Compositions and methods for enhanced gene expression in cone cells

• US 10,000,741 B2
• Filed: 03/17/2015
• Issued: 06/19/2018
• Est. Priority Date: 03/17/2014
• Status: Active Grant

• Current Assignee: Adverum Biotechnologies, Inc. , INC., University of Washington
• Original Assignee: Adverum Biotechnologies, Inc. , INC., University of Washington
• Inventors: Chalberg, Thomas W., Neitz, Jay, Neitz, Maureen
• Primary Examiner(s): Kelly, Robert M
• Application Number:
  US14/660,657
• Publication Number:
  US 20150259395A1
• Time in Patent Office: 1,190 Days
• Field of Search: None
• CPC Class Codes: A61K 35/761 Adenovirus
• CPC Class Codes: A61K 38/00 Medicinal preparations containing peptides peptides containing beta-lactam rings A61K31/00; cyclic dipeptides not having in their molecule any other peptide link than those which form their ring, e.g. piperazine-2,5-diones, A61K31/00; ergot alkaloids of the cyclic peptide type A61K31/48; containing macromolecular compounds having statistically distributed amino acid units A61K31/74; medicinal preparations containing antigens or antibodies A61K39/00; medicinal preparations characterised by the non-active ingredients, e.g. peptides as drug carriers, A61K47/00
• CPC Class Codes: A61K 48/00 Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
• CPC Class Codes: A61P 27/00 Drugs for disorders of the senses
• CPC Class Codes: C12N 15/86 Viral vectors
• CPC Class Codes: C12N 2750/14143 viral genome or elements thereof as genetic vector
• CPC Class Codes: C12N 2800/24 Vectors characterised by the absence of particular element, e.g. selectable marker, viral origin of replication
• CPC Class Codes: C12N 2830/008 cell type or tissue specific enhancer/promoter combination
• CPC Class Codes: C12N 2830/30 being an enhancer not forming part of the promoter region
• CPC Class Codes: C12N 2830/42 being an intron or intervening sequence for splicing and/or stability of RNA
• CPC Class Codes: C12N 7/00 Viruses; Bacteriophages; Compositions thereof; Preparation or purification thereof preparing medicinal viral antigen or antibody compositions, e.g. virus vaccines, A61K39/00

Abstract

The present disclosure provides polynucleotide cassettes, expression vectors and methods for the expression of a gene in cone cells.

Claims

1. A polynucleotide cassette for enhanced expression of a transgene in cone cells of a mammalian retina, comprising

(a) a promoter region consisting of a truncated M-Opsin promoter having a sequence identity of 85% or more to SEQ ID NO:

80;

(b) a coding sequence operatively linked to the promoter region; and

(c) a polyadenylation site operatively linked to the coding sequence.

2. The polynucleotide cassette of claim 1, further comprising a 5′

untranslated region (5′

UTR) consisting essentially of a sequence having a sequence identity of 85% or more to a sequence selected from the group consisting of SEQ ID NO;

56, SEQ ID NO;

57, SEQ ID NO;

58, SEQ ID NO;

84, SEQ ID NO;

85, SEQ ID NO;

86, SEQ ID NO;

87, SEQ ID NO;

88, and SEQ ID NO;

89.

3. The polynucleotide cassette of claim 2, wherein the 5′

UTR does not comprise a polynucleotide ATG.

4. The polynucleotide cassette of claim 2, wherein the 5′

UTR consists essentially of a sequence having a sequence identity of 85% or more to the full length of SEQ ID NO;

85 or SEQ ID NO;

86.

5. The polynucleotide cassette of claim 2, further comprising an intron.

6. The polynucleotide cassette of claim 5, wherein the intron comprises a sequence having a sequence identity of 85% or more to a sequence selected from the group consisting of SEQ ID NO:

5, SEQ ID NO;

59, and SEQ ID NO;

60.

7. The polynucleotide cassette of claim 6, wherein the intron is located within the polynucleotide sequence comprising the 5′

UTR.

8. The polynucleotide cassette of claim 1, further comprising a translation initiation sequence.

9. The polynucleotide cassette of claim 8, wherein the translation initiation sequence comprises a polynucleotide sequence consisting essentially of SEQ ID NO:

72 or SEQ ID NO;

73.

10. The polynucleotide cassette of claim 1, further comprising an enhancer sequence having a sequence identity of 85% or more to SEQ ID NO:

52 or a functional fragment thereof.

11. The polynucleotide cassette of claim 10, wherein the enhancer sequence consists essentially of a sequence having a sequence identity of 85% or more to the full length of SEQ ID NO:

51.

12. The polynucleotide cassette of claim 1, wherein expression of the coding sequence is greater than expression of the coding sequence when the promoter region is replaced with the promoter region of SEQ ID NO:

1 when introduced into a mammalian cone cell.

13. A recombinant adeno-associated virus (rAAV) comprising:

a) an AAV capsid protein, and

b) the polynucleotide cassette of claim 1 flanked by AAV ITRs.

14. A pharmaceutical composition comprising the rAAV of claim 13 and a pharmaceutical excipient.

15. A method for expressing a transgene in cone cells, comprising:

contacting one or more cone cells with an effective amount of the recombinant adeno-associated virus of claim 13, wherein the transgene is expressed at detectable levels in the one or more cone cells.

16. The method according to claim 15, comprising detecting the expression in the cone cells, wherein expression is detected in 60% or more of the cone cells.

17. A method for the treatment or prophylaxis of a cone cell disorder in a mammal in need thereof, comprising administering to the eye of the mammal an effective amount of the pharmaceutical composition of claim 14, wherein the coding sequence encodes a therapeutic gene product, and the rAAV expresses the therapeutic gene product in the cone cells of the mammal.

18. The method of claim 17, wherein the cone cell disorder is a macular dystrophy, a color vision disorder, or a vision disorder of the central macula.

19. The method of claim 18, wherein the color vision disorder is selected from the group consisting of achromotopsia, blue cone monochromacy, a protan defect, a deutan defect, and a tritan defect.

20. The method of claim 18, wherein the method further comprises detecting a change in the disease symptoms, wherein the change comprises an increase in the ability of the mammal to perceive a color.

21. The method of claim 18, wherein the macular dystrophy is selected from the group consisting of Stargardt'"'"'s macular dystrophy, cone dystrophy, cone-rod dystrophy, Spinocerebellar ataxia type 7, and Bardet-Biedl syndrome-1.

22. The method of claim 18, wherein the vision disorder of the central macula is selected from the group consisting of age-related macular degeneration, macular telangiectasia, retinitis pigmentosa, diabetic retinopathy, retinal vein occlusions, glaucoma, Sorsby'"'"'s fundus dystrophy, adult vitelliform macular dystrophy, Best'"'"'s disease, rod-cone dystrophy, Leber'"'"'s congenital amaurosis, and X-linked retinoschisis.

23. The method of claim 18, wherein the method further comprises detecting a change in the disease symptoms, wherein the change comprises reducing the rate of visual acuity loss of the mammal.

24. The polynucleotide cassette of claim 1, wherein the promoter region consists of SEQ ID NO:

80.

25. The polynucleotide cassette of claim 1, wherein the promoter region consists of a truncated M-Opsin promoter having a sequence identity of 95% or more to SEQ ID NO:

80.

26. A polynucleotide cassette for enhanced expression of a transgene in cone cells of a mammalian retina, comprising SEQ ID NO:

95.