JP-2026514375-A - Adeno-associated virus vector encoding connexin 26 and its use

Abstract

This invention relates to a recombinant adeno-associated virus (rAAV) vector encoding connexin 26 (CX26). The invention further relates to the use of the AAV vector in the treatment of hereditary hearing loss. [Selection Diagram] Figure 5

Inventors

• デジーレ，ローラン
• ギース，アルノー
• ヴィダル，パトリス
• プチ，クリスティーヌ
• エリティエ，アン－ヴァレリー
• ミハルスキー，ニコラ

Assignees

• センソリオン
• インスティテュート パストゥール

Dates

Publication Date

20260511

Application Date

20240328

Priority Date

20231220

Claims (15)

1. An adeno-associated virus (AAV) vector comprising an AAV-DJ capsid or a capsid derived from an AAV-DJ capsid, comprising a polynucleotide comprising (i) a nucleic acid sequence encoding connexin 26 protein (CX26) operably linked to a promoter, and (ii) at least one copy of a miRNA target site of the miR183 family, comprising the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31, or a sequence having at least 90% identity with any one of SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31.
2. The AAV vector according to claim 1, comprising 2 to 6 copies, preferably 3 copies, of the miRNA target site of the miR183 family, which includes the sequence described in SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31, or a sequence having at least 90% identity with any one of SEQ ID NO: 3, SEQ ID NO: 16, or SEQ ID NO: 31.
3. The AAV vector according to claim 1 or 2, wherein the promoter is an smCBA promoter.
4. The AAV vector according to any one of claims 1 to 3, wherein the connexin 26 protein is human CX26.
5. The AAV vector according to any one of claims 1 to 4, wherein the polynucleotide further comprises 5' and 3' inverted terminal repeat sequences (ITRs).
6. The AAV vector according to any one of claims 1 to 5, wherein the polynucleotide further comprises at least one copy of another miRNA target site selected from the miR183 family.
7. A pharmaceutical composition comprising an AAV vector according to any one of claims 1 to 6 and at least one pharmaceutically acceptable excipient or carrier.
8. An AAV vector according to any one of claims 1 to 6, or a pharmaceutical composition according to claim 7, for use as a medicine.
9. An AAV vector according to any one of claims 1 to 6, or a pharmaceutical composition according to claim 7, for use in the treatment of hereditary hearing loss in a subject requiring such treatment.
10. The hereditary hearing loss is non-symptomatic hearing loss (DFNB1), as described in claim 9, for use by the AAV vector or pharmaceutical composition.
11. The AAV vector or pharmaceutical composition for use according to claim 9 or 10, wherein the hereditary hearing loss is severe hereditary hearing loss.
12. The AAV vector or pharmaceutical composition for use according to claim 9 or 10, wherein the hereditary hearing loss is severe hereditary hearing loss.
13. The AAV vector or pharmaceutical composition for use according to claim 9 or 10, wherein the hereditary hearing loss is progressive hereditary hearing loss.
14. The subject is an adult, and the AAV vector or pharmaceutical composition for use according to any one of claims 9 to 13.
15. The subject is an infant or a child, as described in any one of claims 9 to 13, an AAV vector or pharmaceutical composition for use.

Description

This invention relates to a recombinant adeno-associated virus (AAV) vector encoding connexin 26 and its therapeutic use. The gap junction β2 (GJB2) protein, also known as connexin 26 (CX26), is a member of the connexin protein family, which includes 21 members in humans. Connexin proteins consist of one intracellular loop, two extracellular loops, and four transmembrane domains linked by the cytoplasmic N and C terminals. Connexin proteins are responsible for forming intercellular channels called gap junctions. Six connexin proteins assemble to form hexameric hemichannels called connexons, which originate in the cell membrane and bind to the extracellular portion of another connexon on the membrane of an adjacent cell. Gap junctions enable the intercellular diffusion of metabolites, ions, and second messenger molecules. The type of connexin protein forming the gap junction determines its size and the types of particles that pass through it. Connexin 26, in particular, is responsible for the transport of potassium ions (K+) and several small molecules. In humans, connexin 26 is expressed in many tissues throughout the body. In particular, connexin 26 is expressed in the inner ear, specifically in the non-sensory epithelial supporting cells of the cochlea surrounding sensory hair cells, the fibrous cells lining the inside of the cochlear duct, and the spiral ligament region associated with the stria vascularis. The gap junctions formed between the epithelial supporting cells and fibrous cells provide a pathway for potassium ions (K+) passing through the base of the hair cells to return to the endolymph above the hair cells. In 1994, locus 13q12 was first identified as being associated with recessive non-syndromic hearing loss, i.e., recessive hearing loss without other clinically recognizable features (Guilford P et al., "A non-syndrome form of neurosensory, recessive hearing loss maps to the pericentromeric region of chromosome 13q." Nat Genet. 1994 Jan;6(1):24-8). Shortly thereafter, the gene GJB2 encoding CX26 was identified as the causative gene (Kelsell). DP et al., "Connexin 26 mutations in hereditary non-syndromic sensorineural hearing loss. Nature. 1997 May 1;387(6628):80-3." Since then, autosomal recessive mutations in the GJB2 gene have been found to be the most common cause of moderate to severe non-syndromic hereditary hearing impairment (also known as non-syndromic hereditary hearing loss) in most populations. (Denoyelle F et al., "Prelingual hearing loss: high incidence of a 30delG mutation in the Connexin 26 gene.") The connexin 26 gene. Hum Mol Genet. 1997 Nov;6(12):2173-7; Kemperman MH et al., "Hearing loss and connexin 26." J R Soc Med. 2002 Apr;95(4):171-7. Non-symptomatic hearing loss caused by biallelegenic GJB2 variants is known as DFNB1. For many years, hearing aids or cochlear implants were the only available treatment options for individuals suffering from hereditary hearing loss. Today, gene therapy is a promising treatment for hereditary hearing loss (also known as hereditary hearing impairment), where the therapeutic gene is typically delivered to the inner ear via a viral vector, such as adeno-associated virus (AAV) vectors. However, a significant limitation of gene therapy is the potential lack of specificity in the type of cells transduced by the viral vector. As mentioned above, CX26 expression in the inner ear is limited to non-sensory supporting cells present in the epithelium and connective tissue. Consequently, the connexin protein is completely absent in the sensory hair cells of the cochlea. In fact, it has been shown that CX26 expression in cochlear inner ear hair cells is detrimental to the survival of these cells (Guo J et al., "GJB2 gene therapy and conditional deletion reveal developmental stage-dependent effects on inner ear structure and function." Mol The Methods Clin Dev. 2021 Oct). (1;23:319-333). Therefore, there is still a need for AAV vectors that enable controlled expression of CX26 in target cells and are thus suitable for gene therapy. In particular, there is still a need for AAV vectors that can prevent or suppress CX26 expression in cells and tissues that may induce some adverse effects. The inventors have surprisingly demonstrated that a recombinant AAV-DJ vector containing a polynucleotide comprising a sequence encoding CX26 operably linked to a promoter and a so-called "precursor miR183 target site" enables CX26 expression in non-sensory support cells of the cochlear while preventing CX26 expression in inner ear hair cells. Therefore, the inventors have demonstrated that administration of the recombinant AAV-DJ vector prevents hearing loss in a mouse model of hereditary hearing loss. Similar cellular patterns of regulatory effects on CX26 expression can be expected when using other "precursor miRNA target sites" of the miR183 family, namely the so-called "precursor miR182 target site" and the so-called "precursor miR96 target site." Therefore, these