Adeno-associated virus (AAV) system for the treatment of hereditary hearing loss

Abstract

Described herein is an optimally modified GJB2 cDNA and associated genetic elements for use in recombinant adeno-associated virus (rAAV)-based gene therapy for the treatment of inherited hearing loss.

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Application No. 62 / 907,834, filed on September 30, 2019, the content of which is incorporated herein by reference in its entirety, under 35 U.S.C.§119(e).

[0002] Sequence Listing This application contains a Sequence Listing that has been electronically submitted in ASCII format and is hereby incorporated by reference in its entirety. The ASCII copy, created on September 28, 2020, is named 119561 - 01920_SL and is 31,079 bytes in size.

[0003] Field of the Invention The present invention relates to the field of gene therapy and includes AAV vectors for expressing isolated polynucleotides in a subject or cell. The present disclosure also relates to nucleic acid constructs, promoters, vectors, and host cells containing polynucleotides, as well as methods for delivering exogenous DNA sequences to target cells, tissues, organs, or organisms, and methods for use in the treatment or prevention of hereditary hearing loss.

Background Art

[0004] Gene therapy aims to improve the clinical outcome for patients suffering from genetic mutations or acquired diseases caused by abnormal gene expression profiles. Gene therapy includes the treatment or prevention of medical conditions resulting from gene deficiencies, or regulatory or expression abnormalities, such as underexpression or overexpression, for example, gene deficiencies, or disorders, malignancies, etc. that can result in impairments, diseases, malignancies, etc. For example, diseases or disorders caused by gene deficiencies may be treated, prevented, or improved by delivering corrective genetic material to the patient, or alternatively, may be treated, prevented, or improved by changing or silencing the gene deficiency with corrective genetic material that results in the therapeutic expression of genetic material in the patient, for example.

[0005] The basis of gene therapy is, for example, to supply a transcription cassette with an active gene product (sometimes referred to as a transgene or therapeutic nucleic acid) that can result in, for example, a positive gain-of-function effect, a negative loss-of-function effect, or another outcome. Such an outcome can be attributed to the expression of a therapeutic protein, such as an antibody, a functional enzyme, or a fusion protein. Gene therapy can also be used to treat diseases or malignancies caused by other factors. Human monogenic disorders can be treated by delivery and expression of a normal gene to target cells. Delivery and expression of a corrective gene to a patient's target cells can be carried out via a number of methods, including the use of engineered viruses and viral gene delivery vectors.

[0006] Adeno-associated virus (AAV) belongs to the family Parvoviridae and more specifically constitutes the genus Dependoparvovirus. Vectors derived from AAV (i.e., recombinant AAV (rAVV) or AAV vectors) are attractive for gene delivery because (i) they can infect (and be transduced into) a wide variety of non-dividing and dividing cell types, including muscle cells and neurons; (ii) they lack viral structural genes, thus attenuating the host cell response to viral infection, such as the interferon-mediated response; (iii) wild-type virus is thought to be non-pathogenic in humans; (iv) in contrast to wild-type AAV, which can integrate into the host cell genome, replication-deficient AAV vectors lack the rep gene and generally persist as episomes, thus limiting the risk of insertional mutagenesis or genotoxicity; and (v) AAV vectors are generally considered to be relatively low-level immunogens compared to other vector systems, thus not eliciting a significant immune response (see (ii)) and therefore allowing for persistence of the vector DNA and potentially long-term expression of the therapeutic transgene.

[0007] Non-syndromic hearing loss (DFNB1; also known as connexin 26 deafness) is autosomal recessive, congenital, non-progressive, and characterized by mild to severe sensorineural hearing impairment. The GJB2 gene encodes connexin 26, which forms gap junctions that control potassium homeostasis, expressed in cochlear supporting cells and crucial for the survival and function of hair cells and normal hearing. Mutations within GJB2 disrupt gap junctions and cochlear homeostasis, leading to hair cell dysfunction and hearing loss.

[0008] Hearing loss is the most common congenital sensory disorder. In developed countries such as the United States, hereditary mutations contribute to the majority of hearing loss in young children, estimated to affect 1 in 500 children before speech development (Shearer et al., "Hereditary Hearing Loss and Deafness Overview", 2017). Since congenital hearing loss is one of the most prevalent chronic conditions in children, newborn screening is mandated. In cases where a deficit is detected, genetic testing typically follows.

[0009] Genetic testing can be used to diagnose DFNB1 by identifying biallelic pathogenic variants in GJB2, including sequence variants and variants in upstream cis-regulatory elements that alter the expression of gap junction beta-2 protein (connexin 26). If GJB2 pathogenic variants causing DFNB1 are detected in affected family members, carrier testing for at-risk relatives, prenatal testing for high-risk pregnancies, and preimplantation genetic diagnosis are possible (Smith and Jones, "Nonsyndromic Hearing Loss and Deafness, DFNB1", 1998: edited by Adam et al., GeneReviews, University of Washington, Seattle; Kemperman et al., Journal of the Royal Society of Medicine, 2002, 95: 171-177). Since the cochlea is surgically accessible and topical application to an environment with relatively high immune defense is possible, gene therapy using viral vectors is an attractive approach for treating hearing loss.

Summary of the Invention

Problems to be Solved by the Invention

[0010] However, there is still a need for effective treatments for sensorineural hearing loss.

Means for Solving the Problems

[0011] The technology described herein is a method and composition for treating or preventing deafness by the expression of gap junction protein beta 2 (GJB2) from a recombinant adeno-associated virus (rAAV) vector, wherein the rAAV vector comprises a nucleic acid sequence of codon-optimized GJB2. In some embodiments, the rAAV vector comprises a nucleic acid sequence of GJB2 combined with a promoter that is codon-optimized and tested for optimal GJB2 expression. Accordingly, the present disclosure relates to an rAAV vector through which GJB2 can be packaged into the GJB2 gene for targeted delivery to patients suffering from hereditary deafness, including patients with autosomal recessive or dominant mutations. Mutations within GJB2 impair gap junctions and cochlear homeostasis, leading to hair cell dysfunction and deafness. The goal of the GJB2 gene therapy described herein is to restore functional gap junctions, preserve hair cells, and improve hearing.

[0012] In one aspect, the present disclosure presents an isolated polynucleotide comprising a nucleic acid sequence encoding GJB2. According to some embodiments, the nucleic acid sequence is a non-naturally occurring sequence. According to some embodiments, the nucleic acid sequence encodes mammalian GJB2. According to some embodiments, the nucleic acid sequence encodes human, mouse, or rat GJB2. According to some embodiments, the nucleic acid sequence comprises SEQ ID NO: 10. According to some embodiments, the nucleic acid sequence is codon-optimized for expression in mammals. According to some embodiments, the nucleic acid sequence comprises SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13.

[0013] According to some embodiments, the nucleic acid sequence comprises a sequence that is at least 85% identical to SEQ ID NO: 11, a sequence that is at least 85% identical to SEQ ID NO: 12, a sequence that is at least 85% identical to SEQ ID NO: 13, or a sequence that is at least 85% identical to SEQ ID NO: 18. According to some embodiments, the nucleic acid sequence is codon-optimized for expression in human cells, rat cells, or mouse cells. According to some embodiments, the nucleic acid sequence is a cDNA sequence. According to some embodiments, the nucleic acid sequence further comprises a hemagglutinin (HA) C-terminal tag operably linked thereto. According to some embodiments, the nucleic acid sequence is operably linked to a promoter. According to some embodiments, the promoter is a ubiquitous active CBA, a mini CBA (smCBA), EF1a, a CASI promoter, a supporting cell promoter of the snail, a GFAP promoter specific for GJB2 expression, a mini GJB2 promoter, a medium GJB2 promoter, a large GJB2 promoter, or a sequence combination of two to three individual promoters specific for GJB2 expression. According to some embodiments, the promoter is optimized to drive a high level of GJB2 expression. According to some embodiments, the nucleic acid sequence further comprises a 3'UTR regulatory region operably linked thereto and containing a WPRE (Woodchuck Hepatitis Virus Postranscriptional Regulatory Element). According to some embodiments, the nucleic acid sequence further comprises a polyadenylation signal operably linked thereto. According to some embodiments, the polyadenylation signal is an SV40 polyadenylation signal. According to some embodiments, the polyadenylation signal is a human growth hormone (hGH) polyadenylation signal.According to some embodiments, a polynucleotide according to any of the aspects and embodiments herein is operably linked to the following promoter elements optimized to drive high-level GJB2 expression: (a) ubiquitous active CBA, small CBA (smCBA), EF1a, or CASI promoter; (b) one of the snail supporting cell promoter or the 1.68 kb GJB2 expression-specific GFAP promoter, small / medium / large GJB2 promoter, or the sequence combination of two to three individual GJB2 expression-specific promoters; and further comprises a 27-nucleotide hemagglutinin C-terminal tag operably linked to a 3'UTR regulatory region containing the WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element), followed by the polyadenylation signal of SV40 or human growth hormone (hGH).

[0014] According to another aspect, the present disclosure presents a host cell comprising a polynucleotide according to any of the aspects or embodiments herein. According to some embodiments, the host cell is a mammalian cell. According to some embodiments, the host cells are HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5. According to some embodiments, the host cell is a BHK cell.

[0015] According to another aspect, the present disclosure presents a recombinant herpes simplex virus (rHSV) comprising a polynucleotide according to any one of the aspects or embodiments herein.

[0016] According to another aspect, the present disclosure presents a polynucleotide according to any one of the aspects and embodiments herein; and a transgene expression cassette comprising a minimal regulatory element.

[0017] According to another aspect, the present disclosure presents a nucleic acid vector comprising an expression cassette according to any of the aspects or embodiments herein. According to some embodiments, the vector is an adeno-associated virus (AAV) vector.

[0018] According to another aspect, the present disclosure presents a host cell comprising a transgene expression cassette according to any of the aspects or embodiments herein.

[0019] According to another aspect, the present disclosure presents a kit comprising an expression vector according to any of the aspects or embodiments herein and instructions for use.

[0020] An expression vector comprising a polynucleotide according to any of the aspects or embodiments herein. According to some embodiments, the vector is an adeno-associated virus (AAV) vector. According to some embodiments, the serotype of the capsid sequence and the serotype of the ITR of the AAV vector are independently selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0021] In accordance with another aspect, the present disclosure presents a recombinant adeno-associated (rAAV) expression vector comprising a polynucleotide according to any of the aspects or embodiments herein and an AAV genome cassette. In accordance with some embodiments, the AAV genome cassette is flanked by two sequence-modulating inverted terminal repeats (ITRs) that are preferably about 143 bases in length. In accordance with some embodiments, the AAV genome cassette is separated by an scAAV-activating ITR (ITRΔtrs) of about 113 bases and flanked at both ends by two inverted identical repeats (IIRs) that are preferably 2.4 kb or less and are flanked by a self-complementary AAV (scAAV) genome cassette consisting of two IIRs. In accordance with some embodiments, the expression vector further comprises a protein capsid variant that is optimized for delivery into the cochlea.

[0022] According to another aspect, the present disclosure provides a polynucleotide according to any of the aspects or embodiments herein, optionally with a hemagglutinin C-terminal tag, preferably about 27 nucleotides in length, or without it, an optionally about 0.68 kilobase (kb) sized codon / sequence optimized human GJB2 cDNA, optimized to drive high levels of GJB2 expression, operably linked to the following promoter elements: (a) a ubiquitous active CBA, preferably about 1.7 kb in size, a small CBA (smCBA), preferably about 0.96 kb in size, an EF1a, preferably about 0.81 kb in size, or a CASI promoter, preferably about 1.06 kb in size; (b) a snail supporting cell promoter, or preferably a GJB2 expression specific GFAP promoter, preferably about 1.68 kb in size, a small GJB2 promoter, preferably about 0.13 kb in size, a medium GJB2 promoter, preferably about 0.54 kb in size, a large GJB2 promoter, preferably about 1.0 kb in size, or one of the sequence combinations of two to three individual GJB2 expression specific promoters; a codon / sequence optimized human GJB2 cDNA operably linked to a 0.9 kb 3'UTR regulatory region containing a WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) and followed by a polyadenylation signal of SV40 or human growth hormone (hGH); a self-complementary AAV (scAAV) genomic cassette consisting of two inverted identical repeats (IIRs), preferably 2.4 kb or less, separated by two approximately 143 base sequence-modulated inverted terminal repeats (ITRs) or approximately 113 base scAAV-activating ITRs (ITRΔtrs) and flanked at both ends by approximately 143 base sequence-modulated ITRs; and further comprising a protein capsid variant suitable for targeted in vivo delivery to the cochlea. In some embodiments, the polyadenylation signal is a polyadenylation signal of SV40 or human growth hormone (hGH).According to some embodiments, the promoter is optimized to drive high levels of GJB2 expression. According to some embodiments, the rAAV is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, rh-AAV10, AAV10, AAV11, and AAV12. According to some embodiments, the rAAV is AAV1, which is a serotype. According to some embodiments, the rAAV is AAV2, which is a serotype. According to some embodiments, the rAAV is AAV3, which is a serotype. According to some embodiments, the rAAV is AAV4, which is a serotype. According to some embodiments, the rAAV is AAV5, which is a serotype. According to some embodiments, the rAAV is AAV6, which is a serotype. According to some embodiments, the rAAV is AAV7, which is a serotype. According to some embodiments, the rAAV is AAV8, which is a serotype. According to some embodiments, the rAAV is AAV9, which is a serotype. According to some embodiments, the rAAV is rh-AAV10, which is a serotype. According to some embodiments, the rAAV is AAV10, which is a serotype. According to some embodiments, the rAAV is AAV11, which is a serotype. According to some embodiments, the rAAV is AAV12, which is a serotype. According to some embodiments, the rAAV is contained within the AAV virion.

[0023] In another aspect, the disclosure presents a recombinant herpes simplex virus (rHSV) comprising an expression vector according to any of the aspects or embodiments herein.

[0024] In accordance with another aspect, the present disclosure presents a polynucleotide comprising the following sequence: CBA-GJB2(X)-HA-WPRE-pA [wherein, in the sequence, X comprises a nucleic acid sequence that is at least 85% identical to SEQ ID NO: 18]. In accordance with another aspect, the present disclosure presents a polynucleotide comprising the following sequence: CBA-GJB2(X)-HA-WPRE-pA [wherein, in the sequence, X comprises a nucleic acid sequence that is at least 90% identical to SEQ ID NO: 18]. In accordance with another aspect, the present disclosure presents a polynucleotide comprising the following sequence: CBA-GJB2(X)-HA-WPRE-pA [wherein, in the sequence, X comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO: 18]. In accordance with another aspect, the present disclosure presents a polynucleotide comprising the following sequence: CBA-GJB2(X)-HA-WPRE-pA [wherein, in the sequence, X comprises a nucleic acid sequence consisting of SEQ ID NO: 18].

[0025] In accordance with another aspect, the present disclosure presents a host cell comprising an expression vector according to any one of the aspects or embodiments herein. In accordance with some embodiments, the host cell is a mammalian cell. In accordance with some embodiments, the host cell is HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5. In accordance with some embodiments, the host cell is a BHK cell.

[0026] In accordance with another aspect, the present disclosure presents a transgene expression cassette comprising a polynucleotide according to any one of the aspects or embodiments herein; and a minimal regulatory element. In accordance with another aspect, the present disclosure presents a nucleic acid vector comprising an expression cassette according to any of the aspects and embodiments herein. In accordance with some embodiments, the vector is an adeno-associated virus (AAV) vector.

[0027] In accordance with another aspect, the present disclosure presents a kit comprising an expression vector according to any of the aspects and embodiments herein and instructions for use.

[0028] According to another aspect, the present disclosure presents a composition comprising a polynucleotide according to any of the aspects and embodiments herein.

[0029] According to another aspect, the present disclosure presents a composition comprising a host cell according to any of the aspects and embodiments herein.

[0030] According to another aspect, the present disclosure presents a composition comprising a recombinant herpes simplex virus (rHSV) according to any of the aspects and embodiments herein.

[0031] According to another aspect, the present disclosure presents a composition comprising a transgene expression cassette according to any of the aspects and embodiments herein.

[0032] According to another aspect, the present disclosure presents a composition comprising an expression vector according to any of the aspects and embodiments herein. According to some embodiments, the composition is a pharmaceutical composition.

[0033] According to another aspect, the present disclosure presents a method of treating hereditary hearing loss, the method comprising administering a polynucleotide according to any of the aspects and embodiments herein to a subject in need thereof.

[0034] According to another aspect, the present disclosure presents a method of preventing hereditary hearing loss, the method comprising administering a polynucleotide according to any of the aspects and embodiments herein to a subject in need thereof.

[0035] According to another aspect, the present disclosure presents a method of treating or preventing hereditary hearing loss, the method comprising administering a transgene expression cassette according to any of the aspects and embodiments herein to a subject in need thereof.

[0036] According to another aspect, the present disclosure presents a method of treating or preventing hereditary hearing loss, the method comprising administering an expression vector according to any of the aspects and embodiments herein to a subject in need thereof.

[0037] According to another aspect, the present disclosure presents a method of treating or preventing hereditary hearing loss, the method comprising administering a recombinant adeno-associated (rAAV) expression vector according to any of the aspects and embodiments herein to a subject in need thereof.

[0038] According to another aspect, the present disclosure presents a method of treating or preventing hereditary hearing loss, the method comprising administering recombinant adeno-associated (rAAV) viral particles comprising a polynucleotide according to any of the aspects and embodiments herein to a subject in need thereof. According to some embodiments, the hereditary hearing loss is DFNB1 hearing loss. According to some embodiments, the hereditary hearing loss is caused by a mutation in GJB2. According to some embodiments, the hereditary hearing loss is caused by an autosomal recessive GJB2 mutant (DFNB1). According to some embodiments, the hereditary hearing loss is caused by an autosomal dominant GJB2 mutant (DFNA3A). According to some embodiments, the administration is administration to the cochlea. According to some embodiments, the administration is intravenous administration, intraventricular administration, intracochlear administration, intrathecal administration, or a combination thereof.

[0039] According to some embodiments of the aspects and embodiments herein, the subject is a pediatric subject. According to some embodiments, the subject is an infant.

[0040] According to another aspect, the present disclosure provides a method for producing recombinant AAV viral particles, comprising co-infecting suspension cells with a first recombinant herpes virus comprising a nucleic acid encoding an AAV rep gene and an AAV cap gene, each operably linked to a promoter; and a second recombinant herpes virus comprising a GJB2 gene and a promoter operably linked to said gene; and causing the cells to produce recombinant AAV viral particles, thereby producing recombinant AAV viral particles. According to some embodiments, the cells are HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5. According to some embodiments, the cells are infected at a multiplicity of co-infection (MOI) between 3 and 14. According to some embodiments, the cap gene is selected from AAV having a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, rh-AAV10, AAV11, and AAV12. According to some embodiments, the first herpes virus and the second herpes virus are viruses selected from the group consisting of cytomegalovirus (CMV), herpes simplex virus (HSV), varicella zoster virus (VZV), and Epstein-Barr virus (EBV). According to some embodiments, the herpes virus is a replication-deficient virus. According to some embodiments, the co-infection is a simultaneous infection.

Brief Description of the Drawings

[0041]

Figure 1A

Figure 1B

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14

Figure 15

Figure 16

Figure 17

Figure 18A

Figure 18B

Figure 19A

Figure 19B

Figure 19C

Figure 20

Figure 21

Figure 22

Figure 23A

Figure 23B

Figure 24A

Figure 24B

Figure 25

Figure 26

Figure 27A

Figure 27B

Figure 28A

Figure 28B

Figure 29

Figure 30

Figure 31A

Figure 31B

Figure 32

Figure 33A

Figure 33B

Mode for Carrying Out the Invention

[0042] Definitions Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The following references: Singleton et al., "Dictionary of Microbiology and Molecular Biology" (2nd ed., 1994); "Cambridge Dictionary of Science and Technology" (ed. Walker, 1988); "Glossary of Genetics", 5th ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale and Marham, "Harper Collins Dictionary of Biology" (1991) give general definitions for many of the terms used in this invention to those of ordinary skill in the art. Unless otherwise specified, the following terms used herein have the meanings ascribed to them below.

[0043] As used herein, terms such as "administering", "administering of", "administration" are intended to refer to the methods used to enable delivery of a therapeutic agent or pharmaceutical composition to the desired biological site of action.

[0044] As used herein, the term "AAV virion" is intended to broadly refer to intact virus particles, such as wild-type AAV virion particles that contain single-stranded genomic DNA packaged into AAV capsid proteins. The single-stranded nucleic acid molecule can be either the sense strand or the antisense strand, as either strand is equally infectious. The term "rAAV virus particle" refers to recombinant AAV virus particles, i.e., particles that are infectious but replication-deficient. rAAV virus particles contain single-stranded genomic DNA packaged into AAV capsid proteins.

[0045] As used herein, the term "bioreactor" is intended to broadly refer to any device that can be used for the purpose of culturing cells.

[0046] As used herein, the term "carrier" is intended to include any solvent, dispersion medium, vehicle, coating, diluent, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffer solutions, carrier solutions, suspensions, colloids, etc., and all combinations of these carriers. The use of such media and agents for pharmaceutically active substances is well known in the art. Supplementary active ingredients can also be incorporated into the compositions. The phrase "pharmaceutically acceptable" refers to molecular entities and compositions that do not produce toxicity, allergic reactions, or similar undesirable reactions when administered to a host.

[0047] As used herein, the term "gene" or "coding sequence" is intended to broadly refer to a DNA region (transcribed region) that encodes a protein. When placed under the control of appropriate regulatory regions, such as a promoter, the coding sequence is transcribed (DNA) and translated into a polypeptide (RNA). A gene can include several operably linked segments, such as a promoter, a 5'-leader sequence, a coding sequence, and a 3'-untranslated sequence containing a polyadenylation site. The phrase "gene expression" refers to the process by which a gene is transcribed into RNA and / or translated into an active protein.

[0048] The term "flanking" refers to the relative position of one nucleic acid sequence with respect to another nucleic acid sequence. Generally, in the sequence ABC, B is sandwiched between A and C. The same holds true for the arrangement AxBxC. Thus, a flanking sequence precedes or follows the sandwiched sequence, but need not be contiguous or directly adjacent to the sandwiched sequence.

[0049] As used herein, the term "functional variant of a gene" includes variants of a gene that do not significantly alter gene function, such as minor mutations including silent mutations, single nucleotide polymorphisms, missense mutations, and other mutations or deletions.

[0050] As used herein, the term "gene delivery" refers to the process by which foreign DNA is introduced into a host cell for the application of gene therapy.

[0051] As used herein, the term "gene of interest (GOI)" broadly refers to a heterologous sequence introduced into an AAV expression vector, typically a nucleic acid sequence encoding a protein that is used therapeutically in humans or animals.

[0052] As used herein, the term "hearing loss" is intended to refer to a reduction in sensitivity to sounds that are normally audible to a subject. The severity of hearing loss is classified according to the increase in volume above normal levels required before a listener can detect it. According to some embodiments, hearing loss can be characterized by an increase in the threshold volume at which an individual perceives tones of different frequencies.

[0053] As used herein, the terms "herpesvirus" or "Herpesviridae family" are intended to broadly refer to the common family of enveloped double-stranded DNA viruses with relatively large genomes. The Herpesviridae family replicates in the nuclei of a wide range of vertebrate and invertebrate hosts, and in preferred embodiments, mammalian hosts such as humans, horses, cows, mice, and pigs. Exemplary members of the Herpesviridae family include cytomegalovirus (CMV), herpes simplex virus type 1 and herpes simplex virus type 2 (HSV1 and HSV2), and varicella-zoster virus (VZV), and Epstein-Barr virus (EBV).

[0054] As used herein, the term "heterologous" means derived from an entity that is significantly different in genotype from the remainder of the entity to which it is being compared, introduced, or incorporated. For example, a polynucleotide introduced into a different cell type by genetic manipulation methods is a heterologous polynucleotide (and, when expressed, can encode a heterologous polypeptide). Similarly, an intracellular sequence (e.g., a gene or a portion thereof) incorporated into a viral vector is a heterologous nucleotide sequence with respect to the vector.

[0055] As used herein, the terms "increase", "enhance", "raise" (and similar terms) generally refer to the act of directly or indirectly increasing a concentration, level, function, activity, or behavior as compared to natural conditions, predicted conditions, or average conditions, or as compared to control conditions.

[0056] As used herein, the term "infection" is intended to broadly refer to the delivery of foreign DNA into cells by a virus. As used herein, the term "coinfection" means "simultaneous infection", "dual infection", "multiple infection", or "sequential infection" with two or more viruses. The infection of a producer cell with two (or more) viruses is referred to as "coinfection". The term "transfection" refers to the process of delivering foreign DNA into a cell by physical or chemical means, such as plasmid DNA introduced into the cell by electroporation, calcium phosphate precipitation, or other methods well known in the art.

[0057] As used herein, the term "inner ear cell" or "cells of the inner ear" refers to inner hair cells (IHC) and outer hair cells (OHC) of the inner ear, spiral ganglion neurons, vestibular hair cells of the inner ear, vestibular ganglion neurons of the inner ear, supporting cells, and cells within the stria vascularis. A supporting cell refers to a non-excitable cell in the ear, e.g., a cell that is not a hair cell or a neuron.

[0058] As used herein, the term "ITR (inverted terminal repeat)" sequence or "ITR" sequence is intended to refer to a relatively short sequence found at the ends of a viral genome that has an inverted orientation. The term "AAV ITR (inverted terminal repeat)" sequence, which is well understood in the art, is a sequence of approximately 145 nucleotides that exists at both ends of the native single-stranded AAV genome. The outermost nucleotides of the ITR can exist in one of two alternative orientations, resulting in heterogeneity between different AAV genomes and between the two ends of a single AAV genome.

[0059] "Wild-type ITR", "WT-ITR", or "ITR" refers to, for example, the sequence of a naturally occurring ITR sequence in AAV or other genus of Dependovirus that retains Rep binding activity and Rep nicking ability. The nucleotide sequence of WT-ITR derived from any AAV serotype may vary slightly from the canonical naturally occurring sequence due to degeneracy or drift of the genetic code, and thus the WT-ITR sequences included for use herein include WT-ITR sequences as a result of naturally occurring changes (e.g., replication errors) that occur during the production process. According to some embodiments, the ITR is a WT AAV2 ITR.

[0060] As used herein, the term "TR (terminal repeat)" or "TR" includes any viral TR (terminal repeat), or a synthetic sequence that includes at least one minimal essential origin of replication and a region containing a palindromic hairpin structure. The Rep binding sequence ("RBS") (also referred to as RBE (Rep binding element)) and the "TRS" (terminal resolution site) together constitute the "minimal essential origin of replication", and thus the TR includes at least one RBS and at least one TRS. TRs that are reverse complements of each other within a given run of a polynucleotide sequence are typically each referred to as "ITR (inverted terminal repeat)" or "ITR". In the context of a virus, the ITR mediates replication, viral packaging, integration, and proviral rescue.

[0061] The term "in vivo" refers to an assay or process that occurs in or within an organism such as a multicellular animal. In some of the aspects described herein, a method or use is said to occur "in vivo" when a unicellular organism such as a bacterium is used. The term "ex vivo" refers to methods and uses that are carried out, inter alia, using live cells with intact membranes that are outside a multicellular animal or plant, such as explants, cultured cells including one or more primary cells and cell lines, transformed cell lines, and extracted cells including extracted tissue or blood cells. The term "in vitro" refers to assays and methods that do not require the presence of cells with intact membranes, such as cell extracts, and may refer to the introduction of programmable synthetic biological circuits into non-cell systems such as media that do not contain cell systems such as cells or cell extracts.

[0062] As used herein, the term "isolated" molecule (e.g., isolated nucleic acid or isolated protein or isolated cell) means that it has been identified, separated and / or recovered from the components of its natural environment.

[0063] As used herein, the term "middle ear" is intended to refer to the space between the tympanic membrane and the inner ear.

[0064] As used herein, the term "minimal regulatory element" is intended to refer to a regulatory element that is necessary for the efficient expression of a gene in a target cell and that should be included within a transgene expression cassette. Such sequences may include, for example, promoter sequences or enhancer sequences, polylinker sequences that facilitate the insertion of DNA fragments into plasmid vectors, and sequences that contribute to intron splicing and polyadenylation of mRNA transcripts.

[0065] As used herein, the terms "minimize", "reduce", "decrease", and / or "inhibit" (and similar terms) generally refer to the act of directly or indirectly reducing a concentration, level, function, activity, or behavior as compared to natural, predicted, or average conditions, or as compared to a control condition.

[0066] As used herein, the term "non-naturally occurring" is intended to broadly refer to proteins, nucleic acids, ribonucleic acids, or viruses that do not occur in nature. For example, a "non-naturally occurring" entity can be a genetically modified variant, such as a cDNA or a codon-optimized nucleic acid.

[0067] As used herein, "nucleic acid" or "nucleic acid molecule" is intended to refer to a molecule composed of a chain of monomeric nucleotides, such as a DNA molecule (e.g., cDNA or genomic DNA). The nucleic acid can encode, for example, a promoter, the GJB2 gene or a portion thereof, or a regulatory element. The nucleic acid molecule can be single-stranded or double-stranded. "GJB2 nucleic acid" refers to a nucleic acid comprising the GJB2 gene or a portion thereof, or a functional variant of the GJB2 gene or a portion thereof. Functional variants of a gene include variants of the gene that do not significantly alter gene function and that are accompanied by minor mutations such as silent mutations, single nucleotide polymorphisms, missense mutations, and other mutations or deletions.

[0068] The asymmetric ends of a DNA strand and an RNA strand are called the 5' (5 prime) end and the 3' (3 prime) end. The 5' end has a terminal phosphate group, and the 3' end has a terminal hydroxyl group. The 5 prime (5') end has the fifth carbon of the sugar ring of deoxyribose or ribose at its terminus. Since the polymerase used to assemble a new strand joins to the 3'-hydroxyl (-OH) group of each new nucleotide via a phosphodiester bond, nucleic acids are synthesized in the 5'-3' direction in vivo.

[0069] As used herein, the term "nucleic acid construct" refers to a nucleic acid molecule that is single-stranded or double-stranded, isolated from a naturally occurring gene, modified to contain segments of nucleic acids in a form that does not occur naturally, or synthesized. When a nucleic acid construct contains the regulatory sequences required for the expression of the coding sequences of the present disclosure, the term nucleic acid construct is synonymous with the term "expression cassette".

[0070] A DNA sequence that "encodes" a particular GJB2 protein is a nucleic acid sequence that is transcribed into a particular RNA and / or protein. A DNA polynucleotide may encode an RNA (mRNA) that is translated into a protein, or a DNA polynucleotide may encode an RNA that is not translated into a protein (e.g., tRNA, rRNA, or DNA targeting RNA; also referred to as "non-coding" RNA or "ncRNA").

[0071] As used herein, the terms "operably linked" or "operatively linked" or "coupled" may refer to the juxtaposition of genetic elements in a relationship that allows the elements to operate in a predicted manner. For example, a promoter can be operably linked to a coding region if the promoter aids in initiating transcription of the coding sequence. There may be intervening residues between the promoter and the coding region as long as this functional relationship is maintained.

[0072] As used herein, "percent identity (%) of a sequence" relative to a reference polypeptide sequence or a reference nucleic acid sequence is defined as the percentage of amino acid residues or nucleotides in a candidate sequence that are identical to the amino acid residues or nucleotides in the reference polypeptide sequence or reference nucleic acid sequence, after aligning the sequences to achieve the maximum percent sequence identity, introducing gaps if necessary, and considering any conservative substitutions not as part of the sequence identity. Alignments for the purpose of determining the percent identity of amino acid or nucleic acid sequences are within the skill in the art and can be achieved in a variety of ways, for example, as described in "Current Protocols in Molecular Biology" (Ausubel et al., eds., 1987), Supplement 30, Section 7.7.18, Table 7.7.1, and can be accomplished using publicly available computer software programs including BLAST software, BLAST-2 software, ALIGN software, or Megalign (DNASTAR) software. An example of an alignment program is ALIGN Plus (Scientific and Educational Software, Pennsylvania). One of ordinary skill in the art can determine appropriate parameters for measuring an alignment, including any algorithms required to achieve a maximum alignment over the full length of the sequences being compared. For the purposes of this specification, the percent identity of an amino acid sequence A to, with, or against a given amino acid sequence B (which can be rephrased as a given amino acid sequence A having or containing the percent identity of a particular amino acid sequence to, with, or against a given amino acid sequence B) is as follows: 100 × ratio X / Y [where X is the number of amino acid residues scored as identical matches by a sequence alignment program in the alignment of A and B by the program, and Y is the total number of amino acid residues in B]. It will be understood that if the length of amino acid sequence A is not equal to the length of amino acid sequence B, the percent amino acid sequence identity of A to B will not be equal to the percent amino acid sequence identity of B to A.For the purposes of this specification, the percent identity of a given nucleic acid sequence C to, with, or against a given nucleic acid sequence D (which can be rephrased as a given nucleic acid sequence C having or containing the percent identity of a particular nucleic acid sequence to, with, or against a given nucleic acid sequence D) is as follows: 100 × ratio W / Z [where W is the number of nucleotides scored as identical matches by a sequence alignment program in an alignment of C and D by the program, and Z is the total number of nucleotides in D]. It will be understood that if the length of nucleic acid sequence C is not equal to the length of nucleic acid sequence D, the percent nucleic acid sequence identity of C to D will not be equal to the percent nucleic acid sequence identity of D to C.

[0073] As used herein, the terms "pharmaceutical composition" or "composition" are intended to refer to a composition or agent described herein (e.g., a recombinant adeno-associated (rAAV) expression vector) optionally mixed with at least one pharmaceutically acceptable chemical component, such as, but not limited to, a carrier, stabilizer, diluent, dispersant, suspending agent, thickening agent, excipient, and the like.

[0074] As used herein, the terms "polypeptide" and "protein" are used interchangeably to refer to a polymer of amino acid residues and are not limited to a minimum length. Such a polymer of amino acid residues may contain natural amino acid residues or non-natural amino acid residues and includes, but is not limited to, peptides, oligopeptides, dimers, trimers, and multimers of amino acid residues. By definition, both full-length proteins and fragments thereof are included. The terms also include post-expression modifications of polypeptides, such as glycosylation, sialylation, acetylation, phosphorylation, etc. Further, for the purposes of the present disclosure, "polypeptide" refers to a protein that includes modifications such as deletions, additions, and substitutions (generally conservative in nature) to the native sequence, so long as the desired activity is maintained. These modifications may be intentional, through site-directed mutagenesis, or may be accidental, such as through mutations in the host producing the protein or errors resulting from PCR amplification.

[0075] As used herein, the term "promoter" is intended to refer to a region of DNA that facilitates the transcription of a particular gene. As part of the transcription process, an enzyme that synthesizes RNA, known as RNA polymerase, binds to the DNA in the vicinity of the gene. A promoter contains specific DNA sequences and response elements that provide an initial binding site for RNA polymerase and transcription factors that recruit RNA polymerase. According to some embodiments, the promoter is highly specific for the expression of supporting cells in the cochlea. According to some embodiments, the promoter is the endogenous GJB2 promoter. According to some embodiments, the promoter is selected from the group consisting of the CBA promoter, the smCBA promoter, the CASI promoter, the GFAP promoter, and the elongation factor 1 alpha (EF1a) promoter. The term "chicken beta-actin (CBA) promoter" refers to a polynucleotide sequence derived from the chicken beta-actin gene (e.g., Gallus beta-actin represented by GenBank Entrez Gene ID: 396526). The "smCBA" promoter refers to a miniaturized form of a CMV-chicken beta-actin promoter hybrid. The "CASI" promoter refers to a promoter that includes a portion of the CMV enhancer, a portion of the chicken beta-actin promoter, and a portion of the UBC enhancer.

[0076] As used herein, the term "enhancer" refers to a cis-acting regulatory sequence (e.g., 50 to 1,500 base pairs) that binds to one or more proteins (e.g., activator proteins or transcription factors) to increase the transcriptional activation of a nucleic acid sequence. Enhancers may be located up to 1,000,000 base pairs upstream or downstream of the gene start site that they regulate.

[0077] A promoter may be said to drive the expression of a nucleic acid sequence it regulates, or may be said to drive this transcription. The phrases "operably linked", "operatively disposed", "operably connected", "under control", and "under transcriptional control" indicate that the promoter is in the correct functional position and / or orientation in relation to the nucleic acid sequence it regulates so as to control the initiation and / or expression of the transcription of this sequence. As used herein, an "inverted promoter" refers to a promoter in which the nucleic acid sequence is in an inverted orientation such that the sequence that was the coding strand is now the non-coding strand and vice versa. Inverted promoter sequences can be used in various embodiments to regulate the state of a switch. Additionally, in various embodiments, a promoter can be used in conjunction with an enhancer.

[0078] A promoter can be a promoter that naturally associates with a sequence obtainable by isolating a 5' non-coding sequence located upstream of a gene or the coding segment and / or exon of a given gene or sequence. Such a promoter can be referred to as "endogenous". Similarly, in some embodiments, an enhancer can be an enhancer that naturally associates with a nucleic acid sequence located downstream or upstream of this sequence.

[0079] In some embodiments, the coding nucleic acid segments are placed under the control of a "recombinant promoter" or "heterologous promoter", which refers to a promoter that, in their native environment, does not normally associate with the coding nucleic acid sequence to which it is operably linked. A recombinant enhancer or heterologous enhancer refers to an enhancer that, in its native environment, does not normally associate with a given nucleic acid sequence. Such promoters or enhancers can include promoters or enhancers of other genes; promoters or enhancers isolated from any other prokaryote, virus, or eukaryotic cell; and synthetic promoters or synthetic enhancers that are not "naturally occurring", i.e., different elements of different transcriptional regulatory regions and / or mutations that alter expression via genetic manipulation methods known in the art.

[0080] As used herein, the term "recombinant" may refer to (1) a polynucleotide that has been removed from its naturally occurring environment, (2) a polynucleotide that is not associated with all or part of a polynucleotide in which the gene is found in nature, (3) a polynucleotide that is operably linked to a polynucleotide that is not linked in nature, or (4) a biomolecule that does not occur in nature, such as a gene or protein. The term "recombinant" may be used with reference to cloned DNA isolates, chemically synthesized polynucleotide analogs, or polynucleotide analogs biologically synthesized by heterologous systems, as well as proteins and / or mRNAs encoded by such nucleic acids.

[0081] As used herein, the terms "recombinant HSV," "rHSV," and "rHSV vector" are intended to broadly refer to isolated genetically modified forms of herpes simplex virus type 1 (HSV) that contain a heterologous gene incorporated into the viral genome. The terms "rHSV-rep2cap2" or "rHSV-rep2cap1" mean an rHSV in which the AAV rep gene and cap gene, derived from AAV serotype 1 or 2, are incorporated into the rHSV genome, and in certain embodiments, mean an rHSV in which a DNA sequence encoding a therapeutic gene of interest is incorporated into the viral genome.

[0082] As used herein, a "subject" or "patient" or "individual" to be treated by the methods of the invention is intended to refer to a human or non-human animal. "Non-human animal" includes any vertebrate or invertebrate organism. Human subjects can be of any age group, gender group, racial group, or ethnic group, such as Caucasian (white), Asian, African, black, African American, African European, Hispanic, Middle Eastern, etc. In some embodiments, the subject can be a patient, or another subject in a clinical setting. In some embodiments, the subject is already undergoing treatment. In some embodiments, the subject is a neonate, infant, child, juvenile, or adult.

[0083] As used herein, the term "therapeutic effect" refers to the outcome of a treatment whose result is judged to be desirable and beneficial. A therapeutic effect can directly or indirectly include the arrest, reduction, or disappearance of disease manifestation. A therapeutic effect can also directly or indirectly include the arrest, reduction, or disappearance of the progression of disease manifestation.

[0084] For any therapeutic agent described herein, a therapeutically effective amount can first be determined from preliminary in vitro studies and / or animal models. A therapeutically effective dose can also be determined from human data. The dose applied can be adjusted based on the relative bioavailability and efficacy of the compound being administered. Adjusting the dose to achieve maximal efficacy, based on the methods described above and other well-known methods, is within the capabilities of one of ordinary skill in the art. The general principles for determining therapeutic effectiveness, which can be found in Chapter 1 of “Goodman and Gilman’s The Pharmacological Basis of Therapeutics”, 10th Edition, McGraw-Hill (New York) (2001), which is incorporated herein by reference, are summarized below.

[0085] As used herein, the term “transgene” is intended to refer to a polynucleotide that is introduced into a cell and is capable of being transcribed into RNA and, optionally, translated and / or expressed under appropriate conditions. In multiple embodiments, the transgene confers a desired property to the cell into which it is introduced or otherwise results in a desired therapeutic or diagnostic outcome.

[0086] As used herein, “transgene expression cassette” or “expression cassette” are used interchangeably and refer to a linear concatenation of nucleic acids that contains a transgene operably linked to one or more promoters or other regulatory sequences sufficient to direct transcription of the transgene, but does not contain a capsid coding sequence, other vector sequences, or an ITR (inverted terminal repeat) region. An expression cassette can additionally contain one or more cis-acting sequences (e.g., a promoter, enhancer, or suppressor), one or more introns, and one or more post-transcriptional regulatory elements.

[0087] As used herein, the term "treating" or "treatment of" a disease or disorder is intended to mean, whether detectable or undetectable, alleviation of one or more symptoms or manifestations of the disease or disorder, diminution of the extent of the disease or disorder, stabilization (e.g., non-worsening) of the state of the disease or disorder, prevention of spread of the disease or disorder, delay or retardation of progression of the disease or disorder, improvement or alleviation of the disease state or disorder state, and remission (partial or complete). For example, when expressed at an effective amount (or effective dosage), GJB2 is sufficient to prevent, correct, and / or normalize an abnormal physiological response. For example, a therapeutic effect is sufficient to reduce clinically significant features of a disease or disorder by at least about 30 percent, more preferably at least 50 percent, and most preferably at least 90 percent. "Treatment" may also refer to an extension of survival as compared to survival that would be predicted in the absence of treatment.

[0088] As used herein, the term "vector" is intended to mean a recombinant plasmid or recombinant virus that contains a nucleic acid to be delivered to a host cell, either in vitro or in vivo.

[0089] As used herein, the term "expression vector" refers to a vector that directs the expression of an RNA or polypeptide from a sequence that has been ligated to transcriptional regulatory sequences on the vector. The sequence to be expressed is often, but not necessarily, heterologous to the cell. An expression vector may contain additional elements. For example, an expression vector may have two replication systems and thus be capable of being maintained in two organisms, such as human cells for expression and a prokaryotic host for cloning and amplification. The term "expression" refers to the cellular processes involved in the production of RNA and protein and, optionally, the secretion of the protein, and includes, but is not limited to, cellular processes such as transcription, transcript processing, translation, and protein folding, modification, and processing, as appropriate. "Expression product" includes RNA transcribed from a gene and polypeptides obtained by translation of mRNA transcribed from a gene. The term "gene" means a nucleic acid sequence (DNA) that is transcribed into RNA in vitro or in vivo when operably linked to appropriate regulatory sequences. A gene may or may not include intervening sequences (introns) between individual coding segments (exons), as well as regions preceding the coding region and regions following the coding region, such as 5' untranslated (5'UTR) sequences or "leader" sequences and 3'UTR sequences or "trailer" sequences.

[0090] As used herein, the term "recombinant viral vector" is intended to refer to a recombinant polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences not derived from a virus). In the case of a recombinant AAV vector, the recombinant nucleic acid is flanked by at least one ITR (inverted terminal repeat) sequence. In some embodiments, the recombinant nucleic acid is flanked by two ITRs.

[0091] As used herein, the term "recombinant AAV vector (rAAV vector)" is intended to refer to a polynucleotide vector that contains one or more heterologous sequences (i.e., nucleic acid sequences not derived from AAV) flanked by at least one AAV inverted terminal repeat (ITR) sequence. Such rAAV vectors can be replicated and packaged into infectious virus particles when present in a host cell that has been infected with a suitable helper virus (or a helper virus that expresses suitable helper functions) and expresses the AAV rep and cap gene products (i.e., the AAV Rep and Cap proteins). When an rAAV vector is incorporated into a large polynucleotide (e.g., within another vector such as a plasmid used for chromosomal or cloning or transfection), the rAAV vector may be referred to as a "provirus," which can be "rescued" by replication and encapsidation in the presence of the AAV packaging function and suitable helper functions. rAAV vectors can be in any of a number of forms, including, but not limited to, plasmids, linear artificial chromosomes, complexed forms with lipids, encapsulated forms within liposomes, and virus particles, e.g., capsid-encapsulated forms within AAV particles. rAAV vectors can be packaged into an AAV virus capsid to produce "recombinant adeno-associated virus particles (rAAV particles).

[0092] As used herein, the term "rAAV virus" or "rAAV virus particle" is intended to refer to a virus particle composed of at least one AAV capsid protein and a capsid-encapsidated rAAV vector genome.

[0093] As used herein, "reporter" refers to a protein that can be used to provide a detectable readout. Reporters generally provide a measurable signal, such as fluorescence, color, or luminescence. The coding sequence of a reporter protein encodes a protein whose presence in a cell or organism can be readily observed. For example, a fluorescent protein, when excited by light of a specific wavelength, causes the cell to fluoresce, luciferase catalyzes a reaction in the cell that emits light, and an enzyme such as β-galactosidase converts a substrate into a colored product. Exemplary reporter polypeptides useful for experimental or diagnostic purposes include, but are not limited to, β-lactamase, β-galactosidase (LacZ), alkaline phosphatase (AP), thymidine kinase (TK), green fluorescent protein (GFP) and other fluorescent proteins, chloramphenicol acetyltransferase (CAT), luciferase, and reporter polypeptides well known in the art.

[0094] Transcriptional regulators refer to transcriptional activators and transcriptional repressors that activate or repress the transcription of a gene of interest, such as GJB2. A promoter is a region of nucleic acid that initiates the transcription of a specific gene. Transcriptional activators typically bind near a transcription promoter and recruit RNA polymerase to directly initiate transcription. Repressors bind to the transcription promoter and sterically interfere with the initiation of transcription by RNA polymerase. Other transcriptional regulators may also be used as activators or repressors depending on their binding sites and cellular and environmental conditions. Non-limiting examples of the transcriptional regulator class include, but are not limited to, homeodomain proteins, zinc finger proteins, winged helix (forkhead) proteins, and leucine zipper proteins.

[0095] As used herein, the terms "repressive protein" or "inducible protein" refer to proteins that bind to regulatory sequence elements and, respectively, repress or activate the transcription of sequences operably linked to the regulatory sequence elements. Preferred repressive and inducible proteins are described herein that are sensitive to the presence or absence of at least one input agent or environmental input. Preferred proteins are described herein in modular form that include, for example, discrete DNA-binding elements or discrete DNA-binding domains, and input agent-binding elements or input agent-binding domains or input agent-responsive elements or input agent-responsive domains.

[0096] As used herein, the terms "comprising" or "comprises" are used with reference to compositions, methods, and respective components thereof required for a method or composition, and are open to the inclusion of elements not specified, whether required or not.

[0097] As used herein, the term "consisting essentially of" refers to the elements required for a given embodiment. The term permits the presence of elements that do not physically affect the basic and novel or functional characteristics of this embodiment. The use of "comprising" indicates inclusion rather than limitation.

[0098] The term "consisting of" refers to the compositions, methods, and respective components thereof described herein that exclude elements not recited in the description of an embodiment.

[0099] As used herein, the term "consisting essentially of" refers to the elements required for a given embodiment. The term permits the presence of additional elements that do not physically affect the basic and novel or functional characteristics of this embodiment of the invention.

[0100] In this specification, the term "comprising" is used to mean "including but not limited to" and is used interchangeably therewith.

[0101] In this specification, the term "such as" is used to mean "such as but not limited to" and is used interchangeably therewith.

[0102] Unless the context clearly dictates otherwise, the singular forms "a", "an", and "the" as used in this specification and the appended claims include plural referents. Thus, for example, a reference to "a method" includes one or more methods and / or steps of the kind described herein and / or that would be apparent to one of ordinary skill in the art upon reading the present disclosure. Similarly, the word "or" is intended to include "and" unless the context clearly dictates otherwise. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, suitable methods and materials are described below. The abbreviation "e.g." is derived from the Latin "exempli gratia" and is used herein to denote non-limiting examples. Thus, the abbreviation "e.g." is synonymous with the term "for example".

[0103] The grouping of alternative elements or embodiments of the invention disclosed herein is not to be understood as a limitation. Members of each group may be individually recited and claimed, or may be recited and claimed in any combination with other members of the group or other elements found herein. One or more members of a group may be incorporated into or deleted from the group for reasons of convenience and / or patentability. In the event of any such incorporation or deletion, the specification is considered to contain the modified group and thus to realize the written description for all Markush groups used in the appended claims.

[0104] In some embodiments of any of the aspects, the disclosure described herein is not directed to processes for cloning humans, processes for modifying the identity of human germline genes, the use of human embryos for industrial or commercial purposes, or processes for modifying the identity of animal genes that are likely to cause harm to humans or animals without substantial medical benefit to them, nor is it directed to animals obtained from such processes.

[0105] Other terms are defined in the description of the various aspects of the invention herein.

[0106] All patents and other publications, including references, issued patents, published patent applications, and co-pending patent applications, cited through this application are hereby expressly incorporated by reference herein for the purpose of describing and disclosing, for example, the methods described in such publications and which may be used in connection with the technology described herein. These publications are presented only for their disclosure prior to the filing date of this application. Nothing in this regard shall be construed as an admission that the inventors are in any way precluded from the prior invention or for any other reason from making the disclosures herein prior to such publications. All statements as to the dates of these documents or as to the content thereof are based on the information available to the applicants and constitute no admission as to the accuracy of the dates or content of these documents.

[0107] The description of embodiments of the present disclosure is not intended to be exhaustive or to limit the present disclosure to the precise forms disclosed. Specific embodiments and examples of the present disclosure are described herein for purposes of illustration, but as will be recognized by those of ordinary skill in the art, various equivalent modifications are possible within the scope of the present disclosure. For example, method steps or functions are presented in a given order, but alternative embodiments may perform the functions in a different order or substantially concurrently. The teachings of the present disclosure presented herein may be applied to other procedures or methods as necessary. The various embodiments described herein may be combined to present further embodiments. Aspects of the present disclosure may be modified to utilize the above references and the compositions, functions, and concepts of this application to provide still further embodiments of the present disclosure, if necessary. Further, some changes may be made to the protein structure without affecting the type or amount of biological or chemical action through consideration of biological functional equivalence. In light of the detailed description, these and other changes may be made to the present disclosure. All such modifications are intended to be included within the scope of the appended claims.

[0108] Any specific elements of the previous embodiments may be combined with or substituted by elements in other embodiments. Further, in the context of these embodiments, advantages associated with certain embodiments of the present disclosure have been described, but other embodiments may also exhibit such advantages, and not all embodiments necessarily have to exhibit such advantages so as to fall within the scope of the present disclosure.

[0109] The techniques described herein are further illustrated by the following examples and should not be considered in any way to be more limiting. Since such methods, protocols, and reagents can vary, it is understood that the present invention is not limited to the specific methods, protocols, reagents, etc. described herein. The terminology used herein is for the purpose of describing only specific embodiments and is not intended to limit the scope of the present invention, which is defined only by the claims.

[0110] Nucleic acid This specification presents the characterization and development of nucleic acid molecules for potential therapeutic use. The nucleic acids for therapeutic use described herein encode the GJB2 protein, and in this case, the nucleic acids are codon-optimized.

[0111] The present disclosure presents promoters, expression cassettes, vectors, kits, and methods that can be used in the treatment of hereditary hearing impairment. Certain aspects of the present disclosure relate to the delivery of heterologous nucleic acids to inner ear cells of a subject, including delivery by administration of a recombinant adeno-associated virus (rAAV) vector. According to some aspects, the present disclosure presents a method of treating or preventing hereditary deafness, including the delivery of a composition comprising the rAAV vector described herein to a subject, wherein the rAAV vector comprises a heterologous nucleic acid (e.g., a nucleic acid encoding GJB2).

[0112] Among subjects with hereditary hearing impairment (HI), GJB2, the most commonly mutated gene, encodes connexin 26 (Cx26), a gap junction channel protein that underlies both cell - cell communication between supporting cells and the homeostasis of perilymph, endolymph, and outer lymph. GJB2 is located at the DFNB1 locus on 13q12. GJB2 is 5513 bp in length and contains two exons (193 bp and 2141 bp in length, respectively) separated by a 3179 - bp intron (Kiang et al., 1997). Transcription starts from a single start site and results in the synthesis of a 2334 - nucleotide mRNA (GenBank NM_004004.5), which is considered canonical. Loss of GJB2 causes cell death of diverse cell types within the inner ear after the onset of hearing. "GJB2 nucleic acid" refers to a nucleic acid comprising the GJB2 gene or a portion thereof, or a functional variant of the GJB2 gene or a portion thereof.

[0113] Thus, in some embodiments, the AAV vector is used for the expression of GJB2 protein to restore hearing.

[0114] According to some embodiments, the gene of interest (e.g., GJB2) is optimized to have better expression (and / or function) than wild - type GJB2 and further has the ability to be distinguished from wild - type GJB2 (at the DNA / RNA level).

[0115] According to some embodiments, the present disclosure presents a GJB2 therapeutic protein or a fragment thereof (e.g., a functional fragment) encoded by a codon - optimized nucleic acid and to be expressed therefrom within the AAV vectors described herein. Those skilled in the art will understand that the GJB2 therapeutic protein includes all splice variants and orthologs of the GJB2 protein. The GJB2 therapeutic protein includes, in addition to the intact molecule, its cleavage fragments (e.g., functional fragments).

[0116] Figure 2 shows an overview of single-stranded (ss) AAV-GJB2 and self-complementary scAAV-GJB2, which are GJB2 vector (genomic) constructs.

[0117] According to some embodiments, the present disclosure presents a nucleic acid encoding a wild-type GJB2 protein. According to some embodiments, the present disclosure presents a nucleic acid encoding a human wild-type GJB2 protein. According to some embodiments, the nucleic acid sequence encoding the human wild-type GJB2 protein is 678 bp in length. According to one embodiment, the nucleic acid encoding the human wild-type GJB2 protein comprises SEQ ID NO: 10. According to one embodiment, the nucleic acid is at least 85% identical to SEQ ID NO: 10. According to one embodiment, the nucleic acid is at least 90% identical to SEQ ID NO: 10. According to one embodiment, the nucleic acid is at least 95% identical to SEQ ID NO: 10. According to one embodiment, the nucleic acid is at least 99% identical to SEQ ID NO: 10. According to one embodiment, the nucleic acid consists of SEQ ID NO: 10.

[0118] Figure 10 shows the nucleic acid sequence of human wild-type GJB2 (hGJB2wt) (SEQ ID NO: 10).

[0119] According to some embodiments, the present disclosure presents a nucleic acid encoding a GJB2 protein, wherein the nucleic acid sequence is codon-optimized for expression in mammals. Human codon-optimized GJB2 is an important element encoding the major gap junction protein required for normal hearing. Codon optimization is performed to enhance the protein expression of GJB2.

[0120] As used herein, the term "codon-optimized" or "codon optimization" refers to the process of modifying a nucleic acid sequence by replacing at least one, more than one, or a significant number of codons of a native sequence (e.g., a prokaryotic sequence) with codons that are used more frequently, or most frequently, in a gene of a target vertebrate, such as a mouse or a human, for enhanced expression in cells within that vertebrate. Different species exhibit a particular bias for certain codons for a particular amino acid. Typically, codon optimization does not change the amino acid sequence of the original translated protein. Codon optimization can be determined using various commercially available platforms (e.g., OPTIMUMGENE by Genscripts, GENEGPS by Atum, or the codon optimization tool by Blue Heron Biotech) or another publicly available database. In some embodiments, the nucleic acid encoding the GJB2 protein is optimized for expression in humans and / or is a human GJB2 or a functional fragment thereof. Exemplary GJB2 codon-optimized sequences are disclosed herein.

[0121] According to some embodiments, the present disclosure provides a nucleic acid encoding a human codon-optimized GJB2 protein. According to some embodiments, the expressed GJB2 therapeutic protein is functional in the treatment of hereditary hearing loss, including the treatment and / or prevention of congenital hearing loss associated with DFNB1 and DFNA3A. In some embodiments, the GJB2 therapeutic protein does not elicit an immune system response.

[0122] According to some embodiments, the nucleic acid sequence encoding the human codon-optimized GJB2 protein is 678 bp in length. According to one embodiment, the nucleic acid encoding the human codon-optimized GJB2 protein comprises SEQ ID NO: 11. According to one embodiment, the nucleic acid is at least 85% identical to SEQ ID NO: 11. According to one embodiment, the nucleic acid is at least 90% identical to SEQ ID NO: 11. According to one embodiment, the nucleic acid is at least 95% identical to SEQ ID NO: 11. According to one embodiment, the nucleic acid is at least 99% identical to SEQ ID NO: 11. According to one embodiment, the nucleic acid consists of SEQ ID NO: 11.

[0123] Figure 11 shows the nucleic acid sequence of human codon-optimized GJB2 (hGJB2co3) (SEQ ID NO: 11).

[0124] According to some embodiments, the nucleic acid sequence encoding the human codon-optimized GJB2 protein is 678 bp in length. According to one embodiment, the nucleic acid encoding the human codon-optimized GJB2 protein comprises SEQ ID NO: 12. According to one embodiment, the nucleic acid is at least 85% identical to SEQ ID NO: 12. According to one embodiment, the nucleic acid is at least 90% identical to SEQ ID NO: 12. According to one embodiment, the nucleic acid is at least 95% identical to SEQ ID NO: 12. According to one embodiment, the nucleic acid is at least 99% identical to SEQ ID NO: 12. According to one embodiment, the nucleic acid consists of SEQ ID NO: 12.

[0125] Figure 12 shows the nucleic acid sequence of human codon-optimized GJB2 (hGJB2co6) (SEQ ID NO: 12).

[0126] According to some embodiments, the nucleic acid sequence encoding the human codon-optimized GJB2 protein is 678 bp in length. According to one embodiment, the nucleic acid encoding the human codon-optimized GJB2 protein comprises SEQ ID NO: 13. According to one embodiment, the nucleic acid is at least 85% identical to SEQ ID NO: 13. According to one embodiment, the nucleic acid is at least 90% identical to SEQ ID NO: 13. According to one embodiment, the nucleic acid is at least 95% identical to SEQ ID NO: 13. According to one embodiment, the nucleic acid is at least 99% identical to SEQ ID NO: 13. According to one embodiment, the nucleic acid consists of SEQ ID NO: 13.

[0127] Figure 13 shows the nucleic acid sequence of human codon-optimized GJB2 (hGJB2co9) (SEQ ID NO: 13).

[0128] According to some embodiments, the nucleic acid sequence encoding the human codon-optimized GJB2 protein is 681 bp in length. According to one embodiment, the nucleic acid encoding the human codon-optimized GJB2 protein comprises SEQ ID NO: 18. According to one embodiment, the nucleic acid is at least 85% identical to SEQ ID NO: 18. According to one embodiment, the nucleic acid is at least 90% identical to SEQ ID NO: 18. According to one embodiment, the nucleic acid is at least 95% identical to SEQ ID NO: 18. According to one embodiment, the nucleic acid is at least 99% identical to SEQ ID NO: 18. According to one embodiment, the nucleic acid consists of SEQ ID NO: 18.

[0129] Figure 18 shows the nucleic acid sequence of the hybrid codon-optimized construct (CO369) (SEQ ID NO: 18).

[0130] According to some embodiments, the nucleic acid sequence encoding the human codon-optimized GJB2 protein is 678 bp in length. According to one embodiment, the nucleic acid encoding the human codon-optimized GJB2 protein comprises SEQ ID NO: 13. According to one embodiment, the nucleic acid is at least 85% identical to SEQ ID NO: 13. According to one embodiment, the nucleic acid is at least 90% identical to SEQ ID NO: 13. According to one embodiment, the nucleic acid is at least 95% identical to SEQ ID NO: 13. According to one embodiment, the nucleic acid is at least 99% identical to SEQ ID NO: 13. According to one embodiment, the nucleic acid consists of SEQ ID NO: 13.

[0131] According to embodiments of the present disclosure, the AAV vectors described herein comprise one or more codon-optimized nucleic acid sequences, for example, nucleic acid sequences encoding a GJB2 therapeutic protein or a functional fragment thereof. In one embodiment, the closed-end DNA vector comprises a nucleic acid sequence encoding a codon-optimized GJB2 sequence. In some embodiments, the AAV vector for the expression of GJB2 may have a sequence encoding the full-length GJB2 protein. In some other embodiments, the expression of GJB2 by the AAV vector may have a sequence encoding a truncated GJB2 protein.

[0132] According to some embodiments, the AAV vector for the expression of the GJB2 protein may further comprise regulatory sequences such as a promoter, a secretion signal, a polyA region, and an enhancer.

[0133] Promoter The expression of the GJB2 therapeutic protein or a fragment thereof from the AAV vector can be achieved spatially and temporally using one or more of the promoters described herein.

[0134] The expression cassette of the AAV vector for the expression of the GJB2 protein may include a promoter that can affect the overall expression level.

[0135] A variety of promoters are envisioned for use in the present disclosure.

[0136] In accordance with some embodiments, the promoter is an endogenous GJB2 promoter. The GJB2 promoter is a supporting cell-specific promoter and is capable of transducing a heterologous nucleic acid into cells of the inner ear that express the GJB2 gene. This promoter can be used for the production of scAAV when its length is short. In accordance with some embodiments, the promoter comprises SEQ ID NO: 6. In accordance with some embodiments, the promoter consists of SEQ ID NO: 6. FIG. 8 shows the nucleic acid sequence of the GJB2 promoter (SEQ ID NO: 6).

[0137] In accordance with some embodiments, the promoter is a CBA promoter. The CBA promoter is a strong ubiquitous promoter capable of transducing nucleic acids into multiple cell types in the inner ear. In accordance with some embodiments, the promoter comprises SEQ ID NO: 1. In accordance with some embodiments, the promoter consists of SEQ ID NO: 1. FIG. 3 shows the nucleic acid sequence of the CBA promoter (SEQ ID NO: 1).

[0138] In accordance with some embodiments, the promoter is an EF1a promoter. The EF1a promoter is a mammalian-derived strong ubiquitous promoter capable of transducing nucleic acids into multiple cell types in the inner ear and can be used for the production of scAAV when its length is short. In accordance with some embodiments, the promoter comprises SEQ ID NO: 2. In accordance with some embodiments, the promoter consists of SEQ ID NO: 2. FIG. 4 shows the nucleic acid sequence of the EF1a promoter (SEQ ID NO: 2).

[0139] In accordance with some embodiments, the promoter is a CASI promoter. The CASI promoter is a strong ubiquitous promoter capable of transducing nucleic acids into multiple cell types in the inner ear and can be used for the production of scAAV when its length is short. In accordance with some embodiments, the promoter comprises SEQ ID NO: 3. In accordance with some embodiments, the promoter consists of SEQ ID NO: 3. FIG. 5 shows the nucleic acid sequence of the CASI promoter (SEQ ID NO: 3).

[0140] According to some embodiments, the promoter is the smCBA promoter. The smCBA promoter is a strong ubiquitous promoter capable of transducing nucleic acids into multiple cell types in the inner ear and can be used for the production of scAAV when its length is short. According to some embodiments, the promoter comprises SEQ ID NO: 4. According to some embodiments, the promoter consists of SEQ ID NO: 4. FIG. 6 shows the nucleic acid sequence (SEQ ID NO: 4) of the smCBA promoter.

[0141] According to some embodiments, the promoter is the GFAP promoter. The GFAP promoter is cell-specific and has activity in the supporting cells of the inner ear. According to some embodiments, the promoter comprises SEQ ID NO: 5. According to some embodiments, the promoter consists of SEQ ID NO: 5. FIG. 7 shows the nucleic acid sequence (SEQ ID NO: 5) of the GFAP promoter.

[0142] ITR (inverted terminal repeat) The AAV vector for the expression of the GJB2 protein described herein contains a nucleic acid, for example, the nucleic acid sequence of GJB2 (e.g., the nucleic acid sequence of codon-optimized GJB2), disposed between two ITR (inverted terminal repeat) sequences.

[0143] In some embodiments, the ITR sequence can be derived from a virus of the family Parvoviridae, subfamily Parvovirinae, genus Dependovirus, including adeno-associated virus (AAV) that normally infects humans (e.g., serotypes 2, 3A, 3B, 5, and 6) or rodents (e.g., serotypes 1 and 4). There are a number of homologous serotypes, such as AAV2, AAV4, AAV6, AAV8.

[0144] According to some embodiments, the ITR is derived from the genus Dependovirus, such as an AAV (e.g., AAV1, AAV2, AAV3, AAV4, AAV5, AAV5, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, AAV-DJ, and AAV-DJ8) genome, a chimeric ITR, or an ITR derived from any synthetic AAV. According to some embodiments, the serotype of the ITR of the AAV vector is independently selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, and AAV12.

[0145] The ITR (inverted terminal repeat) sequences are required for efficient replication of the AAV genome because of their ability to form hairpin structures that allow synthesis of the second DNA strand. The scAAV short-chain ITR (TRS) forms an intramolecular double-stranded DNA template, thereby bypassing the rate-limiting step of second-strand synthesis.

[0146] Those skilled in the art are familiar with the common structure of double-stranded Holliday junctions, where the ITR sequences typically have a T-shaped or Y-shaped hairpin structure. In this case, each WT-ITR is formed by two palindromic arms or palindromic loops (B-B' and C-C') embedded within a large palindromic arm (A-A') and a single-stranded D sequence (in the sequence, the order of these palindromic sequences defines the flip or flop orientation of the ITR). For example, see the structural analysis and sequence comparison of ITRs derived from different AAV serotypes (AAV1 - AAV6) described in Grimm et al., J. Virology, 2006, 80(1), 426 - 439; Yan et al., J. Virology, 2005, 364 - 379; Duan et al., Virology, 1999, 261, 8 - 14. Based on the sequence comparison of ITRs derived from different AAV serotypes (AAV1 - AAV6, as well as avian AAV (AAAV) and bovine AAV (BAAV)) described in Grimm et al., J. Virology, 2006, 80(1), 426 - 439, which shows the percentage identity of the left ITR of AAV2 with the left ITRs of other serotypes: AAV-1 (84%), AAV-3 (86%), AAV-4 (79%), AAV-5 (58%), AAV-6 (left ITR) (100%), and AAV-6 (right ITR) (82%), those skilled in the art can readily determine the WT-ITR sequence derived from any AAV serotype for use in AAV vectors.

[0147] Figure 9 shows the nucleic acid sequences of the following ITRs (AAV2): 5'-3' for single-stranded (ss) AAV genome and self-complementary (sc) AAV genome (SEQ ID NO: 7); 3'-5' for single-stranded (ss) AAV genome only (SEQ ID NO: 8); 3'-5' for self-complementary (sc) AAV genome only (SEQ ID NO: 9).

[0148] In some embodiments, the nucleotide sequence of the WT-ITR may be modified (e.g., by modifying one, two, three, four, or five, or more nucleotides, or any range thereof), in which case the modification is a substitution with a complementary nucleotide, e.g., substitution of C with G and vice versa, and substitution of A with T and vice versa. Thus, in some embodiments, i.e., an ITR that is substantially WT is used (i.e., the ITR has the basic loop structure of WT, but also has some conservative nucleotide changes that do not alter or affect its properties).

[0149] GJB2 Gene Therapy for Hereditary Hearing Loss The present disclosure generally presents methods for generating recombinant adeno-associated virus (AAV) viral particles comprising a GJB2 gene construct, and their use in gene therapy for hereditary hearing loss. The AAV vectors described herein are particularly efficient in the delivery of nucleic acids (e.g., GJB2 gene constructs) to inner ear cells. Described herein are methods for creating, evaluating, and utilizing a recombinant adeno-associated virus (rAAV) therapeutic vector capable of efficiently delivering GJB2 to cells for expression and subsequent secretion. Also described herein are optimally modified GJB2 / connexin 26 (Cx26) cDNA, and related gene elements, for use in a recombinant adeno-associated virus (rAAV)-based gene therapy for hereditary hearing loss, including the treatment and / or prevention of congenital hearing loss associated with DFNB1 and DFNA3A.

[0150] The recombinant adeno-associated virus (rAAV) vector can efficiently accommodate both the GJB2 target gene and related gene elements. Further, such a vector can be designed to specifically express GJB2 within therapeutically important spiral ganglion cells. The present disclosure describes methods for creating, evaluating, and utilizing an rAAV therapeutic vector capable of efficiently delivering a functional GJB2 gene to a patient.

[0151] The GJB2 gene construct comprises: (1) a 0.68 kb codon / sequence-optimized human GJB2 cDNA, with or without a 27-nucleotide-long hemagglutinin (HA) C-terminal tag; (2) the following promoter elements optimized to drive high-level GJB2 expression: (a) a 1.7 kb ubiquitous activity CBA, a 0.96 kb mini CBA (smCBA), an 0.81 kb EF1a, or a 1.06 kb CASI promoter; (b) one of a snail supporting cell promoter or a 1.68 kb GJB2 expression-specific GFAP promoter, a 0.13 / 0.54 / 1.0 kb mini / medium / large GJB2 promoter, or a sequence combination of two to three individual GJB2 expression-specific promoters; (3) a 0.9 kb 3’UTR regulatory region containing a WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element), followed by a polyadenylation signal of SV40 or human growth hormone (hGH); (4) a self-complementary AAV (scAAV) genomic cassette separated by two 143-base sequence-modulated inverted terminal repeats (ITRs) or 113-base scAAV-activating ITR (ITRΔtrs) sandwiching the AAV genomic cassette, and at one end, consisting of two inverted identical repeats (IIRs) (each being 3.0 kb or less) sandwiched by a 143-base sequence-modulated ITR; and (5) may contain a protein capsid mutant that is optimal for inner ear delivery.

[0152] The HA tag is human influenza hemagglutinin, a surface glycoprotein that is used as a general epitope tag within expression vectors and facilitates the detection of the protein of interest. The WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) is a DNA sequence that enhances the expression of the protein of interest by creating a tertiary structure that stabilizes its mRNA. The poly(A) sequence is an important element that promotes RNA processing and transcript stability. The SV40 / bGH sequence is a terminator sequence that signals the end of the transcription unit.

[0153] According to some embodiments, the AAV vectors described herein are particularly suitable for delivering and expressing GLB2 to, among other things, supporting cells of the cochlea. According to some embodiments, the AAV vectors described herein are particularly suitable for delivering and expressing GLB2 to one or more of external supporting cells and / or supporting cells of the organ of Corti. According to some embodiments, the AAV vectors described herein are particularly suitable for delivering and expressing GLB2 to one or more of outer hair cells of the ear, inner hair cells of the ear, Hensen cells, Dieters cells, pillar cells, inner phalangeal cells, and / or outer phalangeal / border cells.

[0154] Adeno-associated virus (AAV) Adeno-associated virus (AAV) is a non-pathogenic single-stranded DNA parvovirus. AAV has a capsid diameter of approximately 20 nm. Each end of the single-stranded DNA genome contains an ITR (inverted terminal repeat), the only cis-acting element required for genome replication and packaging. The AAV genome carries two viral genes: rep and cap. The virus utilizes two promoters and alternative splicing to produce the four proteins (Rep78, Rep68, Rep52, and Rep40) required for replication. A third promoter produces transcripts for the three structural viral capsid proteins 1, 2, and 3 (VP1, VP2, and VP3) via a combination of an alternative splicing start codon and an alternative translation start codon (Berns and Linden, Bioessays, 1995, 17:237-45). While the three capsid proteins share the same 533 amino acid C-terminus, VP2 and VP1 contain additional N-terminal sequences of 65 and 202 amino acids, respectively. The AAV virion contains a total of 60 copies of VP1, VP2, and VP3 arranged in a 1:1:20 ratio in T-1 icosahedral symmetry (Rose et al., J Virol., 1971, 8:766-70). AAV requires adenovirus (Ad), herpes simplex virus (HSV), or another virus as a helper virus to complete its lytic life cycle (Atchison et al., Science, 1965, 149:754-6; Hoggan et al., Proc Natl Acad Sci USA, 1966, 55:1467-74). In the absence of a helper virus, wild-type AAV establishes latency by integration with the assistance of the Rep protein via interaction of the ITR with the chromosome (Berns and Linden (1995)).

[0155] AAV serotype There are a number of different AAV serotypes, including AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, Anc80L65, and variants or hybrids thereof. In vivo studies have shown that diverse AAV serotypes exhibit different tissue tropisms or cell tropisms. For example, AAV1 and AAV6 are two serotypes that are efficient for transduction of skeletal muscle (Gao et al., Proc Natl Acad Sci USA, 2002, 99:11854-11859; Xiao et al., J Virol., 1999, 73:3994-4003; Chao et al., Mol Ther., 2000, 2:619-623). AAV-3 has been shown to be excellent for transduction of megakaryocytes (Handa et al., J Gen Virol., 2000, 81:2077-2084). AAV5 and AAV6 efficiently infect airway apical cells (Zabner et al., J Virol., 2000, 74:3852-3858; Halbert et al., J Virol., 2001, 75:6615-6624). AAV2, AAV4, and AAV5 are transduced into different types of cells within the central nervous system (Davidson et al., Proc Natl Acad Sci USA., 2000, 97:3428-3432). AAV8 and AAV5 can be transduced into hepatocytes better than AAV-2. An AAV-5-based vector was transduced into certain cell types (cultured airway epithelial cells, cultured skeletal muscle cells, and cultured human umbilical vein endothelial cells) with a higher efficiency than AAV2, whereas for the NIH3T3 cell line, skbr3 cell line, and t-47D cell line, both AAV2 and AAV5 showed low transduction efficiencies (Gao et al., Proc Natl Acad Sci USA., 2002, 99:11854-11859; Mingozzi et al., J Virol., 2002, 76:10497-10502; WO99 / 61601).AAV4 was found to be the most efficiently transduced into the rat retina, followed by AAV5 and AAV1 (Rabinowitz et al., J Virol., 2002, 76:791 - 801; Weber et al., Mol Ther., 2003, 7:774 - 781). In summary, AAV1, AAV2, AAV4, AAV5, AAV8, and AAV9 show tropism for CNS tissues. AAV1, AAV8, and AAV9 show tropism for cardiac tissues. AAV2 exhibits tropism for renal tissues. AAV7, AAV8, and AAV9 exhibit tropism for hepatic tissues. AAV4, AAV5, AAV6, and AAV9 exhibit tropism for lung tissues. AAV8 exhibits tropism for pancreatic cells. AAV3, AAV5, and AAV8 show tropism for photoreceptor cells. AAV1, AAV2, AAV4, AAV5, and AAV8 exhibit tropism for retinal pigment epithelial (RPE) cells. AAV1, AAV6, AAV7, AAV8, and AAV9 show tropism for skeletal muscle.

[0156] For example, further modifications to the virus can be made to enhance the efficiency of gene transfer by improving the tropism of each serotype. One approach is to exchange domains derived from one serotype capsid with another domain, thereby creating hybrid vectors with desired qualities from each parent. Since the viral capsid contributes to binding to cell receptors, understanding the viral capsid domain(s) crucial for binding is important. Most of the mutagenesis studies on viral capsids (mainly AAV2) conducted before crystal structures were available were based on functionalization of the capsid surface by adsorption of exogenous moieties, insertion of peptides at random positions, or global mutagenesis at the amino acid level. Choi et al., Curr Gene Ther., June 2005, 5(3):299 - 310 describe different approaches and considerations for hybrid serotypes.

[0157] Capsids derived from other AAV serotypes provide advantages over rAAV vectors based on the AAV2 capsid in certain in vivo applications. First, appropriate use of rAAV vectors having a particular serotype can increase the efficiency of in vivo gene delivery to certain target cells where infection by AAV2-based vectors is low or absent. Second, when readministration of an rAAV vector is clinically necessary, it may be advantageous to use an rAAV vector based on another AAV serotype. Presumably, it has been demonstrated that readministration of the same rAAV vector with the same capsid can be rendered ineffective due to the production of neutralizing antibodies generated against the vector (Xiao et al., 1999; Halbert et al., 1997). This problem can be circumvented by administration of rAAV particles whose capsids are composed of proteins derived from different AAV serotypes and which are not affected by the presence of neutralizing antibodies against the first rAAV vector (Xiao et al., 1999). For the reasons described above, recombinant AAV vectors constructed using the cap gene from AAV2 and additional serotypes are desired. Similar to rHSV, it will be appreciated that construction of recombinant HSV vectors encoding cap genes from other AAV serotypes, such as AAV1, AAV2, AAV3, AAV5-AAV9, can be accomplished using the methods described herein to generate rHSV. In certain preferred embodiments of the invention described herein, recombinant AAV vectors constructed using cap genes from different AAVs are preferred. A significant advantage of the construction of these additional rHSV vectors is the ease and time savings compared to alternative methods used for large-scale production of rAAV. In particular, the difficult process of constructing new rep- and cap-derived cell lines for each different capsid serotype is avoided.

[0158] Production of Recombinant AAV (rAAV) Vectors The production, purification, and characterization of the rAAV vectors of the present invention can be carried out using any of a number of methods known in the art. For a review of laboratory-scale production methods, see, for example, Clark RK, "Recent advances in recombinant adeno-associated virus vector production", Kidney Int., 61s:9-15 (2002); Choi VW et al., "Production of recombinant adeno-associated viral vectors for in vitro and in vivo use", Current Protocols in Molecular Biology, 16.25.1-16.25.24 (2007) (hereinafter referred to as "Choi et al." in this specification); Grieger JC and Samulski RJ, "Adeno-associated virus as a gene therapy vector: Vector development, production, and clinical applications", Adv Biochem Engin / Biotechnol 99:119-145 (2005) (hereinafter referred to as "Grieger and Samulski" in this specification); Heilbronn R and Weger S, "Viral Vectors for Gene Transfer: Current Status of Gene Therapeutics", M. Schaefer-Korting (ed.), "Drug Delivery, Handbook of Experimental Pharmacology", 197:143-170 (2010) (hereinafter referred to as "Heilbronn" in this specification); Howarth JL et al., "Using viral vectors as gene transfer tools", Cell Biol Toxicol, 26:1-10 (2010) (hereinafter referred to as "Howarth" in this specification). The production methods described below are intended as non-limiting examples.

[0159] The production of AAV vectors can be achieved by co-transfection of packaging plasmids (Heilbronn). The cell line supplies the deleted AAV genes, rep and cap, as well as the required helper virus functions. The adenovirus helper genes, VA-RNA, E2A, and E4 are co-transfected together with the AAV rep and cap genes on two separate plasmids or on a single helper construct. A recombinant AAV vector plasmid in which the AAV capsid gene is replaced by a transgene expression cassette (including the gene of interest, e.g., GJB2 nucleic acid; a promoter; and minimal regulatory elements) flanked by ITRs is also transfected. These packaging plasmids are typically transfected into 293 cells, a human cell line that constitutively expresses the remaining essential Ad helper genes, E1A, and E1B. This results in the amplification and packaging of the AAV vector carrying the gene of interest.

[0160] More than 100 AAV serotypes, including 12 human serotypes and those derived from non-human primates, have been identified (Howarth et al., Cell Biol Toxicol, 26:1-10 (2010)). The AAV vectors of the present invention can include a capsid sequence derived from AAV of any known serotype. As used herein, "known serotype" includes capsid mutants that can be generated using methods known in the art. Such methods include, for example, genetic manipulation of viral capsid sequences, domain swapping of exposed surface domains of capsid regions of different serotypes, and development of AAV chimeras using techniques such as markerless rescue. See Bowles et al., "Marker rescue of adeno-associated virus (AAV) capsid mutants: A novel approach for chimeric AAV production", Journal of Virology, 77(1):423-432 (2003), as well as the references cited therein. Further, the AAV vectors of the present invention can include ITRs derived from AAV of any known serotype. Preferentially, the ITRs are derived from one of human serotypes AAV1-AAV12. In some embodiments of the present invention, a pseudotyping method is utilized in which the genome of one ITR serotype is packaged into a capsid of a different serotype.

[0161] According to some embodiments, the capsid sequence is derived from one of human serotypes AAV1-AAV12. According to some embodiments, the capsid sequence is derived from AAV2, which is a serotype. According to some embodiments, the capsid sequence is derived from an AAV2 mutant with a high tropism for targeting supporting cells (e.g., outer hair cells of the ear, inner hair cells of the ear, Hensen cells, Deiters cells, pillar cells, inner phalangeal cells, outer phalangeal / border cells). Capsids suitable for this purpose include AAV2, as well as AAV2 mutants including AAV2-tYF, AAV2-MeB, AAV2-P2V2, AAV2-MeBtYFTV, AAV2-P2V6, as well as AAV5, AAV8, and Anc80L65.

[0162] According to some embodiments, the recombinant AAV vector may be directly targeted, in particular, by genetic manipulation of the viral capsid sequence within the loop-out region of the AAV three-dimensional structure, may be directly targeted by domain swapping of the exposed surface of the capsid regions of different serotypes, or may be directly targeted by the development of AAV chimeras using techniques such as markerless rescue. See Bowles et al., "Marker rescue of adeno-associated virus (AAV) capsid mutants: A novel approach for chimeric AAV production," Journal of Virology, 77(1):423-432 (2003), as well as the references cited therein.

[0163] One possible protocol for the production, purification, and characterization of recombinant AAV (rAAV) vectors is presented by Choi et al. and generally involves the following steps: designing a transgene expression cassette, designing a capsid sequence for targeting a specific receptor, generating an adenovirus-free AAV vector, purifying it, and titrating it. These steps are summarized below and described in detail by Choi et al.

[0164] The transgene expression cassette may be a single-stranded AAV (ssAAV) vector or, alternatively, a "dimeric" or self-complementary AAV (scAAV) vector that is packaged as a pseudo double-stranded transgene (Choi et al.; Howarth et al.). The use of conventional ssAAV vectors generally results in a delay in the onset of gene expression (several days to weeks until the plateau of transgene expression) due to the obligatory conversion of single-stranded AAV DNA to double-stranded DNA. In contrast, scAAV vectors show onset of gene expression within hours after transduction of quiescent cells and reach plateau within days (Heilbronn). In accordance with some embodiments, scAAV is used, in which case transduction of scAAV starts rapidly and stability is increased as compared to single-stranded AAV. Alternatively, the transgene expression cassette may be split between two AAV vectors, enabling delivery of long-chain constructs. See, e.g., Daya S. and Berns, K.I., "Gene therapy using adeno-associated virus vectors", Clinical Microbiology Reviews, 21(4):583-593 (2008) (hereinafter referred to as "Daya et al." in this specification). ssAAV vectors can be constructed by digesting a suitable plasmid (e.g., a plasmid containing the GJB2 gene, etc.) with a restriction endonuclease to remove the rep and cap fragments and gel-purifying the plasmid backbone containing AAVwt-ITR (Choi et al.). Subsequently, the desired transgene expression cassette can be inserted between appropriate restriction sites to construct a single-stranded rAAV vector plasmid. scAAV vectors can be constructed as described by Choi et al.

[0165] Next, large-scale plasmid preparations (at least 1 mg) of rAAV vectors and appropriate AAV helper plasmids and pXX6 Ad helper plasmids can be purified via fractionation by a double CsCl gradient (Choi et al.). Appropriate AAV helper plasmids can each be selected from the pXR series of pXR1 to pXR5, which enable cross-packaging of the AAV2 ITR genome into the capsids of AAV serotypes 1 to 5. Appropriate capsids can be selected based on the efficiency of targeting of the cells of interest by the capsid. Known methods can be utilized to vary the length of the genome (i.e., the transgene expression cassette) and the AAV capsid to improve expression and / or gene transfer in specific cell types (e.g., retinal cone cells). See, for example, Yang GS, "Virus-mediated transduction of murine retina with adeno-associated virus: Effects of viral capsid and genome size", Journal of Virology, 76(15):7651-7660.

[0166] Next, 293 cells are transfected with the pXX6 helper plasmid, the rAAV vector plasmid, and the AAV helper plasmid (Choi et al.). Subsequently, the fractionated cell lysates are subjected to CsCl gradient purification or heparin sepharose column purification following a multi-step rAAV purification process. The production and quantification of rAAV virions can be determined using a dot blot assay. Transduction of rAAV in cell culture, in vitro, can be used to verify the infectivity of the virus and the functionality of the expression cassette.

[0167] In addition to the methods described by Choi et al., in the context of the present invention, various other transfection methods for the production of AAV can also be used. For example, transient transfection methods are available, including methods that rely on the calcium phosphate precipitation protocol.

[0168] In addition to laboratory-scale methods for generating rAAV vectors, the present invention can utilize techniques known in the art for the bioreactor-scale production of AAV vectors, including, for example, the techniques described in Heilbronn; Clement, N. et al., "Large-scale adeno-associated viral vector production using a herpesvirus-based system enables manufacturing for clinical studies", Human Gene Therapy, 20:796-606.

[0169] Progress towards the desired goal of a scalable production system that can yield large amounts of clinical-grade rAAV vectors has been made largely in production systems that utilize transfection as a means of delivering the genetic elements required for intracellular rAAV production. For example, the removal of contaminating adenovirus helper has been circumvented by replacing adenovirus infection with plasmid transfection in a three-plasmid transfection system in which a third plasmid contains a nucleic acid sequence encoding an adenovirus helper protein (Xiao et al., 1998). Improvements in two-plasmid transfection systems have also simplified the production process and increased the production efficiency of rAAV vectors (Grimm et al., 1998).

[0170] Several strategies for improving the yield of rAAV from cultured mammalian cells are based on the development of specialized producer cells created by genetic engineering. In one approach, the inserted AAV genome may be "rescued" by infecting the cells with helper adenovirus or HSV, and large-scale production of rAAV has been achieved by using genetically engineered "provirus" cell lines. Provirus cell lines may be rescued by simple adenovirus infection and can result in increased efficiency compared to transfection protocols.

[0171] A second cell-based approach to improving the yield of rAAV from cells involves the use of a genetically engineered "packaging" cell line that harbors both the AAV rep and cap genes, or both rep-cap and ITR-destination gene, within their genomes (Qiao et al., 2002). In the former approach, to generate rAAV, the packaging cell line is infected or transfected with the helper function and the AAV ITR-GOI element. The latter approach involves infection or transfection of the cells with only the helper function. Typically, the production of rAAV using a packaging cell line is initiated by infecting the cells with wild-type adenovirus or recombinant adenovirus. Since the packaging cells contain the rep and cap genes, it is not necessary to supply these elements exogenously.

[0172] The rAAV yield from the packaging cell line has been shown to be higher than the rAAV yield obtained by the proviral cell line rescue or transfection protocol.

[0173] Improvements in rAAV yield have been made using an approach based on the delivery of helper functions from herpes simplex virus (HSV) using a recombinant HSV unit replication sequence system. Initially, low-level rAAV vector yields on the order of 150 - 500 viral genomes (vg) per cell were reported (Conway et al., 1997), but more recent improvements in rHSV unit replication sequence-based systems have resulted in substantially higher rAAV yields in terms of vg and infectious particles (ip) per cell (Feudner et al., 2002). The unit replication sequence system is an inherently replication-deficient system, but the use of "gutless" vectors, replication-competent (rcHSV), or replication-deficient rHSV still introduces immunogenic HSV components into the rAAV production system. Therefore, appropriate assays for these components and corresponding purification protocols for their removal must also be implemented.

[0174] In addition to these methods, the present specification also describes a method for producing recombinant AAV virus particles in mammalian cells, which comprises co-infecting a mammalian cell capable of growing in suspension with: a first recombinant herpes virus comprising nucleic acid sequences encoding an AAV rep gene and an AAV cap gene, each operably linked to a promoter; and a second recombinant herpes virus comprising a promoter operably linked to the GJB2 gene and flanked by AAV inverted terminal repeats (ITRs) that facilitate packaging of the gene of interest; and infecting the mammalian cell with the viruses, thereby producing recombinant AAV virus particles in the mammalian cell.

[0175] Any type of mammalian cell capable of supporting herpes virus replication is suitable for use according to the methods of the present invention described herein. Thus, mammalian cells can be considered host cells for herpes virus replication in the methods described herein. Any cell type for use as a host cell is contemplated by the present invention, as long as the cell is capable of supporting herpes virus replication. Examples of suitable non-genetically modified mammalian cells include, but are not limited to, cell lines such as HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.

[0176] The host cells used in various embodiments of the present invention can be derived from mammalian cells such as, for example, human fetal kidney cells or rodent cells. Other cell types can include, but are not limited to, BHK cells, Vero cells, CHO cells, or any eukaryotic cell for which a tissue culture method has been established, provided that the cell is herpes virus permissive. The term "herpes virus permissive" means that a herpes virus or herpes virus vector is capable of completing the entire intracellular viral life cycle within the cellular environment. In certain embodiments, the methods described are performed in BHK, a mammalian cell line, and grown in suspension. The host cells may be derived from an existing cell line, such as the BHK cell line, or may be developed de novo.

[0177] The method for producing the rAAV gene constructs described herein also includes co-infecting mammalian cells capable of growing in suspension with: (i) a first recombinant herpes virus comprising nucleic acids encoding an AAV rep gene and an AAV cap gene, each operably linked to a promoter; and (ii) a second recombinant herpes virus comprising GJB2 and a promoter operably linked to said GJB2 gene; and infecting the mammalian cells with the virus, thereby producing recombinant AAV virus particles within the mammalian cells. Also included are recombinant AAV virus particles produced within mammalian cells by a method comprising infecting mammalian cells with: (i) a first recombinant herpes virus comprising nucleic acids encoding an AAV rep gene and an AAV cap gene, each operably linked to a promoter; and (ii) a second recombinant herpes virus comprising GJB2 and a promoter operably linked to said GJB2 gene. The herpes virus described herein is a virus selected from the group consisting of cytomegalovirus (CMV), herpes simplex virus (HSV), varicella zoster virus (VZV), and Epstein - Barr virus (EBV). The recombinant herpes virus is a replication - defective virus. According to some embodiments, the AAV cap gene is a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, Anc80L65, and having serotypes including variants or hybrids (e.g., capsid hybrids of two or more serotypes).

[0178] U.S. Patent Application Publication No. 2007 / 0202587, which is incorporated herein by reference in its entirety, describes essential elements of an rAAV production system. Recombinant AAV is produced in vitro by introduction of a gene construct into cells known as producer cells. Known systems for producing rAAV utilize three basic elements: (1) a gene cassette containing the gene of interest, (2) a gene cassette containing the AAV rep and cap genes, and (3) a source of "helper" viral proteins.

[0179] The first gene cassette is constructed by the gene of interest flanked by ITRs (inverted terminal repeats) derived from AAV. The ITRs function to direct the integration of the gene of interest into the host cell genome and are essential for encapsidation of the recombinant genome (Hermonat and Muzyczka, 1984; Samulski et al., 1983). The second gene cassette contains rep and cap, which are AAV genes encoding proteins required for rAAV replication and packaging. The rep gene encodes four proteins (Rep 78, Rep 68, Rep 52, and Rep 40) required for DNA replication. The cap gene encodes three structural proteins (VP1, VP2, and VP3) that make up the viral capsid (Muzyczka and Berns, 2001).

[0180] Since AAV does not replicate by itself, a third element is required. The helper function is the protein product from a helper DNA virus that creates a cellular environment conducive to efficient rAAV replication and packaging. Conventionally, adenovirus (Ad) has been used to provide the helper function for rAAV, but as discussed herein, herpes viruses can also perform these functions.

[0181] The production of rAAV vectors for gene therapy is performed in vitro using a suitable producer cell line, such as BHK cells grown in suspension. Other cell lines suitable for use in the present invention include HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.

[0182] Any cell type can be used as a host cell, as long as the cell is capable of supporting the replication of herpes virus. Those skilled in the art will be familiar with a wide range of host cells that can be used in the production of herpes virus from host cells. Examples of suitable non-genetically modified mammalian host cells can include, but are not limited to, cell lines such as HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.

[0183] The host cell can be adapted for growth in suspension culture. The host cell can be baby hamster kidney (BHK) cells. BHK cells grown in suspension are derived from the adaptation of adherent BHK cell lines. Both cell lines are commercially available.

[0184] One strategy for delivering all the elements required for the production of rAAV is to utilize two plasmids and a helper virus. This method relies on the transfection of a plasmid containing a gene cassette encoding the necessary gene products into producer cells, as well as the infection of the cells with Ad that provides helper functions. This system utilizes a plasmid with two different gene cassettes. The first plasmid is a proviral plasmid encoding recombinant DNA that is packaged as rAAV. The second plasmid is a plasmid encoding the rep gene and the cap gene. To introduce these various elements into the cells, the cells are transfected with two plasmids in addition to being infected with Ad. The gene products provided by Ad are encoded by the genes E1a, E1b, E2a, E4orf6, and Va (Samulski et al., 1998; Hauswirth et al., 2000, Muzyczka and Burns, 2001). Alternatively, in more recent protocols, the Ad infection step can be replaced by the transfection of an adenovirus "helper plasmid" containing the VA gene, the E2A gene, and the E4 gene (Xiao et al., 1998; Matsushita et al., 1998).

[0185] Ad has been conventionally used as a helper virus for the production of rAAV, but other DNA viruses such as herpes simplex virus type 1 (HSV-1) can also be used similarly. The minimal set of HSV-1 genes required for the replication and packaging of AAV2 has been identified and includes the early genes UL5, UL8, UL52, and UL29 (Muzyczka and Burns, 2001). These genes encode components of the core replication machinery of HSV-1, namely, helicase, primase, primase accessory protein, and single-stranded DNA-binding protein (Knipe, 1989; Weller, 1991). This rAAV helper property of HSV-1 has been exploited in the design and construction of recombinant herpesvirus vectors that can provide the helper virus gene products required for the production of rAAV (Conway et al., 1999).

[0186] The production of rAAV vectors for gene therapy is carried out in vitro using an appropriate producer cell line such as BHK cells grown in suspension. Other cell lines suitable for use in the present invention include HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.

[0187] Any cell type can be used as a host cell as long as the cell is capable of supporting the replication of the herpes virus. A person skilled in the art would be familiar with a wide range of host cells that can be used in the production of herpes viruses from host cells. Examples of suitable non-genetically modified mammalian host cells can include, but are not limited to, cell lines such as HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.

[0188] The host cells can be adapted for growth in suspension culture. In certain embodiments of the invention, the host cells are baby hamster kidney (BHK) cells. BHK cells grown in suspension are derived from the adaptation of adherent BHK cell lines. Both cell lines are commercially available.

[0189] rHSV-based rAAV production process Described herein is a method for generating recombinant AAV virus particles in cells that grow in suspension. Suspensions or anchorage-independent cultures derived from established continuous cell lines are the most widely used means for large-scale production of one or more cells and cell products. Large-scale suspension cultures based on fermentation technology have distinct advantages for the production of mammalian cell products. Uniform conditions can be achieved in bioreactors that allow for the accurate monitoring and control of temperature, dissolved oxygen, and pH, and ensure that representative samples of the culture can be taken. The rHSV vectors used can be easily increased to high titers on permissive cell lines under conditions where a production protocol suitable for scale-up to the virus production levels required for clinical and commercial production is provided, in either tissue culture flasks or bioreactors.

[0190] The culture of cells in stirred-tank bioreactors results in a highly volumetric-specific culture surface area and has been used for the production of viral vaccines (Griffiths, 1986). Furthermore, stirred-tank bioreactors have been demonstrated in the industry to be scalable. One example is the multi-plate type CELL CUBE cell culture system. The ability to produce infectious viral vectors is becoming increasingly important for the pharmaceutical industry, particularly in the context of gene therapy.

[0191] The growth of cells according to the methods described herein can be carried out in a bioreactor that enables the large-scale production of fully biologically active cells that are susceptible to infection by the herpes vectors of the invention. Bioreactors are widely used for the production of biological products from both suspension animal cell cultures and anchorage-dependent animal cell cultures. Most large-scale suspension cultures are carried out as batch or fed-batch processes because they are the easiest to operate and scale up. However, continuous processes based on the chemostat or perfusion principle are also available. The bioreactor system may be configured to incorporate a system that enables medium exchange. For example, a filter may be incorporated into the bioreactor system to facilitate medium exchange by enabling the separation of cells from the spent medium. In some embodiments of the method for producing herpes virus, medium exchange and perfusion are carried out at the start of cell growth on a particular day. For example, medium exchange and perfusion of cells can be initiated on the third day of growth. The filter may be external to the bioreactor or internal to the bioreactor.

[0192] A method for producing recombinant AAV viral particles may include co - infecting suspension cells with a first recombinant herpesvirus comprising a nucleic acid encoding an AAV rep gene and an AAV cap gene each operably linked to a promoter; and a second recombinant herpesvirus comprising a GJB2 gene construct and a promoter operably linked to the gene of interest; and producing recombinant AAV viral particles in the cells, thereby producing recombinant AAV viral particles. The cells can be HEK - 293(293), Vero, RD, BHK - 21, HT - 1080, A549, Cos - 7, ARPE - 19, and MRC - 5. According to some embodiments, the cap gene is an AAV having a serotype selected from the group consisting of AAV1, AAV2, AAV -, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, Anc80L65, and can be selected from AAVs including variants or hybrids thereof (e.g., capsid hybrids of two or more serotypes). The cells can be infected at a multiplicity of coinfection (MOI) between 3 and 14. The first herpesvirus and the second herpesvirus can be viruses selected from the group consisting of cytomegalovirus (CMV), herpes simplex virus (HSV), varicella - zoster virus (VZV), and Epstein - Barr virus (EBV). The herpesvirus can be a replication - defective virus. The co - infection can be a simultaneous infection.

[0193] In mammalian cells, a method for producing recombinant AAV virus particles may include co - infecting suspension cells with a first recombinant herpes virus comprising a nucleic acid encoding an AAV rep gene and an AAV cap gene each operably linked to a promoter; and a second recombinant herpes virus comprising a GJB2 gene construct and a promoter operably linked to the GJB2 gene construct; and increasing the cells, thereby producing recombinant AAV virus particles. The number of virus particles produced is equal to or greater than the number of virus particles grown in an equal number of cells under adherent conditions. The cells can be HEK - 293 (293), Vero, RD, BHK - 21, HT - 1080, A549, Cos - 7, ARPE - 19, and MRC - 5. The cap gene is selected from AAVs having a serotype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, AAV11, AAV12, AAVrh8, AAVrh10, Anc80L65, including variants or hybrids thereof (e.g., capsid hybrids of two or more serotypes). The cells can be infected at a multiplicity of co - infection (MOI) between 3 and 14. The first herpes virus and the second herpes virus can be viruses selected from the group consisting of cytomegalovirus (CMV), herpes simplex virus (HSV), varicella - zoster virus (VZV), and Epstein - Barr virus (EBV). The herpes virus can be a replication - defective virus. The co - infection can be a simultaneous infection.

[0194] A method for delivering a nucleic acid sequence encoding a therapeutic protein into suspension cells comprises co-infecting BHK cells with a first recombinant herpes virus comprising a nucleic acid encoding an AAV rep gene and an AAV cap gene each operably linked to a promoter; and a second herpes virus comprising a GJB2 gene construct, wherein the gene of interest comprises a coding sequence of a therapeutic protein and a promoter operably linked to the GJB2 gene, wherein the cells are infected at a multiplicity of coinfection (MOI) between 3 and 14; and infecting the cells with the virus to express the therapeutic protein, thereby delivering the nucleic acid sequence encoding the therapeutic protein into the cells. The cells can be HEK-293 (293), Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5. See, for example, U.S. Patent No. 9,783,826.

[0195] Treatment method AAV and gene therapy Gene therapy refers to the treatment of congenital or acquired diseases by replacing, altering, or supplementing a gene that causes the disease. Gene therapy is generally achieved by introducing one or more corrective genes into host cells via a medium or vector. Gene therapy using rAAV is very promising for the treatment of many diseases. Described herein are methods for producing recombinant adeno-associated virus (rAAV), particularly methods for producing large amounts of recombinant AAV that assist in the treatment of hereditary hearing loss.

[0196] Currently, worldwide, more than 500 clinical trials on gene therapy are being conducted. The efforts to use rAAV as a vehicle for gene therapy retain promise for its applicability as a treatment for human diseases. Recombinant AAV (rAAV) has already achieved some success preclinically for the delivery and long-term expression of genes introduced into cells in animals, including clinically important non-dividing cells in the brain, liver, skeletal muscle, and lung. In some tissues, AAV vectors have been shown to integrate into the genome of target cells (Hirata et al., 2000, J. of Virology, 74:4612 - 4620).

[0197] A further advantage of rAAV is its ability to implement this function within non-dividing cell types, including hepatocytes, neurons, and skeletal muscle cells. rAAV has been used successfully as a gene therapy vehicle enabling the expression of erythropoietin in the skeletal muscle of mice (Kessler et al., 1996), tyrosine hydroxylase and aromatic amino acid decarboxylase in the CNS in a monkey model of Parkinson's disease (Kaplitt et al., 1994), and factor IX in skeletal muscle and liver in an animal model of hemophilia. At the clinical level, rAAV vectors have been used in human clinical trials to deliver the CFTR gene to cystic fibrosis patients and the factor IX gene to hemophilia patients (Flotte et al., 1998; Wagner et al., 1998), and furthermore, AAV is a helper-dependent DNA parvovirus not associated with diseases in humans or mammals (Berns and Bohensky, 1987, Advances in Virus Research, Academic Press Inc, 32:243 - 307). Therefore, one of the most important attributes of AAV vectors is their safety profile in phase I clinical trials.

[0198] AAV gene therapy has been carried out to treat a variety of diseases and disorders in a number of different pathological situations. For example, in a Phase I study, administration of an AAV2-FIX vector to skeletal muscle of 8 subjects with hemophilia B was demonstrated to be safe and achieved local gene transfer and expression of factor IX for at least 10 months after vector injection (Jiang et al., Mol Ther., 14(3):452-455, 2006), and a Phase I trial of intramuscular injection of a recombinant adeno-associated virus alpha1-antitrypsin (rAAV2-CB-hAAT) gene vector to AAT-deficient adults has also been described (Flotte et al., Hum Gene Ther., 2004, 15(1):93-128), and in another clinical trial, gene therapy with AAV-GAD to the subthalamic nucleus has been shown to be safe and tolerated by patients with advanced Parkinson's disease (Kaplitt et al., Lancet, 2007, 369(9579):2097-2105). The GJB2 AAV construct provides a gene therapy vehicle for the treatment of DFNB1 type hearing loss phenotypes. The use of the gene therapy constructs and methods with GJB2 AAV described herein provides a treatment for DFNB1 type hearing loss, a need that has not been met for many years as there has been no gene therapy-based treatment available to patients.

[0199] GJB2 / Connexin 26 (Cx26) and Hereditary Hearing Loss Described herein are methods that can be used to treat hearing impairment or prevent deafness (or further deafness) in a subject. Delivery of one or more of the nucleic acids described herein to cells within the inner ear, such as cells within the cochlea (or cells that make up the cochlea or cochlear cells), can be used to treat hearing impairment typically defined by partial or total deafness.

[0200] According to some embodiments, provided herein is a method of using GJB2 AAV-based gene therapy for treating non-symptomatic hearing loss (hearing loss and deafness) characterized by congenital, non-progressive, mild to severe sensorineural hearing impairment. The use of the gene therapy constructs and methods with GJB2 AAV described herein represents the first and only example of a long-term (e.g., lifelong) treatment for correcting congenital hearing loss by gene supplementation. Importantly, the use of the gene therapy constructs and methods with GJB2 AAV described herein preserves native hearing, whereas cochlear implantation does not.

[0201] The methods described herein that enable the production of recombinant AAV viral particles in mammalian cells include co-infecting mammalian cells capable of growing in suspension with a first recombinant herpes virus and a second recombinant herpes virus containing a GJB2 gene construct having therapeutic value in the treatment of hereditary hearing loss.

[0202] GJB2 encodes connexin 26 (Cx26), a major gap junction protein that associates with other gap junction proteins to form an extensive network that enables intercellular coupling between non-sensory cells in the cochlea. However, the molecular mechanism by which mutations in GJB2 cause hereditary hearing loss is not fully understood. The fact that loss of GJB2 is known to cause death of various cell types in the inner ear after the onset of hearing suggests its importance during cochlear development. Furthermore, GJB2 / Cx26 is hypothesized to be essential for the formation of the gap junction network required for normal hearing by maintaining potassium gradient homeostasis within the organ of Corti. Individuals with autosomal recessive mutations within GJB2 exhibit the DFNB1 type hearing loss phenotype, which accounts for nearly half of all cases of hereditary hearing loss and results in a prevalence of approximately 2-3 cases per 1000 births.

[0203] The present invention represents a novel rAAV-based gene therapy for treating or preventing hereditary deafness caused by GJB2 mutations, which accounts for approximately 45% of all cases of congenital deafness. The rAAV constructs detailed herein are suitable for pre-linguistic or post-linguistic treatment for the prevention or treatment of both autosomal recessive GJB2 mutants (DFNB1) and autosomal dominant GJB2 mutants (DFNA3A), and will be administered by any method necessary for inner ear delivery. The gene constructs described herein can be used in methods and / or compositions for treating and / or preventing DFNB1 type deafness.

[0204] According to some embodiments, gene therapy with GJB2 AAV is administered to a subject before the onset of deafness. According to some embodiments, the subject is diagnosed with DFNB1 by molecular genetic testing that identifies the mutation in GJB2 that causes deafness. According to some embodiments, the subject has family members with non-symptomatic hearing loss. According to some embodiments, the subject is a pediatric patient. According to some embodiments, the subject is an infant.

[0205] The rAAV constructs described herein are transduced into inner ear cells, such as cochlear cells, with greater efficiency than conventional AAV vectors. According to some embodiments, the compositions and methods described herein enable highly efficient delivery of nucleic acids to inner ear cells, such as cochlear cells. According to some embodiments, the compositions and methods described herein enable delivery of a transgene to at least 50% (e.g., at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of inner ear hair cells and expression of the transgene therein, or delivery to at least 50% (e.g., at least 50, 55, 60, 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of outer ear hair cells and expression therein. According to some embodiments, the compositions and methods described herein enable delivery of a transgene to at least 70% (e.g., at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of inner ear hair cells and expression of the transgene therein, or delivery to at least 70% (e.g., at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of outer ear hair cells and expression therein.

[0206] According to some embodiments, described herein is a nucleic acid sequence that is directly introduced into a cell and is expressed to produce a coded product prior to administration in vivo of the resulting recombinant cell. This can be accomplished by any of a number of methods known in the art, such as electroporation, lipofection, calcium phosphate-mediated transfection, and the like.

[0207] Similar to currently available cochlear implants, GJB2 gene therapy will provide a lifetime solution and, unlike cochlear implants, will also add the benefit of preserving native hearing.

[0208] Pharmaceutical composition In accordance with some embodiments, the present disclosure presents a pharmaceutical composition optionally comprising any of the vectors described herein in a pharmaceutically acceptable excipient.

[0209] As is well known in the art, pharmaceutically acceptable excipients are relatively inert substances that facilitate the administration of pharmacologically active substances and may be supplied as solutions or suspensions, may be supplied as emulsions, or may be supplied in solid forms suitable for dissolution or suspension in a liquid prior to use. For example, excipients may impart form or homogeneity and may act as diluents. Suitable excipients include, but are not limited to, stabilizers, humectants and emulsifiers, salts for varying osmotic pressure, encapsulating agents, pH buffering substances, and buffering agents. Such excipients may include any pharmaceutical agent that can be administered without undue toxicity and is suitable for direct delivery to the ear (e.g., inner ear or middle ear). Pharmaceutically acceptable excipients include, but are not limited to, sorbitol, any of the various TWEEN® compounds, and liquids such as water, physiological saline, glycerol, and ethanol. Among these may be included pharmaceutically acceptable salts, such as inorganic acid salts such as hydrochloride, hydrobromide, phosphate, sulfate; and organic acid salts such as acetate, propionate, malonate, benzoate. A complete discussion of pharmaceutically acceptable excipients is available in "REMINGTON’S PHARMACEUTICAL SCIENCES" (Mack Pub. Co., N.J., 1991).

[0210] In accordance with some embodiments, the pharmaceutical composition comprises one or more of BSST, PBS, or BSS.

[0211] In accordance with some embodiments, the pharmaceutical composition further comprises a histidine buffer.

[0212] In accordance with some embodiments, the pharmaceutical composition further comprises a poloxamer buffer.

[0213] According to some embodiments, delivery media (e.g., polymers) that facilitate the introduction of agents across the tympanic membrane and / or through the round window are available, and any such delivery media can be used to deliver the viruses described herein. See, for example, Arnold et al., 2005, Audiol. Neurootol., 10:53-63, which is incorporated herein by reference in its entirety.

[0214] Optionally, although not necessarily, the composition can be supplied in unit dosage forms suitable for accurate dosing.

[0215] According to some embodiments, the composition is administered to a subject prior to cochlear implantation.

[0216] Route of Administration Generally, compositions formulated for administration to the ear are described herein. According to some embodiments, the composition is formulated for administration to cells within the organ of Corti (OC) in the cochlea. Cells within the OC include Hensen cells, Dieters cells, pillar cells, inner phalangeal cells, and / or outer phalangeal / border cells. The OC includes two classes of sensory hair cells: inner ear hair cells (IHC) that convert the mechanical information carried by sound into electrical signals that are transmitted to neuronal structures, and outer ear hair cells (OHC) that are used to amplify and tune the cochlear response, a process required for complex auditory functions. According to some embodiments, the composition is formulated for administration to IHCs and / or OHCs.

[0217] Injection into the scala media filled with high potassium endolymph could provide direct access to the hair cells. However, this alteration to the delicate fluid environment can disrupt the endocochlear potential and increase the risk of injection-related toxicity. The perilymph-filled cavities surrounding the scala media, scala tympani, and scala vestibuli can be accessed from the middle ear via the oval window membrane or round window membrane. The round window membrane, the only non-bony opening to the inner ear, is relatively easily accessible in many animal models, and administration of viral vectors using this route has good tolerance. In humans, placement of cochlear implants routinely relies on surgical electrode insertion through the round window membrane. Intratympanic injection of therapeutic agents is a technique for injecting drugs into the middle ear and / or inner ear behind the tympanic membrane. According to some embodiments, the composition is administered by intratympanic injection into the inner ear and / or middle ear. According to some embodiments, the composition is administered by injection through the round window membrane. According to some embodiments, the composition is directly administered to the round window membrane via transtympanic injection. According to some embodiments, the composition is administered by injection into the scala tympani or scala media. According to some embodiments, the delivery system is a syringe / needle device capable of puncturing the tympanic membrane and providing direct access to the round window membrane. According to some embodiments, the delivery system is an ear drop. According to some embodiments, the delivery system is a delivery system as a topical formulation. According to some embodiments, the composition is administered during a surgical procedure, such as during cochleotomy or scala media fenestration. For some administration routes, for example, for injection into the inner ear and / or middle ear, a sustained release system may be used.

[0218] By safely and effectively transducing the cochlear cells described herein with a heterologous nucleic acid, the method of the invention may be used to treat an individual, such as a human, in which case the transduced cells produce GJB2 in an amount sufficient to restore hearing or inner ear vestibular function over a long period (e.g., months, years, decades, lifetime).

[0219] According to the treatment method of the present invention, the capacity of the vector to be delivered can be determined based on the characteristics of the subject to be treated, such as the age of the subject and the volume of the region to which the vector is to be delivered. According to some embodiments, the volume of the composition to be injected is between about 10 μl and about 1000 μl, or between about 100 μl and about 1000 μl, or between about 100 μl and about 500 μl, or between about 500 μl and about 1000 μl. According to some embodiments, the volume of the composition to be injected is approximately 1 μl, 2 μl, 3 μl, 4 μl, 5 μl, 6 μl, 7 μl, 8 μl, 9 μl, 10 μl, 15 μl, 20 μl, 25 μl, 50 μl, 75 μl, 100 μl, 200 μl, 300 μl, 400 μl, 500 μl, 600 μl, 700 μl, 800 μl, 900 μl, or any one amount among 1 mL, or exceeds any amount between these.

[0220] According to the treatment method of the present disclosure, the concentration of the vector to be administered may vary depending on the production method, and may be selected or optimized based on the concentration determined to be therapeutically effective for a specific administration route. According to some embodiments, the concentration of the vector genome per milliliter (vg / ml) is about 10 8 vg / ml, about 10 9 vg / ml, about 10 10 vg / ml, about 10 11 vg / ml, about 10 12 vg / ml, about 10 13 vg / ml, and about 10 14 vg / ml selected from the group consisting of. In a preferred embodiment, the concentration is in the range of 10 10 vg / ml to 10 13 vg / ml in volumes of about 0.1 mL, about 0.2 mL, about 0.4 mL, about 0.6 mL, about 0.8 mL, and about 1.0 mL.

[0221] The effectiveness of the compositions described herein can be monitored by several criteria. For example, after treatment of a subject using the methods of the present disclosure, the subject can be evaluated for improvement and / or stabilization and / or delay in progression of one or more signs or symptoms of a disease state by one or more clinical parameters, including, for example, the clinical parameters described herein. Examples of such tests are known in the art and include subjective measures, in addition to objective measures (e.g., subject-reported measures). According to some embodiments, these tests can include, but are not limited to, auditory brainstem response (ABR) measurements, speech perception, communication modalities, and subjective evaluations of auditory response recognition.

[0222] According to some embodiments, first, subjects presenting with asymptomatic hearing loss (hearing loss and deafness) (DFNB1) are examined to determine their threshold hearing sensitivity across the audible range. The subjects are then treated with the rAAV compositions described herein. A change in threshold hearing level as a function of frequency, measured in dB, is determined. According to some embodiments, an improvement in hearing is determined as an improvement of 10 dB to 50 dB in threshold hearing sensitivity in the mid- to high-frequency range in at least one ear. According to some embodiments, an improvement in hearing is determined as an improvement of 10 dB to 30 dB in threshold hearing sensitivity in the mid- to high-frequency range in at least one ear. According to some embodiments, an improvement in hearing is determined as an improvement of 10 dB to 20 dB in threshold hearing sensitivity in the mid- to high-frequency range in at least one ear.

[0223] In vivo mouse model for hereditary hearing loss According to some embodiments, an in vivo mouse model is used to evaluate the effectiveness of the compositions described herein. One example of an in vivo mouse model is a connexin 26 / GJB2 knockout transgenic mouse as a disease model, such as the disease model detailed in Takada et al., Hearing Research, Volume 309, 2014, which is incorporated herein by reference in its entirety.

[0224] With reference to the following examples, further embodiments of the present invention will be described. The examples contained in this specification are presented for illustrative purposes and are not presented for any purpose of limitation in any form.

Example

[0225] Example 1 Method The present invention was implemented using a method including but not limited to the following. In this specification, the method described in PCT Application No. PCT / US2007 / 017645, filed on August 8, 2007, entitled "Production of Recombinant AAV in Mammalian Cells", which is a continuation-in-part of U.S. Application No. 11 / 503,775, filed on August 14, 2007, entitled "Production of Recombinant AAV in Mammalian Cells", which claims the benefit of U.S. Application No. 10 / 252,182, filed on September 23, 2002, and now U.S. Patent No. 7,091,029, issued on August 15, 2006, is described. The entire contents of the aforementioned applications are hereby incorporated by reference in their entirety into this specification.

[0226] rHSV Coinfection Method The rHSV coinfection method for producing recombinant adeno-associated virus (rAAV) utilizes two ICP27-deficient type 1 recombinant herpes simplex virus (rHSV-1) vectors, where one vector carries the AAV rep gene and cap gene (rHSV-rep2capX, where "capX" refers to any of the AAV serotypes), and the second vector carries the gene of interest (GOI) cassette flanked by AAV inverted terminal repeats (ITRs). The system was developed with the rep, cap, and ITRs of AAV serotype 2 and the humanized green fluorescent protein gene (GFP) as a transgene, but may also be utilized with different transgene serotype / pseudotype elements.

[0227] A mammalian cell is infected with all cis - acting rAAV components and trans - acting rAAV components, as well as an rHSV vector that provides helper functions essential for efficient rAAV infection. The cell is infected with a mixture of rHSV - rep2capX and rHSV - GOI. The cells are harvested and lysed to release AAV - GOI, and the resulting vector stock is titrated by various methods described below.

[0228] Lysis with DOC At the time of harvest, the cells and the medium are separated by centrifugation. While the medium is set aside, the cell - associated rAAV is extracted by using 2 - 3 freeze - thaw cycles and extracting the cell pellet with a lysis buffer (20 mM Tris - HCl, pH 8.0, 150 mM NaCl) containing 0.5% (w / v) deoxycholic acid (DOC). Optionally, the medium and the cell - associated rAAV lysate are recombined.

[0229] In situ lysis An alternative method for harvesting rAAV is by in situ lysis. At the time of harvest, MgCl2 is added to a final concentration of 1 mM, Triton X - 100 is added to a final concentration of 1% (v / v), and benzonase is added to a final concentration of 50 units per mL. This mixture is shaken or stirred at 37 °C for 2 hours.

[0230] Quantitative real - time PCR to determine DRP yield The DNase-resistant particle (DRP) assay uses sequence-specific oligonucleotide primers and dual-labeled hybridizing probes to detect and quantify the amplification of a DNA sequence using real-time quantitative polymerase chain reaction (qPCR) technology. The target sequence hybridizes to the DNA and amplifies in the presence of a fluorescent probe that emits copy number-dependent fluorescence. The DRP titer (DRP / mL) is calculated by direct comparison of the relative fluorescence units (RFU) of the test substance to the fluorescence signal generated from a known plasmid dilution containing the same DNA sequence. The data generated from this assay reflects the quantity of packaged viral DNA sequences and does not indicate the integrity of the sequences or the infectivity of the particles.

[0231] Green cell infectivity assay to determine the yield of infectious particles (rAAV-GFP only) Infectious particle (ip) titration is performed on the rAAV-GFP strain using the green cell assay. C12 cells (a HeLa-derived cell line expressing the Rep and Cap genes of AAV2 (see the following references)) are infected with serial dilutions of rAAV-GFP + adenovirus at a saturating concentration (providing helper functions for AAV replication). Two to three days after incubation, the number of fluorescent green cells (each cell representing 1 infectious event) is counted and used to calculate the titer in ip per mL of the virus sample.

[0232] Clark KR et al. described the production of recombinant adenoviruses in Hum. Gene Ther., 1995, 6:1329 - 1341; and Gene Ther., 1996, 3:1124 - 1132, which are hereby incorporated by reference in their entirety.

[0233] TCID to determine the infectivity of rAAV 50 The infectivity of rAAV particles carrying the gene of interest (rAAV-GOI) is determined by TCID 50(50% tissue culture infectious dose) Assay was determined using 8-fold serial dilutions of rAAV in the presence of human adenovirus type 5 and used to infect HeLa RC32 cells (a HeLa-derived cell line expressing AAV2 rep and cap; purchased from ATCC) in 96-well plates. Three days post-infection, lysis buffer (Tris-HCl pH 8.0 at a final concentration of 1 mM, 1 mM EDTA, 0.25% (w / v) deoxycholic acid, 0.45% (v / v) Tween-20, 0.1% (w / v) sodium dodecyl sulfate, 0.3 mg / mL proteinase K) was added to each well and then incubated at 37°C for 1 hour, 55°C for 2 hours, and 95°C for 30 minutes. Lysates (2.5 μL aliquots) from each well were assayed in the DRP qPCR assay described above. Wells with Ct values below the value of the lowest plasmid amount on the standard curve were scored as positive. The ratio of positive wells in a 10-fold dilution series was used to calculate the TCID 50 infectivity (TCID 50 per mL) using the Spearman-Karber formula.

[0234] Cell Lines and Viruses Appropriate producer cell lines, such as HEK293 cells (293), are used to generate rAAV vectors for gene therapy in vitro. Other cell lines suitable for use in the present invention include Vero, RD, BHK-21, HT-1080, A549, Cos-7, ARPE-19, and MRC-5.

[0235] Unless otherwise noted, mammalian cell lines were maintained in Dulbecco's Modified Eagle Medium (DMEM, Hyclone) containing 2-10% (v / v) fetal bovine serum (FBS, Hyclone). Cell culture and virus propagation were performed at 37°C, 5% CO2 for the indicated time intervals.

[0236] Density of Infected Cells The cells can be grown to various concentrations of at least about 1×10 6 ~4×10 6 cells per mL, up to and including approximately this number, or approximately this number, among others. Next, the cells can be infected with recombinant herpes virus at a predetermined MOI.

[0237] Example 2 Cloning of GJB2 Expression Construct The genomic construct was a repeat of variants of the following: ITR(5’)-promoter-GJB2.tag-3’UTR-ITR(3’) * ; variants for each element are further specified below, including references to their concept and experimental development:

[0238] ITR(5’) and ITR(3’) * ITR elements including: The ITR (inverted terminal repeat) element, 143 nucleotides in length, is derived from AAV2. In a normal capacity (i.e., single-stranded (ss)) AAV genome, ITR(3’) is identical in sequence and length to ITR(5’); in a self-complementary (sc) AAV (i.e., half-capacity) genome, ITR(3’) has a 31-nucleotide deletion (abbreviation: “ITRΔtrs”) corresponding to the TRS (terminal resolution site) region, resulting in a truncated length of 113 nucleotides, while ITR(5’) maintains its original 143-nucleotide length.

[0239] Promoters: CBA, smCBA * , EF1a * , CASI * , GFAP, GJB2-128bp * , GJB2-539bp * , GJB2-1000bp * , or a combination promoter (combining two to three of the listed promoters).

[0240] Figure 3 shows the nucleic acid sequence of the CBA promoter (SEQ ID NO: 1). Figure 4 shows the nucleic acid sequence of the EF1a promoter (SEQ ID NO: 2). Figure 5 shows the nucleic acid sequence of the CASI promoter (SEQ ID NO: 3). Figure 6 shows the nucleic acid sequence of the smCBA promoter (SEQ ID NO: 4). Figure 7 shows the nucleic acid sequence of the GFAP promoter (SEQ ID NO: 5). Figure 8 shows the nucleic acid sequence of the GJB2 promoter (SEQ ID NO: 6).

[0241] GJB2 (gene) * : The GJB2 gene reference sequence can be found at NG_008358.1 (ncbi.nlm.nih.gov / nuccore / NG_008358.1). The GJB2 genes used in the described experiments were as follows: wtGJB2 (wild-type human GJB2 gene, human codon-optimized GJB2 (co1-9) (9 codon-optimized human GJB2 cDNA variants (hGJB2co1, hGJB2co2, hGJB2co3, hGJB2co4, hGJB2co5, hGJB2co6, hGJB2co7, hGJB2co8, hGJB2co9)), or the hybrid codon-optimized construct (co369GJB2); the constructs may also contain a C-terminal HA tag (27-nucleotide hemagglutinin). Design and development (gene and tag elements): Figure 10 shows the nucleic acid sequence of human wild-type GJB2 (hGJB2wt) (SEQ ID NO: 10). Figure 11 shows the nucleic acid sequence of human codon-optimized GJB2 (hGJB2co3) (SEQ ID NO: 11). Figure 12 shows the nucleic acid sequence of human codon-optimized GJB2 (hGJB2co6) (SEQ ID NO: 12). Figure 13 shows the nucleic acid sequence of human codon-optimized GJB2 (hGJB2co9) (SEQ ID NO: 13). Figure 18 shows the nucleic acid sequence of the hybrid codon-optimized construct (co369).

[0242] WPRE * , SV40pA *, and a 3’UTR element containing a DNA stuffer: Figure 14 shows the nucleic acid sequence of the HA tag (SEQ ID NO: 14). Figure 15 shows the nucleic acid sequence of the WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element) (SEQ ID NO: 15). Figure 16 shows the nucleic acid sequence of the SV40 poly(A) (SEQ ID NO: 16). Figure 17 shows the nucleic acid sequence of the SV40 / bGH terminator sequence (SEQ ID NO: 17).

[0243] Elements marked by an asterisk ( * ) were also used for the design of the scAAV vector construct.

[0244] Example 3 rAAV Vector for DFNB-1 Treatment The components of the rAAV vector for effective DFNB-1 treatment include a capsid exhibiting cochlear supporting cell tropism, a promoter driving strong to ubiquitous or moderate to strong cell-type specific transgene expression, and a transgene stably encoding the connexin 26 protein.

[0245] Selection of Capsid Figures 19A and 19B summarize data obtained from cochlea-directed studies to identify AAV2 variant capsids for use in gene therapy according to GJB2. These previous studies involved five individual studies, two in mice, one in guinea pigs, and two in non-human primates (NHPs). In the rodent studies, an initial panel of AAV capsids, each containing a CBA-driven GFP reporter, was administered to the cochlea, and then, after two weeks, the tissues were analyzed to identify candidates with high tropism (in this case, high tropism for supporting cells) for the various cochlear tissues of interest. Four final candidate capsids were selected from the rodent data and examined along with two comparator capsids in NHPs (except that it was 12 weeks later instead of two weeks later) in a manner similar to that in the rodents. This data is summarized in the tables shown in Figure 19A (for rodents) and Figure 19B (for NHPs). P2V2 was selected because it presented a favorable combination of positive features compared to other capsids: these features included excellent tropism for supporting cells, relatively low tropism for hair cells, and a low inflammatory outcome.

[0246] Figure 19C depicts various test AAV2 capsid variants. From this study, AAV2-MeB, AAV2-P2V3, AAV2-P2V2, and AAV-P2V6 were identified as top candidates for use in rAAV vectors for the treatment of DFNB.

[0247] Promoter Selection Promoter development consisted of the identification of strong-ubiquitous promoter sequences (CBA, smCBA, EF1a, CASI) as well as cochlear supporting cell type-specific promoters (GFAP and various length variants of the endogenous mammalian GJB2 promoter, including the 128 bp long basal promoter of GJB2 (GJB2(128)), GJB2(539), and GJB2(1000). The numbers in parentheses are the base pair (bp) lengths estimated from the analysis of the UTRs 5' to the GJB2 gene. Although the true endogenous length of this promoter is not precisely known (i.e., the entire promoter has not yet been defined), these lengths were chosen based on predicted regions / motifs of the promoter. The 1000 bp long promoter is predicted to contain all known functional regions of the large GJB2 promoter (mainly the core promoter plus transcription factor binding sites). The 128 bp long promoter represents a minimal "core" promoter (any further truncation would abolish promoter function entirely). The 539 bp long promoter was chosen as an intermediate between these two, presumably to be used as a "medium strength" promoter. The sequences for these promoters were synthesized by a contract service (Genscript), followed by in-house PCR amplification and extraction, resulting in promoter segments with restriction site segments compatible for insertion into a unique viral packaging vector (pAAV-X-hGFP-pA) containing an ampicillin selection cassette, AAV ITR segments, hGFP reporter gene, and SV40 polyA.

[0248] High-efficiency E. coli cells (SURE2) for amplification were transformed with promoter constructs, and clones were selected for verification. Sanger sequencing (Genewiz) and restriction digestion (using appropriate restriction sites (KpnI-MluI) to check for promoter insertion and integrity of the ITR) were performed to verify the promoter plasmids. Positive clones for each promoter construct were selected for subsequent experiments. The native promoter constructs were examined for their effectiveness in driving the expression of hGFP via transfection of HEK-293 (control) or RT4 cells (high GJB2-expressing cell line) in vitro.

[0249] Briefly, 1E5 cells in 400 μl of medium were seeded into 48-well plates. 250 ng of DNA, with a ratio to GeneXPlus of 1:2, was used for transfection. hGFP expression was determined at the time point 48 - 72 hours later. Figure 20 shows GFP expression in HEK293 cells transfected with the pAAV-X-hGFP-pA vector [in the sequence, X is the test promoter selected from CBA, smCBA, CASI, EF1a, GFAP, GJB2(128), GJB2(539), GJB2(1000) as indicated]. Figure 21 shows GFP expression in RT4 cells transfected with the pAAV-X-hGFP-pA vector [in the sequence, X is the test promoter selected from CBA, smCBA, CASI, EF1a, GFAP, GJB2(128), GJB2(539), GJB2(1000) as indicated].

[0250] This data indicated that CBA and EF1a drive strong GFP expression in both cell lines, while GJB2(1000) drives GFP expression moderately in RT4 cells and slightly in HEK293 cells. These results suggested that the CBA promoter and the Ef1a promoter are strong promoters, while GJB2(1000) is a moderately - strong specific promoter.

[0251] Example 4 Design and Synthesis of Codon-Optimized GJB2 Vector The optimization of the GJB2 transgene consists of efforts to enhance protein expression, stability, and function. Codon-optimized variants of GJB2 were synthesized and evaluated for changes in protein expression compared to the wild type (WT). Nine codon-optimized variants were generated, each containing a 27-bp C-terminal HA tag as an alternative means for protein expression and as a means for detection above endogenous proteins. Each codon-optimized variant contained unique optimizations (i.e., codon usage, GC content, stability of the 5'-side mRNA structure, removal of RNA destabilizing sequences, etc.) generated from different algorithms (Genscript, Atum, and Blueheron Biotech).

[0252] In-house PCR amplification and extraction were performed, resulting in the GJB2 transgene segment with restriction sites (NotI) compatible for insertion into a unique viral packaging vector containing an ampicillin selection cassette and AAV ITR segments. After complete synthesis, high-efficiency E. coli cells (SURE2) for amplification were transformed with the codon-optimized construct, and clones were selected for verification. Sanger sequencing (Genewiz) and restriction digestion (restriction sites appropriate for checking the integrity of the transgene insertion and ITR) were performed to verify the codon-optimized GJB2 plasmid.

[0253] Figure 22 depicts the schematic of the codon-optimized construct (AAV-CBA-GJB2(X)-HA-WPRE-pA).

[0254] Positive clones for each codon-optimized construct were selected for subsequent experiments. To determine the codon-optimized sequences with the best expression (named hGJBco1 - hGJBco9), transgene expression experiments were conducted and analyzed via in-cell ELISA, immunofluorescence, and Western blotting. Figures 23A and 23B show the fold changes in GJB2 protein expression and HA protein expression (respectively) by various GJB2 codon-optimized constructs (AAV-CBA-GJB2(X)-HA-WPRE-pA) compared to the control (WT) when assayed by ELISA. As shown in Figure 23A, constructs containing hGJBco3 (CO3), hGJBco6 (CO6), and hGJBco9 (CO9) showed equivalent protein expression compared to WT when assayed with a connexin 26 mouse monoclonal antibody (ThermoFisher Scientific, CX-12H10). As shown in Figure 23B, CO9 showed significantly higher protein expression when assayed with an HA mouse monoclonal antibody (ThermoFisher Scientific; catalog number: 26183). Western blot analysis showed that co9 had equivalent expression to WT when assayed with a connexin 26 mouse monoclonal antibody, and co2, co3, co5, co6, co8, and co9 showed equivalent expression to WT when assayed with an HA monoclonal antibody (not shown). Immunofluorescence data showed equivalent results when assayed with GJB2 and HA antibodies, where co3, co5, co6, co8, and co9 showed equivalent / higher GJB2 expression compared to WT. Quantification by immunofluorescence study is shown in Figure 24A for anti-connexin 26 antibody (anti-CX26) staining determined by GFP intensity, and in Figure 24B for anti-HA staining determined by RFP intensity.

[0255] Example 5 Codon Optimization Study (Second Generation) From the results described in Example 4, codon-optimized GJB2 #9 (hGJB2co9) was selected and advanced. The sequence homology of hGJB2co9 compared to WT (hGJB2) was determined to be 78% (Figure 25). Among attempts to improve protein expression, codon-optimized variants approximating WT were generated. The parameters for designing the variants were as follows: When all three of the co3 / co6 / co9 positions were aligned, nucleotide changes were preserved. When two of the three co3 / co6 / co9 positions were aligned, nucleotide changes were preserved. Single nucleotide changes were removed from individual co3 / co6 or co9. Figure 26 shows the alignment between the sequences of GJB2 WT, co3, co6, and co9, the consensus sequence determined from the alignment, and the co369 hybrid codon-optimized sequence. The aligned nucleotide changes were examined to determine how well they improved protein expression.

[0256] Figures 27A and 27B show the fold changes in GJB2 protein expression and HA protein expression (respectively) by the co9 codon-optimized construct and the co369 codon-optimized construct (AAV-CBA-GJB2(X)-HA-WPRE-pA) compared to the control (WT) when assayed by ELISA (Pierce Colorimetric In-Cell ELISA Kit (#62200)). Janus Green staining (A450 / A615 values) was performed to account for differences in cell numbers in the various wells. As shown in Figure 27A, the connexin 26 antibody (Cx26; ThermoFisher Scientific, CX-12H10) results showed similar protein expression for WT, co9, and co369. As shown in Figure 27A, the absence of WPRE reduced protein expression. As shown in Figure 27B, the results show a high level of mean protein expression for the co369-HA construct (HA-tag mouse monoclonal antibody (ThermoFisher Scientific; catalog number: 26183)). Quadruplicate measurements were taken (4 plates (n = 4)). Janus Green staining (A450 / A615 values) was performed to account for differences in cell numbers in the various wells.

[0257] Western blot analysis showed that co369, a hybrid construct, was among the highest-expressing vectors when assayed with a connexin 26 mouse monoclonal antibody (Figure 28A). When assayed with an anti-HA mouse monoclonal antibody, WT was the highest-expressing vector (Figure 28B). Beta-actin was used as a loading control.

[0258] In summary, the above evaluations of protein expression supported that the co369 hybrid was equivalent to the WT GJB2 construct. The ELISA results supported that the co369 hybrid exhibited the best protein expression. The Western blot results indicated that both the WT and co369 hybrids showed the best protein expression. Furthermore, the immunofluorescence data will inform about the proper localization of the protein (i.e., membrane localization).

[0259] Therefore, from the studies described herein, a construct with the AAV2-P2V6 capsid, CBA-hGFP promoter, and GJB2 co369 hybrid codon-optimized transgene was selected.

[0260] Example 5 In Vivo Study with rAAV Containing GJB2 Genome Expression Construct The following constructs (the terms CBA and CB are used interchangeably to refer to the CBA promoter): pTR-CB-Hybrid GJB2 (co369)-Flag-WPRE Control: pTR-CB-WT GJB2-Flag-WPRE were designed for use in in vivo tests.

[0261] For the addition of 3×flag to each gene of interest, a two-step PCR method was performed to prepare the constructs. An example using the co369 hybrid is shown in Figure 29. Western blot analysis indicated that both the WT flag construct and the co369 hybrid flag construct showed the predicted restriction digest fragment bands (Figure 30).

[0262] Figures 31A and 31B show the expression of the codon-optimized constructs in HEK293 cells when assayed by ELISA and probed with an anti-connexin 26 antibody (anti-Cx26; Thermo, CX-1E8 (33-5800)) (Figure 31A) or an anti-flag antibody (Figure 31B). The expression of the tagged constructs in HEK293 showed equivalent expression when probing for both Cx26 and 3xflag. Interestingly, probing with Cx26 was thought to be affected by the presence of the tag. This result was similarly observed in Western blot (Figure 32). Janus green staining (A450 / A615 value) was performed to account for differences in cell numbers in the various wells. All groups were examined in triplicate.

[0263] Figure 32 is a Western blot confirming the results of the ELISA analysis in Figures 31A and 31B, which support the expression of the codon-optimized constructs in HEK293 cells. A GAPDH probe was used to show that the loading of the lysates was equal. The loading of the lysates was equal (GAPDH probe), and the expression of the tagged constructs in HEK293 was observed as predicted.

[0264] Figures 33A and 33B show the expression of the constructs in HeLa cells when assayed by ELISA and probed with an anti-connexin 26 antibody (anti-Cx26; Thermofisher 33-5800 (1:500)) (Figure 33A) or an anti-flag antibody (Figure 33B). The expression of the tagged constructs in HeLa showed equivalent expression when probing for both Cx26 and 3xflag. Probing with Cx26 was thought to be affected by the presence of the tag. Overall, the results in HeLa cells were equivalent to the previous experiment in HEK293 cells. Western blot experiments confirmed the ELISA results (not shown).

[0265] In an independent set of experiments, both the WT construct and the co369 hybrid construct passed quality control tests (not shown).

[0266] In vivo studies will be performed with rAAV containing the GJB2 genomic expression construct. The data collected will include the expression of GJB2 from mouse tissues after treatment with the AAV-GJB2 lead candidate (determined from the in vitro studies described in the above examples) and the in vivo distribution analysis of the cochlea for this. The data collected will also include the evaluation of GJB2 function by dye diffusion assay and whole cell patch clamp assay.

[0267] For purposes of illustration and clarity of understanding, the present invention has been described in some detail, but it is to be understood that certain changes and modifications can be made within the scope of the appended claims. Modifications of the above-described manner for carrying out the present invention, which are understood in light of the foregoing disclosure or which are obvious to those of ordinary skill in the art of gene therapy, molecular biology, and / or related fields by routine practice or implementation of the present invention, are intended to be within the scope of the following claims.

[0268] All publications (e.g., non-patent literature), patents, patent application publications, and patent applications referred to herein are indicative of the level of the art to which the present invention pertains. All such publications (e.g., non-patent literature), patents, patent application publications, and patent applications are hereby incorporated by reference into this specification to the same extent as if each individual publication, patent, patent application publication, or patent application was specifically and individually indicated to be incorporated by reference.

[0269] The foregoing invention has been described in connection with this preferred embodiment, but is not limited thereby, but is to be limited only by the scope of the following claims.

Claims

1. An isolated polynucleotide comprising a codon-optimized nucleic acid sequence encoding gap junction protein beta-2 (GJB2), wherein the codon-optimized nucleic acid sequence is at least 95% identical to SEQ ID NO:

18.

2. The isolated polynucleotide according to claim 1, wherein the codon-optimized nucleic acid sequence is 100% identical to SEQ ID NO:

18.

3. The isolated polynucleotide according to any one of claims 1 to 2, wherein the codon-optimized nucleic acid sequence is a cDNA sequence.

4. The isolated polynucleotide according to any one of claims 1 to 3, wherein the codon-optimized nucleic acid sequence is operably linked to a promoter.

5. The isolated polynucleotide according to claim 4, wherein the promoter is a ubiquitous active CBA promoter, a mini CBA (smCBA) promoter, an EF1a promoter, a CASI promoter, a supporting cell promoter of the snail, a GJB2 expression-specific GFAP promoter, a mini GJB2 promoter, a medium-sized GJB2 promoter, a large-sized GJB2 promoter, or a sequence combination of two to three individual GJB2 expression-specific promoters.

6. The isolated polynucleotide according to claim 4 or 5, wherein the promoter is optimized to drive high-level GJB2 expression.

7. The codon-optimized nucleic acid sequence is a 3'UTR regulatory region containing a Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element (WPRE), a polyadenylation signal (pA), and / or a hemagglutinin C-terminal tag The isolated polynucleotide according to any one of claims 1 to 6, further comprising.

8. The isolated polynucleotide according to claim 7, wherein the polyadenylation signal is a human growth hormone (hGH) polyadenylation signal.

9. A polynucleotide comprising, in the order from 5' to 3', a CBA promoter, a codon-optimized nucleic acid sequence encoding GJB2, a WPRE, and a polyadenylation signal, wherein the codon-optimized nucleic acid sequence encoding GJB2 comprises a nucleic acid sequence that is at least 95% identical to SEQ ID NO:

18.

10. A host cell comprising the polynucleotide according to any one of claims 1 to 9.

11. The host cell according to claim 10, which is a mammalian cell.

12. The host cell according to claim 10 or 11, which is a HEK-293 (293) cell, Vero cell, Rhabdomyosarcoma (RD) cell, BHK cell, HT-1080 cell, A549 cell, Cos-7 cell, ARPE-19 cell, or MRC-5 cell.

13. The host cell according to claim 12, which is a BHK-21 cell.

14. A virus comprising the polynucleotide according to any one of claims 1 to 9, which is a recombinant herpes simplex virus (rHSV) or an adeno-associated virus (AAV).

15. (a) The polynucleotide according to any one of claims 1 to 9, and (b) a minimal regulatory element A transgene expression cassette comprising.

16. The transgene expression cassette according to claim 15, wherein the minimal regulatory element is (i) one of a ubiquitous active CBA promoter, a small CBA (smCBA) promoter, an EF1a promoter, a CASI promoter, a supporting cell promoter of the snail, a GFAP promoter specific for GJB2 expression, a small GJB2 promoter, a medium GJB2 promoter, a large GJB2 promoter, or a sequence combination of two to three individual GJB2 expression-specific promoters, (ii) a 3'UTR regulatory region containing a WPRE (Woodchuck Hepatitis Virus Posttranscriptional Regulatory Element), and / or (iii) a polyadenylation signal of SV40 or human growth hormone (hGH).

17. A viral vector comprising the transgene expression cassette according to claim 15 or 16.

18. The viral vector according to claim 17, wherein the viral vector is an adeno-associated virus (AAV) vector, and the codon-optimized nucleic acid sequence encoding GJB2 is sandwiched between a 5' ITR (inverted terminal repeat) and a 3' ITR.

19. A host cell comprising the transgene expression cassette according to claim 15 or 16.

20. A kit comprising the viral vector according to claim 17 or 18 and instructions for use.

21. The viral vector according to claim 18, wherein the serotype of the capsid sequence and the serotype of the ITR of the AAV vector are independently derived from serotypes selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, AAV10, rhAAV10, AAV11, and AAV12.

22. The viral vector according to claim 18 or 21, further comprising a protein capsid variant that is optimal for inner ear delivery.

23. The viral vector according to claim 22, wherein the capsid sequence is derived from AAV2.

24. The viral vector according to claim 22 or 23, wherein the capsid sequence is selected from the group consisting of AAV2-MeB, AAV2-P2V2, AAV2-P2V3, and AAV2-P2V6.

25. The viral vector according to any one of claims 22 to 24, wherein the AAV capsid variant comprises a VP1 protein comprising the sequence of SEQ ID NO:

27.

26. The viral vector according to any one of claims 22 to 25, wherein the AAV capsid variant comprises an amino acid sequence having one or more amino acid substitutions compared to the wild-type AAV2 capsid polypeptide selected from the group consisting of Q263A, T491Q, S492D, D494E, Y500F, and T503P.

27. A pharmaceutical composition comprising the viral vector according to any one of claims 17, 18, or 21 to 25 for use in a method of treating hereditary hearing loss in a subject.

28. A pharmaceutical composition comprising the viral vector according to any one of claims 17, 18, or 21 to 25 for use in a method of preventing hereditary hearing loss in a subject.

29. The pharmaceutical composition according to any one of claims 27 to 28, wherein the hereditary hearing loss is DFNB1 type hearing loss.

30. The pharmaceutical composition according to claim 29, wherein the hereditary hearing loss is caused by one or more mutations in GJB2.

31. The pharmaceutical composition according to any one of claims 28 or 29, wherein the hereditary hearing loss is caused by an autosomal recessive GJB2 mutation (DFNB1).

32. The pharmaceutical composition according to any one of claims 28 or 29, wherein the hereditary hearing loss is caused by an autosomal dominant GJB2 mutation (DFNA3A).

33. The pharmaceutical composition according to any one of claims 28 or 29 to 32, which is administered to a snail.

34. The pharmaceutical composition according to any one of claims 28 or 29 to 33, which is administered by an intravenous route, an intracerebroventricular route, an intrasnail route, or an intrathecal route, or a combination thereof.

Images