# US 2020/0399656 A1 · Compositions and methods for enhancing functional expression of therapeutic genes in photoreceptors

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- Number: US 2020/0399656 A1
- Document ID: US20200399656A1
- Status: Published application
- Kind: A1
- Application number: 16/769,802
- Filed: 2018-12-05
- Published: 2020-12-24
- Earliest claimed: 2017-12-05
- Applicants: University of Washington
- Inventors: Maureen NEITZ; Jay NEITZ
- Web page: https://patentgrep.com/patent/US20200399656A1

## CPC

- C12N 15/86: Viral vectors
- C12N 15/861: Adenoviral vectors
- A61K 39/001109: {Vascular endothelial growth factor receptors [VEGFR]}
- A61P 27/02: Ophthalmic agents
- C07K 14/723: {G protein coupled receptor, e.g. TSHR-thyrotropin-receptor, LH/hCG receptor, FSH receptor}
- C12N 15/63: Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N 15/67: General methods for enhancing the expression
- C12N 2750/14143: viral genome or elements thereof as genetic vector
- C12N 2830/00: Vector systems having a special element relevant for transcription
- C12N 2830/42: being an intron or intervening sequence for splicing and/or stability of RNA
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C07K: PEPTIDES (peptides in foodstuffs A23; obtaining protein compositions for foodstuffs, working-up proteins for foodstuffs A23J; preparations for medicinal purposes A61K; peptides containing beta-lactam rings C07D; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, C07D; ergot alkaloids of the cyclic peptide type C07D519/02; macromolecular compounds having statistically distributed amino acid units in their molecules, i.e. when the preparation does not provide for a specific; but for a random sequence of the amino acid units, homopolyamides and block copolyamides derived from amino acids C08G69/00; macromolecular products derived from proteins C08H1/00; preparation of glue or gelatine C09H; single cell proteins, enzymes C12N; genetic engineering processes for obtaining peptides C12N15/00; compositions for measuring or testing processes involving enzymes C12Q; investigation or analysis of biological material G01N33/00)

## Abstract

Disclosed herein are nucleic acid regulatory elements, expression cassettes, expression vectors, and methods for using the nucleic acid regulatory elements, expression cassettes, expression vectors for treating cone cell disorders.

## Claims

### Claim 1

An isolated nucleic acid comprising the sequence:
(SEQ ID NO: 1)
GGGAGGAG GAGGTCTAAG TCCCAGGCCC AATTAAGAGA
TCAGGTAGTG TAGGGTTTGG GAGCTTTTAA GGTGAAGAGG
CCCGGGCTGA TCCCACAGGC CAGTATAAAG CGCCGTGACC
CTCAGGTGAC GCGCCAGGGC CGGCTGCCGT CGGGGACAGG
GCTTTCC(X);
wherein X is absent, or is selected from the group consisting of
(SEQ ID NO: 104)
ATAGCC
and
.

### Claim 2 (depends on 1)

The isolated nucleic acid of claim 1, wherein the nucleic acid sequence comprises the sequence of SEQ ID NO:3, SEQ ID NO:4, or SEQ ID NO:5; optionally wherein the nucleic acid sequence does not include the sequence of SEQ ID NO:70.

### Claim at position 3

-4. (canceled)

### Claim 5 (depends on 1)

A nucleic acid expression cassette, comprising:
(a) a locus control region (LCR) comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO:2; and
(b) a promoter comprising, consisting essentially of, or consisting of the nucleic acid sequence of claim 1, wherein the promoter is located 3′ to the LCR.

### Claim 6 (depends on 5)

The nucleic acid expression cassette of claim 5, wherein the promoter comprises or consists of the sequence of SEQ ID NO:4 or SEQ ID NO:5.

### Claim 7 (depends on 6)

The nucleic acid expression cassette of claim 6, wherein the promoter comprises or consists of the sequence of SEQ ID NO:4, wherein the cassette further comprises:
(c) an intron comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO:6, wherein the intron is located 3′ to the promoter; and
(d) a Kozak sequence, such as a Kozak sequence comprising, consisting essentially of, or consisting of the sequence GCCGCCACC, wherein the Kozak sequence is located 3′ to the intron.

### Claim 8 (depends on 7)

The nucleic acid expression cassette of claim 7, further comprising
(e) a cloning site located 3′ to the Kozak sequence.

### Claim 9 (depends on 7)

The nucleic acid expression cassette of claim 7, further comprising
(e) a coding region located 3′ to the Kozak sequence.

### Claim 10 (depends on 9)

The nucleic acid expression cassette of claim 9, wherein the coding region does not include any introns.

### Claim 11 (depends on 7)

The nucleic acid expression cassette of claim 7, wherein the promoter comprises or consists of the sequence of SEQ ID NO: 5.

### Claim at position 11

-14. (canceled)

### Claim 15 (depends on 9)

The nucleic acid expression cassette of claim 9, wherein the coding region comprises a nucleic acid sequence encoding a polypeptide selected from the group consisting of OPN1LW (for example, exons 1-6 of OPN1LW), OPN1MW (for example, exons 1-6 of OPN1MW), Sequence 14 (modified Aflibercept) or functional fragment, derivative or variant thereof; RS1 (retinoschisin precursor), antibodies (such as monoclonal antibodies) against VEGFA, anti-vascular endothelial growth factor (VEGF) antibodies, or antibody fragments thereof such as ranibizumab and/or bevacizumab, pigment epithelium-derived factor (PEDF), Soluble fms-like tyrosine kinase-1 (FLT1), CD59, cone opsins, cone ion channels, or a polypeptide selected from the group consisting of
(a) SEQ ID NO: 36 Homo sapiens opsin 1 (cone pigments), short-wave-sensitive (OPN1SW);
(b) SEQ ID NO: 37 Homo sapiens opsin 1 (cone pigments), medium-wave-sensitive (OPN1MW);
(c) SEQ ID NO: 38 Homo sapiens opsin 1 (cone pigments), long-wave-sensitive (OPN1LW);
(d) SEQ ID NO: 39 ATP binding cassette retina gene (ABCR) gene (NM_000350);
(e) SEQ ID NO: 40 retinal pigmented epithelium-specific 65 kD protein gene (RPE65) (NM_000329);
(f) SEQ ID NO: 41 retinal binding protein 1 gene (RLBP1) (NM_000326);
(g) SEQ ID NO: 42 (peripherin/retinal degeneration slow gene, (NM_000322);
(h) SEQ ID NO: 43 arrestin (SAG) (NM_000541);
(i) SEQ ID NO: 44 alpha-transducin (GNAT1) (NM_000172);
(j) SEQ ID NO: 45 guanylate cyclase activator 1A (GUCA1A) (NP_000400.2);
(k) SEQ ID NO: 46 retina specific guanylate cyclase (GUCY2D), (NP_000171.1);
(l) SEQ ID NO: 47 and/or 48 alpha subunit of the cone cyclic nucleotide gated cation channel (CNGA3) (NP_001073347.1 or NP_001289.1);
(m) SEQ ID NO: 49 Human cone transducin alpha subunit (incomplete achromotopsia);
(n) SEQ ID NO: 50 cone cGMP-specific phosphodiesterase subunit alpha′, protein (cone dystrophy type 4);
(o) SEQ ID NO: 51 retinal cone rhodopsin-sensitive cGMP 3′5′-cyclic phosphodiesterase subunit gamma, protein (retinal cone dystrophy type 3A);
(p) SEQ ID NO: 52 cone rod homeobox, protein (Cone-rod dystrophy);
(q) SEQ ID NO: 53 cone photoreceptor cyclic nucleotide-gated channel beta subunit, protein (achromatopsia);
(r) SEQ ID NO: 54 cone photoreceptor cGMP-gated cation channel beta-subunit, protein (total color blindness, for example, among Pingelapese Islanders);
(s) SEQ ID NO: 55 retinitis pigmentosa 1 (autosomal dominant) (RP1);
(t) SEQ ID NO: 57 retinitis pigmentosa GTPase regulator interacting protein 1 (RPGRIP1);
(u) SEQ ID NO: 59 PRP8;
(v) SEQ ID NO: 61 centrosomal protein 290 kDa (CEP290);
(w) SEQ ID NO: 63 IMP (inosine 5′-monophosphate) dehydrogenase 1 (IMPDH1), transcript variant 1;
(x) SEQ ID NO: 65 aryl hydrocarbon receptor interacting protein-like 1 (AIPL1), transcript variant 1;
(y) SEQ ID NO: 66 retinol dehydrogenase 12 (all-trans/9-cis/11-cis) (RDH12);
(z) SEQ ID NO: 67 Leber congenital amaurosis 5 (LCA5), transcript variant 1; and
(aa) exemplary OPN1LW/OPN1MW2 polymorphs (compared to OPN1LW (L opsin) polypeptide sequence; the amino acid to the left of the number is the residue present in the L opsin sequence; the number is the reside number in L opsin, and the reside to the right of the number is the variation from L opsin
(i) Thr65Ile;
(ii) Ile111Val;
(iii) Ser116Tyr;
(iv) Leu153Met;
(v) Ile171Val;
(vi) Ala174Val;
(vii) Ile178Val;
(viii) Ser180Ala;
(ix) Ile230Thr;
(x) Ala233 Ser;
(xi) Val236Met;
(xii) Ile274Val;
(xiii) Phe275Leu;
(xiv) Tyr277Phe;
(xv) Val279Phe;
(xvi) Thr285Ala;
(xvii) Pro298Ala; and
(xviii) Tyr309Phe.

### Claim 16 (depends on 13)

The nucleic acid expression cassette of claim 13 wherein the coding region comprises a nucleic acid encoding OPN1LW (exons 1-6) or OPN1MW (exons 1-6), and wherein:
(a) the coding region does not include endogenous OPN1LW/MW introns; and
(b) the one or more introns comprise:
(i) a first intron comprising the sequence of SEQ ID NO:7 upstream of OPN1LW/MW exon 3, and a second intron comprising the sequence of SEQ ID NO:8 downstream of OPN1LW/MW exon 3; or
(ii) a first intron comprising the sequence of SEQ ID NO:10 upstream of OPN1LW/MW exon 3, and a second intron comprising the sequence of SEQ ID NO:12 downstream of OPN1LW/MW exon 3.

### Claim 17 (depends on 9)

The nucleic acid expression cassette of claim 9, wherein the coding region comprises a nucleic acid sequence encoding a polypeptide have a signal sequence at its N-terminus; and/or wherein the coding region comprises a nucleic acid sequence encoding a polypeptide comprising an outer cell membrane targeting sequence at its C-terminus.

### Claim at position 15

-26. (canceled)

### Claim 27 (depends on 8)

The nucleic acid expression cassette of claim 8, further comprising:
(f) a regulatory element comprising, consisting essentially of, or consisting of the sequence of SEQ ID NO:15, wherein the regulatory element is located 3′ to the cloning site or to the coding region; and
(g) an untranslated region nucleic acid comprising, consisting essentially of, or consisting of SEQ ID NO:16, wherein the untranslated region nucleic acid is located 3′ to the regulatory element.

### Claim 28

A nucleic acid expression cassette, comprising a nucleic acid that encodes an opsin polypeptide operatively linked to a promoter, wherein the nucleic acid encoding the opsin polypeptide comprises one or more introns comprising, consisting essentially of, or consisting of the nucleic acid sequence of SEQ ID NO:10 and/or SEQ ID NO: 12.

### Claim at position 18

-38. (canceled)

### Claim 39 (depends on 5)

The nucleic acid expression cassette of claim 5, wherein
(i) the 5′ and 3′ ends of the cassette comprise inverted terminal repeats;
(ii) the nucleic acid expression cassette is less than about 5 kb in length; and/or
(iii) the nucleic acid expression cassette comprises or consists of a sequence selected from the group consisting of SEQ ID NOs:91-95.

### Claim at position 20

-43. (canceled)

### Claim 44 (depends on 5)

A nucleic acid expression vector comprising the nucleic acid expression cassette of claim 5, or a host cell comprising the nucleic acid expression vector.

### Claim 45

(canceled)

### Claim 46 (depends on 5)

A composition, comprising the nucleic acid expression cassette of claim 5 and
(a) an AAV capsid protein, wherein the nucleic acid expression cassette and AAV capsid protein comprise a recombinant adeno-associated virus (rAAV) particle; and/or
(b) a pharmaceutically acceptable carrier; and/or
(c) viral particles, wherein the nucleic acid expression cassette is packaged in the viral particles.

### Claim at position 24

-48. (canceled)

### Claim 49 (depends on 5)

A method for expressing a gene product, such as a protein in cone cells, comprising contacting one or more cone cells with an effective amount of the nucleic acid expression cassette of claim 5, wherein the gene product, such as a protein, encoded by the coding region is expressed at detectable levels in the one or more cone cells.

### Claim 50 (depends on 5)

A method for the treatment or prophylaxis of a cone cell disorder in a mammal in need of treatment or prophylaxis for a cone cell disorder, comprising administering to the eye of the mammal an effective amount of the nucleic acid expression cassette of claim 5, wherein the coding region comprises a nucleic acid sequence encoding a therapeutic gene product.

### Claim at position 27

-54. (canceled)

### Claim 55

An isolated nucleic acid comprising a sequence selected from the group consisting of:
(i) SEQ ID NO: 10;
(ii) SEQ ID NO:12; and
(iii) a nucleic acid sequence of the general formula A-B, wherein A encodes a polypeptide comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:21, and wherein B encodes a gene product for treating a cone cell disorder; or
an isolated comprising, consisting essentially of, or consisting of the amino acid sequence of SEQ ID NO:103.

### Claim at position 29

-63. (canceled)