Methods of treating hearing loss using a secreted target protein
Provided herein are compositions that include a single nucleic acid vector or two different nucleic acid vectors, and the use of these compositions to treat hearing loss in a subject.
1 . A construct comprising:
(i) a cytomegalovirus (CMV) enhancer;
(ii) a chicken β-actin (CBA) promoter;
(iii) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 19;
(iv) a coding sequence comprising a first nucleic acid sequence encoding a Norrin Cystine Knot Growth Factor (NDP) protein; and
(v) a polyadenylation sequence.
2 . The construct of claim 1 , wherein the first nucleic acid sequence encoding the NDP protein is an NDP gene.
3 . The construct of claim 1 , further comprising two adeno-associated virus (AAV) inverted terminal repeats (ITRs), wherein the two AAV ITRs flank the CMV enhancer and the polyadenylation sequence.
4 . An AAV particle comprising a construct, wherein the construct comprises:
(i) a cytomegalovirus (CMV) enhancer;
(ii) a chicken β-actin (CBA) promoter;
(iii) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO 19;
(iv) a coding sequence comprising a first nucleic acid sequence encoding a Norrin Cystine Knot Growth Factor (NDP) protein; and
(v) a polyadenylation sequence.
5 . A composition comprising a construct and a pharmaceutically acceptable carrier, diluent or excipient, wherein the construct comprises:
(i) a cytomegalovirus (CMV) enhancer;
(ii) a chicken β-actin (CBA) promoter;
(iii) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO 19;
(iv) a coding sequence comprising a first nucleic acid sequence encoding a Norrin Cystine Knot Growth Factor (NDP) protein; and
(v) a polyadenylation sequence.
6 . A composition comprising an AAV particle and a pharmaceutically acceptable carrier, diluent or excipient, wherein the AAV particle comprises a construct, wherein the construct comprises:
(i) a cytomegalovirus (CMV) enhancer;
(ii) a chicken β-actin (CBA) promoter;
(iii) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 19;
(iv) a coding sequence comprising a first nucleic acid sequence encoding a Norrin Cystine Knot Growth Factor (NDP) protein; and
(v) a polyadenylation sequence.
7 . The construct of claim 1 , wherein the coding sequence further comprises a second nucleic acid sequence encoding a secretion signal protein.
8 . The construct of claim 7 , wherein the secretion signal protein comprises an NDP secretion signal protein.
9 . The construct of claim 7 , wherein the second nucleic acid sequence encoding the secretion signal protein is operably linked to the first nucleic acid sequence encoding the NDP protein.
10 . The construct of claim 1 , wherein the construct further comprises a 5′ untranslated region (UTR).
11 . The construct of claim 1 , wherein the construct further comprises a 3′ untranslated region (UTR).
12 . The construct of claim 1 , wherein the polyadenylation sequence is a bovine growth hormone polyadenylation (bGHpA) sequence.
13 . The construct of claim 1 , wherein the CMV enhancer comprises the nucleic acid sequence of SEQ ID NO: 17.
14 . The construct of claim 1 , wherein the CBA promoter comprises the nucleic acid sequence of SEQ ID NO: 18.
15 . The construct of claim 10 , wherein the 5′ UTR comprises the nucleic acid sequence of SEQ ID NO: 14.
16 . The construct of claim 8 , wherein the NDP secretion signal protein comprises the amino acid sequence of SEQ ID NO: 5.
17 . The construct of claim 8 , wherein the NDP secretion signal protein is encoded by the nucleic acid sequence of SEQ ID NO: 6.
18 . The construct of claim 1 , wherein the NDP protein comprises the amino acid sequence of SEQ ID NO: 56.
19 . The construct of claim 11 , wherein the 3′ UTR sequence comprises the nucleic acid sequence of SEQ ID NO: 15.
20 . The construct of claim 12 , wherein the bGHpA sequence comprises the nucleic acid sequence of SEQ ID NO: 23.
21 . The AAV particle of claim 4 , further comprising a capsid, wherein the capsid is or is derived from an AAV2 capsid.
22 . The AAV particle of claim 4 , further comprising a capsid, wherein the capsid is or is derived from an Anc80 capsid.
23 . A kit comprising a composition, wherein the composition comprises (a) a construct or (b) an AAV particle comprising the construct, wherein the construct comprises:
(i) a cytomegalovirus (CMV) enhancer;
(ii) a chicken β-actin (CBA) promoter;
(iii) a chimeric intron comprising the nucleic acid sequence of SEQ ID NO: 19;
(iv) a coding sequence comprising a first nucleic acid sequence encoding a Norrin Cystine Knot Growth Factor (NDP) protein; and
(v) a polyadenylation sequence.
24 . The composition of claim 6 , wherein the composition is a pharmaceutical composition.
25 . The kit of claim 23 , wherein the kit further comprises instructions for performing a method.
26 . The kit of claim 25 , wherein the method is a method of introducing the composition into the inner ear of a subject.
27 . The kit of claim 25 , wherein the method is a method of introducing the composition into the cochlea of a subject.
28 . The kit of claim 25 , wherein the method is a method of introducing the composition into a mammalian cell of a subject.
29 . The kit of claim 28 , wherein the mammalian cell is a cochlear inner hair cell or a cochlear outer hair cell.
30 . The kit of claim 25 , wherein the method is a method of treatment.
31 . The kit of claim 25 , wherein the method is a method of treating deafness or hearing loss.
32 . The kit of claim 23 , wherein the kit further comprises a pre-loaded syringe or a vial, wherein the pre-loaded syringe or the vial comprises the composition, wherein the composition is formulated as an aqueous composition, a solid composition, or a lyophilized composition.
33 . The composition of claim 24 , further comprising a pharmaceutically acceptable carrier.