Recombinant adeno-associated virus vector

Abstract

The invention provides adeno-associated virus (rAAV) vectors, in particular rAAV vectors comprising a genetic construct harbouring genes encoding tyrosine receptor kinase B (TrkB) and Brain Derived Neurotrophic Factor (BDNF). The invention also extends to a pharmaceutical composition comprising the rAAV vectors, and to the use of such vectors and compositions in gene therapy methods for preventing or treating a range of optic nerve disorders and cochlear disorders, or for promoting nerve regeneration and / or survival. The invention also provides methods of producing the rAAV vectors.

Description

Abstract This invention provides an adeno-associated virus (rAAV) vector, specifically an rAAV vector comprising a gene construct carrying genes encoding tyrosine receptor kinase B (TrkB) and brain-derived neurotrophic factor (BDNF). The invention also extends to pharmaceutical compositions comprising said rAAV vectors, and the use of these vectors and compositions in gene therapy for the prevention or treatment of a range of optic nerve and cochlear diseases, or for promoting neurogenesis and / or survival. The invention further provides a method for producing rAAV vectors.

Claims

Claims1. A recombinant adeno-associated virus (rAAV) vector comprising a genetic construct comprising, in a 5’ to 3’ orientation: - a cytomegalovirus (CMV) promoter; a first coding sequence, which encodes tyrosine kinase receptor B (TrkB); a nucleotide sequence encoding a linker to generate TrkB and mature brain- derived neurotrophic factor (mBDNF) as individual proteins; and a second coding sequence, which encodes mBDNF, wherein the CMV promoter is operably linked to the first and second coding sequences.

2. The rAAV vector according to claim 1, wherein the CMV promoter comprises a nucleotide sequence as set out in SEQ ID No: 1, or a fragment or variant thereof. 3- The rAAV vector according to claim 1, wherein the first coding sequence encodes naturally occurring TrkB, or a variant having the function thereof.

4. The rAAV vector according to claim 1, wherein the first coding sequence encodes an amino acid sequence as set out in SEQ ID No: 2, or a fragment or variant thereof, and / or wherein the first coding sequence comprises a nucleotide sequence as set out in SEQ ID No: 3, or a fragment or variant thereof.

5. The rAAV vector according to claim 1, wherein the second coding sequence encodes naturally occurring mBDNF.

6. The rAAV vector according to claim 1, wherein the second coding sequence encodes an amino acid sequence as set out in SEQ ID No: 4, or a fragment or variant thereof, and / or wherein the second coding sequence comprises a nucleotide sequence as set out in SEQ ID No: 5, or a fragment or variant thereof.

7. The rAAV vector according to claim 1, wherein the genetic construct further comprises a nucleotide sequence encoding a signal peptide, optionally wherein the signal peptide is positioned on the 5’ side of the nucleotide sequence encoding mBDNF, and / or wherein the the nucleotide sequence encoding the signal peptide is positioned on the 3’ side of the nucleotide sequence encoding the linker.

8. The rAAV vector according to claim 7, wherein the nucleotide sequence encoding the signal peptide encodes an amino acid sequence as set out in SEQ ID No: 6 or SEQ ID No: 20, or a fragment or variant thereof, and / or wherein the signal peptide comprises a nucleotide sequence as set out in SEQ ID No: 7 or SEQ ID No: 21, or a fragment or variant thereof.

9. The rAAV vector according to claim 1, wherein the linker is a P2A peptide.

10. The rAAV vector according to claim 1, wherein the nucleotide sequence encoding the linker encodes an amino acid sequence as set out in SEQ ID No: 8, or a fragment or variant thereof, and / or wherein the linker comprises a nucleotide sequence as set out in SEQ ID No: 9, or a fragment or variant thereof.

11. The rAAV vector according to claim 1, wherein the genetic construct further comprises a nucleotide sequence encoding a woodchuck hepatitis virus post- transcriptional regulatoiy element (WPRE), optionally wherein the WPRE comprises a nucleotide sequence as set out in SEQ ID No: 10, or a fragment or variant thereof.

12. The rAAV vector according to claim 1, wherein the genetic construct further comprises a nucleotide sequence encoding a polyA signal sequence, optionally wherein the polyA signal sequence comprises a nucleotide sequence as set out in SEQ ID No: 11, or a fragment or variant thereof.

13. The rAAV vector according to claim 1, wherein the rAAV vector comprises a genetic construct comprising, in a 5’ to 3’ direction, a CMV promoter sequence, a first coding sequence encoding TrkB, a nucleotide sequence encoding a P2A linker peptide, a nucleotide sequence encoding a signal peptide, a second coding sequence encoding mBDNF, a woodchuck hepatitis virus post-transcriptional regulatory element (WPRE), and a simian virus 40 (SV40) polyA signal sequence.

14. The rAAV vector according to claim 1, wherein the genetic construct comprises a nucleotide sequence as set out in any one of SEQ ID No: 14 to 17, or a variant or fragment thereof.

15. The rAAV vector according to claim 1, wherein the rAAV vector is a rAAV2 vector.

16. The rAAV vector according to claim 1, wherein the rAAV vector is a rAAV2.7m8 vector.

17. The rAAV vector according to claim 1, for use as a medicament or in therapy.

18. The rAAV vector according to claim 1, for use in treating, preventing or ameliorating an optic nerve disorder and / or a retinal degenerative disease involving retinal ganglion cell degeneration.

19. The rAAV vector according to claim 18, wherein the optic nerve disorder and / or retinal degenerative disease involving retinal ganglion cell degeneration is glaucoma or glaucoma optic neuropathy.

20. A method of treating, preventing or ameliorating an optic nerve disorder and / or a retinal degenerative disease involving retinal ganglion cell degeneration in a subject, the method comprising administering, to a subject in need of such treatment, a therapeutically effective amount of the rAAV vector according to claim 1.

21. A pharmaceutical composition comprising the recombinant rAAV vector according to claim 1, and a pharmaceutically acceptable vehicle.

22. A method of preparing the pharmaceutical composition according to claim 21, the method comprising contacting the recombinant rAAV vector according to claim 1, with a pharmaceutically acceptable vehicle.

23. A method for producing the rAAV vector according to claim 1, the method comprising:(i) introducing, into a rAAV vector-producing cell, a genetic construct comprising, in a 5’ to 3’ orientation: a cytomegalovirus (CMV) promoter; a first coding sequence, which encodes tyrosine kinase receptor B (TrkB); a nucleotide sequence encoding a linker to generate TrkB and mature brain- derived neurotrophic factor (mBDNF) as individual proteins; and - a second coding sequence, which encodes mBDNF,wherein the CMV promoter is operably linked to the first and second coding sequence; and(ii) culturing the rAAV vector-producing cell, to thereby produce the rAAV vector according to claim 1.

24. A rAAV vector-producing cell comprising the genetic construct of the rAAV vector of claim 1.

Images