METHODS AND COMPOSITIONS FOR ALLELE SPECIFIC GENE EDITING
20220112504 · 2022-04-14
Assignee
- Children's Medical Center Corporation (Boston, MA)
- President And Fellows Of Harvard College (Cambridge, MA)
Inventors
Cpc classification
C12N2310/20
CHEMISTRY; METALLURGY
C12N9/22
CHEMISTRY; METALLURGY
C07K14/705
CHEMISTRY; METALLURGY
C12N2750/14143
CHEMISTRY; METALLURGY
A61K48/005
HUMAN NECESSITIES
C12N15/1138
CHEMISTRY; METALLURGY
C12N15/1034
CHEMISTRY; METALLURGY
A01K2267/0318
HUMAN NECESSITIES
C12N2320/11
CHEMISTRY; METALLURGY
International classification
C12N15/113
CHEMISTRY; METALLURGY
C12N15/86
CHEMISTRY; METALLURGY
Abstract
The invention provides compositions and methods for allele specific gene editing. In particular, the invention provides methods and compositions for treating dominant progressive hearing loss by selectively inactivating a dominant mutation in TMC1.
Claims
1. A method for allele specific gene editing, the method comprising contacting a double stranded polynucleotide comprising a wild-type allele and a mutant allele with a guide RNA that binds the alleles and a Cas9 polypeptide with a PAM site selective for the mutant allele, such that indels are selectively induced in the mutant target allele.
2. A method for the allele-specific disruption of a dominant mutation, the method comprising contacting a double stranded polynucleotide comprising a wild-type allele and a mutant allele with a guide RNA that binds the alleles and a Cas9 nuclease with a PAM site selective for the mutant allele, such that indels are selectively induced in the mutant allele.
3. The method of claim 1, wherein the double stranded polynucleotide is DNA.
4. The method of claim 3, wherein the DNA is genomic DNA.
5. The method of claim 1, wherein the polynucleotide is present in a cell.
6. The method of claim 5, wherein the cell is a cell in vivo or in vitro.
7. A method for the treatment of a disorder associated with a dominant mutant allele in a target gene, the method comprising: (a) contacting a cell heterozygous for the dominant mutant allele in a target gene with a guide RNA that binds the target gene and a Cas9 nuclease with a PAM site selective for the mutant allele; and (b) selectively inducing an indel in the mutant allele.
8. A method of treating progressive hearing loss in a subject, the method comprising (a) contacting a cell of a subject heterozygous for a p.M418K mutation in TMC1 with a SaCas9-KKH and a guide RNA that targets TMC1; and (b) inducing indels in the TMC1 allele comprising the p.M418K mutation, thereby treating hearing loss in the subject.
9. The method of claim 8, wherein the cell is a cell of the inner ear.
10. The method of claim 9, wherein the cell is an inner or outer hair cell.
11. The method of claim 8, wherein the administering improves or maintains auditory function in the subject.
12. The method of claim 11, wherein an improvement in auditory function is associated with preservation of hair bundle morphology and/or restoration of mechanotransduction.
13. The method of claim 1, wherein the guide RNA and the Cas9 polypeptide are encoded in a single vector.
14. The method of claim 13, wherein the vector is an adeno-associated virus vector or a lentivirus vector.
15. The method of claim 1, wherein the contacting comprises transfecting cells in the subject with a guide RNA and a polynucleotide encoding a Cas9 protein.
16. The method of claim 15, wherein the guide RNA and the Cas9 polypeptide are administered simultaneously.
17. A vector comprising a polynucleotide encoding a SaCas9-KKH polypeptide, or a fragment thereof, and a gRNA having a nucleic acid sequence complementary to a nucleic acid sequence comprising a mutation associated with DFNA36.
18. A pharmaceutical composition comprising the vector of claim 17.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0068]
[0069]
[0070]
[0071]
[0072]
[0073]
[0074]
[0075]
[0076]
[0077]
[0078]
[0079]
[0080]
DETAILED DESCRIPTION OF THE INVENTION
[0081] As described below, the present invention features a Cas9 variant capable of selectively targeting a mutant allele without disrupting the wildtype allele, and methods of using such variants for gene editing. In particular embodiments, the invention provides for the editing of dominant mutations associated with single nucleotide substitutions. The invention further provides methods and compositions for treating a disease or condition or symptoms thereof associated with a dominant mutation.
[0082] The invention is based, at least in part, on the discovery that a Cas9 variant (SaCas9-KKH) recognizes a non-canonical PAM sequence present in a TMC1 allele that carries a dominant mutation associated with progressive deafness and generates double-strand breaks in only the TMC1 alleles that carry the dominant allele. Importantly, SaCas9-KKH does not generate double strand breaks in the wild type allele lacking the PAM sequence. In an effort to identify Cas9 variants having the desired properties, 14 Cas9/gRNA combinations were screened for specific and efficient disruption of a nucleotide substitution that causes the dominant progressive hearing loss, DFNA36. As a model for DFNA36, Beethoven mice were used. Beethoven mice harbor a point mutation in Tmc1, a gene required for hearing that encodes a pore-forming subunit of mechanosensory transduction channels in inner ear hair cells. A PAM variant of Staphylococcus aureus Cas9 (SaCas9-KKH) was identified that selectively and efficiently disrupted the mutant allele, but not the wild-type Tmc1/TMC1 allele, in Beethoven mice and in a DFNA36 human cell line. AAV-mediated SaCas9-KKH delivery prevented deafness in Beethoven mice up to one year post transduction. Analysis of current ClinVar entries revealed that ˜21% of dominant human mutations could be targeted using a similar approach.
Cas9
[0083] Cas9 is a nuclease, an enzyme specialized for cutting DNA, with two active cutting sites, one for each strand of the double helix. Jinek et al. (2012) combined tracrRNA and spacer RNA into a “single-guide RNA” molecule that, mixed with Cas9, could find and cut the correct DNA targets. It has been proposed that such synthetic guide RNAs might be able to be used for gene editing (Jinek et al., Science. 2012 Aug 17;337(6096):816-21).
[0084] Cas9 proteins are known in the art, such as Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), and Francisella novicida Cas9 (FnCas9). In general, Cas9 proteins preferentially interrogate and act upon DNA sequences containing a protospacer adjacent motif (PAM) sequence, and different Cas9 proteins have affinities for different PAMs. The canonical PAM sequence is 5′-NGG-3′, which is recognized by multiple Cas9 proteins, where N can be any nucleotide. For example, SpCas9 and FnCas9 recognize the canonical NGG PAM sequence. Streptococcus thermophilus Cas9 recognizes a 5′-NGA-3′ PAM sequence, and SaCas9 recognizes a 5′-NNGRR(N)-3′ PAM sequence. Additionally, Cas9 proteins can be modified to recognize PAM sequences that are distinct from the PAM sequences recognized by the unmodified Cas9 protein. For example, SaCas9-KKH recognizes a 5′-RRT-3′, where R denotes an adenosine or guanine nucleotide. The Cas9 nuclease used in the presently described methods will recognize a PAM sequence that is present only in the allele to be inactivated (i.e., the allele carrying a deleterious mutation). Thus, the nuclease activity of the Cas9 will act only upon the allele to be inactivated.
gRNA
[0085] A Cas9 protein, having an affinity for a particular PAM sequence can be directed to a particular locus in a genome by a guide RNA. In some embodiments, the guide RNA is a single guide RNA, which comprises a tracrRNA and a spacer RNA. The short spacer RNA, comprising a nucleic acid sequence that specifically binds to the target genomic locus, directs the Cas9 protein to the target, which is then cleaved by the Cas9 protein's nuclease activity. In some embodiments, synthetic gRNAs are about 18, 19, 20, 21, 22, 25, 30, 40, 50, 60, 70, 80, 90, 100, over 100 bp and comprise a nucleic acid sequence complementary to protospacer nucleotides near the PAM sequence
[0086] In some embodiments, the guide RNA will bind a nucleic acid sequence comprising a PAM sequence that is present in an allele carrying a mutation, but is not present in an allele that does not carry the mutation. In some embodiments, the guide RNA binds a nucleic acid sequence that is in close proximity to a PAM sequence that is present only in an allele to be inactivated (i.e., an allele carrying a deleterious mutation). The PAM sequence may be 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides upstream or downstream of the sequence to which with guide RNA binds.
[0087] The following US patents and patent publications are incorporated herein by reference in their entireties: U.S. Pat. No. 8,697,359, 20140170753, 20140179006, 20140179770, 20140186843, 20140186958, 20140189896, 20140227787, 20140242664, 20140248702, 20140256046, 20140273230, 20140273233, 20140273234, 20140295556, 20140295557, 20140310830, 20140356956, 20140356959, 20140357530, 20150020223, 20150031132, 20150031133, 20150031134, 20150044191, 20150044192, 20150045546, 20150050699, 20150056705, 20150071898, 20150071899, 20150071903, 20150079681, 20150159172, 20150165054, 20150166980, and 20150184139.
Polynucleotide Delivery
[0088] Therapeutic success in these approaches relies significantly on the safe and efficient delivery of exogenous gene constructs to the relevant therapeutic cell targets in the organ of Corti in the cochlea. The organ of Corti includes two classes of sensory hair cells: inner hair cells, which convert mechanical information carried by sound into electrical signals transmitted to neuronal structures and outer hair cells which serve to amplify and tune the cochlear response, a process required for complex hearing function.
[0089] Methods of delivering nucleic acids to cells generally are known in the art, and methods of delivering viruses (which also can be referred to as viral particles) containing a transgene to inner ear cells in vivo are described herein. As described herein, about 10.sup.8 to about 10.sup.12 viral particles can be administered to a subject, and the virus can be suspended within a suitable volume (e.g., 10 μL, 50 μL, 100 μL, 500 μL, or 1000 μL) of, for example, artificial perilymph solution.
[0090] A virus containing a promoter (e.g., an Espin promoter, a PCDH15 promoter, a PTPRQ promoter, a Myo6 promoter, a KCNQ4 promoter, a 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) and a polynucleotide encoding a Cas9 protein (e.g., SaCas9-KKH), and in some embodiments, a guide RNA, as described herein can be delivered to inner ear cells (e.g., cells in the cochlea) using any number of means. For example, a therapeutically effective amount of a composition including virus particles containing one or more different types of transgenes as described herein can be injected through the round window or the oval window, or the utricle, typically in a relatively simple (e.g., outpatient) procedure. In some embodiments, a composition comprising a therapeutically effective number of virus particles containing a transgene (e.g., a polynucleotide encoding a transgene and a gRNA), or containing one or more sets of different virus particles, wherein each particle in a set can contain the same type of transgene, but wherein each set of particles contains a different type of transgene than in the other sets, as described herein can be delivered to the appropriate position within the ear during surgery (e.g., a cochleostomy or a canalostomy).
[0091] In addition, delivery vehicles (e.g., polymers) are available that facilitate the transfer of agents across the tympanic membrane and/or through the round window or utricle, and any such delivery vehicles can be used to deliver the viruses described herein. See, for example, Arnold et al., 2005, Audiol. Neurootol., 10:53-63.
[0092] The compositions and methods described herein enable the highly efficient delivery of nucleic acids to inner ear cells, e.g., cochlear cells. For example, a polynucleotide encoding a Cas9 protein, variant (e.g., SaCas9-KKH), or a fragment thereof, can be cloned into a retroviral vector and expression can be driven from its endogenous promoter, from the retroviral long terminal repeat, or from a promoter specific for a target cell type of interest. Other viral vectors that can be used include, for example, a vaccinia virus, a bovine papilloma virus, or a herpes virus, such as Epstein-Barr Virus. Retroviral vectors are particularly well developed and have been used in clinical settings. In some embodiments, a viral vector is used to administer a Cas9 polynucleotide systemically. In some embodiments, a viral vector is used to administer a Cas9 polynucleotide to a particular region of the body.
[0093] For example, the compositions and methods described herein enable the delivery to, and expression of, a KKH-Cas9 polynucleotide in at least 65% (e.g., at least 65, 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99%) of inner and/or outer hair cells or delivery to, and expression in, at least 80% (e.g., at least 85, 90, 91, 92, 93, 94, 95, 96, 97, 98, or 99) of outer hair cells.
[0094] As demonstrated herein, expression of a KKH-Cas9 polynucleotide delivered using an AAV-vector can result in improved structure and function of inner and outer hair cells such that hearing is restored for an extended period of time (e.g., months, years, decades, a life time).
[0095] As described herein, an adeno-associated virus (AAV) are particularly efficient at delivering nucleic acids (e.g., polynucleotides encoding a Cas9 polypeptide and, in some embodiments, a gRNA) to inner ear cells. The Anc80 vector is an example of an Inner Ear Hair Cell Targeting AAV that advantageously transduced greater than about 60%, 70%, 80%, 90%, 95%, or even 100% of inner or outer hair cells. One particular ancestral capsid protein that falls within the class of Anc80 ancestral capsid protein is Anc80-0065 (SEQ ID NO:2) described in International Application No. PCT/US2018/017104, which is incorporated herein by reference in its entirety. However, WO 2015/054653, which is also incorporated herein by reference in its entirety, describes a number of additional ancestral capsid proteins that fall within the class of Anc80 ancestral capsid proteins.
[0096] In particular embodiments, the adeno-associated virus (AAV) contains an ancestral AAV capsid protein that has a natural or engineered tropism for hair cells. In some embodiments, the virus is an Inner Ear Hair Cell Targeting AAV, which delivers a transgene (e.g., a polynucleotide encoding a Cas9 polypeptide and, in some embodiments, a gRNA) to the inner ear in a subject. In some embodiments, the virus is an AAV that comprises purified capsid polypeptides. In some embodiments, the virus is artificial. In some embodiments, the virus is an AAV that has lower seroprevalence than AAV2. In some embodiments, the virus is an exome-associated AAV. In some embodiments, the virus is an exome-associated AAV1. In some embodiments, the virus comprises a capsid protein with at least 95% amino acid sequence identity or homology to Anc80 capsid proteins.
[0097] Expression of a Cas9 polynucleotide may be directed by a heterologous promoter (e.g., CMV promoter, Espin promoter, a PCDH15 promoter, a PTPRQ promoter and a TMHS (LHFPL5) promoter). As used herein, a “heterologous promoter” refers to a promoter that does not naturally direct expression of that sequence (i.e., is not found with that sequence in nature).
[0098] Methods for packaging a transgene into a virus that contains an Anc80 capsid protein are known in the art, and utilize conventional molecular biology and recombinant nucleic acid techniques. In one embodiment, a construct that includes a nucleic acid sequence encoding an Anc80 capsid protein and a construct carrying the polynucleotide encoding a Cas9 (and in some cases a Cas9 and a gRNA) flanked by suitable Inverted Terminal Repeats (ITRs) are provided, which allows for the transgene to be packaged within the Anc80 capsid protein.
[0099] The Cas9 polynucleotide (and in some embodiments, a Cas9 and a gRNA) can be packaged into an AAV containing an Anc80 capsid protein using, for example, a packaging host cell. The components of a virus particle (e.g., rep sequences, cap sequences, inverted terminal repeat (ITR) sequences) can be introduced, transiently or stably, into a packaging host cell using one or more constructs as described herein.
[0100] In some embodiments, a AAVs containing a AAV9-php.b vector is used to efficiently target inner ear cells. AAV9-php.b is described in International Application No. PCT/US2019/020794, the contents of which are incorporated herein by reference in their entirety. AAV-PHP.B encodes the 7-mer sequence TLAVPFK and efficiently delivers transgenes to the cochlea, where it showed remarkably specific and robust expression in the inner and outer hair cells. An AAV-PHP.B vector can comprise, but is not limited to, any of the promoters described herein.
[0101] Non-viral approaches can also be employed for the introduction of therapeutic to a cell of a patient requiring inactivation of an allele carrying a mutation associated with a disease or condition or symptom thereof. For example, a nucleic acid molecule can be introduced into a cell by administering the nucleic acid in the presence of lipofection.
[0102] Gene transfer can also be achieved using non-viral means involving transfection in vitro. Such methods include the use of calcium phosphate, DEAE dextran, electroporation, and protoplast fusion. Liposomes can also be potentially beneficial for delivery of DNA into a cell. Transplantation of normal genes into the affected tissues of a patient can also be accomplished by transferring a normal nucleic acid into a cultivatable cell type ex vivo (e.g., an autologous or heterologous primary cell or progeny thereof), after which the cell (or its descendants) are injected into a targeted tissue.
[0103] cDNA expression for use in polynucleotide therapy methods can be directed from any suitable promoter (e.g., the human cytomegalovirus (CMV), simian virus 40 (SV40), or metallothionein promoters), and regulated by any appropriate mammalian regulatory element. For example, if desired, enhancers known to preferentially direct gene expression in specific cell types can be used to direct the expression of a nucleic acid. The enhancers used can include, without limitation, those that are characterized as tissue- or cell-specific enhancers. Alternatively, if a genomic clone is used as a therapeutic construct, regulation can be mediated by the cognate regulatory sequences or, if desired, by regulatory sequences derived from a heterologous source, including any of the promoters or regulatory elements described above.
[0104] Another therapeutic approach included in the invention involves administration of a recombinant therapeutic, such as a recombinant a Cas9 protein, variant, or fragment thereof, either directly to the site of a potential or actual disease-affected tissue or systemically (for example, by any conventional recombinant protein administration technique). The dosage of the administered protein depends on a number of factors, including the size and health of the individual patient. For any particular subject, the specific dosage regimes should be adjusted over time according to the individual need and the professional judgment of the person administering or supervising the administration of the compositions.
Inactivating Alleles
[0105] Compositions and methods are provided herein for altering a cell to inactivate a mutant allele associated with a disease or condition using a CRISPR-Cas system. In some embodiments, a Cas9 (e.g., SaCas9-KKH), in combination with a single guide RNA is used to target an allele comprising a mutation. To selectively inactivate the allele carrying the mutation, while not inactivating the wild type or other non-deleterious forms of the allele, a Cas protein is used that recognizes a PAM sequence present in the mutant allele but not in the wild type (or other non-mutant form) allele. Upon target recognition, the Cas protein (e.g., Cas9) induces at least one double strand break in the target mutant allele. Repair of the double-strand break by non-homologous end joining (NHEJ) increases the probability of an indel at the double-strand break site. In some embodiments, an indel at the double-strand break site generates a premature stop codon in the mutant allele that inactivates the mutant allele. In some embodiments, the indel can be in a regulatory region of the allele that results in inhibited expression of the allele. In some embodiments, the indel generates a protein product that is lacks a deleterious nature (i.e., the edited allele does not interfere with the expression and function of the wildtype (or non-mutant form) allele.
Compositions and Methods of Treatment
[0106] The present invention provides methods of treating disease and/or disorders or symptoms thereof which comprise administering a therapeutically effective amount of a pharmaceutical composition comprising a Cas9 nuclease and a guide RNA that specifically binds a nucleic acid sequence in the genome comprising a mutation and a PAM sequence recognized by the Cas9 to a subject (e.g., a mammal such as a human), wherein the PAM sequence is not present in the allele that does not carry the mutation. Thus, one embodiment is a method of treating a subject suffering from or susceptible to a disease or disorder or symptom thereof associated with a mutation. The method includes administering to the mammal a therapeutic amount of an amount of a compound herein sufficient to treat the disease or disorder or symptom. In some embodiments, the mutation is a dominant mutation.
[0107] The therapeutic methods of the invention (which include prophylactic treatment), in general, comprise administration of a therapeutically effective amount of the compounds herein, such as a compound of the formulae herein to a subject (e.g., animal, human) in need thereof, including a mammal, particularly a human. Such treatment will be suitably administered to subjects, particularly humans, suffering from, having, susceptible to, or at risk for a disease, disorder, or symptom thereof. Determination of those subjects “at risk” can be made by any objective or subjective determination by a diagnostic test or opinion of a subject or health care provider (e.g., genetic test, enzyme or protein marker, Marker (as defined herein), family history, and the like).
[0108] Compositions are contemplated herein for the treatment of diseases or conditions associated with a mutation. For therapeutic purposes, compositions comprising a Cas9 polypeptide, or a polynucleotide encoding a Cas9 polypeptide and a guide RNA that specifically binds to a nucleic acid sequence in a genome that comprises a mutation that causes or contributes to a disease or condition (e.g., dominant progressive hearing loss) as described herein may be administered directly to a region of the body (e.g., cochlea) that is affected by the disease or condition. In some embodiments, the compositions are formulated in a pharmaceutically-acceptable buffer such as physiological saline. Non-limiting methods of administration include injecting into the cochlear duct or the perilymph-filled spaces surrounding the cochlear duct (e.g., scala tympani and scala vestibuli). Injecting into the cochlear duct, which is filled with high potassium endolymph fluid, could provide direct access to hair cells. However, alterations to this delicate fluid environment may disrupt the endocochlear potential, heightening the risk for injection-related toxicity. The perilymph-filled spaces surrounding the cochlear duct, scala tympani and scala vestibuli, can be accessed from the middle ear, either through the oval or round window membrane. The round window membrane, which is the only non-bony opening into the inner ear, is relatively easily accessible in many animal models and administration of viral vector using this route is well tolerated. In humans, cochlear implant placement routinely relies on surgical electrode insertion through the round window membrane.
[0109] Treatment of human patients or non-human animals are carried out using a therapeutically effective amount of a combination therapeutic in a physiologically-acceptable carrier. The phrase “pharmaceutically acceptable” refers to those compounds of the invention, compositions containing such compounds, and/or dosage forms which are, within the scope of sound medical judgment, suitable for use in contact with the tissues of human beings and animals without excessive toxicity, irritation, allergic response, or other problem or complication, commensurate with a reasonable benefit/risk ratio.
[0110] The phrase “pharmaceutically-acceptable excipient” includes pharmaceutically-acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, carrier, solvent or encapsulating material, involved in carrying or transporting the subject compound from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be “acceptable” in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: (1) sugars, such as lactose, glucose and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository waxes; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; (10) glycols, such as propylene glycol; (11) polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffering agents, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) phosphate buffer solutions; and (21) other non-toxic compatible substances employed in pharmaceutical formulations.
[0111] The compositions may conveniently be presented in unit dosage form and may be prepared by any methods well known in the art. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will vary depending upon the host being treated, the particular mode of administration. The amount of active ingredient which can be combined with a carrier material to produce a single dosage form will generally be that amount of the compound which produces a therapeutic effect. Generally, out of one hundred per cent, this amount will range from about 1 per cent to about ninety-nine percent of active ingredient, preferably from about 5 per cent to about 70 per cent, most preferably from about 10 per cent to about 30 per cent.
[0112] Additional suitable carriers and their formulations are described, for example, in the most recent edition of Remington's Pharmaceutical Sciences by E. W. Martin. The amount of the therapeutic agent to be administered varies depending upon the manner and mode of administration, the age and disease status (e.g., the extent of hearing loss present prior to treatment).
[0113] Compositions are administered at a dosage that controls the clinical or physiological symptoms of the disease or condition, as may in some cases be determined by a diagnostic method known to one skilled in the art.
[0114] Therapeutic compounds and therapeutic combinations are administered in an effective amount. For example, about 10.sup.8 to about 10.sup.12 viral particles can be administered to a subject, and the virus can be suspended within a suitable volume (e.g., 10 μL, 50 μL, 100 μL, 500 μL, or 1000 μL) of, for example, artificial perilymph solution.
Methods of Treating Dominant Progressive Hearing Loss
[0115] Compositions and methods for treating dominant progressive hearing loss (e.g., Deafness, Autosomal Dominant 36, or dominant progressive deafness 36, (DFNA36)) are provided.
[0116] DFNA36 is associated with dominant mutations (acquired or inherited) in the TMC1 gene of affected individuals. To inactivate the dominant mutation in a heterozygous subject, a SaCas9-KKH protein along with a guide RNA that recognizes the genomic locus containing the TMC1 dominant mutant, is administered to a subject as described above. The SaCas9=KKH protein that recognizes the RRT PAM sequence present in the TMC1 allele carrying the dominant mutation. Mutations within the TMC1 gene can cause Deafness, Autosomal Dominant 36 (DFNA36), a dominant progressive form of deafness. The SaCas9-KKH protein binds to a cleaves the TMC1 allele carrying the dominant mutation (but not the wild type allele), which promotes indel formation at the break site during non-homologous end joining. Resulting premature stop codons generate truncated, non-functional TMC1 proteins that are not dominant to the expressed wild type protein.
[0117] In some embodiments, the SaCas9-KKH nuclease is administered to a subject by directly injecting a vector (e.g., AAV or lentiviral vector) encoding the SaCas9-KKH protein and a guide RNA into the cochlea of the subject. In some embodiments, the vector only encodes the SaCas9-KKH protein and the guide RNA is administered in the injection as RNA For therapeutic purposes, compositions comprising a Cas9 polypeptide, or a polynucleotide encoding a Cas9 polypeptide and a guide RNA that specifically binds to a nucleic acid sequence in a genome that comprises a mutation that causes or contributes to dominant progressive hearing loss as described herein may be administered directly to a region of the body (e.g., cochlea) that is affected by the disease or condition. Non-limiting methods of administration include injecting into the cochlear duct or the perilymph-filled spaces surrounding the cochlear duct (e.g., scala tympani and scala vestibuli). Injecting into the cochlear duct, which is filled with high potassium endolymph fluid, could provide direct access to hair cells. However, alterations to this delicate fluid environment may disrupt the endocochlear potential, heightening the risk for injection-related toxicity. The perilymph-filled spaces surrounding the cochlear duct, scala tympani and scala vestibuli, can be accessed from the middle ear, either through the oval or round window membrane. The round window membrane, which is the only non-bony opening into the inner ear, is relatively easily accessible in many animal models and administration of viral vector using this route is well tolerated. In humans, cochlear implant placement routinely relies on surgical electrode insertion through the round window membrane.
[0118] In some embodiments, inactivating the mutant allele that causes DFNA36 while expressing the wildtype allele can restore auditory function in a subject. In some embodiments, the auditory function restored to a subject is about 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90%, or even about 100%.
EXAMPLES
Example 1: Screening Cas9 and gRNA Combinations In Vitro
[0119] In order to develop allele specific genome editing strategies for dominant hearing loss, the Beethoven (Bth) mouse was used. The Bth mouse provides an excellent model for DFNA36 hearing loss in humans. The Bth mutation results in an amino acid substition (p.M412K, c.T1253A) in Tmc1. The mutation causes hair cell degeneration and progressive hearing loss in mice. In humans, the p.M418K substitution is identical to the Bth mutation in the orthologous position and causes DFNA36, dominant progressive hearing loss.
[0120] To selectively disrupt the Bth allele in fibroblasts from Tmc1.sup.Bth/WT mice (Tmc1.sup.WT/WT cells were used as controls), various Cas9 and gRNA combinations were screened in vitro. Plasmids encoding SpCas9-2A-GFP, along with the different gRNAs, were transfected into fibroblasts in duplicate. Four days after transfection, GFP-positive cells were sorted by fluorescence assisted cell sorting (FACS). SpCas9 in combination with 12 different gRNAs were tested, including full-length and truncated forms targeting either Tmc1.sup.Bth or Tmc1.sup.WT (
[0121] Targeted deep sequencing was performed on control (SpCas9-2A-GFP only) cells, WT gRNA and the 3 most specific gRNAs (1.1, 2.1 and 2.4) in Tmc1.sup.Bth/WT (top) Tmc1.sup.WT/WT (bottom) cells. Indels were quantified after segregating Tmc1.sup.Bth and Tmc1 .sup.WT reads by CRISPResso (only insertions and deletions were quantified, substitutions were ignored). None of the gRNAs are specific to the Tmc1.sup.Bth allele, and mediate efficient indel formation on the Tmc1.sup.WT allele as well. Sequencing was performed one time from pooled cells, transfected in triplicates. Specificity was defined as the indel percentage towards the targeted allele among total indels. The gRNA with the highest selectivity towards the Tmc1.sup.Bth allele was gRNA 2.4 (
[0122] To improve allele selectivity, high-fidelity SpCas9 enzymes were also evaluated; however, none mediated selective targeting of the Bth allele (
Example 2: SaCas9-KKH Recognizes a PAM Site Selective for the TMC.SUP.Bth .Allele
[0123] It was hypothesized that a Cas9 nuclease with a PAM site selective for the mutant sequence might show specific targeting of the Tmc1.sup.Bth allele. The Bth mutation is a T to A change; thus, the GGAAGT sequence present in Tmc1.sup.Bth, but not in Tmc1.sup.Bth (GGATGT), may allow the PAM site of the SaCas9-KKH variant (NNNRRT) to distinguish the Tmc1.sup.Bth from the Tmc1.sup.WT allele. Full-length and truncated gRNAs were designed (
[0124] Allele-specific indel formation in Tmc1.sup.Bth/WT cells was analyzed to avoid potential differences in transfection efficiency between Tmc1.sup.Bth/WT and Tmc1.sup.WT/WT fibroblasts. Tmc1.sup.Bth and Tmc1 .sup.WTsequencing reads were segregated using a Python script and indel percentages were analyzed for each allele. (
Example 3: SaCas9-KKH-Mediated Indel Formation in Sensory Hair Cells
[0125] To assess the capability of SaCas9-KKH and gRNA 4.2 to introduce indels into the TMC1 gene, the Cas9 protein and guide RNA were packaged into Anc80L65 capsids (
Example 4: SaCas9-KKH-Guide RNA Significantly Reduces Auditory Brainstem Responses
[0126] SaCas9-KKH-mediated disruption of the Bth allele was evaluated using hair cell mechanosensory transduction current. Although the Bth mutation eventually causes cell death, the mutation does not cause a loss of mechanosensitivity. Single-cell electrophysiology was performed on hair cells from either Tmc1.sup.WT/WT or Tmc1.sup.Bth/Δ mouse pups on a Tmc2.sup.Δ/Δ background because Tmc2 contributes to mechanosensory currents and is expressed transiently at neonatal stages. After injection of AAV-SaCas9-KKH-gRNA-4.2 at P1, cochleas were dissected at P5-P7 and cultured 8-10 days, or the equivalent of P14-P16. Both inner and outer hair cells from injected Tmc1.sup.WT/WT mice showed normal current amplitudes (
Example 5: SaCas9-KKH-Guide RNA Improves Auditory Brain Responses
[0127] Auditory brainstem responses (ABR) and distortion product otoacoustic emissions (DPOAE) were evaluated in Bth mice using the allele-specific SaCas9-KKH nuclease (
[0128] Since the Bth mutation causes progressive hearing loss, the time course of hearing sensitivity in Tmc1.sup.Bth/WT and Tmc1.sup.WT/WT animals was measured at 4, 8, 12 and 24 weeks after injection at frequencies from 5.6 to 32 kHz (
[0129] ABR peak 1 (P1) amplitudes were in the normal range for most of the injected Bth mice at 8 weeks, in contrast to non-injected animals, which showed small P1 amplitudes only at high sound intensities (
[0130] Whether AAV-SaCas9-KKH-gRNA-4.2 injection disrupts hearing in wild-type animals was also investigated. ABRs were performed and no ABR or DPOAE threshold shifts were observed, even 24 weeks post-injection (
[0131] In one cohort of four Bth mice, ABR thresholds were measured at 40 weeks post-injection. Thresholds (8 kHz) between 4 and 40 weeks were tracked and data from two mice with failed injections was excluded, based on histological examination (below). Thresholds were stable over time and only slightly elevated relative to WT mice (
[0132] Next, hair cell survival was evaluated in injected and non-injected Tmc1.sup.Bth/WT and Tmc1.sup.WT/WT animals. Following ABR and DPOAE evaluations, mice were sacrificed at 24 weeks of age. Surviving hair cells were identified with an antibody for MYO7A and phalloidin staining for actin. Inner and outer hair cells were present in uninjected Tmc1.sup.WT/WT mice and those injected with AAV-SaCas9-KKH-gRNA-4.2(
Example 6: Preservation of Normal Hair Bundle Morphology
[0133] Hair bundle morphology was evaluated with scanning electron microscopy, in Bth and WT hair cells. In uninjected Tmc1.sup.WT/WT animals at 24 weeks of age, inner and outer hair cells showed classical staircase organization of hair bundles (
Example 7: Human Haploid Cells
[0134] To validate the strategy for targeting the human p.M418K mutation, a haploid human cell line was generated containing the p.M418K mutation in TMC1 (c.T1253A). Tmc1.sup.DFNA36 and TMC1.sup.WT cells were transfected with SaCas9-KKH and 3 different gRNAs targeting the mutant allele (
Example 8: SaCas9-KKH PAM is Present in Other Dominant Mutations
[0135] In addition to the TMC1 p.M418K mutation (DFNA36), 15 other dominant mutations in deafness genes that are targetable with SaCas9-KKH were identified (
TABLE-US-00011 TABLE 1 Dominant deafness variants potentially targetable with SaCas9-KKH Deafness locus OMIM Disease SNP ID WT Variant Protein Gene Link DFNA11 .0015 DEAFNESS, RS = 121965084 CAATG CATTG ASN458ILE MYO7A www.omim.org/entry/ AUTOSOMAL DOMINANT 11 276903#0015 DFNA12 .0001 DEAFNESS, RS = 281865415 AGCTC AGTTC GLY1824ASP TECTA www.omim.org/entry/ AUTOSOMAL DOMINANT 12 602574#0001 DFNA13 .0006 DEAFNESS, RS = 121912947 GCGCC GCACC ARG549CYS COL11A2 www.omim.org/entry/ AUTOSOMAL DOMINANT 13 120290#0005 DFNA17 .0008 DEAFNESS, RS = 80338828 GGCGG GGTGG ARG705HIS MYH9 www.omim.org/entry/ AUTOSOMAL DOMINANT 17 160775#0008 DFNA20 .0002 DEAFNESS, RS = 104894544 TCTTC TCATC LYS118MET ACTG1 www.omim.org/entry/ AUTOSOMAL DOMINANT 20 102560#0002 DFNA22 .0001 DEAFNESS, RS = 121912557 GTGTT GTATT CYS442TYR MYO6 www.omim.org/entry/ AUTOSOMAL DOMINANT 22 600970#0001 DFNA22 .0006 DEAFNESS, RS = 121912561 AACGA AATGA ARG849TER MYO6 www.omim.org/entry/ AUTOSOMAL DOMINANT 22 600970#0006 DFNA25 .0001 DEAFNESS, RS = 121918339 GGCAC GGTAC ALA211VAL SLC17A8 www.omim.org/entry/ AUTOSOMAL DOMINANT 25 607557#0001 DFNA36 .0007 DEAFNESS, RS = 786201027 GATGT GAAGT MET418LYS TMC1 www.omim.org/entry/ AUTOSOMAL DOMINANT 36 606706#0007 DFNA39 .0004 DEAFNESS, RS = 121912987 AGGTT AGTTT VAL18PHE DSPP www.omim.org/entry/ AUTOSOMAL DOMINANT 125485#0004 NONSYNDROMIC SENSORINERAL 39, WITH DENTINOGENESIS IMPERFECTA 1 DFNA3b .0001 DEAFNESS, RS = 104894414 GACGC GATGC THR5MET GJB6 www.omim.org/entry/ AUTOSOMAL DOMINANT 3B 604418#0001 DFNA41 .0001 DEAFNESS, RS = 587777692 ACGTA ACTTA VAL60LEU P2RX2 www.omim.org/entry/ AUTOSOMAL DOMINANT 41 600844#0001 DFNA48 .0004 RECLASSIFIED- RS = 61753849 GACAT GAAAT GLU385ASP MYO1A www.omim.org/entry/ VARIANT OF UNKNOWN 601478#0004 SIGNIFICANCE DFNA66 .0001 DEAFNESS, RS = 876661402 TCGTT TCATT ARG192TER CD164 www.omim.org/entry/ AUTOSOMAL DOMINANT 66 603356#0001 DFNA68 .0001 DEAFNESS, RS = 864309524 GCCGT GCGGT ARG185PRO HOMER2 www.omim.org/entry/ AUTOSOMAL DOMINANT 68 604799#0001 DFNA9 .0001 DEAFNESS, RS = 121908927 AGTAT AGGAT VAL66LGY COCH www.omim.org/entry/ AUTOSOMAL DOMINANT 9 603196#0001 DFNA9 .0005 DEAFNESS, RS = 121908930 CATCC CAACC ILE109ASN COCH www.omim.org/entry/ AUTOSOMAL DOMINANT 9 603196#0005 DFNA9 .0006 DEAFNESS, RS = 12190831 CTGCT CTACT ALA119THR COCH www.omim.org/entry/ AUTOSOMAL DOMINANT 9 603196#0006
[0136] The results reported herein were generated using the following methods and materials.
Plasmids and Cloning
[0137] Table 2 provides information on the specifics of the CRISPR/Cas9 plasmids used in the above examples. Table 3 shows the sequences of the gRNAs and the Cas9 plasmids used together with the gRNA vectors. 11 different gRNAs were tested with SpCas9 (
TABLE-US-00012 TABLE 2 Specification of plasmids used in this study Plasmid Specification Origin (reference) pX458 U6-sgRNA-CMV-3xFLAG-NLS(SV40)- Addgene ID: 48138, Ran et al. 2013 SpCas9(BB)-NLS(nucleoplasmin)-T2A-GFP-bGHpA MLM3636 U6-sgRNA Addgene ID: 43860 BPK3258 CMV-T7-eSpCas9(1.1)(K848A, K1003A, Addgene ID: 101176, Chen et al. R1060A)-NLS(SV40)-3xFLAG (derived from Slaymaker et al.) BPK4410 CMV-T7-HypaCas9- (N692A, M694A, Addgene ID: 101178, Chen et al. Q695A, H698A)-NLS(SV40)-3xFLAG VP12 CMV-T7-SpCas9-HF1(N497A, R661A, Addgene ID: 72247, Q695A, Q926A)-NLS(SV40)-3xFLAG Kleinstiver BP et al. pBG201 AAV-CMV-NLS(SV40)- Derived from pX601, SaCas9(E782K/N968K/R1015H)- Addgene ID: 61591, Ran et al 2015 NLS(nucleoplasmin)-3xHA-bGHpA0-U6-Bsal-sgRNA
TABLE-US-00013 TABLE 3 gRNA sequences and usage gRNA ID Cas9 gRNA plasmid Cas9 plasmid gRNA sequence.sup.1 + PAM (5′-3′) 1.1 SpCas9 pX458 pX458 GGGTGGGACAGAACTTCCCCAGG 1.1 eSpCas9(1.1) MLM3636 BPK3258 GGGTGGGACAGAACTTCCCCAGG 1.1 HypaCas9 MLM3636 BPK4410 GGGTGGGACAGAACTTCCCCAGG 1.1 SpCas9-HF1 MLM3636 VP12 GGGTGGGACAGAACTTCCCCAGG 1.2 SpCas9 pX458 pX458 GGTGGGACAGAACTTCCCCAGG 1.3 SpCas9 pX458 pX458 GTGGGACAGAACTTCCCCAGG 1.4 SpCas9 pX458 pX458 GGGACAGAACTTCCCCAGG 2.1 SpCas9 pX458 pX458 GTGGGACAGAACTTCCCCAGGAGG 2.2 SpCas9 pX458 pX458 GGGACAGAACTTCCCCAGGAGG 2.3 SpCas9 pX458 pX458 GGACAGAACTTCCCCAGGAGG 2.4 SpCas9 pX458 pX458 GACAGAACTTCCCCAGGAGG 3.1 SpCas9 pX458 pX458 GTGGTAATGTCCCTCCTGGGGAAG 3.2 SpCas9 pX458 pX458 GGTAATGTCCCTCCTGGGGAAG 3.3 SpCas9 pX458 pX458 GTAATGTCCCTCCTGGGGAAG 4.1 SaCas9-KKH pBG201 pBG201 GAACATGGTAATGTCCCTCCTGGGGAAGT 4.2 SaCas9-KKH pBG201 pBG201 GACATGGTAATGTCCCTCCTGGGGAAGT 4.3 SaCas9-KKH pBG201 pBG201 GCATGGTAATGTCCCTCCTGGGGAAGT WT SpCas9 pX458 pX458 GGGTGGGACAGAACATCCCCAGG H1 SaCas9-KKH pBG201 pBG201 GAACATGGTTATGTCCCTCCTAGGGAAGT H2 SaCas9-KKH pBG201 pBG201 GACATGGTTATGTCCCTCCTAGGGAAGT H3 SaCas9-KKH pBG201 pBG201 GCATGGTTATGTCCCTCCTAGGGAAGT Non-matching 5′ G nucleotides are marked underlined
[0138] For gRNAs 1.1-1.4 and 2.1-2.4, a PAM site was adjacent to the mutation. gRNA 1.1 is identical to the Tmc1-mut3 gRNA in the study of Gao et al., Nature, 553: 217-21 (2018). In the case of gRNAs 3.1-3.3, an AAG PAM site created by the mutation was used in order to specifically recognize the mutant allele, as it has been shown that SpCas9 can also cleave at
[0139] NAG PAM sites with somewhat lower efficiency. Several truncated gRNAs were also used because previous studies reported enhanced specificity (Fu, Y. et al., Nat. Biotechnol. 32: 279-84 (2014)). One gRNA specific for the Tmc1WT allele was synthesized as a control. gRNA 1.1 was used (
Cell Culture, Transfection and Sorting
[0140] Mouse primary dermal fibroblasts were established from neonatal C57BL/6 Tmc1WT/WT and Tmc1Bth/WT animals. Briefly, after euthanasia, a small amount of skin was dissected into small pieces and washed with PBS. Next, cells were treated at 37° C. with 1 mg/ml collagenase I (Worthington) for 30 minutes followed by 0.05% trypsin-EDTA treatment for 15 minutes. Cells were cultured in 10% fetal bovine serum containing DMEM (Gibco) supplemented with lx penicillin/streptomycin (Gibco). Cell lines were validated by Sanger sequencing (see below). Mycoplasma screening (MycoAlert, Lonza, Basel) was performed regularly and before transfection experiments. For transfection of fibroblasts, Nucleofection (Lonza) (CZ-167 program, P2 Primary Cell 4D-Nucleofector X Kit) was used. Every transfection reaction was performed in duplicate and experiments were performed on at least two separate occasions. Four days after transfection of pX458 plasmids, cells were sorted based on GFP fluorescence using a FACS Aria Cell Sorter (BD), and genomic DNA was isolated and analyzed by Sanger sequencing and targeted deep sequencing (see below). In the case of high fidelity SpCas9s (eSpCas9(1.1), HypaCas9, SpCas9-HF1) and SaCas9-KKH transfections, cells were not sorted, but genomic DNA was isolated from all cells 4 days after transfection for deep-sequencing analysis.
Mouse Genomic DNA Isolation and PCR
[0141] Genomic DNA was isolated from fibroblasts 4 days after transfection using a Qiagen Blood and Tissue Kit (Qiagen). In the case of cochlear tissue, organs were harvested at different ages (
Sanger Sequencing and TIDE Analysis
[0142] Sanger sequencing was performed in the MGH DNA Core. Sequence traces were analyzed by deconvolution (TIDE, Tracking Indels by Decomposition, Desktop genetics, UK). Aberrant sequences were quantified downstream of the CRISPR cut site. Analysis was performed on forward versus reverse traces and efficiency was averaged.
Targeted Deep Sequencing Data Analysis
[0143] CRISPR-induced indels were analyzed by CRISPResso using two separate methods (
Global CRISPResso Indel Analysis
[0144] To analyze CRISPR action on both Tmc1.sup.Bth and Tmc1WT alleles, the fastq files were subjected to CRISPResso analysis without segregating them to mutant and WT reads (
[0145] Allele-Specific CRISPResso Indel Analysis
[0146] To analyze CRISPR action on Tmc1Bth and Tmc1WT alleles separately, fastq files were first split into read 1 and read 2, and then merged using flash v1.2.11, as described above. The reads from heterozygous samples were segregated based on the presence of wild-type sequence (“TGGGACAGAACA” and its reverse complement “TGTTCTGTCCCA”) and mutant sequence (“TGGGACAGAACT” and its reverse complement “AGTTCTGTCCCA”; mutation site is underlined) using a custom Python script (version 3.4.2) used previously (György, B. et al., Mol. Ther.-Nucleic Acids 11: 429-40 (2018), the contents of which are incorporated herein by reference in their entirety). Reads were segregated based on a sequence downstream of the projected CRISPR cut site so that indels have minor influence on the segregation (
[0147] For mRNA analysis, the reads were first merged with flash as described above. CRISPResso analysis was performed similarly to global indel analysis (see above). To quantify intact, non-edited reads, the following sequences were used: 5′-CATCCCCAGGAGGG-3′ and 5′-CCCTCCTGGGGATG-3′ for WT reads, and 5′-CTTCCCCAGGAGGG-3′ and 5′-CCCTCCTGGGGAAG-3′ for mutant reads.
Off-Target Analysis
[0148] To detect genome-wide Cas9 nuclease activity, a GUIDE-Seq assay was performed in fibroblasts. Briefly, 2 μg of Cas9-2A-GFP-U6-gRNA-2.4 or 2 μg of pAAV-CMV-SaCas9-KKH-U6-gRNA-4.2 along with 50 pmol annealed GUIDE-Seq oligo (forward:/5Phos/G*T*TTAATTGAGTTGTCATATGTTAATAACGGT*A*T, reverse:/5Phos/A*T*ACCGTTATTAACATATGACAACTCAATTAA*A*C, stars indicate thioate bonds) were transfected into Tmc1Bth/WT fibroblasts using electroporation (see above). Four days after transfection, genomic DNA was isolated with a Qiagen DNA Blood and Tissue kit, and a library was constructed as described previously (Tsai, S. Q. et al., Nat. Biotechnol. 33: 187-98 (2015), the contents of which are incorporated herein by reference in their entirety). Sequencing was performed on an Illumina MiSeq machine. As a control, GUIDE-Seq oligo was transfected without CRISPR/Cas9 plasmids. GUIDE-Seq data were analyzed with the guideseq pipeline v1.1b4 (github.com/aryeelab/guideseq) using mm10 as the reference mouse genome.
AAV Vector Production
[0149] AAV vectors were produced by the Boston Children's Hospital Viral Core (Boston, MA, USA). Plasmid containing SaCas9-KKH and gRNA 4.2 was sequenced before packaging (MGH DNA Core, complete plasmid sequencing) into AAV2/Anc8028. Vector titer was 4.8×10.sup.14 gc/ml as determined by qPCR specific for the inverted terminal repeat of the virus.
Inner Ear Injections
[0150] Inner ears of Tmc1Bth/WT or Tmc1WT//WT mouse pups were injected at P1 with 1 μl of Anc80-AAV-CMV-SaCas9-KKH-U6-gRNA-4.2 virus at a rate of 60 nl/min. Pups were anesthetized using hypothermia exposure in ice water for 2-3 min. Upon anesthesia, a post-auricular incision was made to expose the otic bulla and visualize the cochlea. Injections were made manually with a glass micropipette. After injection, a suture was used to close the skin cut. Then, the injected mice were placed on a 42° C. heating pad for recovery. Pups were returned to the mother after they fully recovered within ˜10 min. Standard post-operative care was applied after surgery. Sample sizes for in vivo studies were determined on a continuing basis to optimize the sample size and decrease the variance. At P5 to P7, organs of Corti were excised from injected ears. Organ of Corti tissues were incubated at 37° C., 5% CO.sub.2 for 8-10 days, the tectorial membrane was removed immediately before electrophysiology recording.
Hair Cell Electrophysiology
[0151] Mechanotransduction currents were recorded from cochlear IHCs and OHCs at P14-16. Organ of Corti tissues were bathed in external solution containing: 140 mM NaCl, 5.8 mM KCl, 0.7 mM NaH.sub.2PO.sub.4, 10 mM HEPES, 1.3 mM CaCl.sub.2, 0.9 mM MgCl.sub.2, 5.6 mM glucose, and vitamins and essential amino acids (Thermo Fisher Scientific, Waltham, Mass.), adjusted to pH 7.4 with NaOH, ˜310 mmol/kg. Recording electrodes were pulled from R6 capillary glass (King Precision Glass). The intracellular solution contained: 140 mM CsCl, 5 mM EGTA, 5 mM HEPES, 2.5 mM Na.sub.2-ATP, 0.1 mM CaCl.sub.2, and 3.5 mM MgCl.sub.2, and was adjusted to pH 7.4 with CsOH, ˜285 mmol/kg. Mechanotransduction currents were recorded under whole-cell voltage-clamp configuration using an Axopatch 200B (Molecular Devices) amplifier. Cells were held at −80 mV for all electrophysiology recordings. Data were low-pass filtered at 5 kHz (Bessel filter), then sampled at 20 kHz with a 16-bit acquisition board (Digidata 1440A). Data were corrected for a −4 mV liquid junction potential in standard extracellular solutions. Cochlea IHC and OHC bundles were deflected using stiff glass probes mounted on a PICMA chip piezo actuator (Physik Instruments) driven by an LPZT amplifier (Physik Instruments) and filtered with an 8-pole Bessel filter at 40 kHz to eliminate residual pipette resonance. Fire-polished stimulus pipettes with 3-5 μm tip diameter were designed to fit into the concave aspect of hair cell bundle as previously described (Stauffer and Holt, 2007). Hair bundle deflections were monitored using a C2400 CCD camera (Hamamatsu, Japan).
Hearing Tests
[0152] ABR and DPOAE measurements were recorded using the EPL Acoustic system (Massachusetts Eye and Ear, Boston). Stimuli were generated with 24-bit digital I-O cards (National Instruments PXI-4461) in a PXI-1042Q chassis, amplified by a SA-1 speaker driver (Tucker-Davis Technologies, Inc.), and delivered from two electrostatic drivers (CUI CDMG15008-03A) in a custom acoustic system. An electret microphone (Knowles FG-23329-P07) at the end of a small probe tube was used to monitor ear-canal sound pressure. ABRs and DPOAEs were recorded from mice during the same session. Mice were anesthetized with intraperitoneal injection of xylazine (5-10 mg/kg) and ketamine (60-100 mg/kg), and the base of the pinna was trimmed away to expose the ear canal. Three subcutaneous needle electrodes were inserted into the skin, including a) dorsally between the two ears (reference electrode); b) behind the left pinna (recording electrode); and c) dorsally at the rump of the animal (ground electrode). Additional aliquots of ketamine (60-100 mg/kg i.p.) were given throughout the session to maintain anesthesia if needed. DPOAEs were recorded first. F1 and f2 primary tones (f2/f1=1.2) were presented with f2 varied between 5.6 and 32.0 kHz in half-octave steps and L1−L2=10 dB SPL. At each f2, L2 was varied between 10 and 80 dB in 10 dB increments. DPOAE threshold was defined from the average spectra as the L2-level eliciting a DPOAE of magnitude 5 dB above the noise floor. The mean noise floor level was under 0 dB across all frequencies. ABR recordings were then recorded, with stimuli of broadband “click” tones as well as the pure tones between 5.6 and 32.0 kHz in half-octave steps, all presented as 5 ms tone pips. The responses were amplified (10,000 times), filtered (0.1-3 kHz), and averaged with an analog-to-digital board in a PC based data-acquisition system (EPL, Cochlear function test suite, MEE, Boston). Across various trials, the sound level was raised in 5 to 10 dB steps from 0 to 110 dB sound pressure level (decibels SPL). At each level, 512 responses were averaged (with stimulus polarity alternated) after “artifact rejection”. Threshold was determined by visual inspection of the appearance of Peak 1 relative to background noise. Data were analyzed and plotted using Origin-2015 (OriginLab Corporation, MA). Thresholds averages±standard deviations are presented unless otherwise stated. The majority of these experiments were not performed under blind conditions.
Confocal Microscopy
[0153] The temporal bones of 24-week-old adult mice were harvested, cleaned, and placed in 4% PFA for 1 hour, followed by decalcification for 24 to 36 h with 120 mM EDTA (pH=7.4). The sensory epithelium was then dissected and remained in PBS until staining. Tissues were permeabilized with 0.01% Triton X-100 for one hour, blocked with 2.5% NDS and 2.5% BSA in 0.01% Triton X-100 for one hour, and then incubated with anti-MYO7A primary antibody (Proteus Biosciences) overnight (1:500 dilution). Tissues were then washed and counterstained with phalloidin for 2-3 hours. Images were acquired on a Zeiss LSM 800 laser confocal microscope. Full cochlear maps were reconstructed in Adobe Photoshop and tonotopically mapped using an ImageJ plugin.
Scanning Electron Microscopy
[0154] The temporal bones of 24-week-old adult mice were harvested, and cleaned temporal bones were placed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer (EMS) supplemented with 2 mM CaCl.sub.2 for 45 minutes. Whole-mount tissues were dissected in distilled water and then dehydrated over the course of 4 hours to pure ethanol. Tissues were critical-point dried (Autosamdri-815, series A, Tousimis) and mounted on carbon tape attached to SEM specimen stubs. The mounted tissues were coated with 4 nm of platinum (Leica EM ACE600) and then imaged at 5 kV with a scanning electron microscope (Hitachi S-4700 FESEM).
ClinVar Database Analysis
[0155] The ClinVar database from Apr. 25, 2019 was downloaded from ftp.ncbi.nlm.nih.gov/pub/clinvar/vcf_GRCh38/archive_2.0/2019/clinvar 20190325.vcf.gz. The database was filtered for ‘dominant’ diseases, resulting in 17,783 entries. The possibility of generating a PAM site from single nucleotide mutations was analyzed for the SaCas9 (GRRT) and SaCas9-KKH (RRT) recognition motifs. The GRCh38 human reference genome was queried for 7-nt or 5-nt sequences surrounding the site of interest, i.e., 3 (GRRT) or 2 (RRT) nucleotides on either side of the mutation. These sequences were analyzed with a sliding window of length 4 (GRRT) or 3 (RRT) nucleotides. Entries that had a putative PAM site in both the ‘variant’ nucleotide string and the ‘reference’ (i.e. wild-type) string were excluded from further analysis. The same procedure was used for the reverse complement strand. The resulting databases (all dominant entries, dominant entries with PAM site formation for SaCas9, and dominant entries with PAM site formation for SaCas9-KKH recognition) are uploaded as Supplementary Tables 3-5. All ClinVar entries were analyzed without filtering from dominant diseases. This analysis was done, as several dominant variants are not annotated as ‘dominant’ in the ClinVar database.
TMC1 Gene Inactivation in Human Haploid Cells
[0156] A human cell line carrying the equivalent of the Beethoven mutation in the human TMC1 gene (ATG to AAG mutation, encoding the M418K amino acid substitution) was engineered from the HAP1 parental cell line derived from the KBM-7 haploid cells (Horizon Genomics GmbH, Vienna, Austria)29. Briefly, a T to A point mutation was introduced in the exon 16 of the TMC1 gene (ENSG00000165091; genomic location: chr9: 72,791,914) to generate the TCCCTCCTAGGGAAGTTC sequence. For insertion of the mutation by gene editing, the HAP1 cell line was modified with the CRISPR/Cas9 nuclease using two guide RNA sequences (5′-CATCGCTTTGAAATGGCTAC-3′ and 5′-AACCATGTTCATCTACAAGG-3′) and a 1 kb donor template encompassing TMC1 exon 6 and which contained the T to A Beethoven mutation. The genetic identity of the cells was verified by Sanger sequencing of a PCR amplicon.
[0157] The parental and HAP1 cell lines were cultured as monolayer at 37° C. in a humidified atmosphere with 5% CO.sub.2, using IMDM medium plus GlutaMAX (Gibco) supplemented with 10% FBS, 100 U/ml penicilin and 100 μg/ml streptomycin. Cells were passaged every 2-3 days when reaching 70-75% confluency. For transfection, the cells were grown in 6-well plates at a 70% confluency. One day later, cells were transfected with 2.5 μg pDNA using Lipofectamine 3000 (ThermoFisher), following manufacturer's instructions. Two days after transfection, the cells were collected by trypsinization, and the pellet was stored at −20° C. Total genomic DNA was extracted from the cells with the NucleoSpin® Tissue kit (Macherey-Nagel AG, Switzerland). A PCR amplicon was amplified for next-generation sequencing, using the Phusion High-Fidelity DNA Polymerase (ThermoFisher). For TMC1DFNA36 cells, the primers used were 5′-AGCCTAGCTCAGAATCTTCCA-3′ and 5′-AAAATGCGTCCCAGTAGCCA-3′. For TMCWT cells, the 5′-AAAATGCGTCCAAGTAGCCA-3′ was used due to a point mutation in the primer binding region. The PCR protocol was based on manufacturer's instruction, with 35 cycles (5 s at 98° C.; 20 s at 59° C.; 15 s at 72° C.). The PCR product was visualized on a 2% agarose gel and purified with the PCR clean-up and gel extraction kit (Macherey-Nagel AG, Switzerland).
[0158] Next-generation sequencing was performed by the Massachusetts General Hospital DNA Core facility.
[0159] To verify transfection efficacy in each sample, TaqMan real-time PCR was used to quantify the number of plasmid copies of the sequence contained in the AAV inverted terminal repeats using the following primers: forward: 5′-GGA ACC CCT AGT GAT GGA GTT-3′; reverse: 5′-CGG CCT CAG TGA GCG A-3′; probe: 5′-FAM-CAC TCC CTC TCT GCG CGC TCG-BHQ1-3′. The amount of cellular gDNA was quantified using a set of primers specific for the human albumin gene: forward: 5′-TGA AAC ATA CGT TCC CAA AGA GTT T-3′; reverse: 5′-CTC TCC TTC TCA GAA AGT GTG CAT AT-3′; probe: 5′-FAM-TGC TGA AAC ATT CAC CTT CCA TGC AGA-BHQ1-3′. Absolute number of copies were determined according to standards and used to calculate the number of plasmid copies per cell.
Statistical Analysis
[0160] GraphPad Prism 7.0 for Mac OS and OriginPro (2015) were used for statistical analysis. To compare means, an unpaired two tailed t-test (after Shapiro-Wilk normality testing) was used; to compare multiple groups, ANOVA followed by Tukey's post-hoc test (to compare every mean to every other mean) or Dunnett's test (to compare every mean to a control group mean) was used. p values <0.05 were accepted as significant.
Other Embodiments
[0161] From the foregoing description, it will be apparent that variations and modifications may be made to the invention described herein to adopt it to various usages and conditions. Such embodiments are also within the scope of the following claims.
[0162] The recitation of a listing of elements in any definition of a variable herein includes definitions of that variable as any single element or combination (or subcombination) of listed elements. The recitation of an embodiment herein includes that embodiment as any single embodiment or in combination with any other embodiments or portions thereof.
[0163] All patents and publications mentioned in this specification are herein incorporated by reference to the same extent as if each independent patent and publication was specifically and individually indicated to be incorporated by reference.