COMPOSITIONS AND METHODS TO TREAT BIETTI CRYSTALLINE DYSTROPHY

BACKGROUND

Bietti crystalline dystrophy (BCD) is an autosomal recessive disorder in which numerous small, yellow or white crystalline-like deposits of lipid accumulate in the retina, which is followed by chorioretinal atrophy and progressive vision loss. Subjects with BCD typically begin noticing vision problems in their teens or twenties. They often experience night blindness in addition to a reduction in visual acuity. They also usually lose areas of vision, most often peripheral vision. Color vision may also be impaired.

The vision problems may worsen at different rates in each eye, and the severity and progression of symptoms varies widely among affected subjects, even within the same family. However, most subjects with BCD become legally blind by 40 or 50 years of age. Most affected subjects retain some degree of vision, usually in the center of the visual field, although it is typically blurry and cannot be corrected by prescription lenses.

BCD is caused by mutations in the CYP4V2 gene. The gene, located on the long arm of human chromosome 4, encodes cytochrome P450 family 4 subfamily V member 2. As a member of the cytochrome P450 family of enzymes, the w-hydroxylase is involved in lipid metabolism, specifically oxidation of polyunsaturated fatty acids such as docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA). At least 80 different CYP4V2 gene mutations have been identified in subjects with BCD (Zhang et al., Mol Vis 24:700-711, 2018). CYP4V2 gene mutations that cause BCD impair or eliminate the function of the enzyme and are believed to affect lipid breakdown. However, it is unknown how they lead to the specific signs and symptoms of BCD.

BCD is estimated to affect approximately 65,000 people worldwide (Xiao et al., Biochem Biophys Res Comm 409:181-186, 2011; and Mataftsi et al., Retina 24:416-426, 2004). It is more common in people of East Asian descent, especially those of Chinese and Japanese background. Currently, there is no treatment available for BCD.

SUMMARY

The present invention relates generally to recombinant viral vectors and methods of using recombinant viral vectors to express proteins in the retina, e.g., retinal pigment epithelium (RPE) cells, of subjects suffering from retinal diseases and blindness, e.g., BCD.

The present invention, in one aspect, relates to viral vectors that are capable of delivering a heterologous gene to the retina. The present invention also relates to viral vectors that are capable of directing a heterologous gene to the retina, e.g., RPE cells of the retina.

The present invention further relates to viral vectors that are recombinant adeno-associated viral vectors (rAAV). In certain embodiments the rAAV viral vector may be selected from among any AAV serotype known in the art, including without limitation, AAV1 to AAV12. In certain embodiments, the rAAV vector capsid is an AAV8 serotype. In certain other embodiments, the rAAV vector capsid is an AAV9 serotype. In certain embodiments, the rAAV vector capsid is an AAV2 serotype. In certain embodiments, the rAAV vector capsid is an AAV5 serotype. In certain embodiments, the rAAV vector is a novel synthetic AAV serotype derived from modified wild-type AAV capsid sequences.

In one aspect, viral vectors are provided, wherein the viral vectors comprise a vector genome comprising, in a 5′ to 3′ direction:

(i) a 5′ ITR;

(ii) a promoter;

(iii) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence;

(iv) a polyadenylation (polyA) signal sequence; and

(v) a 3′ ITR.

In one embodiment, the vector genome comprises, in the 5′ to 3′ direction:

(i) a 5′ ITR;

(ii) a promoter;

(iii) an intron;

(iv) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence;

(v) a polyA signal sequence; and

(vi) a 3′ ITR.

In some embodiments, the vector genome comprises, in the 5′ to 3′ direction:

(i) a 5′ ITR;

(ii) a promoter;

(iii) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence;

(iv) a regulatory element;

(v) a polyA signal sequence; and

(vi) a 3′ ITR.

In one embodiment, the vector genome comprises, in the 5′ to 3′ direction:

(i) a 5′ ITR;

(ii) a promoter;

(iii) an intron;

(iv) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence;

(v) a regulatory element;

(vi) a polyA signal sequence; and

(vii) a 3′ ITR.

In some embodiments, the vector genome comprises a length greater than or about 4.1 kb and less than or about 4.9 kb. In another embodiments, the vector genome comprises a length less than or about 5 kb.

In one embodiment, the vector genome comprises a stuffer sequence positioned between the polyA signal sequence and the 3′ ITR. In some embodiments, the stuffer sequence is between about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,500, or 2,500-3,000 nucleotides in length.

In one embodiment, the 5′ ITR comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:1.

In some embodiments, the promoter is a ubiquitous promoter, e.g., a cytomegalovirus (CMV) promoter, CBA promoter, or CAG promoter, e.g., wherein the promoter comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

In one embodiment, the promoter is a retinal pigment epithelium (RPE)-specific promoter, e.g., a ProC2 promoter, VMD2 promoter, CYP4V2 promoter, or RPE65 promoter, e.g., wherein the promoter comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, and promotes expression of the CYP4V2 preferentially in RPE cells, e.g., human RPE cells.

The present invention hence provides an isolated nucleic acid molecule comprising, or consisting of, the nucleic acid sequence of SEQ ID NO: 5 or a nucleic acid sequence having at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to said nucleic acid sequence of SEQ ID NO: 5. The isolated nucleic acid of SEQ ID NO: 5 leads to the expression in human or NHP retinal cells, e.g., human or NHP RPE cells, of a gene operatively linked to the nucleic acid sequence of SEQ ID NO: 5.

In some embodiments, the CYP4V2 coding sequence comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, or SEQ ID NO:49.

In one embodiment, the polyA signal sequence comprises a bovine growth hormone or simian virus 40 polyA nucleotide sequence, e.g., wherein the polyA signal sequence comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:18 or SEQ ID NO:19.

In some embodiments, the 3′ ITR comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:22.

In one embodiment, the intron comprises a human growth hormone, simian virus 40, or human beta gobin intron sequence, e.g., wherein the intron comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

In some embodiments, the regulatory element comprises a hepatitis B virus or woodchuck hepatitis virus sequence, e.g., wherein the regulatory element comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:16 or SEQ ID NO:17.

In one embodiment, the vector genome comprises a Kozak sequence positioned immediately upstream of the recombinant nucleotide sequence comprising the CYP4V2 coding sequence, e.g., wherein the Kozak sequence comprises the nucleotide sequence of SEQ ID NO:12, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.

In some embodiments, the vector genome comprises, in the 5′ to 3′ direction, nucleotide sequences selected from the group consisting of:

i) SEQ ID NOs:1, 2, 13, 18, and 22;

ii) SEQ ID NOs:1, 3, 13, 18, and 22;

iii) SEQ ID NOs:1, 4, 13, 18, and 22;

iv) SEQ ID NOs:1, 5, 13, 18, and 22;

v) SEQ ID NOs:1, 6, 13, 18, and 22;

vi) SEQ ID NOs:1, 7, 13, 18, and 22;

vii) SEQ ID NOs:1, 8, 13, 18, and 22;

viii) SEQ ID NOs:1, 2, 14, 18, and 22;

ix) SEQ ID NOs:1, 3, 14, 18, and 22;

x) SEQ ID NOs:1, 4, 14, 18, and 22;

xi) SEQ ID NOs:1, 5, 14, 18, and 22;

xii) SEQ ID NOs:1, 6, 14, 18, and 22;

xiii) SEQ ID NOs:1, 7, 14, 18, and 22;

xiv) SEQ ID NOs:1, 8, 14, 18, and 22;

xv) SEQ ID NOs:1, 2, 13, 19, and 22;

xvi) SEQ ID NOs:1, 3, 13, 19, and 22;

xvii) SEQ ID NOs:1, 4, 13, 19, and 22;

xviii) SEQ ID NOs:1, 5, 13, 19, and 22;

xix) SEQ ID NOs:1, 6, 13, 19, and 22;

xx) SEQ ID NOs:1, 7, 13, 19, and 22;

xxi) SEQ ID NOs:1, 8, 13, 19, and 22;

xxii) SEQ ID NOs:1, 2, 14, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 14, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 14, 19, and 22;

xxv) SEQ ID NOs:1, 5, 14, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 14, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 14, 19, and 22; and

xxviii) SEQ ID NOs:1, 8, 14, 19, and 22.

In one embodiment, the vector genome comprises, in the 5′ to 3′ direction, nucleotide sequences selected from the group consisting of:

i) SEQ ID NOs:1, 2, 9, 13, 18, and 22;

ii) SEQ ID NOs:1, 3, 9, 13, 18, and 22;

iii) SEQ ID NOs:1, 4, 9, 13, 18, and 22;

iv) SEQ ID NOs:1, 5, 9, 13, 18, and 22;

v) SEQ ID NOs:1, 6, 9, 13, 18, and 22;

vi) SEQ ID NOs:1, 7, 9, 13, 18, and 22;

vii) SEQ ID NOs:1, 8, 9, 13, 18, and 22;

viii) SEQ ID NOs:1, 2, 9, 14, 18, and 22;

ix) SEQ ID NOs:1, 3, 9, 14, 18, and 22;

x) SEQ ID NOs:1, 4, 9, 14, 18, and 22;

xi) SEQ ID NOs:1, 5, 9, 14, 18, and 22;

xii) SEQ ID NOs:1, 6, 9, 14, 18, and 22;

xiii) SEQ ID NOs:1, 7, 9, 14, 18, and 22;

xiv) SEQ ID NOs:1, 8, 9, 14, 18, and 22;

xv) SEQ ID NOs:1, 2, 9, 13, 19, and 22;

xvi) SEQ ID NOs:1, 3, 9, 13, 19, and 22;

xvii) SEQ ID NOs:1, 4, 9, 13, 19, and 22;

xviii) SEQ ID NOs:1, 5, 9, 13, 19, and 22;

xix) SEQ ID NOs:1, 6, 9, 13, 19, and 22;

xx) SEQ ID NOs:1, 7, 9, 13, 19, and 22;

xxi) SEQ ID NOs:1, 8, 9, 13, 19, and 22;

xxii) SEQ ID NOs:1, 2, 9, 14, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 9, 14, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 9, 14, 19, and 22;

xxv) SEQ ID NOs:1, 5, 9, 14, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 9, 14, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 9, 14, 19, and 22; and

xxviii) SEQ ID NOs:1, 8, 9, 14, 19, and 22.

In some embodiments, the vector genome comprises, in the 5′ to 3′ direction, nucleotide sequences selected from the group consisting of:

i) SEQ ID NOs:1, 2, 13, 16, 18, and 22;

ii) SEQ ID NOs:1, 3, 13, 16, 18, and 22;

iii) SEQ ID NOs:1, 4, 13, 16, 18, and 22;

iv) SEQ ID NOs:1, 5, 13, 16, 18, and 22;

v) SEQ ID NOs:1, 6, 13, 16, 18, and 22;

vi) SEQ ID NOs:1, 7, 13, 16, 18, and 22;

vii) SEQ ID NOs:1, 8, 13, 16, 18, and 22;

viii) SEQ ID NOs:1, 2, 14, 16, 18, and 22;

ix) SEQ ID NOs:1, 3, 14, 16, 18, and 22;

x) SEQ ID NOs:1, 4, 14, 16, 18, and 22;

xi) SEQ ID NOs:1, 5, 14, 16, 18, and 22;

xii) SEQ ID NOs:1, 6, 14, 16, 18, and 22;

xiii) SEQ ID NOs:1, 7, 14, 16, 18, and 22;

xiv) SEQ ID NOs:1, 8, 14, 16, 18, and 22;

xv) SEQ ID NOs:1, 2, 13, 16, 19, and 22;

xvi) SEQ ID NOs:1, 3, 13, 16, 19, and 22;

xvii) SEQ ID NOs:1, 4, 13, 16, 19, and 22;

xviii) SEQ ID NOs:1, 5, 13, 16, 19, and 22;

xix) SEQ ID NOs:1, 6, 13, 16, 19, and 22;

xx) SEQ ID NOs:1, 7, 13, 16, 19, and 22;

xxi) SEQ ID NOs:1, 8, 13, 16, 19, and 22;

xxii) SEQ ID NOs:1, 2, 14, 16, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 14, 16, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 14, 16, 19, and 22;

xxv) SEQ ID NOs:1, 5, 14, 16, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 14, 16, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 14, 16, 19, and 22; and

xxviii) SEQ ID NOs:1, 8, 14, 16, 19, and 22.

In one embodiment, the vector genome comprises, in the 5′ to 3′ direction, nucleotide sequences selected from the group consisting of:

i) SEQ ID NOs:1, 2, 9, 13, 16, 18, and 22;

ii) SEQ ID NOs:1, 3, 9, 13, 16, 18, and 22;

iii) SEQ ID NOs:1, 4, 9, 13, 16, 18, and 22;

iv) SEQ ID NOs:1, 5, 9, 13, 16, 18, and 22;

v) SEQ ID NOs:1, 6, 9, 13, 16, 18, and 22;

vi) SEQ ID NOs:1, 7, 9, 13, 16, 18, and 22;

vii) SEQ ID NOs:1, 8, 9, 13, 16, 18, and 22;

viii) SEQ ID NOs:1, 2, 9, 14, 16, 18, and 22;

ix) SEQ ID NOs:1, 3, 9, 14, 16, 18, and 22;

x) SEQ ID NOs:1, 4, 9, 14, 16, 18, and 22;

xi) SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22;

xii) SEQ ID NOs:1, 6, 9, 14, 16, 18, and 22;

xiii) SEQ ID NOs:1, 7, 9, 14, 16, 18, and 22;

xiv) SEQ ID NOs:1, 8, 9, 14, 16, 18, and 22;

xv) SEQ ID NOs:1, 2, 9, 13, 16, 19, and 22;

xvi) SEQ ID NOs:1, 3, 9, 13, 16, 19, and 22;

xvii) SEQ ID NOs:1, 4, 9, 13, 16, 19, and 22;

xviii) SEQ ID NOs:1, 5, 9, 13, 16, 19, and 22;

xix) SEQ ID NOs:1, 6, 9, 13, 16, 19, and 22;

xx) SEQ ID NOs:1, 7, 9, 13, 16, 19, and 22;

xxi) SEQ ID NOs:1, 8, 9, 13, 16, 19, and 22;

xxii) SEQ ID NOs:1, 2, 9, 14, 16, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 9, 14, 16, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 9, 14, 16, 19, and 22;

xxv) SEQ ID NOs:1, 5, 9, 14, 16, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 9, 14, 16, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 9, 14, 16, 19, and 22; and

xxviii) SEQ ID NOs:1, 8, 9, 14, 16, 19, and 22.

In some embodiments, the vector comprises an adeno-associated virus (AAV) serotype 8, 9, 2, or 5 capsid. In one embodiment, the AAV8 capsid comprises VP1, VP2, and VP3 amino acid sequences with greater than or about 90% identity to SEQ ID NOs:24, 25, and 26, respectively. In some embodiments, the AAV8 capsid is encoded by a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:23. In one embodiment, the AAV9 capsid comprises VP1, VP2, and VP3 amino acid sequences with greater than or about 90% identity to SEQ ID NOs:28, 29, and 30, respectively. In some embodiments, the AAV9 capsid is encoded by a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:27. In one embodiment, the AAV2 capsid comprises VP1, VP2, and VP3 amino acid sequences with greater than or about 90% identity to SEQ ID NOs:32, 33, and 34, respectively. In some embodiments, the AAV2 capsid is encoded by a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:31. In one embodiment, the AAV5 capsid comprises VP1, VP2, and VP3 amino acid sequences with greater than or about 90% identity to SEQ ID NOs:36, 37, and 38, respectively. In some embodiments, the AAV5 capsid is encoded by a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:35.

In another aspect, the present disclosure provides compositions comprising a viral vector described herein. In one embodiment, the compositions further comprise a pharmaceutically acceptable excipient. In some embodiments, the compositions are for use in treating a subject with BCD, e.g., for use in improving visual acuity in a subject with BCD.

Also provided herein is a method of expressing a heterologous CYP4V2 gene in a retinal cell, wherein the method comprises contacting the retinal cell with a viral vector described herein. In some embodiments, the retinal cell is a RPE cell.

In another aspect, a method of treating a subject with Bietti crystalline dystrophy (BCD) is provided, wherein the method comprises administering to the subject an effective amount of a composition comprising a viral vector described herein, e.g., wherein the composition further comprises a pharmaceutically acceptable excipient.

In yet another aspect, a method of improving visual acuity, improving visual function or functional vision, or inhibiting decline of visual function or functional vision in a subject with BCD is provided, wherein the method comprises administering to the subject an effective amount of a composition comprising a viral vector described herein, e.g., wherein the composition further comprises a pharmaceutically acceptable excipient.

In one aspect, a nucleic acid comprising a gene cassette is provided, wherein the gene cassette comprises, in the 5′ to 3′ direction:

(i) a 5′ ITR;

(ii) a promoter;

(iii) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence;

(iv) a polyA signal sequence; and

(v) a 3′ ITR.

In one embodiment, the nucleic acid comprising the gene cassette is a plasmid.

In some embodiments, the gene cassette comprises, in the 5′ to 3′ direction, nucleotide sequences selected from the group consisting of:

i) SEQ ID NOs:1, 2, 13, 18, and 22;

ii) SEQ ID NOs:1, 3, 13, 18, and 22;

iii) SEQ ID NOs:1, 4, 13, 18, and 22;

iv) SEQ ID NOs:1, 5, 13, 18, and 22;

v) SEQ ID NOs:1, 6, 13, 18, and 22;

vi) SEQ ID NOs:1, 7, 13, 18, and 22;

vii) SEQ ID NOs:1, 8, 13, 18, and 22;

viii) SEQ ID NOs:1, 2, 14, 18, and 22;

ix) SEQ ID NOs:1, 3, 14, 18, and 22;

x) SEQ ID NOs:1, 4, 14, 18, and 22;

xi) SEQ ID NOs:1, 5, 14, 18, and 22;

xii) SEQ ID NOs:1, 6, 14, 18, and 22;

xiii) SEQ ID NOs:1, 7, 14, 18, and 22;

xiv) SEQ ID NOs:1, 8, 14, 18, and 22;

xv) SEQ ID NOs:1, 2, 13, 19, and 22;

xvi) SEQ ID NOs:1, 3, 13, 19, and 22;

xvii) SEQ ID NOs:1, 4, 13, 19, and 22;

xviii) SEQ ID NOs:1, 5, 13, 19, and 22;

xix) SEQ ID NOs:1, 6, 13, 19, and 22;

xx) SEQ ID NOs:1, 7, 13, 19, and 22;

xxi) SEQ ID NOs:1, 8, 13, 19, and 22;

xxii) SEQ ID NOs:1, 2, 14, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 14, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 14, 19, and 22;

xxv) SEQ ID NOs:1, 5, 14, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 14, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 14, 19, and 22;

xxviii) SEQ ID NOs:1, 8, 14, 19, and 22;

xxix) SEQ ID NOs:1, 2, 9, 13, 18, and 22;

xxx) SEQ ID NOs:1, 3, 9, 13, 18, and 22;

xxxi) SEQ ID NOs:1, 4, 9, 13, 18, and 22;

xxxii) SEQ ID NOs:1, 5, 9, 13, 18, and 22;

xxxiii) SEQ ID NOs:1, 6, 9, 13, 18, and 22;

xxxiv) SEQ ID NOs:1, 7, 9, 13, 18, and 22;

xxxv) SEQ ID NOs:1, 8, 9, 13, 18, and 22;

xxxvi) SEQ ID NOs:1, 2, 9, 14, 18, and 22;

xxxvii) SEQ ID NOs:1, 3, 9, 14, 18, and 22;

xxxviii) SEQ ID NOs:1, 4, 9, 14, 18, and 22;

xxxix) SEQ ID NOs:1, 5, 9, 14, 18, and 22;

xl) SEQ ID NOs:1, 6, 9, 14, 18, and 22;

xli) SEQ ID NOs:1, 7, 9, 14, 18, and 22;

xlii) SEQ ID NOs:1, 8, 9, 14, 18, and 22;

xliii) SEQ ID NOs:1, 2, 9, 13, 19, and 22;

xliv) SEQ ID NOs:1, 3, 9, 13, 19, and 22;

xlv) SEQ ID NOs:1, 4, 9, 13, 19, and 22;

xlvi) SEQ ID NOs:1, 5, 9, 13, 19, and 22;

xlvii) SEQ ID NOs:1, 6, 9, 13, 19, and 22;

xlviii) SEQ ID NOs:1, 7, 9, 13, 19, and 22;

xlix) SEQ ID NOs:1, 8, 9, 13, 19, and 22;

l) SEQ ID NOs:1, 2, 9, 14, 19, and 22;

li) SEQ ID NOs:1, 3, 9, 14, 19, and 22;

lii) SEQ ID NOs:1, 4, 9, 14, 19, and 22;

liii) SEQ ID NOs:1, 5, 9, 14, 19, and 22;

liv) SEQ ID NOs:1, 6, 9, 14, 19, and 22;

lv) SEQ ID NOs:1, 7, 9, 14, 19, and 22;

lvi) SEQ ID NOs:1, 8, 9, 14, 19, and 22;

lvii) SEQ ID NOs:1, 2, 13, 16, 18, and 22;

lviii) SEQ ID NOs:1, 3, 13, 16, 18, and 22;

lix) SEQ ID NOs:1, 4, 13, 16, 18, and 22;

lx) SEQ ID NOs:1, 5, 13, 16, 18, and 22;

lxi) SEQ ID NOs:1, 6, 13, 16, 18, and 22;

lxii) SEQ ID NOs:1, 7, 13, 16, 18, and 22;

lxiii) SEQ ID NOs:1, 8, 13, 16, 18, and 22;

lxiv) SEQ ID NOs:1, 2, 14, 16, 18, and 22;

lxv) SEQ ID NOs:1, 3, 14, 16, 18, and 22;

lxvi) SEQ ID NOs:1, 4, 14, 16, 18, and 22;

lxvii) SEQ ID NOs:1, 5, 14, 16, 18, and 22;

lxviii) SEQ ID NOs:1, 6, 14, 16, 18, and 22;

lxix) SEQ ID NOs:1, 7, 14, 16, 18, and 22;

lxx) SEQ ID NOs:1, 8, 14, 16, 18, and 22;

lxxi) SEQ ID NOs:1, 2, 13, 16, 19, and 22;

lxxii) SEQ ID NOs:1, 3, 13, 16, 19, and 22;

lxxiii) SEQ ID NOs:1, 4, 13, 16, 19, and 22;

lxxiv) SEQ ID NOs:1, 5, 13, 16, 19, and 22;

lxxv) SEQ ID NOs:1, 6, 13, 16, 19, and 22;

lxxvi) SEQ ID NOs:1, 7, 13, 16, 19, and 22;

lxxvii) SEQ ID NOs:1, 8, 13, 16, 19, and 22;

lxxviii) SEQ ID NOs:1, 2, 14, 16, 19, and 22;

lxxix) SEQ ID NOs:1, 3, 14, 16, 19, and 22;

lxxx) SEQ ID NOs:1, 4, 14, 16, 19, and 22;

lxxxi) SEQ ID NOs:1, 5, 14, 16, 19, and 22;

lxxxii) SEQ ID NOs:1, 6, 14, 16, 19, and 22;

lxxxiii) SEQ ID NOs:1, 7, 14, 16, 19, and 22;

lxxxiv) SEQ ID NOs:1, 8, 14, 16, 19, and 22;

lxxxv) SEQ ID NOs:1, 2, 9, 13, 16, 18, and 22;

lxxxvi) SEQ ID NOs:1, 3, 9, 13, 16, 18, and 22;

lxxxvii) SEQ ID NOs:1, 4, 9, 13, 16, 18, and 22;

lxxxviii) SEQ ID NOs:1, 5, 9, 13, 16, 18, and 22;

lxxxix) SEQ ID NOs:1, 6, 9, 13, 16, 18, and 22;

xc) SEQ ID NOs:1, 7, 9, 13, 16, 18, and 22;

xci) SEQ ID NOs:1, 8, 9, 13, 16, 18, and 22;

xcii) SEQ ID NOs:1, 2, 9, 14, 16, 18, and 22;

xciii) SEQ ID NOs:1, 3, 9, 14, 16, 18, and 22;

xciv) SEQ ID NOs:1, 4, 9, 14, 16, 18, and 22;

xcv) SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22;

xcvi) SEQ ID NOs:1, 6, 9, 14, 16, 18, and 22;

xcvii) SEQ ID NOs:1, 7, 9, 14, 16, 18, and 22;

xcviii) SEQ ID NOs:1, 8, 9, 14, 16, 18, and 22;

xcix) SEQ ID NOs:1, 2, 9, 13, 16, 19, and 22;

c) SEQ ID NOs:1, 3, 9, 13, 16, 19, and 22;

ci) SEQ ID NOs:1, 4, 9, 13, 16, 19, and 22;

cii) SEQ ID NOs:1, 5, 9, 13, 16, 19, and 22;

ciii) SEQ ID NOs:1, 6, 9, 13, 16, 19, and 22;

civ) SEQ ID NOs:1, 7, 9, 13, 16, 19, and 22;

cv) SEQ ID NOs:1, 8, 9, 13, 16, 19, and 22;

cvi) SEQ ID NOs:1, 2, 9, 14, 16, 19, and 22;

cvii) SEQ ID NOs:1, 3, 9, 14, 16, 19, and 22;

cviii) SEQ ID NOs:1, 4, 9, 14, 16, 19, and 22;

cix) SEQ ID NOs:1, 5, 9, 14, 16, 19, and 22;

cx) SEQ ID NOs:1, 6, 9, 14, 16, 19, and 22;

cxi) SEQ ID NOs:1, 7, 9, 14, 16, 19, and 22; and

cxii) SEQ ID NOs:1, 8, 9, 14, 16, 19, and 22.

Definitions

Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which this invention pertains.

The term “capsid” refers to the protein coat of the virus or viral vector. The term “AAV capsid” refers to the protein coat of the adeno-associated virus (AAV), which is composed of a total of 60 subunits; each subunit is an amino acid sequence, which can be viral protein 1 (VP1), VP2, or VP3 (Muzyczka N and Berns K I (2001) Chapter 69, Fields Virology. Lippincott Williams & Wilkins).

The term “gene cassette” refers to a manipulatable fragment of DNA carrying, and capable of expressing, one or more genes or coding sequences of interest, for example, between one or more sets of restriction sites, though straddling restriction sites are not required. A gene cassette, or a portion thereof, can be transferred from one DNA sequence (often in a plasmid vector) to another by cutting the fragment out using restriction enzymes and ligating it back into a new context, for example, into a new plasmid backbone.

The term “heterologous gene” or “heterologous nucleotide sequence” will typically refer to a gene or nucleotide sequence that is not naturally-occurring in the virus. Alternatively, a heterologous gene or heterologous nucleotide sequence may refer to a viral sequence that is placed into a non-naturally occurring environment (e.g., by association with a promoter with which it is not naturally associated in the virus).

The terms “inverted terminal repeat” or “ITR” refer to a stretch of nucleotide sequences that exist in adeno-associated viruses (AAV) and/or recombinant adeno-associated viral vectors (rAAV) that can form a T-shaped palindromic structure, which is required for completing wild-type AAV lytic and latent life cycles (Muzyczka N and Berns K I (2001) Chapter 69, Fields Virology. Lippincott Williams & Wilkins). In rAAV, these sequences play a functional role in genome packaging and in second-strand synthesis.

The term “operably linked” refers to a functional relationship between two or more polynucleotide (e.g., DNA) segments. Typically, the term refers to the functional relationship of a transcriptional regulatory sequence to a sequence to be transcribed. For example, a promoter or enhancer sequence is operably linked to a coding sequence if it stimulates or modulates the transcription of the coding sequence in an appropriate host cell or other expression system. Generally, promoter transcriptional regulatory sequences that are operably linked to a transcribable sequence are contiguous to the transcribable sequence, i.e., they are cis-acting. However, some transcriptional regulatory sequences, such as enhancers, need not be physically contiguous or located in close proximity to the coding sequences whose transcription they enhance.

As used herein, the term “percent sequence identity” refers to the degree of identity between any given query sequence and a subject sequence. A subject sequence typically has a length that is from about 80 percent to 250 percent of the length of the query sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 115, or 120, 130, 140, 150, 160, 170, 180, 190, 200, 210, 220, 230, 240, or 250 percent of the length of the query sequence. To determine the percent identity of two nucleotide sequences, or of two amino acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in one or both of a first and a second amino acid or nucleotide sequence for optimal alignment and non-homologous sequences can be disregarded for comparison purposes). The nucleotides or amino acid residues at corresponding nucleotide positions or amino acid positions are then compared. When a position in the first sequence is occupied by the same nucleotide or amino acid residue as the corresponding position in the second sequence, then the molecules are identical at that position (as used herein, nucleotide or amino acid “identity” is equivalent to nucleotide or amino acid “homology”). The percent identity between the two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps, and the length of each gap, which needs to be introduced for optimal alignment of the two sequences.

In another embodiment, the percent identity of two amino acid sequences can be assessed as a function of the conservation of amino acid residues within the same family of amino acids (e.g., positive charge, negative charge, polar and uncharged, hydrophobic) at corresponding positions in both amino acid sequences (e.g., the presence of an alanine residue in place of a valine residue at a specific position in both sequences shows a high level of conservation, but the presence of an arginine residue in place of an aspartate residue at a specific position in both sequences shows a low level of conservation). For purposes of the present invention, the comparison of sequences and determination of percent identity between two sequences can be accomplished using a Blossum 62 scoring matrix with a gap penalty of 12, a gap extend penalty of 4, and a frameshift gap penalty of 5.

The term “promoter” refers to a sequence that regulates transcription of an operably linked gene, or nucleotide sequence encoding a protein. Promoters provide the sequence sufficient to direct transcription, as well as, the recognition sites for RNA polymerase and other transcription factors required for efficient transcription and can direct cell specific expression. In addition to the sequence sufficient to direct transcription, a promoter sequence of the invention can also include sequences of other regulatory elements that are involved in modulating transcription (e.g., enhancers, minimal promoters, Kozak sequences, and introns). Examples of promoters known in the art and useful in the viral vectors described herein include ubiquitous promoters such as the CMV promoter (e.g., SEQ ID NO:2), CBA promoter (e.g., SEQ ID NO:3), and CAG promoter (e.g., SEQ ID NO:4). Alternatively, a RPE-specific promoter may be used to target expression of CYP4V2 preferentially in RPE cells of the retina. Examples of RPE-specific promoters include a ProC2 promoter (e.g., SEQ ID NO:5), and VMD2 promoter (SEQ ID NO:6). In some embodiments, the CYP4V2 promoter (SEQ ID NO:7) or RPE65 promoter (SEQ ID NO:8) can be used as a RPE-specific promoter. In addition, standard techniques are known in the art for creating functional promoters by mixing and matching known regulatory elements. “Truncated promoters” may also be generated from promoter fragments or by mixing and matching fragments of known regulatory elements.

The term “CYP4V2” refers to cytochrome P450 family 4 subfamily V member 2. The human CYP4V2 gene is found on chromosome 4 and has the nucleotide coding sequence as set out, for example, in SEQ ID NO:13. In one embodiment, a codon-optimized sequence of the human CYP4V2 gene can be used. One example of such a codon-optimized CYP4V2 gene has the nucleotide coding sequence as set out in SEQ ID NO:14. The “CYP4V2 gene product” is the protein encoded by a CYP4V2 gene. In one embodiment, an exemplary human CYP4V2 gene product has an amino acid sequence as set out in SEQ ID NO:15. In one embodiment, a CYP4V2 coding sequence encodes the amino acid sequence of SEQ ID NO:15 or a functional variant or fragment thereof. Examples of CYP4V2 coding sequences and CYP4V2 gene products from other species can be found in Table 2 (e.g., SEQ ID NOs:39-50). The term “CYP4V2 coding sequence” or “CYP4V2 GENE CDS” or “CYP4V2 CDS” refers to a nucleotide sequence that encodes a CYP4V2 gene product. One of skill in the art will understand that a CYP4V2 coding sequence may include any nucleotide sequence that encodes a CYP4V2 gene product or a functional variant or fragment thereof. In one embodiment, the CYP4V2 coding sequence encodes the amino acid sequence of SEQ ID NO:15, 40, 42, 44, 46, 48, 50, or a functional variant or fragment thereof. The CYP4V2 coding sequence may or may not include intervening regulatory elements (e.g., introns, enhancers, or other non-coding sequences).

The term “subject” includes human and non-human animals. Non-human animals include all vertebrates (e.g., mammals and non-mammals) such as, non-human primates (e.g., cynomolgus monkey), mice, rats, sheep, dogs, cows, chickens, amphibians, and reptiles. Except when noted, the terms “patient” or “subject” are used herein interchangeably.

As used herein, the term “treating” or “treatment” of any disease or disorder (e.g., BCD) refers to ameliorating the disease or disorder such as by slowing or arresting or reducing the development of the disease or at least one of the clinical symptoms thereof. “Treating” or “treatment” can also refer to alleviating or ameliorating at least one physical parameter, including those that may not be discernible by the subject. “Treating” or “treatment” can also refer to modulating the disease or disorder, either physically, (e.g., stabilization of a discernible symptom), physiologically, (e.g., stabilization of a physical parameter), or both. More specifically, “treatment” of BCD means any action that results in the improvement or preservation of visual function, functional vision, retinal anatomy, and/or Quality of Life in a subject having BCD. As used herein, “treatment” may mean any manner in which one or more of the symptoms of BCD are ameliorated or otherwise beneficially altered. As used herein, amelioration of the symptoms of BCD refers to any lessening, whether permanent or temporary, lasting or transient, that can be attributed to or associated with treatment by the compositions and methods of the present invention. “Preventing or “prevention” as used herein, refers to preventing or delaying the onset or development or progression of the disease or disorder. “Prevention” as it relates to BCD means any action that prevents or slows a worsening in visual function, functional vision, retinal anatomy, Quality of Life, and/or a BCD disease parameter, as described below, in a patient with BCD and at risk for said worsening. Methods for assessing treatment and/or prevention of disease are known in the art and described herein below.

The term “virus vector” or “viral vector” is intended to refer to a non-wild-type recombinant viral particle (e.g., a parvovirus, etc.) that functions as a gene delivery vehicle and which comprises a recombinant viral genome packaged within a viral (e.g., AAV) capsid. A specific type of virus vector may be a “recombinant adeno-associated virus vector”, or “rAAV vector”. The recombinant viral genome packaged in the viral vector is also referred to herein as the “vector genome”.

DESCRIPTION OF DRAWINGS

FIG. 1 are photomicrographs showing ChR2d-eGFP expression in flatmounts of the posterior eyecup. Eyecups were isolated from PFA-fixed eyes, cut into petals, and analyzed for eGFP fluorescence.

FIG. 2A and FIG. 2B are graphs showing mRNA expression levels of ChR2d-eGFP as measured by ddPCR. Fold change in expression relative to TM073 is shown for both the (FIG. 2A) posterior eyecup and (FIG. 2B) neural retina. ChR2d-eGFP expression was normalized to Rab7 control expression for each sample.

FIG. 3 shows confocal images of a NHP retina infected with AAV-ProC2-CatCh-GFP.

FIGS. 3A and 3B: retina sections showing CatCh-GFP (green or gray area in a grayscale image at the top) and nuclear stain (Hoechst, white). FIG. 3C: confocal images of AAV-infected retinas (top view), CatCh-GFP (black). FIGS. 3D and 3E: quantification of CatCh-GFP+ cell density as a percentage of target cell-type or cell class density; values are the mean±s.e.m. from n=10 confocal images. Quantification of AAV-targeting specificity is shown as a percentage of the major (black) cell types among cells expressing the transgene. T, temporal retina quarter; N, nasal retina quarter.

DETAILED DESCRIPTION

The present disclosure is based in part on the discovery that expression of CYP4V2 from recombinant adeno-associated viral vectors (rAAV) having a combination of selected promoter, AAV genome, and capsid serotype provides a potent and efficacious treatment for BCD, e.g., to subjects with a mutation in their CYP4V2 gene (Table 1). Accordingly, the present disclosure provides recombinant viral vectors that direct expression of the CYP4V2 coding sequence to the retina, viral vector compositions, plasmids useful for generating the viral vectors, methods of delivering a CYP4V2 coding sequence to the retina, methods of expressing a CYP4V2 coding sequence in RPE cells of the retina, and methods of use of such viral vectors.

TABLE 1

CYP4V2 mutations associated with BCD

Nucleotide change
Polypeptide change

c.31C > T
p.Q11X

c.64C > G
p.L22V

c.65T > A
p.L22H

c.71T > C
p.L24P

c.130T > A
p.W44R

c.134A > C
p.Q45P

c.181G > A
p.G61S

c.197T > G
p.M66R

c.215 − 2A > G
Splicing acceptor

c.219T > A
p.F73L

c.237G > T
p.E79D

c.253C > T
p.R85C

c.254G > A
p.R85H

c.283G > A
p.G95R

c.328 − 1G > A
Exon3del

c.332T > C
p.I111T

c.335T > G
p.L112X - termination

c.367A > G
p.M123V

c.368T > G
p.M123R

c.400G > T
p.G134X - termination

c.413 + 2T > G
Mis-splicing - Splicing acceptor

c.677T > A
p.M226L

c.694C > T
p.R232X (substitution - nonsense)

c.732G > A
p.W244X

c.791del T
deletion

c.801 + 5G > A
Exon6del

c.802 − 9A > G
Altered splicing

c.802 − 8_810del17insGC
Mis-splicing - Splicing acceptor

c.838G > T
p.E280X

c.958C > T
p.R320X

c.992A > C
p.H331P

c.994G > A
p.D332N

c.1020G > A
p.W340X

c.1027T > G
p.Y343D

c.1061 − 1062insA
frameshift

c.1091 − 2A > G
Mis-splicing - Splicing acceptor

c.1168C > T
p.R390C

c.1169G > A
p.R390H

c.1187C > T
p.P396L

c.1198C > T
p.R400C

c.1199G > A
p.R400H

c.1216T > C
p.C406R

c.1278G > T
p.L426F

c.1400G > A
p.C467Y

c.1508G > A
p.G503E

Except as otherwise indicated, standard methods known to those skilled in the art may be used for the construction of recombinant parvovirus and rAAV vectors, using recombinant plasmids carrying a viral gene cassette, packaging plasmids expressing the parvovirus rep and/or cap sequences, as well as transiently and stably transfected packaging cells. Such techniques are known to those skilled in the art. (e.g., Sambrook et al., MOLECULAR CLONING: A LABORATORY MANUAL 2nd Ed. (Cold Spring Harbor, N.Y., 1989); Choi et al., CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (2007)).

When a viral vector expresses a particular protein or activity, it is not necessary that the relevant gene(s) be identical to the corresponding gene(s) found in nature or disclosed herein. So long as the protein is functional, it may be used in accordance with one aspect of the present invention. One of skill in the art could readily determine if a CYP4V2 coding sequence encodes a functional w-hydroxylase by detecting hydroxylase activity. Briefly, a protein of interest is mixed with fatty acids and other required factors and incubated to allow the hydroxylation reaction to occur. Then, the hydroxylated fatty acids can be measured by mass spectrometry. See, e.g., a functional assay, as described in Nakano et al., Drug Metab Dispos 37:2119-2122, 2009. Very high sequence identity with the natural protein, however, is generally preferred. For instance, large deletions (e.g., greater than about 50 amino acids) should generally be avoided according to certain embodiments of the invention. Therefore, skilled practitioners will appreciate that the present viral vector sequences can vary from the sequences described herein. In some embodiments, the viral nucleotide or amino acid sequence has greater than or about 80% identity to the sequences provided herein, e.g., greater than or about 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequences provided herein.

In some embodiments, a sequence change is a conservative substitution. Such a change includes substituting any of isoleucine (I), valine (V), and leucine (L) for any other of these hydrophobic amino acids; aspartic acid (D) for glutamic acid (E) and vice versa; glutamine (Q) for asparagine (N) and vice versa; and serine (S) for threonine (T) and vice versa. Other substitutions can also be considered conservative depending on the environment of the particular amino acid and its role in the three-dimensional structure of the protein. For example, glycine (G) and alanine (A) can frequently be interchangeable, as can alanine (A) and valine (V). Methionine (M), which is relatively hydrophobic, can frequently be interchanged with leucine and isoleucine, and sometimes with valine. Lysine (K) and arginine (R) are frequently interchangeable in locations in which the significant feature of the amino acid residue is its charge and the differing pK's of these two amino acid residues are not significant. Still other changes can be considered “conservative” in particular environments (see, e.g., Table III of US 20110201052; pages 13-15 “Biochemistry” 2nd Ed. Stryer ed (Stanford University); Henikoff et al., Proc Natl Acad Sci USA 89:10915-10919, 1992; Lei et al., J Biol Chem 270:11882-11886, 1995).

Viral Vectors

The present invention is related to viral vectors that direct expression of a heterologous gene to the retina. In certain aspects of the invention, expression is directed preferentially to RPE cells of the retina. A variety of viral vectors known in the art may be adapted by one of skill in the art for use in the present invention, for example, recombinant adeno-associated viruses, recombinant adenoviruses, recombinant retroviruses, recombinant poxviruses, and recombinant baculoviruses.

In particular, it is contemplated that the viral vector of the invention may be a recombinant adeno-associated (rAAV) vector. AAVs are small, single-stranded DNA viruses that require helper virus to facilitate efficient replication (Muzyczka N and Berns K I (2001) Chapter 69, Fields Virology. Lippincott Williams & Wilkins). The viral vector comprises a vector genome and a protein capsid. The viral vector capsid may be supplied from any of the AAV serotypes known in the art, including presently identified human and non-human AAV serotypes and AAV serotypes yet to be identified (see, e.g., Choi et al., Curr Gene Ther 5:299-310, 2005; Schmidt et al., J Virol 82:1399-1406, 2008; U.S. Pat. Nos. 9,193,956; 9,186,419; 8,632,764; 8,663,624; 8,927,514; 8,628,966; 8,263,396; 8,734,809; 8,889,641; 8,632,764; 8,691,948; 8,299,295; 8,802,440; 8,445,267; 8,906,307; 8,574,583; 8,067,015; 7,588,772; 7,867,484; 8,163,543; 8,283,151; 8,999,678; 7,892,809; 7,906,111; 7,259,151; 7,629,322; 7,220,577; 8,802,080; 7,198,951; 8,318,480; 8,962,332; 7,790,449; 7,282,199; 8,906,675; 8,524,446; 7,712,893; 6,491,907; 8,637,255; 7,186,522; 7,105,345; 6,759,237; 6,984,517; 6,962,815; 7,749,492; 7,259,151; and 6,156,303; U.S. Publication Nos. 2013/0295614; 2015/0065562; 2014/0364338; 2013/0323226; 2014/0359799; 2013/0059732; 2014/0037585; 2014/0056854; 2013/0296409; 2014/0335054 2013/0195801; 2012/0070899; 2011/0275529; 2011/0171262; 2009/0215879; 2010/0297177; 2010/0203083; 2009/0317417; 2009/0202490; 2012/0220492; 2006/0292117; and 2004/0002159; European Publication Numbers 2692731 AI; 2383346 BI; 2359865 BI; 2359866 BI; 2359867 BI; and 2357010 BI; 1791858 BI; 1668143 BI; 1660678 BI; 1664314 BI; 1496944 BI; 1456383 BI; 2341068 BI; 2338900 BI; 1456419 BI; 1310571 BI; 1456383 BI; 1633772 BI; and 1135468 BI; and PCT Publication Nos. WO 2014/124282; WO 2013/170078; WO 2014/160092; WO 2014/103957; WO 2014/052789; WO 2013/174760; WO 2013/123503; WO 2011/038187; WO 2008/124015; and WO 2003/054197).

For the purposes of the disclosure herein, AAV refers to the virus itself and derivatives thereof. Except where otherwise indicated, the terminology refers to all subtypes or serotypes and both replication-competent and recombinant forms. The term “AAV” includes, without limitation, AAV type 1 (AAV1), AAV type 2 (AAV2), AAV type 3A (AAV3A), AAV type 3B (AAV3B), AAV type 4 (AAV4), AAV type 5 (AAV5), AAV type 6 (AAV6), AAV type 7 (AAV7), AAV type 8 (AAV8), AAV type 9 (AAV9), AAV type 10 (AAV 10 or AAVrh10), avian AAV, bovine AAV, canine AAV, caprine AAV, equine AAV, primate AAV, non-primate AAV, and ovine AAV. “Primate AAV” refers to AAV that infect primates, “non-primate AAV” refers to AAV that infect non-primate mammals, “bovine AAV” refers to AAV that infect bovine mammals, etc.

The genomic sequences of various serotypes of AAV, as well as the sequences of the native inverted terminal repeats (ITRs), Rep proteins, and capsid subunits are known in the art. Such sequences may be found in the literature or in public databases such as GenBank.

See, e.g., GenBank Accession Numbers NC_002077.1 (AAV1), AF063497.1 (AAV1), NC_001401.2 (AAV2), AF043303.1 (AAV2), J01901.1 (AAV2), U48704.1 (AAV3A), NC_001729.1 (AAV3A), AF028705.1 (AAV3B), NC_0.001829.1 (AAV4), U89790.1 (AAV4), NC_006152.1 (AA5), AF085716.1 (AAV-5), AF028704.1 (AAV6), NC_006260.1 (AAV7), AF513851.1 (AAV7), AF513852.1 (AAV8) NC_006261.1 (AAV8), AY530579.1 (AAV9), AAT46337 (AAV10), and AA088208 (AAVrh10); the disclosures of which are incorporated by reference herein for teaching AAV nucleic acid and amino acid sequences. See also, e.g., Srivastava et al., J Virol. 45:555-564, 1983; Chiorini et al., J Virol 71:6823-6833, 1998; Chiorini et al., J Virol 73:1309-1319, 1999; Bantel-Schaal et al., J Virol 73:939-947, 1999; Xiao et al., J Virol 73:3994-4003, 1999; Muramatsu et al., Virology 221:208-217, 1996; Shade et al., J Virol 58:921-936, 1986; Gao et al., Proc Natl Acad Sci USA 99:11854-11859, 2002; PCT Publication Nos. WO 00/28061, WO 99/61601, and WO 98/11244; and U.S. Pat. No. 6,156,303.

Virus capsids may be mixed and matched with other vector components to form a hybrid pseudotype viral vector, for example the ITRs and capsid of the viral vector may come from different AAV serotypes. In one aspect, the ITRs can be from an AAV2 serotype while the capsid is from, for example, an AAV8, AAV9, AAV2, or AAV5 serotype. In addition, one of skill in the art will recognize that the vector capsid may also be a mosaic capsid (e.g., a capsid composed of a mixture of capsid proteins from different serotypes), or even a chimeric capsid (e.g., a capsid protein containing a foreign or unrelated protein sequence for generating markers and/or altering tissue tropism). It is contemplated that the viral vector of the invention may comprise an AAV8 capsid (e.g., SEQ ID NOs:24, 25, and 26, encoded by, for example, SEQ ID NO:23). It is also contemplated that the viral vector of the invention may comprise an AAV9 capsid (e.g., SEQ ID NOs:28, 29, and 30, encoded by, for example, SEQ ID NO:27). It is also contemplated that the viral vector of the invention may comprise an AAV2 capsid (e.g., SEQ ID NOs:32, 33, and 34, encoded by, for example, SEQ ID NO:31). It is further contemplated that the invention may comprise an AAV5 capsid (e.g., SEQ ID NOs:36, 37, and 38, encoded by, for example, SEQ ID NO:35).

In one aspect, the AAV is a self-complementary adeno-associated virus (scAAV).

In further particular aspects, the vector genome, e.g., single stranded vector genome, has a length greater than or about 4.1 kb and less than or about 4.9 kb, e.g., greater than or about 4.2 kb and less than or about 4.9 kb, greater than or about 4.3 kb and less than or about 4.9 kb, greater than or about 4.4 kb and less than or about 4.9 kb, greater than or about 4.5 kb and less than or about 4.9 kb, greater than or about 4.6 kb and less than or about 4.9 kb, greater than or about 4.7 kb and less than or about 4.9 kb, greater than or about 4.8 kb and less than or about 4.9 kb, greater than or about 4.1 kb and less than or about 4.8 kb, greater than or about 4.1 kb and less than or about 4.7 kb, greater than or about 4.1 kb and less than or about 4.6 kb, greater than or about 4.1 kb and less than or about 4.5 kb, greater than or about 4.1 kb and less than or about 4.4 kb, greater than or about 4.1 kb and less than or about 4.3 kb, greater than or about 4.1 kb and less than or about 4.2 kb, greater than or about 4.2 kb and less than or about 4.8 kb, greater than or about 4.3 kb and less than or about 4.7 kb, greater than or about 4.4 kb and less than or about 4.6 kb, about 4.1 kb, about 4.2 kb, about 4.3 kb, about 4.4 kb, about 4.5 kb, about 4.6 kb, about 4.7 kb, about 4.8 kb, or about 4.9 kb.

In certain aspects, the invention is related to a vector genome, e.g., single stranded vector genome, comprising, in the 5′ to 3′ direction: (i) a 5′ ITR, (ii) a promoter, (iii) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence, (iv) a polyadenylation (polyA) signal sequence, and (v) a 3′ ITR. In certain aspects of the invention, the vector genome, e.g., single stranded vector genome, comprises in the 5′ to 3′ direction: (i) a 5′ ITR, (ii) a promoter, (iii) an intron, (iv) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence, (v) a polyA signal sequence, and (vi) a 3′ ITR. In some embodiments, the vector genome, e.g., single stranded vector genome, comprises in the 5′ to 3′ direction: (i) a 5′ ITR, (ii) a promoter, (iii) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence, (iv) a regulatory element, (v) a polyA signal sequence, and (vi) a 3′ ITR. In certain aspects of the invention, the vector genome, e.g., single stranded vector genome, comprises in the 5′ to 3′ direction: (i) a 5′ ITR, (ii) a promoter, (iii) an intron, (iv) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence, (v) a regulatory element, (vi) a polyA signal sequence, and (vii) a 3′ ITR. Elements of the vector can have sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to the sequences described in Table 2.

TABLE 2

Nucleotide and amino acid sequences

of viral vector elements

SEQUENCE IDENTIFIER

(SEQ ID NO:)

ELEMENT
AND SEQUENCE

5′ ITR
1

CTGCGCGCTCGCTC

GCTCACTGAGGCCGC

CCGGGCAAAGCCCGG

GCGTCGGGCGACCTT

TGGTCGCCCGGCCTC

AGTGAGCGAGCGAGC

GCGCAGAGAGGGAGT

GGCCAACTCCATCAC

TAGGGGTTCCT

CMV promoter
2

TGTTTACATAACTTA

CGGTAAATGGCCCGC

CTGGCTGACCGCCCA

ACGACCCCCGCCCAT

TGACGTCAATAATGA

CGTATGTTCCCATAG

TAACGCCAATAGGGA

CTTTCCATTGACGTC

AATGGGTGGACTATT

TACGGTAAACTGCCC

ACTTGGCAGTACATC

AAGTGTATCATATGC

CAAGTACGCCCCCTA

TTGACGTCAATGACG

GTAAATGGCCCGCCT

GGCATTATGCCCAGT

ACATGACCTTATGGG

ACTTTCCTACTTGGC

AGTACATCTACGTAT

TAGTCATCGCTATTA

CCATGGTGATGCGGT

TTTGGCAGTACATCA

ATGGGCGTGGATAGC

GGTTTGACTCACGGG

GATTTCCAAGTCTCC

ACCCCATTGACGTCA

ATGGGAGTTTGTTTT

GGCACCAAAATCAAC

GGGACTTTCCAAAAT

GTCGTAACAACTCCG

CCCCATTGACGCAAA

TGGGCGGTAGGCGTG

TACGGTGGGAGGTCT

ATATAGCAGAGCTCT

CTGGCTAACTAGAGA

ACCACTGCTTACTGG

CTTAG

CBA promoter
3

CGTTACATAACTTAC

GGTAAATGGCCCGCC

TGGCTGACCGCCCAA

CGACCCCCGCCCATT

GACGTCAATAATGAC

GTATGTTCCCATAGT

AACGCCAATAGGGAC

TTTCCATTGACGTCA

ATGGGTGGAGTATTT

ACGGTAAACTGCCCA

CTTGGCAGTACATCA

AGTGTATCATATGCC

AAGTACGCCCCCTAT

TGACGTCAATGACGG

TAAATGGCCCGCCTG

GCATTATGCCCAGTA

CATGACCTTATGGGA

CTTTCCTACTTGGCA

GTACATCTACTCGAG

GCCACGTTCTGCTTC

ACTCTCCCCATCTCC

CCCCCCTCCCCACCC

CCAATTTTGTATTTA

TTTATTTTTTAATTA

TTTTGTGCAGCGATG

GGGGCGGGGGGGGGG

GGGGGGCGCGCGCCA

GGCGGGGCGGGGCGG

GGCGAGGGGCGGGGC

GGGGCGAGGCGGAGA

GGTGCGGCGGCAGCC

AATCAGAGCGGCGCG

CTCCGAAAGTTTCCT

TTTATGGCGAGGCGG

CGGCGGCGGCGGCCC

TATAAAAAGCGAAGC

GCGCGGCGGGCGGGA

G

CAG promoter
4

GACATTGATTATTGA

CTAGTTATTAATAGT

AATCAATTACGGGGT

CATTAGTTCATAGCC

CATATATGGAGTTCC

GCGTTACATAACTTA

CGGTAAATGGCCCGC

CTGGCTGACCGCCCA

ACGACCCCCGCCCAT

TGACGTCAATAATGA

CGTATGTTCCCATAG

TAACGCCAATAGGGA

CTTTCCATTGACGTC

AATGGGTGGACTATT

TACGGTAAACTGCCC

ACTTGGCAGTACATC

AAGTGTATCATATGC

CAAGTACGCCCCCTA

TTGACGTCAATGACG

GTAAATGGCCCGCCT

GGCATTATGCCCAGT

ACATGACCTTATGGG

ACTTTCCTACTTGGC

AGTACATCTACGTAT

TAGTCATCGCTATTA

CCATGGGTCGAGGTG

AGCCCCACGTTCTGC

TTCACTCTCCCCATC

TCCCCCCCCTCCCCA

CCCCCAATTTTGTAT

TTATTTATTTTTTAA

TTATTTTGTGCAGCG

ATGGGGGCGGGGGGG

GGGGGGGCGCGCGCC

AGGCGGGGCGGGGCG

GGGCGAGGGGCGGGG

CGGGGCGAGGCGGAG

AGGTGCGGCGGCAGC

CAATCAGAGCGGCGC

GCTCCGAAAGTTTCC

TTTTATGGCGAGGCG

GCGGCGGCGGCGGCC

CTATAAAAAGCGAAG

CGCGCGGCGGGCGGG

AGTCGCTGCGTTGCC

TTCGCCCCGTGCCCC

GCTCCGCGCCGCCTC

GCGCCGCCCGCCCCG

GCTCTGACTGACCGC

GTTACTCCCACAGGT

GAGCGGGCGGGACGG

CCCTTCTCCTCCGGG

CTGTAATTAGCGCTT

GGTTTAATGACGGCT

CGTTTCTTTTCTGTG

GCTGCGTGAAAGCCT

TAAAGGGCTCCGGGA

GGGCCCTTTGTGCGG

GGGGGAGCGGCTCGG

GGGGTGCGTGCGTGT

GTGTGTGCGTGGGGA

GCGCCGCGTGCGGCC

CGCGCTGCCCGGCGG

CTGTGAGCGCTGCGG

GCGCGGCGCGGGGCT

TTGTGCGCTCCGCGT

GTGCGCGAGGGGAGC

GCGGCCGGGGGCGGT

GCCCCGCGGTGCGGG

GGGGCTGCGAGGGGA

ACAAAGGCTGCGTGC

GGGGTGTGTGCGTGG

GGGGGTGAGCAGGGG

GTGTGGGCGCGGCGG

TCGGGCTGTAACCCC

CCCCTGCACCCCCCT

CCCCGAGTTGCTGAG

CACGGCCCGGCTTCG

GGTGCGGGGCTCCGT

GCGGGGCGTGGCGCG

GGGCTCGCCGTGCCG

GGCGGGGGGTGGCGG

CAGGTGGGGGTGCCG

GGCGGGGCGGGGCCG

CCTCGGGCCGGGGAG

GGCTCGGGGGAGGGG

CGCGGCGGCCCCCGG

AGCGCCGGCGGCTGT

CGAGGCGCGGCGAGC

CGCAGCCATTGCCTT

TTATGGTAATCGTGC

GAGAGGGCGCAGGGA

CTTCCTTTGTCCCAA

ATCTGTGCGGAGCCG

AAATCTGGGAGGCGC

CGCCGCACCCCCTCT

AGCGGGCGCGGGGCG

AAGCGGTGCGGCGCC

GGCAGGAAGGAAATG

GGCGGGGAGGGCCTT

CGTGCGTCGCCGCGC

CGCCGTCCCCTTCTC

CCTCTCCAGCCTCGG

GGCTGTCCGCGGGGG

GACGGCTGCCTTCGG

GGGGGACGGGGCAGG

GCGGGGTTCGGCTTC

TGGCGTGTGACCGGC

GGCTCTAGAGCCTCT

GCTAACCATGTTCAT

GCCTTCTTCTTTTTC

CTACAG

ProC2 promoter
5

TCAAGCCTCCTACCG

CCTTTGTTATGCAAA

CATATCAAACGCCCT

CCTTTGTTATGCAAA

AGGGCTGGAACGGGG

CCTTTGTTATGCAAA

TCGCCCTCCCCGATC

CCTTTGTTATGCAAA

TTTGACGAATTCCCA

CCTTTGTTATGCAAA

CAAATCTCCTACCCT

CCTTTGTTATGCAAA

GTGAGAGGGGCTGCA

CCTTTGTTATGCAAA

ATGOGGCCCCTGAGA

CCTTTGTTATGCAAA

AACCATGTACGOTTG

OCTTTGTTATGCAAA

CCGOCTGTTGCTTGG

CCTTTGTTATGCAAA

GCCACGOGATTGGCG

CCTTTGTTATGCAAA

GCTCGGTTATGTACA

CCTTTGTTATGCAAA

GCTACTTTAAACTTG

CCTTTGTTATGCAAA

TCACGACCTGACCGT

CCTTTGTTATGCAAA

AACGGTTGAAATAGT

CCTTTGTTATGCAAA

ATGATATTGAATAGT

CCTTTGTTATGCAAA

AATTTAGATGCCGAC

CCTTTGTTATGCAAA

GGAATGGGCGTGCTG

CCTTTGTTATGCAAA

TTTTCGCTGCGACAG

CCTTTGTTATGCAAA

CATGOTCGCCACTCA

CCTTTGTTATGCAAA

GGTCTAACAATGACC

CCTTTGTTATGCAAA

CTACGTGGAATAGAT

CCTTTGTTATGCAAA

COCCGAGTTTTTGAA

CCTTTGTTATGCAAA

ATCAGTAACTTCATT

CCTTTGTTATGCAAA

ATGTGACTTAACCTC

CCTTTGTTATGCAAA

GCTCGAGATCTGCGA

TCTGCATCTCAATTA

GTCAGCAACCATAGT

CCCGCCCCTAACTCC

GCCCATCCCGCCCCT

AACTCCGCCCAGTTC

CGCCCATTCTCCGCC

CCATCGCTGACTAAT

TTTTTTTATTTATGC

AGAGGCCGAGGCCGC

CTCGGCCTCTGAGCT

ATTCCAGAAGTAGTG

AGGAGGCTTTTTTGG

AGGCCTAGGCTTTTG

CAAA

VMD2 promoter
6

TACGTAATTCTGTCA

TTTTACTAGGGTGAT

GAAATTCCCAAGCAA

CACCATCCTTTTCAG

ATAAGGGCACTGAGG

CTGAGAGAGGAGCTG

AAACCTACCCGGCGT

CACCACACACAGGTG

GCAAGGCTGGGACCA

GAAACCAGGACTGTT

GACTGCAGCCCGGTA

TTCATTCTTTCCATA

GCCCACAGGGCTGTC

AAAGACCCCAGGGCC

TAGTCAGAGGCTOCT

CCTTCCTGGAGAGTT

CCTGGCACAGAAGTT

GAAGCTCAGCACAGC

CCCCTAACCCCCAAC

TCTCTCTGCAAGGCC

TCAGGGGTCAGAACA

CTGGTGGAGCAGATC

CTTTAGCCTCTGGAT

TTTAGGGCCATGGTA

GAGGGGGTGTTGOCC

TAAATTOCAGCCCTG

GTCTCAGCCCAACAC

CCTCCAAGAAGAAAT

TAGAGGGGCCATGGC

CAGGCTGTGCTAGCC

GTTGCTTCTGAGCAG

ATTACAAGAAGGGAC

TAAGACAAGGACTCC

TTTGTGGAGGTCCTG

GCTTAGGGAGTCAAG

TGACGGCGGCTCAGC

ACTCACGTGGGCAGT

GCCAGCCTCTAAGAG

TGGGCAGGGGCACTG

GCCACAGAGTCCCAG

GGAGTCCCACCAGCC

TAGTCGCCAGACC

CYP4V2 promoter
7

CATTACTTTACACAC

TCCGTTCTGCAACTT

GTTTTGTTCACTTGC

TATTTCACGTCAGCC

CATGCAGATGACTTA

AAACAAATTCAAATG

GAGAAAAGCCTTAGG

CATGTTTTTCTCTGC

ACTAAAGAGGCCCTG

CAGGTGGCTGTGGCC

AACTTATGCAGTGGC

TTCGAGTGAACGAAC

GTAATTAGTAATTAA

GCAAAGCGGGCATCC

AGGACATTCAGTTTC

ATCTTCCTGGCACTT

TAGTGTTTAGGAAGG

GCCTGGCCTCTCGGG

GAGCGACACTCCTCG

CCCGGCCACTTTGTA

AGAAGCATCGAGCGT

GGAAAAGGGGCTGGG

GTCAGCACTGGGGCC

CTCTGCCGGGAAGAG

AGACTGGCGGCCAGC

ACACGCGTCTOCCCA

GGCTGAAATGGGGCA

CCCTCTCGCGGCCCT

TCCCTCCCTTCCCGG

GCTGGGTGGCAGGTC

TTGGCTCTTCTGCGA

GTACCCCGCGGCTGC

GGTCTCCCGGCACCC

GCTGAGCAGCCTCGC

CCGCCTTCCTCTCCC

ACCCGCCCCTGCGTG

CTCTCCGGGGCTCCG

GCTTCCTCGGTCTGG

TGCOTGGTGOGTATT

TTGGAAAATACCTGG

CTCATAACCACCTCA

TTGAGCTCCCAGTGT

CCCAGTGGGCCAGCG

GACACACACGGGTAG

CTGACTTCCCTAACG

TGTCCCCAAAGCCTC

CCTGGATTACAGCGC

CCGCTGCTCCATGTG

ACCCCGCGGGAGCCA

GGACGCGCCCCGCCT

CCCGGGGCTGCAAGT

TGCGCAGGGAGCGAG

CGATGCGCAGCAGAG

CTGGGCTCCGAGCCG

GATGCGCCTTCCTTT

CCTCTCCAGAGCAGG

CTGCCCGCCCGCGGA

ATCCCGCACGTAGAG

CAACCTCGCAGCACC

CTCAGAACAGCCCCG

CTGGGGCGCGCCGGG

CTGCCGCGGTGACCT

TTCCGACGCCCCTGA

CCCCGCATCCGGAGG

CGGCCGGAAGTGTCG

CCGGCCTCCTCCCGG

CGCAGCCTCC

RPE65 promoter
8

CGCGTTACGTAATAT

TTATTGAAGTTTAAT

ATTGTGTTTGTGATA

CAGAAGTATTTGCTT

TAATTCTAAATAAAA

ATTTTATGCTTTTAT

TGCTGGTTTAAGAAG

ATTTGGATTATCCTT

GTACTTTGAGGAGAA

GTTTCTTATTTGAAA

TATTTTGGAAACAGG

TCTTTTAATGTGGAA

AGATAGATATTAATC

TCCTCTTCTATTACT

CTCCAAGATCCAACA

AAAGTGATTATACCC

CCCAAAATATGATGG

TAGTATCTTATACTA

CCATCATTTTATAGG

CATAGGGCTCTTAGC

TGCAAATAATGGAAC

TAACTCTAATAAAGC

AGAACGCAAATATTG

TAAATATTAGAGAGC

TAACAATCTCTGGGA

TGGCTAAAGGATGGA

GCTTGGAGGCTACCC

AGCCAGTAACAATAT

TCCGGGCTCCACTGT

TGAATGGAGACACTA

CAACTGCCTTGGATG

GGCAGAGATATTATG

GATGCTAAGCCCCAG

GTGCTACCATTAGGA

CTTCTACCACTGTCC

CTAACGGGTGGAGCC

CATCACATGCCTATG

CCCTCACTGTAAGGA

AATGAAGCTACTGTT

GTATATCTTGGGAAG

CACTTGGATTAATTG

TTATACAGTTTTGTT

GAAGAAGACCCCTAG

GGTAAGTAGCCATAA

CTGCACACTAAATTT

AAAATTGTTAATGAG

TTTCTCAAAAAAAAT

GTTAAGGTTGTTAGC

TGGTATAGTATATAT

CTTGCCTGTTTTCCA

AGGACTTCTTTGGGC

AGTACCTTGTCTGTG

CTGGCAAGCAACTGA

GACTTAATGAAAGAG

TATTGGAGATATGAA

TGAATTGATGCTGTA

TACTCTCAGAGTGCC

AAACATATACCAATG

GACAAGAAGGTGAGG

CAGAGAGCAGACAGG

CATTAGTGACAAGCA

AAGATATGCAGAATT

TCATTCTCAGCAAAT

CAAAAGTCCTCAACC

TGGTTGGAAGAATAT

TGGCACTGAATGGTA

TCAATAAGGTTGCTA

GAGAGGGTTAGAGGT

GCACAATGTGCTTCC

ATAACATTTTATACT

TCTCCAATCTTAGCA

CTAATCAAACATGGT

TGAATACTTTGTTTA

CTATAACTCTTACAG

AGTTATAAGATCTGT

GAAGACAGGGACAGG

GACAATACCCATCTC

TGTCTGGTTCATAGG

TGGTATGTAATAGAT

ATTTTTAAAAATAAG

TGAGTTAATGAATGA

GGGTGAGAATGAAGG

CACAGAGGTATTAGG

GGGAGGTGGGCCCCA

GAGAATGGTGCCAAG

GTCCAGTGGGGTGAC

TGGGATCAGCTCAGG

CCTGACGCTGGCCAC

TCCCACCTAGCTCCT

TTCTTTCTAATCTGT

TCTCATTCTCCTTGG

GAAGGATTGAGGTCT

CTGGAAAACAGCCAA

ACAACTGTTATGGGA

ACAGCAAGCCCAAAT

AAAGCCAAGCATCAG

GGGGATCTGAGAGCT

GAAAGCAACTTCTGT

TCCCCCTCCCTCAGC

TGAAGGGGTGGGGAA

GGGCTCCCAAAGCCA

TAACTCCTTTTAAGG

GATTTAGAAGGCATA

AAAAGGCCCCTGGCT

GAGAACTTCCTTCTT

CATTCTGCAGTTGGT

G

Human growth
9

hormone (hGH)
TTCGAACAGGTAAGC

intron
GCCCCTAAAATCCCT

TTGGGCACAATGTGT

CCTGAGGGGAGAGGC

AGCGACCTGTAGATG

GGACGGGGGCACTAA

CCCTCAGGTTTGGGG

CTTCTGAATGTGAGT

ATCGCCATGTAAGCC

CAGTATTTGGCCAAT

CTCAGAAAGCTCCTG

GTCCCTGGAGGGATG

GAGAGAGAAAAACAA

ACAGCTCCTGGAGCA

GGGAGAGTGCTGGCC

TCTTGCTCTCCGGCT

CCCTCTGTTGCCCTC

TGGTTTCTCCCCAGG

TT

Simian Virus 40
10

(SV40) intron
AACTGAAAAACCAGA

AAGTTAACTGGTAAG

TTTAGTCTTTTTGTC

TTTTATTTCAGGTCC

CGGATCCGGTGGTGG

TGCAAATCAAAGAAC

TGCTCCTCAGTGGAT

GTTGCCTTTACTTCT

AGGCCTGTACGGAAG

TGTTACTTCTGCTCT

AAAAGCTGCGGAATT

GTACCCGCCCCGGGA

TCC

Human beta
11

gobin intron
CGAATCCCGGCCGGG

AACGGTGCATTGGAA

CGCGGATTCCCCGTG

CCAAGAGTGACGTAA

GTACCGCCTATAGAG

TCTATAGGCCCACAA

AAAATGCTTTCTTCT

TTTAATATACTTTTT

TGTTTATCTTATTTC

TAATACTTTCCCTAA

TCTCTTTCTTTCAGG

GCAATAATGATACAA

TGTATCATGCCTCTT

TGCACCATTCTAAAG

AATAACAGTGATAAT

TTCTGGGTTAAGGCA

ATAGCAATATTTCTG

CATATAAATATTTCT

GCATATAAATTGTAA

CTGATGTAAGAGGTT

TCATATTGCTAATAG

CAGCTACAATCCAGC

TACCATTCTGCTTTT

ATTTTATGGTTGGGA

TAAGGCTGGATTATT

CTGAGTCCAAGCTAG

GCCCTTTTGCTAATC

ATGTTCATACCTCTT

ATCTTCCTCCCACAG

CTCCTGGGCAACGTG

CTGGTCTGTGTGCTG

GCCCATCACTTTGGC

AAAGAATTGGGAT

Kozak sequence
12

GCCACC

Homo sapiens

13

CYP4V2
ATGGCGGGGCTCTGG

Wild-type ODS
CTGGGGCTCGTGTGG

NM_207352.4
CAGAAGCTGCTGCTG

TGGGGCGCGGCGAGT

GCCCTTTCCCTGGCC

GGCGCCAGTCTGGTC

CTGAGCCTGCTGCAG

AGGGTGGCGAGCTAC

GCGCGGAAATGGCAG

CAGATGCGGCCCATC

CCCACGGTGGCCCGC

GCCTACCCACTGGTG

GGCCACGCGCTGCTG

ATGAAGCCGGACGGG

CGAGAATTTTTTCAG

CAGATCATTGAGTAC

ACAGAGGAATACCGC

CACATGCCGCTGCTG

AAGCTCTGGGTCGGG

CCAGTGCCCATGGTG

GCCCTTTATAATGCA

GAAAATGTGGAGGTA

ATTTTAACTAGTTCA

AAGCAAATTGACAAA

TCCTCTATGTACAAG

TTTTTAGAACCATGG

CTTGGCCTAGGACTT

CTTACAAGTACTGGA

AACAAATGGCGCTCC

AGGAGAAAGATGTTA

ACACCCACTTTCCAT

TTTACCATTCTGGAA

GATTTCTTAGATATC

ATGAATGAACAAGCA

AATATATTGGTTAAG

AAACTTGAAAAACAC

ATTAACCAAGAAGCA

TTTAACTGCTTTTTT

TACATCACTCTTTGT

GCCTTAGATATCATC

TGTGAAACAGCTATG

GGGAAGAATATTGGT

GCTCAAAGTAATGAT

GATTCCGAGTATGTC

CGTGCAGTTTATAGA

ATGAGTGAGATGATA

TTTCGAAGAATAAAG

ATGCCCTGGCTTTGG

CTTGATCTCTGGTAC

CTTATGTTTAAAGAA

GGATGGGAACACAAA

AAGAGCCTTCAGATC

CTACATACTTTTACC

AACAGTGTCATCGCT

GAACGGGCCAATGAA

ATGAACGCCAATGAA

GACTGTAGAGGTGAT

GGCAGGGGCTCTGCC

CCCTCCAAAAATAAA

CGCAGGGCCTTTCTT

GACTTGCTTTTAAGT

GTGACTGATGACGAA

GGGAACAGGCTAAGT

CATGAAGATATTCGA

GAAGAAGTTGACACC

TTCATGTTTGAGGGG

CACGATACAACTGCA

GCTGCAATAAACTGG

TCCTTATACCTGTTG

GGTTCTAACCCAGAA

GTCCAGAAAAAAGTG

GATCATGAATTGGAT

GACGTGTTTGGGAAG

TCTGACCGTCCCGCT

ACAGTAGAAGACCTG

AAGAAACTTCGGTAT

CTGGAATGTGTTATT

AAGGAGACCCTTCGC

CTTTTTCCTTCTGTT

CCTTTATTTGCCCGT

AGTGTTAGTGAAGAT

TGTGAAGTGGCAGGT

TACAGAGTTCTAAAA

GGCACTGAAGCCGTC

ATCATTCCCTATGCA

TTGCACAGAGATCCG

AGATACTTCCCCAAC

CCCGAGGAGTTCCAG

CCTGAGCGGTTCTTC

CCCGAGAATGCACAA

GGGCGCCATCCATAT

GCCTACGTGCCCTTC

TCTGCTGGCCCCAGG

AACTGTATAGGTCAA

AAGTTTGCTGTGATG

GAAGAAAAGACCATT

CTTTCGTGCATCCTG

AGGCACTTTTGGATA

GAATCCAACCAGAAA

AGAGAAGAGCTTGGT

CTAGAAGGACAGTTG

ATTCTTCGTCCAAGT

AATGGCATCTGGATC

AAGTTGAAGAGGAGA

AATGCAGATGAACGC

TAA

Codon-
14

optimized
ATGGCCGGACTGTGG

CYP4V2
CTGGGACTGGTCTGG

sequence
CAGAAGTTATTACTG

TGGGGAGCTGCCTCC

GCTCTGTCTTTAGCT

GGAGCTTCTTTAGTG

CTGTCTTTACTGCAG

AGGGTCGCCTCCTAT

GCTAGGAAGTGGCAG

CAGATGAGGCCTATT

CCTACCGTGGCCAGA

GCCTATCCTTTAGTG

GGCCACGCTCTGCTG

ATGAAACCCGACGGA

AGGGAGTTCTTCCAG

CAGATCATCGAGTAC

ACCGAAGAGTACAGA

CACATGCCTTTACTG

AAACTGTGGGTGGGA

CCCGTTCCTATGGTG

GCTTTATACAATGCC

GAGAATGTGGAGGTG

ATTTTAACCAGCAGC

AAGCAGATCGACAAG

TCCAGCATGTATAAG

TTTTTAGAGCCTTGG

CTCGGTTTAGGACTG

CTGACCTCCACTGGT

AATAAGTGGAGGTCT

CGTAGGAAAATGCTG

ACCCCCACCTTTCAC

TTCACCATTTTAGAG

GACTTTTTAGATATC

ATGAACGAGCAAGCT

AACATTTTAGTGAAG

AAGCTCGAAAAGCAC

ATTAACCAAGAAGCT

TTCAACTGTTTTTTC

TACATCACTTTATGC

GCCCTCGATATCATC

TGCGAGACAGCCATG

GGCAAGAACATTGGC

GCTCAGAGCAACGAC

GATTCCGAGTACGTG

AGGGCTGTCTACAGA

ATGAGCGAGATGATC

TTCAGAAGAATCAAG

ATGCCTTGGCTGTGG

CTGGACCTCTGGTAT

TTAATGTTTAAGGAA

GGCTGGGAGCATAAG

AAGTCTTTACAGATT

TTACATACATTTACC

AACAGCGTGATCGCC

GAGAGGGCCAATGAA

ATGAACGCCAACGAG

GATTGTCGTGGCGAC

GGAAGAGGCTCCGCT

CCTTCCAAGAACAAG

AGGAGAGCCTTTTTA

GATCTCTTATTATCC

GTGACAGACGATGAG

GGCAATAGGCTGAGC

CACGAGGACATCAGA

GAAGAGGTGGACACC

TTCATGTTCGAGGGA

CACGACACAACCGCC

GCCGCCATCAATTGG

TCTTTATATTTACTC

GGCAGCAACCCCGAG

GTGCAAAAAAAGGTC

GACCACGAGCTCGAC

GACGTGTTCGGCAAG

AGCGATCGTCCCGCC

ACAGTGGAAGATTTA

AAGAAGCTGAGGTAT

CTCGAGTGCGTGATC

AAAGAGACTTTAAGA

CTGTTCCCCAGCGTG

CCTCTGTTTGCTCGT

TCCGTGTCCGAAGAC

TGCGAGGTGGCTGGA

TATCGTGTCCTCAAG

GGCACCGAGGCCGTG

ATCATTCCCTACGCC

CTCCATCGTGATCCC

AGATACTTCCCCAAT

CCCGAGGAGTTCCAG

CCCGAAAGGTTCTTC

CCCGAAAACGCTCAA

GGCAGACACCCTTAC

GCTTACGTGCCTTTC

TCCGCCGGCCCTCGT

AACTGCATTGGCCAG

AAATTCGCCGTCATG

GAGGAAAAGACCATT

TTATCTTGTATTTTA

AGGCACTTCTGGATC

GAAAGCAATCAGAAA

AGGGAGGAACTCGGT

TTAGAAGGACAGCTG

ATTTTAAGACCCAGC

AACGGCATTTGGATC

AAGCTGAAGAGGAGG

AACGCCGACGAGAGG

TGA

Homo sapiens

15

CYP4V2
MAGLWLGLVWQKLLL

Gene Product
WGAASALSLAGASLV

NP_997235.3
LSLLQRVASYARKWQ

QMRPIPTVARAYPLV

GHALLMKPDGREFFQ

QIIEYTEEYRHMPLL

KLWVGPVPMVALYNA

ENVEVILTSSKQIDK

SSMYKFLEPWLGLGL

LTSTGNKWRSRRKML

TPTFHFTILEDFLDI

MNEQANILVKKLEKH

INQEAFNCFFYITLC

ALDIICETAMGKNIG

AQSNDDSEYVRAVYR

MSEMIERRIKMPWLW

LDLWYLMFKEGWEHK

KSLQILHTFTNSVIA

ERANEMNANEDCRGD

GRGSAPSKNKRRAFL

DLLLSVTDDEGNRLS

HEDIREEVDTFMFEG

HDTTAAAINWSLYLL

GSNPEVQKKVDHELD

DVFGKSDRPATVEDL

KKLRYLECVIKETLR

LFPSVPLFARSVSED

CEVAGYRVLKGTEAV

IIPYALHRDPRYFPN

PEEFQPERFFPENAQ

GRHPYAYVPFSAGPR

NCIGQKFAVMEEKTI

LSCILRHFWIESNQK

REELGLEGQLILRPS

NGIWIKLKRRNADER

Hepatitis B
16

virus
TAAACAGGCCTATTG

regulatory
ATTGGAAAGTATGTC

element
AACGAATTGTGGGTC

(HPRE)
TTTTGGGGTTTGCTG

CCCCTTTTACGCAAT

GTGGATATCCTGCTT

TAATGCCTTTATATG

CATGTATACAAGCAA

AACAGGCTTTTACTT

TCTCGCCAACTTACA

AGGCCTTTCTAAGTA

AACAGTATCTGACCC

TTTACCCCGTTGCTC

GGCAACGGCCTGGTC

TGTGCCAAGTGTTTG

CTGACGCAACCCCCA

CTGGTTGGGGCTTGG

CCATAGGCCATCAGC

GCATGCGTGGAACCT

TTGTGTCTCCTCTGC

CGATCCATACTGCGG

AACTCCTAGCCGCTT

GTTTTGCTCGCAGCA

GGTCTGGAGCGAAAC

TCATCGGGACTGACA

ATTCTGTCGTGCTCT

CCCGCAAGTATACAT

CGTTTCCAGGGCTGC

TAGGCTGTGCTGCCA

ACTGGATCCTGCGCG

GGACGTCCTTTGTTT

ACGTCCCGTCGGCGC

TGAATCCCGCGGACG

ACCCCTCCCGGGGCC

GCTTGGGGCTCTACC

GCCCGCTTCTCCGTC

TGCCGTACCGACCGA

CCACGGGGCGCACCT

CTCTTTACGCGGACT

CCCCGTCTGTGCCTT

CTCATCTGCCGGACC

GTGTGCACTTCGCTT

CACCTCTGCACGTCG

CATGGAGACCACCGT

GAACGCCCACCGGAA

CCTGCCCAAGGTCTT

GCATAAGAGGACTCT

TGGACTTTCAGCAAT

GTCAACTCGA

Woodchuck
17

hepatitis
TCAACCTCTGGATTA

virus
CAAAATTTGTGAAAG

regulatory
ATTGACTGGTATTCT

element
TAACTATGTTGCTCC

(WPRE)
TTTTACGCTATGTGG

ATACGCTGCTTTAAT

GCCTTTGTATCATGC

TATTGCTTCCCGTAT

GGCTTTCATTTTCTC

CTCCTTGTATAAATC

CTGGTTGCTGTCTCT

TTATGAGGAGTTGTG

GCCCGTTGTCAGGCA

ACGTGGCGTGGTGTG

CACTGTGTTTGCTGA

CGCAACCCCCACTGG

TTGGGGCATTGCCAC

CACCTGTCAGCTCCT

TTCCGGGACTTTCGC

TTTCCCCCTCCCTAT

TGCCACGGCGGAACT

CATCGCCGCCTGCCT

TGCCCGCTGCTGGAC

AGGGGCTCGGCTGTT

GGGCACTGACAATTC

CGTGGTGTTGTCGGG

GAAATCATCGTCCTT

TCCTTGGCTGCTCGC

CTGTGTTGCCACCTG

GATTCTGCGCGGGAC

GTCCTTCTGCTACGT

CCCTTCGGCCCTCAA

TCCAGCGGACCTTCC

TTCCCGCGGCCTGCT

GCCGGCTCTGCGGCC

TCTTCCGCGTCTTCG

CCTTCGCCCTCAGAC

GAGTCGGATCTCCCT

TTGGGCCGCCTCCCC

GCA

Bovine Growth
18

Hormone (bGH)
GATCTGGATGATGAC

polyA signal
GACAAGTGAGGATCC

sequence
CTGTGCCTTCTAGTT

GCCAGCCATCTGTTG

TTTGCCCCTCCCCCG

TGCCTTCCTTGACCC

TGGAAGGTGCCACTC

CCACTGTCCTTTCCT

AATAAAATGAGGAAA

TTGCATCGCATTGTC

TGAGTAGGTGTCATT

CTATTCTGGGGGGTG

GGGTGGGGCAGGACA

GCAAGGGGGAGGATT

GGGAAGAGAATAGCA

GGCATGCTGGGGAGA

ATTCA

Simian Virus 40
19

(SV40) polyA
GATCATAATCAGCCA

signal
TACCACATTTGTAGA

sequence
GGTTTTACTTGCTTT

AAAAAACCTCCCACA

CCTCCCCCTGAACCT

GAAACATAAAATGAA

TGCAATTGTTGTTGT

TAACTTGTTTATTGC

AGCTTATAATGGTTA

CAAATAAAGCAATAG

CATCACAAATTTCAC

AAATAAAGCATTTTT

TTCACTGCATTCTAG

TTGTGGTTTGTCCAA

ACTCATCAATGTATC

TTATCATGTCT

SYNUCLEIN
20

INTRONIC
GGGCCCCGGTGTTAT

SEQUENCE AS
CTCATTCTTTTTTCT

STUFFER
CCTCTGTAAGTTGAC

SEQUENCE
ATGTGATGTGGGAAC

AAAGGGGATAAAGTC

ATTATTTTGTGCTAA

AATCGTAATTGGAGA

GGACCTCCTGTTAGC

TGGGCTTTCTTCTAT

TTATTGTGGTGGTTA

CTGGAGTTCCTTCTT

CTAGTTTTAGGATAT

ATATATATATTTTTT

TTTTTTCTTTCCCTG

AAGATATAATAATAT

ATATACTTCTGAAGA

TTGAGATTTTTAAAT

TAGTTGTATTGAAAA

CTAGCTAATCAGCAA

TTTAAGGCTAGCTTG

AGACTTATGTCTTGA

ATTTGTTTTTGTAGG

CTCCAAAACCAAGGA

GGGAGTGGTGCATGG

TGTGGCAACAGGTAA

GCTCCATTGTGCTTA

TATCCAAAGATGATA

TTTAAAGTATCTAGT

GATTAGTGTGGCCCA

GTATTCAAGATTCCT

ATGAAATTGTAAAAC

AATCACTGAGCATTC

TAAGAACATATCAGT

CTTATTGAAACTGAA

TTCTTTATAAAGTAT

TTTTAAAAAGGTAAA

TATTGATTATAAATA

AAAAATATACTTGCC

AAGAATAATGAGGGC

TTTGAATTGATAAGC

TATGTTTAATTTATA

GTAAGTGGGCATTTA

AATATTCTGACCAAA

AATGTATTGACAAAC

TGCTGACAAAAATAA

AATGTGAATATTGCC

ATAATTTTAAAAAAA

GAGTAAAATTTCTGT

TGATTACAGTAAAAT

ATTTTGACCTTAAAT

TATGTTGATTACAAT

ATTCCTTTGATAATT

CAGAGTGCATTTCAG

GAAACACCCTTGGAC

AGTCAGTAAATTGTT

TATTGTATTTATCTT

TGTATTGTTATGGTA

TAGCTATTTGTACAA

ATATTATTGTGCAAT

TATTACATTTCTGAT

TATATTATTCATTTG

GCCTAAATTTACCAA

GAATTTGAACAAGTC

AATTAGGTTTACAAT

CAAGAAATATCAAAA

ATGATGAAAAGGATG

ATAATCATCATCAGA

TGTTGAGGAAGATGA

CGATGAGAGTGCCAG

AAATAGAGAAATCAA

AGGAGAACCAAAATT

TAACAAATTAAAAGC

CCACAGACTTGCTGT

AATTAAGTTTTCTGT

TGTAAGTACTCCACG

TTTCCTGGCAGATGT

GGTGAAGCAAAAGAT

ATAATCAGAAATATA

ATTTATATGATCGGA

AAGCATTAAACACAA

TAGTGCCTATACAAA

TAAAATGTTCCTATC

ACTGACTTCTAAAAT

GGAAATGAGGACAAT

GATATGGGAATCTTA

ATACAGTGTTGTGGA

TAGGACTAAAAACAC

AGGAGTCAGATCTTC

TTGGTTCAACTTCCT

GCTTACTCCTTACCA

GCTGTGTGTTTTTTG

CAAGGTTCTTCACCT

CTATGTGATTTAGCT

TCCTCATCTATAAAA

TAATTCAGTGAATTA

ATGTACACAAAACAT

CTGGAAAACAAAAGC

AAACAATATGTATTT

TATAAGTGTTACTTA

TAGTTTTATAGTGAA

CTTTCTTGTGCAACA

TTTTTACAACTAGTG

GAGAAAAATATTTCT

TTAAATGAATACTTT

TGATTTAAAAATCAG

AGTGTAAAAATAAAA

CAGACTCCTTTGAAA

CTAGTTCTGTTAGAA

GTTAATTGTGCACCT

TTAATGGGCTCTGTT

GCAATCCAACAGAGA

AGTAGTTAAGTAAGT

GGACTATGATGGCTT

CTAGGGACCTCCTAT

AAATATGATATTGTG

AAGCATGATTATAAT

AAGAACTAGATAACA

GACAGGTGGAGACTC

CACTATCTGAAGAGG

GTCAACCTAGATGAA

TGGTGTTCCATTTAG

TAGTTGAGGAAGAAC

CCATGAGGTTTAGAA

AGCAGACAAGCATGT

GGCAAGTTCTGGAGT

CAGTGGTAAAAATTA

AAGAACCCAACTATT

ACTGTCACCTAATGA

TCTAATGGAGACTGT

GGAGATGGGCTGCAT

TTTTTTAATCTTCTC

CAGAATGCCAAAATG

TAAACACATATCTGT

GTGTGTGTGTGTGTG

TGTGTGTGTGTGTGA

GAGAGAGAGAGAGAG

AGAGAGAGACTGAAG

TTTGTACAATTAGAC

ATTTTATAAAATGTT

TTCTGAAGGACAGTG

GCTCACAATCTTAAG

TTTCTAACATTGTAC

AATGTTGGGAGACTT

TGTATACTTTATTTT

CTCTTTAGCATATTA

AGGAATCTGAGATGT

CCTACAGTAAAGAAA

TTTGCATTACATAGT

TAAAATCAGGGTTAT

TCAAACTTTTTGATT

ATTGAAACCTTTCTT

CATTAGTTACTAGGG

TTGAATGAAACTAGT

GTTCCACAGAAAACT

ATGGGAAATGTTGCT

AGGCAGTAAGGACAT

GGTGATTTCAGCATG

TGCAATATTTACAGC

GATTGCACCCATGGA

CCACCCTGGCAGTAG

TGAAATAACCAAAAA

TGCTGTCATAACTAG

TATGGCTATGAGAAA

CACATTGGG

RLBP1 INTRONIC
21

SEQUENCE AS
ATTCTCCAGGTTGAG

STUFFER
CCAGACCAATTTGAT

SEQUENCE
GGTAGATTTAGCAAA

TAAAAATACAGGACA

CCCAGTTAAATGTGA

ATTTCCGATGAACAG

CAAATACTTTTTTAG

TATTAAAAAAGTTCA

CATTTAGGCTCACGC

CTGTAATCCCAGCAC

TTTGGGAGGCCGAGG

CAGGCAGATCACCTG

AGGTCAGGAGTTCGA

GACCAGCCTGGCCAA

CATGGTGAAACCCCA

TCTCCACTAAAAATA

CCAAAAATTAGCCAG

GCGTGCTGGTGGGCA

CCTGTAGTTCCAGCT

ACTCAGGAGGCTAAG

GCAGGAGAATTGCTT

GAACCTGGGAGGCAG

AGGTTGCAGTGAGCT

GAGATCGCACCATTG

CACTCTAGCCTGGGC

GACAAGAACAAAACT

CCATCTCAAAAAAAA

AAAAAAAAAAAAAGT

TCACATTTAACTGGG

CATTCTGTATTTAAT

TGGTAATCTGAGATG

GCAGGGAACAGCATC

AGCATGGTGTGAGGG

ATAGGCATTTTTTCA

TTGTGTACAGCTTGT

AAATCAGTATTTTTA

AAACTCAAAGTTAAT

GGCTTGGGCATATTT

AGAAAAGAGTTGCCG

CACGGACTTGAACCC

TGTATTCCTAAAATC

TAGGATCTTGTTCTG

ATGGTCTGCACAACT

GGCTGGGGGTGTCCA

GCCACTGTCCCTCTT

GCCTGGGCTCCCCAG

GGCAGTTCTGTCAGC

CTCTCCATTTCCATT

CCTGTTCCAGCAAAA

CCCAACTGATAGCAC

AGCAGCATTTCAGCC

TGTCTACCTCTGTGC

CCACATACCTGGATG

TCTACCAGCCAGAAA

GGTGGCTTAGATTTG

GTTCCTGTGGGTGGA

TTATGGCCCCCAGAA

CTTCCCTGTGCTTGC

TGGGGGTGTGGAGTG

GAAAGAGCAGGAAAT

GGGGGACCCTCCGAT

ACTCTATGGGGGTCC

TCCAAGTCTCTTTGT

GCAAGTTAGGGTAAT

AATCAATATGGAGCT

AAGAAAGAGAAGGGG

AACTATGCTTTAGAA

CAGGACACTGTGCCA

GGAGCATTGCAGAAA

TTATATGGTTTTCAC

GACAGTTCTTTTTGG

TAGGTACTGTTATTA

TCCTCAGTTTGCAGA

TGAGGAAACTGAGAC

CCAGAAAGGTTAAAT

AACTTGCTAGGGTCA

CACAAGTCATAACTG

ACAAAGCCTGATTCA

AACCCAGGTCTCCCT

AACCTTTAAGGTTTC

TATGACGCCAGCTCT

CCTAGGGAGTTTGTC

TTCAGATGTCTTGGC

TCTAGGTGTCAAAAA

AAGACTTGGTGTCAG

GCAGGCATAGGTTCA

AGTCCCAACTCTGTC

ACTTACCAACTGTGA

CTAGGTGATTGAACT

GACCATGGAACCTGG

TCACATGCAGGAGCA

GGATGGTGAAGGGTT

CTTGAAGGCACTTAG

GCAGGACATTTAGGC

AGGAGAGAAAACCTG

GAAACAGAAGAGCTG

TCTCCAAAAATACCC

ACTGGGGAAGCAGGT

TGTCATGTGGGCCAT

GAATGGGACCTGTTC

TGG

3′ ITR
22

AGGAACCCCTAGTGA

TGGAGTTGGCCACTC

CCTCTCTGCGCGCTC

GCTCGCTCACTGAGG

CCGGGCGACCAAAGG

TCGCCCGACGCCCGG

GCTTTGCCCGGGCGG

CCTCAGTGAGCGAGC

GAGCGCGCAG

AAV8 Capsid
23

Coding Sequence
ATGGCTGCCGATGGT

TATCTTCCAGATTGG

CTCGAGGACAACCTC

TCTGAGGGCATTCGC

GAGTGGTGGGCGCTG

AAACCTGGAGCCCCG

AAGCCCAAAGCCAAC

CAGCAAAAGCAGGAC

GACGGCCGGGGTCTG

GTGCTTCCTGGCTAC

AAGTACCTCGGACCC

TTCAACGGACTCGAC

AAGGGGGAGCCCGTC

AACGCGGCGGACGCA

GCGGCCCTCGAGCAC

GACAAGGCCTACGAC

CAGCAGCTGCAGGCG

GGTGACAATCCGTAC

CTGCGGTATAACCAC

GCCGACGCCGAGTTT

CAGGAGCGTCTGCAA

GAAGATACGTCTTTT

GGGGGCAACCTCGGG

CGAGCAGTCTTCCAG

GCCAAGAAGCGGGTT

CTCGAACCTCTCGGT

CTGGTTGAGGAAGGC

GCTAAGACGGCTCCT

GGAAAGAAGAGACCG

GTAGAGCCATCACCC

CAGCGTTCTCCAGAC

TCCTCTACGGGCATC

GGCAAGAAAGGCCAA

CAGCCCGCCAGAAAA

AGACTCAATTTTGGT

CAGACTGGCGACTCA

GAGTCAGTTCCAGAC

CCTCAACCTCTCGGA

GAACCTCCAGCAGCG

CCCTCTGGTGTGGGA

CCTAATACAATGGCT

GCAGGCGGTGGCGCA

CCAATGGCAGACAAT

AACGAAGGCGCCGAC

GGAGTGGGTAGTTCC

TCGGGAAATTGGCAT

TGCGATTCCACATGG

CTGGGCGACAGAGTC

ATCACCACCAGCACC

CGAACCTGGGCCCTG

CCCACCTACAACAAC

CACCTCTACAAGCAA

ATCTCCAACGGGACA

TCGGGAGGAGCCACC

AACGACAACACCTAC

TTCGGCTACAGCACC

CCCTGGGGGTATTTT

GACTTTAACAGATTC

CACTGCCACTTTTCA

CCACGTGACTGGCAG

CGACTCATCAACAAC

AACTGGGGATTCCGG

CCCAAGAGACTCAGC

TTCAAGCTCTTCAAC

ATCCAGGTCAAGGAG

GTCACGCAGAATGAA

GGCACCAAGACCATC

GCCAATAACCTCACC

AGCACCATCCAGGTG

TTTACGGACTCGGAG

TACCAGCTGCCGTAC

GTTCTCGGCTCTGCC

CACCAGGGCTGCCTG

CCTCCGTTCCCGGCG

GACGTGTTCATGATT

CCCCAGTACGGCTAC

CTAACACTCAACAAC

GGTAGTCAGGCCGTG

GGACGCTCCTCCTTC

TACTGCCTGGAATAC

TTTCCTTCGCAGATG

CTGAGAACCGGCAAC

AACTTCCAGTTTACT

TACACCTTCGAGGAC

GTGCCTTTCCACAGC

AGCTACGCCCACAGC

CAGAGCTTGGACCGG

CTGATGAATCCTCTG

ATTGACCAGTACCTG

TACTACTTGTCTCGG

ACTCAAACAACAGGA

GGCACGGCAAATACG

CAGACTCTGGGCTTC

AGCCAAGGTGGGCCT

AATACAATGGCCAAT

CAGGCAAAGAACTGG

CTGCCAGGACCCTGT

TACCGCCAACAACGC

GTCTCAACGACAACC

GGGCAAAACAACAAT

AGCAACTTTGCCTGG

ACTGCTGGGACCAAA

TACCATCTGAATGGA

AGAAATTCATTGGCT

AATCCTGGCATCGCT

ATGGCAACACACAAA

GACGACGAGGAGCGT

TTTTTTCCCAGTAAC

GGGATCCTGATTTTT

GGCAAACAAAATGCT

GCCAGAGACAATGCG

GATTACAGCGATGTC

ATGCTCACCAGCGAG

GAAGAAATCAAAACC

ACTAACCCTGTGGCT

ACAGAGGAATACGGT

ATCGTGGCAGATAAC

TTGCAGCAGCAAAAC

ACGGCTCCTCAAATT

GGAACTGTCAACAGC

CAGGGGGCCTTACCC

GGTATGGTCTGGCAG

AACCGGGACGTGTAC

CTGCAGGGTCCCATC

TGGGCCAAGATTCCT

CACACGGACGGCAAC

TTCCACCCGTCTCCG

CTGATGGGCGGCTTT

GGCCTGAAACATCCT

CCGCCTCAGATCCTG

ATCAAGAACACGCCT

GTACCTGCGGATCCT

CCGACCACCTTCAAC

CAGTCAAAGCTGAAC

TCTTTCATCACGCAA

TACAGCACCGGACAG

GTCAGCGTGGAAATT

GAATGGGAGCTGCAG

AAGGAAAACAGCAAG

CGCTGGAACCCCGAG

ATCCAGTACACCTCC

AACTACTACAAATCT

ACAAGTGTGGACTTT

GCTGTTAATACAGAA

GGCGTGTACTCTGAA

CCCCGCCCCATTGGC

ACCCGTTACCTCACC

CGTAATCTGTAA

AAV8 Capsid
24

Sequence (VP1)
MAADGYLPDWLEDNL

SEGIREWWALKPGAP

KPKANQQKQDDGRGL

VLPGYKYLGPFNGLD

KGEPVNAADAAALEH

DKAYDQQLQAGDNPY

LRYNHADAEFQERLQ

EDTSFGGNLGRAVFQ

AKKRVLEPLGLVEEG

AKTAPGKKRPVEPSP

QRSPDSSTGIGKKGQ

QPARKRLNFGQTGDS

ESVPDPQPLGEPPAA

PSGVGPNTMAAGGGA

PMADNNEGADGVGSS

SGNWHCDSTWLGDRV

ITTSTRTWALPTYNN

HLYKQISNGTSGGAT

NDNTYFGYSTPWGYF

DFNRFHCHFSPRDWQ

RLINNNWGFRPKRLS

FKLFNIQVKEVTQNE

GTKTIANNLTSTIQV

FTDSEYQLPYVLGSA

HQGCLPPFPADVFMI

PQYGYLTLNNGSQAV

GRSSFYCLEYFPSQM

LRTGNNFQFTYTFED

VPFHSSYAHSQSLDR

LMNPLIDQYLYYLSR

TQTTGGTANTQTLGF

SQGGPNTMANQAKNW

LPGPCYRQQRVSTTT

GQNNNSNFAWTAGTK

YHLNGRNSLANPGIA

MATHKDDEERFFPSN

GILIFGKQNAARDNA

DYSDVMLTSEEEIKT

TNPVATEEYGIVADN

LQQQNTAPQIGTVNS

QGALPGMVWQNRDVY

LQGPIWAKIPHTDGN

FHPSPLMGGFGLKHP

PPQILIKNTPVPADP

PTTFNQSKLNSFITQ

YSTGQVSVEIEWELQ

KENSKRWNPEIQYTS

NYYKSTSVDFAVNTE

GVYSEPRPIGTRYLT

RNL

AAV8 Capsid
25

Sequence (VP2)
MAPGKKRPVEPSPQR

SPDSSTGIGKKGQQP

ARKRLNFGQTGDSES

VPDPQPLGEPPAAPS

GVGPNTMAAGGGAPM

ADNNEGADGVGSSSG

NWHCDSTWLGDRVIT

TSTRTWALPTYNNHL

YKQISNGTSGGATND

NTYFGYSTPWGYFDF

NRFHCHFSPRDWQRL

INNNWGFRPKRLSFK

LFNIQVKEVTQNEGT

KTIANNLTSTIQVFT

DSEYQLPYVLGSAHQ

GCLPPFPADVFMIPQ

YGYLTLNNGSQAVGR

SSFYCLEYFPSQMLR

TGNNFQFTYTFEDVP

FHSSYAHSQSLDRLM

NPLIDQYLYYLSRTQ

TTGGTANTQTLGFSQ

GGPNTMANQAKNWLP

GPCYRQQRVSTTTGQ

NNNSNFAWTAGTKYH

LNGRNSLANPGIAMA

THKDDEERFFPSNGI

LIFGKQNAARDNADY

SDVMLTSEEEIKTTN

PVATEEYGIVADNLQ

QQNTAPQIGTVNSQG

ALPGMVWQNRDVYLQ

GPIWAKIPHTDGNFH

PSPLMGGFGLKHPPP

QILIKNTPVPADPPT

TFNQSKLNSFITQYS

TGQVSVEIEWELQKE

NSKRWNPEIQYTSNY

YKSTSVDFAVNTEGV

YSEPRPIGTRYLTRN

L

AAV8 Capsid
26

Sequence (VP3)
MAAGGGAPMADNNEG

ADGVGSSSGNWHCDS

TWLGDRVITTSTRTW

ALPTYNNHLYKQISN

GTSGGATNDNTYFGY

STPWGYFDFNRFHCH

FSPRDWQRLINNNWG

FRPKRLSFKLFNIQV

KEVTQNEGTKTIANN

LTSTIQVFTDSEYQL

PYVLGSAHQGCLPPF

PADVFMIPQYGYLTL

NNGSQAVGRSSFYCL

EYFPSQMLRTGNNFQ

FTYTFEDVPFHSSYA

HSQSLDRLMNPLIDQ

YLYYLSRTQTTGGTA

NTQTLGFSQGGPNTM

ANQAKNWLPGPCYRQ

QRVSTTTGQNNNSNF

AWTAGTKYHLNGRNS

LANPGIAMATHKDDE

ERFFPSNGILIFGKQ

NAARDNADYSDVMLT

SEEEIKTTNPVATEE

YGIVADNLQQQNTAP

QIGTVNSQGALPGMV

WQNRDVYLQGPIWAK

IPHTDGNFHPSPLMG

GFGLKHPPPQILIKN

TPVPADPPTTFNQSK

LNSFITQYSTGQVSV

EIEWELQKENSKRWN

PEIQYTSNYYKSTSV

DFAVNTEGVYSEPRP

IGTRYLTRNL

AAV9 Capsid
27

Coding Sequence
ATGGCTGCCGATGGT

TATCTTCCAGATTGG

CTCGAGGACAACCTT

AGTGAAGGAATTCGC

GAGTGGTGGGCTTTG

AAACCTGGAGCCCCT

CAACCCAAGGCAAAT

CAACAACATCAAGAC

AACGCTCGAGGTCTT

GTGCTTCCGGGTTAC

AAATACCTTGGACCC

GGCAACGGACTCGAC

AAGGGGGAGCCGGTC

AACGCAGCAGACGCG

GCGGCCCTCGAGCAC

GACAAGGCCTACGAC

CAGCAGCTCAAGGCC

GGAGACAACCCGTAC

CTCAAGTACAACCAC

GCCGACGCCGAGTTC

CAGGAGCGGCTCAAA

GAAGATACGTCTTTT

GGGGGCAACCTCGGG

CGAGCAGTCTTCCAG

GCCAAAAAGAGGCTT

CTTGAACCTCTTGGT

CTGGTTGAGGAAGCG

GCTAAGACGGCTCCT

GGAAAGAAGAGGCCT

GTAGAGCAGTCTCCT

CAGGAACCGGACTCC

TCCGCGGGTATTGGC

AAATCGGGTGCACAG

CCCGCTAAAAAGAGA

CTCAATTTCGGTCAG

ACTGGCGACACAGAG

TCAGTCCCAGACCCT

CAACCAATCGGAGAA

CCTCCCGCAGCCCCC

TCAGGTGTGGGATCT

CTTACAATGGCTTCA

GGTGGTGGCGCACCA

GTGGCAGACAATAAC

GAAGGTGCCGATGGA

GTGGGTAGTTCCTCG

GGAAATTGGCATTGC

GATTCCCAATGGCTG

GGGGACAGAGTCATC

ACCACCAGCACCCGA

ACCTGGGCCCTGCCC

ACCTACAACAATCAC

CTCTACAAGCAAATC

TCCAACAGCACATCT

GGAGGATCTTCAAAT

GACAACGCCTACTTC

GGCTACAGCACCCCC

TGGGGGTATTTTGAC

TTCAACAGATTCCAC

TGCCACTTCTCACCA

CGTGACTGGCAGCGA

CTCATCAACAACAAC

TGGGGATTCCGGCCT

AAGCGACTCAACTTC

AAGCTCTTCAACATT

CAGGTCAAAGAGGTT

ACGGACAACAATGGA

GTCAAGACCATCGCC

AATAACCTTACCAGC

ACGGTCCAGGTCTTC

ACGGACTCAGACTAT

CAGCTCCCGTACGTG

CTCGGGTCGGCTCAC

GAGGGCTGCCTCCCG

CCGTTCCCAGCGGAC

GTTTTCATGATTCCT

CAGTACGGGTATCTG

ACGCTTAATGATGGA

AGCCAGGCCGTGGGT

CGTTCGTCCTTTTAC

TGCCTGGAATATTTC

CCGTCGCAAATGCTA

AGAACGGGTAACAAC

TTCCAGTTCAGCTAC

GAGTTTGAGAACGTA

CCTTTCCATAGCAGC

TACGCTCACAGCCAA

AGCCTGGACCGACTA

ATGAATCCACTCATC

GACCAATACTTGTAC

TATCTCTCAAAGACT

ATTAACGGTTCTGGA

CAGAATCAACAAACG

CTAAAATTCAGTGTG

GCCGGACCCAGCAAC

ATGGCTGTCCAGGGA

AGAAACTACATACCT

GGACCCAGCTACCGA

CAACAACGTGTCTCA

ACCACTGTGACTCAA

AACAACAACAGCGAA

TTTGCTTGGCCTGGA

GCTTCTTCTTGGGCT

CTCAATGGACGTAAT

AGCTTGATGAATCCT

GGACCTGCTATGGCC

AGCCACAAAGAAGGA

GAGGACCGTTTCTTT

CCTTTGTCTGGATCT

TTAATTTTTGGCAAA

CAAGGAACTGGAAGA

GACAACGTGGATGCG

GACAAAGTCATGATA

ACCAACGAAGAAGAA

ATTAAAACTACTAAC

CCGGTAGCAACGGAG

TCCTATGGACAAGTG

GCCACAAACCACCAG

AGTGCCCAAGCACAG

GCGCAGACCGGCTGG

GTTCAAAACCAAGGA

ATACTTCCGGGTATG

GTTTGGCAGGACAGA

GATGTGTACCTGCAA

GGACCCATTTGGGCC

AAAATTCCTCACACG

GACGGCAACTTTCAC

CCTTCTCCGCTGATG

GGAGGGTTTGGAATG

AAGCACCCGCCTCCT

CAGATCCTCATCAAA

AACACACCTGTACCT

GCGGATCCTCCAACG

GCCTTCAACAAGGAC

AAGCTGAACTCTTTC

ATCACCCAGTATTCT

ACTGGCCAAGTCAGC

GTGGAGATCGAGTGG

GAGCTGCAGAAGGAA

AACAGCAAGCGCTGG

AACCCGGAGATCCAG

TACACTTCCAACTAT

TACAAGTCTAATAAT

GTTGAATTTGCTGTT

AATACTGAAGGTGTA

TATAGTGAACCCCGC

CCCATTGGCACCAGA

TACCTGACTCGTAAT

CTGTAA

AAV9 Capsid
28

Sequence (VP1)
MAADGYLPDWLEDNL

SEGIREWWALKPGAP

QPKANQQHQDNARGL

VLPGYKYLGPGNGLD

KGEPVNAADAAALEH

DKAYDQQLKAGDNPY

LKYNHADAEFQERLK

EDTSFGGNLGRAVFQ

AKKRLLEPLGLVEEA

AKTAPGKKRPVEQSP

QEPDSSAGIGKSGAQ

PAKKRLNFGQTGDTE

SVPDPQPIGEPPAAP

SGVGSLTMASGGGAP

VADNNEGADGVGSSS

GNWHCDSQWLGDRVI

TTSTRTWALPTYNNH

LYKQISNSTSGGSSN

DNAYFGYSTPWGYFD

FNRFHCHFSPRDWQR

LINNNWGFRPKRLNF

KLFNIQVKEVTDNNG

VKTIANNLTSTVQVF

TDSDYQLPYVLGSAH

EGCLPPFPADVFMIP

QYGYLTLNDGSQAVG

RSSFYCLEYFPSQML

RTGNNFQFSYEFENV

PFHSSYAHSQSLDRL

MNPLIDQYLYYLSKT

INGSGQNQQTLKFSV

AGPSNMAVQGRNYIP

GPSYRQQRVSTTVTQ

NNNSEFAWPGASSWA

LNGRNSLMNPGPAMA

SHKEGEDRFFPLSGS

LIFGKQGTGRDNVDA

DKVMITNEEEIKTTN

PVATESYGQVATNHQ

SAQAQAQTGWVQNQG

ILPGMVWQDRDVYLQ

GPIWAKIPHTDGNFH

PSPLMGGFGMKHPPP

QILIKNTPVPADPPT

AFNKDKLNSFITQYS

TGQVSVEIEWELQKE

NSKRWNPEIQYTSNY

YKSNNVEFAVNTEGV

YSEPRPIGTRYLTRN

L

AAV9 Capsid
29

Sequence (VP2)
TAPGKKRPVEQSPQE

PDSSAGIGKSGAQPA

KKRLNFGQTGDTESV

PDPQPIGEPPAAPSG

VGSLTMASGGGAPVA

DNNEGADGVGSSSGN

WHCDSQWLGDRVITT

STRTWALPTYNNHLY

KQISNSTSGGSSNDN

AYFGYSTPWGYFDFN

RFHCHFSPRDWQRLI

NNNWGFRPKRLNFKL

FNIQVKEVTDNNGVK

TIANNLTSTVQVFTD

SDYQLPYVLGSAHEG

CLPPFPADVFMIPQY

GYLTLNDGSQAVGRS

SFYCLEYFPSQMLRT

GNNFQFSYEFENVPF

HSSYAHSQSLDRLMN

PLIDQYLYYLSKTIN

GSGQNQQTLKFSVAG

PSNMAVQGRNYIPGP

SYRQQRVSTTVTQNN

NSEFAWPGASSWALN

GRNSLMNPGPAMASH

KEGEDRFFPLSGSLI

FGKQGTGRDNVDADK

VMITNEEEIKTTNPV

ATESYGQVATNHQSA

QAQAQTGWVQNQGIL

PGMVWQDRDVYLQGP

IWAKIPHTDGNFHPS

PLMGGFGMKHPPPQI

LIKNTPVPADPPTAF

NKDKLNSFITQYSTG

QVSVEIEWELQKENS

KRWNPEIQYTSNYYK

SNNVEFAVNTEGVYS

EPRPIGTRYLTRNL

AAV9 Capsid
30

Sequence (VP3)
MASGGGAPVADNNEG

ADGVGSSSGNWHCDS

QWLGDRVITTSTRTW

ALPTYNNHLYKQISN

STSGGSSNDNAYFGY

STPWGYFDFNRFHCH

FSPRDWQRLINNNWG

FRPKRLNFKLFNIQV

KEVTDNNGVKTIANN

LTSTVQVFTDSDYQL

PYVLGSAHEGCLPPF

PADVFMIPQYGYLTL

NDGSQAVGRSSFYCL

EYFPSQMLRTGNNFQ

FSYEFENVPFHSSYA

HSQSLDRLMNPLIDQ

YLYYLSKTINGSGQN

QQTLKFSVAGPSNMA

VQGRNYIPGPSYRQQ

RVSTTVTQNNNSEFA

WPGASSWALNGRNSL

MNPGPAMASHKEGED

RFFPLSGSLIFGKQG

TGRDNVDADKVMITN

EEEIKTTNPVATESY

GQVATNHQSAQAQAQ

TGWVQNQGILPGMVW

QDRDVYLQGPIWAKI

PHTDGNFHPSPLMGG

FGMKHPPPQILIKNT

PVPADPPTAFNKDKL

NSFITQYSTGQVSVE

IEWELQKENSKRWNP

EIQYTSNYYKSNNVE

FAVNTEGVYSEPRPI

GTRYLTRNL

AAV2 Capsid
31

Coding Sequence
ATGGCTGCCGATGGT

TATCTTCCAGATTGG

CTCGAGGACACTCTC

TCTGAAGGAATAAGA

CAGTGGTGGAAGCTC

AAACCTGGCCCACCA

CCACCAAAGCCCGCA

GAGCGGCATAAGGAC

GACAGCAGGGGTCTT

GTGCTTCCTGGGTAC

AAGTACCTCGGACCC

TTCAACGGACTCGAC

AAGGGAGAGCCGGTC

AACGAGGCAGACGCC

GCGGCCCTCGAGCAC

GACAAAGCCTACGAC

CGGCAGCTCGACAGC

GGAGACAACCCGTAC

CTCAAGTACAACCAC

GCCGACGCGGAGTTT

CAGGAGCGCCTTAAA

GAAGATACGTCTTTT

GGGGGCAACCTCGGA

CGAGCAGTCTTCCAG

GCGAAAAAGAGGGTT

CTTGAACCTCTGGGC

CTGGTTGAGGAACCT

GTTAAGACGGCTCCG

GGAAAAAAGAGGCCG

GTAGAGCACTCTCCT

GTGGAGCCAGACTCC

TCCTCGGGAACCGGA

AAGGCGGGCCAGCAG

CCTGCAAGAAAAAGA

TTGAATTTTGGTCAG

ACTGGAGACGCAGAC

TCAGTACCTGACCCC

CAGCCTCTCGGACAG

CCACCAGCAGCCCCC

TCTGGTCTGGGAACT

AATACGATGGCTACA

GGCAGTGGCGCACCA

ATGGCAGACAATAAC

GAGGGCGCCGACGGA

GTGGGTAATTCCTCG

GGAAATTGGCATTGC

GATTCCACATGGATG

GGCGACAGAGTCATC

ACCACCAGCACCCGA

ACCTGGGCCCTGCCC

ACCTACAACAACCAC

CTCTACAAACAAATT

TCCAGCCAATCAGGA

GCCTCGAACGACAAT

CACTACTTTGGCTAC

AGCACCCCTTGGGGG

TATTTTGACTTCAAC

AGATTCCACTGCCAC

TTTTCACCACGTGAC

TGGCAAAGACTCATC

AACAACAACTGGGGA

TTCCGACCCAAGAGA

CTCAACTTCAAGCTC

TTTAACATTCAAGTC

AAAGAGGTCACGCAG

AATGACGGTACGACG

ACGATTGCCAATAAC

CTTACCAGCACGGTT

CAGGTGTTTACTGAC

TCGGAGTACCAGCTC

CCGTACGTCCTCGGC

TCGGCGCATCAAGGA

TGCCTCCCGCCGTTC

CCAGCAGACGTCTTC

ATGGTGCCACAGTAT

GGATACCTCACCCTG

AACAACGGGAGTCAG

GCAGTAGGACGCTCT

TCATTTTACTGCCTG

GAGTACTTTCCTTCT

CAGATGCTGCGTACC

GGAAACAACTTTACC

TTCAGCTACACTTTT

GAGGACGTTCCTTTC

CACAGCAGCTACGCT

CACAGCCAGAGTCTG

GACCGTCTCATGAAT

CCTCTCATCGACCAG

TACCTGTATTACTTG

AGCAGAACAAACACT

CCAAGTGGAACCACC

ACGCAGTCAAGGCTT

CAGTTTTCTCAGGCC

GGAGCGAGTGACATT

CGGGACCAGTCTAGG

AACTGGCTTCCTGGA

CCCTGTTACCGCCAG

CAGCGAGTATCAAAG

ACATCTGCGGATAAC

AACAACAGTGAATAC

TCGTGGACTGGAGCT

ACCAAGTACCACCTC

AATGGCAGAGACTCT

CTGGTGAATCCGGGC

CCGGCCATGGCAAGC

CACAAGGACGATGAA

GAAAAGTTTTTTCCT

CAGAGCGGGGTTCTC

ATCTTTGGGAAGCAA

GGCTCAGAGAAAACA

AATGTGGACATTGAA

AAGGTCATGATTACA

GACGAAGAGGAAATC

AGGACAACCAATCCC

GTGGCTACGGAGCAG

TATGGTTCTGTATCT

ACCAACCTCCAGAGA

GGCAACAGACAAGCA

GCTACCGCAGATGTC

AACACACAAGGCGTT

CTTCCAGGCATGGTC

TGGCAGGACAGAGAT

GTGTACCTTCAGGGG

CCCATCTGGGCAAAG

ATTCCACACACGGAC

GGACATTTTCACCCC

TCTCCCCTCATGGGT

GGATTCGGACTTAAA

CACCCTCCTCCACAG

ATTCTCATCAAGAAC

ACCCCGGTACCTGCG

AATCCTTCGACCACC

TTCAGTGCGGCAAAG

TTTGCTTCCTTCATC

ACACAGTACTCCACG

GGACAGGTCAGCGTG

GAGATCGAGTGGGAG

CTGCAGAAGGAAAAC

AGCAAACGCTGGAAT

CCCGAAATTCAGTAC

ACTTCCAACTACAAC

AAGTCTGTTAATGTG

GACTTTACTGTGGAC

ACTAATGGCGTGTAT

TCAGAGCCTCGCCCC

ATTGGCACCAGATAC

CTGACTCGTAATCTG

TAA

AAV2 Capsid
32

Sequence (VP1)
MAADGYLPDWLEDTL

SEGIRQWWKLKPGPP

PPKPAERHKDDSRGL

VLPGYKYLGPFNGLD

KGEPVNEADAAALEH

DKAYDRQLDSGDNPY

LKYNHADAEFQERLK

EDTSFGGNLGRAVFQ

AKKRVLEPLGLVEEP

VKTAPGKKRPVEHSP

VEPDSSSGTGKAGQQ

PARKRLNFGQTGDAD

SVPDPQPLGQPPAAP

SGLGTNTMATGSGAP

MADNNEGADGVGNSS

GNWHCDSTWMGDRVI

TTSTRTWALPTYNNH

LYKQISSQSGASNDN

HYFGYSTPWGYFDFN

RFHCHFSPRDWQRLI

NNNWGFRPKRLNFKL

FNIQVKEVTQNDGTT

TIANNLTSTVQVFTD

SEYQLPYVLGSAHQG

CLPPFPADVFMVPQY

GYLTLNNGSQAVGRS

SFYCLEYFPSQMLRT

GNNFTFSYTFEDVPF

HSSYAHSQSLDRLMN

PLIDQYLYYLSRTNT

PSGTTTQSRLQFSQA

GASDIRDQSRNWLPG

PCYRQQRVSKTSADN

NNSEYSWTGATKYHL

NGRDSLVNPGPAMAS

HKDDEEKFFPQSGVL

IFGKQGSEKTNVDIE

KVMITDEEEIRTTNP

VATEQYGSVSTNLQR

GNRQAATADVNTQGV

LPGMVWQDRDVYLQG

PIWAKIPHTDGHFHP

SPLMGGFGLKHPPPQ

ILIKNTPVPANPSTT

FSAAKFASFITQYST

GQVSVEIEWELQKEN

SKRWNPEIQYTSNYN

KSVNVDFTVDTNGVY

SEPRPIGTRYLTRNL

AAV2 Capsid
33

Sequence (VP2)
MAPGKKRPVEHSPVE

PDSSSGTGKAGQQPA

RKRLNFGQTGDADSV

PDPQPLGQPPAAPSG

LGTNTMATGSGAPMA

DNNEGADGVGNSSGN

WHCDSTWMGDRVITT

STRTWALPTYNNHLY

KQISSQSGASNDNHY

FGYSTPWGYFDFNRF

HCHFSPRDWQRLINN

NWGFRPKRLNFKLFN

IQVKEVTQNDGTTTI

ANNLTSTVQVFTDSE

YQLPYVLGSAHQGCL

PPFPADVFMVPQYGY

LTLNNGSQAVGRSSF

YCLEYFPSQMLRTGN

NFTFSYTFEDVPFHS

SYAHSQSLDRLMNPL

IDQYLYYLSRTNTPS

GTTTQSRLQFSQAGA

SDIRDQSRNWLPGPC

YRQQRVSKTSADNNN

SEYSWTGATKYHLNG

RDSLVNPGPAMASHK

DDEEKFFPQSGVLIF

GKQGSEKTNVDIEKV

MITDEEEIRTTNPVA

TEQYGSVSTNLQRGN

RQAATADVNTQGVLP

GMVWQDRDVYLQGPI

WAKIPHTDGHFHPSP

LMGGFGLKHPPPQIL

IKNTPVPANPSTTFS

AAKFASFITQYSTGQ

VSVEIEWELQKENSK

RWNPEIQYTSNYNKS

VNVDFTVDTNGVYSE

PRPIGTRYLTRNL

AAV2 Capsid
34

Sequence (VP3)
MATGSGAPMADNNEG

ADGVGNSSGNWHCDS

TWMGDRVITTSTRTW

ALPTYNNHLYKQISS

QSGASNDNHYFGYST

PWGYFDFNRFHCHFS

PRDWQRLINNNWGFR

PKRLNFKLFNIQVKE

VTQNDGTTTIANNLT

STVQVFTDSEYQLPY

VLGSAHQGCLPPFPA

DVFMVPQYGYLTLNN

GSQAVGRSSFYCLEY

FPSQMLRTGNNFTFS

YTFEDVPFHSSYAHS

QSLDRLMNPLIDQYL

YYLSRTNTPSGTTTQ

SRLQFSQAGASDIRD

QSRNWLPGPCYRQQR

VSKTSADNNNSEYSW

TGATKYHLNGRDSLV

NPGPAMASHKDDEEK

FFPQSGVLIFGKQGS

EKTNVDIEKVMITDE

EEIRTTNPVATEQYG

SVSTNLQRGNRQAAT

ADVNTQGVLPGMVWQ

DRDVYLQGPIWAKIP

HTDGHFHPSPLMGGF

GLKHPPPQILIKNTP

VPANPSTTFSAAKFA

SFITQYSTGQVSVEI

EWELQKENSKRWNPE

IQYTSNYNKSVNVDF

TVDTNGVYSEPRPIG

TRYLTRNL

AAV5 Capsid
35

Coding Sequence
ATGTCTTTTGTTGAT

CACCCTCCAGATTGG

TTGGAAGAAGTTGGT

GAAGGTCTTCGCGAG

TTTTTGGGCCTTGAA

GCGGGCCCACCGAAA

CCAAAACCCAATCAG

CAGCATCAAGATCAA

GCCCGTGGTCTTGTG

CTGCCTGGTTATAAC

TATCTCGGACCCGGA

AACGGTCTCGATCGA

GGAGAGCCTGTCAAC

AGGGCAGACGAGGTC

GCGCGAGAGCACGAC

ATCTCGTACAACGAG

CAGCTTGAGGCGGGA

GACAACCCCTACCTC

AAGTACAACCACGCG

GACGCCGAGTTTCAG

GAGAAGCTCGCCGAC

GACACATCCTTCGGG

GGAAACCTCGGAAAG

GCAGTCTTTCAGGCC

AAGAAAAGGGTTCTC

GAACCTTTTGGCCTG

GTTGAAGAGGGTGCT

AAGACGGCCCCTACC

GGAAAGCGGATAGAC

GACCACTTTCCAAAA

AGAAAGAAGGCTCGG

ACCGAAGAGGACTCC

AAGCCTTCCACCTCG

TCAGACGCCGAAGCT

GGACCCAGCGGATCC

CAGCAGCTGCAAATC

CCAGCCCAACCAGCC

TCAAGTTTGGGAGCT

GATACAATGTCTGCG

GGAGGTGGCGGCCCA

TTGGGCGACAATAAC

CAAGGTGCCGATGGA

GTGGGCAATGCCTCG

GGAGATTGGCATTGC

GATTCCACGTGGATG

GGGGACAGAGTCGTC

ACCAAGTCCACCCGA

ACCTGGGTGCTGCCC

AGCTACAACAACCAC

CAGTACCGAGAGATC

AAAAGCGGCTCCGTC

GACGGAAGCAACGCC

AACGCCTACTTTGGA

TACAGCACCCCCTGG

GGGTACTTTGACTTT

AACCGCTTCCACAGC

CACTGGAGCCCCCGA

GACTGGCAAAGACTC

ATCAACAACTACTGG

GGCTTCAGACCCCGG

TCCCTCAGAGTCAAA

ATCTTCAACATTCAA

GTCAAAGAGGTCACG

GTGCAGGACTCCACC

ACCACCATCGCCAAC

AACCTCACCTCCACC

GTCCAAGTGTTTACG

GACGACGACTACCAG

CTGCCCTACGTCGTC

GGCAACGGGACCGAG

GGATGCCTGCCGGCC

TTCCCTCCGCAGGTC

TTTACGCTGCCGCAG

TACGGTTACGCGACG

CTGAACCGCGACAAC

ACAGAAAATCCCACC

GAGAGGAGCAGCTTC

TTCTGCCTAGAGTAC

TTTCCCAGCAAGATG

CTGAGAACGGGCAAC

AACTTTGAGTTTACC

TACAACTTTGAGGAG

GTGCCCTTCCACTCC

AGCTTCGCTCCCAGT

CAGAACCTCTTCAAG

CTGGCCAACCCGCTG

GTGGACCAGTACTTG

TACCGCTTCGTGAGC

ACAAATAACACTGGC

GGAGTCCAGTTCAAC

AAGAACCTGGCCGGG

AGATACGCCAACACC

TACAAAAACTGGTTC

CCGGGGCCCATGGGC

CGAACCCAGGGCTGG

AACCTGGGCTCCGGG

GTCAACCGCGCCAGT

GTCAGCGCCTTCGCC

ACGACCAATAGGATG

GAGCTCGAGGGCGCG

AGTTACCAGGTGCCC

CCGCAGCCGAACGGC

ATGACCAACAACCTC

CAGGGCAGCAACACC

TATGCCCTGGAGAAC

ACTATGATCTTCAAC

AGCCAGCCGGCGAAC

CCGGGCACCACCGCC

ACGTACCTCGAGGGC

AACATGCTCATCACC

AGCGAGAGCGAGACG

CAGCCGGTGAACCGC

GTGGCGTACAACGTC

GGCGGGCAGATGGCC

ACCAACAACCAGAGC

TCCACCACTGCCCCC

GCGACCGGCACGTAC

AACCTCCAGGAAATC

GTGCCCGGCAGCGTG

TGGATGGAGAGGGAC

GTGTACCTCCAAGGA

CCCATCTGGGCCAAG

ATCCCAGAGACGGGG

GCGCACTTTCACCCC

TCTCCGGCCATGGGC

GGATTCGGACTCAAA

CACCCACCGCCCATG

ATGCTCATCAAGAAC

ACGCCTGTGCCCGGA

AATATCACCAGCTTC

TCGGACGTGCCCGTC

AGCAGCTTCATCACC

CAGTACAGCACCGGG

CAGGTCACCGTGGAG

ATGGAGTGGGAGCTC

AAGAAGGAAAACTCC

AAGAGGTGGAACCCA

GAGATCCAGTACACA

AACAACTACAACGAC

CCCCAGTTTGTGGAC

TTTGCCCCGGACAGC

ACCGGGGAATACAGA

ACCACCAGACCTATC

GGAACCCGATACCTT

ACCCGACCCCTTTAA

AAV5 Capsid
36

Sequence (VP1)
MSFVDHPPDWLEEVG

EGLREFLGLEAGPPK

PKPNQQHQDQARGLV

LPGYNYLGPGNGLDR

GEPVNRADEVAREHD

ISYNEQLEAGDNPYL

KYNHADAEFQEKLAD

DTSFGGNLGKAVFQA

KKRVLEPFGLVEEGA

KTAPTGKRIDDHFPK

RKKARTEEDSKPSTS

SDAEAGPSGSQQLQI

PAQPASSLGADTMSA

GGGGPLGDNNQGADG

VGNASGDWHCDSTWM

GDRVVTKSTRTWVLP

SYNNHQYREIKSGSV

DGSNANAYFGYSTPW

GYFDFNRFHSHWSPR

DWQRLINNYWGFRPR

SLRVKIFNIQVKEVT

VQDSTTTIANNLTST

VQVFTDDDYQLPYVV

GNGTEGCLPAFPPQV

FTLPQYGYATLNRDN

TENPTERSSFFCLEY

FPSKMLRTGNNFEFT

YNFEEVPFHSSFAPS

QNLFKLANPLVDQYL

YRFVSTNNTGGVQFN

KNLAGRYANTYKNWF

PGPMGRTQGWNLGSG

VNRASVSAFATTNRM

ELEGASYQVPPQPNG

MTNNLQGSNTYALEN

TMIFNSQPANPGTTA

TYLEGNMLITSESET

QPVNRVAYNVGGQMA

TNNQSSTTAPATGTY

NLQEIVPGSVWMERD

VYLQGPIWAKIPETG

AHFHPSPAMGGFGLK

HPPPMMLIKNTPVPG

NITSFSDVPVSSFIT

QYSTGQVTVEMEWEL

KKENSKRWNPEIQYT

NNYNDPQFVDFAPDS

TGEYRTTRPIGTRYL

TRPL

AAV5 Capsid
37

Sequence (VP2)
TAPTGKRIDDHFPKR

KKARTEEDSKPSTSS

DAEAGPSGSQQLQIP

AQPASSLGADTMSAG

GGGPLGDNNQGADGV

GNASGDWHCDSTWMG

DRVVTKSTRTWVLPS

YNNHQYREIKSGSVD

GSNANAYFGYSTPWG

YFDFNRFHSHWSPRD

WQRLINNYWGFRPRS

LRVKIFNIQVKEVTV

QDSTTTIANNLTSTV

QVFTDDDYQLPYVVG

NGTEGCLPAFPPQVF

TLPQYGYATLNRDNT

ENPTERSSFFCLEYF

PSKMLRTGNNFEFTY

NFEEVPFHSSFAPSQ

NLFKLANPLVDQYLY

RFVSTNNTGGVQFNK

NLAGRYANTYKNWFP

GPMGRTQGWNLGSGV

NRASVSAFATTNRME

LEGASYQVPPQPNGM

TNNLQGSNTYALENT

MIFNSQPANPGTTAT

YLEGNMLITSESETQ

PVNRVAYNVGGQMAT

NNQSSTTAPATGTYN

LQEIVPGSVWMERDV

YLQGPIWAKIPETGA

HFHPSPAMGGFGLKH

PPPMMLIKNTPVPGN

ITSFSDVPVSSFITQ

YSTGQVTVEMEWELK

KENSKRWNPEIQYTN

NYNDPQFVDFAPDST

GEYRTTRPIGTRYLT

RPL

AAV5 Capsid
38

Sequence (VP3)
MSAGGGGPLGDNNQG

ADGVGNASGDWHCDS

TWMGDRVVTKSTRTW

VLPSYNNHQYREIKS

GSVDGSNANAYFGYS

TPWGYFDFNRFHSHW

SPRDWQRLINNYWGF

RPRSLRVKIFNIQVK

EVTVQDSTTTIANNL

TSTVQVFTDDDYQLP

YVVGNGTEGCLPAFP

PQVFTLPQYGYATLN

RDNTENPTERSSFEC

LEYFPSKMLRTGNNF

EFTYNFEEVPFHSSF

APSQNLFKLANPLVD

QYLYRFVSTNNTGGV

QFNKNLAGRYANTYK

NWFPGPMGRTQGWNL

GSGVNRASVSAFATT

NRMELEGASYQVPPQ

PNGMTNNLQGSNTYA

LENTMIFNSQPANPG

TTATYLEGNMLITSE

SETQPVNRVAYNVGG

QMATNNQSSTTAPAT

GTYNLQEIVPGSVWM

ERDVYLQGPIWAKIP

ETGAHFHPSPAMGGF

GLKHPPPMMLIKNTP

VPGNITSFSDVPVSS

FITQYSTGQVTVEME

WELKKENSKRWNPEI

QYTNNYNDPQFVDFA

PDSTGEYRTTRPIGT

RYLTRPL

Macaca mulatta

39

(RHESUS MONKEY)
ATGGCGGGCATCTGG

CYP4V2 ODS
CTGGGGCTCGTGTGG

NM_001193838.1
CAGAAGCTGCTGCTG

TGGGGCGCGGCGAGT

GCCGTGTCCCTGGCC

GGCGCCAGTCTGGTC

CTGAGCCTGCTGCAG

AGGGTGGCGAGCTAC

GTGAGGAAATGGCAG

CAGATGCGGCCCATC

CCCACGGTGGCCCGC

GCCTACCCACTGGTG

GGCCACGCGCTGCTG

ATGAAGCGGGACGGG

CGAGAATTTTTTCAG

CAGATCATTGAGTAC

ACAGAGGAATACCGC

CACATGCCACTCCTG

AAGCTCTGGGTCGGG

CCGGTGCCCATGGTG

GCCCTTTATAATGCA

GAAAATGTGGAGGTA

ATTTTAACTAGTTCA

AAGCAAATTGACAAA

TCCTCTATGTACAAG

TTTTTAGAACCATGG

CTTGGCCTAGGACTT

CTTACAAGTACTGGA

AACAAATGGCGCTCC

AGGAGAAAGATGTTA

ACACCCACTTTCCAT

TTTACCATTCTGGAA

GATTTCTTAGATATC

ATGAATGAACAAGCA

AATATATTGGTTAAG

AAACTTGAAAAACAT

GTTAACCAAGAAGCA

TTTAACTGCTTTGTT

TACATCACTCTTTGT

GCCTTAGATATCATC

TGTGAAACAGCTATG

GGGAAGAATATTGGT

GCTCAAAGTAACGAT

GATTCCGAGTATGTC

CGTGCAGTTTATAGA

ATGAGTGAGATGATA

TTTCGAAGAATAAAG

ATGCCGTGGCTTTGG

CTTGACCTCTGGTAC

CTTATGTTTAAAGAG

GGATGGGAACACAAA

AAGAGCCTTAAGATC

CTACATGCTTTTACC

AACAATGTTATCGCT

GAACGGGCCAATGAA

ATGAACGTGGATGAA

GACTGTAGAGGTGAT

GGCAGGGACTCCGCC

CCCTCCAAAAATAAA

CGCAGGGCCTTTCTT

GACTTGCTTTTAAGT

GTGACTGACGACGAA

GGGAACAGGCTAAGT

CATGAAGATATTCGA

GAAGAAGTTGACACC

TTCATGTTTGAGGGC

CACGACACAACTGCA

GCTGCAATGAACTGG

TCCTTATACCTGTTG

GGGTCTAACCCAGAA

GTCCAGAAAAAAGTG

GACCATGAACTGGAT

GACGTGTTTGGGAGG

TCTGACCGTCCCGCT

ACTGTAGAAGACCTG

AAGAAACTTCGGTAT

CTGGAATGTGTTATT

AAGGAGACCCTTCGC

CTTTTTCCTTCTGTT

CCTTTATTTGCCCGC

AGTGTTAGTGAAGAT

TGTGAAGTGGCAGGT

TACAGAGTTCTGAAA

GGCACTGAAGCCGTC

ATCATTCCCTATGCA

TTGCATAGAGATCCA

AGATACTTCCCCAAC

CCTGAGGAGTTCCGG

CCTGAGCGGTTCTTC

CCCGAGAATGCACAA

GGGCGCCATCCATAT

GCCTACGTGCCCTTC

TCTGCTGGCCCCAGG

AACTGTATAGGTCAA

AAGTTTGCTGTGATG

GAAGAAAAGACCATT

CTTTCGTGCATCCTA

AGGCACTTTTGGATA

GAATCCAACCAGAAA

AGAGAAGAACTTGGT

CTAGAAGGACAGTTG

ATTCTTCGTCCAACT

AATGGCATCTGGATC

AAGTTGAAGAGGAGA

AATGCAGATGAACCC

TAA

Macaca mulatta

40

(RHESUS MONKEY)
MAGIWLGLVWQKLLL

CYP4V2 GENE
WGAASAVSLAGASLV

PRODUCT
LSLLQRVASYVRKWQ

NP_001180767.1
QMRPIPTVARAYPLV

GHALLMKRDGREFFQ

QIIEYTEEYRHMPLL

KLWVGPVPMVALYNA

ENVEVILTSSKQIDK

SSMYKFLEPWLGLGL

LTSTGNKWRSRRKML

TPTFHFTILEDFLDI

MNEQANILVKKLEKH

VNQEAFNCFVYITLC

ALDIICETAMGKNIG

AQSNDDSEYVRAVYR

MSEMIERRIKMPWLW

LDLWYLMFKEGWEHK

KSLKILHAFTNNVIA

ERANEMNVDEDCRGD

GRDSAPSKNKRRAFL

DLLLSVTDDEGNRLS

HEDIREEVDTFMFEG

HDTTAAAMNWSLYLL

GSNPEVQKKVDHELD

DVFGRSDRPATVEDL

KKLRYLECVIKETLR

LFPSVPLFARSVSED

CEVAGYRVLKGTEAV

IIPYALHRDPRYFPN

PEEFRPERFFPENAQ

GRHPYAYVPFSAGPR

NCIGQKFAVMEEKTI

LSCILRHFWIESNQK

REELGLEGQLILRPT

NGIWIKLKRRNADEP

Bos taurus

41

CYP4V2 ODS
ATGCTGGCGCCGTGG

NM_001034373.2
TTGCTGAGCGTCGGG

CCGAAGCTGCTGCTC

TGGAGCGGGCTGTGC

GCCGTCTCCCTGGCA

GGCGCCACCCTCACC

CTGAACCTCCTGAAG

ATGGTGGCGAGCTAT

GCGCGGAAATGGCGT

CAGATGCGTCCCGTC

CCGACCATTGGGGAC

CCCTACCCCTTGGTG

GGACACGCGCTGATG

ATGAAGCCCGATGCA

AGAGATTTTTTTCAG

CAGATAATTGATTTC

ACTGAAGAATGCCGA

CACCTGCCACTGCTG

AAACTCTGGCTCGGG

CCTGTGCCTCTCGTG

GCCCTTTATAACGCA

GAAACTGTGGAAGTA

ATTTTAAGCAGTTCA

AAGCACATTGAAAAA

TCCTATATGTACAAG

TTCTTAGAACCGTGG

CTTGGACTAGGACTT

CTTACAAGTACTGGA

AACAAATGGCGATCT

AGGAGAAAAATGTTA

ACACCCACTTTCCAT

TTTACAATTCTGGAG

GATTTCTTAGATGTC

ATGAATGAACAAGCA

AATATATTGGTTACT

AAGCTTGAAAAGCAT

GTTAACCAAGAAGCG

TTTAACTGCTTTTTT

TACGTCACTCTTTGT

ACCTTAGATATAATC

TGTGAAACAGCTATG

GGAAAGAACATTGGT

GCTCAAAGAAATGAT

GATTCCGAGTATGTT

CGAGCCGTTTATAGG

ATGAGTGATTCGATA

CATCAAAGAATGAAG

ATGCCCTGGCTCTGG

CTTGACCTTATATTC

TATATGTTTAAAAAT

GGACGAGAACACAGA

AGGAGCCTAAAGATT

GTACATGATTTTACC

AACAATGTCATCACT

GAACGGGCCAATGAA

ATGAAGAGACATGAA

GAAGGTACGAGTAAC

GACAAGGAGAAGGAC

TTTCCTCCACGCAAA

ACTAAATGCAGGGCT

TTTCTTGACTTGCTT

TTAAATGTGACTGAT

GACCAAGGGAACAAG

CTGAGTCATGAAGAT

ATAAGAGAAGAAGTC

GACACCTTTATGTTT

GAGGGCCATGATACA

ACTGCAGCTGCAATA

AACTGGTCCTTGTAT

CTGTTGGGTTGGTAT

CCAGAAGTCCAGCAG

AGAGTGGACACTGAG

CTGGAAGAAGTGTTT

GGGAAGTCTGACCGT

CCTGTTACCCTAGAA

GACCTGAAGAAACTT

AAATATCTGGACTGT

GTTATTAAGGAGAGC

CTTCGCCTTTTCCCG

TCTGTTCCTTTCTTT

GCCCGTAATCTTACC

GAAGACTGTGAAGTT

GCGGGTCACAAAATC

GTGCAAGGCTGTCAA

GTAATCATTGTGCCC

TACGCACTGCATAGA

GATCCAAAGTACTTC

CCGGATCCTGAGGAA

TTCAAGCCAGAACGG

TTCTTTCCCGAGAAT

TTGAAAGGACGTCAT

ACATACGCATATGTG

CCCTTCTCTGCAGGC

CCCCGAAACTGTATA

GGTCAAAAGTTTGCC

ATAATGGAAGAAAAG

ACCATTCTTTCCTGC

ATCCTTAGGCACTTT

TGGGTAGAATCCAAC

CAAAAAAGAGAAGAA

CTTGGTCTAGCAGGA

GAGCTCATTCTTCGT

CCAAGTAACGGCATC

TGGATCAAGTTGAAG

AGGAGAAACACAGAT

GAATCCTAA

Bos taurus

42

CYP4V2 GENE
MLAPWLLSVGPKLLL

PRODUCT
WSGLCAVSLAGATLT

NP_001029545.1
LNLLKMVASYARKWR

QMRPVPTIGDPYPLV

GHALMMKPDARDFFQ

QIIDFTEECRHLPLL

KLWLGPVPLVALYNA

ETVEVILSSSKHIEK

SYMYKFLEPWLGLGL

LTSTGNKWRSRRKML

TPITHETILEDFLDV

MNEQANILVTKLEKH

VNQEAFNCFFYVTLC

TLDIICETAMGKNIG

AQRNDDSEYVRAVYR

MSDSIHQRMKMPWLW

LDLIFYMFKNGREHR

RSLKIVHDFTNNVIT

ERANEMKRHEEGTSN

DKEKDFPPRKTKCRA

FLDLLLNVTDDQGNK

LSHEDIREEVDTFMF

EGHDTTAAAINWSLY

LLGWYPEVQQRVDTE

LEEVFGKSDRPVTLE

DLKKLKYLDCVIKES

LRLFPSVPFFARNLT

EDCEVAGHKIVQGCQ

VIIVPYALHRDPKYF

PDPEEFKPERFFPEN

LKGRHTYAYVPFSAG

PRNCIGQKFAIMEEK

TILSCILRHFWVESN

QKREELGLAGELILR

PSNGIWIKLKRRNTD

ES

Rattus

43

norvegicus

ATGTTGTGGCTGTGG

CYP4V3 CDS
TTAGGGCTCAGCGGG

NM_001135600.1
CAGAAGCTATTGCTT

TGGGGCGCAGCGAGC

GCGGTCTCCGTGGCC

GGCGCCACTGTCTTG

CTCAACATCCTGCAG

ATGTTGGTAAGCTAT

GCACGAAAGTGGCAG

CAGATGCGGCCAATC

CCGTCGGTGGCTCGC

GCTTACCCCTTGGTG

GGACATGCGCTGTTT

ATGAAGCCCAACAAC

ACAGAATTTTTTCAG

CAGATAATTCAGTAC

ACAGAAGAATTCCGA

CACCTGCCCATCATT

AAACTCTGGATTGGA

CCAGTGCCCCTGGTG

GCACTTTATAAGGCA

GAGAATGTGGAGGTG

ATTTTGACCAGTTCG

AAGCAAATTGATAAA

TCCTTTATGTACAAG

TTCCTACAGCCATGG

CTGGGACTAGGACTT

CTTACAAGTACTGGG

AGCAAATGGCGTGCC

AGGAGGAAGATGTTA

ACACCCAGTTTCCAT

TTTACAATTCTGGAG

GATTTCTTAGATGTC

ATGAATGAGCAAGCA

AATATATTGGTTAAC

AAGCTTGAAAAACAT

GTCAATCAAGAGGCC

TTTAACTGCTTTTTC

CCCATCACTCTTTGT

GCTCTGGATATAATC

TGTGAAACGGCTATG

GGGAAGAACATTGGA

GCTCAAAGTAATGGT

GATTCTGAGTATGTC

CGTACAGTGTATAGG

ATGAGCGATATGATA

TACAGAAGAATGAAG

ATGCCCTGGTTTTGG

TTTGACCTTTGGTAC

CTTATGTTTAAAGAA

GGAAGGGACCACAAA

AAGGGACTAAAGAGT

CTACATACTTTTACC

AACAATGTCATTGCT

GAACGGGTTAATGCA

AGGAAGGCAGAGCAA

GACTGCATAGGTGCT

GGGAGGGGTCCTCTC

CCCTCGAAAACTAAG

CGCAAGGCCTTTCTT

GACTTGCTTTTGAGT

GTGACTGATGAGGAA

GGAAACAAATTAAGC

CATGAAGACATCCGA

GAGGAAGTTGACACC

TTCATGTTTGAGGGT

CACGATACAACTGCT

GCTGCCATAAACTGG

TCCTTATACCTCCTG

GGCTCTAATCCAGAA

GTCCAGAGGAAAGTG

GACAAGGAGCTGGAT

GATGTGTTTGGAAGA

TCCCATCGCCCTGTC

ACCTTGGAAGACCTG

AAGAAACTTAAATAT

CTGGATTGTGTCATT

AAGGAGACCCTCCGT

GTTTTCCCATCTGTC

CCTTTATTTGCCCGG

AGTCTTAGCGAAGAC

TGTGAAGTGGCGGGT

TACAAAATCTCAAAA

GGAACGGAAGCAGTC

ATCATTCCCTATGCA

CTACATCGAGACCCT

AGATACTTCCCAGAC

CCTGAGGAATTCCAG

CCAGAGCGGTTCTTT

CCTGAAAACTCCCAA

GGACGCCACCCCTAT

GCCTATGTGCCATTC

TCTGCTGGACCTAGA

AACTGCATTGGTCAA

AAGTTTGCGGTCATG

GAGGAAAAGACCATT

CTTGCCTGTATCCTG

AGGGAGTTTTGGATA

GAATCCAACCAGAAG

AGAGAAGAACTCGGC

CTGGCTGGAGATTTG

ATTCTTAGGCCAAAT

AATGGCATCTGGATC

AAGCTGAAGAGGAGG

CATGAAGATGACCCC

TAA

Rattus norvegicus

44

CYP4V3 GENE
MLWLWLGLSGQKLLL

PRODUCT
WGAASAVSVAGATVL

NP_001129072.1
LNILQMLVSYARKWQ

QMRPIPSVARAYPLV

GHALFMKPNNTEFFQ

QIIQYTEEFRHLPII

KLWIGPVPLVALYKA

ENVEVILTSSKQIDK

SFMYKFLQPWLGLGL

LTSTGSKWRARRKML

TPSFHFTILEDFLDV

MNEQANILVNKLEKH

VNQEAFNCFFPITLC

ALDIICETAMGKNIG

AQSNGDSEYVRTVYR

MSDMIYRRMKMPWFW

FDLWYLMFKEGRDHK

KGLKSLHTFTNNVIA

ERVNARKAEQDCIGA

GRGPLPSKTKRKAFL

DLLLSVTDEEGNKLS

HEDIREEVDTFMFEG

HDTTAAAINWSLYLL

GSNPEVQRKVDKELD

DVFGRSHRPVTLEDL

KKLKYLDCVIKETLR

VFPSVPLFARSLSED

CEVAGYKISKGTEAV

IIPYALHRDPRYFPD

PEEFQPERFFPENSQ

GRHPYAYVPFSAGPR

NCIGQKFAVMEEKTI

LACILREFWIESNQK

REELGLAGDLILRPN

NGIWIKLKRRHEDDP

Mus musculus

45

CYP4V3 ODS
ATGTTGTGGCTGTGG

NM_133969.3
TTAGGGCTCAGTGGG

CAGAAACTATTGCTT

TGGGGCGCAGCGAGC

GCGGTCTCCCTGGCC

GGCGCCACTATCCTG

ATCAGCATCTTTCCG

ATGCTGGTAAGCTAC

GCGCGGAAATGGCAG

CAGATGCGGTCAATC

CCGTCGGTGGCCCGC

GCCTACCCCTTGGTG

GGACACGCGCTTTAT

ATGAAGCCCAACAAC

GCAGAATTTTTTCAG

CAGCTAATTTATTAC

ACAGAAGAATTTCGA

CACCTGCCGATCATT

AAACTTTGGATTGGA

CCCGTGCCCCTGGTG

GCACTTTATAAGGCA

GAGAATGTGGAGGTG

ATTTTGACCAGTTCT

AAGCAAATTGATAAA

TCGTTTTTGTACAAG

TTCCTACAGCCATGG

CTGGGACTAGGACTT

CTTACAAGTACGGGG

AGCAAATGGCGCACC

AGGAGGAAGATGCTA

ACGCCCACTTTCCAT

TTTACCATTCTGGAG

AACTTCTTGGATGTC

ATGAATGAGCAAGCA

AATATATTGGTTAAT

AAGCTTGAAAAACAC

GTCAACCAAGAAGCC

TTTAATTGTTTTTTT

TACATCACTCTTTGT

GCTCTGGATATAATC

TGTGAAACGGCTATG

GGGAAGAACATCGGA

GCTCAAAGCAATAAT

GATTCCGAGTATGTC

CGTACAGTGTATAGG

ATGAGTGATATGATA

TATAGAAGAATGAAG

ATGCCCTGGCTTTGG

TTTGACCTTTGGTAC

CTTGTGTTTAAAGAA

GGACGGGACCACAAA

AGGGGACTCAAATGC

CTACATACTTTCACC

AACAATGTCATTGCT

GAACGAGTCAAAGAA

AGGAAGGCAGAGGAA

GACTGGACGGGTGCT

GGCAGGGGTCCTATC

CCCTCCAAAAATAAG

CGCAAGGCTTTCCTT

GACTTGCTTTTGAGT

GTGACTGATGAGGAA

GGAAACAGATTAAGC

CAGGAAGACATCCGA

GAGGAAGTTGACACC

TTCATGTTTGAGGGT

CACGATACAACTGCT

GCTGCAATCAACTGG

TCCTTATACCTATTG

GGCACGAATCCAGAA

GTCCAGAGGAAAGTG

GATCAGGAGCTGGAT

GAAGTGTTTGGAAGA

TCCCATCGTCCTGTC

ACCTTGGAAGACCTG

AAGAAACTTAAATAT

TTGGATTGCGTCATT

AAGGAGACTCTCCGA

GTTTTCCCATCTGTC

CCTTTATTTGCCCGG

AGTCTTAGCGAGGAC

TGTGAAGTGGGCGGT

TACAAAGTCACAAAA

GGAACGGAAGCAATC

ATCATTCCCTACGCA

CTACACCGAGACCCC

AGATACTTCCCAGAT

CCAGAGGAATTCCGA

CCAGAGCGGTTCTTT

CCTGAAAATTCCCAA

GGACGCCATCCCTAT

GCCTATGTGCCATTT

TCTGCTGGACCTCGA

AACTGTATTGGTCAA

AAGTTTGCTGTCATG

GAGGAGAAGACCATT

CTTGCCTGTATCCTG

AGGCAGTTTTGGGTA

GAATCCAACCAGAAG

AGAGAAGAACTCGGC

CTGGCTGGAGATTTG

ATTCTTAGGCCAAAT

AATGGCATCTGGATC

AAGCTGAAGAGGAGA

CATGAAGATGACCCC

TAA

Mus musculus

46

CYP4V3 GENE
MLWLWLGLSGQKLLL

PRODUCT
WGAASAVSLAGATIL

NP_598730.1
ISIFPMLVSYARKWQ

QMRSIPSVARAYPLV

GHALYMKPNNAEFFQ

QLIYYTEEFRHLPII

KLWIGPVPLVALYKA

ENVEVILTSSKQIDK

SFLYKFLQPWLGLGL

LTSTGSKWRTRRKML

TPITHETILENFLDV

MNEQANILVNKLEKH

VNQEAFNCFFYITLC

ALDIICETAMGKNIG

AQSNNDSEYVRTVYR

MSDMIYRRMKMPWLW

FDLWYLVFKEGRDHK

RGLKCLHTFTNNVIA

ERVKERKAEEDWTGA

GRGPIPSKNKRKAFL

DLLLSVTDEEGNRLS

QEDIREEVDTFMFEG

HDTTAAAINWSLYLL

GTNPEVQRKVDQELD

EVFGRSHRPVTLEDL

KKLKYLDCVIKETLR

VFPSVPLFARSLSED

CEVGGYKVTKGTEAI

IIPYALHRDPRYFPD

PEEFRPERFFPENSQ

GRHPYAYVPFSAGPR

NCIGQKFAVMEEKTI

LACILRQFWVESNQK

REELGLAGDLILRPN

NGIWIKLKRRHEDDP

Gallus gallus

47

CYP4V2 ODS1
ATGGCAATGGAGATC

NM_001001879.
ACGCTAGGATCCATG

GAGGGAACACAGCTG

CTGCCCTGGGTGGCT

GGAGCCATCACCCTG

CTGCTGACGGTGGTG

ACTGTACACTTCCTA

CCCTCTTTGCTGAAC

TACTGGTGGTGGTGG

TGGGTGATGAAGCCC

ATCCCAGGCATCCGC

CCATGCTACCCCTTT

GTGGGAAATGCTCTC

CTGTTGGAGCGAAAT

GGAGAAGGTTTTTTT

AAACAGCTACAACAG

TATGCTGATGAGTTC

AGGAAAATGCCAATG

TTCAAACTCTGGTTA

GGTCCACTGCCTGTC

ACAGTATTGTTCCAT

CCTGATAGTGTGGAG

GTTATTCTGAGCAGT

TCAAAGCATATTAAA

AAATCATTCCTGTAC

ACATTTCTGCACCCA

TGGCTGGGGACTGGA

CTTTTGACAAGCACT

GGAGACAAGTGGCGG

TCACGGAGGAAGATG

ATAACTCCTACATTC

CACTTTGCAATCTTA

AATGACTTTCTTGAG

GTTATGAATGAACAA

GGGGGTGTTTTGTTG

GAGAAACTTGAGAAG

CATGTTGACAAGGAA

CCATTTAATATCTTT

ACAGACATCACTCTG

TGTGCACTTGATATT

ATCTGTGAAACTGCA

ATGGGCAAGAATCTG

GGTGCTCAAGACAAT

AAGGATTCTGAGTAT

GTTCGTGCTGTCTAC

AGGATGAGTGATCTA

ATCCAACAGCGACAG

AAGAGCCCTTGGCTT

TGGCATGATCTTATG

TATCTTCTGTTCAAG

GAAGGAAGAGAGCAT

GAGCGGAATCTTAAG

ATTCTGCATGGTTTT

ACAGATACGGTAATT

GCAGAAAAAGTTGCA

GAACTTGAAAACACC

AAGCTAACAAAACAC

GATACTGACGTGAAC

ACTGAAGAAGAAAGT

GGTTCCAAAAAGAGA

GAAGCTTTCTTAGAC

ATGCTGCTGAATGCC

ACAGATGATGAAGGG

AAAAAACTCAGCTAC

AAGGACATTCGTGAA

GAAGTGGATACTTTT

ATGTTTGAGGGTCAT

GATACAACAGCAGCT

GCTATGAACTGGGTC

CTATACTTGCTTGGT

CATCATCCTGAAGCC

CAGAAGAAGGTTCAC

CAAGAACTGGATGAG

GTGTTTGGCAACACA

GAGCGTCCTGTTACA

GTGGATGATTTGAAG

AAACTTCGATACCTC

GAGTGTGTTGTGAAA

GAAGCCCTGAGGCTC

TTCCCTTCAGTTCCC

ATGTTCGCCCGTTCC

TTGCAAGAGGATTGC

TACATTAGTGGATAT

AAGCTACCAAAAGGC

ACGAATGTCCTTGTC

TTAACTTATGTGCTG

CACAGAGATCCTGAG

ATCTTCCCTGAGCCA

GATGAATTCAGGCCT

GAGCGCTTCTTCCCT

GAAAATAGCAAAGGA

AGGCACCCATATGCT

TATGTGCCCTTCTCT

GCTGGCCCCAGGAAC

TGCATTGGCCAACGC

TTTGCACAAATGGAA

GAGAAAACTCTTCTA

GCCCTCATCCTGCGG

CGCTTTTGGGTGGAC

TGTTCTCAAAAGCCA

GAAGAGCTTGGTCTG

TCAGGAGAACTAATT

CTTCGTCCAAATAAT

GGCATCTGGGTTCAA

CTGAAGAGGAGACCA

AAAACTGTAACAGAA

TGA

Gallus gallus

48

CYP4V2 GENE
MAMEITLGSMEGTQL

PRODUCT
LPWVAGAITLLLTVV

NP_001001879.1
TVHFLPSLLNYWWWW

WVMKPIPGIRPCYPF

VGNALLLERNGEGFF

KQLQQYADEFRKMPM

FKLWLGPLPVTVLFH

PDSVEVILSSSKHIK

KSFLYTFLHPWLGTG

LLTSTGDKWRSRRKM

ITPTFHFAILNDFLE

VMNEQGGVLLEKLEK

HVDKEPFNIFTDITL

CALDIICETAMGKNL

GAQDNKDSEYVRAVY

RMSDLIQQRQKSPWL

WHDLMYLLFKEGREH

ERNLKILHGFTDTVI

AEKVAELENTKLTKH

DTDVNTEEESGSKKR

EAFLDMLLNATDDEG

KKLSYKDIREEVDTF

MFEGHDTTAAAMNWV

LYLLGHHPEAQKKVH

QELDEVFGNTERPVT

VDDLKKLRYLECVVK

EALRLFPSVPMFARS

LQEDCYISGYKLPKG

TNVLVLTYVLHRDPE

IFPEPDEFRPERFFP

ENSKGRHPYAYVPFS

AGPRNCIGQRFAQME

EKTLLALILRRFWVD

CSQKPEELGLSGELI

LRPNNGIWVQLKRRP

KTVTE

Canis lupus

49

familiaris

ATGTTAACACCCACT

CYP4V2 CDS
TTCCATTTTACGATT

XM_022404181.1
CTGGAAGATTTCTTA

GATGTCATGAATGAA

CACGCAAATATATTG

GTTAATAAGCTTGAA

AAACATGTTAACCAA

GAAGCATTTAACTGC

TTTTTTTACATCACT

CTTTGTGCATTAGAT

ATAATTTGTGAAACA

GCTATGGGGAAGAAT

ATTGGGGCTCAAAAT

AATGAGGATTCTGAG

TATGTTCGTGCCATC

TACAGAATGAGTGAT

ACGATACATCGAAGA

ATGAAGATGCCCTGG

CTCTGGCTTGACTTT

TTGTTTCTTATGTTT

AAAGAAGGCCGGGAA

CACAAAAGGAACCTA

GAGATCCTACATAAT

TTTACCAATAATGTC

ATCACTGAACGGGCC

AGTGAACTGAAGAGA

GACGAAGAACATGGA

AGTGCTGACAAGGAC

TGCTCCCCCTCCAAA

AATAAACGCAGAGCT

TTTCTTGACTTGCTT

TTAAATGTGACTGAT

GATGAAGGGAACAAG

CTACGTCATGAAGAT

GTTCGAGAAGAAGTT

GACACCTTCATGTTT

GAGGGCCATGATACG

ACAGCAGCGGCGATA

AACTGGTCCTTATAT

CTCTTGGGTTCTTAC

CCAGAAGTCCAGAAA

CAAGTGGACAGTGAA

CTGGAGGACGTGTTT

GGGAAGTCTGATCGT

CCTGCTACCTTAGAA

GACCTGAAGAAACTC

AAATACCTGGAGTGT

GTCATTAAGGAGAGC

CTTCGCCTTTTTCCT

TCAGTTCCCTTATTT

GCCCGTAATCTTAAC

GAAGATTGTGTAGTT

GCGGGTTACAAGGTT

GTGAAAGGCTCCCAA

GCGATCATCATTCCC

TACGCACTTCATAGA

GATCCAAGATATTTC

CCAAATCCCGAGGAG

TTCCAGCCAGAGCGG

TTCTTTCCTGAAAAT

TTGCAAGGACGCCAC

CCATATGCATACATT

CCCTTTTCTGCTGGA

CCCAGAAACTGTATA

GGTCAAAGGTTTGCC

ATAATGGAAGAAAAG

ACTGTTCTTTCCTGT

GTCCTGAGGCATTTT

TGGGTAGAATCCAAC

CAGAAAAGAGAAGAA

CTTGGTCTGGCAGGA

GAGTTGATTCTTCGT

CCAACTAATGGCATC

TGGATCAAGTTGAAG

AGGAGAAATGCAGAT

GAATCTTAA

Canis lupus

50

familiaris

MLTPITHETILEDFL

CYP4V2
DVMNEHANILVNKLE

GENE PRODUCT
KHVNQEAFNCFFYIT

XP_022259889.1
LCALDIICETAMGKN

IGAQNNEDSEYVRAI

YRMSDTIHRRMKMPW

LWLDFLFLMFKEGRE

HKRNLEILHNFTNNV

ITERASELKRDEEHG

SADKDCSPSKNKRRA

FLDLLLNVTDDEGNK

LRHEDVREEVDTFMF

EGHDTTAAAINWSLY

LLGSYPEVQKQVDSE

LEDVFGKSDRPATLE

DLKKLKYLECVIKES

LRLFPSVPLFARNLN

EDCVVAGYKVVKGSQ

AIIIPYALHRDPRYF

PNPEEFQPERFFPEN

LQGRHPYAYIPFSAG

PRNCIGQRFAIMEEK

TVLSCVLRHFWVESN

QKREELGLAGELILR

PTNGIWIKLKRRNAD

ES

Kozak sequence
51

GCCGCC

Kozak sequence
52

GACACC

Kozak sequence
53

GCCACG

In some embodiments, the 5′ and 3′ ITRs comprise about 130 to about 145 nucleotides each. The ITRs are required for efficient multiplication of the AAV genome, and the symmetrical feature of these sequences gives them an ability to form a hairpin, which contributes to so-called self-priming that allows primase-independent synthesis of the second DNA strand. It is contemplated that the 5′ and 3′ ITRs of AAV serotype 2 may be used (e.g., nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs:1 and 22, respectively). In other aspects, ITRs from other suitable serotypes may be selected from among any AAV serotype known in the art, as described herein, e.g., the ITRs may be from AAV8, AAV9, or AAV5.

These ITRs or other AAV components may be readily isolated using techniques available to those of skill in the art from any AAV serotype known, or yet to be identified serotypes, for example, the AAV sequences may be synthetic or obtained through other suitable means by reference to published sequences such as are available in the literature or in databases such as, e.g., GenBank, PubMed, or the like. Alternatively, such AAV components may also be isolated or obtained from academic, commercial, or public sources (e.g., the American Type Culture Collection, Manassas, Va.).

In some embodiments, the 5′ or 3′ ITR region of an AAV vector is mutated to form a ΔITR, e.g., by deleting/mutating the terminal resolution site (trs), and the resulting AAV genome becomes self-complementary (sc) by forming dimeric inverted repeat DNA molecules. In one embodiment, a ΔITR sequence comprises SEQ ID NO: 54. Additional ΔITR sequences are known in the art, e.g., as described in Wang et al., Gene Therapy, 2003, 10: 2105-2111; McCarty et al., Gene Therapy, 2003, 10: 2112-2118; and McCarty et al., Gene Therapy, 2001, 8: 1248-1254, each one of which is incorporated by reference in its entirety.

In certain embodiments, the promoter may be a ubiquitous promoter, e.g., a CMV promoter, CBA promoter, or CAG promoter. For example, the CMV promoter can have a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:2, the CBA promoter can have a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:3, and the CAG promoter can have a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:4. Alternatively, a RPE-specific promoter may be used to target expression of CYP4V2 preferentially in RPE cells, e.g., human RPE cells, of the retina. Examples of RPE-specific promoters include ProC2 promoter, VMD2 promoter, CYP4V2 promoter, and RPE65 promoter. For example, the ProC2 promoter can have a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:5. In one embodiment, the VMD2 promoter can have a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:6. In some embodiments, the CYP4V2 promoter can have a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:7 or a fragment thereof, e.g., fragments of 100, 200, 300, 400, 500, 600, 700, 800, or 900 nucleotides of SEQ ID NO:7. In certain embodiments, the RPE65 promoter can have a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:8.

In some embodiments, an AAV vector genome comprises a promoter operably linked to a recombinant nucleotide sequence comprising a CYP4V2 coding sequence, wherein the promoter can target the expression of CYP4V2 in retinal cells, e.g., non-human primate or human retinal cells, and the promoter is selected from Table 3 below:

TABLE 3

Nucleic acid sequence of retinal-

specific promoters

Promoter & Ref.
SEQ ID NO & SEQUENCE

SynP159 (ProD3)
55

WO 2017/093931
ATCCTGGGGTTTAAACAGGA

CTAGGTCCACTGGGAGAAGA

GGAAGTCAAACAGGACTAGT

CATCGATAGGCTAGCCAGCT

TAATTAATCAGATTGTCTTT

CCTCCCGTCTAAGCATGGAA

GCCCTAAATGGCCTACAATT

CCTATAGCTTAGTTGCAAAT

TTGAGAAGTTCCCCCACTGA

CTCTGGTGGAGGTGGTCTAT

TCTGTGTCCCTGGTGAAAAG

TCTACCCCCTCGGGTTTGTT

ATGGGGCCCAGAACTTGTAC

AATGCCCCGTAAGCTTCTTA

GAGGAGGCGGGTATTAGGCA

GGATTGGATCTGCGTCCCCT

GACATGCCCTTCATACCCTT

GAATTAGGATTCCTAAGGGA

GCCGGCAGGAGGAGAATGGG

GTGTTCCTTGTGGTTCCACT

TCACACCTACTCTTCGAGAC

CCCGGATTCTGACAAGTTTC

CCGAACTTGGAAGACCCAGA

TGTCAAGGTCTGAAGGTAAA

TGATGTGTTTAGGCAGGAGG

GTTCAGCCAGGCTCAGCTCT

CCTCCCCTCTCGCAGGGGAA

GGAGAGAGACCCTCTAGAAT

TCCTGTAGGAGGTGCACGTG

GGCTGATCCTCACATGGTCC

TGCTGGAGTTAGTAGAGGGT

ATATAATGGAAGCTCGACTT

CCAGCTATCACATCCACTGT

GTTGTTGTGAACTGGAATCC

ACTATAGGCCA

SynP160 (ProD4)
56

WO 2017/093934
ATCCTGGGGTTTAAACAGGA

CTAGGTCCACTGGGAGAAGA

GGAAGTCAAACAGGACTAGT

CATCGATAGGCTAGCCAGCT

TAATTAATCAGATCTCCAGT

GTTTACCCAGGGCAAGAAGT

CCCATTTTTGTTCTTTTACA

AGCTGAAGACCAGAAACACA

TGCGGCCCATTCCCGTCCGA

GGCAGAAATGCTGAGGGATA

AGTCTCAGGCTCTGAGCTGA

GTTCAGGAAAAGGTCAGTGT

CCCGATAGAGCACTCAGGTA

GAGTGAATCCGCTTATCACA

GAGTGAATCCAGCTTAGTTG

TCCTTTGGAACCTCAGTTGA

TTGGATAAGTCCTCCAAGTG

TGAAAGAGGCATCTCCACTC

AGGCCTTGGTTCCAATGGTC

TCTCAGGGAACTGCCCTCAG

CAGTGCATAGAGCTGAGCCA

GCTCTCCGGGAAGTTCAGTC

GAGTTTATCCTCACATGGTC

CTGCTGGAGTTAGTAGAGGG

TATATAATGGAAGCTCGACT

TCCAGCTATCACATCCACTG

TGTTGTTGTGAACTGGAATC

CACTATAGGCCA

SynP161 (ProD5)
57

WO 2017/093935
ATCCTGGGGTTTAAACAGGA

CTAGGTCCACTGGGAGAAGA

GGAAGTCAAACAGGACTAGT

CATCGATAGGCTAGCCAGCT

TAATTAATCAGATTTCCAGG

AAGAGGCACCAGAGCCCTCT

CCCTATTGCTCAAAGCGCAG

GTGGAATAATCTTGGCCGTG

TGACCGGAATTCAAAGCAGT

TTTGGGAACTGAGCCCCAAC

CTGTAAGCCCTGATGTTATT

TCAAGAAAGATGGTGTCAGA

ATTAAATTGGATTAGAGAGG

AGGATATGGATTAGAGCAGA

GCAGCATCCTCACATGGTCC

TGCTGGAGTTAGTAGAGGGT

A

SynP162 (ProD6)
58

WO 2017/093936
CACACAAAACACAAATACAA

CAAATCTTTTAAAACTGCGA

TAATCCTATGTTGATTCTCT

TACCTAAATTAAAAAACAAA

ACAAAATATGATAGCCTTGA

ATAAATTTTTATATGATATT

TCATACTGTAGCCCAGGCTA

GCCTCAAACTTGTAGCAATT

CTCTTGTCTTCATCTCCTGA

ACACTGGAATTACAGGTGAG

ATCTAGAATGCTTTTCTTTT

TTATGAGATACATCATCTTT

AAATGAATTTAACTAACTTT

ATGTGTATGGATGTTTTGCC

TGAATGTATGTGCAGGGCCG

AGGGAGTGGGAGAGAACCCA

CTGGCTGTGTTAAGCCACTG

ACATCCTCACATGGTCCTGC

TGGAGTTAGTAGAGGGTATA

TAATGGAAGCTCGACTTCCA

GCTATCACATCCACTGTGTT

GTTGTGAACTGGAATCCACT

ATAGGCCA

SynP198 (ProD1)
59

WO 2018/083607
ATCCTGGGGTTTAAACAGGA

CTAGGTCCACTGGGAGAAGA

GGAAGTCAAACAGGACTAGT

CATCGATAGGCTAGCCAGCT

TAATTAATCAGATGGTTCAT

TGTAAGCCACTGTAGTCGTG

GGTGACTCACATCAAACCAC

CCCTTCGGGAACACGATGCC

GACACTGAAACTACATAGGG

GAGAGCAAATAAAACTGTCT

TCTCTAGTTTGGGGAAATTG

GAGCCTGCCTTAGGGAAACA

GTGACTAATTTACAGCTAGT

GTTTGGAATGAGATCATCTG

TCAAAATAAATGTATCTTTA

CTTCCTTCTTGAGAGGAAGC

AAGCTGTTTTATGATGTTCC

TTGGAATCCTTAAAAATACA

GAGCAACATTTACATTATTA

ATGATACGCTTTATTGCTGC

AGGCTAACTAGGTCCAAAAT

TGTCCTTCCATAGATGGAGC

TGGAGAGTTACACAGAAGTT

TGCATATCGAGCTCTTAGGT

CTGCATGTACAGCTAATGTA

CTTGTGGACCCTGTCACATA

TCCTCACATGGTCCTGCTGG

AGTTAGTAGAGGGTATATAA

TGGAAGCTCGACTTCCAGCT

ATCACATCCACTGTGTTGTT

GTGAACTGGAATCCACTATA

GGCCA

SynP107
60

(ProC17)
AACAAGTCTATCATAATAAT

WO 2018/099974
TACAGGATGTGAACCTGGGA

GGATAATTACAGGACCCCGG

GCGCAATAAATAATTACAGG

AATCGAATATGGAATTATAA

TTACAGGATCTGAAGAATCA

AGTATAATTACAGGAGCCGT

TGTGTGCTGTATAATTACAG

GAATGATCATTTATTGCATA

ATTACAGGATAGGTGTGCCG

CTACATAATTACAGGAGGTT

TCAGCCCAACTATAATTACA

GGACTAGGCAAGTTCGGTAT

AATTACAGGATGGCGCTGCC

CCCCAATAATTACAGGATAG

CTGACTTCGGTAATAATTAC

AGGACATTCTCGCCAGACTA

TAATTACAGGATGCAACTAG

ACTCTGATAATTACAGGATC

GTCTTAAATTGCCATAATTA

CAGGAACAAGCATACGTTGC

ATAATTACAGGACCCCTATT

CTAGTGTATAATTACAGGAT

GTGCACAACCAAAAATAATT

ACAGGATAATTGTCTTGACT

GATAATTACAGGAGGGTGTG

ATACACCTATAATTACAGGA

GCTCGAGATCTGCGATCTGC

ATCTCAATTAGTCAGCAACC

ATAGTCCCGCCCCTAACTCC

GCCCATCCCGCCCCTAACTC

CGCCCAGTTCCGCCCATTCT

CCGCCCCATCGCTGACTAAT

TTTTTTTATTTATGCAGAGG

CCGAGGCCGCCTCGGCCTCT

GAGCTATTCCAGAAGTAGTG

AGGAGGCTTTTTTGGAGGCC

TAGGCTTTTGCAAA

SynPI (ProA6)
61

WO 2018/099975
AGCTAGCACAGCACTAGGCT

WO 2017/046084
AAAGCGTACTGAGCCCTTGT

CTTCCGTGGGAGCTGCAGAG

TGGGATGCATGCGTTGTGAG

CTGAGGCTCAAGCTGCGCTG

GCAGAAGAGCAGGGGTTGCC

TTGTCAGACTCCAGGGTCTC

TTTCTCTCTGAGCCTGGGAA

AGTGCCACTTTATTGGATCT

ATAAAGCCGGGGGGGGGGGG

GGGGGAGGAATCTCAAGGTG

AAGAGGAAGTTCACAGACCC

CTCTAACGCCTCTATTAGAA

CCTTCCAGCTATTCTCTCAT

ACTTGTACACTGAGCTGGCA

CACAGTATAGGCAAGTTCTA

TTCGCATCACCCCTCTAGTT

CCTGTCTCCCTGGTTATGCA

AGCCTCATATTTAGGTAGAT

GTGACCTTAGGAAACCAAAA

TATCCTTTAAGATCTTACTA

ACTGGTTGCCTGTTCAGCTT

TTCCACATTGATCCTGTAGC

CCCCTCGAGGAGGTGAAGGA

AAAAAATCTCCTCTTTGTTT

CTCTAACTCATTAATGAATT

TTAAGGGCACTCTGTAAGGT

TCCTTTCCCATTCTGGTCTG

GTTCGTACATTCTGAGAAAC

ACACTGTGTTTGTGTTGAGA

GTTGGCTCCCTAGCTACACT

GTCTGTCACATTGATGCTCT

GAGTAGGGACAGGGTTCATC

TAGGAAATATATTTTCACTC

ACACTCTGTATCTTTTCCTA

GTTTGGCATATTCTAGTCTG

CATTTGGCTCTCTGTTTAAA

TATAAAAGAAAACTAAAACA

CACCCTTCAGACGCCTATGT

CTGAAAAATCTGGCATTTCC

GTGGGTTTTTCTTTAAGGAG

GCCTTCATTTGTAACCAACA

CCATGCTCTCCTTAAGGAAA

TCAATCTCAATGCCCTATTA

TCCTTCCCTTTTCTTTCCTC

CCAGTTTGAGGCTGCAGTTG

CCTTTTTTTTTCTTATCCCC

TGCTGAACCTGAAAAACCCT

CTCTTTTCTACAGTTTTCTG

TTCCCAGGCCCCGCTGACTT

CCTTTAGAGCATGGGGGGGG

GGGGGATCAGGATTGTGATG

TGTGAACTGGGAGGATCTTG

ACCTACTCCGCTAACCCAGT

GGCCTGAGCAAATCACAAGG

AGGATTGGAGCCATCTGCCC

AGCCCCTCCCCCACGGCAGC

CTGCTGGAAAGAGACAAGTT

AGTCATTCAAATGATTGGCT

TTTTGCCCGCTTCTTCTCTA

AATAAGAAGGCAGCAGCTTC

TGCTGAGGT

SynP88 (ProA5)
62

WO 2018/146588
GGTGCCCAGGCAGTGGGAGC

AGGGCTGACCAGAGTTCTGC

AGAGATTGCCTGGAGGCCTT

CCTGGAAGAAGAGATCCTGG

CACCGCACAAAGAGAAGCAC

AGGCTTTCCAGGGCTGAGGA

GAGGGAGGTCAAGTGAGGCC

CAGGTGCCCCTGCCTGAGCC

TGTGTCCCCAGAAACCTCCT

CTCCCTCTCATCACCCCCAC

ATCCTCCCTGCCACTCCCCG

CAGCTCCCTGTGGCCAAGTG

CACTGCAGCACTCGGCTCTG

CTCCACAAACGGTCTGCTCC

ACTCCAGGAAGGCCACCTCC

TCCCCCCCCCCCCACCTCCG

GCTGTCACCACTCACCGCTC

TAGCCTCCAGGGGGTGGGGA

CCCCAGAGCTGGACACACCC

CATCGAAGCCCCACAGCTCA

GCCAGCCGGACAGACTCACG

GTCGGACTCAAGACCCCGGA

GCCCTGAGGTGGGCAGCGCG

CCAGGGTTCCTCGCAGCCTC

TTCAAGGTCAGTGCAAGTTG

GGTGTGCAGCCCTTTGGAGC

CTTTGAGGTCTGTGGGACTC

AGCTCAGAACCCTCATCCCA

GACCACGAGCCCCAAGGTGG

GCTTCACTCCAGGTGCACCT

GAGCCAAGTGCTGGGCAAAG

GGCGCCCAGGTCCACAGTCA

GCACAGGTCCAGGGGTCCAT

CAGGAGGGGCCCCAGGCTGG

GGGCCAACCCCTTGCTGAGG

CCTCTTTGGGGACACTCCCC

ACCGGCCATGGGGAGCTAAA

ATTGGAAAGTGGCGAGTGGG

AGGCAGCTGACACAGCTATT

TTGTGCGCTCTTCATCCGCT

CCGGTTTCACTCTCCTGCTC

CACTAACCTGATCCTGTGCT

TCCCTCCCCCACCCTGCCGA

TGAAGAGGTGTCTCAGCCCT

CCAGCCACAGTGCAGAGGCT

GCTCAGGAGGGCCAGGCCGG

GAGCGAGCAGGGCCAGCGTG

GTCTGGTCTGAGCCGGGACT

CACCAGGAGCACCTGCCCTT

CAGAGAAGCTCACTGGATGG

CCTCCCGGGTCCCTGACCCT

TTGTGACCCACACCTCTCTG

TCAGTGTAGCAAGGAGCGGC

CTCCCAGAGCCCCAACTACA

GGGCTGGAGGAGACAGATCT

GCAACTGGGAGATCTTGGGT

TGGGAGAGATGGGGTGACCC

TACCCATCTCCAAAAGGGCA

CTGCACCGTGTTCCCCTTTC

CCTCGGTTCCAGGTCTGGAC

CTAAGCAACCACTGGAAGAC

TGGCGGCCAGGCTGAAGAGG

GTGGGAGGGGCTTAGGTACT

GGCCCAGACCCCAGGGATGG

CCCAGCAGGGTAGCCAGAGG

GAGTGACCAGACACATCCTG

GACTTGATACCAGTCAGCAC

CTTGGACAGCAGCCAGCACC

CTCCCAGTTGCTGCCTGGCC

CTGCCTGGTCCAGCTCTGGC

CACTCTGGGGCCTCAAAGAG

CCCAACATCCAGCCTGCAGC

AAGAGCTTAGACTACACAGC

ACTTCAAGGGGGACAGTGGG

TGAACACAGGCAGGGACCTG

AGATAAAACAGACTCACGGC

TTGGCGGAGGAATGGAGTCA

GCAGAGCCCGGAGTTGAGCG

AGTCTGGGAAGTTAGTGCTA

GAGGAGTTCACGGGATCTCA

GGGGACCTCGTGGGCAGGGC

TTTGCTGAGGTTAAGAGAGG

CTCACTGTGAGACGGGAGAA

CTGACAGAGTCTTCCACCAT

GGGAAAGACCCCATGGTTGG

GGAAGTCTTGCAGGAACAGT

TTGGTGTCCCAAACTTGTCA

GCCCATCAGAGAGGTATGAA

GGAGAGGAAGCTAGTCTGTG

CGAAGTCTGGGCTTTGGAGA

ATCTTCAAAGGAAGCAAGAT

TGAGAGCTGCAGAGGAAAGG

AAGAAGGTGTCTGGGTGTGT

TGGGGGGGGTAGATTTACTG

ACAGCATCCCCAGGGGATGG

CTGAGTGGACCAGTCTCCTC

AGAGGGTCCACCAGGGGAGG

CGAGGCAAGGCTGGGACTGG

GGGTTCTCACTCATGCTTCT

CCTTCAGCCCTCTCCAGGCC

SynPVI (ProA7)
63

WO 2017/046084
CATCCTGAGAGATGAGCCAG

GACAAAGAACCAGTAATAGC

TCCTGGAGCAGCACATCTGT

TTTGCCAGGATTATCCCTTG

GATCTCTTAAAACCGAGACC

TTGTAATCTGAAGACTCAAC

TTGGGCTGTACCCTTAACCT

TCAGCTCTATGATGCAAGTG

AGTCCACAGGACCGGAGGCT

TTGAGATGAGCTTTTCAGAA

GGGAGGAGTTGGCCGCTTGC

TCCCAGAGCTCCAGCACCTG

CATTCTTCTGGCTATGTCAG

AAGCCAGATCATTTCCCTCG

TTAAAAACAAAAACAAAAAA

ACAAACAAACAAAATGTTAG

TCTTTGCCCTTTATCTGCCT

GGCAAAGCTTTTAATTGGCT

TGATCTGTCATTCCGCTAGA

CATAAAGGGGACAATCCCCG

GATTAGGAAGGAGCTCTCCA

GCTCGGGTAAGGAGTCTCAA

GGCAAGGTAGGCAAGCACCA

CCGGTCCGCACTCTCGCCCA

GCTTTTACGGGAAGAAGAGA

SynP136 (ProA1)
64

WO 2017/046084
AGAGGCAGGCCGAGTTTGAG

GCCAGCCTGGTCTACACAGG

CGTTCTAGGAGAACCTGTCT

CATATGCACATGGGCCTGGG

ATTACACATACAACTGCAAT

GGCAGCACTTGGAAAGCGGA

AGCAGGAGAATCGGAATTTC

ACACTCATCCTTCATTATAG

AAGTCCAAACCTGTGCTAAG

CTACTTGGACATCGCTAAAA

AACAGCAAAAATCTTTCTAG

GAATTGCCAATGTATACACA

CCAAGTTGTATTTTTGTAGG

GCAGACTTTTCCAACTTGCC

AGATGATAATAATCATTTAT

ATTAGCTACATTTCAGGCTC

CAGAATTAACACTGTTACTG

AAATGTATAGGTTCTAAAAC

ATACCATTTCCAAATATTTT

AAAGGATTAACATTTTTGAA

AAGCATGTGATCTTCCAACC

ATATTTTAGGGAACAGACAT

AAGAAGTAGGCAAGTTTGGG

AAGAAACAGTTCCTGAGGGC

ATTTCTTCCCAACCTTGAAT

GCCCTGTGGGTTAACTGAGG

TCTTGGTAAAATGTAGATTA

TTTACTGGACTTTTTGAACT

GAGAGTGCATTTCTAACAAA

ACTCCAGGGTATGTGGTCCT

TGCATTACATATGGGGTAGC

AACATTCTAAAGCAGTGTTT

TTCAACCTGTGGATCAAGAC

CCACAGAAGTCACATAGCAG

ATATCCTGCCTATTAGATAT

TTACATTAAGATTCAGAGCA

GTAGCAGAATAGGAGTCATC

CCAACCTGAGGAACTGCATC

AGAGTCCCAGCATCAGGAAG

GGTGAGAGCCACTGAGCTAA

AGTCCTCTAGGTGAGGGGGC

TGCCCCGGAAAGACTCATTT

AAATGAAACCACTGACACAG

AGAGCTGACAGATGAGGTGG

GTTCCGTGTCTGTGAGGCTC

TGCTGGTCGTCTCCTCACTC

CCATGGAAGAACCACCGAGA

TGAGGGCGAGGGGCAGAGCT

AACCCAGCCTCTAAGTAGGC

AGAGTCTAGTGTCCAGCTGC

CCAAGGAAGAAGTTTGCTGT

GTGAGGTGGCCCTGATGTCC

GCACACACAAAATGCCAGTG

AAGTCTACTTGACCAAGTGA

AGCTGGTGTGGAATGGGAAG

AAGCACACACAGTCAGTCTC

TCTGCACACACTCTGTCCTT

CACTTCTTCACTTACCAGAG

ATTTGATGAGAACCTACTAG

CAGATCAGATTTGATCCCTG

AGTGGAAAAAACGTACAGTG

GGAGATAAAAGAGGAAAACA

ACGGATCTGAGTTTGAGGTT

AGCCTAGTCTGAGCAAGCCG

GATACACAGTAAGACCCTGT

CTCACATACATCCACTCGCA

CGCGCGCGCACACAAACACA

CACACACACACACACACACA

CACACACACACACACACATC

GTGCTAAGGACAAGATAGGC

ATCCTGAGAGATGAGCCAGG

ACAAAGAACCAGTAATAGCT

CCTGGAGCAGCACATCTGTT

TTGCCAGGATTATCCCTTGG

ATCTCTTAAAACCGAGACCT

TGTAATCTGAAGACTCAACT

TGGGCTGTACCCTTAACCTT

CAGCTCTATGATGCAAGTGA

GTCCACAGGACCGGAGGCTT

TGAGATGAGCTTTTCAGAAG

GGAGGAGTTGGCCGCTTGCT

CCCAGAGCTCCAGCACCTGC

ATTCTTCTGGCTATGTCAGA

AGCCAGATCATTTCCCTCGT

TAAAAACAAAAACAAAAAAA

CAAACAAACAAAATGTTAGT

CTTTGCCCTTTATCTGCCTG

GCAAAGCTTTTAATTGGCTT

GATCTGTCATTCCGCTAGAC

ATAAAGGGGACAATCCCCGG

ATTAGGAAGGAGCTCTCCAG

CTCGGGTAAGGAGTCTCAAG

GCAAGGTAGGCAAGCACCAC

CGGTCCGCACTCTCGCCCAG

CTTTTACGGGAAGAAGAGAA

TGTTACTCTATCCTAACATA

TTTTTCCTTTTCCTCTATCT

CACAGATAGGAAAAATTTAA

GAGCCAGAGGGAACGTCCCT

TCTCAGAGGAGACAGCAGAA

SynP155 (ProA4)
65

WO 2017/046084
CTCTCCTCCCATTGATGACT

GACTAGGCCATCCTCTGCTA

CATAGGTGGCTGGAGCCTGA

GTCCCTCCTTGTGTACTCTT

TGGTTGGTGGTTTACTCTGG

GGGTACTGGTTAGTTCGTAT

TGTTGTTCCTCCTAGGGGAC

TGCAAACCCCTTCAGCTCCT

TGGGTCCTTTCTCTAGTTCC

TTCTTTGGGGACCCTGTGCT

CAGTTCAATGGATGGCCAAT

TTCCTTCTTAAATGCCCCTA

GCAGTAACTGTTAGGTCTCA

ATCCCAAGACAAATGTCTGA

GGTGCCTATTTAACAGATCA

AAGCGGACCTGGCCTCAGGT

TATCCCAGTCCCTCCCTGTA

CCTCAGTCCCTACCCATCAC

CAACTCTCCAGCCCAGAGCT

TGGGCTGCACTTCCCCCACG

GTTCTTCCCATTTTGGCTAC

ATGGTCTTTTTTTTTACCTT

TTTGGTTCCTTTGGCCTTTT

GGCTTTTGGCTTCCAGGGCT

TCTGGATCCCCCCCAACCCC

TCCCATACACATACACATGT

GCACTCGTGCACTCAACCCA

GCACAGGATAATGTTCATTC

TTGACCTTTCCACATACATC

TGGCTATGTTCTCTCTCTTA

TCTACAATAAATCTCCTCCA

CTATACTTAGGAGCAGTTAT

GTTCTTCTTCTTTCTTTCTT

TTTTTTTTTTTTCATTCAGT

AACATCATCAGAATCCCCTA

GCTCTGGCCTACCTCCTCAG

TAACAATCAGCTGATCCCTG

GCCACTAATCTGTACTCACT

AATCTGTTTTCCAAACTCTT

GGCCCCTGAGCTAATTATAG

CAGTGCTTCATGCCACCCAC

CCCAACCCTATTCTTGTTCT

CTGACTCCCACTAATCTACA

CATTCAGAGGATTGTGGATA

TAAGAGGCTGGGAGGCCAGC

TTAGCAACCAGAGCTGGAGG

CTGATGCGAGCTTCATCTCT

TCCCTCAGGTAATATTCTAA

ATCTCTCTGCTTCTAGCCCA

TACTAAGTCCACCTCCTTTG

CCTTTAGCATCTTCTTTAGG

AGGAGAGGGGCATCTTTTCT

GATGCAAGCAATGGATTTTT

CAGGCTGATGTAAGAGACTT

CTAGAACTCAGCACCCCCCC

CCAACTCATCCCTCTGACCA

AGCCTACTATGTTTTGAAGG

AAAGCACCAGAAAGACATTT

CCCTCTCTATGCTTCCCTAG

CACCTTAAGCGTGGGGACAG

GATGGAAATGTTTGGGGAAA

GTGACAAGAGAGATGATGAG

TAAGGGCAAAGAGGGCTTTC

GGCCTCCACAGAGGCTTCTA

CTGCCACCCCCCAAGAAAGT

ATGAGCGCAACCCTTTCTGT

TCTACAGCTTTCCCTCTTCT

TTCCCCCACCTCCCCCCTGT

TCTTCCTTCTGAGCTGGAGG

TCAAAAGCATCCCAATTCAG

GGTCAAGTCCAGTACAGGGA

CAAAAAGAAATTTCATCCAT

TCATTTATTTATTCATATAG

TTAACTGACTGTGTGCCTGT

TGAAGTTCAGTATCAATGCA

GCCCAAGGAACGTATTTAAG

AGATGGAGTATGAGCAAGTA

GGATAAATAGAAGGGGTTAA

AAAATAAGATGAGGAGCAAT

CAAGAAAGACACTGGACATT

CCTCTAGCTTTCACACACAT

GCACCTGTGCATGCATGGAT

ACATGCACAGGCATTCGCTC

ATGCACATACACATGAGAGA

GAGAGAGAATGAGAGAGAGA

GAGAGAGAGAGAGAGAGAGA

GAGAGAGAGAGAGAGAGAGA

GAGAGAGAGAGAAAGAGAGA

AAGAGAGAAAGAGAGAAAGA

GAATGTAGTGCAATGGTTGG

GTAAGTGTGTAACTGAAGAG

TTGCCCTAAGAAGAGGAAAA

AAAAAAAGGAAGCATTGAGT

GTTGGTGTGGTATCTCAGGC

AGTTAATTTATATTACACTA

GGTGTATCATTATGGAATAT

TATAGTGCACTAAAAAAAAA

AGGATTAAATAACTGAGTGT

TCCTCTTCCCCTCATCTCAG

GAAAGATTCAACTGGCCAGC

SynP123 (ProC1)
66

WO 2017/046084
AGATCTATTAGTAAAATTAA

CTACACCTGGTCCTTTATGT

CATTAACTACACGTCAGTCG

TTCTATTATTAACTACACAG

AACTTACTATATAATTAACT

ACACCGCGCATTTGGATATA

TTAACTACACAACTTTGCTT

AATAAATTAACTACACCCGT

AACGGATTCTCATTAACTAC

ACGTTTCATTACGAGGGATT

AACTACACATAGCCCCCGGA

GGCATTAACTACACTTTTTG

AGGTCGCGAATTAACTACAC

TTCGACTCGCAGGACATTAA

CTACACTATACCGATAACGC

AATTAACTACACAAATTTTT

TATGAAGATTAACTACACTG

TTCGCTCCTGCCTATTAACT

ACACGAGGCCGTATTTCCAA

TTAACTACACCCGAGACACA

AGATAATTAACTACACTTAA

CCAGATGGCAGATTAACTAC

ACGTACGGGCAGGCCCGATT

AACTACACCGGCTGTCATTC

CTCATTAACTACACATATCA

CGACTTGTGATTAACTACAC

GCTCGAGATCTGCGATCTGC

ATCTCAATTAGTCAGCAACC

ATAGTCCCGCCCCTAACTCC

GCCCATCCCGCCCCTAACTC

CGCCCAGTTCCGCCCATTCT

CCGCCCCATCGCTGACTAAT

TTTTTTTATTTATGCAGAGG

CCGAGGCCGCCTCGGCCTCT

GAGCTATTCCAGAAGTAGTG

AGGAGGCTTTTTTGGAGGCC

TAGGCTTTTGCAAA

SynP114 (ProC22)
67

WO 2017/046084
GTACTGCGGACATCCGCTAA

TTAGCATAACCCGCAAGGAT

GTAGCTAATTAGCATATCAC

GGGAGACTGGGGCTAATTAG

CATAAAAAGTCTCCCGCCTG

CTAATTAGCATACGAGACTC

TAGAATCGCTAATTAGCATA

TCAGTCAAACCACTTGCTAA

TTAGCATAAGCATCGCGCTA

TTTGCTAATTAGCATAAATG

TTACATACATCGCTAATTAG

CATATGATAGCACTGTCAAG

CTAATTAGCATATCAGCCGC

ACGCATGGCTAATTAGCATA

CGAATACCACAAGCTGCTAA

TTAGCATACGGTTAAAATAA

TACGCTAATTAGCATATCAA

AGACGACAGAAGCTAATTAG

CATAGATCACCATCACCACG

CTAATTAGCATACGTTAATG

CTGTTCTGCTAATTAGCATA

GAAATTGTTGATCTGGCTAA

TTAGCATACCCGCCTTCCTG

AACGCTAATTAGCATACGGT

ACGTCGAGATTGCTAATTAG

CATATGTACTGAACCCATAG

CTAATTAGCATAATAAATTA

CTAATCCGCTAATTAGCATA

GCTCGAGATCTGCGATCTGC

ATCTCAATTAGTCAGCAACC

ATAGTCCCGCCCCTAACTCC

GCCCATCCCGCCCCTAACTC

CGCCCAGTTCCGCCCATTCT

CCGCCCCATCGCTGACTAAT

TTTTTTTATTTATGCAGAGG

CCGAGGCCGCCTCGGCCTCT

GAGCTATTCCAGAAGTAGTG

AGGAGGCTTTTTTGGAGGCC

TAGGCTTTTGCAAA

SynP132 (ProB2)
68

WO 2016/174624
GACCATTTAAAAGGGGTATA

TAAAGATTGCATACAAAAGC

TGAGGGGCCTCACCCTGAAT

GGATTCTTTCTTGAAAGCCA

CTTTTGTTCTTTAAGAGGCG

CGGTCCAAATAATGTGCAAG

CATGATTGGCTGGAGGCACA

TTTCGTAGATTAATCCTCTC

CCTTGAAAGGATCCAAGTCT

GGAAAATAGCCAAAAACGTG

GCTGTTATGCAACCCTTACC

CAAGCTCCTCCTCCCAGCTG

CCATCCCTCTTACTAAGCAG

TGTCATGAGGCTGGGCCAGG

CTGGAGATTAATTCTTGACC

ATTTAAAAGGGGTATATAAA

GATTGCATACAAAAGCTGAG

GGGCCTCACCCTGAATGGAT

TCTTTCTTGAAAGCCACTTT

TGTTCTTTAAGAGGCGCGGT

CCAAATAATGTGCAAGCATG

ATTGGCTGGAGGCACATTTC

GTAGATTAATCCTCTCCCTT

GAAAGGATCCAAGTCTGGAA

AATAGCCAAAAACGTGGCTG

TTATGCAACCCTTACCCAAG

CTCCTCCTCCCAGCTGCCAT

CCCTCTTACTAAGCAGTGTC

ATGAGGCTGGGCCAGGCTGG

AGATTAATTCTT

SynP156
69

WO 2017/064642
GTAGGCCTAGAGCGGTGCTG

ACGTCAGCAATTCCGATCTA

GCTGTGCTGACGTCAGCAGT

TTAATTGAGCTGCTGCTGAC

GTCAGCACATACCATAGCAT

CGTGCTGACGTCAGCAGTGA

GGCTACTATCCTGCTGACGT

CAGCATATATGTCGCTCTAC

TGCTGACGTCAGCAGCACCA

ACTGTAAATTGCTGACGTCA

GCAATAAGAGACGGGCTTTG

CTGACGTCAGCACGAGTACA

TAGCAATTGCTGACGTCAGC

ACTAGTGCGGCCATCGTGCT

GACGTCAGCAAGCTTTCAAA

GAGTCTGCTGACGTCAGCAT

CTTCCTGACGCAACTGCTGA

CGTCAGCATTGAGGGCTATG

GCTTGCTGACGTCAGCAGCA

CACGCTATGGGGTGCTGACG

TCAGCAGCTAAGGAGACATG

TTGCTGACGTCAGCATTCGT

GTCAGACATATGCTGACGTC

AGCAGGGGCTAGCCACTAAT

GCTGACGTCAGCACAGTTGT

CGTTGATATGCTGACGTCAG

CAATAGACACTTTTATGTGC

TGACGTCAGCAGTTAGGGTC

AGAGGCTGCTGACGTCAGCA

GCTCGAGATCTGCGATCTGC

ATCTCAATTAGTCAGCAACC

ATAGTCCCGCCCCTAACTCC

GCCCATCCCGCCCCTAACTC

CGCCCAGTTCCGCCCATTCT

CCGCCCCATCGCTGACTAAT

TTTTTTTATTTATGCAGAGG

CCGAGGCCGCCTCGGCCTCT

GAGCTATTCCAGAAGTAGTG

AGGAGGCTTTTTTGGAGGCC

TAGGCTTTTGCAAA

SynP17 (ProB1)
70

PCT/IB2019/0590
TCACCAAGTAGGAGTCCTTC

AGTAGATGAAAGGGGTATTT

TAAA88CCGTTGAAGCTATC

TTGGTGGCAATCTAGGATGT

TGAGACCTCAGAGAAAATGT

CGAAACCTTGGAAAATTTAT

TTTGACGATTAGAAATTGTT

TAATAATAAAACAAAAGACT

TCCTTTCTCCAGCCCCACTT

TCCCTGTTTCTTTTGTCATA

GGTTGTTGTGAACTCGATCT

GGGAGGGAATCCTGGACTCG

CAACCCGACTCCGCTCGGCG

TTGATTGGCTCCTGCCTCAG

GACCCCGCCGGACCCGCCCC

CCGGCCGTGGGAATCGCCGC

CTAGCAGGCGGGCTGCGGCT

GCCACTCAGTCGGAGTGGCG

GAGGCCGTAGCCCCGCCTCC

TCCCCCCTAATTGATA

SynP27 (ProB12)
71

PCT/IB2019/0590
ACCCTCCTCAGGGGAAGAAA

CAGTACATTCTCTGAAGCCT

CCTG89GTTAAATGGGGGAA

GTATTTGTATTCCTATCACC

AAGTAGGAGTCCTTCAGTAG

ATGAAAGGGGTATTTTAAAC

CGTTGAAGCTATCTTGGTGG

CAATCTAGGATGTTGAGACC

TCAGAGAAAATGTCGAAACC

TTGGAAAATTTATTTTGACG

ATTAGAAATTGTTTAATATC

TCCTTCCAAGATTAATCTAT

GATTAAAGGGCACTTAAGAG

ACTTTTATAATTTCTAAAAT

ATTCAATAGCATAGATGATT

TGGCCATTACAAACTGCCTA

AGATTTCTCCTCAGGCAGAA

GCCAGGCCACCCACGGAGAG

ACCCAGGAACTGGGCCAAGA

GCCTAAACAAAAGACTTCCT

TTCTCCAGCCCCACTTTCCC

TGTTTCTTTTGTCATAGGTT

GTTGTGAACTAAAAAAATAG

CTCAGTTTCACAAAAGCGAT

CTGGGAGGGAATCCTGGACT

CGCAACCCGACTCCGCTCGG

CGTTGATTGGCCCCGCCTCC

CAGCAGCTCAATAGTTACAG

GGGAGTGGCAGGTCCCTCCC

GGCCACGTCTTGGACCCGCC

CCCCGGCCGTGGGAATCGCC

GCCTAGCAGGCGGACAGACT

GGAGAGGTGGATTAATGACC

CCAGCAAAAAGCTGGCATGT

CCAAGAGATTTAAAATATTT

TTTTTATGTAACAGCTTTAG

CCAAAGCTGTTCTAGGGTTC

AGAGGAACTGCCACTCAGTC

GGAGTGGCGGAGGCCGTAGC

CCCGCCTCCTCCCCCCTAAT

TGATAGACTGACTTCTTGAC

TAATGGCCAATCTCTAAGCA

GAGGGGAGGACCTTCCTATT

GGCCAGAGAGTCAGCTTTCA

AATAAAAGAGGCGGCTCTTC

CGGCAACAAAGAAAACAAGG

CGAGCCTATAAACAAAGCCA

CGTGGGCTTCAAGTTTCCAC

GGACGCAAGGTTGTGCAACG

CAGTAGTCACTTATTC

SynP57 (ProA14)
72

PCT/IB2019/0590
GCTCAGGCTCTTGGGGACTG

GGCTCCAGCCCTCTGGGATC

ATCA90TTTGCTCTAAGAAC

TGGCCTGGGTGCAGCTCCAG

ACCAAAGGCAGCAATTGTTC

AGAGCCCTGAAAGCGCCAAG

GCGCCAAGGCTTCTTCTACA

TACTCACCTCTGACCCACCA

GCCCCCCACCCCAGCCCAGG

TCTGACGAAAGGTACCTCTC

TCCACTGCAACAACTGGGGT

GTGGCAGGCTCTGGTTTATT

CGCCTTGTTCTCCCTTCCCC

AACCCCCCTTTTCTCATCCC

CCTAGCAACCAAACTAGATC

CATCAAAGAGCAGGACCTGG

CAGCCGAGCTGGGAGAGACT

AATAGCCTGGAAGGAAGGCG

GGGCCTGGAGAGGAACGGAA

GCCTAGGGATGCAAGCCAGC

ACTGGGCGTTGGCTCTGACC

CATCTCGGAGGACACACGGA

AGGTGGGGGAGTTCTCTGCT

CTGCAGTCTGCAGGGAGCCA

TCCTCCTTATCCCAGTCAGG

CATCCAGCCTAGAACCCCAA

GCCTTCTTCTCTTACACCCG

TCTCTTTCTCAGGACCCAAC

TGAGGTAGACTCATCCTGTT

TGAGAGTCCCAGGGTCCCCA

GTGGTAGCAGACACATGGCT

CTCAGCAAACCCAAAGGGCT

TCAGCATCCTTTCTCCTGCA

GAGAATCCAGACGGCCTCTG

TCCACTCCTGGGACTGCCTG

TGCTGCATTCTGGAAGTAGT

GTGTCACACAAAGGTCAGAC

ACCAGCCTTTCTGCTAACTG

GGGTGTGGGGGCGCTGTTAA

GGGGTGTAGCTGTGTATTCC

TGTCATGTCTGTGCACACAT

GCATATTTGTAGCCTCTACA

AAGCTGGCTCAGTGAGTATT

GGGCAAGTTATCTGTGGACC

TGTCGGAGGACTTCTCTCTC

TAACAGGCTGTAGTGGCTGG

GTATCTCTCCCATCTCATCT

CCCTTTATCTGCACCATGTC

TGGGTACCTGCATCTCCTCT

GCACTGGAGACTGGTGCCTA

CTAGTCTATATGTCTTTCAG

CCCTGGCAGCTGCTATCCCC

CACCCCCCTCCCCTTCCTAC

TTCAGGAATTCCTCTGGTTC

CCGTAAGGCCCGTGACTGCC

CAGCAGATGGTGTGGAGGGG

GCACCAATCCAGTAAAGGCT

GAAAGTGTACCACAGGCCCA

CTCAGCCCCAACAAGAGTGG

GCACCTCACAGGCCCTTTCA

TGGCACAGACCCTTGGAACC

CCGACATCCTCAGCACCCTG

TGAGGTGCCCACTCTTGTTG

GGGTGGGTGTTACGTCCGAG

TTTGGGGGCTGTGTCTTTAA

GATGGAAACATCACCATGCA

ACTTCTGCTGGTCCAAGGGC

GGGGGTGGGGGTGGGAGAGC

TGGTCAGTCCATTAGCTGCA

GAGCTGGCGCCAATCACCAG

CCCTTTACCGTGCCCTGGGG

AGTAGGCAGAGATAAGCTCT

TCCCCAGCTCCCTCTGCCTC

AGCCCTCGGTTGTGGCCAAT

GATGGGGGGCAGTTGACAAC

AGGTGAAAGGAGAACCCCAG

TTTCAGGAGACAGGAGGAGG

CACGAATTCCCTGGCTTAGG

CCAGGTTAGCTCTCCCTCCA

CCTACCCCACTTCTCATTGC

TCAAAACTTGCCCTTTTCCT

CAGGTCCTCATATTCCCTAA

TTTTTACCCCCTCTTCTGAG

AGGGCACCCCAGGTCAAGCC

ATGTCCTCCCATTCTAGGCT

CCAGCGTTGGATGCATGCTC

TAAGGTAGACCTTAGCCCAC

CTCCATCACATCCCGGATCT

CAGCCAGCAACAAGGGGGAA

TCAAGCAGGCAGGGTGCCAG

CAACCAGGAGAGGGAAGGGG

TGGTGTCCTCTCTCTGCAGG

GTGGGGCATCCCCCTCCCCA

CACAGCCCAAGGCTGAAGTC

AGGCCAGTGGGAGGAGCTGT

CGTGGCCCCCCACCCCCCCT

CCCCGGAGACCGCAGGGCTA

TAAAGCCGCCCCGCATCGGT

CTGCAGCTCCTTGCCACCCG

GCCTAGTTCTGCCAAGCGCT

GA

SynP78 (ProA27)
73

PCT/IB2019/0590
GGTCAGTAAAGTGAATGAGA

TGAGGTCATGTTCCATGTGG

GATA91AACATGGTTAGTAG

ACTGCACCAGGTGATGGGGG

AAAGGAGAGAAAAAGATGTG

AGAGGGGAGGGGACCAGGAG

AAGGGACCAAGAGCGAAGAG

AATCAAGATAGCCAAGAGAG

CAAATGGCCAAAAATGGCAG

GGTTACATAGGAAAGAGAAG

CTTGCAGAAGAGTAGCCAGG

GCCCCAGGGAGGAGATGTTT

AGTTCAGGGGAGAGGTGAGA

AAAACTGAGAAGAGCTACAG

GTACTGATGGAAGATAGAAG

CCAGAATGAATTTTGGTGTG

CTAATAGGTACCACAGTTAG

CCAGTTGCTTCTTTGGAACC

CAACATAAAGGACTTTTTTT

CCTCCTCCTCCTCCTCCTCC

TCCTCTTCCTCTTCCTCTTC

CTCCTCCTCCTCTTCCTTCT

CTCTCTCTCTCTCTCTCTCT

CTCTCTCTCTCTCATACATA

GGTGTAGCTAGAAACTGGAG

CACTGTGACCTTATTCAAGG

GGCCACAAAGGTGCCTCTAC

ACCCCCATGCACACACCTTA

CAAAAAAGGATCTCTAGCAT

TTTTGCAGTCTTTCAGTGGA

ATTCAAAAAGGAATCATTTG

ATTGGTAATGAGAATGTTTT

TCCTCTCGGCTTTTCACAGC

TCAGGAAGGAGTAATTTATC

AAGTACTCTGCTTGGAGAAG

TAGAGCATAGCCTGGAATGG

AGGAACATGGATCCCTGTGG

GCGCTTGCAATCTTCTGCTT

TCATTTTGTCAGTCATGCAA

GCACAGTTTTAAGTACTCAC

CAGGGGAGAAAAAGCTCTCT

TCCCAGCTTGTGCTTGCCAG

TAAGACACTCTCCTTTCAGA

GTCAAGGCTGCAGCTGGCCT

TGGGAGCTTCTTCCTAGGTC

TCTAGCAAGTGGAGCCAGAG

CTGCTACAGAGGAGGTAAGG

GCCATGGGCAGCATCCCAGG

AAGGTGGTTGGCAAGAGGGT

GGGAGACCTCTCTGCACGTC

CTCCTGGGACCTGCATGCTC

TCCCCATGCCTCACCCTCAC

CCTAGTTGTGACACCAGACC

AAGAAAGGCGATGTGGTTCC

CCAGCCACATCCAAAGCCAA

GCCTTTGGCCAGAGGAAGGA

AGGCTGTCCCAGTCATGGTT

TTGCTTTTGTTTTTCCCCTC

AGCTTGCTTTTTGAACCTCT

ATCTGAGAGGACAGTGCCCT

GCTGCTCCATAGCTTCAATC

CTGGCTGCAGGGATGGAGGT

TGGGAAGGAAGCTGGTGAGA

TCCTAGATCCTCAAAAATGA

TAACTGGATGTAGCCCTGGT

AGCTAAACATCCACAGATAC

ACAGCTGTGAATGGCCTGTA

CTTGGACAAACTGGAGTTCT

GAGTTTAGGATCCAGATGGA

TTAGGAGTGAGTCCCAAGCA

GGACTTGCTTACCCACTTCA

GGGACCAGCTTCCTTCCAAA

TAGACACAGGAGAGTGTCTC

TCTTCCCTGTCACCCATAGC

CTGACTAGGGAGAGTGAGTG

GCAAACCCAAATTTGCATAT

TTCATTCCAGCTGAGACTAA

AGCCTTGACCTCAGGCATGG

AGGACTGTAACCACTGAGAA

CTAATATGATAACATTGTTA

TCTTACGAGAATTCTCCTGT

ATGCAGAGTAGGTCCATATG

GTTTTCTGGATTCATTGCAC

AGATGAATTCGAACCTGACA

TCCCACAGGGAGAGAGATGT

GTGAGCTCCAGACAGAAGGC

TGTGTAGTGTGGGCACTGTG

CCAGGACCACCAGCATAATT

TCTCCTAAGACACACCTGTC

TAGAAAGTTAGAGCCATAAG

ACAACCTTGGCCCTGGGCCA

TGTCACTGCTCATATCTGGC

CCCAGCTTTGTGTGCCTAGA

GGAGTATTCTTGCCTCTTTG

GAAAAATTGGAGGGAGCCGG

ATTAACCTACTGCAGGTGCT

AGGGGTGGGTGGCAAAGCTG

TGATAGGTGTTGGTAGTGTT

GTAGCTGGCAAAGCTGATCT

CCTGTCTGTTGCAGCCTTCA

CC

SynP151
74

(ProC29)PCT/
CTTACCGATTGCAGACGAAA

IB2019/059092
CCGAAACTTAGCTGACATCG

AGTCGAAACCGAAACTTTTC

ATGGCCGAAGGCGAAACCGA

AACTTGCGTCCGTGTAACGC

GAAACCGAAACTTCCCGAGC

ATCCTTACGAAACCGAAACT

GCAACCGGCTACACTCGAAA

CCGAAACTACTAATGATACC

AACCGAAACCGAAACTCGGT

AAGTGAAACTGCGAAACCGA

AACTTGGTGGACAGCCTTCC

GAAACCGAAACTGGACGATG

TTCGTTCCGAAACCGAAACT

ATATTCGTGCCCAAGCGAAA

CCGAAACTAGGGGGCCGATA

TCCCGAAACCGAAACTACAG

GCCCCGCCCGTCGAAACCGA

AACTTACGTCTCAGGAAGTC

GAAACCGAAACTGGCTACCA

CTGACCACGAAACCGAAACT

TCTACTGTTCCGTGACGAAA

CCGAAACTGACGGACAAGAG

TACCGAAACCGAAACTTAGT

CAGGCGTCCATCGAAACCGA

AACTCAAAGATTCAAAAAAC

GAAACCGAAACTTTCTGTTG

GGATGTCCGAAACCGAAACT

GCTCGAGATCTGCGATCTGC

ATCTCAATTAGTCAGCAACC

ATAGTCCCGCCCCTAACTCC

GCCCATCCCGCCCCTAACTC

CGCCCAGTTCCGCCCATTCT

CCGCCCCATCGCTGACTAAT

TTTTTTTATTTATGCAGAGG

CCGAGGCCGCCTCGGCCTCT

GAGCTATTCCAGAAGTAGTG

AGGAGGCTTTTTTGGAGGCC

TAGGCTTTTGCAAA

SynP194
75

(ProB15)PCT/
GCCCCTGCCTGCGCGAGGGC

IB2019/059093
GGGAAGACAGCCCCCGGGCC

CTCCTCCTCCCTCTGCCTTT

TTAAGGGACGCCCTCCAGGG

CGACCCCGGAGGGCGGACTT

GCCAAGCTGAAGAGAATCAG

TCAAAAATCCGCCCACAGGG

GACACATCATTTAAATAAAT

GTGTTTCTTTGCCCGAACAG

AAGTTCAGATAGGCTCGATT

ATCATTAATTCTGGGTTTCA

CGTAACGAGAGGAAACACAG

GTTGCAATAAAAATAAAAAA

ATGGTTTGAAATCAATTTTA

ACTCATTTTGAACGTCCTCA

CACGTTTGACAAACCGATTT

GTTTCAGGAGACTTGCTAAT

ATCTAAATCGGTGACAGGGT

GTTTGCTGTGAGTGTGGCTC

TGGAAAAGTTATTAAGCGTT

ATAAAAAAAATGATGTAATG

AAATTCTAATTAATGGGAGG

GAAGTGCCAACAAATCACTC

CTTAAAATATTAACGCTATC

AAAGAACAGCTGGAGAAGGA

GGAAACTTGACCCTTGGGGG

GGAAAAGAACCCCGAGCACC

CTCTGAATAAGTCAAATAGA

CAAGGGCTCTAAATGAGGAA

ATTAATTATTATTTTCCAAG

TTGCACCATTGGCAGGGCAC

ACTCTCCGTAGGCAAACAAC

AAAAACCTCTCTCACTCACA

CAAAAGCCCACCTTGCAAAT

GAATGGAATATTAATGATTA

GGAATTTGGGGGCGGGCCCT

GGCCTGGCGAGCTGGTGATC

CTTGCAAAACAACAACTCCC

TGGTACCCAGATGCACGCTG

CCAATGCTCCAGGGAAGATA

ACCCAGTGGAAAGAAGGAAG

AGACTGCAGAAATAACTTCT

GGGGCATCGCTAACTTATTC

AGCAGGTCTACTTTATGGGT

TTAATGAGAAACCCAGGCCA

AAGAAGTCCCCAGATGGATA

AACTGATCTTTTATTCTGAA

AGAGTTAAGGCTTTGCCAGC

TCTGCTTAACTGCTTTGCCA

GTTTGGTTGAATCCAAAGTG

TATTAAAGCGGCCGAATTCT

AAATCTTTCAAAGGTGGGAT

TTATAAGGGAAGTGCTAACA

CGACCTCAACCATGCCAGGC

TGCCAGGAATTATACTGGGA

CCAAGAGCATGCTTTTCCAC

ATTAGAGCCAGAGATTGTGC

AACTGTCAAACAATGCGGGG

CGCTGGAGGAGGCGGTCAGG

CTGAGGACCAGTGATCTCCC

AGCCCCCTCTGTTTGGAACA

GGGAATTTTGGACATGCAAA

AACACACCTTGTTTTAAACG

CAGAGGGCACTCTAAGATCA

AGGCTGAAATTCCCCCTTCC

CTCCTTAAGAAGATAAATCA

CCTGACTTCCTCAAAAGCTC

TGAAACACACCCTTTGGCCT

CACCTTGGCAGCCAAGCCAG

TCCAAGTTGGAGCCGGAGCT

GGTAGGGTTATCTGTGTCAG

GTTCAGATCTATTCAGGGGA

ATAGTTCTAGACCCTGACGC

AGGCAGGCTGAGGCCCTCTG

GGAACCCCACTCCAGCTACT

GGCGACTGTGCCTTTAAATT

GCTGCTTTCAGAGGGTGCAT

CTAGGGGGATGTGGGAGGGA

GAGAAACATCTTTCTACTGG

TCTCTTTCCTCCCTCCCCTC

CAGGACTTCCAGGGTTTGGA

GAGTGATTGCTACCCAAAGA

GAATCAGCAGCCCAAGCTCC

TCCCGAGTTGGAGGCTGGAC

CTAACACCCTTGACTCCAGG

CTAAAA

SynP5 (ProA9)
76

PCT/IB2020/
GCTGATGTGTCTTACTGATG

050538
ATGCATCTTTCAGGTGTGTG

CTGGTGGCCGGAGGATGCTG

TGAGTAGCCTAAGGTTGATA

CTCAGATGGTACAGCCACCG

CAGGCTGCTGCTACCATAGA

CTTCCAACAGCACTGGACTT

CCCCTTTTGTGTCTTTGTTT

TTTCTGGGTGCAGCTTGGTA

GCTCCTGTCTTCCACAGACC

TTTGTCTAGAGCCTATGTAC

AGCCTCAGAGGGTCCTTCAC

TGAAGCCGAGCTTGCAGCCC

CCCCTCCCATGCCTCAGAGA

CCCTTCAGGGCTAAACCTGT

GCCTTGTTCCCATCTGCCTC

TAAGGCCTATAGAAGCATAC

AGCACACCCTCTGTCCACAG

TGCATCCATGTTTAGAAAGC

CAGAAGGAACAGTTAGAAAC

TTAGAAGCCATCTATTTTTG

TCCCCTTAATTTCAGGGGAT

GGAAAGTGGGGCCTTGAGGT

GAAGTGAGTTGCCTAAGGTC

TCCCAACTAGATAGGTTTCT

CTCAGCTTGCTGGCCAGCAC

TTGTACTGCGAGGGTGATAG

CTGTAGAGTTTAGCTTTGCC

TGAAGCATATTCAGGCAGAG

GCCAGTCAGCCAACCTGCCC

TTGGGGAAGGGCTTTTGGGG

GTCAGGGAGGGACATGTTTC

TGGAAACTTCCACAGGAGCC

TGACCAGTTCAGACCCTACT

CAACTCAGGAAATCCCTTCC

CAACTCCTGGTTCCCAAGAC

TCTACCTTTAGCTAGCAGCT

GCTCTGACCCTTCCGACCTT

CTAATGTTCCCAGAGCTGCT

CTGACTGCATTTCATTTGCC

TGTCGGAGGCATGGCCTGCC

TTGCTGGTTATGCTCTGCTG

GCCTTTCCAGAGGGCCTCCC

GAATCTGCTTTGCCCCAGGC

AAGGCTACCCCTTCCCTCTT

AGAGCCATTCTGCCTCTTCA

TTCCACCCAACCAGCAGATG

ATCTCTGGATAATTTATGCA

GCAAATCCTCTTTAATCTTT

AATCTGGGGTCTTGAACAAA

AGGAACCTGCCGCTAACCTG

TGTCTTTTTCACAGCTTGAG

TGGAGGTGGGGAATGTCTGA

CATTTCCCTCATAGGAGGTG

TAGTGTGGGTATGAGCAATG

AGTAACTTGGATGTTCAGTC

AGCTCACTCCCCTAAATCCT

TGTTGAATCTCCGGGAGGTA

ACTCCAACTCTTGCCACAAC

TCACCCTCCCAGCAGTCTCT

CTTCCAGGGATGCCAGGTGG

GCAGGTTTATTTTTCTGTGA

GAACTGACTCGAGTTCCTAC

GGTGCAACACTGCCTGGAGA

GACTATTTTAGAAAAAAAAA

AAAAAAAAGCAGTTGCCCGG

TGCAGCCCTGGGCTTCAGGA

GAAGCTCACGCAGGCACCGG

TTGTGGCTGCAGAGGTACAG

GCCCCGCCCCAGGCCCGACT

AGCCCCGCCCCACCCTGCCA

CCAGCGGACCGCACTCTTGC

AAGGCTAGTGGGGGCCCCCG

CGGTGCAAAGAGGTCGGTGC

GCAGCTCGCATCACGGGTGA

CTGGGGCGCTGTCCAGGAGG

AGCTGGCCCGGCTCGGGCTG

CAACCATGGTAAGGAGAGAG

GACAGCCAGAAACGCAGGGC

CTGCCACTCCGGGGCTGCTC

CGAGGGGACACTCGGCGCTG

CAGCTCTGGCTGGGCAGGGC

GGGAGGTACTGTCCCCGGCT

CCGGGTTCCGAAATGCACTG

CTGGCGCTAGGCGCGCCGCG

TTGCCGTTCTCAAGTTATTC

TTTCTGAGCAGAGCTGCTTT

GAGCGGGCCGAGACCCCCAG

GGTGCTGCCCAAGGTCTTGG

AGGAAGGACTAGGTCAGTTT

TGTGGGAGGTGCCTGCTGGT

AGCCTCTCGTGATGGAGATA

AGGATGGTAGGGGGTCCCCG

CCCCGTGCCCGAATAAGCTG

TGTTACCTGGATGGCCCTTC

CTCACCTACCTGGGGTACAG

AGACCAGCACCAACTTCCTT

TGGAAAACACAGAGGAAATC

CGTAAACACAGCCAGCCTCA

CTAGAAGGGATGATGACAGA

SynP35 (ProC8)
77

PCT/IB2020/
CGAATCCGCGATAGCACCGC

050539
CTGAGGGGATTGAGGACTCC

GTCAGACCGCCTGAGGGGAT

GGAAGAAAGATAATCACCGC

CTGAGGGGATGGCTCGGTTT

TGCACACCGCCTGAGGGGAT

ACGAAATTCTATCGCACCGC

CTGAGGGGATAAGCTCGTTC

CTATGACCGCCTGAGGGGAT

AATCTCCTCAATACGACCGC

CTGAGGGGATTATAGAATGC

ATTCAACCGCCTGAGGGGAT

GAACTACCGGTTTTCACCGC

CTGAGGGGATTGAGCTCCCA

CGGCTACCGCCTGAGGGGAT

AAGGCCAGAACTGTCACCGC

CTGAGGGGATTCTGACTAAT

TCGGGACCGCCTGAGGGGAT

AGTTTCGGTCTGGATACCGC

CTGAGGGGATAACCTCTCCG

CGGTGACCGCCTGAGGGGAT

GCGCCATGGCGGTTAACCGC

CTGAGGGGATATTCCGCCTT

CTACAACCGCCTGAGGGGAT

AAAGAGCGAGCCGAGACCGC

CTGAGGGGATCATCCACGGG

TCTCTACCGCCTGAGGGGAT

TCCCCTACGAGTTGGACCGC

CTGAGGGGATTCTAACTCAG

TGGCAACCGCCTGAGGGGAT

GCTCGAGATCTGCGATCTGC

ATCTCAATTAGTCAGCAACC

ATAGTCCCGCCCCTAACTCC

GCCCATCCCGCCCCTAACTC

CGCCCAGTTCCGCCCATTCT

CCGCCCCATCGCTGACTAAT

TTTTTTTATTTATGCAGAGG

CCGAGGCCGCCTCGGCCTCT

GAGCTATTCCAGAAGTAGTG

AGGAGGCTTTTTTGGAGGCC

TAGGCTTTTGCAAA

SynP66 (ProA21)
78

PCT/IB2020/
AAGGAATCCCAAGTTTTAAA

050540
AATTCGTAGAGAAGCCAAGA

GGTGGCGCCAGGCGAGACGC

ACTTCCTGGGAGGCTGAAAG

TCGGTCGTAGATGCTGCAGG

GGAGCTCTGTCCTGATAGGG

GTTATTCTATCCAAACCTCC

TCTTTCCTACCTTGTGAAAT

ATGTGTCAATTCTCCTTTCA

CCCCCACCCCACCCCCCTCT

TAACTTTCCGGACACCAAAG

AAATCTTAACTTGTATTCAC

ATCTTTTATTCTTCCTAGAT

GGGACTGGATGAGGCTTTGG

AAGAACTTTAAGACGGAAAC

TTTTTTTTAGGGGATGAAGT

ATTCGCAAAGCGATAAAACG

TTGTGTGTGTGTGTGTGTGT

GTGTGTGTGTGTGTGTGTGT

GTATTTTAAATATATATATA

TATTTTTAAAAAGTCCTTAC

TCTGAAGCTCAGTCGTGGGA

CAGAAAAGCACTCTGAGCTC

CTGTCTCCGCAGGAGTGATG

ACCTGGCACATGCATTATTC

TGTTCATTTGGCTTTTATCC

CGTGCCCTTGTTTGGCTTCT

GCTCCTTTGTTTTGGCTATT

TATTGCTACTTGATAGGAGT

AAGGAGGGCAGAAAGACCTC

ACAGAGGTTATGGAGGAGAG

ATTTCACCATCTAGCTTGAA

ATTATCTGTGGTCTTCACAC

TCACTAATGAGGGACTTAGG

ATGTACATTCCGATCGGGAT

TTCTAGTCCCGTGGGGGAGC

GCACACCAGCCACATTCCTG

AGACGCCACGCAGCCTCCAT

GCTCCACTAGCAAACTCAGT

TCCACCACAGCAAGTTTCAC

TTGATCCCACAGGACCTGCC

TGGACCCTTTGTTGGGTGGG

CGGGAAGCCCCTGGACCCCT

CACCCACCTGGTCACATGGC

CTTTCTATGGCCCCTCCCCT

CTCTTTCTTTGGAGTCCGCC

TTTTTCTCATGGTGACAGTA

ATTGTTTCTACAGTCACCCA

TCACTCCTCCTGCCCGGCCT

CTCTGCTAGGGGTCAGTGTC

CTCTTCAGTGACAAAAGGCG

CCCTAGATCTGGGCTCCCTG

GGGAGGGAGGTGGCTGGATC

TGGTAGTTAGAATGCTGTCT

CTCCCTGGTGCTGCGACAAC

CCCTCCCCCGCTGTGCACAC

AAATGGGCTTTATCCGGCAA

ACAAAACCGCACCCCCACCC

CAGGCTACACAGGCACGTGG

ACCTAAGCCTCACCAAGGGA

AGGGGGTGGTGACAGGCGCC

AGAGAAAGACGAGGCAGAAG

ACAAATATTCTCCTCGCCCC

ACTTCTTGCTCTCCACAATC

CAGATTTTAAAGTTTGACTT

TCCACTCCCCACCTGCTCCC

TGAGCCTCCAACTTCTGTTC

TCCTATCTCCCGAATCTGAA

TTCTTTCTGGTTCCAACCAG

CTCAACTTCTCTTGGTTTCA

GAAACAGGTTCCCCACCAGC

AGTCCCATACCACCCAGGGA

TGTGCTCCTCAGGCAGGGGA

ATGAGAACACGGTGTCCTGG

GAGGGAGAGGTGCGAAACCC

CCCTTCCCTGACCGAGAGTT

CCAGGGGACACCCGTACGAG

CGGCAGTGTAAATAGCAAAG

GAAGAGCTCACTCCAGGGTC

CTGAGCCAAGGCCAGGGCGC

TGGGCTCTGCTGGGCCATCC

TGCATGCCTAATCCGCTATT

TAAGGAGCTGCTCGGCTGCA

GCCCAGGCACCACCCCCCTC

CTCACGTACACCGGTTTCGA

GCCACCTTAAAAGCGGGAGG

CAACTGTGAAGTTGCTGGCC

AGTAAGTGGTGTGGAGATTA

CGAAGGGACATACGGAGAGG

GCAGAGAGAGGAAGAGAGAG

AAAGTGAGAGAGGAAGAAAT

TTATCCTGGGATCCAGAAGT

TCGTGTTAACCTGTGGAAAA

TCAAGTCCCTGGAAGTCTGC

AGAGACAGAGACAAGAAGAA

AAGAGATAGAACGCCAGAAA

CTCCTCTCTCTCCCTCCCTC

TCCACCTCTCTCTTCTAAAC

CCCAAATTCCTGGTCCCTTG

TACCCCATTGTGGGGATAAT

SynP166
79

(ProA36)PCT/
GGGAATACGTATGTAGTTCC

IB2020/050541
TGGGGATGCCTTGAAAAAGA

GAAATCCCAGTGTGTAAGGT

CAGTATTGTATGGAGTACCA

GGTAATAGGGTGGCATCAGA

AGGAAGGGGTGGGGTTTGCT

TTCTAAAACTTACTATACAC

CCCAACAGTCCTCTAGTCTC

TAAGGAAATGGCAGGGGCAG

CTATGAATAACACATAAACA

CAACACCAAAATGTATCTAA

AATTACAACCCCTGAGACTA

CACTCAAATGACACATTCAC

CATTGCCAATGTGACAGGCC

TCTCTGTCTCTTGCCACCTG

CCTGGTCTGCCTGCTCACTC

CTCCTAGGCAGCTTTATTCT

ACCTTTGTTACTTAGGAACA

ATAACACCAGGGCTTAACTT

ATAATAGAGACTCACAACGG

ACCCAGGTCCCCATGACCCA

GTCTTGCCTTGAACACACCA

TAAGTAAACATGTATAGAGC

GCATCCAACACACTGAACTT

TGTAACTCAGGCTTACCCAC

CTCTGTCCGCTCAGAACACT

CACGTTGGACAAAATCCCGG

AGAAATAATCTCCTGTGGTT

GTATAAGGCATGTTGTGGTA

CTGTGGTAAAGTCAAAACAT

TTAAACTGTGGTAGATTCAA

AACATTTAGGTGAGCCTATA

GTGAGTCTGGAGCCGTGTTT

GTGTGTGTGTGTGTGTGTGT

GTGTGTGTGTGTAAATGAAT

GTATATATGTGTATCTGTGT

GTGCATGATGTAAATGTGTG

TATGTAAATGTGTGTATATG

TGTATATGTATGAATTTGTA

TGTACATGTGTATATGTGTG

TATATGTGCATATGTATGTG

TCAGTGTCTGTGTGTGCATG

TATATGTGTGTATGTATATG

TGAGTGTCTGTGTGAATGTA

CATGTGTATCTGTGTGCATG

TAAATGTTTATATGTAAATG

TGTGAATGTGTGTGTGTGTA

TATGTGTATATGTATGAATA

TATGTGTATGTACATGTGTG

TGCATGTGTCTGTATGAATA

TGTGTTTATATGTGCATATG

TATGTGTATGTTTCTGTGTG

TGCATGTATATGTGTGTACA

TCTATGAATATGTGTGTGTG

TGTCTGTGTGTCTGTGTGTC

TGTGTGTGTCTGTGTGTGAA

TGTATATATATGTGTCTCTG

TGTGTATGTAAATGTGTGTA

TGTGTGTATATGTGCATATG

TATGAATGTGTGTATATGTG

TGTATGAATAGGTGTATGTG

TGTGAATGTGTCTGTGTGGG

CATATATATGTATGTATGAA

TGTATGTATATATATATATA

TATATATATATATATATATA

TATATATATATATATTGTGT

GTCTGTGTCTGTGTGTGCAC

GTGTGTGTGTGTGTGCACAT

GTGTGTGCACGTGTGTGTTC

AGCACCTTTTCCAGCCAGTT

TGAGCTATTTTGTACTGTTC

TGGCAGTGGAGATAACAACC

CACAGAACACAATCTTTCCC

TATCTGGCACTTCCATTCTG

GCAGACAGTTTGGGGTTTCC

CAGCAGCGGTGAGGAAGGAG

CCATCAGAGAGTGATGGATA

AAAGGTCTTGCTGAGAAGGT

GAAGTGTCTAGGCCCAAATG

TGACTGGGGGAGGATTAGAA

AAACTCCCATGCAGTGTAGG

TTAAGTGGTAGGGCTGCCTT

TAGACCCTAAGCTCCCAAAC

TCCTGCACTATAAGGTCCCA

TGGGTGCCCCTCAGATGGGG

AAGACAAACAGCAAGCAGTG

TGGCTCTTCGGGGGCTTAAA

TGCCGAGGATGCTTGGTTCA

AGGGTCCCTCCTTGTTCCTT

TCTTCTCAACAAGCACAGGA

AAGGTAAGTGGCTGCTCTTG

GCTCTTTATTTTAATCTCAG

AAGGGTGCCCCTCCTCCCTC

CAAGGACTGGGCAGACCTGG

CCGCAAATCCCCCCTAATCC

ATCAGCACCTGGGTCTCCAC

CCCATAGCAGCACACAGCCT

TGTTAGCCAGGCGACCACAC

TTGTCCCTCCCGCCCCCCGC

GGCGCCTACTCTCCCTCACC

Dalkara
80

WO 2017/093566
CACCCACAAGCCAGTTCCTG

TCCCTGAGGACTTGGCTCAG

GGACTCTGGGAATGTGGTAG

ACATGGGGTGGCCCCACCAA

ATGCATCCTTATGGGAACCT

GCTCCCTGGGAGCCATGAAA

AGAGCGTGGACTTCGAGGTG

GGGCCACAGGAAGTGGTCAG

GTCCATCTCAGGGGACCTGC

TGCCCATCCACACTGCTGGC

CAGGAAATGGGGGGCAATTC

ATGCCTCCTCAGCACCTTCA

GCACTGGGCGGCTCAAAGAA

GGCAAGGGACTATTCTGGGG

TCACACAGCATGCAGCCAGA

GGCCAAGGCATGAGGAAGTC

CTTCATTTCCCCACCCCCAC

CCACCTCAGATCCTCCAACC

GGTTTCATGGCAGCCCAGGG

TCCAGCGGCATCCAGGATGC

TGGTGGGTAGCTGCACAGCC

CAGGCCGCGGGAGGTTGGCT

GCTCTCACCTAACAGGCCTA

TGTGGCCCTGACCCCTACCT

AGGAAGCTGGGGACAATGGC

CAAGGCGCCTCCCCTCTCTG

TGCCTGTCTGTCCAGGTGCA

GCATAGACACAGCACCCCTG

GGGCCAAGAGCACCCAGCCA

GGGCTGCCCCCATGGGTGGG

CAGGGCAGTAAATGAATGAG

GGACAGGTTGGGAGGTGGCC

AGCCCCCTCCAGCCCATGGA

GGGCACGGGGCAGGAGAGCT

GGGCTGAGCCAGCAGGAGCC

CAGGGAGCCTGGTCTCTGCC

TTCCTATCCTGGAGGAAGGT

GAGGCTGAACCTCCTTCCCT

CCCTCCCTCCCTCCCCGCCC

CCACTGCACGCAGGGCTGGC

TGGGCTCCAGCTGGCCTCCG

CATCAATATTTCATCGGCGT

CAATAGGAGGCATCGGGGAC

AGCCGCTGCGGCAGCACTCG

AGCCAGCTCAAGCCCGCAGC

TCGCAGGGAGATCCAGCTCC

GTCCTGCCTGCAGCAGCACA

ACCCTGCACACCC

Each one of the above references is incorporated by reference in its entirety.

In one embodiment, an AAV vector genome comprises a promoter operably linked to a recombinant nucleotide sequence comprising a CYP4V2 coding sequence, wherein the promoter comprises a nucleic acid sequence selected from the group consisting of SEQ ID NOs: 55-80. The promoter can target the expression of CYP4V2 in retinal cells, e.g., non-human primate or human retinal cells.

In some embodiments, the vector genome, e.g., single stranded vector genome, may comprise an intron sequence. For example, the intron may be a human growth hormone (hGH) intron, Simian Virus 40 (SV40) intron, or a human beta gobin intron, e.g., a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:9, 10, or 11, respectively.

In one embodiment, the vector genome, e.g., single stranded vector genome, comprises a recombinant nucleotide sequence comprising a CYP4V2 coding sequence, e.g., human CYP4V2 coding sequence. The CYP4V2 coding sequence can have a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:13, 14, 39, 41, 43, 45, 47, or 49. In one particular embodiment, the vector genome comprises a recombinant nucleotide sequence comprising a CYP4V2 coding sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:14.

In addition, the vector genome, e.g., single stranded vector genome, may include a regulatory element operably linked to the heterologous CYP4V2 gene. The regulatory element may include appropriate transcription initiation, termination, and enhancer sequences, efficient RNA processing signals such as splicing signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency; sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product. A great number of regulatory sequences are known in the art and may be utilized. Regulatory element sequences of the invention include those described in Table 2, for example, Hepatitis B virus regulatory element (HPRE) and Woodchuck hepatitis virus regulatory element (WPRE), which can have a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:16 and 17, respectively.

In one embodiment, the vector genome, e.g., single stranded vector genome, comprises a polyA signal sequence. PolyA signal sequences of the invention include those described in Table 2, for example, Bovine Growth Hormone (bGH) polyA signal sequence and Simian Virus 40 (SV40) polyA signal sequence, which can have a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:18 and 19, respectively. In one particular embodiment, the vector genome comprises a bGH polyA signal sequence, which can have a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:18.

Therefore, in one aspect, the invention is related to a vector genome, e.g., single stranded vector genome, comprising, in the 5′ to 3′ direction: (i) a 5′ ITR (e.g., SEQ ID NO:1), (ii) a promoter (e.g., SEQ ID NO:2, 3, 4, 5, 6, 7, or 8), (iii) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence (e.g., SEQ ID NO:13, 14, 39, 41, 43, 45, 47, or 49), (iv) a polyA signal sequence (e.g., SEQ ID NO:18 or 19), and (v) a 3′ ITR (e.g., SEQ ID NO:22). In certain aspects of the invention, the vector genome, e.g., single stranded vector genome, comprises in the 5′ to 3′ direction: (i) a 5′ ITR (e.g., SEQ ID NO:1), (ii) a promoter (e.g., SEQ ID NO:2, 3, 4, 5, 6, 7, or 8), (iii) an intron (e.g., SEQ ID NO:9, 10, or 11), (iv) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence (e.g., SEQ ID NO:13, 14, 39, 41, 43, 45, 47, or 49), (v) a polyA signal sequence (e.g., SEQ ID NO:18 or 19), and (vi) a 3′ ITR (e.g., SEQ ID NO:22). In some embodiments, the vector genome, e.g., single stranded vector genome, comprises in the 5′ to 3′ direction: (i) a 5′ ITR (e.g., SEQ ID NO:1), (ii) a promoter (e.g., SEQ ID NO:2, 3, 4, 5, 6, 7, or 8), (iii) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence (e.g., SEQ ID NO:13, 14, 39, 41, 43, 45, 47, or 49), (iv) a regulatory element (e.g., SEQ ID NO:16 or 17), (v) a polyA signal sequence (e.g., SEQ ID NO:18 or 19), and (vi) a 3′ ITR (e.g., SEQ ID NO:22). In certain aspects of the invention, the vector genome, e.g., single stranded vector genome, comprises in the 5′ to 3′ direction: (i) a 5′ ITR (e.g., SEQ ID NO:1), (ii) a promoter (e.g., SEQ ID NO:2, 3, 4, 5, 6, 7, or 8), (iii) an intron (e.g., SEQ ID NO:9, 10, or 11), (iv) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence (e.g., SEQ ID NO:13, 14, 39, 41, 43, 45, 47, or 49), (v) a regulatory element (e.g., SEQ ID NO:15 or 17), (vi) a polyA signal sequence (e.g., SEQ ID NO:18 or 19), and (vii) a 3′ ITR (e.g., SEQ ID NO:22).

In some embodiments, the vector genome, e.g., single stranded vector genome, may further comprise a stuffer polynucleotide sequence. The stuffer polynucleotide sequence can be located in the vector sequence at any desired position such that it does not prevent a function or activity of the vector. In one aspect, the stuffer polynucleotide sequence is positioned between the polyA signal sequence and the 3′ ITR. Typically, a stuffer polynucleotide sequence is inert or innocuous and has no function or activity. In various particular aspects, the stuffer polynucleotide sequence is not a bacterial polynucleotide sequence; the stuffer polynucleotide sequence is not a sequence that encodes a protein or peptide; and the stuffer polynucleotide sequence is distinct from an ITR sequence, the promoter, the recombinant nucleotide sequence comprising a CYP4V2 coding sequence, and the polyA signal sequence. In some embodiments, the stuffer sequence can be a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:20 or 21. In various additional aspects, a stuffer polynucleotide sequence is between about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,500, or 2,500-3,000 nucleotides in length.

Therefore, in certain embodiments, the vector genome, e.g., single stranded vector genome, comprises, in the 5′ to 3′ direction: (i) a 5′ ITR (e.g., SEQ ID NO:1), (ii) a promoter (e.g., SEQ ID NO:2, 3, 4, 5, 6, 7, or 8), (iii) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence (e.g., SEQ ID NO:13, 14, 39, 41, 43, 45, 47, or 49), (iv) a polyA signal sequence (e.g., SEQ ID NO:18 or 19), (v) a stuffer sequence (e.g., SEQ ID NO:20 or 21), and (vi) a 3′ ITR (e.g., SEQ ID NO:22). In certain aspects of the invention, the vector genome, e.g., single stranded vector genome, comprises in the 5′ to 3′ direction: (i) a 5′ ITR (e.g., SEQ ID NO:1), (ii) a promoter (e.g., SEQ ID NO:2, 3, 4, 5, 6, 7, or 8), (iii) an intron (e.g., SEQ ID NO:9, 10, or 11), (iv) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence (e.g., SEQ ID NO:13, 14, 39, 41, 43, 45, 47, or 49), (v) a polyA signal sequence (e.g., SEQ ID NO:18 or 19), (vi) a stuffer sequence (e.g., SEQ ID NO:20 or 21), and (vii) a 3′ ITR (e.g., SEQ ID NO:22). In some embodiments, the vector genome, e.g., single stranded vector genome, comprises in the 5′ to 3′ direction: (i) a 5′ ITR (e.g., SEQ ID NO:1), (ii) a promoter (e.g., SEQ ID NO:2, 3, 4, 5, 6, 7, or 8), (iii) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence (e.g., SEQ ID NO:13, 14, 39, 41, 43, 45, 47, or 49), (iv) a regulatory element (e.g., SEQ ID NO:16 or 17), (v) a polyA signal sequence (e.g., SEQ ID NO:18 or 19), (vi) a stuffer sequence (e.g., SEQ ID NO:20 or 21), and (vii) a 3′ ITR (e.g., SEQ ID NO:22). In certain aspects of the invention, the vector genome, e.g., single stranded vector genome, comprises in the 5′ to 3′ direction: (i) a 5′ ITR (e.g., SEQ ID NO:1), (ii) a promoter (e.g., SEQ ID NO:2, 3, 4, 5, 6, 7, or 8), (iii) an intron (e.g., SEQ ID NO:9, 10, or 11), (iv) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence (e.g., SEQ ID NO:13, 14, 39, 41, 43, 45, 47, or 49), (v) a regulatory element (e.g., SEQ ID NO:16 or 17), (vi) a polyA signal sequence (e.g., SEQ ID NO:18 or 19), (vii) a stuffer sequence (e.g., SEQ ID NO:20 or 21), and (viii) a 3′ ITR (e.g., SEQ ID NO:22).

In some embodiments, the vector genome, e.g., single stranded vector genome, may also comprise a Kozak sequence. The Kozak sequence is a sequence that occurs on eukaryotic mRNA and has the consensus (gcc)gccRccAUGG sequence and plays a role in the initiation of the translation process. The Kozak sequence can be positioned immediately upstream of the recombinant nucleotide sequence comprising the CYP4V2 coding sequence. In some embodiments, the Kozak sequence is GCCACC (SEQ ID NO:12). Alternatively, the vector genome, e.g., single stranded vector genome, comprises a Kozak sequence of GCCGCC (SEQ ID NO:51), GACACC (SEQ ID NO:52), or GCCACG (SEQ ID NO:53).

In certain aspects of the invention, the viral vector comprises an AAV8 capsid comprising VP1, VP2, and VP3 amino acid sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs:24, 25, and 26, respectively, encoded by, for example, a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:23 and a vector genome comprising in the 5′ to 3′ direction nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to one of the following sets of nucleotide sequences:

i) SEQ ID NOs:1, 2, 13, 18, and 22;

ii) SEQ ID NOs:1, 3, 13, 18, and 22;

iii) SEQ ID NOs:1, 4, 13, 18, and 22;

iv) SEQ ID NOs:1, 5, 13, 18, and 22;

v) SEQ ID NOs:1, 6, 13, 18, and 22;

vi) SEQ ID NOs:1, 7, 13, 18, and 22;

vii) SEQ ID NOs:1, 8, 13, 18, and 22;

viii) SEQ ID NOs:1, 2, 14, 18, and 22;

ix) SEQ ID NOs:1, 3, 14, 18, and 22;

x) SEQ ID NOs:1, 4, 14, 18, and 22;

xi) SEQ ID NOs:1, 5, 14, 18, and 22;

xii) SEQ ID NOs:1, 6, 14, 18, and 22;

xiii) SEQ ID NOs:1, 7, 14, 18, and 22;

xiv) SEQ ID NOs:1, 8, 14, 18, and 22;

xv) SEQ ID NOs:1, 2, 13, 19, and 22;

xvi) SEQ ID NOs:1, 3, 13, 19, and 22;

xvii) SEQ ID NOs:1, 4, 13, 19, and 22;

xviii) SEQ ID NOs:1, 5, 13, 19, and 22;

xix) SEQ ID NOs:1, 6, 13, 19, and 22;

xx) SEQ ID NOs:1, 7, 13, 19, and 22;

xxi) SEQ ID NOs:1, 8, 13, 19, and 22;

xxii) SEQ ID NOs:1, 2, 14, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 14, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 14, 19, and 22;

xxv) SEQ ID NOs:1, 5, 14, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 14, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 14, 19, and 22;

xxviii) SEQ ID NOs:1, 8, 14, 19, and 22;

xxix) SEQ ID NOs:1, 2, 9, 13, 18, and 22;

xxx) SEQ ID NOs:1, 3, 9, 13, 18, and 22;

xxxi) SEQ ID NOs:1, 4, 9, 13, 18, and 22;

xxxii) SEQ ID NOs:1, 5, 9, 13, 18, and 22;

xxxiii) SEQ ID NOs:1, 6, 9, 13, 18, and 22;

xxxiv) SEQ ID NOs:1, 7, 9, 13, 18, and 22;

xxxv) SEQ ID NOs:1, 8, 9, 13, 18, and 22;

xxxvi) SEQ ID NOs:1, 2, 9, 14, 18, and 22;

xxxvii) SEQ ID NOs:1, 3, 9, 14, 18, and 22;

xxxviii) SEQ ID NOs:1, 4, 9, 14, 18, and 22;

xxxix) SEQ ID NOs:1, 5, 9, 14, 18, and 22;

xl) SEQ ID NOs:1, 6, 9, 14, 18, and 22;

xli) SEQ ID NOs:1, 7, 9, 14, 18, and 22;

xlii) SEQ ID NOs:1, 8, 9, 14, 18, and 22;

xliii) SEQ ID NOs:1, 2, 9, 13, 19, and 22;

xliv) SEQ ID NOs:1, 3, 9, 13, 19, and 22;

xlv) SEQ ID NOs:1, 4, 9, 13, 19, and 22;

xlvi) SEQ ID NOs:1, 5, 9, 13, 19, and 22;

xlvii) SEQ ID NOs:1, 6, 9, 13, 19, and 22;

xlviii) SEQ ID NOs:1, 7, 9, 13, 19, and 22;

xlix) SEQ ID NOs:1, 8, 9, 13, 19, and 22;

l) SEQ ID NOs:1, 2, 9, 14, 19, and 22;

li) SEQ ID NOs:1, 3, 9, 14, 19, and 22; Hi) SEQ ID NOs:1, 4, 9, 14, 19, and 22;

liii) SEQ ID NOs:1, 5, 9, 14, 19, and 22;

liv) SEQ ID NOs:1, 6, 9, 14, 19, and 22;

Iv) SEQ ID NOs:1, 7, 9, 14, 19, and 22;

lvi) SEQ ID NOs:1, 8, 9, 14, 19, and 22;

lvii SEQ ID NOs:1, 2, 13, 16, 18, and 22;

lviii) SEQ ID NOs:1, 3, 13, 16, 18, and 22;

lix) SEQ ID NOs:1, 4, 13, 16, 18, and 22;

lx) SEQ ID NOs:1, 5, 13, 16, 18, and 22;

lxi) SEQ ID NOs:1, 6, 13, 16, 18, and 22;

lxii) SEQ ID NOs:1, 7, 13, 16, 18, and 22;

lxiii) SEQ ID NOs:1, 8, 13, 16, 18, and 22;

lxiv) SEQ ID NOs:1, 2, 14, 16, 18, and 22;

lxv) SEQ ID NOs:1, 3, 14, 16, 18, and 22;

lxvi) SEQ ID NOs:1, 4, 14, 16, 18, and 22;

lxvii) SEQ ID NOs:1, 5, 14, 16, 18, and 22;

lxviii) SEQ ID NOs:1, 6, 14, 16, 18, and 22;

lxix) SEQ ID NOs:1, 7, 14, 16, 18, and 22;

lxx) SEQ ID NOs:1, 8, 14, 16, 18, and 22;

lxxi) SEQ ID NOs:1, 2, 13, 16, 19, and 22;

lxxii) SEQ ID NOs:1, 3, 13, 16, 19, and 22;

lxxiii) SEQ ID NOs:1, 4, 13, 16, 19, and 22;

lxxiv) SEQ ID NOs:1, 5, 13, 16, 19, and 22;

lxxv) SEQ ID NOs:1, 6, 13, 16, 19, and 22;

lxxvi) SEQ ID NOs:1, 7, 13, 16, 19, and 22;

lxxvii) SEQ ID NOs:1, 8, 13, 16, 19, and 22;

lxxviii) SEQ ID NOs:1, 2, 14, 16, 19, and 22;

lxxix) SEQ ID NOs:1, 3, 14, 16, 19, and 22;

lxxx) SEQ ID NOs:1, 4, 14, 16, 19, and 22;

lxxxi) SEQ ID NOs:1, 5, 14, 16, 19, and 22;

lxxxii) SEQ ID NOs:1, 6, 14, 16, 19, and 22;

lxxxiii) SEQ ID NOs:1, 7, 14, 16, 19, and 22;

lxxxiv) SEQ ID NOs:1, 8, 14, 16, 19, and 22;

lxxxv) SEQ ID NOs:1, 2, 9, 13, 16, 18, and 22;

lxxxvi) SEQ ID NOs:1, 3, 9, 13, 16, 18, and 22;

lxxxvii) SEQ ID NOs:1, 4, 9, 13, 16, 18, and 22;

lxxxviii) SEQ ID NOs:1, 5, 9, 13, 16, 18, and 22;

lxxxix) SEQ ID NOs:1, 6, 9, 13, 16, 18, and 22;

xc) SEQ ID NOs:1, 7, 9, 13, 16, 18, and 22;

xci) SEQ ID NOs:1, 8, 9, 13, 16, 18, and 22;

xcii) SEQ ID NOs:1, 2, 9, 14, 16, 18, and 22;

xciii) SEQ ID NOs:1, 3, 9, 14, 16, 18, and 22;

xciv) SEQ ID NOs:1, 4, 9, 14, 16, 18, and 22;

xcv) SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22;

xcvi) SEQ ID NOs:1, 6, 9, 14, 16, 18, and 22;

xcvii) SEQ ID NOs:1, 7, 9, 14, 16, 18, and 22;

xcviii) SEQ ID NOs:1, 8, 9, 14, 16, 18, and 22;

xcix) SEQ ID NOs:1, 2, 9, 13, 16, 19, and 22;

c) SEQ ID NOs:1, 3, 9, 13, 16, 19, and 22;

ci) SEQ ID NOs:1, 4, 9, 13, 16, 19, and 22;

cii) SEQ ID NOs:1, 5, 9, 13, 16, 19, and 22;

ciii) SEQ ID NOs:1, 6, 9, 13, 16, 19, and 22;

civ) SEQ ID NOs:1, 7, 9, 13, 16, 19, and 22;

cv) SEQ ID NOs:1, 8, 9, 13, 16, 19, and 22;

cvi) SEQ ID NOs:1, 2, 9, 14, 16, 19, and 22;

cvii) SEQ ID NOs:1, 3, 9, 14, 16, 19, and 22;

cviii) SEQ ID NOs:1, 4, 9, 14, 16, 19, and 22;

cix) SEQ ID NOs:1, 5, 9, 14, 16, 19, and 22;

cx) SEQ ID NOs:1, 6, 9, 14, 16, 19, and 22;

cxi) SEQ ID NOs:1, 7, 9, 14, 16, 19, and 22; and

cxii) SEQ ID NOs:1, 8, 9, 14, 16, 19, and 22.

In some embodiments, the vector genome comprises in the 5′ to 3′ direction nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs:1, 5, 14, 18, and 22; SEQ ID NOs:1, 5, 9, 14, 18, and 22; SEQ ID NOs:1, 5, 14, 16, 18, and 22; or SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22. In certain embodiments, the AAV8 capsid may comprise subcombinations of capsid proteins VP1, VP2, and/or VP3.

It is also contemplated that the viral vector of the invention may comprise an AAV9 capsid comprising VP1, VP2, and VP3 amino acid sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs:28, 29, and 30, respectively, encoded by, for example, a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:27 and a vector genome comprising in the 5′ to 3′ direction nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to one of the following sets of nucleotide sequences:

i) SEQ ID NOs:1, 2, 13, 18, and 22;

ii) SEQ ID NOs:1, 3, 13, 18, and 22;

iii) SEQ ID NOs:1, 4, 13, 18, and 22;

iv) SEQ ID NOs:1, 5, 13, 18, and 22;

v) SEQ ID NOs:1, 6, 13, 18, and 22;

vi) SEQ ID NOs:1, 7, 13, 18, and 22;

vii) SEQ ID NOs:1, 8, 13, 18, and 22;

viii) SEQ ID NOs:1, 2, 14, 18, and 22;

ix) SEQ ID NOs:1, 3, 14, 18, and 22;

x) SEQ ID NOs:1, 4, 14, 18, and 22;

xi) SEQ ID NOs:1, 5, 14, 18, and 22;

xii) SEQ ID NOs:1, 6, 14, 18, and 22;

xiii) SEQ ID NOs:1, 7, 14, 18, and 22;

xiv) SEQ ID NOs:1, 8, 14, 18, and 22;

xv) SEQ ID NOs:1, 2, 13, 19, and 22;

xvi) SEQ ID NOs:1, 3, 13, 19, and 22;

xvii) SEQ ID NOs:1, 4, 13, 19, and 22;

xviii) SEQ ID NOs:1, 5, 13, 19, and 22;

xix) SEQ ID NOs:1, 6, 13, 19, and 22;

xx) SEQ ID NOs:1, 7, 13, 19, and 22;

xxi) SEQ ID NOs:1, 8, 13, 19, and 22;

xxii) SEQ ID NOs:1, 2, 14, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 14, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 14, 19, and 22;

xxv) SEQ ID NOs:1, 5, 14, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 14, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 14, 19, and 22;

xxviii) SEQ ID NOs:1, 8, 14, 19, and 22;

xxix) SEQ ID NOs:1, 2, 9, 13, 18, and 22;

xxx) SEQ ID NOs:1, 3, 9, 13, 18, and 22;

xxxi) SEQ ID NOs:1, 4, 9, 13, 18, and 22;

xxxii) SEQ ID NOs:1, 5, 9, 13, 18, and 22;

xxxiii) SEQ ID NOs:1, 6, 9, 13, 18, and 22;

xxxiv) SEQ ID NOs:1, 7, 9, 13, 18, and 22;

xxxv) SEQ ID NOs:1, 8, 9, 13, 18, and 22;

xxxvi) SEQ ID NOs:1, 2, 9, 14, 18, and 22;

xxxvii) SEQ ID NOs:1, 3, 9, 14, 18, and 22;

xxxviii) SEQ ID NOs:1, 4, 9, 14, 18, and 22;

xxxix) SEQ ID NOs:1, 5, 9, 14, 18, and 22;

xl) SEQ ID NOs:1, 6, 9, 14, 18, and 22;

xli) SEQ ID NOs:1, 7, 9, 14, 18, and 22;

xlii) SEQ ID NOs:1, 8, 9, 14, 18, and 22;

xliii) SEQ ID NOs:1, 2, 9, 13, 19, and 22;

xliv) SEQ ID NOs:1, 3, 9, 13, 19, and 22;

xlv) SEQ ID NOs:1, 4, 9, 13, 19, and 22;

xlvi) SEQ ID NOs:1, 5, 9, 13, 19, and 22;

xlvii) SEQ ID NOs:1, 6, 9, 13, 19, and 22;

xlviii) SEQ ID NOs:1, 7, 9, 13, 19, and 22;

xlix) SEQ ID NOs:1, 8, 9, 13, 19, and 22;

l) SEQ ID NOs:1, 2, 9, 14, 19, and 22;

li) SEQ ID NOs:1, 3, 9, 14, 19, and 22;

lii) SEQ ID NOs:1, 4, 9, 14, 19, and 22;

liii) SEQ ID NOs:1, 5, 9, 14, 19, and 22;

liv) SEQ ID NOs:1, 6, 9, 14, 19, and 22;

Iv) SEQ ID NOs:1, 7, 9, 14, 19, and 22;

lvi) SEQ ID NOs:1, 8, 9, 14, 19, and 22;

lvii) SEQ ID NOs:1, 2, 13, 16, 18, and 22;

lviii) SEQ ID NOs:1, 3, 13, 16, 18, and 22;

lix) SEQ ID NOs:1, 4, 13, 16, 18, and 22;

lx) SEQ ID NOs:1, 5, 13, 16, 18, and 22;

lxi) SEQ ID NOs:1, 6, 13, 16, 18, and 22;

lxii) SEQ ID NOs:1, 7, 13, 16, 18, and 22;

lxiii) SEQ ID NOs:1, 8, 13, 16, 18, and 22;

lxiv) SEQ ID NOs:1, 2, 14, 16, 18, and 22;

lxv) SEQ ID NOs:1, 3, 14, 16, 18, and 22;

lxvi) SEQ ID NOs:1, 4, 14, 16, 18, and 22;

lxvii) SEQ ID NOs:1, 5, 14, 16, 18, and 22;

lxviii) SEQ ID NOs:1, 6, 14, 16, 18, and 22;

lxix) SEQ ID NOs:1, 7, 14, 16, 18, and 22;

lxx) SEQ ID NOs:1, 8, 14, 16, 18, and 22;

lxxi) SEQ ID NOs:1, 2, 13, 16, 19, and 22;

lxxii) SEQ ID NOs:1, 3, 13, 16, 19, and 22;

lxxiii) SEQ ID NOs:1, 4, 13, 16, 19, and 22;

lxxiv) SEQ ID NOs:1, 5, 13, 16, 19, and 22;

lxxv) SEQ ID NOs:1, 6, 13, 16, 19, and 22;

lxxvi) SEQ ID NOs:1, 7, 13, 16, 19, and 22;

lxxvii) SEQ ID NOs:1, 8, 13, 16, 19, and 22;

lxxviii) SEQ ID NOs:1, 2, 14, 16, 19, and 22;

lxxix) SEQ ID NOs:1, 3, 14, 16, 19, and 22;

lxxx) SEQ ID NOs:1, 4, 14, 16, 19, and 22;

lxxxi) SEQ ID NOs:1, 5, 14, 16, 19, and 22;

lxxxii) SEQ ID NOs:1, 6, 14, 16, 19, and 22;

lxxxiii) SEQ ID NOs:1, 7, 14, 16, 19, and 22;

lxxxv) SEQ ID NOs:1, 8, 14, 16, 19, and 22;

lxxxv) SEQ ID NOs:1, 2, 9, 13, 16, 18, and 22;

lxxxvi) SEQ ID NOs:1, 3, 9, 13, 16, 18, and 22;

lxxxvii) SEQ ID NOs:1, 4, 9, 13, 16, 18, and 22;

lxxxviii) SEQ ID NOs:1, 5, 9, 13, 16, 18, and 22;

lxxxix) SEQ ID NOs:1, 6, 9, 13, 16, 18, and 22;

xc) SEQ ID NOs:1, 7, 9, 13, 16, 18, and 22;

xci) SEQ ID NOs:1, 8, 9, 13, 16, 18, and 22;

xcii) SEQ ID NOs:1, 2, 9, 14, 16, 18, and 22;

xciii) SEQ ID NOs:1, 3, 9, 14, 16, 18, and 22;

xciv) SEQ ID NOs:1, 4, 9, 14, 16, 18, and 22;

xcv) SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22;

xcvi) SEQ ID NOs:1, 6, 9, 14, 16, 18, and 22;

xcvii) SEQ ID NOs:1, 7, 9, 14, 16, 18, and 22;

xcviii) SEQ ID NOs:1, 8, 9, 14, 16, 18, and 22;

xcix) SEQ ID NOs:1, 2, 9, 13, 16, 19, and 22;

c) SEQ ID NOs:1, 3, 9, 13, 16, 19, and 22;

ci) SEQ ID NOs:1, 4, 9, 13, 16, 19, and 22;

cii) SEQ ID NOs:1, 5, 9, 13, 16, 19, and 22;

ciii) SEQ ID NOs:1, 6, 9, 13, 16, 19, and 22;

civ) SEQ ID NOs:1, 7, 9, 13, 16, 19, and 22;

cv) SEQ ID NOs:1, 8, 9, 13, 16, 19, and 22;

cvi) SEQ ID NOs:1, 2, 9, 14, 16, 19, and 22;

cvii) SEQ ID NOs:1, 3, 9, 14, 16, 19, and 22;

cviii) SEQ ID NOs:1, 4, 9, 14, 16, 19, and 22;

cix) SEQ ID NOs:1, 5, 9, 14, 16, 19, and 22;

cx) SEQ ID NOs:1, 6, 9, 14, 16, 19, and 22;

cxi) SEQ ID NOs:1, 7, 9, 14, 16, 19, and 22; and

cxii) SEQ ID NOs:1, 8, 9, 14, 16, 19, and 22.

In some embodiments, the vector genome comprises in the 5′ to 3′ direction nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs:1, 5, 14, 18, and 22; SEQ ID NOs:1, 5, 9, 14, 18, and 22; SEQ ID NOs:1, 5, 14, 16, 18, and 22; or SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22. In certain embodiments, the AAV9 capsid may comprise subcombinations of capsid proteins VP1, VP2, and/or VP3.

In certain aspects of the invention, the viral vector comprises an AAV2 capsid comprising VP1, VP2, and VP3 amino acid sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs:32, 33, and 34, respectively, encoded by, for example, a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:31 and a vector genome comprising in the 5′ to 3′ direction nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to one of the following sets of nucleotide sequences:

i) SEQ ID NOs:1, 2, 13, 18, and 22;

ii) SEQ ID NOs:1, 3, 13, 18, and 22;

iii) SEQ ID NOs:1, 4, 13, 18, and 22;

iv) SEQ ID NOs:1, 5, 13, 18, and 22;

v) SEQ ID NOs:1, 6, 13, 18, and 22;

vi) SEQ ID NOs:1, 7, 13, 18, and 22;

vii) SEQ ID NOs:1, 8, 13, 18, and 22;

viii) SEQ ID NOs:1, 2, 14, 18, and 22;

ix) SEQ ID NOs:1, 3, 14, 18, and 22;

x) SEQ ID NOs:1, 4, 14, 18, and 22;

xi) SEQ ID NOs:1, 5, 14, 18, and 22;

xii) SEQ ID NOs:1, 6, 14, 18, and 22;

xiii) SEQ ID NOs:1, 7, 14, 18, and 22;

xiv) SEQ ID NOs:1, 8, 14, 18, and 22;

xv) SEQ ID NOs:1, 2, 13, 19, and 22;

xvi) SEQ ID NOs:1, 3, 13, 19, and 22;

xvii) SEQ ID NOs:1, 4, 13, 19, and 22;

xviii) SEQ ID NOs:1, 5, 13, 19, and 22;

xix) SEQ ID NOs:1, 6, 13, 19, and 22;

xx) SEQ ID NOs:1, 7, 13, 19, and 22;

xxi) SEQ ID NOs:1, 8, 13, 19, and 22;

xxii) SEQ ID NOs:1, 2, 14, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 14, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 14, 19, and 22;

xxv) SEQ ID NOs:1, 5, 14, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 14, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 14, 19, and 22;

xxviii) SEQ ID NOs:1, 8, 14, 19, and 22;

xxix) SEQ ID NOs:1, 2, 9, 13, 18, and 22;

xxx) SEQ ID NOs:1, 3, 9, 13, 18, and 22;

xxxi) SEQ ID NOs:1, 4, 9, 13, 18, and 22;

xxxii) SEQ ID NOs:1, 5, 9, 13, 18, and 22;

xxxiii) SEQ ID NOs:1, 6, 9, 13, 18, and 22;

xxxiv) SEQ ID NOs:1, 7, 9, 13, 18, and 22;

xxxv) SEQ ID NOs:1, 8, 9, 13, 18, and 22;

xxxvi) SEQ ID NOs:1, 2, 9, 14, 18, and 22;

xxxvii) SEQ ID NOs:1, 3, 9, 14, 18, and 22;

xxxviii) SEQ ID NOs:1, 4, 9, 14, 18, and 22;

xxxix) SEQ ID NOs:1, 5, 9, 14, 18, and 22;

xl) SEQ ID NOs:1, 6, 9, 14, 18, and 22;

xli) SEQ ID NOs:1, 7, 9, 14, 18, and 22;

xlii) SEQ ID NOs:1, 8, 9, 14, 18, and 22;

xliii) SEQ ID NOs:1, 2, 9, 13, 19, and 22;

xliv) SEQ ID NOs:1, 3, 9, 13, 19, and 22;

xlv) SEQ ID NOs:1, 4, 9, 13, 19, and 22;

xlvi) SEQ ID NOs:1, 5, 9, 13, 19, and 22;

xlvii) SEQ ID NOs:1, 6, 9, 13, 19, and 22;

xlviii) SEQ ID NOs:1, 7, 9, 13, 19, and 22;

xlix) SEQ ID NOs:1, 8, 9, 13, 19, and 22;

l) SEQ ID NOs:1, 2, 9, 14, 19, and 22;

li) SEQ ID NOs:1, 3, 9, 14, 19, and 22;

lii) SEQ ID NOs:1, 4, 9, 14, 19, and 22;

liii) SEQ ID NOs:1, 5, 9, 14, 19, and 22;

liv) SEQ ID NOs:1, 6, 9, 14, 19, and 22;

lv) SEQ ID NOs:1, 7, 9, 14, 19, and 22;

lvi) SEQ ID NOs:1, 8, 9, 14, 19, and 22;

lvii) SEQ ID NOs:1, 2, 13, 16, 18, and 22;

lviii) SEQ ID NOs:1, 3, 13, 16, 18, and 22;

lix) SEQ ID NOs:1, 4, 13, 16, 18, and 22;

lx) SEQ ID NOs:1, 5, 13, 16, 18, and 22;

lxi) SEQ ID NOs:1, 6, 13, 16, 18, and 22;

lxii) SEQ ID NOs:1, 7, 13, 16, 18, and 22;

lxiii) SEQ ID NOs:1, 8, 13, 16, 18, and 22;

lxiv) SEQ ID NOs:1, 2, 14, 16, 18, and 22;

lxv) SEQ ID NOs:1, 3, 14, 16, 18, and 22;

lxvi) SEQ ID NOs:1, 4, 14, 16, 18, and 22;

lxvii) SEQ ID NOs:1, 5, 14, 16, 18, and 22;

lxviii) SEQ ID NOs:1, 6, 14, 16, 18, and 22;

lxix) SEQ ID NOs:1, 7, 14, 16, 18, and 22;

lxx) SEQ ID NOs:1, 8, 14, 16, 18, and 22;

lxxi) SEQ ID NOs:1, 2, 13, 16, 19, and 22;

lxxii) SEQ ID NOs:1, 3, 13, 16, 19, and 22;

lxxiii) SEQ ID NOs:1, 4, 13, 16, 19, and 22;

lxxix) SEQ ID NOs:1, 5, 13, 16, 19, and 22;

lxxv) SEQ ID NOs:1, 6, 13, 16, 19, and 22;

lxxvi) SEQ ID NOs:1, 7, 13, 16, 19, and 22;

lxxvii) SEQ ID NOs:1, 8, 13, 16, 19, and 22;

lxxviii) SEQ ID NOs:1, 2, 14, 16, 19, and 22;

lxxix) SEQ ID NOs:1, 3, 14, 16, 19, and 22;

lxxx) SEQ ID NOs:1, 4, 14, 16, 19, and 22;

lxxxi) SEQ ID NOs:1, 5, 14, 16, 19, and 22;

lxxxii) SEQ ID NOs:1, 6, 14, 16, 19, and 22;

lxxxii) SEQ ID NOs:1, 7, 14, 16, 19, and 22;

lxxxiii) SEQ ID NOs:1, 8, 14, 16, 19, and 22;

lxxxix) SEQ ID NOs:1, 2, 9, 13, 16, 18, and 22;

lxxxvi) SEQ ID NOs:1, 3, 9, 13, 16, 18, and 22;

lxxxvii) SEQ ID NOs:1, 4, 9, 13, 16, 18, and 22;

lxxxviii) SEQ ID NOs:1, 5, 9, 13, 16, 18, and 22;

lxxxix) SEQ ID NOs:1, 6, 9, 13, 16, 18, and 22;

xc) SEQ ID NOs:1, 7, 9, 13, 16, 18, and 22;

xci) SEQ ID NOs:1, 8, 9, 13, 16, 18, and 22;

xcii) SEQ ID NOs:1, 2, 9, 14, 16, 18, and 22;

xciii) SEQ ID NOs:1, 3, 9, 14, 16, 18, and 22;

xciv) SEQ ID NOs:1, 4, 9, 14, 16, 18, and 22;

xcv) SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22;

xcvi) SEQ ID NOs:1, 6, 9, 14, 16, 18, and 22;

xcvii) SEQ ID NOs:1, 7, 9, 14, 16, 18, and 22;

xcviii) SEQ ID NOs:1, 8, 9, 14, 16, 18, and 22;

xcix) SEQ ID NOs:1, 2, 9, 13, 16, 19, and 22;

c) SEQ ID NOs:1, 3, 9, 13, 16, 19, and 22;

ci) SEQ ID NOs:1, 4, 9, 13, 16, 19, and 22;

cii) SEQ ID NOs:1, 5, 9, 13, 16, 19, and 22;

ciii) SEQ ID NOs:1, 6, 9, 13, 16, 19, and 22;

civ) SEQ ID NOs:1, 7, 9, 13, 16, 19, and 22;

cv) SEQ ID NOs:1, 8, 9, 13, 16, 19, and 22;

cvi) SEQ ID NOs:1, 2, 9, 14, 16, 19, and 22;

cvii) SEQ ID NOs:1, 3, 9, 14, 16, 19, and 22;

cviii) SEQ ID NOs:1, 4, 9, 14, 16, 19, and 22;

cix) SEQ ID NOs:1, 5, 9, 14, 16, 19, and 22;

cx) SEQ ID NOs:1, 6, 9, 14, 16, 19, and 22;

cxi) SEQ ID NOs:1, 7, 9, 14, 16, 19, and 22; and

cxii) SEQ ID NOs:1, 8, 9, 14, 16, 19, and 22.

In some embodiments, the vector genome comprises in the 5′ to 3′ direction nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs:1, 5, 14, 18, and 22; SEQ ID NOs:1, 5, 9, 14, 18, and 22; SEQ ID NOs:1, 5, 14, 16, 18, and 22; or SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22. In certain embodiments, the AAV2 capsid may comprise subcombinations of capsid proteins VP1, VP2, and/or VP3.

In certain aspects of the invention, the viral vector comprises an AAV5 capsid comprising VP1, VP2, and VP3 amino acid sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs:36, 37, and 38, respectively, encoded by, for example, a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:35 and a vector genome comprising in the 5′ to 3′ direction nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to one of the following sets of nucleotide sequences:

i) SEQ ID NOs:1, 2, 13, 18, and 22;

ii) SEQ ID NOs:1, 3, 13, 18, and 22;

iii) SEQ ID NOs:1, 4, 13, 18, and 22;

iv) SEQ ID NOs:1, 5, 13, 18, and 22;

v) SEQ ID NOs:1, 6, 13, 18, and 22;

vi) SEQ ID NOs:1, 7, 13, 18, and 22;

vii) SEQ ID NOs:1, 8, 13, 18, and 22;

viii) SEQ ID NOs:1, 2, 14, 18, and 22;

ix) SEQ ID NOs:1, 3, 14, 18, and 22;

x) SEQ ID NOs:1, 4, 14, 18, and 22;

xi) SEQ ID NOs:1, 5, 14, 18, and 22;

xii) SEQ ID NOs:1, 6, 14, 18, and 22;

xiii) SEQ ID NOs:1, 7, 14, 18, and 22;

xiv) SEQ ID NOs:1, 8, 14, 18, and 22;

xv) SEQ ID NOs:1, 2, 13, 19, and 22;

xvi) SEQ ID NOs:1, 3, 13, 19, and 22;

xvii) SEQ ID NOs:1, 4, 13, 19, and 22;

xviii) SEQ ID NOs:1, 5, 13, 19, and 22;

xix) SEQ ID NOs:1, 6, 13, 19, and 22;

xx) SEQ ID NOs:1, 7, 13, 19, and 22;

xxi) SEQ ID NOs:1, 8, 13, 19, and 22;

xxii) SEQ ID NOs:1, 2, 14, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 14, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 14, 19, and 22;

xxv) SEQ ID NOs:1, 5, 14, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 14, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 14, 19, and 22;

xxviii) SEQ ID NOs:1, 8, 14, 19, and 22;

xxix) SEQ ID NOs:1, 2, 9, 13, 18, and 22;

xxx) SEQ ID NOs:1, 3, 9, 13, 18, and 22;

xxxi) SEQ ID NOs:1, 4, 9, 13, 18, and 22;

xxxii) SEQ ID NOs:1, 5, 9, 13, 18, and 22;

xxxiii) SEQ ID NOs:1, 6, 9, 13, 18, and 22;

xxxiv) SEQ ID NOs:1, 7, 9, 13, 18, and 22;

xxxv) SEQ ID NOs:1, 8, 9, 13, 18, and 22;

xxxvi) SEQ ID NOs:1, 2, 9, 14, 18, and 22;

xxxvii) SEQ ID NOs:1, 3, 9, 14, 18, and 22;

xxxviii) SEQ ID NOs:1, 4, 9, 14, 18, and 22;

xxxix) SEQ ID NOs:1, 5, 9, 14, 18, and 22;

xl) SEQ ID NOs:1, 6, 9, 14, 18, and 22;

xli) SEQ ID NOs:1, 7, 9, 14, 18, and 22;

xlii) SEQ ID NOs:1, 8, 9, 14, 18, and 22;

xliii) SEQ ID NOs:1, 2, 9, 13, 19, and 22;

xliv) SEQ ID NOs:1, 3, 9, 13, 19, and 22;

xlv) SEQ ID NOs:1, 4, 9, 13, 19, and 22;

xlvi) SEQ ID NOs:1, 5, 9, 13, 19, and 22;

xlvii) SEQ ID NOs:1, 6, 9, 13, 19, and 22;

xlviii) SEQ ID NOs:1, 7, 9, 13, 19, and 22;

xlix) SEQ ID NOs:1, 8, 9, 13, 19, and 22;

l) SEQ ID NOs:1, 2, 9, 14, 19, and 22;

li) SEQ ID NOs:1, 3, 9, 14, 19, and 22;

lii) SEQ ID NOs:1, 4, 9, 14, 19, and 22;

liii) SEQ ID NOs:1, 5, 9, 14, 19, and 22;

liv) SEQ ID NOs:1, 6, 9, 14, 19, and 22;

lv) SEQ ID NOs:1, 7, 9, 14, 19, and 22;

lvi) SEQ ID NOs:1, 8, 9, 14, 19, and 22;

lvii) SEQ ID NOs:1, 2, 13, 16, 18, and 22;

lviii) SEQ ID NOs:1, 3, 13, 16, 18, and 22;

lix) SEQ ID NOs:1, 4, 13, 16, 18, and 22;

lx) SEQ ID NOs:1, 5, 13, 16, 18, and 22;

lxi) SEQ ID NOs:1, 6, 13, 16, 18, and 22;

lxii) SEQ ID NOs:1, 7, 13, 16, 18, and 22;

lxiii) SEQ ID NOs:1, 8, 13, 16, 18, and 22;

lxiv) SEQ ID NOs:1, 2, 14, 16, 18, and 22;

lxv) SEQ ID NOs:1, 3, 14, 16, 18, and 22;

lxvi) SEQ ID NOs:1, 4, 14, 16, 18, and 22;

lxvii) SEQ ID NOs:1, 5, 14, 16, 18, and 22;

lxviii) SEQ ID NOs:1, 6, 14, 16, 18, and 22;

lxix) SEQ ID NOs:1, 7, 14, 16, 18, and 22;

lxx) SEQ ID NOs:1, 8, 14, 16, 18, and 22;

lxxi) SEQ ID NOs:1, 2, 13, 16, 19, and 22;

lxxii) SEQ ID NOs:1, 3, 13, 16, 19, and 22;

lxxiii) SEQ ID NOs:1, 4, 13, 16, 19, and 22;

lxxiv) SEQ ID NOs:1, 5, 13, 16, 19, and 22;

lxxv) SEQ ID NOs:1, 6, 13, 16, 19, and 22;

lxxvi) SEQ ID NOs:1, 7, 13, 16, 19, and 22;

lxxvii) SEQ ID NOs:1, 8, 13, 16, 19, and 22;

lxxviii) SEQ ID NOs:1, 2, 14, 16, 19, and 22;

lxxix) SEQ ID NOs:1, 3, 14, 16, 19, and 22;

lxxx) SEQ ID NOs:1, 4, 14, 16, 19, and 22;

lxxxi) SEQ ID NOs:1, 5, 14, 16, 19, and 22;

lxxxii) SEQ ID NOs:1, 6, 14, 16, 19, and 22;

lxxxiii) SEQ ID NOs:1, 7, 14, 16, 19, and 22;

lxxxiv) SEQ ID NOs:1, 8, 14, 16, 19, and 22;

lxxxv) SEQ ID NOs:1, 2, 9, 13, 16, 18, and 22;

lxxxvi) SEQ ID NOs:1, 3, 9, 13, 16, 18, and 22;

lxxxvii) SEQ ID NOs:1, 4, 9, 13, 16, 18, and 22;

lxxxviii) SEQ ID NOs:1, 5, 9, 13, 16, 18, and 22;

lxxxix) SEQ ID NOs:1, 6, 9, 13, 16, 18, and 22;

xc) SEQ ID NOs:1, 7, 9, 13, 16, 18, and 22;

xci) SEQ ID NOs:1, 8, 9, 13, 16, 18, and 22;

xcii) SEQ ID NOs:1, 2, 9, 14, 16, 18, and 22;

xciii) SEQ ID NOs:1, 3, 9, 14, 16, 18, and 22;

xciv) SEQ ID NOs:1, 4, 9, 14, 16, 18, and 22;

xcv) SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22;

xcvi) SEQ ID NOs:1, 6, 9, 14, 16, 18, and 22;

xcvii) SEQ ID NOs:1, 7, 9, 14, 16, 18, and 22;

xcviii) SEQ ID NOs:1, 8, 9, 14, 16, 18, and 22;

xcix) SEQ ID NOs:1, 2, 9, 13, 16, 19, and 22;

c) SEQ ID NOs:1, 3, 9, 13, 16, 19, and 22;

ci) SEQ ID NOs:1, 4, 9, 13, 16, 19, and 22;

cii) SEQ ID NOs:1, 5, 9, 13, 16, 19, and 22;

ciii) SEQ ID NOs:1, 6, 9, 13, 16, 19, and 22;

civ) SEQ ID NOs:1, 7, 9, 13, 16, 19, and 22;

cv) SEQ ID NOs:1, 8, 9, 13, 16, 19, and 22;

cvi) SEQ ID NOs:1, 2, 9, 14, 16, 19, and 22;

cvii) SEQ ID NOs:1, 3, 9, 14, 16, 19, and 22;

cviii) SEQ ID NOs:1, 4, 9, 14, 16, 19, and 22;

cix) SEQ ID NOs:1, 5, 9, 14, 16, 19, and 22;

cx) SEQ ID NOs:1, 6, 9, 14, 16, 19, and 22;

cxi) SEQ ID NOs:1, 7, 9, 14, 16, 19, and 22; and

cxii) SEQ ID NOs:1, 8, 9, 14, 16, 19, and 22.

In some embodiments, the vector genome comprises in the 5′ to 3′ direction nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs:1, 5, 14, 18, and 22; SEQ ID NOs:1, 5, 9, 14, 18, and 22; SEQ ID NOs:1, 5, 14, 16, 18, and 22; or SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22. In certain embodiments, the AAV5 capsid may comprise subcombinations of capsid proteins VP1, VP2, and/or VP3.

Methods for generating viral vectors are well known in the art and would allow for the skilled artisan to generate the viral vectors of the invention (see, e.g., U.S. Pat. No. 7,465,583), including the viral vectors described in Table 4. In general, methods of producing rAAV vectors are applicable to producing the viral vectors of the invention; the primary difference between the methods is the structure of the genetic elements to be packaged. To produce a viral vector according to the present invention, sequences of the genetic elements described in Table 2 can be used to produce an encapsidated viral genome.

The genetic elements as described in Table 2 are in the context of a circular plasmid or a viral genome, e.g., a singled stranded or self-complementary viral genome, but one of skill in the art will appreciate that a DNA substrate may be provided in any form known in the art, including but not limited to a plasmid, naked DNA vector, bacterial artificial chromosome (BAC), yeast artificial chromosome (YAC) or a viral vector (e.g., adenovirus, herpesvirus, Epstein-Barr Virus, AAV, baculoviral, retroviral vectors, and the like). Alternatively, the genetic elements in Table 2 necessary to produce the viral vectors described herein may be stably incorporated into the genome of a packaging cell.

The viral vector particles according to the invention may be produced by any method known in the art, e.g., by introducing the sequences to be replicated and packaged into a permissive or packaging cell, as those terms are understood in the art (e.g., a “permissive” cell can be infected or transduced by the virus; a “packaging” cell is a stably transformed cell providing helper functions).

In one embodiment, a method is provided for producing a CYP4V2 viral vector, wherein the method comprises providing to a cell permissive for parvovirus replication: (a) a nucleotide sequence containing the genetic elements for producing a vector genome of the invention (as described in detail below and in Table 2); (b) nucleotide sequences sufficient for replication of the vector genome sequence in (a) to produce a vector genome; (c) nucleotide sequences sufficient to package the vector genome into a parvovirus capsid, under conditions sufficient for virus vectors comprising the vector genome encapsidated within the parvovirus capsid to be produced in the cell. Preferably, the parvovirus replication and/or capsid coding sequences are AAV sequences.

Any method of introducing the nucleotide sequence carrying the gene cassettes described below into a cellular host for replication and packaging may be employed, including but not limited to, electroporation, calcium phosphate precipitation, linear polyethylenimine polymer precipitation, microinjection, cationic or anionic liposomes, and liposomes in combination with a nuclear localization signal.

Viral vectors described herein may be produced using methods known in the art, such as, for example, triple transfection or baculovirus mediated virus production. Any suitable permissive or packaging cell known in the art may be employed to produce the vectors.

Mammalian cells are preferred. Also preferred are trans-complementing packaging cell lines that provide functions deleted from a replication-defective helper virus, e.g., 293 cells or other Ela trans-complementing cells. Also preferred are mammalian cells or cell lines that are defective for DNA repair as known in the art, as these cell lines will be impaired in their ability to correct the mutations introduced into the plasmids described herein.

The gene cassette may contain some or all of the parvovirus (e.g., AAV) cap and rep genes. Preferably, however, some or all of the cap and rep functions are provided in trans by introducing a packaging vector(s) encoding the capsid and/or Rep proteins into the cell. Most preferably, the gene cassette does not encode the capsid or Rep proteins. Alternatively, a packaging cell line is used that is stably transformed to express the cap and/or rep genes (see, e.g., Gao et al., Hum Gene Ther 9:2353-2362, 1998; Inoue et al., J Virol 72:7024-7031, 1998; U.S. Pat. No. 5,837,484; WO 98/27207; U.S. Pat. No. 5,658,785; WO 96/17947).

In addition, helper virus functions are preferably provided for the virus vector to propagate new virus particles. Both adenovirus and herpes simplex virus may serve as helper viruses for AAV. See, e.g., Bernard N. Fields et al., VIROLOGY, volume 2, chapter 69 (3d ed., Lippincott-Raven Publishers). Exemplary helper viruses include, but are not limited to, Herpes simplex (HSV) varicella zoster, cytomegalovirus, and Epstein-Barr virus. The multiplicity of infection (MOI) and the duration of the infection will depend on the type of virus used and the packaging cell line employed. Any suitable helper vector may be employed. Preferably, the helper vector is a plasmid, for example, as described by Xiao et al., J Virol 72:2224, 1998. The vector can be introduced into the packaging cell by any suitable method known in the art, as described above.

Vector stocks free of contaminating helper virus may be obtained by any method known in the art. For example, recombinant single stranded or self complementary virus and helper virus may be readily differentiated based on size. The viruses may also be separated away from helper virus based on affinity for a heparin substrate (Zolotukhin et al., Gene Ther 6:973-985, 1999). Preferably, deleted replication-defective helper viruses are used so that any contaminating helper virus is not replication competent. As a further alternative, an adenovirus helper lacking late gene expression may be employed, as only adenovirus early gene expression is required to mediate packaging of the duplexed virus. Adenovirus mutants defective for late gene expression are known in the art (e.g., ts100K and ts149 adenovirus mutants).

One method for providing helper functions employs a non-infectious adenovirus miniplasmid that carries all of the helper genes required for efficient AAV production (Ferrari et al., Nat Med 3:1295-1297, 1997; Xiao et al., J Virol 72:2224-2232, 1998). The rAAV titers obtained with adenovirus miniplasmids are forty-fold higher than those obtained with conventional methods of wild-type adenovirus infection (Xiao et al., J Virol 72:2224-2232, 1998). This approach obviates the need to perform co-transfections with adenovirus (Hölscher et al., J Virol 68:7169-7177, 1994; Clark et al., Hum Gene Ther 6:1329-1341, 1995; Trempe and Yang, (1993), in, Fifth Parvovirus Workshop, Crystal River, Fla.).

Other methods of producing rAAV stocks have been described, including but not limited to, methods that split the rep and cap genes onto separate expression cassettes to prevent the generation of replication-competent AAV (see, e.g., Allen et al., J Virol 71:6816-6822, 1997), methods employing packaging cell lines (see, e.g., Gao et al., Hum Gene Ther 9:2353-2362, 1998; Inoue et al., J Virol 72:7024-7031, 1998; U.S. Pat. No. 5,837,484; WO 98/27207; U.S. Pat. No. 5,658,785; WO 96/17947), and other helper virus free systems (see, e.g., U.S. Pat. No. 5,945,335).

Herpesvirus may also be used as a helper virus in AAV packaging methods. Hybrid herpesviruses encoding the AAV Rep protein(s) may advantageously facilitate for more scalable AAV vector production schemes. A hybrid herpes simplex virus type I (HSV-1) vector expressing the AAV-2 rep and cap genes has been described (Conway et al., Gene Ther 6:986-993, 1999, and WO 00/17377).

In summary, the gene cassette to be replicated and packaged, parvovirus cap genes, appropriate parvovirus rep genes, and (preferably) helper functions are provided to a cell (e.g., a permissive or packaging cell) to produce rAAV particles carrying the vector genome. The combined expression of the rep and cap genes encoded by the gene cassette and/or the packaging vector(s) and/or the stably transformed packaging cell results in the production of a viral vector particle in which a viral vector capsid packages a viral vector genome according to the invention. The single stranded viral vectors are allowed to assemble within the cell, and may then be recovered by any method known by those of skill in the art and described in the examples. For example, viral vectors may be purified by standard CsCl centrifugation methods (Grieger et al., Nat Protoc 1:1412-1428, 2006), iodixanol centrifugation methods, or by various methods of column chromatography known to the skilled artisan (see, e.g., Lock et al., Hum Gene Ther 21:1259-1271, 2010; Smith et al., Mol Ther 17:1888-1896, 2009; and Vandenberghe et al., Hum Gene Ther 21:1251-1257, 2010).

The reagents and methods disclosed herein may be employed to produce high-titer stocks of the inventive viral vectors, preferably at essentially wild-type titers. It is also preferred that the parvovirus stock has a titer of about 10¹⁰vg/mL to about 10¹³vg/mL, e.g., at least or about 10¹⁰vg/mL, 6.6×10¹⁰vg/mL, 10¹¹vg/mL, 5×10¹¹vg/mL, 10¹²vg/mL, 5×10¹²vg/mL, 10¹³vg/mL, 5×10¹³vg/mL, or more.

Nucleic Acids for use in Generating the Viral Vector

The invention also relates to nucleic acids useful for the generation of viral vectors. In certain aspects of the invention, the nucleic acids useful for the generation of viral vectors may be in the form of plasmids. Plasmids useful for the generation of viral vectors, also referred to as a viral vector plasmid, may contain a gene cassette. At a minimum, a gene cassette of a viral vector plasmid contains: a promoter, a heterologous CYP4V2 gene, a polyA signal sequence, and 5′ and 3′ ITRs.

The composition of the heterologous gene and other elements will depend upon the use to which the resulting vector will be put. For example, one type of heterologous gene sequence includes a reporter sequence, which upon expression produces a detectable signal. Such reporter sequences include, without limitation, DNA sequences encoding β-lactamase, β-galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (GFP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins including, for example, CD2, CD4, CD8, the influenza hemagglutinin protein, and others well known in the art, to which high affinity antibodies directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc. For example, where the reporter sequence is the LacZ gene, the presence of the vector carrying the signal is detected by assays for beta-galactosidase activity. Where the reporter sequence is GFP or luciferase, the vector carrying the signal may be measured visually by color or light production in a luminometer.

The heterologous gene sequences, when associated with elements that drive their expression, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA), and immunohistochemistry.

The heterologous gene may also be a non-marker sequence encoding a product that is useful in biology and medicine, such as proteins, peptides, RNA, enzymes, dominant negative mutants, or catalytic RNAs. Desirable RNA molecules include tRNA, dsRNA, ribosomal RNA, catalytic RNAs, siRNA, small hairpin RNA, trans-splicing RNA, and antisense RNAs. One example of a useful RNA sequence is a sequence that inhibits or extinguishes expression of a targeted nucleotide sequence in the treated animal.

The heterologous gene may also be used to correct or ameliorate gene deficiencies, which may include deficiencies in which normal genes are expressed at less than normal levels or deficiencies in which the functional gene product is not expressed. It is contemplated in the present invention that the heterologous gene sequence may be a CYP4V2 coding sequence. Examples of CYP4V2 coding sequences are provided in Table 2: SEQ ID NOs:13, 14, 39, 41, 43, 45, 47, and 49.

One aspect of the invention relates to nucleic acids that comprise a gene cassette comprising in the 5′ to 3′ direction nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to one of the following sets of nucleotide sequences:

i) SEQ ID NOs:1, 2, 13, 18, and 22;

ii) SEQ ID NOs:1, 3, 13, 18, and 22;

iii) SEQ ID NOs:1, 4, 13, 18, and 22;

iv) SEQ ID NOs:1, 5, 13, 18, and 22;

v) SEQ ID NOs:1, 6, 13, 18, and 22;

vi) SEQ ID NOs:1, 7, 13, 18, and 22;

vii) SEQ ID NOs:1, 8, 13, 18, and 22;

viii) SEQ ID NOs:1, 2, 14, 18, and 22;

ix) SEQ ID NOs:1, 3, 14, 18, and 22;

x) SEQ ID NOs:1, 4, 14, 18, and 22;

xi) SEQ ID NOs:1, 5, 14, 18, and 22;

xii) SEQ ID NOs:1, 6, 14, 18, and 22;

xiii) SEQ ID NOs:1, 7, 14, 18, and 22;

xiv) SEQ ID NOs:1, 8, 14, 18, and 22;

xv) SEQ ID NOs:1, 2, 13, 19, and 22;

xvi) SEQ ID NOs:1, 3, 13, 19, and 22;

xvii) SEQ ID NOs:1, 4, 13, 19, and 22;

xviii) SEQ ID NOs:1, 5, 13, 19, and 22;

xix) SEQ ID NOs:1, 6, 13, 19, and 22;

xx) SEQ ID NOs:1, 7, 13, 19, and 22;

xxi) SEQ ID NOs:1, 8, 13, 19, and 22;

xxii) SEQ ID NOs:1, 2, 14, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 14, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 14, 19, and 22;

xxv) SEQ ID NOs:1, 5, 14, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 14, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 14, 19, and 22;

xxviii) SEQ ID NOs:1, 8, 14, 19, and 22;

xxix) SEQ ID NOs:1, 2, 9, 13, 18, and 22;

xxx) SEQ ID NOs:1, 3, 9, 13, 18, and 22;

xxxi) SEQ ID NOs:1, 4, 9, 13, 18, and 22;

xxxii) SEQ ID NOs:1, 5, 9, 13, 18, and 22;

xxxiii) SEQ ID NOs:1, 6, 9, 13, 18, and 22;

xxxiv) SEQ ID NOs:1, 7, 9, 13, 18, and 22;

xxxv) SEQ ID NOs:1, 8, 9, 13, 18, and 22;

xxxvi) SEQ ID NOs:1, 2, 9, 14, 18, and 22;

xxxvii) SEQ ID NOs:1, 3, 9, 14, 18, and 22;

xxxviii) SEQ ID NOs:1, 4, 9, 14, 18, and 22;

xxxix) SEQ ID NOs:1, 5, 9, 14, 18, and 22;

xl) SEQ ID NOs:1, 6, 9, 14, 18, and 22;

xli) SEQ ID NOs:1, 7, 9, 14, 18, and 22;

xlii) SEQ ID NOs:1, 8, 9, 14, 18, and 22;

xliii) SEQ ID NOs:1, 2, 9, 13, 19, and 22;

xliv) SEQ ID NOs:1, 3, 9, 13, 19, and 22;

xlv) SEQ ID NOs:1, 4, 9, 13, 19, and 22;

xlvi) SEQ ID NOs:1, 5, 9, 13, 19, and 22;

xlvii) SEQ ID NOs:1, 6, 9, 13, 19, and 22;

xlviii) SEQ ID NOs:1, 7, 9, 13, 19, and 22;

xlix) SEQ ID NOs:1, 8, 9, 13, 19, and 22;

l) SEQ ID NOs:1, 2, 9, 14, 19, and 22;

li) SEQ ID NOs:1, 3, 9, 14, 19, and 22;

lii) SEQ ID NOs:1, 4, 9, 14, 19, and 22;

liii) SEQ ID NOs:1, 5, 9, 14, 19, and 22;

liv) SEQ ID NOs:1, 6, 9, 14, 19, and 22;

lv) SEQ ID NOs:1, 7, 9, 14, 19, and 22;

lvi) SEQ ID NOs:1, 8, 9, 14, 19, and 22;

lvii) SEQ ID NOs:1, 2, 13, 16, 18, and 22;

lviii) SEQ ID NOs:1, 3, 13, 16, 18, and 22;

lix) SEQ ID NOs:1, 4, 13, 16, 18, and 22;

lx) SEQ ID NOs:1, 5, 13, 16, 18, and 22;

lxi) SEQ ID NOs:1, 6, 13, 16, 18, and 22;

lxii) SEQ ID NOs:1, 7, 13, 16, 18, and 22;

lxiii) SEQ ID NOs:1, 8, 13, 16, 18, and 22;

lxiv) SEQ ID NOs:1, 2, 14, 16, 18, and 22;

lxv) SEQ ID NOs:1, 3, 14, 16, 18, and 22;

lxvi) SEQ ID NOs:1, 4, 14, 16, 18, and 22;

lxvii) SEQ ID NOs:1, 5, 14, 16, 18, and 22;

lxviii) SEQ ID NOs:1, 6, 14, 16, 18, and 22;

lxix) SEQ ID NOs:1, 7, 14, 16, 18, and 22;

lxx) SEQ ID NOs:1, 8, 14, 16, 18, and 22;

lxxi) SEQ ID NOs:1, 2, 13, 16, 19, and 22;

lxxii) SEQ ID NOs:1, 3, 13, 16, 19, and 22;

lxxiii) SEQ ID NOs:1, 13, 16, 19, and 22;

lxxiv) SEQ ID NOs:1, 13, 16, 19, and 22;

lxxv) SEQ ID NOs:1, 13, 16, 19, and 22;

lxxvi) SEQ ID NOs:1, 7, 13, 16, 19, and 22;

lxxvii) SEQ ID NOs:1, 8, 13, 16, 19, and 22;

lxxviii) SEQ ID NOs:1, 2, 14, 16, 19, and 22;

lxxix) SEQ ID NOs:1, 3, 14, 16, 19, and 22;

lxxx) SEQ ID NOs:1, 4, 14, 16, 19, and 22;

lxxxi) SEQ ID NOs:1, 5, 14, 16, 19, and 22;

lxxxii) SEQ ID NOs:1, 6, 14, 16, 19, and 22;

lxxxiii) SEQ ID NOs:1, 7, 14, 16, 19, and 22;

lxxxiv) SEQ ID NOs:1, 8, 14, 16, 19, and 22;

lxxxv) SEQ ID NOs:1, 2, 9, 13, 16, 18, and 22;

lxxxvi) SEQ ID NOs:1, 3, 9, 13, 16, 18, and 22;

lxxxvii) SEQ ID NOs:1, 4, 9, 13, 16, 18, and 22;

lxxxviii) SEQ ID NOs:1, 5, 9, 13, 16, 18, and 22;

lxxxix) SEQ ID NOs:1, 6, 9, 13, 16, 18, and 22;

xc) SEQ ID NOs:1, 7, 9, 13, 16, 18, and 22;

xci) SEQ ID NOs:1, 8, 9, 13, 16, 18, and 22;

xcii) SEQ ID NOs:1, 2, 9, 14, 16, 18, and 22;

xciii) SEQ ID NOs:1, 3, 9, 14, 16, 18, and 22;

xciv) SEQ ID NOs:1, 4, 9, 14, 16, 18, and 22;

xcv) SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22;

xcvi) SEQ ID NOs:1, 6, 9, 14, 16, 18, and 22;

xcvii) SEQ ID NOs:1, 7, 9, 14, 16, 18, and 22;

xcviii) SEQ ID NOs:1, 8, 9, 14, 16, 18, and 22;

xcix) SEQ ID NOs:1, 2, 9, 13, 16, 19, and 22;

c) SEQ ID NOs:1, 3, 9, 13, 16, 19, and 22;

ci) SEQ ID NOs:1, 4, 9, 13, 16, 19, and 22;

cii) SEQ ID NOs:1, 5, 9, 13, 16, 19, and 22;

ciii) SEQ ID NOs:1, 6, 9, 13, 16, 19, and 22;

civ) SEQ ID NOs:1, 7, 9, 13, 16, 19, and 22;

cv) SEQ ID NOs:1, 8, 9, 13, 16, 19, and 22;

cvi) SEQ ID NOs:1, 2, 9, 14, 16, 19, and 22;

cvii) SEQ ID NOs:1, 3, 9, 14, 16, 19, and 22;

cviii) SEQ ID NOs:1, 4, 9, 14, 16, 19, and 22;

cix) SEQ ID NOs:1, 5, 9, 14, 16, 19, and 22;

cx) SEQ ID NOs:1, 6, 9, 14, 16, 19, and 22;

cxi) SEQ ID NOs:1, 7, 9, 14, 16, 19, and 22; and

cxii) SEQ ID NOs:1, 8, 9, 14, 16, 19, and 22.

In some embodiments, the gene cassette comprises in the 5′ to 3′ direction nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs:1, 5, 14, 18, and 22; SEQ ID NOs:1, 5, 9, 14, 18, and 22; SEQ ID NOs:1, 5, 14, 16, 18, and 22; or SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22. In certain embodiments, the nucleic acid comprising the gene cassette may be a plasmid.

Methods for incorporating the elements in Table 2 are well known in the art and would allow for the skilled artisan to generate the nucleic acids and plasmids of the invention using the methods described herein.

Pharmaceutical Compositions

In one aspect, the invention provides pharmaceutical compositions comprising the viral vectors of the invention formulated together with a pharmaceutically acceptable carrier. The compositions can additionally contain one or more other therapeutic agents that are suitable for treating or preventing BCD. Pharmaceutically acceptable carriers enhance or stabilize the composition, or can be used to facilitate preparation of the composition. Pharmaceutically acceptable carriers include solvents, surfactants, dispersion media, coatings, antibacterial and antifungal agents, isotonic and absorption delaying agents, and the like that are physiologically compatible.

A pharmaceutical composition of the present invention can be administered by a variety of methods known in the art. The route and/or mode of administration vary depending upon the desired results. It is preferred that administration be subretinal. The pharmaceutically acceptable carrier should be suitable for subretinal, intravitreal, intravenous, sub-cutaneous, or topical administration.

The composition should be sterile and fluid. Proper fluidity can be maintained, for example, by use of coating such as lecithin, by maintenance of required particle size in the case of dispersion, and by use of surfactants. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, and sodium chloride in the composition. In one embodiment, the composition can include a buffer with 1×PBS and 0.001% PLURONIC™ F-68 as a surfactant, with a pH of about 6.5 to 8.0, e.g., pH 6.5 to 7.5 and pH 6.5 to 7.0.

Pharmaceutical compositions of the invention can be prepared in accordance with methods well known and routinely practiced in the art. See, e.g., Remington: The Science and Practice of Pharmacy, Mack Publishing Co., 20th ed., 2000; and Sustained and Controlled Release Drug Delivery Systems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978. Pharmaceutical compositions are preferably manufactured under GMP conditions. Typically, a therapeutically effective dose or efficacious dose of the viral vector is employed in the pharmaceutical compositions of the invention. The viral vectors may be formulated into pharmaceutically acceptable dosage forms by conventional methods known to those of skill in the art. Dosage regimens are adjusted to provide the optimum desired response (e.g., a therapeutic response). For example, a single bolus may be administered, several doses may be administered over time, or the dose may be proportionally reduced or increased as indicated by the exigencies of the therapeutic situation. It is especially advantageous to formulate parenteral compositions in dosage unit form for ease of administration and uniformity of dosage. Dosage unit form as used herein refers to physically discrete units suited as unitary dosages for the subjects to be treated; each unit contains a predetermined quantity of active compound calculated to produce the desired therapeutic effect in association with the required pharmaceutical carrier.

Actual dosage levels of the active ingredients in the pharmaceutical compositions of the present invention can be varied so as to obtain an amount of the active ingredient that is effective to achieve the desired therapeutic response for a particular patient, composition, and mode of administration, without being toxic to the patient. The selected dosage level depends upon a variety of pharmacokinetic factors including the activity of the particular compositions of the present invention employed, the route of administration, the time of administration, other drugs, compounds and/or materials used in combination with the particular compositions employed, the age, sex, weight, condition, general health and prior medical history of the patient being treated, and like factors.

A physician or veterinarian can start doses of the viral vectors of the invention employed in the pharmaceutical composition at levels lower than that required to achieve the desired therapeutic effect and gradually increase the dosage until the desired effect is achieved. In general, effective doses of the compositions of the present invention, for the treatment of BCD as described herein vary depending upon different factors, including means of administration, target site, physiological state of the patient, whether the patient is human or an animal, other medications administered, and whether treatment is prophylactic or therapeutic. Treatment dosages need to be titrated to optimize safety and efficacy. For subretinal administration with a viral vector, the dosage may range from about 1×10⁸vector genomes (vg)/eye to about 1×10¹²vg/eye. For example, the dosage may be greater than or about 1×10⁸vg/eye, 2.5×10⁸vg/eye, 5×10⁸vg/eye, 7.5×10⁸vg/eye, 1×10⁹vg/eye, 2.5×10⁹vg/eye, 5×10⁹vg/eye, 7.5×10⁹vg/eye, 1×10¹⁰vg/eye, 2.5×10¹⁰vg/eye, 5×10¹⁰vg/eye, 7.5×10¹⁰vg/eye, 1×10¹¹vg/eye, 2×10¹¹vg/eye, 2.5×10¹¹vg/eye, 5×10¹¹vg/eye, 7.5×10¹¹vg/eye, or 1×10¹²vg/eye.

The viral vectors described herein are mainly used as one time doses per eye, with the possibility of repeat dosing to treat regions of the retina that are not covered in the previous dosing. The dosage of administration may vary depending on whether the treatment is prophylactic or therapeutic.

The various features and embodiments of the present invention, referred to in individual sections and embodiments above apply, as appropriate, to other sections and embodiments, mutatis mutandis. Consequently, features specified in one section or embodiment may be combined with features specified in other sections or embodiments, as appropriate.

Therapeutic Uses

Viral vectors as described herein, can be used at a therapeutically useful concentration for the treatment of eye related diseases, by administering to a subject in need thereof, an effective amount of the viral vectors of the invention. For example, the viral vector may comprises an AAV8 capsid comprising VP1, VP2, and VP3 amino acid sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs:24, 25, and 26, respectively, encoded by, for example, a nucleotide sequence with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NO:23 and a vector genome comprising in the 5′ to 3′ direction nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to one of the following sets of nucleotide sequences:

i) SEQ ID NOs:1, 2, 13, 18, and 22;

ii) SEQ ID NOs:1, 3, 13, 18, and 22;

iii) SEQ ID NOs:1, 4, 13, 18, and 22;

iv) SEQ ID NOs:1, 5, 13, 18, and 22;

v) SEQ ID NOs:1, 6, 13, 18, and 22;

vi) SEQ ID NOs:1, 7, 13, 18, and 22;

vii) SEQ ID NOs:1, 8, 13, 18, and 22;

viii) SEQ ID NOs:1, 2, 14, 18, and 22;

ix) SEQ ID NOs:1, 3, 14, 18, and 22;

x) SEQ ID NOs:1, 4, 14, 18, and 22;

xi) SEQ ID NOs:1, 5, 14, 18, and 22;

xii) SEQ ID NOs:1, 6, 14, 18, and 22;

xiii) SEQ ID NOs:1, 7, 14, 18, and 22;

xiv) SEQ ID NOs:1, 8, 14, 18, and 22;

xv) SEQ ID NOs:1, 2, 13, 19, and 22;

xvi) SEQ ID NOs:1, 3, 13, 19, and 22;

xvii) SEQ ID NOs:1, 4, 13, 19, and 22;

xviii) SEQ ID NOs:1, 5, 13, 19, and 22;

xix) SEQ ID NOs:1, 6, 13, 19, and 22;

xx) SEQ ID NOs:1, 7, 13, 19, and 22;

xxi) SEQ ID NOs:1, 8, 13, 19, and 22;

xxii) SEQ ID NOs:1, 2, 14, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 14, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 14, 19, and 22;

xxv) SEQ ID NOs:1, 5, 14, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 14, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 14, 19, and 22;

xxviii) SEQ ID NOs:1, 8, 14, 19, and 22;

xxix) SEQ ID NOs:1, 2, 9, 13, 18, and 22;

xxx) SEQ ID NOs:1, 3, 9, 13, 18, and 22;

xxxi) SEQ ID NOs:1, 4, 9, 13, 18, and 22;

xxxii) SEQ ID NOs:1, 5, 9, 13, 18, and 22;

xxxiii) SEQ ID NOs:1, 6, 9, 13, 18, and 22;

xxxiv) SEQ ID NOs:1, 7, 9, 13, 18, and 22;

xxxv) SEQ ID NOs:1, 8, 9, 13, 18, and 22;

xxxvi) SEQ ID NOs:1, 2, 9, 14, 18, and 22;

xxxvii) SEQ ID NOs:1, 3, 9, 14, 18, and 22;

xxxviii) SEQ ID NOs:1, 4, 9, 14, 18, and 22;

xxxix) SEQ ID NOs:1, 5, 9, 14, 18, and 22;

xl) SEQ ID NOs:1, 6, 9, 14, 18, and 22;

xli) SEQ ID NOs:1, 7, 9, 14, 18, and 22;

xlii) SEQ ID NOs:1, 8, 9, 14, 18, and 22;

xliii) SEQ ID NOs:1, 2, 9, 13, 19, and 22;

xliv) SEQ ID NOs:1, 3, 9, 13, 19, and 22;

xlv) SEQ ID NOs:1, 4, 9, 13, 19, and 22;

xlvi) SEQ ID NOs:1, 5, 9, 13, 19, and 22;

xlvii) SEQ ID NOs:1, 6, 9, 13, 19, and 22;

xlviii) SEQ ID NOs:1, 7, 9, 13, 19, and 22;

xlix) SEQ ID NOs:1, 8, 9, 13, 19, and 22;

l) SEQ ID NOs:1, 2, 9, 14, 19, and 22;

li) SEQ ID NOs:1, 3, 9, 14, 19, and 22;

lii) SEQ ID NOs:1, 4, 9, 14, 19, and 22;

liii) SEQ ID NOs:1, 5, 9, 14, 19, and 22;

liv) SEQ ID NOs:1, 6, 9, 14, 19, and 22;

lv) SEQ ID NOs:1, 7, 9, 14, 19, and 22;

lvi) SEQ ID NOs:1, 8, 9, 14, 19, and 22;

lvii) SEQ ID NOs:1, 2, 13, 16, 18, and 22;

lviii) SEQ ID NOs:1, 3, 13, 16, 18, and 22;

lix) SEQ ID NOs:1, 4, 13, 16, 18, and 22;

lx) SEQ ID NOs:1, 5, 13, 16, 18, and 22;

lxi) SEQ ID NOs:1, 6, 13, 16, 18, and 22;

lxii) SEQ ID NOs:1, 7, 13, 16, 18, and 22;

lxiii) SEQ ID NOs:1, 8, 13, 16, 18, and 22;

lxiv) SEQ ID NOs:1, 2, 14, 16, 18, and 22;

lxv) SEQ ID NOs:1, 3, 14, 16, 18, and 22;

lxvi) SEQ ID NOs:1, 4, 14, 16, 18, and 22;

lxvii) SEQ ID NOs:1, 5, 14, 16, 18, and 22;

lxviii) SEQ ID NOs:1, 6, 14, 16, 18, and 22;

lxix) SEQ ID NOs:1, 7, 14, 16, 18, and 22;

lxx) SEQ ID NOs:1, 8, 14, 16, 18, and 22;

lxxi) SEQ ID NOs:1, 2, 13, 16, 19, and 22;

lxxii) SEQ ID NOs:1, 3, 13, 16, 19, and 22;

lxxiii) SEQ ID NOs:1, 13, 16, 19, and 22;

lxxiv) SEQ ID NOs:1, 13, 16, 19, and 22;

lxxv) SEQ ID NOs:1, 13, 16, 19, and 22;

lxxvi) SEQ ID NOs:1, 7, 13, 16, 19, and 22;

lxxvii) SEQ ID NOs:1, 8, 13, 16, 19, and 22;

lxxviii) SEQ ID NOs:1, 2, 14, 16, 19, and 22;

lxxix) SEQ ID NOs:1, 3, 14, 16, 19, and 22;

lxxx) SEQ ID NOs:1, 4, 14, 16, 19, and 22;

lxxxi) SEQ ID NOs:1, 5, 14, 16, 19, and 22;

lxxxii) SEQ ID NOs:1, 6, 14, 16, 19, and 22;

lxxxiii) SEQ ID NOs:1, 7, 14, 16, 19, and 22;

lxxxiv) SEQ ID NOs:1, 8, 14, 16, 19, and 22;

lxxxv) SEQ ID NOs:1, 2, 9, 13, 16, 18, and 22;

lxxxvi) SEQ ID NOs:1, 3, 9, 13, 16, 18, and 22;

lxxxvii) SEQ ID NOs:1, 4, 9, 13, 16, 18, and 22;

lxxxviii) SEQ ID NOs:1, 5, 9, 13, 16, 18, and 22;

lxxxix) SEQ ID NOs:1, 6, 9, 13, 16, 18, and 22;

xc) SEQ ID NOs:1, 7, 9, 13, 16, 18, and 22;

xci) SEQ ID NOs:1, 8, 9, 13, 16, 18, and 22;

xcii) SEQ ID NOs:1, 2, 9, 14, 16, 18, and 22;

xciii) SEQ ID NOs:1, 3, 9, 14, 16, 18, and 22;

xciv) SEQ ID NOs:1, 4, 9, 14, 16, 18, and 22;

xcv) SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22;

xcvi) SEQ ID NOs:1, 6, 9, 14, 16, 18, and 22;

xcvii) SEQ ID NOs:1, 7, 9, 14, 16, 18, and 22;

xcviii) SEQ ID NOs:1, 8, 9, 14, 16, 18, and 22;

xcix) SEQ ID NOs:1, 2, 9, 13, 16, 19, and 22;

c) SEQ ID NOs:1, 3, 9, 13, 16, 19, and 22;

ci) SEQ ID NOs:1, 4, 9, 13, 16, 19, and 22;

cii) SEQ ID NOs:1, 5, 9, 13, 16, 19, and 22;

ciii) SEQ ID NOs:1, 6, 9, 13, 16, 19, and 22;

civ) SEQ ID NOs:1, 7, 9, 13, 16, 19, and 22;

cv) SEQ ID NOs:1, 8, 9, 13, 16, 19, and 22;

cvi) SEQ ID NOs:1, 2, 9, 14, 16, 19, and 22;

cvii) SEQ ID NOs:1, 3, 9, 14, 16, 19, and 22;

cviii) SEQ ID NOs:1, 4, 9, 14, 16, 19, and 22;

cix) SEQ ID NOs:1, 5, 9, 14, 16, 19, and 22;

cx) SEQ ID NOs:1, 6, 9, 14, 16, 19, and 22;

cxi) SEQ ID NOs:1, 7, 9, 14, 16, 19, and 22; and

cxii) SEQ ID NOs:1, 8, 9, 14, 16, 19, and 22.

In some embodiments, the vector genome comprises in the 5′ to 3′ direction nucleotide sequences with greater than or about 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identity to SEQ ID NOs:1, 5, 14, 18, and 22; SEQ ID NOs:1, 5, 9, 14, 18, and 22; SEQ ID NOs:1, 5, 14, 16, 18, and 22; or SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22. In certain other embodiments, the AAV8 capsid may comprise subcombinations of capsid proteins VP1, VP2, and/or VP3.

Subjects in need of treatment may include those who have one or more mutation in their CYP4V2 gene, e.g., Table 1. More specifically, the present invention provides a method of treating BCD, by administering to a subject in need thereof an effective amount of a viral vector comprising a CYP4V2 coding sequence (e.g., a nucleotide sequence encoding a human CYP4V2 protein, e.g., SEQ ID NO:15). In some aspects, provided herein is a method of improving vision in a subject with BCD, by administering to a subject in need thereof an effective amount of a viral vector comprising a CYP4V2 coding sequence (e.g., a nucleotide sequence encoding a human CYP4V2 protein, e.g., SEQ ID NO:15). In some aspects, provided herein is a method of preventing the decline of vision in a subject with BCD, by administering to a subject in need thereof an effective amount of a viral vector comprising a CYP4V2 coding sequence (e.g., a nucleotide sequence encoding a human CYP4V2 protein, e.g., SEQ ID NO:15). In specific aspects, the present invention provides viral vectors comprising a CYP4V2 coding sequence for use in treating BCD in a subject. In one embodiment, the viral vectors described herein can be administered subretinally or intravitreally using methods known to those of skill in the art. In one embodiment, the methods may include genotypying a subject to determine whether they possess one or more CYP4V2 mutation associated with BCD (see, e.g., Table 1), and treating a subject with one or more CYP4V2 mutation associated with BCD for BCD by administering a viral vector as described herein. In specific embodiments, a viral vector provided herein for use with methods of treating BCD comprises a CYP4V2 coding sequence (e.g., a nucleotide sequence encoding a human CYP4V2 protein, e.g., SEQ ID NO:15) operably linked to a promoter, e.g., ProC2 promoter.

Use of recombinant AAV has been shown to be feasible and safe for the treatment of retinal disease. See, e.g., Bainbridge et al., N Engl J Med 358:2231-2239, 2008; Bainbridge et al., Gene Ther 15:1191-1192, 2008; Hauswirth et al., Hum Gene Ther 19:979-990, 2008; Maguire et al., N Engl J Med 358:2240-2248, 2008; Bennett et al., Lancet 388:661-672, 2016; and Russell et al., Lancet 390:849-860, 2017. The viral vectors described herein can be used, inter alia, to treat and prevent progression of BCD and reduce vision loss.

The present invention also relates to a method of expressing a CYP4V2 coding sequence in RPE cells by administering viral vectors of the invention to a subject in need thereof, e.g., a subject with one or more mutations in their CYP4V2 gene, e.g., Table 1. The present invention also relates to viral vectors of the invention for use in expressing a CYP4V2 coding sequence in RPE cells of the retina of the subject in need thereof. The invention also contemplates a method of delivering and expressing a CYP4V2 coding sequence to the retina, specifically to RPE cells in the retina, of a subject having BCD. It is contemplated that the CYP4V2 coding sequence is delivered to the subject in need thereof by contacting the retina and/or RPE cells of the subject (e.g., administering subretinally or intravitreally) with a viral vector as described herein. Alternatively, a CYP4V2 coding sequence is delivered to a subject by administering to the subject a viral vector as described herein.

In some aspects, the present invention further includes methods of expressing a CYP4V2 coding sequence in RPE cells in the retina of a subject having BCD, by contacting the retina of the subject with viral vectors of the invention. In certain aspects, RPE cells of the retina of the subject are contacted with viral vectors of the invention.

Treatment and/or prevention of ocular disease such as BCD can be determined by an ophthalmologist, optometrist, or health care professional using clinically relevant measurements of visual function, functional vision, retinal anatomy, and/or Quality of Life. Treatment of BCD means any action (e.g., administration of a viral vector described herein) contemplated to improve or preserve visual function, functional vision, retinal anatomy, and/or Quality of Life. In addition, prevention as it relates to BCD means any action (e.g., administration of a viral vector described herein) that inhibits, prevents, or slows a worsening in visual function, functional vision, retinal anatomy, and/or BCD phenotype, as defined herein, in a subject at risk for said worsening, e.g., decrease number and/or size of yellow or white crystalline-like deposits in the retina.

Visual function may include, for example, visual acuity, visual acuity with low illumination, visual field, central visual field, peripheral vision, contrast sensitivity, dark adaptation, photostress recovery, color discrimination, reading speed, dependence on assistive devices (e.g., large typeface, magnifying devices, telescopes), facial recognition, proficiency at operating a motor vehicle, ability to perform one or more activities of daily living, and/or patient-reported satisfaction related to visual function. Thus, in certain embodiments, treatment of BCD can be said to occur where a subject has at least a 10%, 20%, or 30% decrease or lack of a 10%, 20%, or 30% or more increase in time to a pre-specified degree of dark adaptation. In addition, treatment of BCD can be said to occur where a subject exhibits early severe night blindness and slow dark adaptation at a young age, followed by progressive loss of visual acuity, visual fields and color vision, leading to legal blindness, determined by a qualified health care professional, e.g., ophthalmologist and optometrist.

Exemplary measures of visual function include Snellen visual acuity, ETDRS visual acuity, low-luminance visual acuity, Amsler grid, Goldmann visual field, standard automated perimetry, microperimetry, Pelli-Robson charts, SKILL card, Ishihara color plates, Farnsworth D15 or D100 color test, standard electroretinography, multifocal electroretinography, validated tests for reading speed, facial recognition, driving simulations, and patient reported satisfaction. Thus, treatment of BCD can be said to be achieved upon a gain of or failure to lose 2 or more lines (or 10 letters) of vision on an ETDRS scale. In addition, in certain aspects, treatment of BCD can be said to occur upon improvement or slowing of loss of retinal function as measured by, for example, electroretinography; improvement or slowing of progression of retinal architecture as measured by, for example, optical coherence tomography (OCT); improvement or slowing of loss in ambulatory navigation, e.g., through a maze at various illumination intensities; and/or at least a 10%, 20%, or 30% increase or lack of 10%, 20%, or 30% decrease in reading speed (words per minute). In addition, in some aspects, treatment of BCD can be said to occur where a subject exhibits at least a 20% increase or lack of a 20% decrease in the proportion of correctly identified plates on an Ishihara test or correctly sequenced disks on a Farnsworth test. Thus, treatment of BCD can be determined by, for example, improvement of rate of dark adaptation, an improvement in visual acuity, or slowing the rate of visual acuity loss.

Undesirable aspects of retinal anatomy that may be treated or prevented include, for example, accumulation of small, yellow or white crystalline-like deposits of lipids in the retina, retinal atrophy, retinal pigment epithelium atrophy, narrowing of retinal vessels, pigmentary clumping, and subretinal fluid. Exemplary means of assessing retinal anatomy include fundoscopy, fundus photography, fluorescein angiography, indocyanine green angiography, OCT, spectral domain optical coherence tomography, scanning laser ophthalmoscopy, confocal microscopy, adaptive optics, fundus autofluorescence, biopsy, necropsy, and immunohistochemistry. Thus, the viral vectors described herein can be used to treat BCD in a subject, as determined by, for example, a reduction in the rate of development of retinal atrophy and/or a reduction in the number and/or size of yellow or white crystalline-like deposits in the retina.

Treatment of BCD can also be determined by, for example, improvement or preservation of Quality of Life. Skilled practioners will appreciate that Quality of Life can be determined by many different tests, e.g., National Eye Institute NEI-VFQ25 questionnaire.

Subjects to be treated with therapeutic agents of the present invention can also be administered other therapeutic agents or devices with known efficacy for treating retinal dystrophy such as vitamin and mineral preparations, low-vision aids, guide dogs, or other devices known to assist patients with low vision.

Currently, there are no other approved therapeutic agents for the treatment of BCD.

As other new therapies emerge, the present compositions and newer therapies can be administered sequentially in either order or simultaneously as clinically indicated.

The following are exemplary embodiments of the present invention.

1. A viral vector comprising a vector genome comprising, in a 5′ to 3′ direction:

(i) a 5′ ITR;

(ii) a promoter;

(iii) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence;

(iv) a polyadenylation (polyA) signal sequence; and

(v) a 3′ ITR.

2. The viral vector of embodiment 1, wherein the vector genome comprises, in the 5′ to 3′ direction:

(i) a 5′ ITR;

(ii) a promoter;

(iii) an intron;

(iv) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence;

(v) a polyA signal sequence; and

(vi) a 3′ ITR.

3. The viral vector of embodiment 1, wherein the vector genome comprises, in the 5′ to 3′ direction:

(i) a 5′ ITR;

(ii) a promoter;

(iii) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence;

(iv) a regulatory element;

(v) a polyA signal sequence; and

(vi) a 3′ ITR.

4. The viral vector of embodiment 1, wherein the vector genome comprises, in the 5′ to 3′ direction:

(i) a 5′ ITR;

(ii) a promoter;

(iii) an intron;

(iv) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence;

(v) a regulatory element;

(vi) a polyA signal sequence; and

(vii) a 3′ ITR.

5. The viral vector of any one of embodiments 1 to 4, wherein the vector genome comprises a length greater than or about 4.1 kb and less than or about 4.9 kb.

6. The viral vector of any one of embodiments 1 to 5, wherein the vector genome comprises a stuffer sequence positioned between the polyA signal sequence and the 3′ ITR.

7. The viral vector of embodiment 6, wherein the stuffer sequence is between about 1-10, 10-20, 20-30, 30-40, 40-50, 50-60, 60-75, 75-100, 100-150, 150-200, 200-250, 250-300, 300-400, 400-500, 500-750, 750-1,000, 1,000-1,500, 1,500-2,000, 2,000-2,500, or 2,500-3,000 nucleotides in length.

8. The viral vector of any one of embodiments 1 to 7, wherein the 5′ ITR comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:1.

9. The viral vector of any one of embodiments 1 to 8, wherein the promoter is a ubiquitous promoter.

10. The viral vector of embodiment 9, wherein the promoter is a cytomegalovirus (CMV) promoter, CBA promoter, or CAG promoter.

11. The viral vector of embodiment 10, wherein the promoter comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

12. The viral vector of any one of embodiments 1 to 8, wherein the promoter is a retinal pigment epithelium (RPE)-specific promoter.

13. The viral vector of embodiment 12, wherein the promoter is a ProC2 promoter, VMD2 promoter, CYP4V2 promoter, or RPE65 promoter.

14. The viral vector of embodiment 13, wherein the promoter comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, or SEQ ID NO:8, and promotes expression of the CYP4V2 in RPE cells.

15. The viral vector of any one of embodiments 1 to 14, wherein the CYP4V2 coding sequence comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:39, SEQ ID NO:41, SEQ ID NO:43, SEQ ID NO:45, SEQ ID NO:47, or SEQ ID NO:49.

16. The viral vector of any one of embodiments 1 to 15, wherein the polyA signal sequence comprises a bovine growth hormone or simian virus 40 polyA nucleotide sequence.

17. The viral vector of embodiment 16, wherein the polyA signal sequence comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:18 or SEQ ID NO:19.

18. The viral vector of any one of embodiments 1 to 17, wherein the 3′ ITR comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:22.

19. The viral vector of any one of embodiments 2 and 4, wherein the intron comprises a human growth hormone, simian virus 40, or human beta gobin intron sequence.

20. The viral vector of embodiment 19, wherein the intron comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:9, SEQ ID NO:10, or SEQ ID NO:11.

21. The viral vector of any one of embodiments 3 and 4, wherein the regulatory element comprises a hepatitis B virus or woodchuck hepatitis virus sequence.

22. The viral vector of embodiment 21, wherein the regulatory element comprises a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:16 or SEQ ID NO:17.

23. The viral vector of any one of embodiments 1 to 22, wherein the vector genome comprises a Kozak sequence positioned immediately upstream of the recombinant nucleotide sequence comprising the CYP4V2 coding sequence.

24. The viral vector of embodiment 23, wherein the Kozak sequence comprises the nucleotide sequence of SEQ ID NO:12, SEQ ID NO:51, SEQ ID NO:52, or SEQ ID NO:53.

25. The viral vector of embodiment 1, wherein the vector genome comprises, in the 5′ to 3′ direction, nucleotide sequences selected from the group consisting of:

i) SEQ ID NOs:1, 2, 13, 18, and 22;

ii) SEQ ID NOs:1, 3, 13, 18, and 22;

iii) SEQ ID NOs:1, 4, 13, 18, and 22;

iv) SEQ ID NOs:1, 5, 13, 18, and 22;

v) SEQ ID NOs:1, 6, 13, 18, and 22;

vi) SEQ ID NOs:1, 7, 13, 18, and 22;

vii) SEQ ID NOs:1, 8, 13, 18, and 22;

viii) SEQ ID NOs:1, 2, 14, 18, and 22;

ix) SEQ ID NOs:1, 3, 14, 18, and 22;

x) SEQ ID NOs:1, 4, 14, 18, and 22;

xi) SEQ ID NOs:1, 5, 14, 18, and 22;

xii) SEQ ID NOs:1, 6, 14, 18, and 22;

xiii) SEQ ID NOs:1, 7, 14, 18, and 22;

xiv) SEQ ID NOs:1, 8, 14, 18, and 22;

xv) SEQ ID NOs:1, 2, 13, 19, and 22;

xvi) SEQ ID NOs:1, 3, 13, 19, and 22;

xvii) SEQ ID NOs:1, 4, 13, 19, and 22;

xviii) SEQ ID NOs:1, 5, 13, 19, and 22;

xix) SEQ ID NOs:1, 6, 13, 19, and 22;

xx) SEQ ID NOs:1, 7, 13, 19, and 22;

xxi) SEQ ID NOs:1, 8, 13, 19, and 22;

xxii) SEQ ID NOs:1, 2, 14, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 14, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 14, 19, and 22;

xxv) SEQ ID NOs:1, 5, 14, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 14, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 14, 19, and 22; and

xxviii) SEQ ID NOs:1, 8, 14, 19, and 22.

26. The viral vector of embodiment 2, wherein the vector genome comprises, in the 5′ to 3′ direction, nucleotide sequences selected from the group consisting of:

i) SEQ ID NOs:1, 2, 9, 13, 18, and 22;

ii) SEQ ID NOs:1, 3, 9, 13, 18, and 22;

iii) SEQ ID NOs:1, 4, 9, 13, 18, and 22;

iv) SEQ ID NOs:1, 5, 9, 13, 18, and 22;

v) SEQ ID NOs:1, 6, 9, 13, 18, and 22;

vi) SEQ ID NOs:1, 7, 9, 13, 18, and 22;

vii) SEQ ID NOs:1, 8, 9, 13, 18, and 22;

viii) SEQ ID NOs:1, 2, 9, 14, 18, and 22;

ix) SEQ ID NOs:1, 3, 9, 14, 18, and 22;

x) SEQ ID NOs:1, 4, 9, 14, 18, and 22;

xi) SEQ ID NOs:1, 5, 9, 14, 18, and 22;

xii) SEQ ID NOs:1, 6, 9, 14, 18, and 22;

xiii) SEQ ID NOs:1, 7, 9, 14, 18, and 22;

xiv) SEQ ID NOs:1, 8, 9, 14, 18, and 22;

xv) SEQ ID NOs:1, 2, 9, 13, 19, and 22;

xvi) SEQ ID NOs:1, 3, 9, 13, 19, and 22;

xvii) SEQ ID NOs:1, 4, 9, 13, 19, and 22;

xviii) SEQ ID NOs:1, 5, 9, 13, 19, and 22;

xix) SEQ ID NOs:1, 6, 9, 13, 19, and 22;

xx) SEQ ID NOs:1, 7, 9, 13, 19, and 22;

xxi) SEQ ID NOs:1, 8, 9, 13, 19, and 22;

xxii) SEQ ID NOs:1, 2, 9, 14, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 9, 14, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 9, 14, 19, and 22;

xxv) SEQ ID NOs:1, 5, 9, 14, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 9, 14, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 9, 14, 19, and 22; and

xxviii) SEQ ID NOs:1, 8, 9, 14, 19, and 22.

27. The viral vector of embodiment 3, wherein the vector genome comprises, in the 5′ to 3′ direction, nucleotide sequences selected from the group consisting of:

i) SEQ ID NOs:1, 2, 13, 16, 18, and 22;

ii) SEQ ID NOs:1, 3, 13, 16, 18, and 22;

iii) SEQ ID NOs:1, 4, 13, 16, 18, and 22;

iv) SEQ ID NOs:1, 5, 13, 16, 18, and 22;

v) SEQ ID NOs:1, 6, 13, 16, 18, and 22;

vi) SEQ ID NOs:1, 7, 13, 16, 18, and 22;

vii) SEQ ID NOs:1, 8, 13, 16, 18, and 22;

viii) SEQ ID NOs:1, 2, 14, 16, 18, and 22;

ix) SEQ ID NOs:1, 3, 14, 16, 18, and 22;

x) SEQ ID NOs:1, 4, 14, 16, 18, and 22;

xi) SEQ ID NOs:1, 5, 14, 16, 18, and 22;

xii) SEQ ID NOs:1, 6, 14, 16, 18, and 22;

xiii) SEQ ID NOs:1, 7, 14, 16, 18, and 22;

xiv) SEQ ID NOs:1, 8, 14, 16, 18, and 22;

xv) SEQ ID NOs:1, 2, 13, 16, 19, and 22;

xvi) SEQ ID NOs:1, 3, 13, 16, 19, and 22;

xvii) SEQ ID NOs:1, 4, 13, 16, 19, and 22;

xviii) SEQ ID NOs:1, 5, 13, 16, 19, and 22;

xix) SEQ ID NOs:1, 6, 13, 16, 19, and 22;

xx) SEQ ID NOs:1, 7, 13, 16, 19, and 22;

xxi) SEQ ID NOs:1, 8, 13, 16, 19, and 22;

xxii) SEQ ID NOs:1, 2, 14, 16, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 14, 16, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 14, 16, 19, and 22;

xxv) SEQ ID NOs:1, 5, 14, 16, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 14, 16, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 14, 16, 19, and 22; and

xxviii) SEQ ID NOs:1, 8, 14, 16, 19, and 22.

28. The viral vector of embodiment 4, wherein the vector genome comprises, in the 5′ to 3′ direction, nucleotide sequences selected from the group consisting of:

i) SEQ ID NOs:1, 2, 9, 13, 16, 18, and 22;

ii) SEQ ID NOs:1, 3, 9, 13, 16, 18, and 22;

iii) SEQ ID NOs:1, 4, 9, 13, 16, 18, and 22;

iv) SEQ ID NOs:1, 5, 9, 13, 16, 18, and 22;

v) SEQ ID NOs:1, 6, 9, 13, 16, 18, and 22;

vi) SEQ ID NOs:1, 7, 9, 13, 16, 18, and 22;

vii) SEQ ID NOs:1, 8, 9, 13, 16, 18, and 22;

viii) SEQ ID NOs:1, 2, 9, 14, 16, 18, and 22;

ix) SEQ ID NOs:1, 3, 9, 14, 16, 18, and 22;

x) SEQ ID NOs:1, 4, 9, 14, 16, 18, and 22;

xi) SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22;

xii) SEQ ID NOs:1, 6, 9, 14, 16, 18, and 22;

xiii) SEQ ID NOs:1, 7, 9, 14, 16, 18, and 22;

xiv) SEQ ID NOs:1, 8, 9, 14, 16, 18, and 22;

xv) SEQ ID NOs:1, 2, 9, 13, 16, 19, and 22;

xvi) SEQ ID NOs:1, 3, 9, 13, 16, 19, and 22;

xvii) SEQ ID NOs:1, 4, 9, 13, 16, 19, and 22;

xviii) SEQ ID NOs:1, 5, 9, 13, 16, 19, and 22;

xix) SEQ ID NOs:1, 6, 9, 13, 16, 19, and 22;

xx) SEQ ID NOs:1, 7, 9, 13, 16, 19, and 22;

xxi) SEQ ID NOs:1, 8, 9, 13, 16, 19, and 22;

xxii) SEQ ID NOs:1, 2, 9, 14, 16, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 9, 14, 16, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 9, 14, 16, 19, and 22;

xxv) SEQ ID NOs:1, 5, 9, 14, 16, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 9, 14, 16, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 9, 14, 16, 19, and 22; and

xxviii) SEQ ID NOs:1, 8, 9, 14, 16, 19, and 22.

29. The viral vector of any one of embodiments 1 to 28, wherein the vector comprises an adeno-associated virus (AAV) serotype 8, 9, 2, or 5 capsid.

30. The viral vector of embodiment 29, wherein the AAV8 capsid comprises VP1, VP2, and VP3 amino acid sequences with greater than or about 90% identity to SEQ ID NOs:24, 25, and 26, respectively.

31. The viral vector of embodiment 29, wherein the AAV8 capsid is encoded by a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:23.

32. The viral vector of embodiment 29, wherein the AAV9 capsid comprises VP1, VP2, and VP3 amino acid sequences with greater than or about 90% identity to SEQ ID NOs:28, 29, and 30, respectively.

33. The viral vector of embodiment 29, wherein the AAV9 capsid is encoded by a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:27.

34. The viral vector of embodiment 29, wherein the AAV2 capsid comprises VP1, VP2, and VP3 amino acid sequences with greater than or about 90% identity to SEQ ID NOs:32, 33, and 34, respectively.

35. The viral vector of embodiment 29, wherein the AAV2 capsid is encoded by a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:31.

36. The viral vector of embodiment 29, wherein the AAV5 capsid comprises VP1, VP2, and VP3 amino acid sequences with greater than or about 90% identity to SEQ ID NOs:36, 37, and 38, respectively.

37. The viral vector of embodiment 29, wherein the AAV5 capsid is encoded by a nucleotide sequence with greater than or about 90% identity to SEQ ID NO:35.

38. A composition comprising the viral vector of any of the preceding embodiments.

39. The composition of embodiment 38, wherein the composition further comprises a pharmaceutically acceptable excipient.

40. A method of expressing a heterologous CYP4V2 gene in a retinal cell, the method comprising contacting the retinal cell with the viral vector of any of the preceding embodiments.

41. The method of embodiment 40, wherein the retinal cell is a RPE cell.

42. A method of treating a subject with Bietti crystalline dystrophy (BCD), the method comprising administering to the subject an effective amount of the composition of embodiment 39.

43. A method of improving visual acuity, improving visual function or functional vision, or inhibiting decline of visual function or functional vision in a subject with BCD, the method comprising administering to the subject an effective amount of the composition of embodiment 39.

44. The composition of embodiment 39 for use in treating a subject with BCD.

45. The composition of embodiment 39 for use in improving visual acuity in a subject with BCD.

46. A nucleic acid comprising a gene cassette, wherein the gene cassette comprises, in the 5′ to 3′ direction:

(i) a 5′ ITR;

(ii) a promoter;

(iii) a recombinant nucleotide sequence comprising a CYP4V2 coding sequence;

(iv) a polyA signal sequence; and

(v) a 3′ ITR.

47. The nucleic acid of embodiment 46, wherein the nucleic acid is a plasmid.

48. The nucleic acid of embodiment 46, wherein the gene cassette comprises, in the 5′ to 3′ direction, nucleotide sequences selected from the group consisting of:

i) SEQ ID NOs:1, 2, 13, 18, and 22;

ii) SEQ ID NOs:1, 3, 13, 18, and 22;

iii) SEQ ID NOs:1, 4, 13, 18, and 22;

iv) SEQ ID NOs:1, 5, 13, 18, and 22;

v) SEQ ID NOs:1, 6, 13, 18, and 22;

vi) SEQ ID NOs:1, 7, 13, 18, and 22;

vii) SEQ ID NOs:1, 8, 13, 18, and 22;

viii) SEQ ID NOs:1, 2, 14, 18, and 22;

ix) SEQ ID NOs:1, 3, 14, 18, and 22;

x) SEQ ID NOs:1, 4, 14, 18, and 22;

xi) SEQ ID NOs:1, 5, 14, 18, and 22;

xii) SEQ ID NOs:1, 6, 14, 18, and 22;

xiii) SEQ ID NOs:1, 7, 14, 18, and 22;

xiv) SEQ ID NOs:1, 8, 14, 18, and 22;

xv) SEQ ID NOs:1, 2, 13, 19, and 22;

xvi) SEQ ID NOs:1, 3, 13, 19, and 22;

xvii) SEQ ID NOs:1, 4, 13, 19, and 22;

xviii) SEQ ID NOs:1, 5, 13, 19, and 22;

xix) SEQ ID NOs:1, 6, 13, 19, and 22;

xx) SEQ ID NOs:1, 7, 13, 19, and 22;

xxi) SEQ ID NOs:1, 8, 13, 19, and 22;

xxii) SEQ ID NOs:1, 2, 14, 19, and 22;

xxiii) SEQ ID NOs:1, 3, 14, 19, and 22;

xxiv) SEQ ID NOs:1, 4, 14, 19, and 22;

xxv) SEQ ID NOs:1, 5, 14, 19, and 22;

xxvi) SEQ ID NOs:1, 6, 14, 19, and 22;

xxvii) SEQ ID NOs:1, 7, 14, 19, and 22;

xxviii) SEQ ID NOs:1, 8, 14, 19, and 22;

xxix) SEQ ID NOs:1, 2, 9, 13, 18, and 22;

xxx) SEQ ID NOs:1, 3, 9, 13, 18, and 22;

xxxi) SEQ ID NOs:1, 4, 9, 13, 18, and 22;

xxxii) SEQ ID NOs:1, 5, 9, 13, 18, and 22;

xxxiii) SEQ ID NOs:1, 6, 9, 13, 18, and 22;

xxxiv) SEQ ID NOs:1, 7, 9, 13, 18, and 22;

xxxv) SEQ ID NOs:1, 8, 9, 13, 18, and 22;

xxxvi) SEQ ID NOs:1, 2, 9, 14, 18, and 22;

xxxvii) SEQ ID NOs:1, 3, 9, 14, 18, and 22;

xxxviii) SEQ ID NOs:1, 4, 9, 14, 18, and 22;

xxxix) SEQ ID NOs:1, 5, 9, 14, 18, and 22;

xl) SEQ ID NOs:1, 6, 9, 14, 18, and 22;

xli) SEQ ID NOs:1, 7, 9, 14, 18, and 22;

xlii) SEQ ID NOs:1, 8, 9, 14, 18, and 22;

xliii) SEQ ID NOs:1, 2, 9, 13, 19, and 22;

xliv) SEQ ID NOs:1, 3, 9, 13, 19, and 22;

xlv) SEQ ID NOs:1, 4, 9, 13, 19, and 22;

xlvi) SEQ ID NOs:1, 5, 9, 13, 19, and 22;

xlvii) SEQ ID NOs:1, 6, 9, 13, 19, and 22;

xlviii) SEQ ID NOs:1, 7, 9, 13, 19, and 22;

xlix) SEQ ID NOs:1, 8, 9, 13, 19, and 22;

l) SEQ ID NOs:1, 2, 9, 14, 19, and 22;

li) SEQ ID NOs:1, 3, 9, 14, 19, and 22;

lii) SEQ ID NOs:1, 4, 9, 14, 19, and 22;

liii) SEQ ID NOs:1, 5, 9, 14, 19, and 22;

liv) SEQ ID NOs:1, 6, 9, 14, 19, and 22;

lv) SEQ ID NOs:1, 7, 9, 14, 19, and 22;

lvi) SEQ ID NOs:1, 8, 9, 14, 19, and 22;

lvii) SEQ ID NOs:1, 2, 13, 16, 18, and 22;

lviii) SEQ ID NOs:1, 3, 13, 16, 18, and 22;

lix) SEQ ID NOs:1, 4, 13, 16, 18, and 22;

lx) SEQ ID NOs:1, 5, 13, 16, 18, and 22;

lxi) SEQ ID NOs:1, 6, 13, 16, 18, and 22;

lxii) SEQ ID NOs:1, 7, 13, 16, 18, and 22;

lxiii) SEQ ID NOs:1, 8, 13, 16, 18, and 22;

lxiv) SEQ ID NOs:1, 2, 14, 16, 18, and 22;

lxv) SEQ ID NOs:1, 3, 14, 16, 18, and 22;

lxvi) SEQ ID NOs:1, 4, 14, 16, 18, and 22;

lxvii) SEQ ID NOs:1, 5, 14, 16, 18, and 22;

lxviii) SEQ ID NOs:1, 6, 14, 16, 18, and 22;

lxix) SEQ ID NOs:1, 7, 14, 16, 18, and 22;

lxx) SEQ ID NOs:1, 8, 14, 16, 18, and 22;

lxxi) SEQ ID NOs:1, 2, 13, 16, 19, and 22;

lxxii) SEQ ID NOs:1, 3, 13, 16, 19, and 22;

lxxiii) SEQ ID NOs:1, 4, 13, 16, 19, and 22;

lxxiv) SEQ ID NOs:1, 5, 13, 16, 19, and 22;

lxxv) SEQ ID NOs:1, 6, 13, 16, 19, and 22;

lxxvi) SEQ ID NOs:1, 7, 13, 16, 19, and 22;

lxxvii) SEQ ID NOs:1, 8, 13, 16, 19, and 22;

lxxviii) SEQ ID NOs:1, 2, 14, 16, 19, and 22;

lxxix) SEQ ID NOs:1, 3, 14, 16, 19, and 22;

lxxx) SEQ ID NOs:1, 4, 14, 16, 19, and 22;

lxxxi) SEQ ID NOs:1, 5, 14, 16, 19, and 22;

lxxxii) SEQ ID NOs:1, 6, 14, 16, 19, and 22;

lxxxiii) SEQ ID NOs:1, 7, 14, 16, 19, and 22;

lxxxiv) SEQ ID NOs:1, 8, 14, 16, 19, and 22;

lxxxv) SEQ ID NOs:1, 2, 9, 13, 16, 18, and 22;

lxxxvi) SEQ ID NOs:1, 3, 9, 13, 16, 18, and 22;

lxxxvii) SEQ ID NOs:1, 4, 9, 13, 16, 18, and 22;

lxxxviii) SEQ ID NOs:1, 5, 9, 13, 16, 18, and 22;

lxxxix) SEQ ID NOs:1, 6, 9, 13, 16, 18, and 22;

xc) SEQ ID NOs:1, 7, 9, 13, 16, 18, and 22;

xci) SEQ ID NOs:1, 8, 9, 13, 16, 18, and 22;

xcii) SEQ ID NOs:1, 2, 9, 14, 16, 18, and 22;

xciii) SEQ ID NOs:1, 3, 9, 14, 16, 18, and 22;

xciv) SEQ ID NOs:1, 4, 9, 14, 16, 18, and 22;

xcv) SEQ ID NOs:1, 5, 9, 14, 16, 18, and 22;

xcvi) SEQ ID NOs:1, 6, 9, 14, 16, 18, and 22;

xcvii) SEQ ID NOs:1, 7, 9, 14, 16, 18, and 22;

xcviii) SEQ ID NOs:1, 8, 9, 14, 16, 18, and 22;

xcix) SEQ ID NOs:1, 2, 9, 13, 16, 19, and 22;

c) SEQ ID NOs:1, 3, 9, 13, 16, 19, and 22;

ci) SEQ ID NOs:1, 4, 9, 13, 16, 19, and 22;

cii) SEQ ID NOs:1, 5, 9, 13, 16, 19, and 22;

ciii) SEQ ID NOs:1, 6, 9, 13, 16, 19, and 22;

civ) SEQ ID NOs:1, 7, 9, 13, 16, 19, and 22;

cv) SEQ ID NOs:1, 8, 9, 13, 16, 19, and 22;

cvi) SEQ ID NOs:1, 2, 9, 14, 16, 19, and 22;

cvii) SEQ ID NOs:1, 3, 9, 14, 16, 19, and 22;

cviii) SEQ ID NOs:1, 4, 9, 14, 16, 19, and 22;

cix) SEQ ID NOs:1, 5, 9, 14, 16, 19, and 22;

cx) SEQ ID NOs:1, 6, 9, 14, 16, 19, and 22;

cxi) SEQ ID NOs:1, 7, 9, 14, 16, 19, and 22; and

cxii) SEQ ID NOs:1, 8, 9, 14, 16, 19, and 22.

49. A viral vector comprising a vector genome comprising a promoter operably linked to a recombinant nucleotide sequence comprising a CYP4V2 coding sequence, wherein the promoter is a ProC2 promoter.

EXAMPLES

The following examples are provided to further illustrate the invention but not to limit its scope. Other variants of the invention will be readily apparent to one of ordinary skill in the art and are encompassed by the appended claims.

Example 1: Construction of AAV-ITR Plasmids
1.1. Cloning of AAV-ITR Plasmids:

The nucleotide sequences of the individual plasmid elements are described in Table 2. The sequences were either synthesized or purchased commercially. Table 4 describes the elements that exist in each plasmid that was constructed. Standard molecular biology cloning techniques were used in generating the plasmids described in Table 4. A plasmid backbone with kanamycin resistance was used as the backbone and starting material. The individual sequence elements were cloned in at restriction enzyme sites or using blunt end cloning.

Because the antibiotic resistance gene cassette contained in the plasmid backbone does not play a role in the production of the AAV vectors, one of skill in the art could use alternate plasmid backbones and/or antibiotic resistance gene cassettes and yield the same viral vectors.

1.2. Triple Plasmid Transfection to Produce rAAV Vectors:

Recombinant AAV (rAAV) viral vectors were generated by triple transfection methods. Methods for triple transfection are known in the art (Ferrari et al., Nat Med 3:1295-1297, 1997). Briefly, AAV-ITR-containing plasmids (described in Table 4), AAV-RepCap containing plasmid (carrying Rep2 and Cap8) and Adeno-helper plasmid (carrying genes that assist in completing AAV replication cycle) were co-transfected into 293 cells. Cells were cultured for 4 days. At the end of the culture period, the cells were lysed and the vectors in the culture supernatant and in the cell lysate were purified by column chromatography using AVB sepharose affinity columns (GE Healthcare Life Sciences). Skilled practioners will appreciate that a standard CsCl gradient centrifugation method (method based on Grieger et al., Nat Protoc 1:1412-1428, 2006) may also be used.

Alternatively, GMP-like rAAV vectors can be generated by the cell transfection and culture methods described above. The harvested cell culture material is then processed by column chromatography based on methods described by Lock et al., Hum Gene Ther 21:1259-1271, 2010; Smith et al., Mol Ther 17:1888-1896, 2009; and Vandenberghe et al., Hum Gene Ther 21:1251-1257, 2010.

TABLE 4

Plasmid Compositions

SEQUENCE IDENTIFIER

Element
(SEQ ID NO:)
Cloning Strategy

Plasmid NVS1 Composition

5′ ITR
1
MluI and PmlI digested

AAV transfer plasmid

insert is ligated into

MluI and PmlI sites of

AAV plasmid backbone

containing WT AAV2

5′and 3′ITRs.

ProC2 Promoter
5
PCR amplify ProC2

promoter adding MluI

and SacII restriction

sites. Ligate

SacII/MluI digested

amplicon into

SacII/MluI cut AAV

plasmid backbone.

CYP4V2 coding
14
Cyp4V2 synthesis

sequence

fragment flanked by

XbaI and HindIII

sites. Ligate

XbaI/HindIII fragment

into XbaI/HindIII

sites of AAV plasmid

backbone.

bGH polyA signal
18
Ligate HindIII/SgrA1

sequence

fragment containing

bGH polyA signal

sequence into AAV

plasmid digested with

HindIII/SgrAI.

3′ ITR
22
MluI and PmlI digested

AAV transfer plasmid

insert is ligated into

MluI and PmlI sites of

AAV plasmid backbone

containing WT AAV2

5′and 3′ITRs.

Plasmid NVS2 Composition

5′ ITR
1
MluI and PmlI digested

AAV transfer plasmid

insert is ligated into

MluI and PmlI sites of

AAV plasmid backbone

containing WT AAV2

5′and 3′ITRs.

ProC2 Promoter
5
PCR amplify promoter

adding MluI and SacII

restriction sites.

Ligate SacII/MluI

digested amplicon into

SacII/MluI cut AAV

plasmid backbone.

hGH intron
9
PCR amplify hGHintron

adding SacII and XbaI

restriction sites.

Ligate SacII/XbaI cut

amplicon into

SacII/XbaI cut AAV

plasmid backbone.

CYP4V2 coding
14
Cyp4V2 synthesis

sequence

fragment flanked by

XbaI and HindIII

sites. Ligate

XbaI/HindIII cut

fragment into

XbaI/HindIII sites of

AAV plasmid backbone.

bGH polyA signal
18
Ligate HindIII/SgrA1

sequence

fragment containing

bGH polyA signal

sequence into AAV

plasmid digested with

HindIII/SgrAI.

3′ ITR
22
MluI and PmlI digested

AAV transfer plasmid

insert is ligated into

MluI and PmlI sites of

AAV plasmid backbone

containing WT AAV2

5′and 3′ ITRs.

Plasmid NVS3 Composition

5′ ITR
1
MluI and PmlI digested

AAV transfer plasmid

insert is ligated into

MluI and PmlI sites of

AAV plasmid backbone

containing WT AAV2

5′and 3′ ITRs.

ProC2 Promoter
5
PCR amplify promoter

adding MluI and SacII

restriction sites.

Ligate SacII/MluI

digested amplicon into

SacII/MluI cut AAV

plasmid backbone.

CYP4V2 coding
14
Cyp4V2 synthesis

sequence

fragment flanked by

XbaI and HindIII

sites. Ligate

XbaI/HindIII cut

fragment into

XbaI/HindIII sites of

AAV plasmid backbone.

HPRE regulatory
16
Digest plasmid

element

containing HPRE with

XhoI and blunt. Digest

with SalI. Ligate

into AAV plasmid cut

first with BglII,

blunted and cut with

SalI.

bGH polyA signal
18
Ligate HindIII/SgrA1

sequence

fragment containing

bGH polyA signal

sequence into AAV

plasmid digested with

HindIII/SgrAI.

3′ ITR
22
MluI and PmlI digested

AAV transfer plasmid

insert is ligated into

MluI and PmlI sites of

AAV plasmid backbone

containing WT AAV2

5′and 3′ ITRs.

Plasmid NVS4 Composition

5′ ITR
1
MluI and PmlI digested

AAV transfer plasmid

insert is ligated into

MluI and PmlI sites of

AAV plasmid backbone

containing WT AAV2

5′and 3′ ITRs.

ProC2 Promoter
5
PCR amplify promoter

adding MluI and SacII

restriction sites.

Ligate SacII/MluI cut

amplicon into

SacII/MluI digested

AAV plasmid backbone.

hGH intron
9
PCR amplify hGHintron

adding SacII and XbaI

restriction sites.

Ligate SacII/XbaI cut

amplicon into

SacII/XbaI cut AAV

plasmid backbone.

CYP4V2 coding
14
Cyp4V2 synthesis

sequence

fragment flanked by

XbaI and HindIII

sites. Ligate

XbaI/HindIII cut

fragment into

XbaI/HindIII sites of

AAV plasmid backbone.

HPRE regulatory
16
Cut plasmid containing

element

HPRE element with XhoI

then blunt ends and

cut with SalI. Digest

AAV plasmid backbone

with BglII then blunt

ends and cut with

SalI. Ligate.

bGH polyA signal
18
Ligate HindIII/SgrA1

sequence

fragment containing

bGH polyA signal

sequence into AAV

plasmid digested with

HindIII/SgrAI.

3′ ITR
22
MluI and PmlI digested

AAV transfer plasmid

insert is ligated into

MluI and PmlI AAV

plasmid backbone

containing WT AAV2

5′and 3′ ITRs.

Example 2: Inclusion of the hGH Intron, bGH polyA Signal Sequence, and HPRE Provide Enhanced Expression of eGFP

AAV8 vectors expressing ChR2d-eGFP were designed containing different elements, including different introns, different polyA signal sequences, and with or without a HPRE, to determine the best combination for optimal gene expression in the retina.

Methods

C57BL/6 mice were injected subretinally with 1×10⁹vg of AAV8 vectors expressing channelrhodopsin fused to eGFP (ChR2d-eGFP) and containing different elements, e.g., different introns (e.g., hGH intron and SV40 intron), polyA signal sequences (e.g., bGH polyA signal sequence and SV40 polyA signal sequence), and with or without a HPRE. Four weeks later, eyes were harvested from the mice and some were fixed with 1 ml 4% paraformaldehyde (PFA) fixative at 4° C. overnight and then placed in phosphate-buffered saline (PBS). These eyes were then dried with a paper towel and transferred to a 35 mm dish. Extraneous tissue was removed from the outside of the eye, as well as the cornea and lens, and then the eyecup was submerged in 1 ml PBS. The retina was then removed from the eyecup, and both were cut into petals and mounted on slides. eGFP fluorescent images were obtained using a Zeiss Axio Imager M1 fluorescent microscope and AxioVision software. All images were taken at 2.5× magnification and with the same exposure time.

The other harvested eyes were separated into neural retina and posterior eyecup and frozen for analysis of ChR2d-eGFP mRNA expression using droplet digital PCR (ddPCR). Briefly, RNA was isolated from the tissue samples using the Qiagen RNeasy Mini Kit and then 200 ng RNA was used to generate cDNA using the High-Capacity cDNA Reverse Transcription Kit from ThermoFisher Scientific. 1 ng of cDNA for each sample was added to ddPCR Supermix and primers and probe that recognize eGFP (Mr04097229_mr; ThermoFisher Scientific) and mouse Rab7 (Mm00784318_sH; ThermoFisher Scientific) were added to each reaction. ddPCR was performed according to the manufacturer's protocol (Bio-Rad). ChR2d-eGFP expression was normalized to Rab7 control expression for each sample.

Results and Conclusions

Five different AAV8 vectors were constructed and injected subretinally into mice. Four weeks post-injection, eyes were harvested from the mice and ChR2d-eGFP expression was examined by both flatmount and ddPCR. Flatmounts of the posterior eyecup showed eGFP fluorescence was highest in the eyes injected with vectors containing the hGH intron (FIG. 1). Also, inclusion of the bGH polyA signal sequence showed slightly higher fluorescence compared to the same vector with the SV40 polyA signal sequence (AAV8-TM078 versus AAV8-TM073).

To obtain a more quantitative analysis of ChR2d-eGFP expression level, ddPCR was performed on mRNA isolated from separated neural retina and posterior eyecup samples. The results showed that in the posterior eyecup, expression was most significantly enhanced by addition of the HPRE (AAV8-TM075) (FIG. 2A). Also, vectors containing the bGH polyA signal sequence showed higher expression compared to the respective vectors containing the SV40 polyA signal sequence (compare AAV8-TM078 to AAV8-TM073, and AAV8-TM079 to AAV8-TM074). In the neural retina, addition of the HPRE again led to an enhancement in expression level of eGFP (AAV8-TM075), but the greatest increase in expression was observed from addition of the bGH polyA signal sequence (AAV8-TM078 and AAV8-TM079) (FIG. 2B).

Taken together, these results show that the optimal expression cassette for gene expression in the retina may include the hGH intron, bGH polyA signal sequence, and HPRE elements. Therefore, vectors containing one, two, or all three of those elements were constructed for delivery of the CYP4V2 cDNA for the treatment of BCD.

Example 3: Subretinal Injection of AAV-CYP4V2 Vectors into Cyp4v3 Knockout Mice Prevents Progression of Disease

Cyp4v3 is the mouse ortholog of human CYP4V2 (82% identity). The Cyp4v3 gene was knocked out using CRISPR/Cas9, and Cyp4v3 knockout mice are injected with AAV vector expressing CYP4V2 at approximately 1×10⁹vg per eye. At different time points post-injection, mice are examined by fundus imaging and optical coherence tomography to determine the number and size of crystalline deposits present in the retina. In addition, the mice are evaluated for visual function (e.g., visual acuity, dark adaptation) and functional vision (e.g., mobility test). Compared to control-injected eyes (AAV-eGFP vector), eyes injected with the vector expressing CYP4V2 show a reduction in number and/or size of crystalline deposits and/or an improvement in visual function and/or functional vision, demonstrating successful expression of CYP4V2 in the RPE cells and restoration of CYP4V2 protein function.

Example 4: Subretinal Injection of AAV-ProC2 Vectors into Non-Human Primates Leads to Gene Expression in RPE Cells

ProC2 promoter sequence was chemically synthesized by GENEWIZ, with short flanks containing MluI/NheI/AscI and BamHI/EcoRI/BgIII restriction sites. ProC2 promoter sequence was subcloned using an appropriate restriction site combination into pAAV-EF1a-CatCh-GFP replacing the EF1a or hRO promoters. The pAAV-EF1a-CatCh-GFP plasmid was constructed by adapter PCR and the Clontech In-Fusion kit using pcDNA3.1(−)-CatCh-GFP. HEK293T cells were co-transfected with an AAV transgene plasmid, an AAV helper plasmid encoding the AAV Rep2 and Cap proteins for the selected capsid (BP2), and the pHGT1-Adenol helper plasmid harboring the adenoviral genes using branched polyethyleneimine (Polysciences). One cell culture dish 15 cm in diameter was co-transfected with the plasmid mixture at 80% confluence of the HEK293T cells. A cell transfection mixture containing 7 μg AAV transgene plasmid, 7 μg Rep2 and Cap-encoding plasmid, 20 μg AAV helper plasmid and 6.8 μM polyethyleneimine in 5 ml of DMEM was incubated at room temperature for 15 min before being added to a cell culture dish containing 10 ml of DMEM. At 60 h post-transfection, cells were collected and resuspended in buffer containing 150 mM NaCl and 20 mM Tris-HCl, pH 8.0. Cells were lysed by repeated freeze-thaw cycles and MgCl2 was added to make a final concentration of 1 mM. Plasmid and genomic DNA were removed by treatment with 250 U ml−1 of TurboNuclease at 37° C. for 10 min. Cell debris was removed by centrifugation at 4,000 r.p.m. for 30 min. AAV particles were purified and concentrated in Millipore Amicon 100 K columns (catalog no. UFC910008; Merck Millipore). Encapsidated viral DNA was quantified by TaqMan reverse transcription PCR following denaturation of the AAV particles using protease K; titers were calculated as genome copies per ml.

For AAV administration in mice, ocular injections were performed on mice anesthetized with 2.5% isoflurane. A small incision was made with a sharp 30-G needle in the sclera near the lens and 2 μl of AAV suspension was injected through this incision into the subretinal/intravitreal space using a blunt 5-μl Hamilton syringe (Hamilton Company) held in a micromanipulator.

For AAV administration in non-human primates, 50 microliter of AAV particle suspension were injected subretinally in collaboration with an ophthalmologist and a third party contractor in Kunming, China. After 3 month, the isolated eyecups were fixed overnight in 4% PFA in PBS, followed by a washing step in PBS at 4 C. After receiving the fixed eyecups, the infected retinal region was dissected out and treated with 10% normal donkey serum (NDS), 1% BSA, 0.5% Triton X-100 in PBS for 1 h at room temperature. Treatment with monoclonal rat anti-GFP Ab (Molecular Probes Inc.; 1:500) and polyclonal goat anti-ChAT (Millipore: 1:200) in 3% NDS, 1% BSA, 0.5% Triton X-100 in PBS was carried out for 5 days at room temperature. Treatment with secondary donkey anti-rat Alexa Fluor-488 Ab (Molecular Probes Inc.; 1:200), anti-goat Alexa Fluor-633 and Hoechst, was done for 2 hr. Sections were washed, mounted with ProLong Gold antifade reagent (Molecular Probes Inc.) on glass slides, and photographed using a Zeiss LSM 700 Axio Imager Z2 laser scanning confocal microscope (Carl Zeiss Inc.).

FIG. 3 shows that subretinal injection of AAV-ProC2-CatCh-GFP in cynomolgus monkeys (NHP) induced expression in RPE cells (gray areas of grayscale image at the top of FIGS. 3A & 3B).

Table 5 below summarizes the ability of the synthetic promoter ProC2 to drive expression in mouse and NHP retinal cells.

TABLE 5

Cell Specificity Expression in Mouse and NHP Retinal Cells

Targeted Cell Types

Targeted cell

density as a

percentage of

target
Target Expression

In order of
Targeting
population
Outer
Inner

abundance
specificity
density
Retina
Retina

Mouse
All AC,
All AC (75%),
All AC
0
1

s-MG
s-MG (25%)
(34 ± 6.9%)

NHP
RPE
RPE (100%)
RPE (50 ± 7%)

MG = Muller glia; AC = amacrine cells; All AC = All amacrine cells; s- (as prefix) = sparse expression; RPE = retinal pigment epithelium cells.

Example 5: Subretinal Injection of AAV Vectors into Cynomolgus Monkeys to Determine the Best Capsid Serotype for Targeting RPE Cells

AAV2, AAV6, AAV8, and AAV9 vectors expressing GFP from a CMV promoter were injected subretinally in cynomolgus monkeys at a dose of 1×10¹¹vg per eye. Four weeks post-injection, GFP expression in the monkey eyes was examined in-life by fundus autofluorescence imaging and post-harvest by immunohistochemistry. In addition, the level and localization of GFP mRNA and AAV genomic DNA were assessed by in-situ hybridization.

All four serotypes tested facilitated GFP expression in photoreceptor and RPE cells. Expression was observed near the injection site with varying degree of spread to peripheral regions. GFP expression was not detected in optic nerve or brain sections for any of the serotypes tested.

Example 6: Subretinal Injection of AAV Vectors into Cynomolgus Monkeys to Determine the Best Promoter for RPE-Specific Expression

AAV vectors expressing GFP from RPE-specific promoters are injected subretinally in cynomolgus monkeys at a dose of 1×10¹¹vg per eye. The RPE-specific promoters include ProC2 and VMD2. Four weeks post-injection, GFP expression in the monkey eyes is examined in-life by fundus autofluorescence imaging and post-harvest by immunohistochemistry. In addition, the level and localization of GFP mRNA and AAV genomic DNA are assessed by in-situ hybridization. The two different promoters both show RPE-specific GFP expression but the level of expression is variable.

Example 7: AAV-Driven Gene Expression in Human Retinal Tissues

Human retinal tissues are prepared from enucleated human eyeballs from which the retina is dissected suing fine scissors. AAV vectors expressing GFP from RPE-specific promoters are incubated with human retinal tissues. The RPE-specific promoters include ProC2 and VMD2. AAV-induced GFP expression is examined 6-8 weeks after virus administration. Six weeks post-injection, GFP expression in the human retinal tissues are examined by via immunofluorescence and imaging. The two different promoters both show RPE-specific GFP expression.

Example 8: AAV-Driven Expression of CYP4V2 Under the ProC2 Promoter in NHP and Human Tissues

AAV vectors expressing human CYP4V2 under the ProC2 promoter (“AAV-ProC2-CYP4V2 vector”) are incubated with human retinal tissues as described in Example 7. CYP4V2 gene expression is examined 6-8 weeks after virus administration. Six weeks post-injection, CYP4V2 expression in the human retinal tissues is detected.

AAV-ProC2-CYP4V2 vector is injected subretinally in cynomolgus monkeys at a dose of 1×10¹¹vg per eye. Four weeks post-injection, CYP4V2 expression in the monkey eyes is examined post-harvest by immunohistochemistry. In addition, the level and localization of CYP4V2 mRNA and AAV genomic DNA are assessed by in-situ hybridization. The ProC2 promoter induces RPE-specific expression of the CYP4V2 gene in the monkey eyes.

Having described the present disclosure in detail, it will be apparent that modifications, variations, and equivalent aspects are possible without departing from the spirit and scope of the present disclosure as described herein and in the appended claims. Furthermore, it should be appreciated that all examples in the present disclosure are provided as non-limiting examples. Any references cited herein, including, e.g., all patents, published patent applications, and non-patent publications, are incorporated by reference in their entirety.

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