  • the therapeutic agent comprises a nucleic acid encoding a therapeutic protein or an inhibitory nucleic acid.
  • the inhibitory nucleic acid comprises a siRNA, miRNA, shRNA, or dsRNA.
  • the inhibitory nucleic acid comprises a siRNA, miRNA, shRNA, of dsRNA targeting a neurodegenerative disease related gene.
  • the neurodegenerative disease is spinocerebellar ataxia-2, amyotrophic lateral sclerosis, frontotemporal dementia, 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, or Alzheimer’s disease.
  • the neurodegenerative disease is a polyglutamine repeat disease.
  • the inhibitory nucleic acid targets ATXN2.
  • ATXN2 refers to mammalian ATNX2, including human ATXN2.
  • the expression construct comprises a heterologous nucleic acid encoding an artificial miRNA targeting ATXN2. Examples of heterologous nucleic acids encoding guide sequences and passenger sequences and artificial miRNAs targeting ATXN2 are provided in Table 5. RNA formats of the encoded guide, passenger, and artificial miRNA sequences are provided, as well as the DNA formats for insertion into cis plasmids and rAAV vectors.
  • a heterologous nucleic acid encodes an artificial miRNA targeting ATXN2 and comprises a guide sequence selected from SEQ ID NOS:1-4 and 71. In some embodiments, a heterologous nucleic acid encodes an artificial miRNA comprising a guide sequence provided by SEQ ID NO:1 and a passenger sequence provided by SEQ ID NO:5. In some embodiments, a heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO:2 and a passenger sequence provided by SEQ ID NO:6. In some embodiments, a heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO:3 and a passenger sequence provided by SEQ ID NO:7.
  • a heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO:4 and a passenger sequence provided by SEQ ID NO:8. In some embodiments, a heterologous nucleic acid comprises a guide sequence provided by SEQ ID NO:71 and a passenger sequence provided by SEQ ID NO:72. In some embodiments, the heterologous nucleic acid comprises a guide sequence and passenger sequence embedded in a miRNA backbone (or scaffold). In some embodiments, the miRNA backbone is miR-100 or miR-1-1. In some embodiments, the heterologous nucleic acid encodes an artificial miRNA sequence provided by any one of SEQ ID NOs:9-12 and 73.
  • rAAV vector operably linked to the heterologous nucleic acid (e.g., encoding an inhibitory nucleic acid), including one or more of transcription initiation sequences, termination sequences, promoter sequences, enhancer sequences, repressor sequences, splice site sequences, polyadenylation (polyA) signal sequences, or any combination thereof.
  • heterologous nucleic acid e.g., encoding an inhibitory nucleic acid
  • rAAV vector comprises a H1 promoter.
  • the H1 promoter is a native H1 promoter, such as a promoter comprising or consisting of the sequence set forth in SEQ ID NO:53.
  • the promoter is a short H1 promoter, such as a promoter comprising or consisting of the sequence set forth in SEQ ID NO:54 or nucleotides 113-203 of any one of SEQ ID NOS:17, 25-28, and 37-44.
  • the promoter is a long H1 promoter, such as a promoter comprising or consisting of the sequence set forth in SEQ ID NO:52 or nucleotides 113-343 of any one of SEQ ID NOS:13-16 and 18-24.
  • the H1 promoter is oriented in the 5’ to 3’ direction in the expression construct, particularly when the 5’ ITR lacks a terminal resolution site.
  • the rAAV vector comprises a SV40 termination sequence. Examples of SV40 termination sequence are set forth in SEQ ID NO:77; nucleotides 489-710 of any one of SEQ ID NOS:20, 21, 23, and 24; nucleotides 358- 579 of any one of SEQ ID NOS:31, 32, 35, and 36; nucleotides 349-570 of any one of SEQ ID NOS:40, 41, and 44. rAAV vectors may have one or more AAV wild type genes deleted in whole or in part.
  • the rAAV vector is replication defective. In some embodiments, the rAAV vector lacks a functional Rep protein and/or capsid protein. In some embodiments, the rAAV vector is a self-complementary AAV (scAAV) vector. In some embodiments, the rAAV vector comprises: a 5’ ITR, a promoter operably linked to a heterologous nucleic acid encoding an ATXN2 specific artificial miRNA, a vector stuffer sequence, and a 3’ ITR. In some embodiments, the 5’ ITR is modified to lack a terminal resolution site. In some embodiments, the promoter is orientated in the 5’ to 3’ direction.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in any one of SEQ ID NOS:13-24, 29-44, and 78-80. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:13, wherein nucleotides 344-481 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:14, wherein nucleotides 344-481 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:15, wherein nucleotides 344-481 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO:16, wherein nucleotides 344-481 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO:17, wherein nucleotides 204-335 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises the nucleotide sequence set forth in SEQ ID NO:18, wherein nucleotides 344-481 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:19, wherein nucleotides 344-481 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:20, wherein nucleotides 344-481 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:21, wherein nucleotides 344-481 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:22, wherein nucleotides 344-481 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:23, wherein nucleotides 344-481 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:24, wherein nucleotides 344-481 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:29, wherein nucleotides 213-350 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:30, wherein nucleotides 213-350 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:31, wherein nucleotides 213-350 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:32, wherein nucleotides 213-350 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:33, wherein nucleotides 213-350 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:34, wherein nucleotides 213-350 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:35, wherein nucleotides 213-350 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:36, wherein nucleotides 213-350 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:37, wherein nucleotides 204-341 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:38, wherein nucleotides 204-341 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:39, wherein nucleotides 204-341 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:40, wherein nucleotides 204-341 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:41, wherein nucleotides 204-341 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:42, wherein nucleotides 204-341 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:43, wherein nucleotides 204-341 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:44, wherein nucleotides 204-341 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:78, wherein nucleotides 204-342 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:79, wherein nucleotides 213-351 are substituted with a sequence encoding an artificial miRNA of interest.
  • the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:80, wherein nucleotides 204-335 are substituted with a sequence encoding an artificial miRNA of interest. In some embodiments, the rAAV vector comprises from 5’ ITR to 3’ ITR the nucleotide sequence set forth in SEQ ID NO:81, wherein nucleotides 204-335 are substituted with a sequence encoding an artificial miRNA of interest. Recombinant AAV vectors of the present disclosure may be encapsidated by one or more AAV capsid proteins to form a rAAV particle.
  • a “rAAV particle” or “rAAV virion” refers to an infectious, replication-defective virus including an AAV protein shell, encapsidating a rAAV vector comprising a transgene of interest, which is flanked on each side by a 5’ AAV ITR and 3’ AAV ITR.
  • a rAAV particle is produced in a suitable host cell which has had sequences specifying a rAAV vector, AAV helper functions and accessory functions introduced therein to render the host cell capable of encoding AAV polypeptides that are required for packaging the rAAV vector (containing the transgene sequence of interest) into infectious rAAV particles for subsequent gene delivery.
  • one or more of the required components for packaging the rAAV vector may be provided by a stable host cell that has been engineered to to contain the one or more required components (e.g., by a vector).
  • Expression of the required components for AAV packaging may be under control of an inducible or constitutive promoter in the host packaging cell.
  • AAV helper vectors are commonly used to provide transient expression of AAV rep and/or cap genes, which function in trans, to complement missing AAV functions that are necessary for AAV replication.
  • AAV helper vectors lack AAV ITRs and can neither replicate nor package themselves.
  • AAV helper vectors can be in the form of a plasmid, phage, transposon, cosmid, virus, or virion.
  • rAAV particles may be produced using the triple transfection method (see, e.g., U.S. Patent No.6,001,650, incorporated herein by reference in its entirety). In this approach, the rAAV particles are produced by transfecting a host cell with a rAAV vector (comprising a transgene) to be packaged into rAAV particles, an AAV helper vector, and an accessory function vector.
  • the AAV helper function vector supports efficient AAV vector production without generating any detectable wild-type AAV virions (e.g., AAV virions containing functional rep and cap genes).
  • the accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (e.g., “accessory functions”).
  • the accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly.
  • Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.
  • helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.
  • a double transfection method wherein the AAV helper function and accessory function are cloned on a single vector, which is used to generate rAAV particles.
  • the AAV capsid is an important element in determining these tissue-specificity of the rAAV particle.
  • a rAAV particle having a capsid tissue specificty can be selected.
  • the rAAV particle comprises a capsid protein selected from a AAV serotype of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV9.47, AAV9(hu14), AAV10, AAV11, AAV 12, AAVrh8, AAVrh10, AAV-DJ8, AAV-DJ, AAV- PHP.A, AAV-PHP.B, AAVPHP.B2, AAVPHP.B3, AAVPHP.N/PHP.B-DGT, AAVPHP.B- EST, AAVPHP.B-GGT, AAVPHP.B-ATP, AAVPHP.B-ATT-T, AAVPHP.B-DGT-T, AAVPHP.B-GGT-T, AAVPHP.B-SGS, AAVPHP.B-AQP, AAVPHP.B-QQP, AAVPHP.B- SNP(3),
  • the AAV capsid is selected from a serotype that is capable of crossing the blood-brain barrier, e.g., AAV9, AAVrh.10, AAV-PHP-B, or a variant thereof.
  • AAV9 capsid sequences are provided in US Patent No.7,906,111, incorporated by reference in its entirety.
  • the AAV capsid is a chimeric AAV capsid.
  • the AAV particle is a pseudotyped AAV, having capsid and genome from different AAV serotypes.
  • the rAAV particle is capable of transducing cells of the CNS.
  • the rAAV particle is capable of transducing non-neuronal cells or neuronal cells of the CNS.
  • the CNS cell is a neuron, glial cell, astrocyte, or microglial cell.
  • the present disclosure provides host cells transfected with the rAAV particles comprising the vector stuffer sequences described herein.
  • the host cell is a prokaryotic cell or a eukaryotic cell.
  • the host cell is a mammalian cell (e.g., HEK293T, COS cells, HeLa cells, KB cells), bacterial cell (E.
  • the present disclosure provides methods of delivering a therapeutic agent to a subject, comprising administering a composition of the present disclosure (e.g., rAAV particle comprising a rAAV vector comprising the vector stuffer sequence provided hereinand an expression construct comprising heterologous nucleic acid sequence encoding a therapeutic agent).
  • a composition of the present disclosure e.g., rAAV particle comprising a rAAV vector comprising the vector stuffer sequence provided hereinand an expression construct comprising heterologous nucleic acid sequence encoding a therapeutic agent.
  • 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 term "treat” refers to preventing or delaying onset of neurodegenerative disease (e.g., ALS/FTD, 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.
  • the subject has a neurodegenerative disease or is at risk of developing a neurodegenerative disease.
  • neurodegenerative diseases include spinocerebellar ataxia-2, amyotrophic lateral sclerosis, frontotemporal dementia, 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.
  • 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.
  • Examples of symptoms characteristic of neurodegenerative disease include motor dysfunction, cognitive dysfunction, emotional/behavioral dysfunction, or any combination thereof. Paralsysis, shaking, unsteadiness, rigidity, twitching, muscle weakness, muscle cramping, muscle stiffness, muscle atrophy, difficulty swallowing, difficulty breathing, speech and language difficulties (e.g., slurred speech), slowness of movement, difficulty with walking, dementia, depression, anxiety, or any combination thereof.
  • the methods for treatment of the present disclosure of the present disclosure comprise administration as a monotherapy or in combination with one or more additional therapies for the treatment of the neurodegenerative disease.
  • Combination therapy may mean administration of the compositions of the present disclosure to the subject concurrently, prior to, subsequent to one or more additional therapies.
  • Concurrent administration of combination therapy may mean that the the compositions of the present disclosure and additional therapy are formulated for administration in the same dosage form or administered in separate dosage forms.
  • 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 may be administered to the subject by any route, including enteral (e.g., oral), parenteral, intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intra-cerebral, intracerebral ventricular, intraocular, intraventricular, intralumbar, subcutaneous, transdermal, interdermal, rectal, intravaginal, intraperitoneal, topical (as by powders, ointments, creams, and/or drops), mucosal, nasal, bucal, sublingual; by intratracheal instillation, bronchial instillation, and/or inhalation; and/or as an oral spray, nasal spray, and/or aerosol.
  • enteral e.g., oral
  • parenteral intravenous, intramuscular, intra-arterial, intramedullary, intrathecal, subpial, intra
  • compositions are directly injected into the CNS of the subject.
  • compositions are injected by intrathecal, subpial, intraparenchymal, intrastriatal, intracranial, intracisternal, intra-cerebral, intracerebral ventricular, intraocular, intraventricular, intralumbar administration, or any combination thereof.
  • a composition of the present disclosure is directly injected into the CNS of the subject.
  • direct injection into the CNS is 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
  • EXAMPLE 1 DESIGN OF VECTOR “STUFFER” SEQUENCE Selection of “dispensable” human genomic DNA for base stuffer sequence
  • “dispensable” regions of the human genome were identified having two intended properties: (a) sequences with minimal impact if integration in the genome occurs (although this is thought to be very rare for AAV); and (b) sequences with minimal risk of initiating unexpected transcription. Therefore, regions of the genome in which i) deletions and duplications were common in the population and not associated with disease-relevant phenotypes (no evidence of evolutionary pressure) were examined; and/or ii) RNA expression across human tissues was low or undetectable (lack strong intrinsic enhancers/promoter elements).
  • Region I AMELY gene region (chrY:6,865,918-6,874,027, HG38)
  • AMELY gene region is not under evolutionary selection.
  • the AMELY region arose from an ancient duplication event and have functional homologs on chromosome X that are under selection. (Lahn, & Page, Science (1999) 286, 964–967).
  • AMELY has not been associated with any rare human diseases (Online Mendelian Inheritance in Man, OMIM.org.)
  • AMELY mRNA is not detectably expressed in most human tissues (GTEX, The GTEx Consortium. (Science (2020) 369:1318-1330), and only present in chromosome Y carriers (males).
  • AMELX the functional homolog of AMELY
  • Region II PSG gene region (chr19:43,511,809-43,530,631, HG38)
  • PSG Pregnancy-specific Glycoprotein
  • the base sequences (chrY:6,865,918-6,874,027, HG38) and (chr19:43,511,809-43,530,631, HG38) were modified in the following manner (see FIG.1): i) Remove expressed regions: Exons + 10bp on either side (exons were defined using gene models in GENCODEv19), human expressed sequence tags (EST’s: defined using “Human ESTs Including Unspliced” track on the UCSC genome browser, based off of Genbank data: (Nucleic Acids Res.
  • a set of 4 vectors were designed, with two variants each of AMELY and PSG11 derived-stuffers (SEQ ID NOS:25-28).
  • Each vector contained a H1 short promoter at the 5’ side immediately downstream of the left ITR (SEQ ID NO:57), and a non-miRNA control sequence was included instead of a miRNA downstream of the H1 short promoter.
  • a Pol III terminator is positioned 3’ of the control sequence.
  • the artificial miRNA which was shown previously to exhibit toxicity was chosen to serve as a positive control for toxicity, presumably through an off-target effect of the specific artificial miRNA sequence.
  • Vectors were dosed intravenously at 1.3E11 total viral genome (vg) via tailvein to 16 weeks of age wild-type C57Bl/6 mice. Titer was determined based on DNA concentration of column-purified vector. This concentration was compared to the concentration of a similarly purified vector that was also titered by vector genome qPCR, to calculate corresponding titers for the stuffer vectors described in this example.
  • the vector expressing the toxic positive control artificial miRNA elicited a substantial rise in liver enzymes ALT and AST.
  • none of the vectors with the stuffer sequences elicited a rise in liver enzymes (FIGS.2A-2B).
  • a similar peak is present if a control sequence is used in the place of the amiRNA but containing secondary structure such as palindromic sequences.
  • FIGS.3E-3F compare two rAAV preparations (scAAV9_H1_MCS_PSG11_V1 (SEQ ID NO:27) and scAAV9_H1_MCS_PSG11_V2 (SEQ ID NO:28)) derived from cis plasmids containing one of two versions of stuffer sequence derived from PSG11 (PSG11_V1 (SEQ ID NO:45) and PSG11_V2 (SEQ ID NO:46)). These sequences were structurally similar, though ATG sites were edited to eliminate ATG sequences in PSG11_V2.
  • FIGS.3G-3H compare a similar set of two rAAV preparations (scAAV9_H1_miR-1-1-XD-14792_PSG11_V1 (SEQ ID NO:78) and scAAV9_H1_native_miR-1-1-XD-14792_PSG11_V2 (SEQ ID NO:79)) derived from cis plasmids containing either of stuffer sequence PSG11_V1 (SEQ ID NO:45) or PSG11_V2 (SEQ ID NO:46).
  • both vectors contain an amiRNA element downstream of an H1 promoter (there are 3 nucleotides inserted in the promoter sequence on the PSG11_V2-containing vector versus the V1 containing vector, but otherwise the 5’ sequences are identical).
  • fragment analyer trace FIG.3H
  • the replication of self-complementary AAV (scAAV) vectors such as those described here involves initiation of replication from the wild-type ITR. In the sequences described here, this is at the 3’ side in the cis plasmids.
  • PSG11_V1 sequence structure was favored.
  • a “PSG11_V5” stuffer sequence (SEQ ID NO:48) was generated by using the favorable sequence structure from the PSG11_V1 sequence. This is envisioned to yield vector preparations with reduced truncated vector genomes.
  • the _V5 stuffer incorporated edits to CG sequences and ATG sequences to reduce the potential for hypomethylated CpG dinucleotides or unintended open reading frames.
  • EXAMPLE 4 FURTHER EVALUATION OF SAFETY OF STUFFER SEUQENCES WHEN COMBINED WITH ATXN2 AMIRNA EXPRESSION CASSETTES
  • H1 short SEQ ID NO:54
  • H1 native SEQ ID NO:53
  • H1 long SEQ ID NO:52
  • two different ATXN2 targeting artificial miRNA 3330 or 14792 (also referred to herein as 1784)
  • miR1.1 miRNA
  • miR100_3330 encode SEQ ID NO:12
  • miR1.1_14792 encode SEQ ID NO:73
  • the presence or absence of a Pol-II transcriptional terminator SV40 polyadenylation sequence and different versions of the stuffer sequences (AMELY_V3 (SEQ ID NO:51), PSG11_V2 (SEQ ID NO:46), PSG11_V3 (SEQ ID NO:47), PSG11_V5 (SEQ ID NO:48))were assembled in various combinations (see, Table 1).
  • the H1 promoter is oriented from 5’ to 3’ in the cis plasmid intended for packaging as a self-complementary AAV vector, where the mutant ITR (lacking a terminal resolution site) is on the 5’ side (e.g., SEQ ID NO:57 or nucleotides 1-106 of any one of SEQ ID NOS:13-24, 29-44, and 78-82) and a wild-type AAV ITR is on the 3’ side (e.g., SEQ ID NO:58 or nucleotides 2214-2358 of any one of SEQ ID NOS:13-16).
  • the mutant ITR lacking a terminal resolution site
  • a wild-type AAV ITR is on the 3’ side (e.g., SEQ ID NO:58 or nucleotides 2214-2358 of any one of SEQ ID NOS:13-16).
  • the latter three stains can be used to evaluate astrocytic reactivity, microglial reactivity, and neuronal death, respectively.
  • liver sections from animals treated with i.v. administered vector there were no test article effects identified in the examined sections of brain. Handling artifacts limited interpretation in some tissues. In general, changes were observed at the injection site and included slight disruption of the tissue with light gliosis (identified on H&E-stained slides), slight microgliosis (identified on GFAP-labeled slides), and subtle hemosiderin pigment.
  • the hemosiderin pigment is a common change with intraparenchymal injections.
  • Test articles were not interpreted to cause an exacerbation or changes associated with the experimental procedures in any of the examined sections at the level of the basal nuclei/striatum from any animal.
  • Transcriptional Analysis was performed on striatal punch biopsies from animals dosed with the above vectors. Striatal tissue was column purified to extract RNA (after DNase-treatment) using Qiagen’s AllPrep DNA/RNA/Protein Mini Kit (Qiagen, P/N 80004).
  • Stranded RNA-seq libraries were prepared with the Stranded Total RNA Prep Ligation with Ribo-Zero Plus kit (Illumina, P/N 20040525) followed by paired end 2 x 100 bp sequencing on the Illumina Novaseq 6000 system. These reads were then aligned to the respective cis plasmid used to package the rAAV administered to that animal. Separately, DNA was purified from the same punch biopsy that had been used to extract the sequenced RNA (Qiagen, P/N 80004), and the vector exposure confirmed. Table 3 lists the exposures for each sample tested, along with the vector.
  • FIG.6 shows pileup of reads from representative vectors.
  • the use of the H1 short promoter and miR 1.1 backbone along with the PSG11 V5 stuffer sequence led to an increased number of reads aligning to the cis plasmid downstream of the expected transcription of the pri-miRNA.
  • the use of the H1 long promoter with the PSG11 V5 stuffer led to a beneficial minimal amount of unintended transcription.
  • Atxn2 levels were calculated as the mean of these ATXN2/control transcript ratios, and further normalized to the ratios obtained for animals dosed with vehicle.
  • Vector genome exposures were also measured, using ddPCR with probes against the vector genome and a mouse genome probe (Tert). As seen in Table 4, robust Atxn2 knockdown occurred in animals treated with all combinations of stuffer sequences and miRNA vector components.
  • Example 4 shows the unexpected benefit in reducing transcriptional activity downstream of the artificial miRNA sequence when the “H1 long” variant of the promoter was inserted upstream of the PSG11_V5 stuffer sequence versus the “H1 short” variant of the promoter.
  • Production yields of combinations of H1 long and H1 short promoters and the AMELY and PSG11 derived stuffer sequences were also assessed. Production yields were assessed by ddPCR of vector genomes using primer/probe sets specific to the stuffer regions.
  • cis plasmids with H1 long format promoter (SEQ ID NO:52) combinations with the stuffer sequences assessed, which included AMELY_V3 (SEQ ID NO:51), PSG11_V5 (SEQ ID NO:48), PSG11_V3 (SEQ ID NO:47), and PSG11_V2 (SEQ ID NO:46), produced higher production yields (FIG.7).
  • An analysis of vector truncations (FIGS.8A-8C) further suggested that there are fewer truncations when the H1 long version of the promoter was included upstream compared to the H1 short promoter.
  • EXAMPLE 7 PRODUCTION OF VECTORS INCORPORATING STUFFER SEQUENCES WITH ADDITIONAL COMBINATIONS OF ARTIFICIAL MIRNAS Stuffer sequences created according to Example 1 and containing combinations of promoter and specific stuffer sequences as described in Example 4 were assessed for their ability, as part of AAV vector genomes, to be packaged in rAAV. The ability to consistently achieve good productivity in the context of different payloads in the vector was also tested for these stuffer sequences.
  • ITR cis plasmid sequences are set forth in SEQ ID NO:13 (scAAV_H1_long_miR100_1755_PSG11_V5_ITR_to_ITR), SEQ ID NO:14 (scAAV_H1_long_miR100_2586_PSG11_V5_ITR_to_ITR), SEQ ID NO:15 (scAAV_H1_long_miR100_2945_PSG11_V5_ITR_to_ITR), and SEQ ID NO:16 (scAAV_H1_long_miR100_3330_PSG11_V5_ITR_to_ITR). Sequences for the amiRNA guide, passenger, and
  • Vector sequences from 5’ ITR to 3’ ITR that contain an ATXN2 targeting artificial miRNA cassette and an AMELY stuffer sequence are provided for “H1_short_miR16-2- 1755_AMELY_V1” (SEQ ID NO:17) and “H1 – miR1-1_1784_AMELY_V3“ (SEQ ID NO:18).
  • H1_short refers to a 91 bp truncated form of the H1 promoter (SEQ ID NO:54 or nucleotides 113-203 of SEQ ID NO:17). Yields from productions of these vectors are listed in Table 7.
  • vectors listed in Table 8 were evaluated for knockdown of ATXN2 mRNA in human stem-cell derived motor neurons.
  • –Vectors included scAAV_H1_long_miR100_1755_PSG11_V5_ITR_to_ITR.gb (SEQ ID NO:13), scAAV_H1_long_miR100_2586_PSG11_V5_ITR_to_ITR.gb (SEQ ID NO:14), scAAV_H1_long_miR100_2945_PSG11_V5_ITR_to_ITR.gb (SEQ ID NO:15), and scAAV_H1_long_miR100_3330_PSG11_V5_ITR_to_ITR.gb (SEQ ID NO:16).
  • Stem- cell derived motor neurons were treated with indicated vectors packaged in AAV-DJ, and 7 days after transduction, RNA harvested and assessed for knockdown using RT- ddPCR with probes against ATXN2 and housekeeping probes GUSB and B2M. Mean ATXN2 mRNA signal, as normalized against GUSB and B2M probes and against untransduced cells, was measured. Data is listed for cells treated with a dose of 3.16E4 vector genomes/cell. Table 8: ATXN2 Knockdown in amiRNA/AAV Vector Treated Human Stem-Cell Derived Motor Neurons _ _ . .
  • the vector including AMELY_V3 stuffer (SEQ ID NO:51 or nucleotides 488-2177 of SEQ ID NO:18) and an ATXN2 targeting amiRNA under the control of the H1 long promoter was tested by intravenous administration into adult male C57Bl/6 mice and knockdown of the target ATXN2 measured 3 weeks after dosing. Knockdown of Atxn2 was assessed by extracting RNA from liver tissue and performing RT-ddPCR with Atxn2 and the control probes Hprt and Gusb. Table 9 lists average percent knockdown in liver tissue assessed from 3 animals dosed with vector related to animals dosed with vehicle (PBS + 0.001% PF-68).

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Abstract

La présente divulgation concerne des polynucléotides de remplissage de vecteur et des compositions de ceux-ci, comprenant des constructions d'expression et des vecteurs, tels que des vecteurs viraux et des procédés d'administration d'un agent thérapeutique (par exemple, un acide nucléique inhibiteur) à un mammifère ou des méthodes de traitement d'une maladie.
EP22705501.9A 2021-02-05 2022-02-04 Vecteurs comprenant des séquences polynucléotidiques de remplissage Pending EP4288555A1 (fr)

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