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CN-121991947-A - GJB 2-delivering viral vectors and uses thereof

CN121991947ACN 121991947 ACN121991947 ACN 121991947ACN-121991947-A

Abstract

The present invention relates to an expression cassette for expressing a GJB2 protein comprising a cochlear support cell and a cochlear outer sidewall specific promoter and a nucleic acid encoding a GJB2 protein. The invention also relates to a recombinant adeno-associated viral vector comprising in its genome an expression cassette for expressing the GJB2 protein inserted between two ITR sequences, and a combination of at least two of said recombinant adeno-associated viral vectors. The recombinant adeno-associated viral vectors of the invention, or combinations thereof, can be used to treat or prevent hearing loss associated with deficiency in the GJB2 gene.

Inventors

  • CHAI RENJIE
  • CUI ZHIPING
  • SONG HUAIEN
  • SUN SIJIE
  • Qi Jieyu
  • TAN FANGZHI
  • Sun Qiuhan
  • ZHANG LIYAN
  • LU LING
  • ZHANG SHANZHONG
  • XU LEI
  • DING ZEYANG

Assignees

  • 苏州星奥拓维生物技术有限公司

Dates

Publication Date
20260508
Application Date
20241107

Claims (12)

  1. 1. An isolated promoter sequence comprising the nucleotide sequence set forth in SEQ ID No. 3, SEQ ID No.4, SEQ ID No. 5 or variants thereof having at least about 90% identity (e.g., having at least 95%, 96%, 97%, 98%, 99% or more identity), respectively; For driving the specific expression of exogenous nucleic acid of interest operatively linked thereto in inner ear support cells and cochlear outer side walls.
  2. 2. An expression cassette comprising an operably linked in the 5'-3' direction: (a) Promoter sequence elements, which are variant promoter sequences comprising the promoter sequences shown in SEQ ID NO.3, SEQ ID NO. 4, SEQ ID NO. 5 or having at least about 90% identity (e.g., at least 95%, 96%, 97%, 98%, 99% or more identity) to them, respectively, and (B) Nucleic acid encoding a mammalian, e.g., human, non-human primate, mouse, pig or rat functional GJB2 protein, e.g., the nucleic acid encoding a GJB2 protein has at least 85%, e.g., at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% identity to the sequence of SEQ ID No. 2; Optionally, the expression cassette is inserted between two ITR sequences.
  3. 3. The expression cassette according to claim 2, further comprising an additional expression regulatory element, for example, the expression regulatory element is a woodchuck hepatitis post-transcriptional regulatory element (WPRE) or variant thereof.
  4. 4. A nucleic acid vector, e.g., a nucleic acid expression vector, comprising the expression cassette of claim 2 or 3, e.g., two ITR sequences that are the same or different, e.g., the ITR sequences are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or AAV9.
  5. 5. Use of the expression cassette of claim 2 or 3 or the nucleic acid vector of claim 5 for the preparation of a recombinant adeno-associated virus (rAAV) vector, preferably a recombinant AAV-ie vector or a recombinant AAV1 vector.
  6. 6. A recombinant adeno-associated virus (rAAV) vector, preferably a recombinant AAV-ie vector or a recombinant AAV1 vector, comprising in its genome the expression cassette of claim 2 or 3 interposed between two ITR sequences, e.g. the two ITR sequences are identical or different, e.g. the ITR sequences are derived from AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8 or AAV9, preferably the genome of the recombinant adeno-associated virus vector is self-complementing to form a double stranded DNA molecule.
  7. 7. A combination of recombinant adeno-associated virus (rAAV) vectors, which is a combination of at least two recombinant adeno-associated virus (rAAV) vectors according to claim 6, preferably which is a combination of two recombinant adeno-associated virus (rAAV) vectors according to claim 6, e.g. which is a combination of a recombinant AAV-ie vector and a recombinant AAV1 vector.
  8. 8. A pharmaceutical composition comprising the nucleic acid vector of claim 4, the recombinant AAV viral vector of claim 6, or the combination of recombinant AAV viral vectors of claim 7, and a pharmaceutically acceptable excipient.
  9. 9. Use of the nucleic acid vector of claim 4, the recombinant AAV viral vector of claim 6, or the combination of recombinant AAV viral vectors of claim 7, for the preparation of a medicament for treating or preventing hearing loss associated with a deficiency in the GJB2 gene.
  10. 10. A medicament or formulation for treating or preventing hearing loss associated with deficiency of the GJB2 gene, prepared from the recombinant AAV viral vector of claim 6 or the combination of recombinant AAV viral vectors of claim 7, wherein the adeno-associated virus is packaged by transferring a packaging vector system of adeno-associated virus comprising the nucleic acid vector of claim 4 expressing the GJB2 protein, a vector carrying AAV rep and cap genes, and a helper viral vector into a host cell.
  11. 11. The medicament or formulation of claim 10, wherein the medicament or formulation further comprises a neutral salt buffer, an acidic salt buffer, a basic salt buffer, glucose, mannose, mannitol, a protein, a polypeptide, an amino acid, an antibiotic, a chelator, an adjuvant, a preservative, a nanoparticle, a liposome, and a positive lipid particle.
  12. 12. The medicament or formulation according to claim 10 or 11, wherein the administration by injection is through the round window, oval window, semicircular canal, manifold of the cochlea and single or multiple administrations throughout life, the total dose being 1 x 10 9 -1×10 13 viral genomes.

Description

GJB 2-delivering viral vectors and uses thereof Technical Field The present invention relates to AAV viral vectors that deliver gap junction β -2 (GJB 2) coding sequences, and their use in gene therapy, particularly in the treatment of hearing loss. Background Deafness is the most common clinical disability disease, has seriously affected normal life of human beings and has caused great burden to society. WHO reported that almost 15 million people worldwide had varying degrees of hearing loss, 4.66 million people had disabled hearing loss, accounting for 5% of the general population, with 3400 tens of thousands of children. It is expected that 25 hundred million people will suffer from varying degrees of hearing loss worldwide by 2050, and 7 hundred million people will suffer from disabled hearing loss. Genetic and environmental factors are two major factors causing deafness, and the environmental reasons are mainly related to various environmental factors or certain complications such as ototoxic drugs, pregnancy infection, neonatal hypoxia and radiation irradiation. The genetic factors are mainly caused by the defect of individual deafness genes, which leads to different degrees of hearing decline, and pathogenic genes are transmitted to the next generation through different genetic modes, and can occur at any age, and the influence is permanent. Inheritance is a main cause of deafness, the deafness caused by genetic factors accounts for about 60 percent, more than 120 genes are found to be related to the deafness, more than 1500 pathogenic variations are related, and no medicine for treating the hereditary deafness exists clinically until now. Hereditary hearing loss can be classified into a syndrome type hearing disorder (syndromic hearing loss, SHL) and a non-syndrome type hearing disorder (non-syndromic hearing loss, NSHL) according to whether there are clinical symptoms of other organs than the auditory system. Syndrome hearing impairment (SHL) is often accompanied by clinical manifestations of other systems, including eyes, heart, kidneys, nervous system, skin, bones, etc., accounting for 30% of hereditary hearing loss. SHL is most well known for pendered syndrome, usher Syndrome (USH) and Waadenburg Syndrome (WS), whereas USH, pendered syndrome and Jervell and Lange-Nielsen syndrome (JLNS) have successfully achieved inner ear gene therapy in preclinical animal model studies. There are four types of non-syndromic hearing disorders (NSHL), autosomal Dominant (DFNA), autosomal recessive (DFNB), X-chromosome linkage (DFNX) and mitochondrial non-syndromic deafness. About 70% of patients with hereditary hearing loss are non-syndromic. NSHL the most common genetic patterns are autosomal recessive inheritance (75% -80%), followed by autosomal dominant inheritance (20%), X chromosome linked inheritance (< 2%) and mitochondrial inheritance (< 1%). To date, 120 genes have been reported to be related to NSHL, of which 51 genes related to DFNA, 78 genes related to DFNB, and 10 genes related to DFNA and DFNB, CLOL A2, GJB6, MYO3A, MYO6, MYO7A, PTPRQ, TCB1D24, TECTA, TMC1 genes, and 5 genes related to DFNX, respectively. The GJB2 gene is the most common causative cause in NSHL, and about 50% of autosomal recessive inherited deafness patients are caused by GJB2 gene mutation. The GJB2 gene is a first-line detection gene for clinical deafness gene diagnosis. The GJB2 gene is located on human chromosome 13q12, encodes Gap Junction protein 26 (Connexin 26, also known as "Cx26 protein" or "GJB2 protein"), and is highly expressed in the cochlear Gap Junction (GJ). In normal people, the GJB2 protein encoded by the GJB2 gene and gap junction proteins of adjacent cells form a complete gap junction channel. This channel plays an important role in signal transduction and substance exchange, and is also an important channel for intercellular switching of electrolytes, second messengers and metabolites, and potassium ion circulation of cochlear hair cells and cochlear lymph is regulated by gap junction protein channels. Potassium ions are connected through the interstitials into the vessel vein and released by the intermediate cells into the interstices of the vessel vein where they return to the endolymph. When the coding region of the GJB2 gene is mutated, and the function of the GJB2 protein is changed, the structure of the gap junction protein is changed, so that the normal opening and closing of the gap junction protein channel are affected. Abnormal structure of gap junction proteins can cause the circulation of potassium ions back to endolymph fluid to be affected. When the concentration of potassium ions is changed and reaches a certain concentration, potassium poisoning and damage to cochlea hair cells can be caused, and sensorineural deafness can be caused. The human GJB2 gene is expressed in the support cells of the cochlea apparatus and in the outer wall of the cochlea. In view of the deficiency of GJB2, there rem