GENE THERAPIES FOR USHER SYNDROME (USH2A)
Aspects of the disclosure relate to compositions and methods useful for delivering minigenes to a subject. Accordingly, the disclosure is based, in part, on isolated nucleic acids and gene therapy vectors, such as viral (e.g., rAAV) vectors, comprising one or more gene fragments encoding a therapeutic gene product, such as a protein or peptide (e.g., a minigene). In some embodiments, the disclosure relates to gene therapy vectors encoding a USH2A protein (e.g., the gene product of USH2A gene) or a portion thereof. In some embodiments, compositions described by the disclosure are useful for treating diseases associated with mutations in the USH2A gene, for example Usher Syndrome.
1 . An isolated nucleic acid comprising a transgene encoding a USH2A minigene having the nucleic acid sequence set forth in any one of SEQ ID NOs: 3-14.
2 . The isolated nucleic acid of claim 1 , wherein the transgene further comprises a promoter operably linked to the USH2A minigene sequence.
3 . The isolated nucleic acid of claim 2 , wherein the promoter is a constitutive promoter, inducible promoter, or a tissue-specific promoter, optionally wherein the tissue specific promoter is a photoreceptor-specific promoter.
4 . The isolated nucleic acid of any one of claims 1 to 3 , wherein the transgene is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).
5 . The isolated nucleic acid of claim 4 , wherein at least one of the ITRs is an AAV2 ITR.
6 . The isolated nucleic acid of claim 4 or 5 , wherein at least one ITR lacks a terminal resolution site, optionally wherein the ITR is a ΔITR.
7 . An isolated nucleic acid comprising a transgene having a nucleic acid sequence encoding a USH2A protein, wherein the USH2A protein comprises an amino acid sequence as set forth in any one of SEQ ID NOs: 15-26.
8 . The isolated nucleic acid of claim 7 , wherein the transgene further comprises a promoter operably linked to the nucleic acid sequence encoding the USH2A protein.
9 . The isolated nucleic acid of claim 8 , wherein the promoter is a constitutive promoter, inducible promoter, or a tissue-specific promoter, optionally wherein the tissue specific promoter is a photoreceptor-specific promoter.
10 . The isolated nucleic acid of any one of claims 7 to 9 , wherein the transgene is flanked by adeno-associated virus (AAV) inverted terminal repeats (ITRs).
11 . The isolated nucleic acid of claim 10 , wherein at least one of the ITRs is an AAV2 ITR.
12 . The isolated nucleic acid of claim 10 or 11 , wherein at least one ITR lacks a terminal resolution site, optionally wherein the ITR is a ΔITR.
13 . A vector comprising the isolated nucleic acid of any one of claims 1 to 12 .
14 . The vector of claim 13 , wherein the vector is a plasmid DNA, closed-ended DNA, lipid/DNA nanoparticle, or a viral vector.
15 . The vector of claim 14 , wherein the viral vector is an adeno-associated virus (AAV) vector, adenoviral (Ad) vector, lentiviral vector, retroviral vector, or Baculovirus vector.
16 . A host cell comprising the isolated nucleic acid of any one of claims 1 to 12 , or the vector of any one of claims 13 to 15 .
17 . The host cell of claim 16 , wherein the cell is a mammalian (human) cell, bacterial cell, yeast cell, or insect cell.
18 . A recombinant adeno-associated virus (rAAV) comprising:
(i) the isolated nucleic acid of any one of claims 1 to 12 ; and
(ii) an AAV capsid protein.
19 . The rAAV of claim 18 , wherein the capsid protein has a tropism for ocular cells.
20 . The rAAV of claim 18 or 19 , wherein the capsid protein is AAV8 capsid protein.
21 . The rAAV of any one of claims 18 to 20 , wherein the rAAV is formulated for delivery to the eye, optionally wherein the rAAV is formulated for delivery to photoreceptor cells or retinal pigmented epithelium (RPE).
22 . A composition comprising the isolated nucleic acid of any one of claims 1 to 12 , or the vector of any one of claims 13 to 15 , or the rAAV of any one of claims 18 to 21 , and a pharmaceutically acceptable excipient.
23 . A method for delivering a transgene to a cell, the method comprising administering the isolated nucleic acid of any one of claims 1 to 12 , or the vector of any one of claims 13 to 15 , or the rAAV of any one of claims 18 to 21 , or the composition of claim 22 , to a cell.
24 . The method of claim 23 , wherein the cell is in a subject, optionally a mammalian subject.
25 . The method of claim 24 , wherein the mammalian subject is a human subject.
26 . The method of any one of claims 23 to 25 , wherein the cell is an eye cell.
27 . The method of claim 26 , wherein the eye cell is a photoreceptor cell or retinal pigmented epithelium (RPE).
28 . A method for treating Usher Syndrome in a subject in need thereof, the method comprising administering the isolated nucleic acid of any one of claims 1 to 12 , or the vector of any one of claims 13 to 15 , or the rAAV of any one of claims 18 to 21 , or the composition of claim 22 , to the subject.
29 . The method of claim 28 , wherein the subject is a mammal, optionally wherein the subject is a human.
30 . The method of claim 28 or 29 , wherein the subject is characterized by having one or more mutations in a USH2A gene.
31 . The method of any one of claims 28 to 30 , wherein the administration is via injection, optionally subretinal injection or intravitreal injection or suprachoroidal injection.
32 . The method of any one of claims 28 to 30 , wherein the administration is topical administration to the eye of the subject.