WO2024182378A1 - Methods and materials for treating prader-willi syndrome - Google Patents

Methods and materials for treating prader-willi syndrome Download PDF

Info

Publication number
WO2024182378A1
WO2024182378A1 PCT/US2024/017458 US2024017458W WO2024182378A1 WO 2024182378 A1 WO2024182378 A1 WO 2024182378A1 US 2024017458 W US2024017458 W US 2024017458W WO 2024182378 A1 WO2024182378 A1 WO 2024182378A1
Authority
WO
WIPO (PCT)
Prior art keywords
snorna
polypeptide
nucleotide sequence
sequence encoding
adeno
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/US2024/017458
Other languages
French (fr)
Other versions
WO2024182378A9 (en
Inventor
Robert David Nicholls
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
University of Pittsburgh
Original Assignee
University of Pittsburgh
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by University of Pittsburgh filed Critical University of Pittsburgh
Publication of WO2024182378A1 publication Critical patent/WO2024182378A1/en
Anticipated expiration legal-status Critical
Publication of WO2024182378A9 publication Critical patent/WO2024182378A9/en
Ceased legal-status Critical Current

Links

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/46—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • C07K14/47—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
    • C12N15/113—Non-coding nucleic acids modulating the expression of genes, e.g. antisense oligonucleotides; Antisense DNA or RNA; Triplex- forming oligonucleotides; Catalytic nucleic acids, e.g. ribozymes; Nucleic acids used in co-suppression or gene silencing
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2830/00—Vector systems having a special element relevant for transcription
    • C12N2830/20—Vector systems having a special element relevant for transcription transcription of more than one cistron

Definitions

  • adeno-associated virus (AAV) vectors can be designed to express one or more nucleic acids associated with PWS (PWS nucleic acids) and administered to a mammal (e.g., a human) having PWS to treat the mammal.
  • PWS nucleic acids PWS nucleic acids
  • PWS is a neurodevelopmental disorder, characterized by numerous behavioral abnormalities and negative impacts on life expectancy and/or life quality.
  • PWS is genetically complex with loss of function of a 2-3 Mb domain of approximately 10 paternally-expressed, imprinted genes (Nicholls et al., Anna. Rev. Genomics Hum. Genet., 2:153-175 (2001); Chamberlain et al., NeurobioL Dis., 39: 13-20 (2010); and Chung et al., Open Biol., 10: 200195 (2020)), all with different and poorly understood, or even unknown, functions.
  • the actual causes of PWS clinical features are not known.
  • This document provides methods and materials for treating a mammal (e.g., a human) having PWS.
  • this document provides AAV vectors designed to express one or more (e.g., one, two, three, four, five, six, seven, eight, or more) PWS nucleic acids.
  • AAV vectors can be designed to express one or more PWS nucleic acids and can be administered to a mammal (e.g., a human) having PWS to treat the mammal.
  • an AAV vector having a genome (e.g., a single-stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a nucleotide sequence including a nucleotide sequence encoding a necdin (NDN) polypeptide, (b) a nucleotide sequence encoding a SNRPN upstream reading frame (SNURF) polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a small nuclear ribonucleoprotein-associated protein N (SNRPN) polypeptide, (d) a nucleotide sequence encoding a SNORD107 small nucleolar RN A (snoRNA), (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SN0RD116 snoRNA, (
  • AAV vectors e.g., a population of a single AAV vector having a genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 s
  • a nucleic acid sequence e.g., an engineered DNA sequence
  • a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide
  • AAV vectors provided herein e.g., one or more AAV vectors designed to express one or more PWS nucleic acids
  • AAV vectors provided herein can be produced using cost-effective methods.
  • a population of a single AAV vector provided herein can be used to increase expression of one or more PWS nucleic acids to treat PWS.
  • one aspect of this document features adeno-associated viruses having a single- stranded DNA, where the single-stranded DNA comprises a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence comprises (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 sno
  • the mammal can be a human.
  • the cell can be a pancreatic islet endocrine cell.
  • the cell can be a gut endocrine cell.
  • the cell can be a pituitary neuroendocrine cell.
  • the adeno-associated virus can be AAV6, AAV8, AAV3, AAVDJ, AAVKP1, or AAV9.
  • the first ITR sequence can be directly followed by the engineered DNA sequence.
  • the first ITR sequence can be indirectly followed by the engineered DNA sequence.
  • the engineered DNA sequence can be directly followed by the second ITR sequence.
  • the engineered DNA sequence can be indirectly followed by the second ITR sequence.
  • the first ITR sequence can include the DNA sequence set forth in SEQ ID NO:32 and the first ITR sequence can include the DNA sequence set forth in SEQ ID NO:34.
  • the first promotor sequence can include four of the motifs.
  • the second promotor sequence can include six of the motifs.
  • Each of the motifs can include a DNA sequence set forth in any one of SEQ ID NOs: 11 to 20.
  • Each of the motifs can include a DNA sequence set forth in any one of SEQ ID NOs: 11 to 14.
  • Each of the 8 to 12 intervening nucleotides can include a DNA sequence set forth in any one of SEQ ID NOs: 21 to 29.
  • Each of the 8 to 12 intervening nucleotides can include a DNA sequence set forth in any one of SEQ ID NOs: 21 to 23.
  • the necdin polypeptide can be a human necdin polypeptide.
  • the necdin polypeptide can include the amino acid sequence set forth in SEQ ID NO: 1.
  • the SNURF polypeptide can be a human SNURF polypeptide.
  • the SNURF polypeptide can include the amino acid sequence set forth in SEQ ID NO:2.
  • the SNRPN polypeptide can be a human SNRPN polypeptide.
  • the SNRPN polypeptide can include the amino acid sequence set forth in SEQ ID NO:3.
  • the SNORD107 snoRNA can be a human SNORD107 snoRNA.
  • the SNORD107 snoRNA can include the RNA sequence set forth in SEQ ID NO:4.
  • the SNORD64 snoRNA can be a human SNORD64 snoRNA.
  • the SNORD64 snoRNA can include the RNA sequence set forth in SEQ ID NO:5.
  • the SNORD116 snoRNA can be a human SNORD116 snoRNA.
  • the SNORD116 snoRNA can include the RNA sequence set forth in SEQ ID NO: 6 or SEQ ID NO:7.
  • the SNORD115 snoRNA can be a human SNORD115 snoRNA.
  • the SNORD115 snoRNA can include the RNA sequence set forth in SEQ ID NO:8.
  • the SNQRD109 snoRNAcan include the RNA sequence set forth in SEQ ID NO:9.
  • the first DNA segment can include, downstream of the nucleotide sequence encoding the necdin polypeptide, a poly(A) signal.
  • the poly(A) signal can include an SV40 poly(A) signal.
  • the poly(A) signal can include the DNA sequence AATAAA or the DNA sequence ATTAAA.
  • the second DNA segment can include, downstream of the nucleotide sequence encoding the SNURF polypeptide, the nucleotide sequence encoding the SNRPN polypeptide, the nucleotide sequence encoding the SNQRD107 snoRNA, the nucleotide sequence encoding the SNORD64 snoRNA, the nucleotide sequence encoding the SNORD116 snoRNA, the nucleotide sequence encoding the SNORD115 snoRNA, and the nucleotide sequence encoding the SNORD109 snoRNA, a poly(A) signal.
  • the poly(A) signal can include an SV40 poly(A) signal.
  • the poly(A) signal can include the DNA sequence AATAAA or the DNA sequence ATTAAA.
  • this document features isolated nucleic acid molecules comprising the engineered DNA sequence having (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a
  • this document features in vitro host cells, where the host cell comprises an adeno-associated virus having a single-stranded DNA, where the singlestranded DNA comprises a first ITR sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence comprises (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 s
  • this document features in vitro host cells, where the host cell comprises a nucleic acid having a (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109
  • compositions comprising an adeno- associated virus having a single-stranded DNA, where the single-stranded DNA comprises a ITR sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence comprises (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence
  • this document features methods for increasing expression of a necdin polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA by cells within a mammal.
  • the methods can include, or consist essentially of, administering an adeno-associated virus having a single-stranded DNA, where the singlestranded DNA comprises a first ITR sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence comprises (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA,
  • this document features methods for treating Prader-Willi syndrome.
  • the methods can include, or consist essentially of, administering an adeno-associated virus having a single-stranded DNA, where the single-stranded DNA comprises a first ITR sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence comprises (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleo
  • Figures 1A - 1D PWS-minigene design for single AAV delivery.
  • Figure 1A Engineered DNA sequences including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 snoRNA, and (h) a nucleotide sequence encoding a SNORD109 snoRNA fit within the 4.75
  • Figure 1B A schematic showing an exemplary design of a nucleotide sequence encoding a SNURF polypeptide, a SNRPN polypeptide, and five snoRNAs (i.e., a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA), where the five snoRNAs are located within individual synthetic introns between protein-coding exons.
  • snoRNAs i.e., a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA
  • Figure 1C A schematic showing an exemplary design of a nucleotide sequence encoding a SNURF polypeptide, a SNRPN polypeptide, and five snoRNAs (i.e., a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA), where the five snoRNAs are clustered within a single segment between protein-coding exons.
  • snoRNAs i.e., a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA
  • Figure 1D A schematic showing an exemplary design of a nucleotide sequence encoding a SNURF polypeptide, a SNRPN polypeptide, and five snoRNAs (i.e., a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA), where the five snoRNAs are located 3' of SNURF-SNRPN as in the endogenous locus.
  • snoRNAs i.e., a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA
  • NRF1- binding sites in promoters are shown by hash marks.
  • FIG. 1 Expression levels of mNeonGreen in transfected INS-1 ⁇ -cells.
  • mNeonGreen driven by the CMV promoter left panel
  • a 4xNRF1 promoter array right panel
  • a 10XNRF1 promoter array right panel
  • FIGs 3A - 3D PWS INS-1 ⁇ -cell model.
  • Figures 3 A Genome editing led to isolation of PWS-lines (3, 19-1, and 19-4) having 3.16 Mb deletions on the paternal allele with loss of expression of PWS nucleic acids (Snrpn, Snord116, and not shown), while control lines (2, 16, and 5-9+) expressed the PWS nucleic acids. All lines were clonally expanded from single cells.
  • the insulin antibody detects all forms of insulin (PrePro, Pro, and fully processed), and mouse proINS2- or C-peptide (CP)- mCherry.
  • the KDEL antibody detects GRP94 (endoplasmin) and GRP78 (BiP).
  • This document provides methods and materials for treating a mammal (e.g., a human) having PWS.
  • this document provides AAV vectors designed to express one or more (e.g., one, two, three, four five, six, seven, eight, or more) PWS nucleic acids.
  • an AAV vector designed to express one or more PWS nucleic acids can have a genome (e.g., a single-stranded or double-stranded DNA genome) including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 snoRNA, and (h) a nucleotide sequence encoding a genome (e
  • an AAV vector designed to express one or more PWS nucleic acids can have a genome (e.g., a single-stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 s
  • AAV vectors provided herein can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA within a cell (e.g., within a cell within a mammal such as a human).
  • one or more AAV vectors provided herein can be administered to a mammal (e.g., a human) having PWS to treat the mammal.
  • a population of a single AAV vector provided herein can be used to increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within a mammal (e.g., a human) having PWS to treat the mammal.
  • a mammal e.g., a human
  • Any appropriate AAV can be designed to express one or more PWS nucleic acids (e.g., can be designed to have a genome including a nucleic acid sequence including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA).
  • a nucleic acid sequence including (a) a DNA sequence including a nucle
  • an AAV vector designed to express one or more PWS nucleic acids can have a single-stranded or doublestranded DNA genome. When containing a single-stranded DNA genome, the AAV vector designed to express one or more PWS nucleic acids can be a positive-strand virus or a negative-strand virus. In some cases, an AAV vector designed to express one or more PWS nucleic acids can infect dividing cells. In some cases, an AAV vector designed to express one or more PWS nucleic acids can infect non-dividing cells.
  • AAV vectors that can be designed to express one or more PWS nucleic acids as described herein (e.g., designed to have a genome including a nucleic acid sequence including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA) include, without limitation, serotype 9 AAV (AAV9) vectors, A
  • An AAV vector provided herein can include any appropriate nucleic acid encoding a NDN polypeptide.
  • an AAV vector having a genome e.g., a single-stranded or double-stranded DNA genome
  • a nucleic acid sequence e.g., an engineered DNA sequence
  • a nucleic acid encoding a NDN polypeptide in an AAV vector provided herein can encode any appropriate NDN polypeptide.
  • an AAV vector having a genome e.g., a singlestranded or double-stranded DNA genome
  • a nucleic acid sequence e.g., an engineered DNA sequence
  • a NDN polypeptide that can be encoded by a nucleic acid encoding a NDN polypeptide in an AAV vector provided herein can have the amino acid sequence set forth in the National Center for Biotechnology Information (NCBI) databases at Accession No. NM 002487.
  • NBI National Center for Biotechnology Information
  • a NDN polypeptide that can be encoded by a nucleic acid encoding a NDN polypeptide in an AAV vector provided herein e.g., an AAV vector designed to express one or more PWS nucleic acids
  • SEQ ID NO:1 amino acid sequence set forth in SEQ ID NO:1.
  • a NDN polypeptide that can be encoded by a nucleic acid encoding a NDN polypeptide in an AAV vector provided herein can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 1.
  • a NDN polypeptide that can be encoded by a nucleic acid encoding a NDN polypeptide in an AAV vector provided herein can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:1 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof.
  • a NDN polypeptide that can be encoded by a nucleic acid encoding a NDN polypeptide in an AAV vector provided herein can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 1 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof.
  • An AAV vector provided herein can include any appropriate nucleic acid encoding a SNURF polypeptide.
  • an AAV vector having a genome e.g., a single-stranded or doublestranded DNA genome
  • a nucleic acid sequence e.g., an engineered DNA sequence
  • a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence
  • a nucleic acid encoding a SNURF polypeptide in an AAV vector provided herein can encode any appropriate SNURF polypeptide.
  • an AAV vector having a genome e.g., a single- stranded or double-stranded DNA genome
  • a nucleic acid sequence e.g., an engineered DNA sequence
  • a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 s
  • a SNURF polypeptide that can be encoded by a nucleic acid encoding a SNURF polypeptide in an AAV vector provided herein can have the amino acid sequence set forth in the NCBI databases at Accession No. NP 005669.2.
  • a SNURF polypeptide that can be encoded by a nucleic acid encoding a SNURF polypeptide in an AAV vector provided herein can have the amino acid sequence set forth in SEQ ID NO:2.
  • a SNURF polypeptide that can be encoded by a nucleic acid encoding a SNURF polypeptide in an AAV vector provided herein can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:2.
  • a SNURF polypeptide that can be encoded by a nucleic acid encoding a SNURF polypeptide in an AAV vector provided herein can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:2 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof.
  • a SNURF polypeptide that can be encoded by a nucleic acid encoding a SNURF polypeptide in an AAV vector provided herein can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:2 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof.
  • An AAV vector provided herein can include any appropriate nucleic acid encoding a SNRPN polypeptide.
  • an AAV vector having a genome e.g., a single-stranded or doublestranded DNA genome
  • a nucleic acid sequence e.g., an engineered DNA sequence
  • a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence
  • a nucleic acid encoding a SNRPN polypeptide in an AAV vector provided herein can encode any appropriate SNRPN polypeptide.
  • an AAV vector having a genome e.g., a single- stranded or double-stranded DNA genome
  • a nucleic acid sequence e.g., an engineered DNA sequence
  • a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 s
  • a SNRPN polypeptide that can be encoded by a nucleic acid encoding a SNRPN polypeptide in an AAV vector provided herein can have the amino acid sequence set forth in the NCBI databases at Accession No. NP 003088.1.
  • a SNRPN polypeptide that can be encoded by a nucleic acid encoding a SNRPN polypeptide in an AAV vector provided herein can have the amino acid sequence set forth in SEQ ID NO:3.
  • a SNRPN polypeptide that can be encoded by a nucleic acid encoding a SNRPN polypeptide in an AAV vector provided herein can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:3.
  • a SNRPN polypeptide that can be encoded by a nucleic acid encoding a SNRPN polypeptide in an AAV vector provided herein can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:3 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof.
  • a SNRPN polypeptide that can be encoded by a nucleic acid encoding a SNRPN polypeptide in an AAV vector provided herein can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:3 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof.
  • An AAV vector provided herein can include a nucleic acid encoding any appropriate snoRNA.
  • a snoRNA can be a C/D box small nucleolar RNA (SNORD).
  • snoRNAs that can be encoded by a nucleic acid encoding any appropriate snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) include, without limitation, SNORD 107 snoRNAs, SNORD64 snoRNAs, SNORD116 snoRNAs, SNORD115 snoRNAs, and SNORD 109 snoRNAs (e.g., SNORD 109A snoRNAs and SNORD 109B snoRNAs).
  • an AAV vector provided herein can include nucleic acid encoding a SNORD107 snoRNA.
  • An AAV vector provided herein e.g., an AAV vector designed to express one or more PWS nucleic acids
  • a nucleic acid encoding a SNORD107 snoRNA in an AAV vector provided herein can encode any appropriate SNORD107 snoRNA.
  • a SNORD107 snoRNA that can be encoded by a nucleic acid encoding a SNORD107 snoRNA in an AAV vector provided herein can have the nucleic acid sequence set forth in the NCBI databases at Accession No. NR_001293.
  • a SNORD107 snoRNA that can be encoded by a nucleic acid encoding a SNORD107 snoRNA in an AAV vector provided herein e.g., an AAV vector designed to express one or more PWS nucleic acids
  • a SNORD107 snoRNA that can be encoded by a nucleic acid encoding a SNORD107 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:4.
  • a SNORD107 snoRNA that can be encoded by a nucleic acid encoding a SNORD107 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:4 with one, two, three, four, five, six, seven, eight, nine, or ten nucleotide deletions, additions, substitutions, or combinations thereof.
  • a SNORD107 snoRNA that can be encoded by a nucleic acid encoding a SNORD107 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:4 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less nucleotide deletions, additions, substitutions, or combinations thereof.
  • an AAV vector provided herein can include nucleic acid encoding a SNORD64 snoRNA.
  • An AAV vector provided herein e.g., an AAV vector designed to express one or more PWS nucleic acids
  • a nucleic acid encoding a SNORD64 snoRNA in an AAV vector provided herein can encode any appropriate SNORD64 snoRNA.
  • a SNORD64 snoRNA that can be encoded by a nucleic acid encoding a SNORD64 snoRNA in an AAV vector provided herein can have the nucleic acid sequence set forth in the NCBI databases at Accession No. NR_001294.
  • a SNORD64 snoRNA that can be encoded by a nucleic acid encoding a SNORD64 snoRNA in an AAV vector provided herein can have the RNA sequence set forth in SEQ ID NO: 5.
  • a SNORD64 snoRNA that can be encoded by a nucleic acid encoding a SNORD64 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:5.
  • a SNORD64 snoRNA that can be encoded by a nucleic acid encoding a SNORD64 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 5 with one, two, three, four, five, six, seven, eight, nine, or ten nucleotide deletions, additions, substitutions, or combinations thereof.
  • a SNORD64 snoRNA that can be encoded by a nucleic acid encoding a SNORD64 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 5 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less nucleotide deletions, additions, substitutions, or combinations thereof.
  • an AAV vector provided herein can include nucleic acid encoding a SNORD116 snoRNA.
  • An AAV vector provided herein e.g., an AAV vector designed to express one or more PWS nucleic acids
  • a nucleic acid encoding a SNORD116 snoRNA in an AAV vector provided herein can encode any appropriate SNORD116 snoRNA.
  • a SNORD116 snoRNA that can be encoded by a nucleic acid encoding a SNORD116 snoRNA in an AAV vector provided herein can have the nucleic acid sequence set forth in the NCBI databases at Accession Nos. NR 003324 and NR 003332.
  • a SNORD116 snoRNA that can be encoded by a nucleic acid encoding a SNORD116 snoRNA in an AAV vector provided herein can have the RNA sequence set forth in SEQ ID NO: 6 or SEQ ID NO:7.
  • a SNORD116 snoRNA that can be encoded by a nucleic acid encoding a SNORD116 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:6 or SEQ ID NO:7.
  • a SNORD116 snoRNA that can be encoded by a nucleic acid encoding a SNORD116 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:6 or SEQ ID NO:7 with one, two, three, four, five, six, seven, eight, nine, or ten nucleotide deletions, additions, substitutions, or combinations thereof.
  • a SNORD116 snoRNA that can be encoded by a nucleic acid encoding a SNORD116 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less nucleotide deletions, additions, substitutions, or combinations thereof.
  • an AAV vector provided herein can include nucleic acid encoding a SNORD115 snoRNA.
  • An AAV vector provided herein e.g., an AAV vector designed to express one or more PWS nucleic acids
  • a nucleic acid encoding a SNORD115 snoRNA in an AAV vector provided herein can encode any appropriate SNORD115 snoRNA.
  • a SNORD115 snoRNA that can be encoded by a nucleic acid encoding a SNORD115 snoRNA in an AAV vector provided herein can have the nucleic acid sequence set forth in the NCBI databases at Accession No. NR_003304.
  • a SNORD115 snoRNA that can be encoded by a nucleic acid encoding a SNORD115 snoRNA in an AAV vector provided herein can have the RNA sequence set forth in SEQ ID NO:8.
  • a SNORD115 snoRNA that can be encoded by a nucleic acid encoding a SNORD115 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:8.
  • a SNORD115 snoRNA that can be encoded by a nucleic acid encoding a SNORD115 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 8 with one, two, three, four, five, six, seven, eight, nine, or ten nucletoide deletions, additions, substitutions, or combinations thereof.
  • a SNORD115 snoRNA that can be encoded by a nucleic acid encoding a SNORD 115 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 8 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less nucleotide deletions, additions, substitutions, or combinations thereof.
  • an AAV vector provided herein can include nucleic acid encoding a SNORD109 snoRNA.
  • An AAV vector provided herein e.g., an AAV vector designed to express one or more PWS nucleic acids
  • a nucleic acid encoding a SNORD109 snoRNA in an AAV vector provided herein can encode any appropriate SNORD 109 snoRNA.
  • a SNORD109 snoRNA that can be encoded by a nucleic acid encoding a SNORD109 snoRNA in an AAV vector provided herein can have the nucleic acid sequence set forth in the NCBI databases at Accession No. NR_001295.
  • a SNORD109 snoRNA that can be encoded by a nucleic acid encoding a SNORD109 snoRNA in an AAV vector provided herein e.g., an AAV vector designed to express one or more PWS nucleic acids
  • a SNORD109 snoRNA that can be encoded by a nucleic acid encoding a SNORD109 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:9.
  • a SNORD109 snoRNA that can be encoded by a nucleic acid encoding a SNORD109 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 9 with one, two, three, four, five, six, seven, eight, nine, or ten nucleotide deletions, additions, substitutions, or combinations thereof.
  • a SNORD109 snoRNA that can be encoded by a nucleic acid encoding a SNORD109 snoRNA in an AAV vector provided herein can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 9 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less nucleotide deletions, additions, substitutions, or combinations thereof.
  • a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and/or a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include one or more regulatory elements operably linked to the nucleotide sequence encoding a NDN polypeptide and/or to the nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNURF poly
  • regulatory elements can include, without limitation, promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid.
  • the choice of regulatory element(s) that can be included in an AAV vector provided herein can depend on several factors, including, without limitation, inducibility, targeting, and the level of expression desired.
  • a promoter can be included in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) to facilitate transcription of a nucleic acid encoding a polypeptide, and/or a nucleotide sequence encoding a snoRNA.
  • a promoter can be a naturally occurring promoter, a synthetic promoter, or a recombinant promoter.
  • a promoter can be ubiquitous or inducible (e.g., in the presence of tetracycline), and can affect the expression of a nucleic acid encoding a polypeptide or a nucleic acid encoding a snoRNA in a general or tissue-specific manner.
  • an AAV vector provided herein e.g., an AAV vector designed to express one or more PWS nucleic acids
  • the promoter can be operably linked to the nucleic acid encoding a NDN polypeptide such that it drives expression of the NDN polypeptide in cells.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include at least four (e.g., 4, 5, 6, 7, 8, 9, 10, or more) motifs having a DNA sequence of 5'-YGCGCANGCGCR-3' (SEQ ID NO: 10), each separated by an intervening nucleotide sequence. Examples of motifs having a DNA sequence of 5'- YGCGCANGCGCR-3' (SEQ ID NO: 10) are set forth in Table 1.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include 10 motifs having a DNA sequence of 5 -YGCGCANGCGCR- 3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include motifs set forth in each of SEQ ID NOs: 11 to 20, with each adjacent motif being separated by an intervening nucleotide sequence.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include 4 motifs having a DNA sequence of 5 -YGCGCANGCGCR-3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include motifs set forth in each of SEQ ID NOs: 11 to 14, with each adjacent motif being separated by an intervening nucleotide sequence.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include at least four (e.g., 4, 5, 6, 7, 8, 9, 10, or more) motifs having a DNA sequence of 5'-YGCGCANGCGCR-3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence, can include any appropriate intervening nucleotide sequences.
  • An intervening nucleotide sequence can include any appropriate number of nucleotides. In some cases, an intervening nucleotide sequence can include from about 8 to about 12 nucleotides.
  • intervening nucleotide sequences that can be present between each adjacent motif having a DNA sequence of 5 -YGCGCANGCGCR-3' (SEQ ID NO: 10) in a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells are set forth in Table 2.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include 10 motifs having a DNA sequence of 5 -YGCGCANGCGCR- 3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include 10 motifs having a DNA sequence of 5'- YGCGCANGCGCR-3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence set forth in each of SEQ ID NOs: 21 to 29.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include 4 motifs having a DNA sequence of 5 -YGCGCANGCGCR-3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include 4 motifs having a DNA sequence of 5'-YGCGCANGCGCR-3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence set forth in each of SEQ ID NOs: 21 to 23.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include the sequence set forth in SEQ ID NO: 30.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include the sequence set forth in SEQ ID NO: 31.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can comprise, consist essentially of, or consist of one of the amino acid sequences set forth in Example 2.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can comprise, consist essentially of, or consist of one of the amino acid sequences set forth in Example 2 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid or nucleotide deletions, additions, substitutions, or combinations thereof.
  • a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can comprise, consist essentially of, or consist of one of the amino acid sequences set forth in Example 2 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid or nucleotide deletions, additions, substitutions, or combinations thereof.
  • nucleotide sequences encoding snoRNAs can be located between polypeptide-encoding portions (e.g., exons) of a nucleotide sequence encoding a SNURF polypeptide and/or polypeptide-encoding portions (e.g., exons)
  • polypeptide-encoding portions e.g., exons
  • polypeptide-encoding portions e.g., exons
  • nucleotide sequences encoding snoRNAs in a nucleic acid sequence (e.g., an engineered DNA sequence) in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can be clustered within non-coding portions (e.g., introns) of a nucleotide sequence encoding a SNURF polypeptide and/or non-coding portions (e.g., introns) a nucleo
  • nucleotide sequences encoding snoRNAs can be located upstream (5' of) and/or downstream (3' of) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide and a nucleotide sequence encoding a SNRPN polypeptide.
  • a nucleic acid sequence including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can be flanked by a first ITR sequence and a second ITR sequence.
  • a first ITR sequence can be directly or indirectly followed by a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA.
  • a nucleic acid sequence e.g., an engineered DNA sequence
  • a nucleic acid sequence including
  • a nucleic acid sequence including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can be directly or indirectly followed by a second ITR sequence.
  • ITR sequences that can flank a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA within an AAV vector provided herein are set forth in Table 3.
  • a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA within an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can be flanked by a first I
  • one or more AAV vectors provided herein can be formulated into a composition (e.g., a pharmaceutically acceptable composition).
  • a composition e.g., a pharmaceutically acceptable composition
  • one or more AAV vectors provided herein e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids
  • Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin
  • compositions containing one or more AAV vectors provided herein can be formulated for oral or parenteral (including, without limitation, intramuscular, intravenous, intradermal, intra-cerebral, intrathecal, or intraperitoneal (i.p.) injection) administration to the mammal.
  • parenteral including, without limitation, intramuscular, intravenous, intradermal, intra-cerebral, intrathecal, or intraperitoneal (i.p.) injection
  • compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient.
  • nucleic acid molecules including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA.
  • a nucleic acid sequence e.g., an engineered DNA sequence
  • a nucleic acid sequence e.g., an engine
  • cells e.g., host cells
  • AAV vectors provided herein
  • a host cell containing one or more AAV vectors provided herein can be an in vitro cell (e.g., a cell in an in vitro culture).
  • cells e.g., host cells containing a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA.
  • a nucleic acid sequence e.g., an engineered DNA sequence
  • a nucleic acid sequence e.
  • cells e.g., host cells
  • AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) and/or a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence en
  • AAV vectors provided herein e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids.
  • one or more AAV vectors provided herein e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids
  • a mammal e.g., a human having PWS to treat the mammal.
  • one or more AAV vectors provided herein can be used to increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within a mammal (e.g., a human) having PWS to treat the mammal.
  • a mammal e.g., a human
  • one or more AAV vectors provided herein can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having PWS) to increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within the mammal.
  • a mammal e.g., a human
  • a mammal e.g., a human
  • a mammal e.g., a human in need thereof (e.g., a human having PWS) to increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a
  • the materials and methods described herein can be used to increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within a mammal having PWS by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
  • One or more AAV vectors provided herein can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA in any appropriate type of cells within a mammal (e.g., a human).
  • a mammal e.g., a human
  • one or more AAV vectors designed to express one or more PWS nucleic acids can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA in pancreatic islet endocrine cells (e.g., pancreatic islet ⁇ -cells and pancreatic islet ⁇ -cells), gut endocrine cells, neurons (e.g., hypothalamic endocrine cells), pituitary neuroendocrine cells, or any combinations thereof.
  • pancreatic islet endocrine cells e.g., pancreatic islet ⁇ -cells and pancreatic islet ⁇ -cells
  • gut endocrine cells e.g., hypothalamic endocrine cells
  • one or more AAV vectors provided herein can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having PWS) to reduce or eliminate one or more symptoms of PWS.
  • Examples of symptoms of PWS include, without limitation, hypotonia, distinct facial features (e.g., almond-shaped eyes, a narrowing of the head at the temples, a turned-down mouth, and a thin upper lip), poor infant sucking reflex, underdeveloped genitals, constant craving for food, rapid weight gain, short adult height, low muscle mass, high body fat, hypothyroidism, central adrenal insufficiency, intellectual disability, delayed motor development, speech problems (e.g., speech delays and poor articulation), scoliosis, hip problems, reduced saliva flow, nearsightedness, problems regulating body temperature, a high pain tolerance, hypopigmentation, hypoglycemia episodes, and hormone abnormalities (e.g., reductions in hormones such as growth hormone, insulin, pancreatic hormones, gut hormones, pituitary hormones, and/or hypothalamic hormones and increases in hormones such as ghrelin).
  • the materials and methods described herein can be used to reduce one or more symptoms of PWS within a
  • any appropriate mammal having PWS can be treated as described herein (e.g., by administering a population of a single AAV vector provided herein such as an AAV vector having genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 sno
  • Examples of mammals that can have PWS and that can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, pigs, sheep, mice, and rats. In some cases, a human having PWS can be treated as described herein.
  • the methods described herein can include identifying a mammal (e.g., a human) as having PWS by, for example, identifying that cells within the mammal include one or more mutations in an endogenous PWS locus. Any appropriate method can be used to identify the presence of one or more mutations in an endogenous PWS locus. For example, chromosomal analysis techniques can be used to identify the presence of one or more mutations in an endogenous PWS locus.
  • any appropriate method can be used to deliver one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered to a mammal (e.g., a human) having PWS.
  • a composition e.g., a pharmaceutically acceptable composition
  • AAV vectors provided herein e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids
  • a composition e.g., a pharmaceutically acceptable composition
  • AAV vectors provided herein e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids
  • composition containing one or more AAV vectors provided herein can be administered locally via endoscopic retrograde cholangio-pancreatography (ERCP).
  • ERCP endoscopic retrograde cholangio-pancreatography
  • Any appropriate amount (e.g., any appropriate dose) of one or more AAV vectors provided herein can be administered to a mammal (e.g., a human) having PWS.
  • An effective amount of a composition containing one or more AAV vectors provided herein can be any amount that can treat a mammal having PWS as described herein (e.g., that can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within the mammal) without producing significant toxicity to the mammal.
  • a composition containing one or more AAV vectors provided herein can be any amount that can treat a mammal having PWS as described herein (e.g., that can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA,
  • an effective amount of one or more AAV vectors provided herein can be from about 1E+11 viral genomes to about 1E+13 viral genomes.
  • the effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment.
  • Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and/or severity of the PWS in the mammal being treated may require an increase or decrease in the actual effective amount administered.
  • One or more AAV vectors provided herein can be administered to a mammal (e.g., a human) having PWS in any appropriate frequency.
  • the frequency of administration can be any frequency that can treat a mammal having PWS as described herein (e.g., that can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within the mammal) without producing significant toxicity to the mammal.
  • the frequency of administration can be from about once a day to about once a week, from about once a week to about once a month, or from about twice a month to about once a month.
  • the frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, and/or route of administration may require an increase or decrease in administration frequency.
  • One or more AAV vectors provided herein can be administered to a mammal (e.g., a human) having PWS for any appropriate duration.
  • An effective duration for administering or using a composition containing one or more AAV vectors provided herein can be any duration that can treat a mammal having PWS as described herein (e.g., that can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within the mammal) without producing significant toxicity to the mammal.
  • the effective duration can vary from several weeks to several months, from several months to several years, or from several years to a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, and/or route of administration.
  • methods for treating a mammal (e.g., a human) having PWS can include administering to the mammal one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) as the sole active ingredient to treat the mammal.
  • a composition containing one or more AAV vectors provided herein can include the one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) as the sole active ingredient in the composition that is effective to treat a mammal having PWS.
  • methods for treating a mammal e.g., a human having PWS as described herein (e.g., by administering one or more AAV vectors provided herein such as AAV vectors designed to express one or more PWS nucleic acids) also can include administering to the mammal one or more (e.g., one, two, three, four, five or more) agents that can treat one or more symptoms of PWS.
  • agents that can be used to treat one or more symptoms of PWS and can be administered together with one or more AAV vectors provided herein include, without limitation, GH treatments (e.g., human growth hormone (HGH) treatments), sex hormone treatments, psychiatric agents, and any combinations thereof.
  • GH treatments e.g., human growth hormone (HGH) treatments
  • sex hormone treatments e.g., sex hormone treatments
  • psychiatric agents e.g., psychiatric agents
  • agents that can be used to treat one or more symptoms of PWS can be as described elsewhere (see, e.g., Mahmoud et al. , Int. J. Mol.
  • one or more AAV vectors provided herein e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids
  • the one or more agents used to treat one or more symptoms of PWS can be administered at the same time (e.g., in a single composition containing both the one or more AAV vectors provided herein and the one or more agents used to treat one or more symptoms of PWS) or independently.
  • one or more AAV vectors provided herein can be administered first, and the one or more agents used to treat one or more symptoms of PWS administered second, or vice versa.
  • methods for treating a mammal (e.g., a human) having PWS as described herein e.g., by administering one or more AAV vectors provided herein such as AAV vectors designed to express one or more PWS nucleic acids
  • methods for treating a mammal e.g., a human
  • methods for treating a mammal e.g., a human
  • AAV vectors provided herein e.g., by administering one or more AAV vectors provided herein such as AAV vectors designed to express one or more PWS nucleic acids
  • therapies that can be used to treat PWS include, without limitation, nutrition coaching (e.g., a healthy, reduced-calorie diet, a restricted-calorie diet, and supplemental vitamins and/or mineral), increasing physical activity and exercise, physical therapy (e.g., to improve movement skills and strength), speech therapy (e.g., to improve verbal skills), occupational therapy (e.g., to learn everyday skills), and combinations thereof.
  • nutrition coaching e.g., a healthy, reduced-calorie diet, a restricted-calorie diet, and supplemental vitamins and/or mineral
  • increasing physical activity and exercise e.g., physical therapy (e.g., to improve movement skills and strength), speech therapy (e.g., to improve verbal skills), occupational therapy (e.g., to learn everyday skills), and combinations thereof.
  • one or more AAV vectors provided herein e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids
  • the one or more additional therapies can be performed at the same time or independently of the administration of one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids).
  • one or more AAV vectors provided herein e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids
  • Example 1 AAV vectors with miniaturized promoters
  • AAV vectors that can express one or more PWS nucleic acids (e.g., AAV vectors designed to have a genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 snoRNA, and (h) a nucleic acid sequence
  • NRF1 -array mini-promoters allow nucleic acid sequences that can encode 8 of the 10 major PWS genes to fit into a single AAV ( Figure 1 A).
  • Each vector insert includes SNURF (216-nt with stop codon; 71 amino acids) and SNRPN (723 -nt; 240 amino acids) in the endogenous bicistronic layout with a 188-nt intercistronic region, as well as SNORD107, SNORD64, SNORD116, SNORD115, and SNORD109 snoRNAs (each 70-120 nt).
  • AAV vectors containing inserts that contain nucleic acid that can encode one or more PWS nucleic acids are also referred to herein as AAV-PWS minigenes.
  • the CMV promoter-enhancer (613 -nt) in an AAV vector was substituted with minimal synthetic NRF1 -array mini-promoters.
  • the 4xNRF1 (84-nt) and 10xNRF1 (216-nt) promoters were characterized, and it was shown that these promoters drive high levels of mNeonGreen expression in a variety of tested cell lines including INS1 ⁇ -cells ( Figure 2), fibroblasts, myoblasts, schwannoma, and others.
  • NRF1 -array mini- promoters mimic endogenous regulation as in vivo expression of SNURF-SNRPN-snoRNA long intergenic non-coding RNAs (lincRNAs) is controlled by alternate 5'-promoters driven by several NRF1 binding sites and an intron 1 enhancer featuring NRF1.
  • in vivo expression of NDN is primarily regulated by NRF1.
  • PWS ⁇ - (INS-1) cell models show a cell-autonomous defect in insulin secretion along with deficits in RNA and protein production of multiple hormones and ER chaperones (Figure 3 A).
  • AAV-PWS minigene vectors are packaged as serotype AAV6 or AAV8 for transduction of control and PWS ⁇ -cell lines (3 each), and transduced cells were assessed for changes in levels of insulin secretion (Figure 3B), mRNA levels for Ins1, Ins2, lapp, Npy, and ER chaperones Sdf2ll, Dnajbll, Creld2, Grp78, Pdia4, Grp94, and Hyou1 using ddRT- PCR, and protein levels for peptide hormones (e.g., insulin) and major ER chaperones (Figure 3C and Figure 3D).
  • peptide hormones e.g., insulin
  • Figure 3C and Figure 3D protein levels for peptide hormones (e.g., insulin) and major ER chaperones
  • Designed vector inserts are produced by in vitro DNA synthesis and standard molecular cloning techniques used to insert between the AAV inverted terminal repeats (ITRs).
  • ITRs AAV inverted terminal repeats
  • a multiplicity of infection (MOI) of 100,000 AAV genome copies/cell is used for cell transduction, with equivalent ddRT-PCR and protein (western blot) assays confirming the expression of each cistron.
  • mRNA (SNURF-SNRPN, NDN) and snoRNA expression is measured by droplet digital ddRT-PCR (with PCR primers specific for human exogenous vs. rat endogenous genes).
  • Polypeptide expression for SNURF, SmN/SNRPN, and NDN is assessed by western blot analysis. Constructs that express all gene components are converted to AAV vectors.
  • a human identified as having PWS is administered a composition including a population of a single AAV vector designed to express one or more PWS nucleic acids (e.g., designed to have a genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 s
  • the single AAV vector designed to express one or more PWS nucleic acids is effective to increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA by cells within that mammal.
  • a human identified as having PWS is administered a composition including a population of a single AAV vector designed to express one or more PWS nucleic acids (e.g., designed to have a genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 s

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Engineering & Computer Science (AREA)
  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Zoology (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Wood Science & Technology (AREA)
  • Biophysics (AREA)
  • Biochemistry (AREA)
  • Medicinal Chemistry (AREA)
  • Plant Pathology (AREA)
  • Physics & Mathematics (AREA)
  • Microbiology (AREA)
  • Virology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Toxicology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Epidemiology (AREA)
  • Animal Behavior & Ethology (AREA)
  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)
  • Micro-Organisms Or Cultivation Processes Thereof (AREA)

Abstract

This document provides methods and materials for treating a mammal (e.g., a human) having Prader-Willi syndrome (PWS). For example, adeno-associated virus (AAV) vectors can be designed to express one or more nucleic acids associated with PWS (PWS nucleic acids) and administered to a mammal (e.g., a human) having PWS to treat the mammal.

Description

METHODS AND MATERIALS FOR TREATING PRADER-WILLI SYNDROME
CROSS-REFERENCE TO RELATED APPLICATIONS
This application claims the benefit of U.S. Patent Application Serial No. 63/448,587, filed on February 27, 2023. The disclosure of the prior application is considered part of, and is incorporated by reference in, the disclosure of this application.
SEQUENCE LISTING
This application contains a Sequence Listing that has been submitted electronically as an XML file named “48881-0052W01_SL.xml.” The XML file, created on February 13, 2024, is 44000 bytes in size. The material in the XML file is hereby incorporated by reference in its entirety.
TECHNICAL FIELD
This document relates to methods and materials for treating a mammal (e.g., a human) having Prader-Willi syndrome (PWS). For example, adeno-associated virus (AAV) vectors can be designed to express one or more nucleic acids associated with PWS (PWS nucleic acids) and administered to a mammal (e.g., a human) having PWS to treat the mammal.
BACKGROUND
PWS is a neurodevelopmental disorder, characterized by numerous behavioral abnormalities and negative impacts on life expectancy and/or life quality. PWS is genetically complex with loss of function of a 2-3 Mb domain of approximately 10 paternally-expressed, imprinted genes (Nicholls et al., Anna. Rev. Genomics Hum. Genet., 2:153-175 (2001); Chamberlain et al., NeurobioL Dis., 39: 13-20 (2010); and Chung et al., Open Biol., 10: 200195 (2020)), all with different and poorly understood, or even unknown, functions. Despite advances in understanding genetic causes of PWS and having animal models that mimic some clinical components, the actual causes of PWS clinical features are not known. Consequently, current therapeutic approaches for PWS utilize behavioral modification, such as change in diet and growth hormone (GH)-treatment. Although such therapeutic approaches can improve outcomes, none is a cure, and significant clinical morbidity persists (Chung et al., Open Biol., 10: 200195 (2020); and Tauber et al., Lancet Diabetes Endocrinol ., 9: 235- 246 (2021)). Development of new therapies to reactivate or replace silent or missing PWS genes is complicated by heterochromatic silencing or the large number of genes (Chung et al., Open Biol., 10: 200195 (2020); Wang et al., Trends Pharmacol. Sci., 40: 605-608 (2019); and Kim et al., Transl. Res., 208: 105-118 (2019)).
SUMMARY
This document provides methods and materials for treating a mammal (e.g., a human) having PWS. In some cases, this document provides AAV vectors designed to express one or more (e.g., one, two, three, four, five, six, seven, eight, or more) PWS nucleic acids. As described herein, AAV vectors can be designed to express one or more PWS nucleic acids and can be administered to a mammal (e.g., a human) having PWS to treat the mammal. For example, an AAV vector having a genome (e.g., a single-stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a nucleotide sequence including a nucleotide sequence encoding a necdin (NDN) polypeptide, (b) a nucleotide sequence encoding a SNRPN upstream reading frame (SNURF) polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a small nuclear ribonucleoprotein-associated protein N (SNRPN) polypeptide, (d) a nucleotide sequence encoding a SNORD107 small nucleolar RN A (snoRNA), (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SN0RD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 snoRNA, and (h) a nucleotide sequence encoding a SNORD109 snoRNA can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SN0RD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA within a cell (e.g., within a cell within a mammal such as a human).
Having the ability to increase expression of one or more PWS nucleic acids within a cell (e.g., within a cell within a mammal such as a human) using one or more AAV vectors provided herein (e.g., a population of a single AAV vector having a genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 snoRNA, and (h) a nucleotide sequence encoding a SNORD109 snoRNA) provides a unique and unrealized opportunity to treat having PWS. In addition, AAV vectors provided herein (e.g., one or more AAV vectors designed to express one or more PWS nucleic acids) can be produced using cost-effective methods. In some cases, a population of a single AAV vector provided herein can be used to increase expression of one or more PWS nucleic acids to treat PWS.
In general, one aspect of this document features adeno-associated viruses having a single- stranded DNA, where the single-stranded DNA comprises a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence comprises (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA, where each of the first promotor sequence and the second promotor sequence comprises at least four motifs having the DNA sequence set forth in SEQ ID NO: 10 (5 -YGCGCANGCGCR-3'), where 8 to 12 intervening nucleotides are located between each adjacent motif of the at least four motifs, and where delivery of the adeno-associated virus to a cell within the mammal results in expression of the necdin polypeptide, the SNURF polypeptide, the SNRPN polypeptide, the SNORD107 snoRNA, the SNORD64 snoRNA, the SNORD116 snoRNA, the SNORD115 snoRNA, and the SNORD109 snoRNA. The mammal can be a human. The cell can be a pancreatic islet endocrine cell. The cell can be a gut endocrine cell. The cell can be a pituitary neuroendocrine cell. The adeno-associated virus can be AAV6, AAV8, AAV3, AAVDJ, AAVKP1, or AAV9. The first ITR sequence can be directly followed by the engineered DNA sequence. The first ITR sequence can be indirectly followed by the engineered DNA sequence. The engineered DNA sequence can be directly followed by the second ITR sequence. The engineered DNA sequence can be indirectly followed by the second ITR sequence. The first ITR sequence can include the DNA sequence set forth in SEQ ID NO:32 and the first ITR sequence can include the DNA sequence set forth in SEQ ID NO:34. The first promotor sequence can include four of the motifs. The second promotor sequence can include six of the motifs. Each of the motifs can include a DNA sequence set forth in any one of SEQ ID NOs: 11 to 20. Each of the motifs can include a DNA sequence set forth in any one of SEQ ID NOs: 11 to 14. Each of the 8 to 12 intervening nucleotides can include a DNA sequence set forth in any one of SEQ ID NOs: 21 to 29. Each of the 8 to 12 intervening nucleotides can include a DNA sequence set forth in any one of SEQ ID NOs: 21 to 23. The necdin polypeptide can be a human necdin polypeptide. The necdin polypeptide can include the amino acid sequence set forth in SEQ ID NO: 1. The SNURF polypeptide can be a human SNURF polypeptide. The SNURF polypeptide can include the amino acid sequence set forth in SEQ ID NO:2. The SNRPN polypeptide can be a human SNRPN polypeptide. The SNRPN polypeptide can include the amino acid sequence set forth in SEQ ID NO:3. The SNORD107 snoRNA can be a human SNORD107 snoRNA. The SNORD107 snoRNA can include the RNA sequence set forth in SEQ ID NO:4. The SNORD64 snoRNA can be a human SNORD64 snoRNA. The SNORD64 snoRNA can include the RNA sequence set forth in SEQ ID NO:5. The SNORD116 snoRNA can be a human SNORD116 snoRNA. The SNORD116 snoRNA can include the RNA sequence set forth in SEQ ID NO: 6 or SEQ ID NO:7. The SNORD115 snoRNA can be a human SNORD115 snoRNA. The SNORD115 snoRNA can include the RNA sequence set forth in SEQ ID NO:8. The SNORD109 snoRNAcan be a human SNORD109 snoRNA. The SNQRD109 snoRNAcan include the RNA sequence set forth in SEQ ID NO:9. The first DNA segment can include, downstream of the nucleotide sequence encoding the necdin polypeptide, a poly(A) signal. The poly(A) signal can include an SV40 poly(A) signal. The poly(A) signal can include the DNA sequence AATAAA or the DNA sequence ATTAAA. The second DNA segment can include, downstream of the nucleotide sequence encoding the SNURF polypeptide, the nucleotide sequence encoding the SNRPN polypeptide, the nucleotide sequence encoding the SNQRD107 snoRNA, the nucleotide sequence encoding the SNORD64 snoRNA, the nucleotide sequence encoding the SNORD116 snoRNA, the nucleotide sequence encoding the SNORD115 snoRNA, and the nucleotide sequence encoding the SNORD109 snoRNA, a poly(A) signal. The poly(A) signal can include an SV40 poly(A) signal. The poly(A) signal can include the DNA sequence AATAAA or the DNA sequence ATTAAA. In another aspect, this document features isolated nucleic acid molecules comprising the engineered DNA sequence having (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA, where each of the first promotor sequence and the second promotor sequence comprises at least four motifs having the DNA sequence set forth in SEQ ID NO: 10 (5 -YGCGCANGCGCR-3'), where 8 to 12 intervening nucleotides are located between each adjacent motif of the at least four motifs, and where delivery of the adeno-associated virus to a cell within the mammal results in expression of the necdin polypeptide, the SNURF polypeptide, the SNRPN polypeptide, the SNORD107 snoRNA, the SNORD64 snoRNA, the SNORD116 snoRNA, the SNORD115 snoRNA, and the SNORD109 snoRNA.
In another aspect, this document features in vitro host cells, where the host cell comprises an adeno-associated virus having a single-stranded DNA, where the singlestranded DNA comprises a first ITR sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence comprises (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA, where each of the first promotor sequence and the second promotor sequence comprises at least four motifs having the DNA sequence set forth in SEQ ID NO: 10 (5'-YGCGCANGCGCR-3'), where 8 to 12 intervening nucleotides are located between each adjacent motif of the at least four motifs, and where delivery of the adeno-associated virus to a cell within the mammal results in expression of the necdin polypeptide, the SNURF polypeptide, the SNRPN polypeptide, the SNORD107 snoRNA, the SNORD64 snoRNA, the SNORD116 snoRNA, the SNORD115 snoRNA, and the SNORD109 snoRNA. The host cell can be an epithelial cell.
In another aspect, this document features in vitro host cells, where the host cell comprises a nucleic acid having a (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA, where each of the first promotor sequence and the second promotor sequence comprises at least four motifs having the DNA sequence set forth in SEQ ID NO: 10 (5 -YGCGCANGCGCR-3'), where 8 to 12 intervening nucleotides are located between each adjacent motif of the at least four motifs, and where delivery of the adeno-associated virus to a cell within the mammal results in expression of the necdin polypeptide, the SNURF polypeptide, the SNRPN polypeptide, the SNORD107 snoRNA, the SNORD64 snoRNA, the SNORD116 snoRNA, the SNORD115 snoRNA, and the SNORD109 snoRNA. The host cell can be an epithelial cell.
In another aspect, this document features compositions comprising an adeno- associated virus having a single-stranded DNA, where the single-stranded DNA comprises a ITR sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence comprises (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA, where each of the first promotor sequence and the second promotor sequence comprises at least four motifs having the DNA sequence set forth in SEQ ID NO:10 (5'-YGCGCANGCGCR-3'), where 8 to 12 intervening nucleotides are located between each adjacent motif of the at least four motifs, and where delivery of the adeno-associated virus to a cell within the mammal results in expression of the necdin polypeptide, the SNURF polypeptide, the SNRPN polypeptide, the SNORD107 snoRNA, the SNORD64 snoRNA, the SNORD116 snoRNA, the SNORD115 snoRNA, and the SNORD109 snoRNA.
In another aspect, this document features methods for increasing expression of a necdin polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA by cells within a mammal. The methods can include, or consist essentially of, administering an adeno-associated virus having a single-stranded DNA, where the singlestranded DNA comprises a first ITR sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence comprises (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA, where each of the first promotor sequence and the second promotor sequence comprises at least four motifs having the DNA sequence set forth in SEQ ID NO: 10 (5'-YGCGCANGCGCR-3'), where 8 to 12 intervening nucleotides are located between each adjacent motif of the at least four motifs, and where delivery of the adeno-associated virus to a cell within the mammal results in expression of the necdin polypeptide, the SNURF polypeptide, the SNRPN polypeptide, the SNORD107 snoRNA, the SNORD64 snoRNA, the SNORD116 snoRNA, the SNORD115 snoRNA, and the SNORD109 snoRNA to the mammal, thereby increasing expression of the necdin polypeptide, the SNURF polypeptide, the SNRPN polypeptide, the SNORD107 snoRNA, the SNORD64 snoRNA, the SNORD116 snoRNA, the SNORDU5 snoRNA, and the SNORD109 snoRNAby the cells within the mammal. The mammal can be a human.
In another aspect, this document features methods for treating Prader-Willi syndrome. The methods can include, or consist essentially of, administering an adeno-associated virus having a single-stranded DNA, where the single-stranded DNA comprises a first ITR sequence followed by an engineered DNA sequence followed by a second ITR sequence, where the engineered DNA sequence comprises (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA, where each of the first promotor sequence and the second promotor sequence comprises at least four motifs having the DNA sequence set forth in SEQ ID NO:10 (5'-YGCGCANGCGCR-3'), where 8 to 12 intervening nucleotides are located between each adjacent motif of the at least four motifs, and where delivery of the adeno-associated virus to a cell within the mammal results in expression of the necdin polypeptide, the SNURF polypeptide, the SNRPN polypeptide, the SNORD107 snoRNA, the SNORD64 snoRNA, the SNORD116 snoRNA, the SNORD115 snoRNA, and the SNORD109 snoRNA to a mammal having Prader-Willi syndrome, where expression of a necdin polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNAby cells within the mammal is increased following the administering step, and where severity of a symptom of the Prader-Willi syndrome is reduced following the administering step. The mammal can be a human.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. Although methods and materials similar or equivalent to those described herein can be used to practice the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The details of one or more embodiments of the invention are set forth in the accompanying drawings and the description below. Other features, objects, and advantages of the invention will be apparent from the description and drawings, and from the claims.
BRIEF DESCRIPTION OF THE DRAWINGS
Figures 1A - 1D. PWS-minigene design for single AAV delivery. Figure 1A) Engineered DNA sequences including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 snoRNA, and (h) a nucleotide sequence encoding a SNORD109 snoRNA fit within the 4.75 kb packaging capacity of a single AAV vector. Figure 1B) A schematic showing an exemplary design of a nucleotide sequence encoding a SNURF polypeptide, a SNRPN polypeptide, and five snoRNAs (i.e., a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA), where the five snoRNAs are located within individual synthetic introns between protein-coding exons. Figure 1C) A schematic showing an exemplary design of a nucleotide sequence encoding a SNURF polypeptide, a SNRPN polypeptide, and five snoRNAs (i.e., a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA), where the five snoRNAs are clustered within a single segment between protein-coding exons. Figure 1D) A schematic showing an exemplary design of a nucleotide sequence encoding a SNURF polypeptide, a SNRPN polypeptide, and five snoRNAs (i.e., a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA), where the five snoRNAs are located 3' of SNURF-SNRPN as in the endogenous locus. Numbered boxes encode protein-coding exons of SNURF-SNRPN; NDN coding exon; snoRNAs are individually labeled (as is NDN protein-coding exon in Fig. 1 A); unlabeled black boxes are non-coding rapidly degraded microexons (from SNHG1); the left most box is the 5'-UTRand the right most box is the 3'-UTR with polyA (pA) sites; in Fig. 1A, NRF1- binding sites in promoters are shown by hash marks.
Figure 2. Expression levels of mNeonGreen in transfected INS-1 β-cells. mNeonGreen driven by the CMV promoter (left panel), by a 4xNRF1 promoter array (middle panel), or by a 10XNRF1 promoter array (right panel).
Figures 3A - 3D. PWS INS-1 β-cell model. Figures 3 A) Genome editing led to isolation of PWS-lines (3, 19-1, and 19-4) having 3.16 Mb deletions on the paternal allele with loss of expression of PWS nucleic acids (Snrpn, Snord116, and not shown), while control lines (2, 16, and 5-9+) expressed the PWS nucleic acids. All lines were clonally expanded from single cells. Figure 3B) PWS β-cell lines have insulin secretion deficits at low (2.2 mM) and high (22 mM) glucose levels, based on the pooled insulin secretory rates from n=36 for each of PWS and control groups (n=12 per cell line). Although glucose-stimulated secretion increased 1.75-fold for control (p<0.0001) and 1.85-fold for PWS (p<0.0001), loss of expression of PWS genes decreased the secretory rate by 36% (p<0.0001) in basal 2.8 rnM glucose conditions and by 32% (p<0.0001) in stimulatory 22 mM glucose conditions. Figures 3C and 3D) Reduced insulins and ER chaperones in PWS INS1 β-cell lines. Insulin (Figure 3C), mCherry, KDEL (Figure 3D), and control (TUBG or GPI) antibodies were used in western blots of whole cell lysates from β-cell lines under control (DMSO) conditions for 5 hours. The insulin antibody detects all forms of insulin (PrePro, Pro, and fully processed), and mouse proINS2- or C-peptide (CP)- mCherry. The KDEL antibody detects GRP94 (endoplasmin) and GRP78 (BiP).
DETAILED DESCRIPTION
This document provides methods and materials for treating a mammal (e.g., a human) having PWS. In some cases, this document provides AAV vectors designed to express one or more (e.g., one, two, three, four five, six, seven, eight, or more) PWS nucleic acids. For example, an AAV vector designed to express one or more PWS nucleic acids can have a genome (e.g., a single-stranded or double-stranded DNA genome) including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 snoRNA, and (h) a nucleotide sequence encoding a SNORD109 snoRNA. In some cases, an AAV vector designed to express one or more PWS nucleic acids can have a genome (e.g., a single-stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA. In some cases, AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA within a cell (e.g., within a cell within a mammal such as a human). In some cases, one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered to a mammal (e.g., a human) having PWS to treat the mammal. For example, a population of a single AAV vector provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be used to increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within a mammal (e.g., a human) having PWS to treat the mammal.
Any appropriate AAV can be designed to express one or more PWS nucleic acids (e.g., can be designed to have a genome including a nucleic acid sequence including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA). In some cases, an AAV vector designed to express one or more PWS nucleic acids can have a single-stranded or doublestranded DNA genome. When containing a single-stranded DNA genome, the AAV vector designed to express one or more PWS nucleic acids can be a positive-strand virus or a negative-strand virus. In some cases, an AAV vector designed to express one or more PWS nucleic acids can infect dividing cells. In some cases, an AAV vector designed to express one or more PWS nucleic acids can infect non-dividing cells. Examples AAV vectors that can be designed to express one or more PWS nucleic acids as described herein (e.g., designed to have a genome including a nucleic acid sequence including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA) include, without limitation, serotype 9 AAV (AAV9) vectors, AAV6 vectors, AAV8 vectors, AAV3 vectors, AAVDJ vectors, and AAVKP1 vectors.
An AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include any appropriate nucleic acid encoding a NDN polypeptide. For example, an AAV vector having a genome (e.g., a single-stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include any appropriate nucleic acid encoding a NDN polypeptide.
A nucleic acid encoding a NDN polypeptide in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can encode any appropriate NDN polypeptide. For example, an AAV vector having a genome (e.g., a singlestranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include nucleic acid encoding any appropriate NDN polypeptide. In some cases, a NDN polypeptide that can be encoded by a nucleic acid encoding a NDN polypeptide in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the amino acid sequence set forth in the National Center for Biotechnology Information (NCBI) databases at Accession No. NM 002487. In some cases, a NDN polypeptide that can be encoded by a nucleic acid encoding a NDN polypeptide in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the amino acid sequence set forth in SEQ ID NO:1. In some cases, a NDN polypeptide that can be encoded by a nucleic acid encoding a NDN polypeptide in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 1. In some cases, a NDN polypeptide that can be encoded by a nucleic acid encoding a NDN polypeptide in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:1 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof. In some cases, a NDN polypeptide that can be encoded by a nucleic acid encoding a NDN polypeptide in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO: 1 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof.
An AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include any appropriate nucleic acid encoding a SNURF polypeptide. For example, an AAV vector having a genome (e.g., a single-stranded or doublestranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include any appropriate nucleic acid encoding a SNURF polypeptide.
A nucleic acid encoding a SNURF polypeptide in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can encode any appropriate SNURF polypeptide. For example, an AAV vector having a genome (e.g., a single- stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include nucleic acid encoding any appropriate SNURF polypeptide. In some cases, a SNURF polypeptide that can be encoded by a nucleic acid encoding a SNURF polypeptide in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the amino acid sequence set forth in the NCBI databases at Accession No. NP 005669.2. In some cases, a SNURF polypeptide that can be encoded by a nucleic acid encoding a SNURF polypeptide in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the amino acid sequence set forth in SEQ ID NO:2. In some cases, a SNURF polypeptide that can be encoded by a nucleic acid encoding a SNURF polypeptide in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:2. In some cases, a SNURF polypeptide that can be encoded by a nucleic acid encoding a SNURF polypeptide in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:2 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof. In some cases, a SNURF polypeptide that can be encoded by a nucleic acid encoding a SNURF polypeptide in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:2 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof.
An AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include any appropriate nucleic acid encoding a SNRPN polypeptide. For example, an AAV vector having a genome (e.g., a single-stranded or doublestranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include any appropriate nucleic acid encoding a SNRPN polypeptide.
A nucleic acid encoding a SNRPN polypeptide in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can encode any appropriate SNRPN polypeptide. For example, an AAV vector having a genome (e.g., a single- stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include nucleic acid encoding any appropriate SNRPN polypeptide. In some cases, a SNRPN polypeptide that can be encoded by a nucleic acid encoding a SNRPN polypeptide in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the amino acid sequence set forth in the NCBI databases at Accession No. NP 003088.1. In some cases, a SNRPN polypeptide that can be encoded by a nucleic acid encoding a SNRPN polypeptide in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the amino acid sequence set forth in SEQ ID NO:3. In some cases, a SNRPN polypeptide that can be encoded by a nucleic acid encoding a SNRPN polypeptide in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:3. In some cases, a SNRPN polypeptide that can be encoded by a nucleic acid encoding a SNRPN polypeptide in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:3 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid deletions, additions, substitutions, or combinations thereof. In some cases, a SNRPN polypeptide that can be encoded by a nucleic acid encoding a SNRPN polypeptide in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the amino acid sequence set forth in SEQ ID NO:3 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid deletions, additions, substitutions, or combinations thereof.
An AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include a nucleic acid encoding any appropriate snoRNA. In some cases, a snoRNA can be a C/D box small nucleolar RNA (SNORD). Examples of snoRNAs that can be encoded by a nucleic acid encoding any appropriate snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) include, without limitation, SNORD 107 snoRNAs, SNORD64 snoRNAs, SNORD116 snoRNAs, SNORD115 snoRNAs, and SNORD 109 snoRNAs (e.g., SNORD 109A snoRNAs and SNORD 109B snoRNAs).
In some cases, an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include nucleic acid encoding a SNORD107 snoRNA. An AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include any appropriate nucleic acid encoding a SNORD 107 snoRNA. For example, an AAV vector having a genome (e.g., a single- stranded or doublestranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include any appropriate nucleotide sequence encoding a SNORD107 snoRNA.
A nucleic acid encoding a SNORD107 snoRNA in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can encode any appropriate SNORD107 snoRNA. For example, an AAV vector having a genome (e.g., a single- stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include nucleic acid encoding any appropriate SNORD107 snoRNA. In some cases, a SNORD107 snoRNA that can be encoded by a nucleic acid encoding a SNORD107 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the nucleic acid sequence set forth in the NCBI databases at Accession No. NR_001293. In some cases, a SNORD107 snoRNA that can be encoded by a nucleic acid encoding a SNORD107 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the RNA sequence set forth in SEQ ID NO:4. In some cases, a SNORD107 snoRNA that can be encoded by a nucleic acid encoding a SNORD107 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:4. In some cases, a SNORD107 snoRNA that can be encoded by a nucleic acid encoding a SNORD107 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:4 with one, two, three, four, five, six, seven, eight, nine, or ten nucleotide deletions, additions, substitutions, or combinations thereof. In some cases, a SNORD107 snoRNA that can be encoded by a nucleic acid encoding a SNORD107 snoRNA in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:4 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less nucleotide deletions, additions, substitutions, or combinations thereof.
In some cases, an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include nucleic acid encoding a SNORD64 snoRNA. An AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include any appropriate nucleic acid encoding a SNORD64 snoRNA. For example, an AAV vector having a genome (e.g., a single- stranded or doublestranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include any appropriate nucleotide sequence encoding a SNORD64 snoRNA.
A nucleic acid encoding a SNORD64 snoRNA in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can encode any appropriate SNORD64 snoRNA. For example, an AAV vector having a genome (e.g., a single- stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include nucleic acid encoding any appropriate SNORD64 snoRNA. In some cases, a SNORD64 snoRNA that can be encoded by a nucleic acid encoding a SNORD64 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the nucleic acid sequence set forth in the NCBI databases at Accession No. NR_001294. In some cases, a SNORD64 snoRNA that can be encoded by a nucleic acid encoding a SNORD64 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the RNA sequence set forth in SEQ ID NO: 5. In some cases, a SNORD64 snoRNA that can be encoded by a nucleic acid encoding a SNORD64 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:5. In some cases, a SNORD64 snoRNA that can be encoded by a nucleic acid encoding a SNORD64 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 5 with one, two, three, four, five, six, seven, eight, nine, or ten nucleotide deletions, additions, substitutions, or combinations thereof. In some cases, a SNORD64 snoRNA that can be encoded by a nucleic acid encoding a SNORD64 snoRNA in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 5 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less nucleotide deletions, additions, substitutions, or combinations thereof.
In some cases, an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include nucleic acid encoding a SNORD116 snoRNA. An AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include any appropriate nucleic acid encoding a SNORD116 snoRNA. For example, an AAV vector having a genome (e.g., a single-stranded or doublestranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNQRD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNQRD109 snoRNA can include any appropriate nucleotide sequence encoding a SNORD116 snoRNA. A nucleic acid encoding a SNORD116 snoRNA in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can encode any appropriate SNORD116 snoRNA. For example, an AAV vector having a genome (e.g., a single- stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include nucleic acid encoding any appropriate SNORD116 snoRNA. In some cases, a SNORD116 snoRNA that can be encoded by a nucleic acid encoding a SNORD116 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the nucleic acid sequence set forth in the NCBI databases at Accession Nos. NR 003324 and NR 003332. In some cases, a SNORD116 snoRNA that can be encoded by a nucleic acid encoding a SNORD116 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the RNA sequence set forth in SEQ ID NO: 6 or SEQ ID NO:7. In some cases, a SNORD116 snoRNA that can be encoded by a nucleic acid encoding a SNORD116 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:6 or SEQ ID NO:7. In some cases, a SNORD116 snoRNA that can be encoded by a nucleic acid encoding a SNORD116 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:6 or SEQ ID NO:7 with one, two, three, four, five, six, seven, eight, nine, or ten nucleotide deletions, additions, substitutions, or combinations thereof. In some cases, a SNORD116 snoRNA that can be encoded by a nucleic acid encoding a SNORD116 snoRNA in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 6 or SEQ ID NO: 7 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less nucleotide deletions, additions, substitutions, or combinations thereof.
In some cases, an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include nucleic acid encoding a SNORD115 snoRNA. An AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include any appropriate nucleic acid encoding a SNORD115 snoRNA. For example, an AAV vector having a genome (e.g., a single-stranded or doublestranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include any appropriate nucleotide sequence encoding a SNORD115 snoRNA.
A nucleic acid encoding a SNORD115 snoRNA in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can encode any appropriate SNORD115 snoRNA. For example, an AAV vector having a genome (e.g., a single- stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include nucleic acid encoding any appropriate SNORD115 snoRNA. In some cases, a SNORD115 snoRNA that can be encoded by a nucleic acid encoding a SNORD115 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the nucleic acid sequence set forth in the NCBI databases at Accession No. NR_003304. In some cases, a SNORD115 snoRNA that can be encoded by a nucleic acid encoding a SNORD115 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the RNA sequence set forth in SEQ ID NO:8. In some cases, a SNORD115 snoRNA that can be encoded by a nucleic acid encoding a SNORD115 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:8. In some cases, a SNORD115 snoRNA that can be encoded by a nucleic acid encoding a SNORD115 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 8 with one, two, three, four, five, six, seven, eight, nine, or ten nucletoide deletions, additions, substitutions, or combinations thereof. In some cases, a SNORD115 snoRNA that can be encoded by a nucleic acid encoding a SNORD 115 snoRNA in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 8 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less nucleotide deletions, additions, substitutions, or combinations thereof.
In some cases, an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include nucleic acid encoding a SNORD109 snoRNA. An AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can include any appropriate nucleic acid encoding a SNORD109 snoRNA. For example, an AAV vector having a genome (e.g., a single-stranded or doublestranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD 109 snoRNA can include any appropriate nucleotide sequence encoding a SNORD109 snoRNA.
A nucleic acid encoding a SNORD109 snoRNA in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can encode any appropriate SNORD 109 snoRNA. For example, an AAV vector having a genome (e.g., a single- stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include nucleic acid encoding any appropriate SNORD109 snoRNA. In some cases, a SNORD109 snoRNA that can be encoded by a nucleic acid encoding a SNORD109 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the nucleic acid sequence set forth in the NCBI databases at Accession No. NR_001295. In some cases, a SNORD109 snoRNA that can be encoded by a nucleic acid encoding a SNORD109 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can have the RNA sequence set forth in SEQ ID NO: 9. In some cases, a SNORD109 snoRNA that can be encoded by a nucleic acid encoding a SNORD109 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO:9. In some cases, a SNORD109 snoRNA that can be encoded by a nucleic acid encoding a SNORD109 snoRNA in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 9 with one, two, three, four, five, six, seven, eight, nine, or ten nucleotide deletions, additions, substitutions, or combinations thereof. In some cases, a SNORD109 snoRNA that can be encoded by a nucleic acid encoding a SNORD109 snoRNA in an AAV vector provided herein (e.g., a single AAV vector designed to express one or more PWS nucleic acids) can comprise, consist essentially of, or consist of the RNA sequence set forth in SEQ ID NO: 9 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less nucleotide deletions, additions, substitutions, or combinations thereof.
In some cases, a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and/or a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include one or more regulatory elements operably linked to the nucleotide sequence encoding a NDN polypeptide and/or to the nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA. For example, an AAV vector having a genome (e.g., a single-stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include one or more regulatory elements operably linked to the nucleotide sequence encoding a NDN polypeptide. For example, an AAV vector having a genome (e.g., a single-stranded or double-stranded DNA genome) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include one or more regulatory elements operably linked to the nucleotide sequence encoding a SNURF polypeptide, the nucleotide sequence encoding a SNRPN polypeptide, the nucleotide sequence encoding a SNORD107 snoRNA, the nucleotide sequence encoding a SNORD64 snoRNA, the nucleotide sequence encoding a SNORD116 snoRNA, the nucleotide sequence encoding a SNORD115 snoRNA, and the nucleotide sequence encoding a SNORD109 snoRNA. Such regulatory elements can include, without limitation, promoter sequences, enhancer sequences, response elements, signal peptides, internal ribosome entry sequences, polyadenylation signals, terminators, and inducible elements that modulate expression (e.g., transcription or translation) of a nucleic acid. The choice of regulatory element(s) that can be included in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can depend on several factors, including, without limitation, inducibility, targeting, and the level of expression desired. For example, a promoter can be included in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) to facilitate transcription of a nucleic acid encoding a polypeptide, and/or a nucleotide sequence encoding a snoRNA. A promoter can be a naturally occurring promoter, a synthetic promoter, or a recombinant promoter. A promoter can be ubiquitous or inducible (e.g., in the presence of tetracycline), and can affect the expression of a nucleic acid encoding a polypeptide or a nucleic acid encoding a snoRNA in a general or tissue-specific manner.
The term “operably linked” as used herein with respect to a regulatory element and a nucleic acid encoding a polypeptide and/or a nucleic acid encoding a snoRNA refers to positioning of the regulatory element relative to the nucleic acid encoding a polypeptide and/or a snoRNA in such a way as to permit or facilitate expression of the polypeptide and/or snoRNA. For example, an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can contain a promoter and nucleic acid encoding a NDN polypeptide. In this case, the promoter can be operably linked to the nucleic acid encoding a NDN polypeptide such that it drives expression of the NDN polypeptide in cells.
In some cases, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include at least four (e.g., 4, 5, 6, 7, 8, 9, 10, or more) motifs having a DNA sequence of 5'-YGCGCANGCGCR-3' (SEQ ID NO: 10), each separated by an intervening nucleotide sequence. Examples of motifs having a DNA sequence of 5'- YGCGCANGCGCR-3' (SEQ ID NO: 10) are set forth in Table 1.
Table 1. Exemplary motifs having a DNA sequence of 5'-YGCGCANGCGCR-3' (SEQ ID NO: 10).
Figure imgf000026_0001
Figure imgf000027_0001
In some cases, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include 10 motifs having a DNA sequence of 5 -YGCGCANGCGCR- 3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence. For example, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include motifs set forth in each of SEQ ID NOs: 11 to 20, with each adjacent motif being separated by an intervening nucleotide sequence. In some cases, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include 4 motifs having a DNA sequence of 5 -YGCGCANGCGCR-3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence. For example, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include motifs set forth in each of SEQ ID NOs: 11 to 14, with each adjacent motif being separated by an intervening nucleotide sequence.
A promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include at least four (e.g., 4, 5, 6, 7, 8, 9, 10, or more) motifs having a DNA sequence of 5'-YGCGCANGCGCR-3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence, can include any appropriate intervening nucleotide sequences. An intervening nucleotide sequence can include any appropriate number of nucleotides. In some cases, an intervening nucleotide sequence can include from about 8 to about 12 nucleotides. Examples of intervening nucleotide sequences that can be present between each adjacent motif having a DNA sequence of 5 -YGCGCANGCGCR-3' (SEQ ID NO: 10) in a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells are set forth in Table 2.
Table 2. Exemplary intervening nucleotide sequence.
Figure imgf000027_0002
Figure imgf000028_0001
In some cases, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include 10 motifs having a DNA sequence of 5 -YGCGCANGCGCR- 3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence. For example, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include 10 motifs having a DNA sequence of 5'- YGCGCANGCGCR-3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence set forth in each of SEQ ID NOs: 21 to 29. In some cases, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include 4 motifs having a DNA sequence of 5 -YGCGCANGCGCR-3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence. For example, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include 4 motifs having a DNA sequence of 5'-YGCGCANGCGCR-3' (SEQ ID NO: 10), with each adjacent motif being separated by an intervening nucleotide sequence set forth in each of SEQ ID NOs: 21 to 23.
In some cases, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include the sequence set forth in SEQ ID NO: 30.
In some cases, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can include the sequence set forth in SEQ ID NO: 31.
In some cases, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can comprise, consist essentially of, or consist of one of the amino acid sequences set forth in Example 2. In some cases, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can comprise, consist essentially of, or consist of one of the amino acid sequences set forth in Example 2 with one, two, three, four, five, six, seven, eight, nine, or ten amino acid or nucleotide deletions, additions, substitutions, or combinations thereof. In some cases, a promoter that can be used to drive expression of a polypeptide and/or a snoRNA in cells can comprise, consist essentially of, or consist of one of the amino acid sequences set forth in Example 2 with two or less, three or less, four or less, five or less, six or less, seven or less, eight or less, nine or less, or ten or less amino acid or nucleotide deletions, additions, substitutions, or combinations thereof.
In some cases, nucleotide sequences encoding snoRNAs (e.g., a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA) in a nucleic acid sequence (e.g., an engineered DNA sequence) in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can be located between polypeptide-encoding portions (e.g., exons) of a nucleotide sequence encoding a SNURF polypeptide and/or polypeptide-encoding portions (e.g., exons) a nucleotide sequence encoding a SNRPN polypeptide. For example, a nucleic acid sequence (e.g., an engineered DNA sequence) including a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include (1) a first polypeptide-encoding portion of a nucleotide sequence encoding a SNURF polypeptide, followed by (2) a nucleotide sequence encoding a SNORD116 snoRNA, followed by (3) a second polypeptide-encoding portion of a nucleotide sequence encoding a SNURF polypeptide, followed by (4) a nucleotide sequence encoding a SNORD107 snoRNA, followed by (5) a third polypeptide-encoding portion of a nucleotide sequence encoding a SNURF polypeptide, followed by (6) a nucleotide sequence encoding a SNORD64 snoRNA, followed by (7) a first polypeptide-encoding portion of a nucleotide sequence encoding a SNRPN polypeptide, followed by (8) a nucleotide sequence encoding a SNORD109 snoRNA, followed by (9) a second polypeptide-encoding portion of a nucleotide sequence encoding a SNRPN polypeptide, followed by (10) a nucleotide sequence encoding a SNORD115 snoRNA, followed by 11) a third polypeptide-encoding portion of a nucleotide sequence encoding a SNRPN polypeptide.
In some cases, nucleotide sequences encoding snoRNAs (e.g., a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA) in a nucleic acid sequence (e.g., an engineered DNA sequence) in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can be clustered within non-coding portions (e.g., introns) of a nucleotide sequence encoding a SNURF polypeptide and/or non-coding portions (e.g., introns) a nucleotide sequence encoding a SNRPN polypeptide. For example, a nucleic acid sequence (e.g., an engineered DNA sequence) including a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include (1) a nucleotide sequence encoding a SNURF polypeptide, followed by (2) a first polypeptide-encoding portion of a nucleotide sequence encoding a SNRPN polypeptide, followed by (3) a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA all clustered within a non-coding portion of a nucleotide sequence encoding a SNRPN polypeptide, followed by (4) a second polypeptide-encoding portion of a nucleotide sequence encoding a SNRPN polypeptide.
In some cases, nucleotide sequences encoding snoRNAs (e.g., a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA) in a nucleic acid sequence (e.g., an engineered DNA sequence) in an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can be located upstream (5' of) and/or downstream (3' of) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide and a nucleotide sequence encoding a SNRPN polypeptide. For example, a nucleic acid sequence (e.g., an engineered DNA sequence) including a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can include (1) a nucleotide sequence encoding a SNURF polypeptide, followed by (2) a first polypeptide-encoding portion of a nucleotide sequence encoding a SNRPN polypeptide, followed by (3) a second polypeptide-encoding portion of a nucleotide sequence encoding a SNRPN polypeptide, followed by (4) a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA all clustered within a non-coding portion of a nucleotide sequence encoding a SNRPN polypeptide located 3' of the second polypeptide-encoding portion of a nucleotide sequence encoding a SNRPN polypeptide.
In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can be flanked by a first ITR sequence and a second ITR sequence. In some cases, a first ITR sequence can be directly or indirectly followed by a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA. In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can be directly or indirectly followed by a second ITR sequence. Examples of ITR sequences that can flank a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA within an AAV vector provided herein are set forth in Table 3.
Table 3. Exemplary ITR sequences.
Figure imgf000032_0001
In some cases, a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA within an AAV vector provided herein (e.g., an AAV vector designed to express one or more PWS nucleic acids) can be flanked by a first ITR sequence having a DNA sequence set forth in SEQ ID NO:32 and a second ITR sequence having DNA sequence set forth in SEQ ID NO:34. In some cases, one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be formulated into a composition (e.g., a pharmaceutically acceptable composition). For example, one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be formulated together with one or more pharmaceutically acceptable carriers (additives), excipients, and/or diluents. Examples of pharmaceutically acceptable carriers, excipients, and diluents that can be used in a composition described herein include, without limitation, sucrose, lactose, starch (e.g., starch glycolate), cellulose, cellulose derivatives (e.g., modified celluloses such as microcrystalline cellulose, and cellulose ethers like hydroxypropyl cellulose (HPC) and cellulose ether hydroxypropyl methylcellulose (HPMC)), xylitol, sorbitol, mannitol, gelatin, polymers (e.g., polyvinylpyrrolidone (PVP), polyethylene glycol (PEG), crosslinked polyvinylpyrrolidone (crospovidone), carboxymethyl cellulose, polyethylene-polyoxypropylene-block polymers, and crosslinked sodium carboxymethyl cellulose (croscarmellose sodium)), titanium oxide, azo dyes, silica gel, fumed silica, talc, magnesium carbonate, vegetable stearin, magnesium stearate, aluminum stearate, stearic acid, antioxidants (e.g., vitamin A, vitamin E, vitamin C, retinyl palmitate, and selenium), citric acid, sodium citrate, parabens (e.g., methyl paraben and propyl paraben), petrolatum, dimethyl sulfoxide, mineral oil, serum proteins (e.g., human serum albumin), glycine, sorbic acid, potassium sorbate, water, salts or electrolytes (e.g., saline, protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, and zinc salts), colloidal silica, magnesium trisilicate, polyacrylates, waxes, wool fat, lecithin, and corn oil.
In some cases, a composition containing one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be formulated for oral or parenteral (including, without limitation, intramuscular, intravenous, intradermal, intra-cerebral, intrathecal, or intraperitoneal (i.p.) injection) administration to the mammal. Compositions suitable for parenteral administration include, without limitation, aqueous and non-aqueous sterile injection solutions that can contain antioxidants, buffers, bacteriostats, and solutes that render the formulation isotonic with the blood of the intended recipient.
Also provided herein are nucleic acid molecules (e.g., isolated nucleic acid molecules) including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA.
Also provided herein are cells (e.g., host cells) containing one or more AAV vectors provided herein (e.g., host cells containing a population of a single AAV vector designed to express one or more PWS nucleic acids). In some cases, a host cell containing one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be an in vitro cell (e.g., a cell in an in vitro culture).
Also provided herein are cells (e.g., host cells) containing a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA. In some cases, a host cell containing a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA can be an in vitro cell (e.g., a cell in an in vitro culture).
Examples of cells (e.g., host cells) that can contain one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) and/or a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a DNA sequence including a nucleotide sequence encoding a NDN polypeptide and (b) a DNA sequence including a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 snoRNA, a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SNORD116 snoRNA, a nucleotide sequence encoding a SNORD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA include, without limitation, epithelial cells such as HEK293 cells, endocrine cells, and neurons.
Also provided herein are methods for using one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids). In some cases, one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered to a mammal (e.g., a human) having PWS to treat the mammal. For example, one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be used to increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within a mammal (e.g., a human) having PWS to treat the mammal.
In some cases, one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having PWS) to increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within the mammal. In some cases, the materials and methods described herein can be used to increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within a mammal having PWS by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
One or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA in any appropriate type of cells within a mammal (e.g., a human). In some cases, one or more AAV vectors designed to express one or more PWS nucleic acids can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA in pancreatic islet endocrine cells (e.g., pancreatic islet β-cells and pancreatic islet α-cells), gut endocrine cells, neurons (e.g., hypothalamic endocrine cells), pituitary neuroendocrine cells, or any combinations thereof.
In some cases, one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered to a mammal (e.g., a human) in need thereof (e.g., a human having PWS) to reduce or eliminate one or more symptoms of PWS. Examples of symptoms of PWS include, without limitation, hypotonia, distinct facial features (e.g., almond-shaped eyes, a narrowing of the head at the temples, a turned-down mouth, and a thin upper lip), poor infant sucking reflex, underdeveloped genitals, constant craving for food, rapid weight gain, short adult height, low muscle mass, high body fat, hypothyroidism, central adrenal insufficiency, intellectual disability, delayed motor development, speech problems (e.g., speech delays and poor articulation), scoliosis, hip problems, reduced saliva flow, nearsightedness, problems regulating body temperature, a high pain tolerance, hypopigmentation, hypoglycemia episodes, and hormone abnormalities (e.g., reductions in hormones such as growth hormone, insulin, pancreatic hormones, gut hormones, pituitary hormones, and/or hypothalamic hormones and increases in hormones such as ghrelin). In some cases, the materials and methods described herein can be used to reduce one or more symptoms of PWS within a mammal having PWS by, for example, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, or more percent.
Any appropriate mammal having PWS can be treated as described herein (e.g., by administering a population of a single AAV vector provided herein such as an AAV vector having genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 snoRNA, and (h) a nucleotide sequence encoding a SNORD109 snoRNA). Examples of mammals that can have PWS and that can be treated as described herein include, without limitation, humans, non-human primates (e.g., monkeys), dogs, cats, pigs, sheep, mice, and rats. In some cases, a human having PWS can be treated as described herein.
In some cases, the methods described herein can include identifying a mammal (e.g., a human) as having PWS by, for example, identifying that cells within the mammal include one or more mutations in an endogenous PWS locus. Any appropriate method can be used to identify the presence of one or more mutations in an endogenous PWS locus. For example, chromosomal analysis techniques can be used to identify the presence of one or more mutations in an endogenous PWS locus.
Any appropriate method can be used to deliver one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered to a mammal (e.g., a human) having PWS. In some cases, a composition (e.g., a pharmaceutically acceptable composition) including one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered locally or systemically. In some cases, a composition containing one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered locally via endoscopic retrograde cholangio-pancreatography (ERCP).
Any appropriate amount (e.g., any appropriate dose) of one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered to a mammal (e.g., a human) having PWS. An effective amount of a composition containing one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be any amount that can treat a mammal having PWS as described herein (e.g., that can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within the mammal) without producing significant toxicity to the mammal. In some cases, an effective amount of one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be from about 1E+11 viral genomes to about 1E+13 viral genomes. The effective amount can remain constant or can be adjusted as a sliding scale or variable dose depending on the mammal’s response to treatment. Various factors can influence the actual effective amount used for a particular application. For example, the frequency of administration, duration of treatment, use of multiple treatment agents, route of administration, and/or severity of the PWS in the mammal being treated may require an increase or decrease in the actual effective amount administered.
One or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered to a mammal (e.g., a human) having PWS in any appropriate frequency. The frequency of administration can be any frequency that can treat a mammal having PWS as described herein (e.g., that can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within the mammal) without producing significant toxicity to the mammal. For example, the frequency of administration can be from about once a day to about once a week, from about once a week to about once a month, or from about twice a month to about once a month. The frequency of administration can remain constant or can be variable during the duration of treatment. As with the effective amount, various factors can influence the actual frequency of administration used for a particular application. For example, the effective amount, duration of treatment, use of multiple treatment agents, and/or route of administration may require an increase or decrease in administration frequency.
One or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered to a mammal (e.g., a human) having PWS for any appropriate duration. An effective duration for administering or using a composition containing one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be any duration that can treat a mammal having PWS as described herein (e.g., that can increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and/or a SNORD109 snoRNA by cells within the mammal) without producing significant toxicity to the mammal. For example, the effective duration can vary from several weeks to several months, from several months to several years, or from several years to a lifetime. Multiple factors can influence the actual effective duration used for a particular treatment. For example, an effective duration can vary with the frequency of administration, effective amount, use of multiple treatment agents, and/or route of administration. In some cases, methods for treating a mammal (e.g., a human) having PWS can include administering to the mammal one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) as the sole active ingredient to treat the mammal. For example, a composition containing one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can include the one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) as the sole active ingredient in the composition that is effective to treat a mammal having PWS.
In some cases, methods for treating a mammal (e.g., a human) having PWS as described herein (e.g., by administering one or more AAV vectors provided herein such as AAV vectors designed to express one or more PWS nucleic acids) also can include administering to the mammal one or more (e.g., one, two, three, four, five or more) agents that can treat one or more symptoms of PWS. Examples of agents that can be used to treat one or more symptoms of PWS and can be administered together with one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) include, without limitation, GH treatments (e.g., human growth hormone (HGH) treatments), sex hormone treatments, psychiatric agents, and any combinations thereof. In some cases, agents that can be used to treat one or more symptoms of PWS can be as described elsewhere (see, e.g., Mahmoud et al. , Int. J. Mol. Sci., 24(3):2150 (2023); Miller et al., Diseases, 3(2):78-85 (2015); and Tan et al., Obes. Rev., 21(5):el2992 (2020)). In cases where one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) are used in combination with one or more agents used to treat one or more symptoms of PWS, the one or more agents used to treat one or more symptoms of PWS can be administered at the same time (e.g., in a single composition containing both the one or more AAV vectors provided herein and the one or more agents used to treat one or more symptoms of PWS) or independently. For example, one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered first, and the one or more agents used to treat one or more symptoms of PWS administered second, or vice versa. In some cases, methods for treating a mammal (e.g., a human) having PWS as described herein (e.g., by administering one or more AAV vectors provided herein such as AAV vectors designed to express one or more PWS nucleic acids) also can include subjecting the mammal one or more (e.g., one, two, three, four, five or more) additional therapies used to treat PWS. Examples of therapies that can be used to treat PWS include, without limitation, nutrition coaching (e.g., a healthy, reduced-calorie diet, a restricted-calorie diet, and supplemental vitamins and/or mineral), increasing physical activity and exercise, physical therapy (e.g., to improve movement skills and strength), speech therapy (e.g., to improve verbal skills), occupational therapy (e.g., to learn everyday skills), and combinations thereof. In cases where one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) are used in combination with one or more additional therapies used to treat PWS, the one or more additional therapies can be performed at the same time or independently of the administration of one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids). For example, one or more AAV vectors provided herein (e.g., a population of a single AAV vector designed to express one or more PWS nucleic acids) can be administered before, during, or after the one or more additional therapies are performed.
The invention will be further described in the following examples, which do not limit the scope of the invention described in the claims.
EXAMPLES
Example 1: AAV vectors with miniaturized promoters
This Example describes the design of AAV vectors that can express one or more PWS nucleic acids (e.g., AAV vectors designed to have a genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 snoRNA, and (h) a nucleotide sequence encoding a SNORD109 snoRNA).
NRF1 -array mini-promoters allow nucleic acid sequences that can encode 8 of the 10 major PWS genes to fit into a single AAV (Figure 1 A). Each vector insert includes SNURF (216-nt with stop codon; 71 amino acids) and SNRPN (723 -nt; 240 amino acids) in the endogenous bicistronic layout with a 188-nt intercistronic region, as well as SNORD107, SNORD64, SNORD116, SNORD115, and SNORD109 snoRNAs (each 70-120 nt). For each of the duplicated SNORD116 and SNORD115 snoRNA families, a single “best matching sequence” was designed to maintain the conserved box C, box D, and putative antisense boxes for that specific snoRNA family. Three alternative vector inserts have been designed that include the nucleic acid encoding snoRNAs in the introns between protein-coding exons (Figure 1B), clustered in SNURF-SNRPN introns (Figure 1C), or located 3' of SNURF- SNRPN (Figure 1D). Additional vector design elements for each protein-coding transcription unit include a 5'-UTR (60-nt) and a 3'-UTR with a poly(A) motif (129-nt). AAV vectors containing inserts that contain nucleic acid that can encode one or more PWS nucleic acids are also referred to herein as AAV-PWS minigenes.
The CMV promoter-enhancer (613 -nt) in an AAV vector was substituted with minimal synthetic NRF1 -array mini-promoters. The 4xNRF1 (84-nt) and 10xNRF1 (216-nt) promoters were characterized, and it was shown that these promoters drive high levels of mNeonGreen expression in a variety of tested cell lines including INS1 β-cells (Figure 2), fibroblasts, myoblasts, schwannoma, and others. Furthermore, these NRF1 -array mini- promoters mimic endogenous regulation as in vivo expression of SNURF-SNRPN-snoRNA long intergenic non-coding RNAs (lincRNAs) is controlled by alternate 5'-promoters driven by several NRF1 binding sites and an intron 1 enhancer featuring NRF1. Likewise, in vivo expression of NDN is primarily regulated by NRF1.
The ability of the AAV-PWS minigenes to treat PWS is tested in a cellular PWS model. PWS β- (INS-1) cell models show a cell-autonomous defect in insulin secretion along with deficits in RNA and protein production of multiple hormones and ER chaperones (Figure 3 A). AAV-PWS minigene vectors are packaged as serotype AAV6 or AAV8 for transduction of control and PWS β-cell lines (3 each), and transduced cells were assessed for changes in levels of insulin secretion (Figure 3B), mRNA levels for Ins1, Ins2, lapp, Npy, and ER chaperones Sdf2ll, Dnajbll, Creld2, Grp78, Pdia4, Grp94, and Hyou1 using ddRT- PCR, and protein levels for peptide hormones (e.g., insulin) and major ER chaperones (Figure 3C and Figure 3D).
Designed vector inserts are produced by in vitro DNA synthesis and standard molecular cloning techniques used to insert between the AAV inverted terminal repeats (ITRs).
A multiplicity of infection (MOI) of 100,000 AAV genome copies/cell is used for cell transduction, with equivalent ddRT-PCR and protein (western blot) assays confirming the expression of each cistron. mRNA (SNURF-SNRPN, NDN) and snoRNA expression is measured by droplet digital ddRT-PCR (with PCR primers specific for human exogenous vs. rat endogenous genes). Polypeptide expression (for SNURF, SmN/SNRPN, and NDN) is assessed by western blot analysis. Constructs that express all gene components are converted to AAV vectors.
Example 2: Exemplary Promoter Sequences
Exemplary 4xNRF1 promoter sequence
Figure imgf000042_0001
(SEQ ID NO:30) Exemplary 10xNRFl promoter sequence
Figure imgf000043_0001
(SEQIDN0:31)
Example 3: Exemplary NDN Sequences
Exemplary NDN polypeptide sequence
Figure imgf000043_0002
(SEQIDNO:1)
Nucleic acid sequence encoding SEQ ID NO:1
Figure imgf000043_0003
Figure imgf000044_0001
first underlined section = start (ATG) codon Second underlined section = stop (TAG) codon third underlined section = poly A signal (AATAAA)
Example 4: Exemplary SNURF Sequences
Exemplary SNURF polypeptide sequence
Figure imgf000044_0003
Nucleic acid sequence encoding SEQ ID NO:2
Figure imgf000044_0002
first underlined section = start (ATG) codon Second underlined section = stop (TAG) codon third underlined section = polyA signal (AATAAA) fourth underlined section = polyA signal (ATT AAA)
Example 5: Exemplary SNRPN Sequences
Exemplary SNRPN polypeptide sequence
Figure imgf000045_0001
Nucleic acid sequence encoding SEQ ID NO:3
Figure imgf000045_0002
first underlined section = start (ATG) codon Second underlined section = stop (TAG) codon third underlined section = polyA signal (AATAAA) fourth underlined section = poly A signal (ATTAAA)
Figure imgf000047_0001
Figure imgf000048_0001
Figure imgf000049_0001
Example 7: Treating PWS
A human identified as having PWS is administered a composition including a population of a single AAV vector designed to express one or more PWS nucleic acids (e.g., designed to have a genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 snoRNA, and (h) a nucleotide sequence encoding a SNORD109 snoRNA). The single AAV vector designed to express one or more PWS nucleic acids is effective to increase expression of a NDN polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SNORD116 snoRNA, a SNORD115 snoRNA, and a SNORD109 snoRNA by cells within that mammal.
Example 8: Treating PWS
A human identified as having PWS is administered a composition including a population of a single AAV vector designed to express one or more PWS nucleic acids (e.g., designed to have a genome including a nucleic acid sequence (e.g., an engineered DNA sequence) including (a) a nucleotide sequence including a nucleotide sequence encoding a NDN polypeptide, (b) a nucleotide sequence encoding a SNURF polypeptide, (c) a nucleotide sequence including a nucleotide sequence encoding a SNRPN polypeptide, (d) a nucleotide sequence encoding a SNORD107 snoRNA, (e) a nucleotide sequence encoding a SNORD64 snoRNA, (f) a nucleotide sequence encoding a SNORD116 snoRNA, (g) a nucleotide sequence encoding a SNORD115 snoRNA, and (h) a nucleotide sequence encoding a SNORD109 snoRNA). The single AAV vector designed to express one or more PWS nucleic acids is effective to reduce one or more symptoms of PWS within that mammal. OTHER EMBODIMENTS
It is to be understood that while the invention has been described in conjunction with the detailed description thereof, the foregoing description is intended to illustrate and not limit the scope of the invention, which is defined by the scope of the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

WHAT IS CLAIMED IS:
1. An adeno-associated virus comprising a single-stranded DNA, wherein said singlestranded DNA comprises a first inverted terminal repeat (ITR) sequence followed by an engineered DNA sequence followed by a second ITR sequence, wherein said engineered DNA sequence comprises (a) a first DNA sequence comprising a first promotor sequence operably linked to first DNA segment comprising a nucleotide sequence encoding a necdin polypeptide and (b) a second DNA sequence comprising a second promotor sequence operably linked to a second DNA segment comprising a nucleotide sequence encoding a SNURF polypeptide, a nucleotide sequence encoding a SNRPN polypeptide, a nucleotide sequence encoding a SNORD107 small nucleolar RNA (snoRNA), a nucleotide sequence encoding a SNORD64 snoRNA, a nucleotide sequence encoding a SN0RD116 snoRNA, a nucleotide sequence encoding a SN0RD115 snoRNA, and a nucleotide sequence encoding a SNORD109 snoRNA, wherein each of said first promotor sequence and said second promotor sequence comprises at least four motifs having the DNA sequence set forth in SEQ ID NO: 10 (5 -YGCGCANGCGCR-3'), wherein 8 to 12 intervening nucleotides are located between each adjacent motif of said at least four motifs, and wherein delivery of said adeno- associated virus to a cell within said mammal results in expression of said necdin polypeptide, said SNURF polypeptide, said SNRPN polypeptide, said SNORD107 snoRNA, said SNORD64 snoRNA, said SNORD116 snoRNA, said SNORD115 snoRNA, and said SNORD109 snoRNA.
2. The adeno-associated virus of claim 1, said mammal is a human.
3. The adeno-associated virus of any one of claims 1-2, wherein said cell is a pancreatic islet endocrine cell.
4. The adeno-associated virus of any one of claims 1-2, wherein said cell is a gut endocrine cell.
5. The adeno-associated virus of any one of claims 1-2, wherein said cell is a pituitary neuroendocrine cell.
6. The adeno-associated virus of any one of claims 1-5, wherein said adeno-associated virus is AAV6, AAV8, AAV3, AAVDJ, AAVKP1, or AAV9.
7. The adeno-associated virus of any one of claims 1-6, wherein said first ITR sequence is directly followed by said engineered DNA sequence.
8. The adeno-associated virus of any one of claims 1-6, wherein said first ITR sequence is indirectly followed by said engineered DNA sequence.
9. The adeno-associated virus of any one of claims 1-8, wherein said engineered DNA sequence is directly followed by said second ITR sequence.
10. The adeno-associated virus of any one of claims 1-8, wherein said engineered DNA sequence is indirectly followed by said second ITR sequence.
11. The adeno-associated virus of any one of claims 1-10, wherein said first ITR sequence comprises the DNA sequence set forth in SEQ ID NO:32 wherein said first ITR sequence comprises the DNA sequence set forth in SEQ ID NO:34.
12. The adeno-associated virus of any one of claims 1-11, wherein said first promotor sequence comprises four of said motifs.
13. The adeno-associated virus of any one of claims 1-11, wherein said second promotor sequence comprises six of said motifs.
14. The adeno-associated virus of any one of claims 1-13, wherein each of said motifs comprise a DNA sequence set forth in any one of SEQ ID NOs: 11 to 20.
15. The adeno-associated virus of any one of claims 1-13, wherein each of said motifs comprise a DNA sequence set forth in any one of SEQ ID NOs: 11 to 14.
16. The adeno-associated virus of any one of claims 1-15, wherein each of said 8 to 12 intervening nucleotides comprise a DNA sequence set forth in any one of SEQ ID NOs: 21 to 29.
17. The adeno-associated virus of any one of claims 1-15, wherein each of said 8 to 12 intervening nucleotides comprise a DNA sequence set forth in any one of SEQ ID NOs: 21 to 23.
16. The adeno-associated virus of any one of claims 1-15, wherein said necdin polypeptide is a human necdin polypeptide.
17. The adeno-associated virus of any one of claims 1-15, wherein said necdin polypeptide comprises the amino acid sequence set forth in SEQ ID NO:1.
18. The adeno-associated virus of any one of claims 1-17, wherein said SNURF polypeptide is a human SNURF polypeptide.
19. The adeno-associated virus of any one of claims 1-17, wherein said SNURF polypeptide comprises the amino acid sequence set forth in SEQ ID NO:2.
20. The adeno-associated virus of any one of claims 1-19, wherein said SNRPN polypeptide is a human SNRPN polypeptide.
21. The adeno-associated virus of any one of claims 1-19, wherein said SNRPN polypeptide comprises the amino acid sequence set forth in SEQ ID NO:3.
22. The adeno-associated virus of any one of claims 1-19, wherein said SNQRD107 snoRNA is a human SNQRD107 snoRNA.
23. The adeno-associated virus of any one of claims 1-19, wherein said SNORD107 snoRNA comprises the RNA sequence set forth in SEQ ID NO:4.
24. The adeno-associated virus of any one of claims 1-23, wherein said SNORD64 snoRNA is a human SNORD64 snoRNA.
25. The adeno-associated virus of any one of claims 1-23, wherein said SNORD64 snoRNA comprises the RNA sequence set forth in SEQ ID NO: 5.
26. The adeno-associated virus of any one of claims 1-25, wherein said SNORD116 snoRNA is a human SNORD116 snoRNA.
27. The adeno-associated virus of any one of claims 1-25, wherein said SNORD116 snoRNA comprises the RNA sequence set forth in SEQ ID NO:6 or SEQ ID NO:7.
28. The adeno-associated virus of any one of claims 1-27, wherein said SNORD115 snoRNA is a human SNORD115 snoRNA.
29. The adeno-associated virus of any one of claims 1-27, wherein said SNORD115 snoRNA comprises the RNA sequence set forth in SEQ ID NO: 8.
30. The adeno-associated virus of any one of claims 1-29, wherein said SNORD109 snoRNA is a human SNORD109 snoRNA.
31. The adeno-associated virus of any one of claims 1-29, wherein said SNORD109 snoRNA comprises the RNA sequence set forth in SEQ ID NO:9.
32. The adeno-associated virus of any one of claims 1-31, wherein said first DNA segment comprises, downstream of said nucleotide sequence encoding said necdin polypeptide, a poly(A) signal.
33. The adeno-associated virus of claim 32, wherein said poly(A) signal comprises an SV40 poly(A) signal.
34. The adeno-associated virus of claim 32, wherein said poly(A) signal comprises the DNA sequence AATAAA or the DNA sequence ATTAAA.
35. The adeno-associated virus of any one of claims 1-34, wherein said second DNA segment comprises, downstream of said nucleotide sequence encoding said SNURF polypeptide, said nucleotide sequence encoding said SNRPN polypeptide, said nucleotide sequence encoding said SNORD107 snoRNA, said nucleotide sequence encoding said SNORD64 snoRNA, said nucleotide sequence encoding said SNORD116 snoRNA, said nucleotide sequence encoding said SNORD115 snoRNA, and said nucleotide sequence encoding said SNORD109 snoRNA, a poly(A) signal.
36. The adeno-associated virus of claim 35, wherein said poly(A) signal comprises an SV40 poly(A) signal.
37. The adeno-associated virus of claim 35, wherein said poly(A) signal comprises the DNA sequence AATAAA or the DNA sequence ATTAAA.
38. An isolated nucleic acid molecule comprising the engineered DNA sequence of any one of claims 1-37.
39. An in vitro host cell, wherein said host cell comprises an adeno-associated virus of any one of claims 1-37.
40. An in vitro host cell, wherein said host cell comprises a nucleic acid of claim 38.
41. The host cell of any one of claims 39-40, wherein said host cell is an epithelial cell.
42. A composition comprising an adeno-associated virus of any one of claims 1-37.
43. A method for increasing expression of a necdin polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SN0RD116 snoRNA, a SN0RD115 snoRNA, and a SNORD109 snoRNA by cells within a mammal, wherein said method comprises administering an adeno-associated virus of any one of claims 1-37 to said mammal, thereby increasing expression of said necdin polypeptide, said SNURF polypeptide, said SNRPN polypeptide, said SNORD107 snoRNA, said SNORD64 snoRNA, said SN0RD116 snoRNA, said SN0RD115 snoRNA, and said SNORD109 snoRNA by said cells within said mammal.
44. The method of claim 43, wherein said mammal is a human.
45. A method for treating Prader-Willi syndrome, wherein said method comprises administering an adeno-associated virus of any one of claims 1-37 to a mammal having Prader-Willi syndrome, wherein expression of a necdin polypeptide, a SNURF polypeptide, a SNRPN polypeptide, a SNORD107 snoRNA, a SNORD64 snoRNA, a SN0RD116 snoRNA, a SN0RD115 snoRNA, and a SNORD109 snoRNA by cells within said mammal is increased following said administering step, and wherein severity of a symptom of said Prader-Willi syndrome is reduced following said administering step.
46. The method of claim 45, wherein said mammal is a human.
PCT/US2024/017458 2023-02-27 2024-02-27 Methods and materials for treating prader-willi syndrome Ceased WO2024182378A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US202363448587P 2023-02-27 2023-02-27
US63/448,587 2023-02-27

Publications (2)

Publication Number Publication Date
WO2024182378A1 true WO2024182378A1 (en) 2024-09-06
WO2024182378A9 WO2024182378A9 (en) 2026-03-19

Family

ID=92590197

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US2024/017458 Ceased WO2024182378A1 (en) 2023-02-27 2024-02-27 Methods and materials for treating prader-willi syndrome

Country Status (1)

Country Link
WO (1) WO2024182378A1 (en)

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210171977A1 (en) * 2017-03-13 2021-06-10 Massachusetts Institute Of Technology Synthetic promoters
WO2022076901A1 (en) * 2020-10-09 2022-04-14 Duke University Novel targets for reactivation of prader-willi syndrome-associated genes

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20210171977A1 (en) * 2017-03-13 2021-06-10 Massachusetts Institute Of Technology Synthetic promoters
WO2022076901A1 (en) * 2020-10-09 2022-04-14 Duke University Novel targets for reactivation of prader-willi syndrome-associated genes

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
ALTHAMMER FERDINAND, MUSCATELLI FRANCOISE, GRINEVICH VALERY, SCHAAF CHRISTIAN P.: "Oxytocin-based therapies for treatment of Prader-Willi and Schaaf-Yang syndromes: evidence, disappointments, and future research strategies", TRANSLATIONAL PSYCHIATRY, GB, vol. 12, no. 1, GB , XP093210043, ISSN: 2158-3188, DOI: 10.1038/s41398-022-02054-1 *
BURNETT LISA COLE, HUBNER GABRIELA, LEDUC CHARLES A, MORABITO MICHAEL V, CARLI JAYNE F MARTIN, LEIBEL RUDOLPH L: "Loss of the imprinted, non-coding Snord116 gene cluster in the interval deleted in the Prader Willi syndrome results in murine neuronal and endocrine pancreatic developmental phenotypes", HUMAN MOLECULAR GENETICS, OXFORD UNIVERSITY PRESS, GB, vol. 26, no. 23, 1 December 2017 (2017-12-01), GB , pages 4606 - 4616, XP093210046, ISSN: 0964-6906, DOI: 10.1093/hmg/ddx342 *
KIM SU JIN, CHO SUNG YOON, JIN DONG-KYU: "Prader-Willi syndrome: an update on obesity and endocrine problems", ANNALS OF PEDIATRIC ENDOCRINOLOGY & METABOLISM, 대한소아내분비학회, vol. 26, no. 4, 31 December 2021 (2021-12-31), pages 227 - 236, XP093210045, ISSN: 2287-1012, DOI: 10.6065/apem.2142164.082 *

Also Published As

Publication number Publication date
WO2024182378A9 (en) 2026-03-19

Similar Documents

Publication Publication Date Title
EP3218386B1 (en) Modulatory polynucleotides
WO2023240236A1 (en) Compositions and methods for the treatment of spinal muscular atrophy related disorders
CN114848850B (en) Application of RS1 gene in the preparation of XLRS therapeutic agents and therapeutic agents
BR112021002202A2 (en) mini-gde for the treatment of glycogen storage disease iii
JP2022533448A (en) UBE3A genes and expression cassettes and their uses
KR20230054419A (en) Viral vectors encoding GLP-1 receptor agonist fusions and their use in the treatment of feline metabolic diseases
US20240058477A1 (en) Methods and compositions for treatment of friedreich&#39;s ataxia
US11981912B2 (en) Adeno associated virus vectors for the treatment of hunter disease
CN115029360B (en) Transgenic expression cassettes of type IIIA for the treatment of mucopolysaccharidosis
WO2024182378A1 (en) Methods and materials for treating prader-willi syndrome
US20220226502A1 (en) Adeno-associated virus vector delivery of cystathionine beta-synthase (cbs) enzyme for treating cbs deficiency
WO2020211843A1 (en) A new type of enzyme composition
WO2024131237A1 (en) Recombinant viral vector, recombinant adeno-associated virus comprising the same, and uses thereof in treating sialidosis
US20230108025A1 (en) Kir 7.1 gene therapy vectors and methods of using the same
US20250312489A1 (en) Novel methods and composition of aav vectors for the treatment of friedreich&#39;s ataxia
US20240182925A1 (en) Insulin gene therapy to treat diabetes
US12331323B1 (en) Composition for regulating production of fusion proteins
EP4326339B1 (en) Compositions and methods for treating retinal degenerative disorders
WO2024245120A1 (en) Recombinant aav vectors for treating proteinopathies in central nervous system
WO2023034966A1 (en) Compositions and methods of using the same for treating disorders associated with thymosin βeta 4
US20250263745A1 (en) PLASMID ENCODING AN INSULIN PEPTIDE AND Fc FUSION PROTEIN
HK40062715A (en) Modulatory polynucleotides
EA051405B1 (en) Adenosine-associated virus-based vectors for the treatment of Hunter disease
EA046903B1 (en) MINI-GDE FOR TREATMENT OF GLYCOGENosis TYPE III
TW202124416A (en) Frataxin expression constructs having engineered promoters and methods of use thereof

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 24764453

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 24764453

Country of ref document: EP

Kind code of ref document: A1