Patent Application
20250114480
The contents of sequence the electronic listing (BGTR_004_01WO_SeqList_ST26.xml; Size: 63,410 bytes; and Date of Creation: Jan. 25, 2023) are herein incorporated by reference in their entirety.
The disclosure relates to adeno-associated virus-mediated gene therapy for genetic hearing loss.
About 50% to 60% of hearing loss in babies is due to genetic causes. Of these, more than 10,000 patients suffer from genetic hearing loss that is associated with the deficiency or dysfunction of transmembrane channel-like 1 (TMC1). Pathophysiology studies show that mutated TMC1 disrupts electrical response to sound-evoked displacement of hair cell stereocilia, and therefore causes moderate to profound sensorineural hearing loss.
Around 60% of patients with TMC1-associated hearing loss experience profound, pre-lingual deafness and are identified through newborn screening. The remaining proportion of patients experience mild-moderate hearing loss in early childhood and progress to severe hearing loss by adolescence. These children often fall in a state of hearing called the “donut hole” where the child is poorly aided but not deaf enough to be indicated for a cochlear implant. These patients are likely to progress to severe or profound deafness. Although patients who have profound hearing loss are indicated for a cochlear implant, these implants often do not fully restore hearing.
Thus, there is an unmet need for compositions and methods, such as gene therapy compositions and methods that can be used to treat genetic hearing loss, such as TMC1-associated hearing loss.
The present disclosure provides compositions and methods for use in the treatment of TMC1-associated hearing loss. In an aspect, the present disclosure provides nucleic acid molecules comprising an adeno-associated virus (AAV) expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′: a 5′ AAV inverted terminal repeat (ITR); a promoter comprising a CB6 promoter; a transgene encoding transmembrane channel-like 1 (TMC1); and a 3′ AAV ITR. The present disclosure also provides plasmids, comprising any one of the nucleic acid molecules disclosed herein, and cells comprising any one of the nucleic acid molecules or plasmids disclosed herein.
The present disclosure also provides recombinant adeno-associated viruses (rAAVs), comprising the AAV expression cassettes disclosed herein, and methods of use thereof in treating a genetic hearing loss in a subject in need thereof, comprising administering to the ear of the subject for an administration period, a therapeutically effective amount of any one of the nucleic acid molecules, plasmids, cells, rAAVs, or compositions disclosed herein.
In some embodiments, the subject suffers from, or is at a risk of developing the genetic hearing loss. In some embodiments, the genetic hearing loss is an autosomal recessive non-syndromic hearing loss (ARNSHL). In some embodiments, the genetic hearing loss is associated with, promoted by, or caused by a mutation in the transmembrane channel-like 1 (TMC1)-encoding gene. In some embodiments, the subject has a hearing threshold in the range of about 25 decibels (dB) to about 80 dB.
In some embodiments, the hearing threshold of the subject during or after the administration period is lower, as compared to before the administration period. In some embodiments, the survival of inner hair cells and/or outer hair cells in the ear of the subject during or after the administration period is improved as compared to the survival of inner hair cells and/or outer hair cells in the ear prior to the administration period.
In some embodiments, the nucleic acid molecule, the plasmid, the cell, the rAAV, or the composition is administered via intracochlear delivery. In some embodiments, the subject is a human subject. In some embodiments, the subject is a neonate or an infant.
The present disclosure provides gene therapy compositions and methods for treating genetic hearing loss. In particular, the disclosure provides compositions, comprising recombinant adeno-associated viruses (rAAVs) comprising an AAV capsid protein, and an AAV expression cassette encoding transmembrane channel-like 1 (TMC1), and methods of use thereof.
It is to be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.
Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood to one of ordinary skill in the art to which the present application belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present application, representative methods and materials are herein described.
The terms “a”, “an”, and “the” refer to “one or more” when used in this application, including the claims. Thus, for example, reference to “a carrier” includes mixtures of one or more carriers, two or more carriers, and the like and reference to “the method” includes reference to equivalent steps and/or methods known to those skilled in the art, and so forth.
In the present description, any concentration range, percentage range, ratio range, or integer range is to be understood to include the value of any integer within the recited range and, when appropriate, fractions thereof (such as one tenth and one hundredth of an integer), unless otherwise indicated. The term “about”, when immediately preceding a number or numeral, means that the number or numeral ranges plus or minus 10%, such as plus or minus 5%.
Also as used herein, “and/or” refers to and encompasses any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative (“or”). The use of the alternative (e.g., “or”) should be understood to mean either one, both, or any combination thereof of the alternatives.
As used herein, “carrier” includes any and all solvents, dispersion media, vehicles, coatings, diluents, antibacterial and antifungal agents, isotonic and absorption delaying agents, buffers, carrier solutions, suspensions, colloids, and the like.
The term “pharmaceutically acceptable”, unless otherwise noted, is used to characterize a moiety (e.g., a salt, dosage form, or excipient) as being appropriate for use in accordance with sound medical judgment. In general, a pharmaceutically acceptable moiety has one or more benefits that outweigh any deleterious effect that the moiety may have. Deleterious effects may include, for example, excessive toxicity, irritation, allergic response, and other problems and complications.
As used herein, “treatment,” “treating,” “palliating,” and “ameliorating” are used interchangeably. These terms refer to an approach for obtaining beneficial or desired results including but not limited to a therapeutic benefit and/or a prophylactic benefit. Therapeutic benefit refers to any therapeutically relevant improvement in or effect on one or more diseases, conditions, or symptoms under treatment. The term “treating” in one embodiment, includes: (1) preventing or delaying the appearance of clinical symptoms of the state, disorder or condition developing in the patient that may be afflicted with or predisposed to the state, disorder or condition but does not yet experience or display clinical or subclinical symptoms of the state, disorder or condition; (2) inhibiting the state, disorder or condition (e.g., arresting, reducing or delaying the development of the disease, or a relapse thereof in case of maintenance treatment, of at least one clinical or subclinical symptom thereof); (3) relieving the condition (for example, by causing regression, or reducing the severity of the state, disorder or condition or at least one of its clinical or subclinical symptoms).
The term “effective amount” or “therapeutically effective amount” refers to the amount of an agent that is sufficient to achieve an outcome, for example, to effect beneficial or desired results, such as treatment of genetic hearing loss or of a symptom thereof. The therapeutically effective amount may vary depending upon one or more of: the subject and disease condition being treated, the weight and age of the subject, the severity of the disease condition, the manner of administration and the like. A therapeutically effective amount may be an amount sufficient to treat hearing loss and/or to ameliorate, diminish the severity of, eliminate, and/or delay the onset of one or more symptoms of genetic hearing loss.
The terms “subject,” “individual,” and “patient” are used interchangeably herein to refer to a vertebrate, such as a mammal. The mammal may be, for example, a mouse, a rat, a rabbit, a cat, a dog, a pig, a sheep, a horse, a non-human primate (e.g., cynomolgus monkey, chimpanzee), or a human. A subject's tissues, cells, or derivatives thereof, obtained in vivo or cultured in vitro are also encompassed. A human subject may be an adult, a teenager, a child (2 years to 14 years of age), an infant (1 month to 24 months), or a neonate (up to 1 month). In some embodiments, the adults are seniors about 65 years or older, or about 60 years or older. In some embodiments, a subject is an infant or neonate. In some embodiments, a subject is up to 24 months old. In some embodiments, a subject is less than 6 years of age. In some embodiments, a subject is between about 6 and about 12 years of age. In some embodiments, a subject is between about 12 and about 18 years of age. In some embodiments, a subject is less than 18 years of age. In some embodiments, a subject is at least 18 years of age. In some embodiments, the subject is a pregnant woman or a woman intending to become pregnant.
An “adeno-associated virus (AAV) expression cassette” is a nucleic acid that gets packaged into a recombinant AAV vector, and comprises a sequence encoding one or more transgenes, such as one or more transgenes flanked by a 5′ inverted terminal repeat (ITR) and a 3′ITR.
As used herein, the terms “virus vector,” “viral vector,” or “gene delivery vector” refer to a virus particle that functions as a nucleic acid delivery vehicle, and which comprises a nucleic acid (e.g., an AAV expression cassette) packaged within a virion. Exemplary virus vectors include adeno-associated virus vectors (AAVs).
As used herein, the term “adeno-associated virus” (AAV), includes but is not limited to, AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV type rh32.33, AAV type rh8, AAV type rh10, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV218, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, AAV9-php.b, and any other AAV now known or later discovered.
As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotides or polypeptide sequences are invariant throughout a window of alignment of components, e.g. nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e. the entire reference sequence or a smaller defined part of the reference sequence. “Percent identity” is the identity fraction times 100. The extent of identity (homology) between two sequences can be ascertained using a computer program and mathematical algorithm. Percentage identity can be calculated using the alignment program Clustal Omega, available at www.ebi.ac.uk/Tools/msa/clustalo using default parameters. See, Sievers et al., “Fast, scalable generation of high-quality protein multiple sequence alignments using Clustal Omega.” (2011 Oct. 11) Molecular systems biology 7:539 For the purposes of calculating identity to a sequence, extensions such as tags are not included.
As used herein, a nucleic acid sequence (e.g., coding sequence) and regulatory sequences are said to be “operably linked” when they are covalently linked in such a way as to place the expression or transcription of the nucleic acid sequence under the influence or control of the regulatory sequences. If it is desired that the nucleic acid sequences be translated into a functional protein, two DNA sequences are said to be operably linked if induction of a promoter in the 5′ regulatory sequences results in the transcription of the coding sequence and if the nature of the linkage between the two DNA sequences does not (1) result in the introduction of a frame-shift mutation, (2) interfere with the ability of the promoter region to direct the transcription of the coding sequences, or (3) interfere with the ability of the corresponding RNA transcript to be translated into a protein.
As used herein, the hearing threshold is the sound level below which a person's ear is unable to detect any sound. The hearing threshold of a control subject exhibiting “normal hearing” is in the range of about 0 dB to about 15 dB.
As used herein, “genetic hearing loss” refers to an impairment or reduction of hearing in a subject and a higher hearing threshold in the subject, as compared to a control subject exhibiting normal hearing. In some embodiments, the genetic hearing loss is associated with, promoted by, or caused by a dysfunction of the inner ear, the outer ear, or a combination thereof. In some embodiments, genetic hearing loss is progressive (e.g., worsens over time). In some embodiments, genetic hearing loss is non-progressive (e.g., does not worsen over time). In some embodiments, genetic hearing loss is familial. In some embodiments, genetic hearing loss is sporadic or de novo. In some embodiments, genetic hearing loss is syndromic (e.g., associated with one or more other conditions). In some embodiments, genetic hearing loss is non-syndromic (e.g., not-associated with other conditions). In some embodiments, genetic hearing loss is acquired. In some embodiments, genetic hearing loss is congenital (e.g., present at birth). In some embodiments, the genetic hearing loss is associated with one or more mutations (e.g., recessive mutations) of TMC1.
In some embodiments, a subject with a hearing threshold of about 25 dB to about 40 dB is said to be experiencing “mild hearing loss”. In some embodiments, a subject with a hearing threshold of about 40 dB to about 55 dB is said to be experiencing “moderate hearing loss”. In some embodiments, a subject with a hearing threshold of about 55 dB to about 70 dB is said to be experiencing “moderate to severe hearing loss”. In some embodiments, a subject with a hearing threshold of about 70 dB to about 90 dB is said to be experiencing “severe hearing loss”. In some embodiments, a subject with a hearing threshold of about 90 dB to about 120 dB is said to be experiencing “profound hearing loss”.
In the present disclosure, dosages may be presented using scientific notation, which can be converted into a decimal number by a person of ordinary skill in the art. For instance, a dosage written in the scientific notation of 310E refers to a decimal number of 3λ1010.
The present disclosure provides gene therapy compositions and methods for treating genetic hearing loss (e.g., autosomal recessive non-syndromic hearing loss (ARNSHL)). For example, the present disclosure provides nucleic acid molecules comprising adeno-associated virus (AAV) expression cassettes. In some embodiments, the AAV expression cassette of a nucleic acid molecule comprises, from 5′ to 3′: a 5′ AAV inverted terminal repeat (ITR); a promoter; a transgene (e.g., a transgene encoding transmembrane channel-like 1 (TMC1)); and a 3′ AAV ITR.
In some embodiments, the AAV expression cassette comprises cis-acting 5′ and 3′ inverted terminal repeat sequences, as described further in B. J. Carter, in “Handbook of Parvoviruses”, ed., P. Tijsser, CRC Press, pp. 155 168 (1990), which is incorporated herein by reference in its entirety for all purposes. The AAV ITR sequences may be obtained from any known or presently unknown AAV, including presently identified mammalian AAV types disclosed herein.
In some embodiments, the AAV expression cassette comprises a 5′ ITR and/or a 3′ ITR derived from AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV type rh32.33, AAV type rh8, AAV type rh10, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV2i8, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, or AAV9-php.b. In some embodiments, the AAV expression cassette comprises a 5′ ITR derived from AAV2, a 3′ ITR derived from AAV2, or a combination thereof.
In some embodiments, the 5′ AAV ITR sequence comprises a nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 6. In some embodiments, the 5′ AAV ITR sequence comprises, or consists of, the sequence of SEQ ID NO: 6.
In some embodiments, the 3′ AAV ITR sequence comprises a nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 7. In some embodiments, the 3′ AAV ITR sequence comprises, or consists of, the sequence of SEQ ID NO: 7.
In some embodiments, the AAV expression cassette comprises expression control elements which are operably linked to the transgene. Expression control elements include appropriate transcription initiation, termination, and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (polyA) signals; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency; sequences that enhance protein stability; and, in some cases, sequences that enhance secretion of the encoded product.
In some embodiments, the AAV expression cassette comprises an intron. In some embodiments, the intron is located between a promoter/enhancer sequence and a transgene. In some embodiments, the intron is derived from SV-40, and is referred to as the SV-40 T intron sequence. In some embodiments, the AAV expression cassette comprises an internal ribosome entry site (IRES). In some embodiments, the AAV expression cassette comprises a nucleic acid encoding a 2A self-cleaving peptide. Illustrative 2A self-cleaving peptides include P2A, E2A, F2A, and T2A. In some embodiments, the AAV expression cassette comprises an element described in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., and references cited therein, at, for example, pages 3.18, 3.26, 16.17, and 16.27 and Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, New York, 1989, each of which is incorporated herein by reference in its entirety for all purposes.
In some embodiments, the AAV expression cassette comprises a woodchuck hepatitis virus post-transcriptional element (WPRE). (See, e.g., Wang and Verma, Proc. Natl. Acad. Sci., USA, 96:3906-3910 (1999)). In some embodiments, the AAV expression cassette comprises a hepatitis B virus posttranscriptional regulatory element (HBVPRE) and/or a RNA transport element (RTE). In some embodiments, the WPRE or HBVPRE sequence is any of the WPRE or HBVPRE sequences disclosed in U.S. Pat. Nos. 6,136,597 and 6,287,814. In some embodiments, the WPRE comprises a nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 5. In some embodiments, the WPRE comprises the nucleic acid sequence of SEQ ID NO: 5.
In some embodiments, the AAV expression cassette comprises one or more 5′-non-transcribed and/or 5′-non-translated sequences involved with the initiation of transcription and translation, respectively, such as a TATA box, capping sequence, CAAT sequence, enhancer element, or the like. In some embodiments, the AAV expression cassette comprises an enhancer sequence and/or upstream activator sequence. In some embodiments, the AAV expression cassette comprises one or more 5′ leader and/or signal sequences.
In some embodiments, the AAV expression cassette comprises one or more promoters. In some embodiments, the AAV expression cassette comprises a chicken β-actin promoter. In some embodiments, the AAV expression cassette comprises a CB6 promoter. In some embodiments, the CB6 promoter comprises a nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 2. In some embodiments, the CB6 promoter comprises, or consists of, the nucleic acid sequence of SEQ ID NO: 2.
In some embodiments, the AAV expression cassette comprises a CMV promoter. In some embodiments, the CMV promoter comprises a nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 18. In some embodiments, the CMV promoter comprises, or consists of, the nucleic acid sequence of SEQ ID NO: 18.
In some embodiments, the AAV expression cassette comprises a promoter selected from an Espin promoter, a protocadherin 15 (PCDH15) promoter, a PTPRQ promoter, a My06 promoter, a KCNQ4 promoter, a myosin 7a (Myo7a) promoter, a synapsin promoter, a GFAP promoter, a CMV promoter, a CAG promoter, a CBH promoter, a CBA promoter, a U6 promoter, and a TMHS (LHFPL5) promoter.
In some embodiments, the AAV expression cassette comprises a constitutive promoter. Examples of constitutive promoters include, without limitation, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the cytomegalovirus (CMV) promoter (optionally with the CMV enhancer), the CMV-IE enhancer, the SV40 promoter, the dihydrofolate reductase promoter, the β-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EF1a promoter.
In some embodiments, the AAV expression cassette comprises an inducible promoter. Non-limiting examples of inducible promoters include the zinc-inducible sheep metallothionine (MT) promoter, the dexamethasone (Dex)-inducible mouse mammary tumor virus (MMTV) promoter, the T7 polymerase promoter system, the ecdysone insect promoter, the tetracycline-repressible system, the tetracycline-inducible system, the RU486-inducible system, and the rapamycin-inducible system. Other types of inducible promoters include those that are regulated by a specific physiological state, e.g., temperature, acute phase, a particular differentiation state of the cell, or a specific cell cycle phase.
In some embodiments, the AAV expression cassette comprises the native promoter, or fragment thereof, or the native expression control element, operably linked to the transgene encoding TMC1. In some embodiments, the AAV expression cassette comprises one or more regulatory sequences that impart tissue-specific gene expression capabilities (e.g., tissue-specific regulatory sequences). In some cases, a tissue-specific regulatory sequence binds one or more tissue-specific transcription factors that induce transcription in a tissue-specific manner. Examples of tissue-specific regulatory sequences include, but are not limited to, the following tissue specific promoters: neuronal promoters such as the neuron-specific enolase (NSE) promoter, the neurofilament light chain gene promoter, and the neuron-specific vgf gene promoter.
In some embodiments, the AAV expression cassette comprises a CMV-IE enhancer. In some embodiments, the enhancer is a CMV-IE enhancer. In some embodiments, the CMV-IE enhancer comprises a nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 16 or SEQ ID NO: 17. In some embodiments, the CMV-IE enhancer comprises, or consists of, the nucleic acid sequence of SEQ ID NO: 16 or SEQ ID NO: 17.
In some embodiments, the AAV expression cassette comprises a consensus sequence, such as a Kozak sequence (for example, a DNA sequence transcribed to an RNA Kozak sequence). As used herein, a “Kozak sequence” refers to a DNA element encoding an “RNA Kozak sequence” which regulates translational initiation.” In some embodiments, the AAV expression cassette comprises a Kozak sequence. In some embodiments, the Kozak sequence comprises a nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 4. In some embodiments, the Kozak sequence comprises, or consists of, the nucleic acid sequence of SEQ ID NO: 4.
In some embodiments, the AAV expression cassette comprises a Kozak sequence upstream of the transgene. In some embodiments, the Kozak sequence (e.g., RNA Kozak sequence) comprises or consists of ACCAUGG (SEQ ID NO: 100), GCCGCCACCAUGG (SEQ ID NO: 101), CCACCAUG (SEQ ID NO: 102), or CCACCAUGG (SEQ ID NO: 103).
In some embodiments, the AAV expression cassette comprises one or more binding sites for one or more miRNAs. Furthermore, the TMC1 transgene may be designed such that multiple miRNAs regulate mRNA by recognizing the same or multiple sites. The presence of multiple miRNA binding sites may result in the cooperative action of multiple RNA-induced silencing complexes (RISCs) and provide highly efficient inhibition of expression. The target site sequence may comprise a total of at least 5, 10, or more nucleotides, such as between 5-100, or between 10-60 nucleotides. The target site sequence may comprise at least 5 nucleotides of the sequence of a target gene binding site. In some embodiments, the AAV expression cassette comprises an miR-1 binding site, an miR-133a binding site, an miR-122 binding site, or any combination thereof. Further details on the miRNAs are provided in Geisler and Fechner, World J Exp Med 2016 May 20; 6 (2): 37-54, which is incorporated herein by reference in its entirety for all purposes.
In some embodiments, the AAV expression cassette comprises a polyadenylation (poly A) sequence. As used herein, the “polyA sequence” refers to a DNA sequence that when transcribed regulates the addition of a polyA tail to the mRNA transcript. PolyA signals may be derived from many suitable species, including, without limitation SV-40, human, and bovine. In some embodiments, the polyA sequence is a β-globin polyA sequence, such as a mammalian β-globin polyA sequence. In some embodiments, the poly A sequence is a human polyA sequence or a bovine β-globin polyA sequence. In some embodiments, the AAV expression cassette comprises a rabbit β-globin polyA sequence. In some embodiments, the β-globin poly A sequence comprises the nucleic acid sequence of SEQ ID NO: 3.
In some embodiments, the transgene comprises a sequence encoding the TMC1 protein. In some embodiments, the transgene comprises a codon-optimized sequence encoding the TMC1 protein. In some embodiments, the transgene comprises a codon-optimized nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 1. In some embodiments, the transgene comprises a codon-optimized sequence comprising or consisting of the nucleic acid sequence of SEQ ID NO: 1.
In some embodiments, the AAV expression cassette comprises, from 5′ to 3′: (i) a 5′ ITR (e.g. an AAV2-based ITR), (ii) a CB6 promoter, (iii) a Kozak sequence, (iv) a codon optimized transgene encoding human TMC1 protein, (v) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), (vi) a beta globin polyadenylation sequence, and (vii) a 3′ ITR (e.g. an AAV2-based ITR).
In some embodiments, the AAV expression cassette comprises, from 5′ to 3′: (i) a 5′ ITR comprising the nucleic acid sequence of SEQ ID NO: 6, (ii) a CB6 promoter comprising the nucleic acid sequence of SEQ ID NO: 2, (iii) a Kozak sequence comprising the nucleic acid sequence of SEQ ID NO: 4, (iv) a codon optimized transgene encoding human TMC1 protein comprising the nucleic acid sequence of SEQ ID NO: 1, (v) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 5, (vi) a beta globin polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 3, and (vii) a 3′ ITR comprising the nucleic acid sequence of SEQ ID NO: 7.
In some embodiments, the AAV expression cassette comprises a codon-optimized nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 8. In some embodiments, the AAV expression cassette comprises or consists of the nucleic acid sequence of SEQ ID NO: 8.
In some embodiments, the AAV expression cassette comprises a codon-optimized nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 9. In some embodiments, the AAV expression cassette comprises or consists of the nucleic acid sequence of SEQ ID NO: 9.
In some embodiments, the AAV expression cassette comprises a codon-optimized nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 10. In some embodiments, the AAV expression cassette comprises or consists of the nucleic acid sequence of SEQ ID NO: 10.
Recombinant Adeno-Associated Virus (rAAV) for Treating Genetic Hearing Loss
The present disclosure also provides plasmids, comprising any one of the nucleic acid molecules disclosed herein, and cells comprising any one of the nucleic acid molecules or plasmids disclosed herein.
The present disclosure further provides methods of producing a recombinant adeno-associated virus (rAAV). In some embodiments, a method of producing an rAAV comprises contacting an AAV producer cell with any one of the nucleic acid molecules or plasmids disclosed herein. Accordingly, the present disclosure also provides recombinant adeno-associated viruses (rAAVs) produced by the methods of producing rAAVs disclosed herein. In some embodiments, the rAAV comprises an AAV type 1, AAV type 2, AAV type 3 (including types 3A and 3B), AAV type 4, AAV type 5, AAV type 6, AAV type 7, AAV type 8, AAV type 9, AAV type 10, AAV type 11, AAV type 12, AAV type 13, AAV type rh32.33, AAV type rh8, AAV type rh10, AAV type rh74, AAV type hu.68, avian AAV, bovine AAV, canine AAV, equine AAV, ovine AAV, snake AAV, bearded dragon AAV, AAV218, AAV2g9, AAV-LK03, AAV7m8, AAV Anc80, or AAV9-php.b capsid protein.
In some embodiments, the rAAV comprises an AAV9 capsid protein, an AAV Anc80 capsid protein, and/or an AAV9-php.b capsid protein. In some embodiments, the rAAV comprises an AAV9 capsid protein. In some embodiments, the rAAV comprises an AAV9-php.b capsid protein.
In some embodiments, the AAV9-php.b capsid protein is encoded by a nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 13. In some embodiments, the AAV9-php.b capsid protein is encoded by the nucleic acid sequence of SEQ ID NO: 13.
In some embodiments, the AAV9-Anc80 capsid protein has an amino acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 14 or SEQ ID NO: 15. In some embodiments, the AAV9-Anc80 capsid protein has the amino acid sequence of SEQ ID NO: 14 or SEQ ID NO: 15. Further details on the AAV9-php.b and AAV Anc80 capsids are provided in WO2019173367A1, WO2018145111A1, and WO2017100791A1, the contents of which are herein incorporated by reference in their entirety for all purposes.
The present disclosure also provides recombinant adeno-associated viruses (rAAVs). In some embodiments, an rAAV comprises an AAV9-php.b capsid protein and any one of the nucleic acid molecules disclosed herein. In some embodiments, an rAAV comprises an AAV9-php.b capsid protein and a nucleic acid molecule, wherein the nucleic acid molecule comprises an AAV expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′: (i) a 5′ ITR (e.g. an AAV2-based ITR), (ii) a CB6 promoter, (iii) a Kozak sequence, (iv) a codon optimized transgene encoding human TMC1 protein, (v) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), (vi) a beta globin polyadenylation sequence, and (vii) a 3′ ITR (e.g. an AAV2-based ITR).
In some embodiments, an rAAV comprises an AAV9-php.b capsid protein and a nucleic acid molecule, wherein the nucleic acid molecule comprises an AAV expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′: (i) a 5′ ITR comprising the nucleic acid sequence of SEQ ID NO: 6, (ii) a CB6 promoter comprising the nucleic acid sequence of SEQ ID NO: 2, (iii) a Kozak sequence comprising the nucleic acid sequence of SEQ ID NO: 4, (iv) a codon optimized transgene encoding human TMC1 protein comprising the nucleic acid sequence of SEQ ID NO: 1, (v) a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) comprising the nucleic acid sequence of SEQ ID NO: 5, (vi) a beta globin polyadenylation sequence comprising the nucleic acid sequence of SEQ ID NO: 3, and (vii) a 3′ ITR comprising the nucleic acid sequence of SEQ ID NO: 7.
In some embodiments, an rAAV comprises an AAV9-php.b capsid protein; and a nucleic acid molecule, wherein the nucleic acid molecule comprises an AAV expression cassette, wherein the AAV expression cassette comprises a codon-optimized nucleic acid sequence having at least about 80% (for example, at least about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, about 99%, about 99.5%, or greater, such as 100%, including all values and subranges that lie therebetween) identity to the sequence of SEQ ID NO: 8. In some embodiments, an rAAV comprises an AAV9-php.b capsid protein; and a nucleic acid molecule, wherein the nucleic acid molecule comprises an AAV expression cassette, wherein the AAV expression cassette comprises a codon-optimized nucleic acid sequence of SEQ ID NO: 8.
In some embodiments, an rAAV is a self-complementary AAV. In some embodiments, an rAAV is a single-stranded AAV.
In some embodiments, preparation of rAAV particles involves culturing a host cell that contains a nucleic acid sequence encoding an AAV capsid protein or fragment thereof; a functional rep gene; a recombinant AAV vector composed of AAV inverted terminal repeats (ITRs) and the AAV expression cassette encoding TMC1; and sufficient helper functions to permit packaging of the recombinant AAV vector into the AAV capsid proteins. In some embodiments, the components to be cultured in the host cell to package a rAAV vector in an AAV capsid are provided to the host cell in trans. In some embodiments, any one or more of the required components (e.g., recombinant AAV vector, rep sequences, cap sequences, and/or helper functions) are provided by a stable host cell that has been engineered to contain one or more of the required components. In some embodiments, a stable host cell will contain the required component(s) under the control of an inducible promoter or a constitutive promoter. In some embodiments, a selected stable host cell contains selected component(s) under the control of a constitutive promoter and other selected component(s) under the control of one or more inducible promoters. For example, a stable host cell may be generated which is derived from 293 cells (which contain El helper functions under the control of a constitutive promoter), but which contain the rep and/or cap proteins under the control of inducible promoters. The recombinant AAV vector, rep sequences, cap sequences, and helper functions required for producing the rAAVs disclosed herein may be delivered to the packaging host cell using any appropriate genetic element (for example, a vector). Further details on methods of preparing rAAV particles are provided in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.; K. Fisher et al, J. Virol., 70:520-532 (1993) and U.S. Pat. No. 5,478,745, the contents of each of which are herein incorporated in its entirety for all purposes.
In some embodiments, recombinant AAVs are produced using the triple transfection method, as described in U.S. Pat. No. 6,001,650, the contents of which are herein incorporated in its entirety for all purposes. In some embodiments, the recombinant AAVs are produced by transfecting a host cell with a recombinant AAV vector (comprising the AAV expression cassette encoding TMC1) to be packaged into AAV particles, an AAV helper function vector, and an accessory function vector. An AAV helper function vector encodes the “AAV helper function” sequences (i.e., rep and cap), which function in trans for productive AAV replication and encapsidation. Non-limiting examples of AAV helper function vectors include pHLP19 and pRep6cap6 vector, described in U.S. Pat. Nos. 6,001,650 and 6,156,303, respectively, the contents of each of which are herein incorporated in its entirety for all purposes. The accessory function vector encodes nucleotide sequences for non-AAV derived viral and/or cellular functions upon which AAV is dependent for replication (i.e., “accessory functions”). The accessory functions include those functions required for AAV replication, including, without limitation, those moieties involved in activation of AAV gene transcription, stage specific AAV mRNA splicing, AAV DNA replication, synthesis of cap expression products, and AAV capsid assembly. Viral-based accessory functions can be derived from any of the known helper viruses such as adenovirus, herpesvirus (other than herpes simplex virus type-1), and vaccinia virus.
In some embodiments, recombinant AAVs are produced using baculovirus vectors. Baculovirus vectors are used to produce recombinant AAVs in insect cells (e.g., Spodoptera frugiperda (Sf9) cells).
The present disclosure further provides pharmaceutical compositions, comprising: (a) any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the cells disclosed herein, or any one the rAAVs disclosed herein; and (b) a pharmaceutically acceptable carrier.
In some embodiments, the compositions disclosed herein comprise at least one pharmaceutically acceptable carrier, excipient, and/or vehicle, for example, solvents, buffers, solutions, dispersion media, coatings, antibacterial agents, antifungal agents, isotonic agents, and absorption delaying agents. In some embodiments, the pharmaceutically acceptable carrier, excipient, and/or vehicle comprises saline, buffered saline, dextrose, water, glycerol, sterile isotonic aqueous buffer, or a combination thereof. In some embodiments, the pharmaceutically acceptable carrier, excipient, and/or vehicle comprises phosphate buffered saline, sterile saline, lactose, sucrose, calcium phosphate, dextran, agar, pectin, peanut oil, sesame oil, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, polyol (e.g., glycerol, propylene glycol, and liquid polyethylene glycol, and the like), or a suitable mixture thereof. In some embodiments, the compositions disclosed herein further comprise emulsifying or wetting agents, or pH buffering agents. Such species may be present in small amounts (e.g., less than 10% by weight of the composition, such as less than 5% by weight of the composition, 2% by weight of the composition, 1% by weight of the composition, or less).
In some embodiments, the compositions disclosed herein further comprise one or more other pharmaceutical ingredients, such as one or more preservatives or chemical stabilizers. Examples of preservatives and chemical stabilizers include, but are not limited to, chlorobutanol, potassium sorbate, sorbic acid, sulfur dioxide, propyl gallate, parabens, ethyl vanillin, glycerin, phenol, parachlorophenol, and albumin. In some embodiments, the compositions disclosed herein further comprise antibacterial agents and/or antifungal agents, such as parabens, chlorobutanol, phenol, sorbic acid, and thimerosal; isotonic agents, such as sugars and sodium chloride; and/or agents delaying absorption, such as aluminum monostearate and gelatin.
In some embodiments, the compositions disclosed herein are formulated to reduce aggregation of AAV particles in the composition, particularly where high rAAV concentrations are present (e.g., ˜1013 GC/mL or more). Methods for reducing aggregation of rAAVs include addition of surfactants, pH adjustment, and salt concentration adjustment, as further described in Wright, et al., Molecular Therapy (2005) 12, 171-178, the contents of which are incorporated herein by reference in its entirety for all purposes.
In some embodiments, the pharmaceutical compositions are in a form of an injectable solution or dispersion, such as an aqueous solution or dispersion. In some embodiments, the pharmaceutical composition is a sterile powder for the extemporaneous preparation of sterile injectable solutions or dispersions. Dispersions may be prepared in water, glycerol, liquid polyethylene glycols, oils, or any combination thereof. Delivery vehicles such as liposomes, nanocapsules, microparticles, microspheres, lipid particles, vesicles, and the like, may be used for the introduction of the pharmaceutical compositions disclosed herein.
The present disclosure also provides methods of expressing transmembrane channel-like 1 (TMC1) in a cell, comprising: contacting any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, or any one of the compositions disclosed herein with the cell, thereby expressing TMC1 in the cell. In some embodiments, the cell is an ear cell. In some embodiments, the cell is an inner hair cell (IHC), or an outer hair cell (OHC). In some embodiments, the cell is a vestibular hair cell, spiral ganglion, or vestibular ganglion.
In some embodiments, the contacting step is performed in vitro, ex vivo, or in vivo. In some embodiments, the contacting step is performed in vivo in a subject in need thereof. In some embodiments, the contacting step comprises administering a therapeutically effective amount of the nucleic acid molecule, the plasmid, the rAAV, or the composition to the subject.
The present disclosure further provides methods of treating genetic hearing loss or a symptom thereof in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, or any one of the compositions disclosed herein, thereby treating genetic hearing loss or a symptom thereof in the subject. In a related aspect, the present disclosure provides methods of ameliorating, diminishing the severity of, eliminating, and/or delaying the onset of one or more symptoms of genetic hearing loss in a subject in need thereof, comprising: administering to the subject a therapeutically effective amount of any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, or any one of the compositions disclosed herein, thereby ameliorating, diminishing the severity of, eliminating, and/or delaying the onset of the one or more symptoms of genetic hearing loss in the subject.
In some embodiments, the subject suffers from, or is at a risk of developing the genetic hearing loss. In some embodiments, the genetic hearing loss is associated with one or more mutations (e.g., recessive mutations) in TMC1. In some embodiments, the genetic hearing loss is an autosomal recessive non-syndromic hearing loss (ARNSHL). In some embodiments, the subject has autosomal dominant non-syndromic sensorineural hearing loss. In some embodiments, the subject has a mutant allele of TMC1 called DFNA36. In some embodiments, the subject has autosomal recessive non-syndromic neurosensory deafness. In some embodiments, the subject has a mutant allele of TMC1 called DFNB7. In some embodiments, the genetic hearing loss is associated with, promoted by, or caused by a mutation in the transmembrane channel-like 1 (TMC1)-encoding gene. In some embodiments, the mutation in the TMC1-encoding gene is present at the DFNB7/11 locus on chromosome 9q31-21.
In some embodiments, the method comprises diminishing the severity of; delaying the onset or progression of; and/or eliminating a symptom of the genetic hearing loss. In some embodiments, the symptom of the genetic hearing loss comprises: a reduced ability to hear, or an inability to hear. In some embodiments, the genetic hearing loss is: (a) a progressive genetic hearing loss, (b) a pre-lingual genetic hearing loss, (c) a congenital genetic hearing loss, or (d) any combination thereof. In some embodiments, the subject has a hearing threshold in the range of about 25 dB to about 80 dB. In some embodiments, the subject has a hearing threshold of about 40 dB. In some embodiments, the subject has a hearing threshold of about 55 dB. In some embodiments, the subject has a hearing threshold of about 70 dB.
In some embodiments, the method comprises decreasing the hearing threshold of the subject during or after the administration period, as compared to prior to the administration period. In some embodiments, the hearing threshold of the subject during or after the administration period is lower, as compared to before the administration period. In some embodiments, the hearing threshold of the subject during or after the administration period is at least about 2% (for example, about 5%, about 10%, about 15%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or 100%, including values and subranges that lie therebetween) lower, as compared to before the administration period. In some embodiments, the hearing threshold of the subject during or after the administration period is at least about 5% lower, as compared to before the administration period.
In some embodiments, the hearing threshold during or after the administration period of a subject, who is administered an rAAV, comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′: a 5′ AAV ITR, a CB6 promoter, a Kozak sequence, a transgene, encoding a codon-optimized human transmembrane channel-like 1 (co-hTMC1), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), a beta globin polyadenylation sequence, and a 3′ AAV ITR, is lower, as compared to that of a control subject, wherein the control subject is administered an rAAV, comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette lacks a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and comprises, from 5′ to 3′: a 5′ AAV ITR, a CB6 promoter, a Kozak sequence, a transgene, encoding a codon-optimized human transmembrane channel-like 1 (co-hTMC1), a beta globin polyadenylation sequence, and a 3′ AAV ITR.
In some embodiments, the hearing threshold during or after the administration period of a subject, who is administered an rAAV, comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′: a 5′ AAV ITR, a CB6 promoter, a Kozak sequence, a transgene, encoding a codon-optimized human transmembrane channel-like 1 (co-hTMC1), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), a beta globin polyadenylation sequence, and a 3′ AAV ITR, is lower for a longer period of time, as compared to a control subject, wherein the control subject is administered an rAAV, comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette lacks a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and comprises, from 5′ to 3′: a 5′ AAV ITR, a CB6 promoter, a Kozak sequence, a transgene, encoding a codon-optimized human transmembrane channel-like 1 (co-hTMC1), a beta globin polyadenylation sequence, and a 3′ AAV ITR.
In some embodiments, the hearing threshold during or after the administration period of a subject, who is administered an rAAV, comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′: a 5′ AAV ITR, a CB6 promoter, a Kozak sequence, a transgene, encoding a codon-optimized human transmembrane channel-like 1 (co-hTMC1), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), a beta globin polyadenylation sequence, and a 3′ AAV ITR, is lower, as compared to that of a control subject, wherein the control subject is administered an rAAV, comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette lacks a CB6 promoter and comprises, from 5′ to 3′: a 5′ AAV ITR, a CMV promoter, a Kozak sequence, a transgene, encoding a codon-optimized human transmembrane channel-like 1 (co-hTMC1), a beta globin polyadenylation sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and a 3′ AAV ITR.
In some embodiments, the hearing threshold during or after the administration period of the subject, who is administered an rAAV, comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′: a 5′ AAV ITR, a CB6 promoter, a Kozak sequence, a transgene, encoding a codon-optimized human transmembrane channel-like 1 (co-hTMC1), a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), a beta globin polyadenylation sequence, and a 3′ AAV ITR, is lower for a longer period of time, as compared to that of a control subject, wherein the control subject is administered an rAAV, comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette lacks a CB6 promoter and comprises, from 5′ to 3′: a 5′ AAV ITR, a CMV promoter, a Kozak sequence, a transgene, encoding a codon-optimized human transmembrane channel-like 1 (co-hTMC1), a beta globin polyadenylation sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and a 3′ AAV ITR.
In some embodiments, the survival of inner hair cells and/or outer hair cells in the ear of the subject during or after the administration period is improved as compared to the survival of inner hair cells and/or outer hair cells in the ear prior to the administration period. In some embodiments, the number of inner hair cells and/or outer hair cells in the ear of the subject during or after the administration period is higher, as compared to the number of inner hair cells and/or outer hair cells in the ear before the administration period.
In some embodiments of any of the preceding aspects, the administration is intravenous or intravascular. In some embodiments, the administration is by direct delivery to the ear, or a component thereof. In some embodiments of any of the preceding aspects, the administration is by intracochlear injection, or any other mode of administration into the ear, or a mode of administration that is suitable for expression of the transgene in the ear. In some embodiments of any of the preceding aspects, the administration is by intrathecal administration.
In some embodiments of any of the preceding aspects, the administration is by injection into the central nervous system. Other modes of administration that may be used include transmucosal, intranasal, oral, inhalation (e.g., via an aerosol), buccal (e.g., sublingual), intrathecal, intraocular, transdermal, parenteral (e.g., intravenous, subcutaneous, intradermal, intramuscular, intradermal, intrapleural, intracerebral, and intraarticular), topical (e.g., to skin and/or mucosal surfaces, including airway surfaces, and transdermal administration), intralymphatic, and the like, as well as direct tissue injection (e.g., to the ear).
In some embodiments of any of the preceding aspects, the method comprises administering a therapeutically effective amount of an rAAV, wherein the therapeutically effective amount is in a range of about 105 genome copies to 1020 genome copies per cochlea administered, for example, about 106 genome copies/cochlea, about 107 genome copies/cochlea, about 108 genome copies/cochlea, about 109 genome copies/cochlea, about 1010 genome copies/cochlea, about 1011 genome copies/cochlea, about 1012 genome copies/cochlea, about 1013 genome copies/cochlea, about 1014 genome copies/cochlea, about 1015 genome copies/cochlea, about 1016 genome copies/cochlea, about 1017 genome copies/cochlea, about 1018 genome copies/cochlea, or about 1019 genome copies/cochlea, including all values and subranges that lie therebetween. In some embodiments, the method comprises administering a therapeutically effective amount of rAAV, wherein the therapeutically effective amount is in a range of 1010 genome copies to 1014 genome copies per kilogram.
In some embodiments of any of the preceding aspects, the method comprises administering a therapeutically effective amount of an rAAV, wherein the therapeutically effective amount is in a range of about 105 genome copies to 1020 genome copies per kilogram (kg), for example, about 106 genome copies/kg, about 107 genome copies/kg, about 108 genome copies/kg, about 109 genome copies/kg, about 1010 genome copies/kg, about 1011 genome copies/kg, about 1012 genome copies/kg, about 1013 genome copies/kg, about 1014 genome copies/kg, about 1015 genome copies/kg, about 1016 genome copies/kg, about 1017 genome copies/kg, about 1018 genome copies/kg, or about 1019 genome copies/kg, including all values and subranges that lie therebetween. In some embodiments, the method comprises administering a therapeutically effective amount of rAAV, wherein the therapeutically effective amount is in a range of 1010 genome copies to 1014 genome copies per kilogram.
In some embodiments of any of the preceding aspects, the therapeutically effective amount is in the range of about 105 to 1020 genome copies per subject, for example, about 106 genome copies per subject, about 107 genome copies per subject, about 108 genome copies per subject, about 109 genome copies per subject, about 1010 genome copies per subject, about 1011 genome copies per subject, about 1012 genome copies per subject, about 1013 genome copies per subject, about 1014 genome copies per subject, about 1015 genome copies per subject, about 1016 genome copies per subject, about 1017 genome copies per subject, about 1018 genome copies per subject, or about 1019 genome copies per subject, including all values and subranges that lie therebetween. In some embodiments, the therapeutically effective amount is in the range of about 109 to 1016 genome copies per subject.
In some embodiments of any of the preceding aspects, the therapeutically effective amount is administered in a volume of about 1 microliter (μl) to about 100 milliliter (mL) of solution, for example, about 10 μl, about 50 μl, about 100 μl, about 500 μl, about 1 mL, about 20 mL, about 30 mL, about 40 mL, about 50 mL, about 60 mL, about 70 mL, about 80 mL, about 90 mL, or about 100 mL, including all values and subranges that lie therebetween. The volume used may depend on the dose of the rAAV, and the route of administration. For example, for intrathecal administration a volume in the range of about 1 μl to about 10 μl, or about 10 μl to about 100 μl may be used. For intravenous administration a volume in range of about 10 μl to about 100 μl, or about 100 μl to 1 mL, or about 1 mL to about 10 mL, or more may be used.
In some embodiments of any of the preceding aspects, more than one administration (e.g., two, three, four or more administrations) may be employed to achieve the desired level of gene expression over a period of various intervals, e.g., daily, weekly, monthly, yearly, etc.
In some embodiments, the subject is a human subject. In some embodiments, the subject is a neonate or an infant. In some embodiments, a subject is up to 24 months old. In some embodiments, a subject is less than 6 years of age. In some embodiments, a subject is between about 6 and about 12 years of age. In some embodiments, a subject is between about 12 and about 18 years of age. In some embodiments, the subject is less than 18 years of age. In some embodiments, the subject is at least 18 years of age.
The present disclosure also provides kits comprising one or more agents (e.g., any one of the nucleic acid molecules disclosed herein, any one of the plasmids disclosed herein, any one of the rAAVs disclosed herein, or any one of the compositions disclosed herein). In some embodiments, the kits are pharmaceutical or diagnostic or research kits to be used in therapeutic, diagnostic or research applications. A kit may include one or more containers housing the agents disclosed herein and instructions for use. In certain embodiments, agents in a kit are in a pharmaceutical formulation and dosage suitable for a particular application and for a method of administration of the agents. In some embodiments, the container is a syringe, vial, tube, topical application devices, IV needle tubing and bag, and other containers.
Provided below are the nucleic acid and protein sequences disclosed in this application:
The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described. All documents, or portions of documents, cited herein, including but not limited to patents, patent applications, articles, books, and treatises, are hereby expressly incorporated by reference in their entirety for any purpose. In the event that one or more of the incorporated documents or portions of documents define a term that contradicts that term's definition in the application, the definition that appears in this application controls. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment, or any form of suggestion, that they constitute valid prior art or form part of the common general knowledge in any country in the world.
Unless the context indicates otherwise, it is specifically intended that the various features described herein can be used in any combination.
TMC1 knockout mice were administered via intracochlear injection, different doses (vector genomes per cochlea) of a recombinant adeno-associated vector (rAAV), comprising an AAV expression cassette having the nucleic acid sequence of SEQ ID NO: 8 (comprising a 5′ITR, a CB6 promoter, a Kozak sequence, a transgene encoding TMC1, a WPRE, a beta globin poly A sequence, and a 3′ ITR) or SEQ ID NO: 9 (comprising a 5′ ITR, a CB6 promoter, a Kozak sequence, a transgene encoding TMC1, a beta globin poly A sequence, and a 3′ ITR), and the auditory brain stem response (ABR) threshold was measured at different time points (such as, 4 weeks, 8 weeks, or 12 weeks after administration) at different frequencies. Wildtype mice (WT) and TMC1 knockout (TMC1 KO) mice were used as controls. As shown in
The results show that the expression of TMC1 from an expression cassette containing WPRE results in more durable and improved recovery of the hearing function, as compared to the expression of TMC1 from an expression cassette lacking WPRE. Therefore, the presence of WPRE in the expression cassette has an unexpected effect on the rescue of the hearing function.
This surprising effect is also observed when the same dosage of rAAV is injected into the mice. For instance,
TMC1 knockout mice were administered via intracochlear injection, a recombinant adeno-associated vector (rAAV), comprising an AAV expression cassette having the nucleic acid sequence of SEQ ID NO: 8 (comprising a 5′ITR, a CB6 promoter, a Kozak sequence, a transgene encoding TMC1, a WPRE, a beta globin polyA sequence, and a 3′ ITR) or SEQ ID NO: 10 (comprising a 5′ITR, a CMV promoter, a Kozak sequence, a transgene encoding TMC1, a WPRE, a beta globin polyA sequence, and a 3′ ITR), and the auditory brain stem response (ABR) threshold was measured at different time points (such as, 4 weeks, 8 weeks, or 12 weeks after administration) at different frequencies. Wildtype mice (WT) and TMC1 knockout (TMC1 KO) mice were used as controls. As shown in
TMC1 knockout mice were administered via intracochlear injection, the same dosage (3E10 vector genome/cochlea) of a recombinant adeno-associated vector (rAAV), comprising an AAV expression cassette having the nucleic acid sequence of SEQ ID NO: 8 (comprising a WPRE element and a CB6 promoter), SEQ ID NO: 9 (lacking a WPRE element), or SEQ ID NO: 10 (comprising a CMV promoter), and the number of outer hair cells were evaluated at 4 weeks post administration. TMC1 knockout (TMC1 KO) mice were used as control. As shown in
The effect of TMC1 expression on hair cell survival was further evaluated over time. As shown in
Mice carrying the “Tmc1 p.N193I mutation” are referred to herein as “N193I mutant mice” and were used in this experiment. The Tmc1 p.N193I mutation is equivalent to the hypofunctional human TMC1 mutation p.N199I (c.596A>T), which causes progressive moderate-to-severe hearing loss during childhood. N193I mutant mice were administered via intracochlear injection, different doses (vector genomes per cochlea) of a recombinant adeno-associated vector (rAAV), comprising an AAV expression cassette having the nucleic acid sequence of SEQ ID NO: 8 (comprising a WPRE) or SEQ ID NO: 9 (lacking a WPRE), and the auditory brain stem response (ABR) threshold was measured at different time points (such as, 4 weeks, 8 weeks, or 12 weeks after administration) at different frequencies. Wildtype mice (WT) and N193I mutant (N1311) mice were used as controls. As shown in
Furthermore, the effect of TMC1 expression on hair cell survival in N193I mutant mice was further evaluated over time. Wildtype (WT) and N193I mutant mice were used as controls. As shown in
Embodiment 1. A nucleic acid molecule, comprising an adeno-associated virus (AAV) expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′:
Embodiment 2. The nucleic acid molecule of claim 1, wherein the transgene encodes a human transmembrane channel-like 1 (hTMC1).
Embodiment 3. The nucleic acid molecule of claim 1 or claim 2, wherein the transgene encodes a codon-optimized human transmembrane channel-like 1 (co-hTMC1).
Embodiment 4. The nucleic acid molecule of any one of claims 1-3, wherein the transgene comprises a nucleic acid sequence having at least 90% identity to SEQ ID NO: 1.
Embodiment 5. The nucleic acid molecule of any one of claims 1-4, wherein the transgene comprises the nucleic acid sequence of SEQ ID NO: 1.
Embodiment 6. The nucleic acid molecule of any one of claims 1-5, wherein the promoter is operably linked to the transgene.
Embodiment 7. The nucleic acid molecule of any one of claims 1-6, wherein the CB6 promoter comprises the nucleic acid sequence of SEQ ID NO: 2.
Embodiment 8. The nucleic acid molecule of any one of claims 1-7 wherein the AAV expression cassette comprises a beta globin polyadenylation sequence.
Embodiment 9. The nucleic acid molecule of claim 8, wherein the beta globin polyadenylation sequence comprises the nucleic acid sequence of SEQ ID NO: 3.
Embodiment 10. The nucleic acid molecule of any one of claims 1-9, wherein the AAV expression cassette comprises a Kozak sequence.
Embodiment 11. The nucleic acid molecule of claim 10, wherein the Kozak sequence comprises the nucleic acid sequence of SEQ ID NO: 4.
Embodiment 12. The nucleic acid molecule of any one of claims 1-11, wherein the AAV expression cassette comprises a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE).
Embodiment 13. The nucleic acid molecule of claim 12, wherein the WPRE comprises the nucleic acid sequence of SEQ ID NO: 5.
Embodiment 14. The nucleic acid molecule of any one of claims 1-13, wherein the 5′ AAV ITR sequence comprises the nucleic acid sequence of SEQ ID NO: 6.
Embodiment 15. The nucleic acid molecule of any one of claims 1-14, wherein the 3′ AAV ITR sequence comprises the nucleic acid sequence of SEQ ID NO: 7.
Embodiment 16. The nucleic acid molecule of any one of claims 1-15, wherein the AAV expression cassette comprises, from 5′ to 3′:
Embodiment 17. The nucleic acid molecule of any one of claims 1-16, wherein the AAV expression cassette comprises the nucleic acid sequence of SEQ ID NO: 8.
Embodiment 18. A plasmid, comprising the nucleic acid molecule of any one of claims 1-17.
Embodiment 19. A cell, comprising the nucleic acid molecule of any one of claims 1-17, or the plasmid of claim 18.
Embodiment 20. A method of producing a recombinant adeno-associated virus (rAAV), the method comprising:
Embodiment 21. A recombinant adeno-associated virus (rAAV) produced by the method of claim 20.
Embodiment 22. The rAAV of claim 21, wherein the rAAV comprises an AAV9-php.b capsid protein.
Embodiment 23. The rAAV of claim 21, wherein the rAAV comprises a AAV9 capsid protein, comprising an insertion of the amino acid sequence of SEQ ID NO: 11 (TLAVPFK) between amino acid 588 and amino acid 589, wherein the amino acids are numbered according to VP1 capsid protein.
Embodiment 24. A recombinant adeno-associated virus (rAAV), comprising:
Embodiment 25. A recombinant adeno-associated virus (rAAV), comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule comprising an AAV expression cassette, wherein the AAV expression cassette comprises, from 5′ to 3′:
Embodiment 26. A recombinant adeno-associated virus (rAAV), comprising:
Embodiment 27. The rAAV of any one of claims 22-26, wherein the AAV9.php.b capsid protein is encoded by a nucleic acid having at least 90% identity to SEQ ID NO: 13.
Embodiment 28. The rAAV of any one of claims 22-27, wherein the AAV expression cassette comprises an miR-1 binding site, an miR-133a binding site, and/or an miR-122 binding site.
Embodiment 29. The rAAV of any one of claims 22-28, wherein the rAAV is a self-complementary AAV.
Embodiment 30. The rAAV of any one of claims 22-28, wherein the rAAV is a single-stranded AAV.
Embodiment 31. A pharmaceutical composition, comprising:
Embodiment 32. A method of expressing transmembrane channel-like 1 (TMC1) in a cell, comprising:
Embodiment 33. The method of claim 32, wherein the contacting step is performed in vitro, ex vivo, or in vivo.
Embodiment 34. The method of claim 33, wherein the contacting step is performed in vivo in a subject in need thereof.
Embodiment 35. The method of claim 34, wherein the contacting step comprises administering a therapeutically effective amount of the nucleic acid molecule, the plasmid, the rAAV, or the composition to the subject.
Embodiment 36. The method of any one of claims 32-35, wherein the cell is an ear cell.
Embodiment 37. The method of claim 36, wherein the cell is an inner hair cell (IHC), or an outer hair cell (OHC).
Embodiment 38. The method of any one of claims 32-36, wherein the cell is a vestibular hair cell, spiral ganglion, or vestibular ganglion.
Embodiment 39. A method of treating a genetic hearing loss in a subject in need thereof, comprising: administering to the ear of the subject for an administration period, a therapeutically effective amount of the nucleic acid molecule of any one of claims 1-17, the plasmid of claim 18, the cell of claim 19, or the rAAV of any one of claims 21-30, or the composition of claim 31, thereby treating the genetic hearing loss in the subject.
Embodiment 40. A method of treating a genetic hearing loss in a subject in need thereof, comprising: administering to the ear of the subject for an administration period, a therapeutically effective amount of the rAAV of claim 26, thereby treating the genetic hearing loss in the subject.
Embodiment 41. The method of claim 39 or claim 40, wherein the subject suffers from, or is at a risk of developing the genetic hearing loss.
Embodiment 42. The method of any one of claims 39-41, wherein the genetic hearing loss is an autosomal recessive non-syndromic hearing loss (ARNSHL).
Embodiment 43. The method of any one of claims 39-42, wherein the genetic hearing loss is associated with, promoted by, or caused by a mutation in the transmembrane channel-like 1 (TMC1)-encoding gene.
Embodiment 44. The method of claim 43, wherein the mutation in the TMC1-encoding gene is present at the DFNB7/11 locus on chromosome 9q31-21.
Embodiment 45. The method of any one of claims 39-44, wherein the method comprises diminishing the severity of; delaying the onset or progression of; and/or eliminating a symptom of the genetic hearing loss.
Embodiment 46. The method of claim 45, wherein symptom of the genetic hearing loss comprises: a reduced ability to hear, or an inability to hear.
Embodiment 47. The method of any one of claims 39-46, wherein the genetic hearing loss is: (a) a progressive genetic hearing loss, (b) a pre-lingual genetic hearing loss, (c) a congenital genetic hearing loss, or (d) any combination thereof.
Embodiment 48. The method of any one of claims 39-47, wherein the subject has a hearing threshold in the range of about 25 dB to about 80 dB.
Embodiment 49. The method of claim 48, wherein the subject has a hearing threshold of about 40 dB.
Embodiment 50. The method of claim 48, wherein the subject has a hearing threshold of about 55 dB.
Embodiment 51. The method of claim 48, wherein the subject has a hearing threshold of about 70 dB.
Embodiment 52. The method of any one of claims 39-51, wherein the method comprises decreasing the hearing threshold of the subject during or after the administration period, as compared to prior to the administration period.
Embodiment 53. The method of any one of claims 39-52, wherein, the hearing threshold of the subject during or after the administration period is lower, as compared to before the administration period.
Embodiment 54. The method of any one of claims 39-53, wherein, the hearing threshold of the subject during or after the administration period is at least 5% lower, as compared to before the administration period.
Embodiment 55. The method of any one of claims 39-54, wherein the hearing threshold of the subject during or after the administration period is lower, as compared to that of a control subject, wherein the control subject is administered an rAAV, comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette lacks a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and comprises, from 5′ to 3′: a 5′ AAV ITR, a CB6 promoter, a Kozak sequence, a transgene, encoding a codon-optimized human transmembrane channel-like 1 (co-hTMC1), a beta globin polyadenylation sequence, and a 3′ AAV ITR.
Embodiment 56. The method of any one of claims 39-55, wherein the hearing threshold of the subject during or after the administration period is lower for a longer period of time, as compared to a control subject, wherein the control subject is administered an rAAV, comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette lacks a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and comprises, from 5′ to 3′: a 5′ AAV ITR, a CB6 promoter, a Kozak sequence, a transgene, encoding a codon-optimized human transmembrane channel-like 1 (co-hTMC1), a beta globin polyadenylation sequence, and a 3′ AAV ITR.
Embodiment 57. The method of any one of claims 39-56, wherein the hearing threshold of the subject during or after the administration period is lower, as compared to that of a control subject, wherein the control subject is administered an rAAV, comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette lacks a CB6 promoter and comprises, from 5′ to 3′: a 5′ AAV ITR, a CMV promoter, a Kozak sequence, a transgene, encoding a codon-optimized human transmembrane channel-like 1 (co-hTMC1), a beta globin polyadenylation sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and a 3′ AAV ITR.
Embodiment 58. The method of any one of claims 39-57, wherein the hearing threshold of the subject during or after the administration period is lower for a longer period of time, as compared to that of a control subject, wherein the control subject is administered an rAAV, comprising: an AAV9-php.b capsid protein; and a nucleic acid molecule, comprising an AAV expression cassette, wherein the AAV expression cassette lacks a CB6 promoter and comprises, from 5′ to 3′: a 5′ AAV ITR, a CMV promoter, a Kozak sequence, a transgene, encoding a codon-optimized human transmembrane channel-like 1 (co-hTMC1), a beta globin polyadenylation sequence, a woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) and a 3′ AAV ITR.
Embodiment 59. The method of any one of claims 39-58, wherein the survival of inner hair cells and/or outer hair cells in the ear of the subject during or after the administration period is improved as compared to the survival of inner hair cells and/or outer hair cells in the ear prior to the administration period.
Embodiment 60. The method of any one of claims 39-59, wherein the number of inner hair cells and/or outer hair cells in the ear of the subject during or after the administration period is higher, as compared to the number of inner hair cells and/or outer hair cells in the ear before the administration period.
Embodiment 61. The method of any one of claims 39-60, wherein the nucleic acid molecule, the plasmid, the cell, the rAAV, or the composition is administered via intracochlear delivery.
Embodiment 62. The method of any one of claims 39-61, wherein the subject is a human subject.
Embodiment 63. The method of claim 62, wherein the subject is a neonate or an infant.
Embodiment 64. The method of claim 62, wherein the subject is less than 18 years of age.
Embodiment 65. The method of claim 62, wherein the subject is at least 18 years of age.
Embodiment 66. The method of any one of claims 62-65, wherein the subject has autosomal dominant non-syndromic sensorineural hearing loss.
Embodiment 67. The method of any one of claims 62-65, wherein the subject has autosomal recessive non-syndromic neurosensory deafness.
The present Application claims the benefit of priority to U.S. Provisional Application No. 63/302,913, filed on Jan. 25, 2022, the contents of which are hereby incorporated by reference in their entirety.
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/US2023/061304 | 1/25/2023 | WO |
| Number | Date | Country | |
|---|---|---|---|
| 63302913 | Jan 2022 | US |
