TREATMENT OF RPE65-ASSOCIATED EYE DISEASES AND DISORDERS
The disclosure described herein provides recombinant adeno-associated virus (rAAV) viral particle comprising an AAV9 serotype capsid and a vector genome encoding an RPE65 (e.g., hRPE65) polypeptide, as well as related compositions and uses thereof (e.g., use to treat Leber's congenital amaurosis 2 (LCA2)).
1 . A recombinant adeno-associated virus (rAAV) viral particle comprising an AAV9 serotype capsid and a vector genome encoding an RPE65 (e.g., hRPE65) polypeptide, such as a polynucleotide at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% identical to SEQ ID NO: 1.
2 . The rAAV viral particle of claim 1 , wherein the vector genome comprises:
a) a 5′ inverted terminal repeat (ITR);
b) an RPE65 polynucleotide encoding an RPE65 polypeptide, wherein the RPE65 polynucleotide comprises a polynucleotide sequence of SEQ ID NO: 2 or having a sequence identity of at least 90%, 95%, 96%, 97%, 98%, 99%, 99.2%, 99.4%, 99.6%, or 99.8% to the polynucleotide sequence of SEQ ID NO: 2;
c) a promoter operably linked to and drives the transcription of the RPE65 polynucleotide;
d) an optional Kozak sequence upstream of the RPE65 polynucleotide and downstream of the promoter;
e) a polyA signal sequence; and
f) a 3′ ITR,
optionally, wherein the RPE65 polypeptide has the amino acid sequence of SEQ ID NO: 3.
3 . The rAAV viral particle of claim 2 , wherein the 5′ ITR and 3′ ITR are derived from AAV2 or AAV9, optionally, the 5′ ITR comprises the nucleotide sequence of SEQ ID NO: 7, and/or and the 3′ ITR comprises the nucleotide sequence of SEQ ID NO: 8.
4 . The rAAV viral particle of claim 2 , wherein the promoter is a ubiquitous promoter, a tissue-specific promoter, a constitutive promoter, or an inducible promoter.
5 - 7 . (canceled)
8 . The rAAV viral particle of claim 2 , wherein the promoter is selected from the group consisting of a pol I promoter, a pol II promoter, a pol III promoter, a T7 promoter, a U6 promoter, a H1 promoter, retroviral Rous sarcoma virus LTR promoter, a cytomegalovirus (CMV) promoter, a SV40 promoter, a dihydrofolate reductase promoter, a β-actin promoter, an elongation factor 1α short (EFS) promoter, a β glucuronidase (GUSB) promoter, a cytomegalovirus (CMV) immediate-early (IE) enhancer and/or promoter, a chicken β-actin (CBA) promoter or derivative thereof such as a CAG promoter, CB promoter, a (human) elongation factor 1α-subunit (EF1α) promoter, a ubiquitin C (UBC) promoter, a prion promoter, a neuron-specific enolase (NSE), a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a platelet-derived growth factor (PDGF) promoter, a platelet-derived growth factor B-chain (PDGF-β) promoter, a synapsin (Syn) promoter, a synapsin 1 (Syn1) promoter, a methyl-CpG binding protein 2 (MeCP2) promoter, a Ca2+/calmodulin-dependent protein kinase II (CaMKII) promoter, a metabotropic glutamate receptor 2 (mGluR2) promoter, a neurofilament light (NFL) promoter, a neurofilament heavy (NFH) promoter, a β-globin minigene nβ2 promoter, a preproenkephalin (PPE) promoter, an enkephalin (Enk) promoter, an excitatory amino acid transporter 2 (EAAT2) promoter, a glial fibrillary acidic protein (GFAP) promoter, a myelin basic protein (MBP) promoter, or a functional fragment thereof.
9 . The rAAV viral particle of claim 8 , wherein the promoter is the CAG promoter.
10 . The rAAV viral particle of claim 9 , wherein the CAG promoter comprises a sequence having a sequence identity of at least 95%, 96%, 97%, 98%, or 99% to SEQ ID NO: 4.
11 . The rAAV viral particle of claim 10 , wherein the CAG promoter comprises, consists essentially of, or consists of SEQ ID NO: 4.
12 . The rAAV viral particle of claim 2 , wherein the polyA signal sequence is selected from the group consisting of bovine growth hormone polyadenylation signal sequence (bGH polyA), a small polyA signal sequence (SPA), a human growth hormone polyadenylation signal sequence (hGH polyA), a rabbit beta globin polyA signal sequence (rBG polyA), an SV40 polyA signal sequence (SV40 polyA), or a variant thereof.
13 . The rAAV viral particle of claim 12 , wherein the polyA signal sequence is the bGH polyA.
14 . The rAAV viral particle of claim 13 , wherein the bGH polyA comprises, consists essentially of, or consists of SEQ ID NO: 5.
15 . The rAAV viral particle of claim 2 , wherein the Kozak sequence is GCCACC (SEQ ID NO: 6) or a sequence comprising at most 1, 2, 3, or 4 nucleotide differences from GCCACC (SEQ ID NO: 6), and optionally wherein the last three nucleotide is ACC or GCC.
16 . The rAAV viral particle of claim 1 , wherein the vector genome comprises, in 5′ to 3′ direction,
(1) a 5′ ITR of SEQ ID NO: 7,
(2) a CAG promoter of SEQ ID NO: 4,
(3) a Kozak sequence of GCCACC (SEQ ID NO: 6),
(4) a hRPE65 polynucleotide sequence of SEQ ID NO: 2,
(5) a bGH polyA signal sequence of SEQ ID NO: 5, and
(6) a 3′ ITR of SEQ ID NO: 8,
with an optional linker between (1) and (2), between (2) and (3), between (3) and (4), between (4) and (5), and/or between (5) and (6);
optionally wherein the vector genome comprises, consists essentially of, or consists of SEQ ID NO: 1, or a polynucleotide having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% to SEQ ID NO: 1, encoding an RPE65 polypeptide having a sequence identity of at least 95%, 96%, 97%, 98%, or 99% to SEQ ID NO: 3 (e.g., 100% identical to SEQ ID NO: 3).
17 . The rAAV viral particle of claim 16 , wherein the vector genome has a sequence identity of at least 99% to SEQ ID NO: 1.
18 . The rAAV viral particle of claim 16 , wherein the vector genome consists of SEQ ID NO: 1.
19 . The rAAV viral particle of claim 1 , wherein the AAV9 serotype capsid comprises AAV9 VP1, AAV9 VP2, and AAV9 VP3; or VP1, VP2, and VP3 variants independently having a sequence identity of at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% thereto, respectively.
20 . The rAAV viral particle of claim 19 , wherein the AAV9 serotype capsid comprises AAV9 VP1 (SEQ ID NO: 9), AAV9 VP2, and AAV9 VP3.
21 . A pharmaceutical composition comprising the rAAV viral particle of claim 1 , and a pharmaceutically acceptable excipient.
22 . A method of treating a RPE65-associated eye disease or disorder (e.g., (human) RPE65-deficient) in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of the rAAV viral particle of claim 1 , wherein the rAAV viral particle specifically induces expression of the RPE65 polypeptide from the vector genome of the rAAV viral particle (e.g., in retinal pigment epithelial (RPE) cells).
23 - 32 . (canceled)