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WO2024229211A2 - Système modulaire pour convertir des cassettes d'expression de microarn thérapeutique de promoteurs de polymérase iii à des promoteurs de polymérase ii - Google Patents

Système modulaire pour convertir des cassettes d'expression de microarn thérapeutique de promoteurs de polymérase iii à des promoteurs de polymérase ii Download PDF

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WO2024229211A2
WO2024229211A2 PCT/US2024/027396 US2024027396W WO2024229211A2 WO 2024229211 A2 WO2024229211 A2 WO 2024229211A2 US 2024027396 W US2024027396 W US 2024027396W WO 2024229211 A2 WO2024229211 A2 WO 2024229211A2
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promoter
nucleotide sequence
nucleic acid
enhancer
seq
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WO2024229211A3 (fr
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Scott Quenton HARPER
Matthew GUGGENBILLER
Noah Taylor
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Research Institute At Nationwide Children's Hospital
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    • C—CHEMISTRY; METALLURGY
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    • 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
    • C12N15/111—General methods applicable to biologically active non-coding nucleic acids
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • 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
    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/50—Physical structure
    • C12N2310/53—Physical structure partially self-complementary or closed
    • C12N2310/531—Stem-loop; Hairpin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12N2330/00—Production
    • C12N2330/50—Biochemical production, i.e. in a transformed host cell
    • C12N2330/51—Specially adapted vectors

Definitions

  • RNA interference RNA interference
  • RNAi-based systems rely upon first-generation expression strategies that employ ubiquitous RNA polymerase III (pol III)-driven promoters, such as U6 or H1, to drive shRNA or miRNA expression in vivo. These systems are advantageous because they produce primary miRNA transcripts from defined transcription start and termination sites, thereby enabling consistent processing of predictable mature products through endogenous miRNA biogenesis pathways.
  • pol III-based systems do not allow cell or tissue-specific expression.
  • tissue-specific promoters utilize RNA polymerase II (pol II) to drive transcription. These pol II-based systems often have multiple transcription start sites and require poly A signals and transcript poly-adenylation for termination.
  • converting a pol III-driven miRNA to a pol II-driven system can change the secondary structure of the primary miRNA transcript, thereby altering the maturation process by the RNAse enzymes Drosha and Dicer. This is important because even a single nucleotide change in a mature miRNA or shRNA sequence can impact specificity and efficiency.
  • converting an existing pol III- 28335/58942 2023-012-02 based miRNA expression system to one driven by RNA pol II, and still producing the same mature miRNA can be challenging.
  • Many first-generation miRNA or shRNA expression systems use RNA polymerase III-based promoters to drive expression (e.g. U6, H1, tRNA promoters).
  • RNA pol II-based promoters Other first generation systems have employed RNA pol II-based promoters, but these systems are less commonly used and tend to produce primary transcripts that are less predictably processed to mature miRNAs.
  • a system that allows conversion of a pol III-based system to a pol II-based system would allow researchers to leverage pre-clinical data already generated with first- generation RNA pol II-based promoter systems while also restricting expression of a miRNA to specific cell- or tissue-types.
  • This disclosure provides several novel miRNA designs which allow for the conversion from an existing U6 promoter-driven miRNA to a tissue-specific, pol II-based system.
  • Novel secondary structure elements were incorporated in the primary transcript regions flanking Drosha and Dicer processing sites, and efficacy and expression of the mature sequences in vitro were confirmed.
  • This system allows for rapid conversion of ubiquitous to tissue-specific miRNA expression systems without rederivation of lead RNAi triggers, while also providing a new approach to restrict expression of therapeutic miRNAs.
  • [0006] The development of a system and products for convert an existing pol III-based promoter-driven miRNA system to a tissue-specific, pol II-based promoter system while maintaining fidelity of processing and efficacy represents a critical unmet need in the field.
  • the disclosure provides a modular microRNA (miRNA) expression cassette for converting an RNA polymerase III-based promoter system to an RNA polymerase II-based promoter system.
  • miRNA microRNA
  • expression cassette or “expression construct” or “DNA construct” are used interchangeably when discussing the miRNA expression cassette because the expression cassette comprises DNA encoding the miRNA and the various components required for efficient tissue-specific expression of the miRNA for use with a polymerase type II (pol II) promoter.
  • the disclosure provides a nucleic acid encoding a microRNA (miRNA) expression cassette for converting an RNA polymerase III-based promoter system to an RNA polymerase II-based promoter system, wherein the cassette comprises a DNA nucleotide sequence encoding a single stranded stem of a microRNA; 28335/58942 2023-012-02 a 5’ double-stranded stem of a microRNA; a sense strand of a mature microRNA; a modified microRNA loop comprising a nucleotide change to facilitate folding; an antisense strand of a mature microRNA; a 3’ double-stranded stem of a microRNA; a single-stranded stem of a microRNA; and a polyA signal.
  • miRNA microRNA
  • the cassette comprises a DNA nucleotide sequence encoding the single stranded stem of a microRNA; the 5’ double-stranded stem of a microRNA; the sense strand of mature microRNA; the modified microRNA loop comprising a nucleotide change to facilitate folding; the antisense strand of mature microRNA; the 3’ double-stranded stem of a microRNA; the single-stranded stem of a microRNA; and the polyA signal in a 5’ to 3’ order, respectively, in the expression cassette.
  • the cassette comprises a DNA nucleotide sequence encoding a single stranded stem of mir30; a 5’ double-stranded stem of mir30; the sense strand of a mature microRNA; a modified mir30 loop comprising a nucleotide change to facilitate folding; the antisense strand of a mature microRNA; a 3’ double-stranded stem of mir30; a single-stranded stem of mir30; and the polyA signal.
  • the cassette comprises the DNA nucleotide sequence encoding the single stranded stem of mir30; the 5’ double-stranded stem of mir30; the sense strand of a mature microRNA; the modified mir30 loop comprising a nucleotide change to facilitate folding; the antisense strand of a mature microRNA; the 3’ double-stranded stem of mir30; the single-stranded stem of mir30; and the polyA signal in a 5’ to 3’ order, respectively, in the expression cassette.
  • the single-stranded stem of mir30 comprises the nucleotide sequence of any one of SEQ ID NOs: 20-24, or a nucleotide sequence comprising at least or about 90% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 20-24
  • the 5’ double-stranded stem of mir30 comprises the nucleotide sequence of SEQ ID NO: 25, or a nucleotide sequence comprising at least or about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 25.
  • the sense strand of mature miRNA is the sense strand of any mature miRNA.
  • the sense strand of mature miRNA comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence comprising at least or about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 17 or 18.
  • the modified mir30 loop comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 26.
  • the antisense strand of mature miRNA is the antisense strand of any mature miRNA.
  • the antisense strand of mature miRNA comprises the nucleotide sequence of SEQ ID NO: 27 or 28, or a nucleotide sequence comprising at least or about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 27 or 28.
  • the 3’ double-stranded stem of mir30 comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence comprising at least or about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 29.
  • the mutated single stranded stem of mir30 comprises the nucleotide sequence of SEQ ID NO: 30, or a nucleotide sequence comprising at least or about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 30.
  • the polyadenylation (polyA) signal is wild type neuropilin-1 (WT NRP1) polyA signal.
  • WT NRP1 polyA signal comprises the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence comprising at least or about 90% sequence identity to the nucleotide sequence of SEQ ID NO: 32.
  • the cassette further comprises restriction sites for cloning, and wherein the cassette comprises a DNA nucleotide sequence encoding 28335/58942 2023-012-02 a first restriction enzyme site; a single stranded stem of a microRNA or mir30; a 5’ double-stranded stem of a microRNA or mir30; a sense strand of a mature microRNA; a modified microRNA loop or mir30 loop comprising a nucleotide change to facilitate folding; an antisense strand of a mature microRNA; a 3’ double-stranded stem of a microRNA or mir30; a single-stranded stem of a microRNA or mir30; a second restriction enzyme site; a polyA signal; and a third enzyme restriction site.
  • the first restriction enzyme site is an AgeI site. In some aspects, the AgeI site comprises the nucleotide sequence of SEQ ID NO: 19. [0025] in some aspects, the second restriction enzyme site is an AflII site. In some aspects, the AflII site comprises the nucleotide sequence of SEQ ID NO: 31. [0026] In some aspects, the third restriction site is an EcoR1 site. In some aspects, the EcoR1 site comprises the nucleotide sequence of SEQ ID NO: 33. [0027] In some aspects, the third restriction site is an MfeI site. In some aspects, the MfeI site comprises the nucleotide sequence of SEQ ID NO: 34.
  • a nucleic acid of the disclosure comprises a nucleotide sequence encoding the sequence of SEQ ID NO: 35 (ACCGGN 1 N 2 N 3 N 4 N 5 N 6 N 7 GCUN 8 N 9 N 10 N 11 ACAGUGAGCGAN 12 NNNNNNNNNNNNN NNNNNGUAAAGCCACAGAUGGGNNNNNNNNNNNNNNNNNNNNNNUGCUACUGCN 13 N 1 4 UUAN 15 N 16 N 17 N 18 N 19 N 20 AAUAAAAUACGAAAUN 21 N 22 N 23 N 24 CN 25 N 26 AAUUN 27 ), or a nucleotide sequence comprising at least or about 80% sequence identity to a nucleotide sequence encoding the nucleotide sequence of SEQ ID NO: 35, wherein N 1 at position 6 is U or is omitted; wherein N 2 at position 7 is C, A, or U; wherein N 3 at position 8 is C or is omitted; 28335/58942 2023-012-02
  • a nucleic acid of the disclosure comprises a nucleotide sequence encoding an RNA sequence comprising at least or about 80% sequence identity to the nucleotide sequence of any one of SEQ ID NOs: 1-16 or 35-53; or a nucleotide sequence that encodes the RNA sequence comprising the nucleotide sequence of any one of SEQ ID NOs: 1-16 or 35-53. 28335/58942 2023-012-02 [0030]
  • a nucleic acid of the disclosure further comprises a promoter and/or enhancer.
  • the promoter is any polymerase type II (pol II) promoter.
  • the promoter and/or enhancer is any of a U7 promoter and/or enhancer, an RSV promoter and/or enhancer, a human skeletal a-actin (HSA) promoter and/or enhancer, a desmin promoter and/or enhancer, a CMV promoter and/or enhancer, a minimal CMV promoter and/or enhancer, a T7 promoter and/or enhancer, an EF1-alpha promoter and/or enhancer, a minimal EF1-alpha promoter and/or enhancer, an unc45b promoter and/or enhancer, a myosin heavy chain kinase (MHCK) promoter, a muscle creatine kinase (MCK) promoter, a tMCK promoter and/or enhancer, a dMCK promoter and/or enhancer, a CK1 promoter and/or enhancer, a CK6 promoter and/or enhancer, a CK7 promoter and/or
  • the promoter and/or enhancer is a CMV promoter and/or enhancer, is an HSA promoter and/or enhancer, is an MPZ promoter and/or enhancer, or is an MCK promoter and/or enhancer.
  • the synthetic promoter and/or enhancer is an SPc5-12 promoter and/or enhancer, an SP-301 promoter and/or enhancer, an MH promoter and/or enhancer, or a Sk-CRM4/DES promoter and/or enhancer.
  • the neuronal-specific promoter and/or enhancer is a synapsin promoter and/or enhancer.
  • the vector is an adeno-associated virus (AAV) vector. In some aspects, the AAV lacks rep and/or cap genes. In some aspects, the vector is a recombinant AAV (rAAV) vector. In some aspects, the vector is a self-complementary recombinant AAV (scAAV) vector or a single-stranded recombinant AAV (ssAAV) vector.
  • AAV adeno-associated virus
  • rAAV recombinant AAV
  • scAAV self-complementary recombinant AAV
  • ssAAV single-stranded recombinant AAV
  • the AAV serotype is AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-anc80, AAV-B1, AAV-BR1, AAV.PHP.EB, AAVv66, AAV2/1, AAV2/8, or AAV2/9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101, or any derivative thereof.
  • the AAV serotype is AAV9, mAAV9, or AAV-SLB101.
  • the disclosure further provides a nanoparticle, extracellular vesicle, or exosome comprising a nucleic acid of the disclosure.
  • the disclosure provides a composition comprising (a) a nucleic acid of the disclosure; 28335/58942 2023-012-02 (b) a vector of the disclosure; or (c) a nanoparticle, extracellular vesicle, or exosome of the disclosure; and a pharmaceutically acceptable carrier.
  • the disclosure further provides a method of reducing, inhibiting, and/or interfering with expression of a gene in a cell comprising contacting the cell with (a) a nucleic acid of the disclosure; (b) a vector of the disclosure; (c) a nanoparticle, extracellular vesicle, or exosome of the disclosure; and/or (d) a composition of the disclosure.
  • the gene is any gene whose expression in a cell is associated with a pathological disease or condition.
  • the gene is PMP22 or DUX4.
  • the disclosure further provides a method of treating or ameliorating a subject suffering from or at risk of suffering from a disease associated with DUX4 expression or PMP22 expression comprising administering to the subject an effective amount of (a) a nucleic acid of the disclosure; (b) a vector of the disclosure; (c) a nanoparticle, extracellular vesicle, or exosome of the disclosure; and/or (d) a composition of the disclosure.
  • the disease associated with DUX4 expression is facioscapulohumeral muscular dystrophy (FSHD) or cancer.
  • the disease associated with PMP22 expression is Charcot-Marie-Tooth disease type 1A (CMT1A).
  • the disclosure provides a use of (a) a nucleic acid of the disclosure; (b) a vector of the disclosure; (c) a nanoparticle, extracellular vesicle, or exosome of the disclosure; and/or (d) a composition of the disclosure for the preparation of a medicament for reducing or inhibiting expression of DUX4 or PMP22 in a cell.
  • the disclosure provides a use of (a) a nucleic acid of the disclosure; (b) a vector of the disclosure; (c) a nanoparticle, extracellular vesicle, or exosome of the disclosure; and/or (d) a composition of the disclosure for treating or ameliorating facioscapulohumeral muscular dystrophy (FSHD), cancer, or Charcot-Marie-Tooth disease type 1A (CMT1A).
  • FSHD facioscapulohumeral muscular dystrophy
  • CMT1A Charcot-Marie-Tooth disease type 1A
  • the disclosure provides a use of (a) a nucleic acid of the disclosure; (b) a vector of the disclosure; (c) a nanoparticle, extracellular vesicle, or exosome of the disclosure; and/or (d) a composition of the disclosure for the preparation of a medicament for treating or ameliorating FSHD, cancer, or CMT1A.
  • the disclosure provides a use of (a) a nucleic acid of the disclosure; (b) a vector of the disclosure; (c) a nanoparticle, extracellular vesicle, or exosome of the disclosure and/or (d) a composition of the disclosure for reducing, inhibiting, and/or interfering with expression of a gene in a cell.
  • the gene is any gene whose expression in a cell is associated with a pathological disease or condition.
  • the gene is PMP22 or DUX4.
  • the cell is in a subject.
  • the subject is a human subject.
  • the disclosure provides a (a) nucleic acid of the disclosure;; (b) vector of any one of the disclosure; (c) nanoparticle, extracellular vesicle, or exosome of the disclosure; (d) composition of the disclosure; (e) method of the disclosure; or 28335/58942 2023-012-02 (f) use of the disclosure, wherein the nucleic acid, vector, nanoparticle, extracellular vesicle, exosome, composition, or medicament is formulated for oral administration, subcutaneous administration or injection, intradermal administration or injection, intraventricular delivery or injection, intracerebroventricular delivery or injection, intrathecal delivery or injection, transdermal delivery or injection, injection into the blood stream, or aerosol administration.
  • Fig.1 shows the structure of a pol II miRNA expression cassette of the disclosure (i.e., P2.mi871-4).
  • the terminal 5’ end of the nucleic acid construct contains ACCGGT AgeI site (encoding an ACCGGU).
  • the terminal 3’ end contains an EcoRI site (GAATTC, shown here, (encoding a GAAUUC)) in structures 1-4 and an MfeI site in structures 5-8 (CAATTG (encoding a CAAUUG)).
  • the 3’ end of Stem 1 contains an AflII site (CTTAAG (encoding a CUUAAG)).
  • Fig.2 shows the U6.mi871 RNA sequence and predicted structure.
  • Fig.3 shows the Pol II promoter mi871.4 RNA sequence and predicted structure.
  • Fig.4 shows the high levels of off-target expression of mi405 and mi871 in the heart, liver, and kidneys with both Intravenous (IV) and Intrathecal (IT) routes of administration under the control of a U6, pol III, promoter.
  • Fig.5 shows two different muscle-specific or Schwann cell-specific promoters to drive expression of either mi405 or mi871, respectively.
  • Fig.6 shows the structures of eight novel miRNA designs to convert an existing U6 promoter-driven miRNA to a tissue-specific, pol II-based system.
  • Fig.7 shows the structure and sequence (SEQ ID NO: 9) of the mi405-1 construct.
  • Fig.8 shows the structure and sequence (SEQ ID NO: 10) of the mi405-2 construct.
  • Fig.9 shows the structure and sequence (SEQ ID NO: 11) of the mi405-3 construct.
  • Fig.10 shows the structure and sequence (SEQ ID NO: 12) of the mi405-4 construct.
  • Fig.11 shows the structure and sequence (SEQ ID NO: 13) of the mi405-5 construct.
  • Fig.12 shows the structure and sequence (SEQ ID NO: 14) of the mi405-6 construct.
  • Fig.13 shows the structure and sequence (SEQ ID NO: 15) of the mi405-7 construct.
  • Fig.14 shows the structure and sequence (SEQ ID NO: 16) of the mi405-8 construct.
  • Fig.15 shows the structure and sequence (SEQ ID NO: 1) of the mi871-1 construct.
  • Fig.16 shows the structure and sequence (SEQ ID NO: 2) of the mi871-2 construct.
  • Fig.17 shows the structure and sequence (SEQ ID NO: 3) of the mi871-3 construct.
  • Fig.18 shows the structure and sequence (SEQ ID NO: 4) of the mi871-4 construct.
  • Fig.19 shows the structure and sequence (SEQ ID NO: 5) of the mi871-5 construct.
  • Fig.20 shows the structure and sequence (SEQ ID NO: 6) of the mi871-6 construct.
  • Fig.21 shows the structure and sequence (SEQ ID NO: 7) of the mi871-7 28335/58942 2023-012-02 construct.
  • Fig.22 shows the structure and sequence (SEQ ID NO: 8) of the mi871-8 construct.
  • Fig.23 provides a detailed description of the eight novel miPMP22-871 expression construct sequences of the disclosure.
  • Fig.24 provides a detailed description of the eight novel mi405 expression construct sequences of the disclosure. Subparts of each of the sequences are identified.
  • Fig.25 provides a clustal alignment of the 16 novel sequences of the miRNA expression cassettes of the disclosure showing the similarities between the sequences and a consensus sequence (SEQ ID NO: 35).
  • Fig.26 provides a clustal alignment the miRNA expression cassettes comprising the nucleotide sequences of SEQ ID NOs: 1-8 showing the similarities between the sequences and a consensus sequence (SEQ ID NO: 36).
  • Fig.27 provides a clustal alignment the miRNA expression cassettes comprising the nucleotide sequences of SEQ ID NOs: 9-16 showing the similarities between the sequences and a consensus sequence (SEQ ID NO: 37).
  • Fig.28 shows that converting a pol III-driven miRNA to a pol II-driven system can change the secondary structure of the primary miRNA transcript, thereby altering the maturation process by the RNAse enzymes Drosha and Dicer.
  • Fig.29 shows that pol II-driven miRNAs can be efficiently expressed in vitro, and measured by a droplet digital PCR (ddPCR) assay targeting the mature miRNA sequence.
  • ddPCR droplet digital PCR
  • Fig.30 shows that knockdown of gene targets was measured using a dual- luciferase assay where either DUX4 or Human PMP22 was cloned into the 3’ UTR of the Renilla Luciferase gene within the Psicheck2 vector.
  • Fig.31 shows that the eight constructs designed to knockdown human PMP22 were effective in knockdown with the CMV promoter.
  • Fig.32 shows that the eight constructs designed to knockdown human 405 were effective in knockdown with the CMV promoter.
  • Fig.33 shows two different muscle-specific pol II promoters (CK6 and HSA) and two different Schwann cell-specific pol II promoters (rat MPZ and human MPZ) that were used to drive expression of mi405 or mi871. 28335/58942 2023-012-02 [0077]
  • Fig.34 shows that human and rat pol II MPZ promoters were each able to drive expression of mi871 in a rat Schwann cell culture, leading to the reduction of endogenous rat PMP22.
  • Fig.34 also shows that expression of mi871 in rat Schwann Cells was able to reduce relative luciferase assay when co-transfected with Psicheck.HuPMP22 construct.
  • HSA Human skeletal actin
  • CK6 creatine kinase 6
  • the disclosure provides a novel system for the accurate and rapid conversion of an ubiquitous RNA polymerase III-driven microRNA (miRNA) expression system to an ubiquitous or tissue-specific miRNA expression system relying upon RNA polymerase II.
  • miRNA ubiquitous RNA polymerase III-driven microRNA
  • RNA interference is a mechanism of gene regulation in eukaryotic cells that has been considered for the treatment of various diseases. RNAi is a gene silencing mechanism that is mediated by small RNAs. Effective and stable gene knockdown can be achieved by the expression of engineered, artificial miRNAs (also called miRNA shuttles) or short hairpin RNAs (shRNAs), both of which are processed into small interfering RNAs (siRNAs).
  • miRNAs also called miRNA shuttles
  • shRNAs short hairpin RNAs
  • miRNA and shRNA expression cassettes have been designed to mimic natural miRNA genes and to deliver therapeutic miRNAs.
  • the major distinction between engineered miRNAs and shRNAs is the amount of processing required within the cell to yield a mature, siRNA-like product.
  • miRNAs are processed by the enzymes Drosha, Exportin-5 and Dicer, while shRNAs are only processed by Exportin-5 and Dicer.
  • MiRNAs or shRNAs driven by RNA polymerase II or RNA polymerase III promoters, can induce efficient, stable, and regulated silencing in cultured cells as well as in animal models. The expression of such shRNAs is dependent upon the presence of miRNA biogenesis factors.
  • RNAi refers to post-transcriptional control of gene expression mediated by microRNAs (miRNAs).
  • miRNAs are small (21-25 nucleotides), noncoding RNAs that share sequence homology and base-pair with 3' untranslated regions of cognate messenger RNAs (mRNAs).
  • mRNAs messenger RNAs
  • RNAi pathway is summarized in Duan (Ed.), Section 7.3 of Chapter 7 in Muscle Gene Therapy, Springer Science + Business Media, LLC (2010).
  • MiRNAs are small endogenous single-stranded noncoding RNA molecules able to modulate gene expression at the post-transcriptional level. In their mature form, they bind target mRNAs by base pairing their seed sequence to a region located in the target 3′ untranslated region (3′-UTR). This binding leads to repression of gene expression by inhibiting mRNA translation and/or promoting its degradation.
  • Artificial miRNAs can be transcribed from DNA expression cassettes, as disclosed herein.
  • MiRNA biogenesis starts with transcription of a long primary transcript, called a pri- miRNA, that, in the canonical pathway, is processed in the nucleus by Drosha and DiGeorge syndrome critical region gene 8 (DCGR8) enzymes (forming the microprocessor complex) and converted into a shorter transcript, called pre-miRNA, after a stem-loop cropping.
  • DCGR8 DiGeorge syndrome critical region gene 8
  • pre-miRNA a short transcript
  • the pre-miRNA transcript is further processed by the endonuclease Dicer, which generates a small RNA duplex intermediate (about 22 nucleotides).
  • RISC RNA-induced silencing complex
  • seed sequence typically spanning nucleotides 2–7 at the 5′-end of the microRNA sequence.
  • miR-30 microRNA precursor is a small non-coding RNA that regulates gene expression.
  • Animal microRNAs are transcribed as pri-miRNA (primary miRNA) of varying length which in turns are processed in the nucleus by Drosha into ⁇ 70 nucleotide stem-loop precursor called pre-miRNA (precursor miRNA) and subsequently processed by the Dicer enzyme to give a mature ⁇ 22 nucleotide product.
  • pre-miRNA precursor miRNA
  • Dicer enzyme Dicer enzyme to give a mature ⁇ 22 nucleotide product.
  • the mature sequence comes from both the 3' (miR-30)and 5' (mir-97-6) arms of the precursor.
  • Converting a pol III-driven miRNA to a pol II-driven system can change the secondary structure of the primary miRNA transcript, thereby altering the maturation process by the RNAse enzymes Drosha and Dicer.
  • various features of the system need to be converted to accommodate the use of a pol III promoter.
  • a U6 pol III promoter is used with an RNA pol III termination signal whereas a tissue-specific pol II promoter, like CMV, is used with an RNA pol III poly A signal (Fig.5).
  • MiRNA expression cassettes were designed to accurately position the RNAse III enzyme Drosha at a junction between Stem 2 and Stem 1, where Stem 1 is designed to contain as much single-stranded structure as possible.
  • the Drosha/DGCR8 complex (called the microprocessor) is positioned at the junction of single and double-stranded RNA structures (Han et al., Cell 125(5): P887-901, 2006).
  • FIG.6 shows a comparative drawing of the structures of these eight constructs with incorporation of a DNA sequence encoding mi405 (a DUX4-specific miRNA), an exemplary miRNA of interest.
  • the structures of the new mi405 constructs are shown in Figs.7-14, and the structures of the new mi871 constructs are shown in Figs.15-22.
  • RNA nucleotide sequences encoded by the new miPMP22-871 constructs are shown in Fig.23, and the RNA nucleotide sequences encoded by the new miDUX4- 405 constructs (SEQ ID NOs: 9-16) are shown in Fig.24.
  • the disclosed DNA expression cassette(s) is designed to be used to express any miRNA of interest, and is not limited to an expression cassette(s) comprising the exemplary miRNAs used herein to test the efficacy of the disclosed expression cassette(s).
  • miRNAs listed in the microRNA database known as miRBase (https_colon_forward slash_forward slash_www.mirbase.org.
  • the disclosure includes the use of any of these microRNAs in the modular system, i.e., a DNA expression cassette, designed to convert therapeutic miRNA expression cassettes from the use of ubiquitous RNA polymerase III-based promoters to RNA polymerase II-based promoters for tissue specific expression of the miRNAs while maintaining fidelity and efficacy of processing as described herein.
  • Clustal alignment of the constructs was carried out (see Figs.25-27) to show sequence similarity and to derive consensus sequences of the novel constructs (SEQ ID NOs: 35-53) such that these constructs may be used with an miRNA of interest.
  • Each of the miRNA expression cassettes for converting an RNA polymerase III- based promoter system to an RNA polymerase II-based promoter system is a nucleic acid comprising a DNA nucleotide sequence encoding a single stranded stem of mir30; a 5’ double-stranded stem of mir30; a sense strand of a mature microRNA; a modified mir30 loop comprising a nucleotide change to facilitate folding; an antisense strand of mature microRNA; a 3’ double-stranded stem of mir30; a single-stranded stem of mir30; and a polyadenylation (polyA) signal.
  • polyA polyadenylation
  • each of the miRNA expression cassettes for converting an RNA polymerase III-based promoter system to an RNA polymerase II-based promoter system is a nucleic acid comprising in a 5’ to 3’ order a DNA nucleotide sequence encoding a single stranded stem of mir30; a 5’ double-stranded stem of mir30; a sense strand of a mature microRNA; a modified mir30 loop comprising a nucleotide change to facilitate folding; an 28335/58942 2023-012-02 antisense strand of mature microRNA; a 3’ double-stranded stem of mir30; a single-stranded stem of mir30; and a polyadenylation (polyA) signal.
  • polyA polyadenylation
  • This pol II-driven miRNA expression cassette was designed to be modular, thereby allowing for its use with (1) any desired RNA polymerase II-based promoter, and (2) any mature sense and antisense strands of microRNA sequence of interest (not only limited to exemplary miRNA sequences disclosed herein.
  • expression cassettes of the disclosure are chemically synthesized.
  • an expression cassette of the disclosure also may comprise restriction enzyme sites for cloning.
  • the expression cassette is a nucleic acid comprising in a 5’ to 3’ order a DNA nucleotide sequence encoding a first restriction enzyme site; a single stranded stem of mir30; a 5’ double-stranded stem of mir30; a sense strand of a mature microRNA; a modified mir30 loop comprising a nucleotide change to facilitate folding; an antisense strand of mature microRNA; a 3’ double-stranded stem of mir30; a single-stranded stem of mir30; a second restriction enzyme site; a polyadenylation (polyA) signal; and a third enzyme restriction site.
  • polyA polyadenylation
  • a pol II-driven miRNA expression cassette of the disclosure was designed to be modular, thereby allowing for the cutting and pasting in of (1) any desired RNA polymerase II-based promoter at the indicated AgeI site and (2) any mature sense and antisense strands of microRNA sequence of interest (not only limited to exemplary miRNA sequences disclosed herein) in between the AgeI and Afl II sites in the expression cassette.
  • a modular expression cassette as described herein is designed to be used with any microRNA known in the art.
  • the expression cassette of the disclosure not only allows for the cutting and pasting in of (1) any desired RNA polymerase II-based promoter at the indicated AgeI site but also allows for the cutting and pasting in of (2) any mature sense and antisense strands of microRNA sequence of interest.
  • a nucleotide sequence encoding any mature sense and antisense strands of miRNA of interest may simply be swapped into the expression cassette of the disclosure.
  • exemplary microRNA of miDUX4-450 and miPMP22-871 have been described and have been used in the examples provided herein, the modular expression cassette of the disclosure is designed and intended to be used with any microRNA of interest and any pol II-based promoter that is relevant for the particular miRNA.
  • an expression cassette construct as disclosed herein may be chemically synthesized. Therefore, there is no requirement for restriction enzyme sites to be present in the construct.
  • the pol II-driven expression cassette is not restricted only to the use of AgeI and AflII restriction sites.
  • the AflI site was removed to facilitate unfolding of stem 1 into 5’ and 3’ single-stranded regions.
  • microRNAs can be designed to include the Nrp I poly A signal and cloned into AgeI and EcoRI sites.
  • the first restriction enzyme site is an AgeI site.
  • the AgeI site comprises a nucleotide sequence encoding ACCGGU (SEQ ID NO: 19).
  • the single-stranded stem of mir30 comprises a nucleotide sequence encoding GGUUGCUGUUGA (SEQ ID NO: 20), GGUUUGCUGAGGA (SEQ ID NO: 21), GGUUUGCUCCUUA (SEQ ID NO: 22), GGUAAGCUCCUUA (SEQ ID NO: 23), or GGUCCCCAAGCUCCUUA (SEQ ID NO: 24).
  • the 5’ double-stranded stem of mir30 comprises a nucleotide sequence encoding CAGUGAGCGAN (SEQ ID NO: 25), wherein N is A, U, C, or G. In the miPMP22-871 constructs, the N is G. In the miDUX4-405 constructs, the N is U).
  • the sense strand of mature miRNA and the antisense strand of mature miRNA are strands of mature miRNA of any gene of interest. Thus, this is the purpose of the design of the disclosed modular system, i.e., to allow one to simply insert the sequences encoding any miRNA into the construct for pol II tissue-specific expression of the miRNA.
  • the disclosure includes the use of 28335/58942 2023-012-02 any of these microRNAs in the modular system, i.e., a DNA expression cassette, designed to convert therapeutic miRNA expression cassettes from the use of ubiquitous RNA polymerase III-based promoters to RNA polymerase II-based promoters for tissue specific expression of the miRNAs while maintaining fidelity and efficacy of processing as described herein.
  • a DNA expression cassette designed to convert therapeutic miRNA expression cassettes from the use of ubiquitous RNA polymerase III-based promoters to RNA polymerase II-based promoters for tissue specific expression of the miRNAs while maintaining fidelity and efficacy of processing as described herein.
  • the sense strand of mature miRNA comprises a nucleotide sequence encoding miPMP22-871 (i.e., GGGUUGCUGUUGAUUGAAGACU (SEQ ID NO: 17) or miDUX4-405 (i.e., CCAGGAUUCAGAUCUGGUUUCU (SEQ ID NO: 18).
  • the modified mir30 loop comprises a nucleotide sequence encoding GUAAAGCCACAGAUGGG (SEQ ID NO: 26).
  • the antisense strand of mature miRNA are strands of mature miRNA of any gene of interest.
  • the antisense strand of mature miRNA comprises a nucleotide sequence encoding miPMP22-871 (i.e., UCUUCAAUCAACAGCAAUCCCC (SEQ ID NO: 27) or miDUX4-405 (i.e., AAACCAGAUCUGAAUCCUGGAC (SEQ ID NO: 28).
  • the 3’ ds stem of mir30 comprises a nucleotide sequence encoding UGCCUACUG (SEQ ID NO: 29).
  • the ss stem of mir30 (mutated) comprises a nucleotide sequence encoding CCUUUUACUU (SEQ ID NO: 30).
  • the second restriction enzyme site is an AflII site.
  • the AflII site comprises a nucleotide sequence encoding CUUAAG (SEQ ID NO: 31).
  • the polyA signal is wild type neuropilin-1 polyadenylation (WT NRP1 polyA) signal.
  • WT NRP1 poly A signal comprises a nucleotide sequence encoding AAUAAAAUACGAAAUGUGACAGA (SEQ ID NO: 32).
  • the third restriction site is an EcoR1 site.
  • the EcoR1 site comprises a nucleotide sequence encoding GAAUUC (SEQ ID NO: 33).
  • the third restriction site is an MfeI site.
  • the MfeI site comprises a nucleotide sequence encoding AAUUG (SEQ ID NO: 34). 28335/58942 2023-012-02 [00111]
  • the disclosure provides eight exemplary structures designed for the substitution of pol II promoters with pol II promoters, and each of the eight structures was tested with two exemplary miRNAs, miPMP22-871, or mi-871 (GGGUUGCUGUUGAUUGAAGACU (SEQ ID NO: 17), and miDUX4-405, or mi-405 (CCAGGAUUCAGAUCUGGUUUCU (SEQ ID NO: 18). See, for example, WO 2022/119826 and U.S.
  • RNAscope in situ hybridization-based method for detecting DUX4 expression in vitro RNA Sep;25(19):1211- 1217; and Saad et al., (2021) Human microRNA mir-675 inhibits DUX4 expression and may be exploited as a potential treatment for FSHD. Nature Communications. Dec 8;12(1):7128 [00112]
  • Mi871 is used to induce silencing of overexpressed peripheral myelin protein-22 (PMP22) and constructs of the disclosure are designed to do the same.
  • Mi405 is used to induce silencing of DUX4 protein and constructs of the disclosure are designed to do the same.
  • Exemplary embodiments of the system were designed to contain a small poly- adenylation signal derived from the human neuropilin-1 (Nrp1) gene or a modified human neuropilin-1 poly adenylation signal in which the 7 indicated nucleotides (5’-GTGACAG-3’ (SEQ ID NO: 54) , DNA; 5’ GUGACAG (SEQ ID NO: 55), RNA) were mutated to C nucleotides to improve the potential single-strandedness of Stem 1.
  • Exemplary embodiments of the system were designed so that the Stem 1 region of the cassette comprises nucleotide sequences with 5’ and 3’ ends ranging from about 15 to about 46 nucleotides in length.
  • the Stem 1 region may comprise nucleotide sequences less than or exceed these indicated lengths.
  • Exemplary embodiments of the 5’ end of the Stem 1 region comprise base pairs, including G:U RNA wobble base-pairs, ranging from about 0-50% base pairing.
  • the base pairing is about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50%.
  • the Stem 1 region also comprises base-pairing that exceeds about 50% base pairing.
  • Exemplary embodiments of the 3’ end of the Stem 1 region comprise base pairs, including G:U RNA wobble base-pairs, ranging from about 0-30% base pairing.
  • the base pairing is about 5%, about 10%, about 15%, about 20%, about 25%, or about 30%.
  • the Stem 1 region also comprises base-pairing that exceeds about 30% base-pairing, and in some aspects, may exceed about 50% base pairing.
  • Exemplary embodiments of the microRNA (miRNA) region of the cassette comprises miRNA nucleotide sequences ranging from about 100 to about 200 nucleotides, but the miRNA encoding region may comprise sequences less than or greater than the indicated lengths.
  • the miRNA nucleotide sequences ranges in length from about 120 to about 180 nucleotides.
  • the miRNA region ranges in length from about 136-167 nucleotides in length (see Table 1).
  • the cassettes were designed to contain either natural or artificial microRNAs.
  • the disclosure provides nucleic acids comprising nucleotide sequences encoding a microRNA (miRNA) expression cassette for converting an RNA polymerase III-based promoter system to an RNA polymerase II-based promoter system.
  • the cassette comprises a DNA nucleotide sequence encoding a single stranded stem of mir30; a 5’ double-stranded stem of mir30; a sense strand of mature microRNA; a modified mir30 loop comprising a nucleotide change to facilitate folding; an antisense strand of mature microRNA; a 3’ double-stranded stem of mir30; a single-stranded stem of mir30; and a polyA signal.
  • the single-stranded stem of mir30 comprises the nucleotide sequence of any one of SEQ ID NOs: 20-24, or a nucleotide sequence comprising at least or about 90% identity to any one of SEQ ID NOs: 20-24.
  • the 5’ double-stranded stem of mir30 comprises the nucleotide sequence of SEQ ID NO: 25, or a nucleotide sequence comprising at least or about 90% identity to SEQ ID NO: 25.
  • the sense strand of mature miRNA is the sense strand of any mature miRNA.
  • the sense strand of mature miRNA comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 17 or 18. 28335/58942 2023-012-02 [00123]
  • the modified mir30 loop comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 26.
  • the antisense strand of mature miRNA is the antisense strand of any mature miRNA.
  • the antisense strand of mature miRNA comprises the nucleotide sequence of SEQ ID NO: 27 or 28, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 27 or 28.
  • the 3’ double-stranded stem of mir30 comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 29.
  • the mutated single stranded stem of mir30 comprises the nucleotide sequence of SEQ ID NO: 30, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 30.
  • the polyadenylation (polyA) signal is wild type neuropilin-1 polyA signal.
  • the WT NRP1 polyA signal comprises the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 32.
  • the cassette comprises each of the above-recited components in a 5’ to 3’ order.
  • the disclosure provides in a 5’ to 3’ order a single stranded stem of mir30; a 5’ double-stranded stem of mir30; a sense strand of mature microRNA; a modified mir30 loop comprising a nucleotide change to facilitate folding; an antisense strand of mature microRNA; a 3’ double-stranded stem of mir30; a single-stranded stem of mir30; and a polyA signal.
  • the disclosure provides nucleic acids comprising nucleotide sequences encoding a microRNA (miRNA) expression cassette for converting an RNA polymerase III-based promoter system to an RNA polymerase II-based promoter system.
  • miRNA microRNA
  • the cassette comprises a DNA nucleotide sequence encoding a first restriction enzyme site; a single stranded stem of mir30; a 5’ double-stranded stem of mir30; a sense strand of mature microRNA; a modified mir30 loop comprising a nucleotide change to facilitate folding; an antisense strand of mature microRNA; a 3’ double-stranded stem of mir30; a single-stranded stem of mir30; a second restriction enzyme site; a polyA signal; and a third enzyme restriction site.
  • the first restriction enzyme site is an AgeI site.
  • the AgeI site comprises the nucleotide sequence of SEQ ID NO: 19.
  • the single-stranded stem of mir30 comprises the nucleotide sequence of any one of SEQ ID NOs: 20-24, or a nucleotide sequence comprising at least or about 90% identity to any one of SEQ ID NOs: 20-24.
  • the 5’ double-stranded stem of mir30 comprises the nucleotide sequence of SEQ ID NO: 25, or a nucleotide sequence comprising at least or about 90% identity to SEQ ID NO: 25.
  • the sense strand of mature miRNA is the sense strand of any mature miRNA.
  • the sense strand of mature miRNA comprises the nucleotide sequence of SEQ ID NO: 17 or 18, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 17 or 18. 28335/58942 2023-012-02 [00137]
  • the modified mir30 loop comprises the nucleotide sequence of SEQ ID NO: 26, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 26.
  • the antisense strand of mature miRNA is the antisense strand of any mature miRNA.
  • the antisense strand of mature miRNA comprises the nucleotide sequence of SEQ ID NO: 27 or 28, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 27 or 28.
  • the 3’ double-stranded stem of mir30 comprises the nucleotide sequence of SEQ ID NO: 29, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 29.
  • the mutated single stranded stem of mir30 comprises the nucleotide sequence of SEQ ID NO: 30, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 30.
  • the second restriction enzyme site is an AflII site.
  • the AflII site comprises the nucleotide sequence of SEQ ID NO: 31.
  • the polyadenylation (polyA) signal is wild type neuropilin-1 polyA signal.
  • the WT NRP1 polyA signal comprises the nucleotide sequence of SEQ ID NO: 32, or a nucleotide sequence comprising at least or about 90% identity to the nucleotide sequence of SEQ ID NO: 32.
  • the third restriction site is an EcoR1 site. In some aspects, the EcoR1 site comprises the nucleotide sequence of SEQ ID NO: 33. [00146] In some aspects, the third restriction site is an MfeI site. In some aspects, the MfeI site comprises the nucleotide sequence of SEQ ID NO: 34.
  • nucleic acid comprising a nucleotide sequence encoding a microRNA (miRNA) expression cassette for converting an RNA polymerase III- based promoter system to an RNA polymerase II-based promoter system.
  • miRNA microRNA
  • the cassette comprises a nucleotide sequence encoding the sequence of SEQ ID NO: 35 (ACCGGN 1 N 2 N 3 N 4 N 5 N 6 N 7 GCUN 8 N 9 N 10 N 11 ACAGUGAGCGAN 12 NNNNNNNNNNNNN NNNNNGUAAAGCCACAGAUGGGNNNNNNNNNNNNNNNNNNNNNNUGCUACUGCN 13 N 1 4 UUAN 15 N 16 N 17 N 18 N 19 N 20 AAUAAAAUACGAAAUN 21 N 22 N 23 N 24 CN 25 N 26 AAUUN 27 ), or a 28335/58942 2023-012-02 nucleotide sequence comprising at least or about 80% identity to the nucleotide sequence encoding the sequence of SEQ ID NO: 35, wherein N 1 at position 6 is U or is omitted; wherein N 2 at position 7 is C, A, or U; wherein N 3 at position 8 is C or is omitted; wherein N 4 at position 9 is C or is omitted; wherein N
  • the disclosure in exemplary aspects, includes miRNA expression cassettes, wherein the sense strand of the miRNA is 5’ to the antisense strand of the miRNA in the expression cassette.
  • an miRNA expression cassette of the disclosure might also comprise the antisense strand of the miRNA 5’ to the sense strand of the miRNA in the expression cassette.
  • more A:U base pairs were incorporated at the 5’ end of the mature sequence and more G:C base pairs were incorporated at the 3’ end of the antisense sequence to bias incorporation of the antisense strand into the RNA-induced silencing complex (RISC).
  • RISC RNA-induced silencing complex
  • the disclosure provides a nucleic acid comprising a nucleotide sequence encoding a microRNA (miRNA) expression cassette for converting an RNA polymerase III-based promoter system to an RNA polymerase II-based promoter system, wherein the nucleic acid comprises a nucleotide sequence set forth in any of SEQ ID NOs: 1-16 or 35-53, or a nucleotide sequence comprising 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 89%, 88%, 87%, 86%, 85%, 84%, 83%, 82%, 81%, 80%, 79%, 78%, 77%, 28335/58942 2023-012-02 76%, 75%, 74%, 73%,
  • nucleic acid comprises the nucleotide sequence. In some aspects, the nucleic acid consists essentially of the nucleotide sequence. In some aspects, the nucleic acid consists of the nucleotide sequence.
  • miRNA expression cassettes of the disclosure are designed to be used with any miRNA and are not limited to the exemplary miRNA disclosed herein.
  • miRNA include those disclosed in microRNA databases known in the art including, but not limited to, the microRNAs listed in the microRNA database, known as miRBase (https_colon_forward slash_forward slash_www.mirbase.org.
  • the disclosure includes the use of any of these microRNAs in the modular system, i.e., a DNA expression cassette, designed to convert therapeutic miRNA expression cassettes from the use of ubiquitous RNA polymerase III-based promoters to RNA polymerase II-based promoters for tissue specific expression of the miRNAs while maintaining fidelity and efficacy of processing as described herein.
  • the disclosure includes constructs for RNA interference to reduce or inhibit the expression of a gene associated with a detrimental effect when it is expressed.
  • the disclosure provides constructs to be used with any such miRNA to reduce or inhibit expression of the gene for which the miRNA is designed to target.
  • the disclosure provides constructs to be used in inhibiting PMP22 or DUX4 expression.
  • the miRNA targeting PMP22 is mi871.
  • Mi871 is a microRNA which targets the PMP22 gene and is used in treating CMT1A (see WO 2022/119826, incorporated herein by reference in its entirety).
  • the 28335/58942 2023-012-02 disclosure provides constructs and methods for targeting PMP22 and treating CMT1A.
  • CMT1A is thought to depend on gene dosage effects of PMP22 because CMT1A patients have increased PMP22 mRNA [Yoshikawa et al., Ann.
  • CMT1A patients with 1.4 Mb duplication may have variable PMP22 levels in skin biopsies not necessarily correlating with disease severity [Nobbio et al., Brain, 137(Pt 6): 1614-1620 (2014) and Katona et al., Brain, 132(Pt 7): 1734-1740 (2014)]. Nevertheless, supporting the PMP22 gene dosage effect as the driving mechanism of CMT1A, rodent models overexpressing PMP22 reproduced a CMT1A-like phenotype [Sereda et al., Neuron, 16(5): 1049-60 (1996); Huxley et al., Hum. Mol. Genet., 5(5): 563-569 (1996); Huxley et al., Hum. Mol.
  • PMP22 has been shown to saturate the proteasomal capacity for degradation, leading to perinuclear or cytoplasmic PMP22 accumulation, decreased overall proteasomal activity, and ER stress. PMP22 is also involved in early steps of myelinogenesis, in the determination of myelin thickness and maintenance. PMP22 duplication destabilizes the architecture, protein stoichiometry and function of the myelin sheath and SC, leading to demyelination, remyelination, the characteristic onion bulb formation and SC apoptosis.
  • Methods are provided of preventing or treating CMT1A comprising administering a delivery vehicle (such as rAAV) comprising DNA encoding a miPMP22 as described herein
  • a delivery vehicle such as rAAV
  • the methods provided result in restoration of PMP22 expression to at least or about 25 percent, at least or about 30, at least or about 40, at least or about 50, at least or about 60, at least or about 70, at least or about 80, at least or about 90, at least or about 95, at least or about 98 percent, at least or about 99 percent, or 100 percent or more, of normal PMP22 expression in an unaffected subject.
  • the miRNA targeting DUX4 is mi405.
  • the disclosure provides constructs and methods for inhibiting the expression of DUX4 and, thus, for treating facioscapulohumeral muscular dystrophy.
  • the role of DUX4 in FSHD pathogenesis can be explained as follows. First, D4Z4 repeats are not pseudogenes. The DUX4 locus produces 1.7 kb and 2.0 kb full-length mRNAs with identical coding regions, and D4Z4 repeats also harbor smaller sense and antisense transcripts, including some resembling microRNAs. Over-expressed DUX4 transcripts and a ⁇ 50 kDa full-length DUX4 protein are found in biopsies and cell lines from FSHD patients.
  • D4Z4 repeats and DUX4 likely have functional importance, since tandemly-arrayed D4Z4 repeats are conserved in at least eleven different placental mammalian species (non-placental animals lack D4Z4 repeats), with the greatest sequence conservation occurring within the DUX4 ORF.
  • over-expressed DUX4 is toxic to tissue culture cells and embryonic progenitors of developing lower organisms in vivo.
  • This toxicity occurs at least partly through a pro-apoptotic mechanism, indicated by Caspase-3 activation in DUX4 transfected cells, and presence of TUNEL-positive nuclei in developmentally arrested Xenopus embryos injected with DUX4 mRNA at the two-cell stage.
  • Human DUX4 inhibits differentiation of mouse C2C12 myoblasts in vitro, potentially by interfering with PAX3 and/or PAX7, and causes developmental arrest and reduced staining of some muscle markers when delivered to progenitor cells of zebrafish or Xenopus embryos. Finally, aberrant DUX4 function is directly associated with potentially important molecular changes seen in FSHD patient muscles. Specifically, full-length human DUX4 encodes an approximately 50 kDa double homeodomain transcription factor, and its only 28335/58942 2023-012-02 known target, Pitx1, was elevated in DUX4 over-expressing FSHD patient muscles. These data support that DUX4 catalyzes numerous downstream molecular changes that are incompatible with maintaining normal muscle integrity.
  • RNA interference is a mechanism of gene regulation in eukaryotic cells that has been considered for the treatment of various diseases. RNAi refers to post- transcriptional control of gene expression mediated by miRNAs. MiRNAs are small (about 21-25 nucleotides), noncoding RNAs that share sequence homology and base-pair with sequence target sites of cognate messenger RNAs (mRNAs). In exemplary aspects the miRNAs are about 22 nucleotides.
  • RNAi interfering RNA
  • shRNA short (or small) hairpin RNA
  • miRNA microRNA
  • shRNA and miRNA are expressed in vivo from plasmid- or virus-based vectors and may thus achieve long term gene silencing with a single administration, for as long as the vector is present within target cell nuclei and the driving promoter is active (Davidson et al., Methods Enzymol.392:145-73, 2005). Importantly, this vector-expressed approach leverages the decades-long advancements already made in the gene therapy field, but instead of expressing protein coding genes, the vector cargo in RNAi therapy strategies are artificial shRNA or miRNA cassettes targeting disease genes-of-interest. [00164] The disclosure provides new constructs for the delivery of microRNA (miRNA) with a pol II tissue-specific promoter.
  • miRNA microRNA
  • miRNAs are a class of non-coding RNAs that play important roles in RNA silencing and in regulating gene expression. The majority of miRNAs are transcribed from DNA sequences into primary miRNAs and processed into precursor miRNAs, and finally mature miRNAs. In most cases, miRNAs interact with the 3′ untranslated region (3′ UTR) of target mRNAs to induce mRNA degradation and translational repression. However, interaction of miRNAs with other regions, including the 5′ UTR, coding sequence, and gene promoters, have also been reported. Under certain conditions, miRNAs can also activate translation or regulate transcription.
  • 3′ UTR 3′ untranslated region
  • miRNAs bind to a specific sequence at the 3′ UTR of their target mRNAs to induce translational repression and mRNA deadenylation and decapping. miRNA binding sites have also been detected in other mRNA regions including the 5′ UTR and coding sequence, as well as within promoter regions. The binding of miRNAs to 5′ UTR and coding regions have silencing effects on gene expression while miRNA interaction with promoter region has been reported to induce transcription.
  • the miRNA constructs of the disclosure were designed to be modular constructs to substitute any miRNA into the construct for expression with an RNA pol II promoter.
  • the miRNA constructs of the disclosure are expressed under various promoters and/or enhancers including, but not limited to any RNA polymerase type II (pol II) promoters and/or enhancers.
  • the promoter and/or enhancer is a U7 promoter and/or enhancer, an RSV promoter and/or enhancer, a human skeletal a-actin (HSA) promoter and/or enhancer, a desmin promoter and/or enhancer, a CMV promoter and/or enhancer, a minimal CMV promoter and/or enhancer, a T7 promoter and/or enhancer, an EF1-alpha promoter and/or enhancer, a minimal EF1-alpha promoter and/or enhancer, an unc45b promoter and/or enhancer, a myosin heavy chain kinase (MHCK) promoter, a muscle creatine kinase (MCK) promoter, a tMCK promoter and/or enhancer, a dMCK promoter and/or enhancer, a CK1 promoter and/or enhancer, a CK6 promoter and/or enhancer, a CK7 promoter and/or
  • the promoter and/or enhancer is any of the promoters and/or enhancers disclosed by Skopenova et al. (Acta Naturae.2021; 13(1):47-58) incorporated herein by reference in its entirety).
  • the synthetic promoter is an SPc5-12 promoter, an SP-301 promoter, an MH promoter, or a Sk-CRM4/DES promoter.
  • a neuronal-specific or brain-specific promoter is human Synapsin1 (hSyn1), neuron-specific enolase (Nse), MeCP2, mDLX, mDLX5/6, calmodulin- 28335/58942 2023-012-02 dependent kinase II (CaMKII or Camk2a), or an MPZ promoter.
  • the promoter and/or enhancer is any of the promoters and/or enhancers disclosed by Haery et al.
  • the disclosure includes a vector comprising any of the nucleic acids described herein to deliver nucleic acids encoding the miRNA.
  • embodiments of the disclosure utilize vectors (for example, viral vectors, such as adeno- associated virus (AAV), adenovirus, retrovirus, lentivirus, equine-associated virus, alphavirus, pox virus, herpes virus, herpes simplex virus, polio virus, Sindbis virus, vaccinia virus or a synthetic virus, e.g., a chimeric virus, mosaic virus, or pseudotyped virus, and/or a virus that contains a foreign protein, synthetic polymer, nanoparticle, or small molecule) to deliver the nucleic acids disclosed herein.
  • viruses for example, viral vectors, such as adeno- associated virus (AAV), adenovirus, retrovirus, lentivirus, equine-associated virus, alphavirus, pox virus, herpes virus, herpes simplex virus, polio virus, Sindbis virus, vaccinia virus or a synthetic virus, e.g., a chimeric virus, mosaic virus, or pseudotype
  • AAV is a replication-deficient parvovirus, the single- stranded DNA genome of which is about 4.7 kb in length including 145 nucleotide inverted terminal repeat (ITRs).
  • ITRs nucleotide inverted terminal repeat
  • AAV1 is provided in GenBank Accession No. NC_002077
  • the complete genome of AAV2 is provided in GenBank Accession No. NC_001401 and Srivastava et al., J. Virol., 45: 555-564 ⁇ 1983)
  • the complete genome of AAV3 is provided in GenBank Accession No.
  • NC_1829 the complete genome of AAV4 is provided in GenBank Accession No. NC_001829; the AAV5 genome is provided in GenBank Accession No. AF085716; the complete genome of AAV6 is provided in GenBank Accession No. NC_001862; at least portions of AAV7 and AAV8 genomes are provided in GenBank Accession Nos. AX753246 and AX753249, respectively (see also U.S. Patent Nos.7,282,199 and 7,790,449 relating to AAV8); the AAV9 genome is provided in Gao et al., J. Virol., 78: 6381-6388 (2004); the AAV10 genome is provided in Mol.
  • Cis-acting sequences directing viral DNA replication (rep), encapsidation/packaging and host cell chromosome integration are contained within the AAV ITRs.
  • Three AAV promoters (named p5, p19, and p40 for their relative map locations) drive the expression of the two AAV internal open reading frames encoding rep and cap genes.
  • the two rep promoters (p5 and p19), coupled with the 28335/58942 2023-012-02 differential splicing of the single AAV intron (at nucleotides 2107 and 2227), result in the production of four rep proteins (rep 78, rep 68, rep 52, and rep 40) from the rep gene.
  • Rep proteins possess multiple enzymatic properties that are ultimately responsible for replicating the viral genome.
  • the cap gene is expressed from the p40 promoter and it encodes the three capsid proteins VP1, VP2, and VP3. Alternative splicing and non-consensus translational start sites are responsible for the production of the three related capsid proteins.
  • a single consensus polyadenylation site is located at map position 95 of the AAV genome.
  • AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy.
  • AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic.
  • AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo.
  • AAV transduces slowly dividing and non-dividing cells and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element).
  • the AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible. Furthermore, because the signals directing AAV replication, genome encapsidation and integration are contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA. In some aspects, the rep and cap proteins are provided in trans. Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56o to 65oC for several hours), making cold preservation of AAV less critical.
  • AAV may be lyophilized and AAV-infected cells are not resistant to superinfection.
  • AAV possesses unique features that make it attractive as a vector for delivering foreign DNA to cells, for example, in gene therapy.
  • AAV infection of cells in culture is noncytopathic, and natural infection of humans and other animals is silent and asymptomatic.
  • AAV infects many mammalian cells allowing the possibility of targeting many different tissues in vivo.
  • AAV transduces slowly dividing and non-dividing cells, and can persist essentially for the lifetime of those cells as a transcriptionally active nuclear episome (extrachromosomal element).
  • the AAV proviral genome is infectious as cloned DNA in plasmids which makes construction of recombinant genomes feasible.
  • the signals directing AAV replication, genome encapsidation and integration are 28335/58942 2023-012-02 contained within the ITRs of the AAV genome, some or all of the internal approximately 4.3 kb of the genome (encoding replication and structural capsid proteins, rep-cap) may be replaced with foreign DNA.
  • the rep and cap proteins are provided in trans.
  • Another significant feature of AAV is that it is an extremely stable and hearty virus. It easily withstands the conditions used to inactivate adenovirus (56o to 65oC for several hours), making cold preservation of AAV less critical. AAV may be lyophilized and AAV-infected cells are not resistant to superinfection.
  • the viral vector is an adeno-associated virus (AAV), such as an AAV1 (i.e., an AAV containing AAV1 inverted terminal repeats (ITRs) and/or AAV1 capsid proteins), AAV2 (i.e., an AAV containing AAV2 ITRs and/or AAV2 capsid proteins), AAV3 (i.e., an AAV containing AAV3 ITRs and/or AAV3 capsid proteins), AAV4 (i.e., an AAV containing AAV4 ITRs and/or AAV4 capsid proteins), AAV5 (i.e., an AAV containing AAV5 ITRs and/or AAV5 capsid proteins), AAV6 (i.e., an AAV containing AAV6 ITRs and/or AAV6 capsid proteins), AAV7 (i.e., an AAV containing AAV7 ITRs and/or AAV7 capsid proteins), AAV8 (AAV1 (i.e
  • the AAV is a laboratory-engineered AAV not normally isolated in nature.
  • the AAV is an AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-anc80, AAV-B1, AAV-BR1, AAV.PHP.EB, AAVv66, AAV2/1, AAV2/8, 28335/58942 2023-012-02 or AAV2/9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101, or any derivative thereof.
  • rAAV variants for example rAAV with capsid mutations
  • rAAV with capsid mutations are also included in the disclosure. See, for example, Marsic et al., Molecular Therapy 22(11): 1900- 1909 (2014).
  • nucleotide sequences of the genomes of various AAV serotypes are known in the art. Use of cognate components is specifically contemplated. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety.
  • Recombinant AAV genomes of the disclosure comprise one or more AAV ITRs flanking the novel nucleic acid constructs of the disclosure.
  • AAV genome of the disclosure includes any of the various AAV serotypes known in the art.
  • the AAV vectors are single stranded AAV vectors.
  • the AAV is recombinant AAV (rAAV).
  • the rAAV lack rep and cap genes.
  • rAAV are self-complementary (sc)AAV.
  • DNA plasmids of the disclosure comprise rAAV genomes of the disclosure. The DNA plasmids are transferred to cells permissible for infection with a helper virus of AAV (e.g., adenovirus, E1-deleted adenovirus or herpes virus) for assembly of the rAAV genome into infectious viral particles.
  • helper virus of AAV e.g., adenovirus, E1-deleted adenovirus or herpes virus
  • rAAV particles in which an AAV genome to be packaged, rep and cap genes, and helper virus functions are provided to a cell are standard in the art. Production of rAAV requires that the following components are present within a single cell (denoted herein as a packaging cell): a rAAV genome, AAV rep and cap genes separate from (i.e., not in) the rAAV genome, and helper virus functions.
  • the AAV rep genes may be from any AAV serotype for which recombinant virus can be derived and may be from a different AAV serotype than the rAAV genome ITRs, including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV- anc80, AAV-B1, AAV-BR1, AAV.PHP.EB, AAVv66, AAV2/1, AAV2/8, or AAV2/9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV- SLB101, or any derivative thereof.
  • AAV serotypes AAV1, AAV2, AAV3, AAV
  • AAV DNA in the rAAV genomes is from any AAV serotype for which a recombinant virus can be derived including, but not limited to, AAV serotypes AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV.rh74, AAV.rh8, AAV.rh10, AAV11, AAV12, AAV13, AAV14, AAV15, AAV16, AAV-anc80, AAV-B1, AAV-BR1, AAV.PHP.EB, AAVv66, AAV2/1, AAV2/8, or AAV2/9, AAVMYO, MYOAAV, MYOAAV1A, MYOAAV2A, MYOAAV3A, modified AAV9 (mAAV9), or AAV-SLB101, or any 28335/58942 2023-012-02 derivative thereof.
  • rAAV variants for example rAAV with capsid mutations
  • rAAV with capsid mutations are also included in the disclosure. See, for example, Marsic et al., Molecular Therapy 22(11): 1900-1909 (2014).
  • the nucleotide sequences of the genomes of various AAV serotypes are known in the art. Use of cognate components is specifically contemplated. Production of pseudotyped rAAV is disclosed in, for example, WO 01/83692 which is incorporated by reference herein in its entirety.
  • packaging cells are provided. Packaging cells are created in order to have a cell line that stably expresses all the necessary components for AAV particle production.
  • Retroviral vectors are created by removal of the retroviral gag, pol, and env genes. These are replaced by the therapeutic gene.
  • a packaging cell In order to produce vector particles, a packaging cell is essential. Packaging cell lines provide all the viral proteins required for capsid production and the virion maturation of the vector. Thus, packaging cell lines are made so that they contain the gag, pol and env genes.
  • retroviral vectors comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell.
  • AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing [Samulski et al., 1982, Proc. Natl. Acad. S6.
  • the packaging cell line is then infected with a helper virus such as adenovirus.
  • a helper virus such as adenovirus.
  • the advantages of this method are that the cells are selectable and are suitable for large-scale production of rAAV.
  • Other examples of suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and/or rep and cap genes into packaging cells.
  • a method of generating a packaging cell to create a cell line that stably expresses all the necessary components for AAV particle production is provided.
  • a plasmid (or multiple plasmids) comprising a rAAV genome lacking AAV rep and cap genes, AAV rep and cap genes separate from the rAAV genome, and a selectable marker, such as a neomycin resistance gene, are integrated into the genome of a cell.
  • AAV genomes have been introduced into bacterial plasmids by procedures such as GC tailing [Samulski et al., 1982, Proc. Natl. Acad. S6.
  • Suitable methods employ adenovirus or baculovirus rather than plasmids to introduce rAAV genomes and/or rep and cap genes into packaging cells.
  • rAAV production is reviewed in, for example, Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka, 1992, Curr. Topics in Microbiol. and Immunol.158:97-129).
  • Various approaches are described in Ratschin et al., Mol. Cell. Biol.4:2072 (1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984); Tratschin et al., Mo1. Cell.
  • the AAV is a recombinant linear AAV (rAAV), a single-stranded AAV (ssAAV), or a recombinant self-complementary AAV (scAAV).
  • rAAV recombinant linear AAV
  • ssAAV single-stranded AAV
  • scAAV recombinant self-complementary AAV
  • the disclosure thus provides in some embodiments packaging cells that produce infectious rAAV.
  • packaging cells are stably transformed cancer cells, such as HeLa cells, 293 cells and PerC.6 cells (a cognate 293 line).
  • packaging cells are cells that are not transformed cancer cells, such as low passage 293 cells (human fetal kidney cells transformed with E1 of adenovirus), MRC-5 cells (human fetal 28335/58942 2023-012-02 fibroblasts), WI-38 cells (human fetal fibroblasts), Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lung cells).
  • the rAAV in some aspects, are purified by methods standard in the art, such as by column chromatography or cesium chloride gradients. Methods for purifying rAAV vectors from helper virus are known in the art and include methods disclosed in, for example, Clark et al., Hum.
  • the disclosure includes a composition comprising any of the nucleic acids or any of the vectors described herein in combination with a diluent, excipient, or buffer.
  • the disclosure provides a composition comprising a vector, e.g., such as a viral vector, as described herein.
  • compositions comprising delivery vehicles (such as rAAV) described herein are provided.
  • such compositions also comprise a pharmaceutically acceptable carrier.
  • pharmaceutically acceptable carrier means all aqueous and non- aqueous solutions, sterile solutions, solvents, buffers, e.g. phosphate buffered saline (PBS) solutions, water, suspensions, emulsions, such as oil/water emulsions, various types of wetting agents, liposomes, dispersion media and coatings, which are compatible with pharmaceutical administration, in particular with parenteral administration.
  • PBS phosphate buffered saline
  • any composition of the disclosure also comprises other ingredients, such as a diluent, excipients, and/or adjuvant.
  • Acceptable carriers, diluents, excipients, and adjuvants are nontoxic to recipients and are preferably inert at the dosages and concentrations employed, and include buffers such as phosphate, citrate, or other organic acids; antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt- forming counterions such as sodium; and/or nonionic surfactants such
  • the nucleic acids are introduced into the cell via non- vectorized delivery.
  • the disclosure includes non-vectorized delivery 28335/58942 2023-012-02 of any of the miRNA expression cassette constructs disclosed herein.
  • synthetic carriers able to form complexes with nucleic acids, and protect them from extra- and intracellular nucleases are an alternative to viral vectors.
  • non-vectorized delivery includes the use of nanoparticles, extracellular vesicles, or exosomes comprising the nucleic acids of the disclosure.
  • the disclosure also includes compositions comprising any of the constructs described herein alone or in combination.
  • Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze-drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.
  • Titers of rAAV to be administered in methods of the disclosure will vary depending, for example, on the particular rAAV, the mode of administration, the treatment goal, the individual, and the cell type(s) being targeted, and may be determined by methods standard in the art. Titers of rAAV may range from about 1x10 6 , about 1x10 7 , about 1x10 8 , about 1x10 9 , about 1x10 10 , about 1x10 11 , about 1x10 12 , about 1x10 13 to about 1x10 14 or more DNase resistant particles (DRP) per ml.
  • DNase resistant particles DNase resistant particles
  • Dosages may also be expressed in units of viral genomes (vg) (e.g., 1x10 7 vg, 1x10 8 vg, 1x10 9 vg, 1x10 10 vg, 1x10 11 vg, 1x10 12 vg, 1x10 13 vg, and 1x10 14 vg, respectively).
  • vg viral genomes
  • the disclosure provides a method of delivering to a cell or to a subject any one or more of the miRNA expression cassette constructs disclosed herein.
  • the method comprises delivering the nucleic acid constructs in one or more AAV vectors.
  • the method comprises delivering the nucleic acids to the cell in non-vectorized delivery.
  • the method comprises administering to a cell or to a subject an AAV comprising any one or more miRNA expression cassette constructs disclosed herein.
  • the disclosure provides AAV transducing cells for the delivery of nucleic acids encoding the miRNA as described herein. Methods of transducing a target cell with rAAV, in vivo or in vitro, are included in the disclosure. The methods comprise the step 28335/58942 2023-012-02 of administering an effective dose, or effective multiple doses, of a composition comprising a rAAV of the disclosure to a subject, including an animal (such as a human being) in need thereof.
  • an effective dose is a dose that alleviates (eliminates or reduces) at least one symptom of the disease or disorder associated with mutant or pathogenic expression of the gene being targeted by the miRNA, that slows or prevents progression of a symptom of the disease or disorder associated with mutant or pathogenic expression of the gene, and/or that results in remission (partial or total) of the symptom(s) of the disease or disorder associated with mutant or pathogenic expression of the gene.
  • the disclosure provided non-vectorized delivery of nucleic acids encoding the miRNA as described herein.
  • the nucleic acids or compositions comprising the nucleic acids are delivered in nanoparticles, extracellular vesicles, or exosomes.
  • Combination therapies are also contemplated by the disclosure.
  • the disclosure includes possible combination therapy or therapies comprising one or more other compounds or compositions comprising other RNA inhibitory compounds or small molecule compounds for downregulating gene expression.
  • Combination as used herein includes simultaneous treatment or sequential treatments.
  • Combinations of methods of the disclosure with standard medical treatments and supportive care are specifically contemplated, as are combinations of therapies, such as physical and occupational therapies, speech & language therapy, therapy by developmental specialists for their neurodevelopmental delay and autistic symptoms, medications to address behavioral problems (including, but not limited to, alpha-2 adrenergic agonists, antipsychotics, selective serotonin reuptake inhibitors (SSRIs), and the like), medications to address sleep problems (including, but not limited to, melatonin, trazodone, benzodiazepines, doxepine, eszopiclone, lemborexant, ramelteon, suvorexant, zaleplon, zolpidem, and the like) and medications to address seizures and/or EEG abnormalities (including, but not limited to, any of the many anti-seizure medications known in the art including, but not limited to, carbamazepine, eslicarbazepine, ethosuximide, everolimus
  • compositions including AAV, nanoparticles, extracellular vesicles, and exosomes comprising the compositions and nucleic acids of the disclosure
  • routes standard in the art including, but not limited to, intramuscular, parenteral, intravascular, intravenous, oral, buccal, nasal, pulmonary, intracranial, intracerebroventricular, intrathecal, intraosseous, intraocular, rectal, or vaginal.
  • Route(s) of administration and serotype(s) of AAV components of rAAV (in particular, the AAV ITRs and capsid protein) of the disclosure may be chosen and/or matched by those skilled in the art taking into account the disease state being treated and the target cells/tissue(s), such as cells that express a gene to be downregulated, which in some aspects of the disclosure is DUX4 or PMP22.
  • the composition or medicament is formulated for intracerebroventricular injection, intrathecal injection, intramuscular injection, oral administration, subcutaneous, intradermal, or transdermal transport, injection into the blood stream, or for aerosol administration.
  • the route of administration is intracerebroventricular.
  • the route of administration is intravenous.
  • actual administration of rAAV of the present disclosure may be accomplished by using any physical method that will transport the rAAV recombinant vector into the target tissue of an animal.
  • Administration according to the disclosure includes, but is not limited to, injection directly into the brain, the bloodstream, the central nervous system, and/or other organ. Simply resuspending a rAAV in phosphate buffered saline has been demonstrated to be sufficient to provide a vehicle useful for expression in the brain, and there are no known restrictions on the carriers or other components that can be co- administered with the rAAV (although compositions that degrade DNA should be avoided in the normal manner with rAAV).
  • Capsid proteins of a rAAV may be modified so that the rAAV is targeted to a particular target tissue of interest such as the brain. See, for example, WO 02/053703, the disclosure of which is incorporated by reference herein.
  • Pharmaceutical compositions can be prepared for oral administration, as injectable formulations, or as topical formulations to be delivered to the muscles by subcutaneous, intradermal, and/or transdermal transport. Numerous formulations for both intramuscular injection and 28335/58942 2023-012-02 transdermal transport have been previously developed and can be used in the practice of the disclosure.
  • the rAAV can be used with any pharmaceutically acceptable carrier for ease of administration and handling.
  • solutions such as sterile aqueous solutions are used.
  • aqueous solutions can be buffered, if desired, and the liquid diluent first rendered isotonic with saline or glucose.
  • Solutions of rAAV as a free acid (DNA contains acidic phosphate groups) or a pharmacologically acceptable salt can be prepared in water suitably mixed with a surfactant such as hydroxpropylcellulose.
  • a dispersion of rAAV can also be prepared in glycerol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • sterile aqueous media employed are all readily obtainable by standard techniques well-known to those skilled in the art.
  • 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. In all cases the form must be sterile and must be fluid to the extent that easy syringability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol and the like), suitable mixtures thereof, and vegetable oils.
  • polyol for example, glycerol, propylene glycol, liquid polyethylene glycol and the like
  • suitable mixtures thereof and vegetable oils.
  • proper fluidity is maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like.
  • the formulation comprises a stabilizer.
  • stabilizer refers to a substance or excipient which protects the formulation from adverse conditions, such as those which occur during heating or freezing, and/or prolongs the stability or shelf- life of the formulation in a stable state.
  • the formulation comprises an antimicrobial preservative.
  • antimicrobial preservative refers to any substance which is added to the composition that inhibits the growth of microorganisms that may be introduced upon repeated puncture of the vial or container being used.
  • antimicrobial preservatives include, but are not limited to, substances such as thimerosal, 2-phenoxyethanol, benzethonium chloride, and phenol.
  • transduction is used to refer to the administration/delivery of one or more of the nucleic acid constructs encoding a miRNA, as described herein, to a recipient cell either in vivo or in vitro, via a replication-deficient rAAV of the disclosure resulting in decreased expression of the targeted gene by the recipient cell.
  • transduction with rAAV is carried out in vitro.
  • desired target cells are removed from the subject, transduced with rAAV and reintroduced into the subject.
  • syngeneic or xenogeneic cells can be used where those cells will not generate an inappropriate immune response in the subject.
  • Suitable methods for the transduction and reintroduction of transduced cells into a subject are known in the art.
  • cells are transduced in vitro by combining rAAV with cells, e.g., in appropriate media, and screening for those cells harboring the DNA of interest using conventional techniques such as Southern blots and/or PCR, or by using selectable markers.
  • Transduced cells can then be formulated into pharmaceutical compositions, and the composition introduced into the subject by various techniques, such as by intracerebroventricular, intramuscular, intravenous, subcutaneous and intraperitoneal injection, or by injection into the brain or smooth and cardiac muscle, using e.g., a catheter.
  • the disclosure provides methods of administering an effective dose (or doses, administered essentially simultaneously or doses given at intervals) of rAAV that comprise DNA that encodes microRNA designed to reduce or inhibit the expression of a gene targeted for inhibition by the miRNA.
  • the microRNA is designed to reduce or inhibit the expression of any gene whose expression in a cell or in a cell of a subject (including but not limited to a human subject) is associated with a pathological disease or condition.
  • the microRNA is mi405 for targeting DUX4.
  • the microRNA is mi871 for targeting PMP22, to a cell or to a subject in need thereof.
  • the effective dose is therefore a therapeutically 28335/58942 2023-012-02 effective dose.
  • the dose or effective dose of rAAV administered is about 1.0x10 10 vg/kg to about 1.0x10 16 vg/kg.
  • 1.0x10 10 vg/kg is also designated 1.0 E10 vg/kg, which is simply an alternative way of indicating the scientific notation.
  • 10 11 is equivalent to E11, and the like.
  • the dose of rAAV administered is about 1.0x10 11 vg/kg to about 1.0x10 15 vg/kg.
  • the dose of rAAV is about 1.0x10 10 vg/kg, about 2.0x10 10 vg/kg, about 3.0x10 10 vg/kg, about 4.0x10 10 vg/kg, about 5.0x10 10 vg/kg, about 6.0x10 10 vg/kg, about 7.0x10 10 vg/kg, about 8.0x10 10 vg/kg, about 9.0x10 10 about 1.0x10 11 vg/kg, about 2.0x10 11 vg/kg, about 3.0x10 11 vg/kg, about 4.0x10 11 vg/kg, about 5.0x10 11 vg/kg, about 6.0x10 11 vg/kg, about 7.0x10 11 vg/kg, about 8.0x10 11 vg/kg, about 9.0x10 11 vg/kg, about 1.0x10 12 vg/kg, about 2.0x10 12 vg/kg, about 3.0x10 12 vg/kg, about
  • the dose is about 1.0x10 11 vg/kg to about 1.0x10 15 vg/kg. In some aspects, the dose is about 1.0x10 13 vg/kg to about 5.0x10 13 vg/kg. In some aspects, the dose is about 2.0x10 13 vg/kg to about 4.0x10 13 vg/kg. In some aspects, the dose is about 3.0x10 13 vg/kg. [00208] In some aspects, an initial dose is followed by a second greater dose. In some aspects, an initial dose is followed by a second same dose. In some aspects, an initial dose is followed by one or more lesser doses. In some aspects, an initial dose is followed by multiple doses which are the same or greater doses.
  • Methods of transducing a target cell with a delivery vehicle such as rAAV
  • a delivery vehicle such as rAAV
  • Transduction of cells with an rAAV of the disclosure results in sustained expression of the miRNA sequence.
  • the disclosure thus provides rAAV and methods of administering/delivering rAAV which express the miRNA sequence in the cell(s) in vitro or in vivo in a subject.
  • the subject is a mammal.
  • the mammal is a human.
  • These methods include transducing cells and tissues 28335/58942 2023-012-02 (including, but not limited to, tissues such as the brain) with one or more rAAV described herein.
  • Transduction may be carried out with gene cassettes comprising cell-specific control elements.
  • the term “transduction” is used to refer to, as an example, the administration/delivery of a nucleic acid comprising a nucleotide sequence encoding the miRNA sequence, e.g., the DUX4 or PMP22 miRNA, i.e., mi405 or mi871, respectively, to a target cell either in vivo or in vitro, via a replication-deficient rAAV described herein resulting in the manipulating expression of the target gene, e.g., DUX4 or PMP22, by the target cell.
  • the in vivo methods comprise the step of administering an effective dose, or effective multiple doses, of a composition comprising a delivery vehicle (such as rAAV) to a subject (including a human subject) in need thereof.
  • a delivery vehicle such as rAAV
  • methods are provided of administering an effective dose (or doses, administered essentially simultaneously or doses given at intervals) of rAAV described herein to a subject in need thereof. If the dose or doses is administered prior to development of a disorder/disease, the administration is prophylactic. If the dose or doses is administered after the development of a disorder/disease, the administration is therapeutic.
  • An effective dose is a dose that alleviates (eliminates or reduces) at least one symptom associated with the disorder/disease state being treated, that slows or prevents progression to a disorder/disease state, that slows or prevents progression of a disorder/disease state, that diminishes the extent of disease, that results in remission (partial or total) of disease, and/or that prolongs survival.
  • compositions and methods of the disclosure are used in treating, ameliorating, or preventing a disease or disorder associated with expression of a DUX4 or PMP22 gene, such as FSHD or CMT1A disease.
  • the level of target gene expression or protein expression in a cell of the subject is decreased after administration of the nucleic acid encoding the miRNA or the vector, e.g., rAAV, comprising the nucleic acid encoding the miRNA as compared to the level of miRNA gene expression or protein expression before administration of the nucleic acid encoding the miRNA or the vector, e.g. rAAV.
  • expression of the target gene is changed by at least or about 10%, at least or about 20%, at least or about 30%, at least or about 40%, at least or about 50%, at least or about 60%, at least or about 70%, at least or about 80%, at least or about 90%, at least or about 95%, at least or about 98%, at least or about 99%, at least or about 100% percent, or at least about greater than 100%.
  • an effective dose of a nucleic acid, viral vector, nanoparticle, extracellular vesicle, exosome, or composition of the disclosure may be by routes standard 28335/58942 2023-012-02 in the art including, but not limited to, intracerebroventricular, intrathecal, intravenous, intracranial, oral, buccal, nasal, intraosseous, intramuscular, parenteral, intravascular, pulmonary, intraocular, rectal, or vaginal.
  • an effective dose is delivered by a systemic route of administration, i.e., systemic administration.
  • Systemic administration is a route of administration into the circulatory system so that the entire body is affected.
  • Such systemic administration takes place via enteral administration (absorption of the drug through the gastrointestinal tract) or parenteral administration (generally via injection, infusion, or implantation).
  • an effective dose is delivered by a combination of routes.
  • an effective dose is delivered intravenously and/or intramuscularly, or intravenously and intracerebroventricularly, and the like.
  • an effective dose is delivered in sequence or sequentially.
  • an effective dose is delivered simultaneously.
  • the nucleic acid, vector, nanoparticle, extracellular vesicle, exosome, composition, or medicament is formulated for the delivery of an effective dose by oral administration, subcutaneous administration or injection, intradermal administration or injection, intraventricular delivery or injection, intracerebroventricular delivery or injection, intrathecal delivery or injection, transdermal delivery or injection, injection into the blood stream, or aerosol administration.
  • Route(s) of administration and serotype(s) of AAV components of the rAAV (in particular, the AAV ITRs and capsid protein) of the disclosure are chosen and/or matched by those skilled in the art taking into account the condition or state of the disease or disorder being treated, the condition, state, or age of the subject, and the target cells/tissue(s) that are to express the nucleic acid or protein.
  • actual administration of delivery vehicle such as rAAV
  • Administration includes, but is not limited to, injection into the brain, the nervous system, the liver, or the bloodstream.
  • compositions can be prepared as injectable 28335/58942 2023-012-02 formulations or as topical formulations to be delivered to the muscles by transdermal transport.
  • the delivery vehicle (such as rAAV) can be used with any pharmaceutically acceptable carrier for ease of administration and handling.
  • a dispersion of delivery vehicle (such as rAAV) can also be prepared in glycerol, sorbitol, liquid polyethylene glycols and mixtures thereof and in oils. Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms.
  • the sterile aqueous media employed are all readily obtainable by standard techniques known to those skilled in the art.
  • 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.
  • the form must be sterile and must be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating actions of microorganisms such as bacteria and fungi.
  • the carrier can be a solvent or dispersion medium containing, for example, water, ethanol, polyol (for example, glycerol, propylene glycol, liquid polyethylene glycol, sorbitol and the like), suitable mixtures thereof, and vegetable oils.
  • the proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of a dispersion and by the use of surfactants.
  • the prevention of the action of microorganisms can be brought about by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal and the like. In many cases it will be preferable to include isotonic agents, for example, sugars or sodium chloride.
  • Prolonged absorption of the injectable compositions can be brought about by use of agents delaying absorption, for example, aluminum monostearate and gelatin.
  • Sterile injectable solutions are prepared by incorporating rAAV in the required amount in the appropriate solvent with various other ingredients enumerated above, as required, followed by filter sterilization.
  • dispersions are prepared by incorporating the sterilized active ingredient into a sterile vehicle which contains the basic dispersion medium and the required other ingredients from those enumerated above.
  • the preferred methods of preparation are vacuum drying and the freeze drying technique that yield a powder of the active ingredient plus any additional desired ingredient from the previously sterile-filtered solution thereof.
  • Treating includes ameliorating, reducing, or inhibiting any one or more symptoms of the disease or condition associated with expression of the target gene, i.e., the gene that the microRNA is designed to knockdown expression.
  • Preventing includes prohibiting or blocking any one or more symptoms of the disease or condition associated with expression of the target gene, i.e., the gene that the microRNA is designed to knockdown expression. Such preventing, in some aspects, includes prohibiting expression of the gene at a level to not allow the manifestation of symptoms of the disease or condition associated with expression of the gene.
  • methods are provided of administering an effective dose (or doses, administered essentially simultaneously or doses given at intervals) of rAAV described herein to a subject in need thereof. If the dose or doses is administered prior to development of a disorder/disease, the administration is prophylactic. If the dose or doses is administered after the development of a disorder/disease, the administration is therapeutic.
  • compositions and methods of the disclosure are used in treating, ameliorating, or preventing a disease, such as a muscular dystrophy (MD) associated with the expression of the double homeobox 4 (DUX4) gene.
  • MD muscular dystrophy
  • DUX4 double homeobox 4
  • FSHD is caused by aberrant expression of the DUX4 gene which produces a transcription factor that is toxic to skeletal muscle.
  • DUX4 is normally functional during the two-cell stage of human development but repressed thereafter in essentially all other tissues, except perhaps the testes.
  • specific genetic and epigenetic factors conspire to permit DUX4 de-repression, where it then initiates several aberrant gene expression cascades, including those involved in differentiation abnormalities, oxidative stress, inflammatory infiltration, cell death and muscle atrophy.
  • FSHD is among the most commonly inherited muscular dystrophies, estimated to affect as many as 870,000 individuals.
  • FSHD presentation Classical descriptions of FSHD presentation include progressive muscle weakness in the face, shoulder-girdle and arms, but disease can manifest more broadly, including in muscles of the trunk and lower extremities. Variability is also commonly seen within individuals, as asymmetrical weakness is common. Age-at-onset 28335/58942 2023-012-02 can range from early childhood to adulthood, and is usually related to disease severity, where earlier onset is often associated with more severe muscle weakness. Although most patients with FSHD have a normal life span, respiratory insufficiency can occur, and the disease can be debilitating, as approximately 25% of affected individuals may become wheelchair dependent by their fifties, and even earlier in more severe forms of the disease, while others maintain lifelong ambulation.
  • nucleic acids, vectors, nanoparticles, extracellular vesicles, exosomes, and compositions of the disclosure are used in treating, ameliorating, or preventing a disease, such as a cancer.
  • DUX4 has been shown to be activated in some cancer types, where it functions to mask tumor cells from the immune system [Chew et al., Dev. Cell 50(5):658-71 (2019)].
  • DUX4 protein fusions are known to cause cancer, such as rhabdomyosarcoma and Ewing's sarcoma.
  • a CIC-DUX4 gene fusion induces sarcomas and drives sarcoma metastasis [Yoshimoto et al., Cancer Res.2017 Jun 1; 77(11): 2927-2937; Okimoto et al., J Clin Invest.2019; 129(8):3401-3406)].
  • cancer tissues such as those tissues from the adrenal, B-cell lymphoma, bile duct, bladder, breast, cervix, colon, endometrium, esophagus, head/neck, liver, brain (e.g., lower grade glioma), lung, mesothelium, neural crest, ovary, pancreas, prostate, kidney, skin, soft tissue, stomach, testicles, and thymus, also were shown to express DUX4 [Chew et al., Dev. Cell 50(5):658-71 (2019)].
  • nucleic acids, vectors, nanoparticles, extracellular vesicles, exosomes, and compositions of the disclosure described herein are used in inhibiting DUX4 expression in the treatment, amelioration, or prevention of cancer.
  • Molecular, biochemical, histological, and functional outcome measures demonstrate the therapeutic efficacy of the products and methods disclosed herein for decreasing the expression of the DUX4 gene and protein and treating muscular dystrophies, such as FSHD. Outcome measures are described, for example, in Chapters 32, 35 and 43 of Dyck and Thomas, Peripheral Neuropathy, Elsevier Saunders, Philadelphia, PA, 4th Edition, Volume 1 (2005) and in Burgess et al., Methods Mol.
  • Outcome measures include, but are not limited to, reduction or elimination of DUX4 mRNA or protein in affected tissues.
  • the lack of expression of DUX4 and/or the downregulation of expression of DUX4 in the cell is detected by measuring the level of DUX4 protein by methods known in 28335/58942 2023-012-02 the art including, but not limited to, RT-PCR, QRT-PCR, RNAscope, Western blot, immunofluorescence, or immunohistochemistry in muscle biopsied before and after administration of the rAAV to determine the improvement.
  • the level of DUX4 gene expression or protein expression in a cell of the subject is decreased after administration of the nucleic acid encoding the DUX4 miRNA or the vector, e.g., rAAV, comprising the nucleic acid encoding the DUX4 miRNA as compared to the level of DUX4 gene expression or protein expression before administration of the nucleic acid encoding the DUX4 miRNA or the vector, e.g. rAAV.
  • expression of a DUX4 is decreased by at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100% percent, or at least about greater than 100%.
  • improved muscle strength, improved muscle function, and/or improved mobility and stamina show an improvement by at least about 2%, at least about 5%, at least about 10%, at least about 20%, at least about 30%, at least about 40%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 95%, at least about 98%, at least about 99%, at least about 100% percent, or at least about greater than 100%.
  • Other outcome measures include measuring the level of serum creatinine kinase (CK) in the subject before and after treatment. Increased CK levels are a hallmark of muscle damage. In muscular dystrophy patients, CK levels are significantly increased above the normal range (10 to 100 times the normal level since birth).
  • a positive therapeutic outcome for treatment with the methods of the disclosure is a reduction in the level of serum creatinine kinase after administration of the rAAV as compared to the level of serum creatinine kinase before administration of the rAAV.
  • Other outcome measures include, but are not limited to, measuring to determine if there is improved muscle strength, improved muscle function, improved mobility, improved stamina, or a combination of two or more thereof in the subject after treatment. Such outcome measures are important in determining muscular dystrophy progression in the subject and are measured by various tests known in the art.
  • a combination therapy includes both simultaneous treatment(s) and sequential treatment(s).
  • glucocorticoids include, but are not limited to, prednisone, prednisolone, dexamethasone, deflazacort, beclomethasone, betamethasone, budesonide, cortisone, hydrocortisone, methylprednisolone, and triamcinolone.
  • combination therapies included in the disclosure are the DUX4 miRNAs, as described herein, in combination with other miRNAs, or in combination with U7-snRNA- based gene therapy, a small molecule inhibitor of DUX4 expression, oligonucleotides to inhibit DUX4 through RNAi or RNAse H or exon skipping mechanisms, U7-snRNA plus a theoretical CRISPR-based gene therapy approach.
  • nucleic acids, vectors, nanoparticles, extracelllulare vesicles, endosomes, compositions and methods of the disclosure are used in treating, ameliorating, or preventing a disease, such as Charcot-Marie-Tooth disease (CMT), including but not limited to CMT1A, associated with the expression of the PMP22 gene.
  • CMT Charcot-Marie-Tooth disease
  • the subject is treated before the onset of disease and, thus, the disease is prevented.
  • the subject is diagnosed as having a pathological PMP22 gene duplication or mutation, and the subject is treated after the onset of disease and, thus, the disease is treated or ameliorated.
  • CMT disease refers to a heterogeneous group of hereditary peripheral neuropathies that affect 1 in 2500 people.
  • the most common type, CMT Type 1 is a demyelinating peripheral neuropathy.
  • the CMT Type 1 subtype that affects more than 50% of all CMT cases and about 70-80% of CMT Type 1 cases is autosomal dominant demyelinating CMT neuropathy type 1A [CMT1A (MIM 118220)].
  • CMT1A is most frequently caused by a dominantly inherited 1.4 Mb tandem intra-chromosomal duplication on chromosome 17p11.2-p12.
  • CMT1A patients with CMT1A develop slowly progressive distal muscle weakness and atrophy, sensory loss, and absent reflexes with a typical onset at adolescence.
  • CMT1A shows high variability in disease severity even within the same family.
  • Sensory responses are usually absent while motor nerve conduction velocities (MNCVs) are slowed, ranging from 5 to 35 m/s in the forearm, but most average around 20 m/s, with uniform and symmetric findings in different nerves.
  • MNCVs motor nerve conduction velocities
  • Molecular, biochemical, histological, and functional outcome measures demonstrate the therapeutic efficacy of the methods.
  • Outcome measures are described, for example, in Chapters 32, 35 and 43 of Dyck and Thomas, Peripheral Neuropathy, Elsevier Saunders, Philadelphia, PA, 4th Edition, Volume 1 (2005) and in Burgess et al., Methods Mol. Biol., 602: 347-393 (2010).
  • Outcome measures include, but are not limited to, one or more of the reduction or elimination of mutant PMP22 mRNA or protein in affected tissues, PMP22 gene knockdown, increased body weight and improved muscle strength.
  • Others include, but are not limited to, nerve histology (axon number, axon size and myelination), neuromuscular junction analysis, and muscle weights and/or muscle histology.
  • kits comprising a nucleic acid, vector, or composition of the disclosure or produced according to a process of the disclosure.
  • kit means two or more components, one of which corresponds to a nucleic acid, vector, or composition of the disclosure, and the other which corresponds to a container, recipient, instructions, or otherwise.
  • a kit therefore, in various 28335/58942 2023-012-02 aspects, is a set of products that are sufficient to achieve a certain goal, which can be marketed as a single unit.
  • the kit may comprise one or more recipients (such as vials, ampoules, containers, syringes, bottles, bags) of any appropriate shape, size and material containing the nucleic acid, vector, or composition of the disclosure in an appropriate dosage for administration (see above).
  • the kit may additionally contain directions or instructions for use (e.g. in the form of a leaflet or instruction manual), means for administering the nucleic acid, vector, or composition, such as a syringe, pump, infuser or the like, means for reconstituting the nucleic acid, vector, or composition and/or means for diluting the nucleic acid, vector, or composition.
  • the kit comprises a label and/or instructions that describes use of the reagents provided in the kit.
  • the kits also optionally comprise catheters, syringes or other delivering devices for the delivery of one or more of the compositions used in the methods described herein.
  • the disclosure also provides kits for a single dose of administration unit or for multiple doses.
  • the disclosure provides kits containing single- chambered and multi-chambered pre-filled syringes.
  • the disclosure also includes, for instance, all embodiments of the disclosure narrower in scope in any way than the variations specifically mentioned above. With respect to aspects of the disclosure described as a genus, all individual species are considered separate aspects of the disclosure. With respect to aspects of the disclosure described or claimed with “a” or “an,” it should be understood that these terms mean “one or more” unless context unambiguously requires a more restricted meaning. [00243] Unless otherwise indicated, the term “at least” preceding a series of elements is to be understood to refer to every element in the series. Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments of the disclosure described herein. Such equivalents are intended to be encompassed by the disclosure.
  • Example 1 Materials and Methods [00253] Study Design. [00254] U6 promoter-driven systems were previously developed and reported which were shown to express therapeutic miRNAs that target and reduce several dominant disease genes. See at least U.S. Patents No.9,469,851 and 10,301,649 (i.e., DUX4 miRNA, e.g., mi405), which provide miRNAs that target the DUX4 gene, and WO 2022/119826 (PMP22 miRNA, e.g. mi-871) which target the PMP22 gene, which are associated with facioscapulohumeral muscular dystrophy (FSHD) and Charcot-Marie-Tooth disease type 1A (CMT1A), respectively.
  • FSHD facioscapulohumeral muscular dystrophy
  • CMT1A Charcot-Marie-Tooth disease type 1A
  • RNAi therapies targeting DUX4 and PMP22 have shown the ability of specifically designed synthetic miRNAs to reduce mRNA and protein levels while alleviating disease-associated outcomes in targeted tissues.
  • IV Intravenous
  • IT Intrathecal
  • a U6 pol III promoter is used with an RNA pol III termination signal whereas a tissue-specific pol II promoter, like CMV, is used with an RNA pol III poly A signal 28335/58942 2023-012-02 (Fig.5).
  • the use of a pol II promoter is important in the new modular expression cassette construct disclosed herein because it allows for tissue-specific expression of therapeutic miRNAs.
  • the disclosure provides modular expression cassettes which were designed to take these factors into consideration and allow for the substitution of various miRNAs into the cassettes more readily.
  • novel miRNA expression cassettes were designed to accurately position the RNAse III enzyme Drosha at a junction between Stem 2 and Stem 1, where Stem 1 is designed to contain as much single-stranded structure as possible.
  • the Drosha/DGCR8 complex (called the microprocessor) is positioned at the junction of single and double-stranded RNA structures (Han et al., Cell 125(5): P887-901, 2006).
  • eight novel miRNA expression cassettes were designed to convert an existing U6 promoter- driven miRNA to a tissue-specific, pol II-based system. Novel secondary structural elements were incorporated in the primary transcript regions flanking Drosha and Dicer processing sites.
  • Fig.6 shows a comparative drawing of the structures of these eight constructs with incorporation of a DNA sequence encoding mi405 (a DUX4-specific miRNA), an exemplary miRNA of interest.
  • the structures of the new mi405 constructs are shown in Figs.7-14, and the structures of the new mi871 constructs are shown in Figs.15-22.
  • the RNA nucleotide sequences encoded by the new miPMP22-871 constructs (SEQ ID NOs: 1-8) are shown in Fig.23, and the RNA nucleotide sequences encoded by the new miDUX4-405 constructs (SEQ ID NOs: 9-16) are shown in Fig.24.
  • Each of the miRNA expression cassettes for converting an RNA polymerase III- based promoter system to an RNA polymerase II-based promoter system is a nucleic acid comprising in a 5’ to 3’ order a DNA nucleotide sequence encoding a single stranded stem of mir30; a 5’ double-stranded stem of mir30; a sense strand of a mature microRNA; a modified mir30 loop comprising a nucleotide change to facilitate folding; an antisense strand of mature microRNA; a 3’ double-stranded stem of mir30; a single-stranded stem of mir30; and a polyadenylation (polyA) signal.
  • polyA polyadenylation
  • cassettes designed in our laboratory comprised restriction sites for cloning, but these restriction sites are not required in the expression cassettes of the disclosure. It is important to note that an expression cassette construct, as disclosed herein may be chemically synthesized and, thus, the restriction sites discussed in this example are not required in the final product.
  • each of the miRNA expression cassettes comprised in a 5’ to 3’ order a DNA nucleotide sequence encoding a first restriction enzyme site; a single stranded stem of mir30; a 5’ double-stranded stem of mir30; a sense strand of a mature microRNA; a modified mir30 loop comprising a nucleotide change to facilitate folding; an antisense strand of mature microRNA; a 3’ double-stranded stem of mir30; a single-stranded stem of mir30; a second restriction enzyme site; a polyadenylation (polyA) signal; and a third enzyme restriction site.
  • polyA polyadenylation
  • the pol II-driven miRNA expression cassettes of the disclosure were designed to be modular, thereby allowing for the cutting and pasting in of (1) any desired RNA polymerase II-based promoter at the indicated AgeI site and (2) any mature sense and antisense strands of microRNA sequence of interest (not only limited to exemplary miRNA sequences disclosed herein) in between the AgeI and Afl II sites in the expression cassette.
  • the pol II-driven expression cassette is not restricted only to the use of AgeI and AflII restriction sites.
  • the AflI site was removed to facilitate unfolding of stem 1 into 5’ and 3’ single-stranded regions.
  • microRNAs can be designed to include the Nrp I poly A signal and cloned into AgeI and EcoRI sites. This is a strategy which can be applied to the insertion of various miRNAs of interest.
  • the first restriction enzyme site is an AgeI site.
  • the AgeI site comprises a nucleotide sequence encoding ACCGGU (SEQ ID NO: 19).
  • the single-stranded stem of mir30 comprises a nucleotide sequence encoding GGUUGCUGUUGA (SEQ ID NO: 20), GGUUUGCUGAGGA (SEQ ID NO: 21), GGUUUGCUCCUUA (SEQ ID NO: 22), GGUAAGCUCCUUA (SEQ ID NO: 23), or GGUCCCCAAGCUCCUUA (SEQ ID NO: 24).
  • the 5’ double-stranded stem of mir30 comprises a nucleotide sequence encoding CAGUGAGCGAN (SEQ ID NO: 25), wherein N is A, U, C, or G. In the miPMP22-871 constructs, the N is G.
  • the N is U).
  • the sense strand of mature miRNA and the antisense strand of mature miRNA are strands of mature miRNA of any gene of interest.
  • this is the purpose of the design of the disclosed modular system, i.e., to allow one to simply insert the sequences encoding any miRNA into the construct for pol II tissue-specific expression of the 28335/58942 2023-012-02 miRNA.
  • the sense strand of mature miRNA comprises a nucleotide sequence encoding miPMP22-871 (i.e., GGGUUGCUGUUGAUUGAAGACU (SEQ ID NO: 17) or miDUX4-405 (i.e., CCAGGAUUCAGAUCUGGUUUCU (SEQ ID NO: 18).
  • the modified mir30 loop comprises a nucleotide sequence encoding GUAAAGCCACAGAUGGG (SEQ ID NO: 26).
  • the antisense strand of mature miRNA are strands of mature miRNA of any gene of interest.
  • the antisense strand of mature miRNA comprises a nucleotide sequence encoding miPMP22-871 (i.e., UCUUCAAUCAACAGCAAUCCCC (SEQ ID NO: 27) or miDUX4-405 (i.e., AAACCAGAUCUGAAUCCUGGAC (SEQ ID NO: 28).
  • the 3’ ds stem of mir30 comprises a nucleotide sequence encoding UGCCUACUG (SEQ ID NO: 29).
  • the ss stem of mir30 (mutated) comprises a nucleotide sequence encoding CCUUUUACUU (SEQ ID NO: 30).
  • the second restriction enzyme site is an AflII site.
  • the AflII site comprises a nucleotide sequence encoding CUUAAG (SEQ ID NO: 31).
  • the polyA signal is wild type neuropilin-1 polyadenylation (WT NRP1 polyA) signal.
  • WT NRP1 poly A signal comprises a nucleotide sequence encoding AAUAAAAUACGAAAUGUGACAGA (SEQ ID NO: 32).
  • the third restriction site is an EcoR1 site.
  • the EcoR1 site comprises a nucleotide sequence encoding GAAUUC (SEQ ID NO: 33).
  • the third restriction site is an MfeI site.
  • the MfeI site comprises a nucleotide sequence encoding AAUUG (SEQ ID NO: 34).
  • Various exemplary structures were designed for substituting the pol II promoter with a pol II promoter. Each of eight structures were tested with two exemplary miRNAs, miPMP22-871, or mi-871 (GGGUUGCUGUUGAUUGAAGACU (SEQ ID NO: 17), and miDUX4-405, or mi-405 (CCAGGAUUCAGAUCUGGUUUCU (SEQ ID NO: 18).
  • the sense strand of the miPMP22-871 comprises the nucleotide sequence of SEQ ID NO: 17.
  • the 28335/58942 2023-012-02 antisense strand of the miPMP22-871 comprises the nucleotide sequence of SEQ ID NO: 27.
  • the sense strand of the miDUX4-405 comprises the nucleotide sequence of SEQ ID NO: 18.
  • the antisense strand of the miDUX4-405 comprises the nucleotide sequence of SEQ ID NO: 28. See, for example, WO 2022/119826 and U.S. Patent Nos.9,469,851 and 10,301,649; and Marina Stavrou et al., (2022) A miRNA-based gene silencing approach for the treatment of CMT1A.
  • Nature Communications. Dec 8;12(1):7128Mi871 is used to induce silencing of overexpressed peripheral myelin protein-22 (PMP22) and constructs of the disclosure are designed to do the same.
  • PMP22 is a transmembrane glycoprotein component of myelin, important for myelin functioning. Mutation of PMP22 gene cause Charcot-Marie-Tooth Disease.
  • Mi405 is used to induce silencing of double homeobox 4, also known as DUX4, a protein which in humans is encoded by the DUX4 gene and which the misexpression of is the cause of facioscapulohumeral muscular dystrophy (FSHD).
  • DUX4 double homeobox 4
  • FSHD facioscapulohumeral muscular dystrophy
  • the expression cassette construct was designed to contain a small poly- adenylation signal derived from the human neuropilin-1 (Nrp1) gene or a modified human neuropilin-1 poly adenylation signal in which the 7 indicated nucleotides (5’-GTGACAG-3’ (SEQ ID NO: 54) , DNA; 5’ GUGACAG (SEQ ID NO: 55), RNA) were mutated to C nucleotides to improve the potential single-strandedness of Stem 1.
  • the expression cassette construct was designed so that the Stem 1 region of the cassette comprises nucleotide sequences with 5’ and 3’ ends ranging from about 15 to about 46 nucleotides in length.
  • the Stem 1 region may comprise nucleotide sequences less than or exceed these indicated lengths.
  • Exemplary embodiments of the 5’ end of the Stem 1 region comprise base pairs, including G:U RNA wobble base-pairs, ranging from about 0-50% base pairing. In some aspects, however, the Stem 1 region also comprises base-pairing that exceeds about 50% base pairing. 28335/58942 2023-012-02
  • Exemplary embodiments of the 3’ end of the Stem 1 region comprise base pairs, including G:U RNA wobble base-pairs, ranging from about 0-30% base pairing.
  • the Stem 1 region also comprises base-pairing that exceeds about 30% base-pairing, and in some aspects, may exceed about 50% base pairing.
  • Exemplary embodiments of the microRNA (miRNA) region of the cassette comprises miRNA nucleotide sequences ranging from about 100 to about 200 nucleotides, but the miRNA encoding region may comprise sequences less than or greater than the indicated lengths.
  • the miRNA nucleotide sequences ranges in length from about 120 to about 180 nucleotides.
  • the miRNA region ranges in length from 136-167 nucleotides in length (see Table 2).
  • the cassettes were designed to contain either natural or artificial microRNAs. [00280] Table 2.
  • HEK-293 Cell Culture HEK-293 (ATCC) cells were grown at 37°C in Gibco TM DMEM, high glucose, no glutamine (ThermoFisher) supplemented with 1% L-glutamine, 1% Penicillin/Streptomycin, and 10% FBS by volume.
  • RT4-D6P2T Rat Schwann cells were grown at 37°C in Gibco TM DMEM, high glucose, no glutamine (ThermoFisher) supplemented with 1% L-glutamine, 1% Sodium Pyruvate, 1% Penicillin/Streptomycin, and 10% FBS by volume. Cells were passaged at 90% confluency with Gibco TM TrypLETM Express Enzyme (1X), phenol red (ThermoFisher).
  • RNA Extraction For the Dual-Luciferase assay, 65,000 cells were pre-plated in a white 96-well plate, and 200 ng total DNA was transfected using LipofectamineTM 3000 Transfection Reagent (ThermoFisher) according to the manufacturer’s protocol. For the ddPCR expression and knockdown assays, 250,000 cells were pre-plated in a white 96-well plate, and 500 ng total DNA was transfected using LipofectamineTM 3000 Transfection Reagent (ThermoFisher) according to the manufacturer’s protocol. [00285] RNA Extraction.
  • RNA samples were placed in a DNA Speed Vac and spun on low speed for 3 min.50 ⁇ L of upH2O was used to resuspend samples and the concentration was subsequently measured by Nanodrop.200 ng of RNA were DNase treated according the manufacturer’s protocol for the DNA-freeTM DNA Removal kit (Invitrogen) with the addition of 20 U of RNase Inhibitor, Human Placenta (New England Biolabs) and using heat inactivation of the enzyme at 75 o C for 10 min instead of the included inactivation reagent.
  • cDNA was generated using the High- Capacity cDNA Reverse Transcription Kit (Applied Biosystems) and oligo(dT)18 Primer (Thermo Scientific) instead of 10x RT random primers following manufacturer’s instructions.
  • ddPCR Droplet Digital PCR
  • the ddPCR reaction mixture (25 ⁇ L) contained 1x ddPCR Supermix for Probes (No dUTP), 1x TaqMan probes for Pmp22 and Rpl13a (ThermoFisher) and 0.5 ng of synthesized cDNA.
  • DUX4.V5 or Human full-length PMP22 cDNA were cloned into the XhoI and NotI sites located within the 3’ UTR region of the Renilla Luciferase gene before the synthetic polyA sequence. Each construct was then co-transfected with its corresponding set of test miRNAs as described before. After 24-48 hrs, media was removed from the cells, 30 ⁇ l of Passive Lysis Buffer was added, and the cells were placed on a plate shaker at 425 rpm for 20 min at RT. The plate was then transferred to a GloMax Discover (Promega) plate reader where the Renilla and Firefly expression was quantified according to the Dual-Luciferase® Reporter Assay System Technical Manual (Promega).
  • Pol II-driven miRNAs were efficiently expressed in vitro, as measured by droplet digital PCR (ddPCR) assay targeting the mature miRNA sequence. Briefly, HEK-293 cells were transfected with plasmids expressing mi871/mi405 structures 1-8 driven by a CMV promoter.
  • Control conditions included HEK-293 cells undergoing a mock transfection, transfection with a plasmid expressing mi871/mi405 under the pol III U6 promoter system, and transfection of a non-targeting miRNA, miGFP, driven by the pol III U6 promoter system. Each condition was run in triplicate according to the conditions described above. At 48 hours post-transfection, total RNA was isolated, samples were rDNase-treated, and cDNA was synthesized following the aforementioned protocols. Finally, the expression of mi871/mi405 28335/58942 2023-012-02 (in addition to that of the normalizer gene Rpl13a) was measured for each sample using ddPCR.
  • Renilla and Firefly luciferase expression was measured by a GloMax Discover Luminescence Plate Reader (Promega) and percent knockdown was calculated by normalizing each ratio of Renilla:Firefly expression to the positive control, and empty U6T6 vector (Figs.31, 32).
  • CMV.mi871-4 exhibited the highest knockdown of the human PMP22 target at 83%, compared to 90% knockdown by the Pol III U6.mi871 system (Fig 31).
  • CMV.mi405-4 had the highest knockdown of the DUX4 target at 64%, compared to 58% for U6.mi405 (Fig.32).
  • Pol II-driven miRNA systems thus, show comparable target knockdown to their Pol III counterparts.
  • Example 3 Testing miRNA expression and knockdown efficiency from tissue-specific promoters
  • two different muscle-specific or Schwann cell-specific promoters were tested for their ability to drive expression of either mi405 or mi871, respectively. Briefly, Rat Schwann Cells or C2C12 cells were transfected with plasmids expressing mi871 or mi405 structures 4 and 8, respectively, driven by the hMPZ/rMPZ or CK6/HSA promoters, respectively.
  • Control conditions included HEK-293 cells undergoing a mock transfection, transfection with a plasmid expressing mi871/mi405 under the pol III U6 promoter system, and transfection of a non-targeting miRNA, miLacZ, driven by the pol III U6 promoter system. Each condition was run in triplicate according to the conditions described above. At 48 hours post-transfection, total RNA was isolated, samples were rDNase-treated, and cDNA was synthesized following the aforementioned protocols disclosed herein above. [00291] Finally, the expression of mi871/mi405, target genes rPmp22 and DUX4, and the normalizer gene Rpl13a were measured for each sample using ddPCR.
  • the copies of mi871/mi405 and target genes rPmp22/DUX4 per ⁇ L were normalized to copies of Rpl13a per ⁇ L and plotted using Prism 9 (Graphpad).
  • Human and rat MPZ promoters were each able to drive expression of mi871 in a rat Schwann cell culture (Fig.34).
  • the pol II tissue 28335/58942 2023-012-02 specific systems demonstrated comparable knockdown (hMPZ.mi871-4: 49% knockdown; rMPZ.mi871-4: 51% knockdown) of rat Pmp22 to the pol III system (U6.mi871: 66% knockdown) (Fig.34).
  • the CMV promoter in the aforementioned mi871-4 and mi871-8 constructs were replaced with either human or rat MPZ promoter sequences with the 3’ end of the promoter sequence being cloned into the AgeI site of each miRNA construct (Fig.33).
  • the resulting constructs were co-transfected with Psicheck.Human- PMP22 into rat Schwann cells in addition to testing their CMV-driven predecessors, U6.mi871, an empty U6T6 vector, and a control U6-driven miRNA.
  • Renilla and Firefly luciferase expression was measured by a GloMax Discover Luminescence Plate Reader (Promega) and percent knockdown was calculated by normalizing each ratio of Renilla:Firefly expression to the positive control -- the empty U6T6 vector (Fig.34).
  • hMPZ.mi871-4 showed a knockdown efficiency of 85% compared with 93% for U6.mi871 (Fig.34). This showed that we were able to achieve a similar level of knockdown while driving mi871 expression from Schwann cell-specific promoter compared to the Pol III system.

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Abstract

L'invention concerne un système modulaire, c'est-à-dire une cassette d'expression d'ADN, spécifiquement conçu pour convertir des cassettes d'expression de miARN thérapeutiques de l'utilisation de promoteurs d'ARN polymérase III ubiquiste à l'utilisation de promoteurs d'ARN polymérase Il pour permettre l'expression spécifique de tissu des miARN tout en maintenant la fidélité et l'efficacité du traitement. L'invention concerne également des compositions, des vecteurs, des nanoparticules, des vésicules extracellulaires et des exosomes comprenant la cassette d'expression d'acide nucléique, ainsi que des procédés d'utilisation et des procédés de traitement faisant appel à la cassette d'expression d'ADN.
PCT/US2024/027396 2023-05-02 2024-05-02 Système modulaire pour convertir des cassettes d'expression de microarn thérapeutique de promoteurs de polymérase iii à des promoteurs de polymérase ii WO2024229211A2 (fr)

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