Gene therapy constructs and methods for treating hearing loss
阅读说明：本技术 用于治疗听力丧失的基因治疗构建体和方法 (Gene therapy constructs and methods for treating hearing loss ) 是由 H·施特克尔 X·Z·刘 陈正一 C·J·阿亚拉 于 2018-03-16 设计创作，主要内容包括：本发明公开了可用于治疗和/或预防由TMPRSS3基因或LOXHD1基因的遗传突变所导致的听力丧失的组合物和方法。本文公开的组合物和方法使用腺相关病毒(AAV)载体基因递送TRMPSS3或LOXHD1至内耳以分别恢复TMPRSS3基因或LOXHD1基因的活性,促进毛细胞存活并恢复患有听力丧失的患者的听力。(Compositions and methods useful for treating and/or preventing hearing loss caused by a genetic mutation in the TMPRSS3 gene or the LOXHD1 gene are disclosed. The compositions and methods disclosed herein use adeno-associated virus (AAV) vector genes to deliver TRMPSS3 or LOXHD1 to the inner ear to restore the activity of the TMPRSS3 gene or LOXHD1 gene, respectively, to promote hair cell survival and restore hearing in patients with hearing loss.)
1. An expression vector comprising:
a nucleic acid sequence selected from the group consisting of SEQ ID NO 1, a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID NO 1, SEQ ID NO 3 or a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID NO 3; and
a promoter operably linked to a nucleic acid sequence.
2. The expression vector of claim 1, wherein the expression vector is an adeno-associated viral vector.
3. The expression vector of claim 2, wherein the adeno-associated viral vector is selected from the group consisting of AAV2, AAV2/Anc80, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or Anc80.
4. The expression vector of claim 1, wherein the promoter is selected from the group consisting of TMPRSS3 promoter, Myo7a promoter, Human Cytomegalovirus (HCMV) promoter, cytomegalovirus/chicken β -actin (CBA) promoter, and Pou4f3 promoter.
5. A pharmaceutical composition for use in a method of treating or preventing hearing loss, the pharmaceutical composition comprising an expression vector comprising a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID No. 1, SEQ ID No. 3, or a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID No. 3; and a promoter operably linked to the nucleic acid sequence.
6. The pharmaceutical composition of claim 5, wherein the expression vector is an adeno-associated viral vector.
7. The pharmaceutical composition of claim 6, wherein the adeno-associated viral vector is selected from the group consisting of AAV2, AAV2/Anc80, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or Anc80.
8. The pharmaceutical composition of claim 5, wherein the promoter is selected from the group consisting of: TMPRSS3 promoter, Myo7a promoter, Human Cytomegalovirus (HCMV) promoter, cytomegalovirus/chicken β -actin (CBA) promoter, and Pou4f3 promoter.
9. A cell transfected with an expression vector comprising a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID No. 1, SEQ ID No. 3, or a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID No. 3; and a promoter operably linked to the nucleic acid sequence.
10. The cell of claim 9, wherein the expression vector is an adeno-associated viral vector.
11. The cell of claim 10, wherein the adeno-associated viral vector is selected from the group consisting of AAV2, AAV2/Anc80, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, and Anc80.
12. The cell of claim 9, wherein the promoter is selected from the group consisting of TMPRSS3 promoter, Myo7a promoter, Human Cytomegalovirus (HCMV) promoter, cytomegalovirus/chicken β -actin (CBA) promoter, and Pou4f3 promoter.
13. The cell of claim 9, wherein the cell is a stem cell.
14. The cell of claim 13, wherein the stem cell is an induced pluripotent stem cell.
15. A method of treating or preventing hearing loss comprising administering to a subject in need thereof an effective amount of an expression vector comprising a nucleic acid sequence selected from the group consisting of SEQ ID No. 1, a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID No. 1, SEQ ID No. 3, or a nucleic acid sequence having at least 90% sequence identity to the nucleic acid of SEQ ID No. 3; and a promoter operably linked to the nucleic acid sequence.
16. The method of claim 15, wherein the expression vector is an adeno-associated viral vector.
17. The method of claim 16, wherein the adeno-associated viral vector is selected from the group consisting of AAV2, AAV2/Anc80, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or Anc80.
18. The method of claim 15, wherein the promoter is selected from the group consisting of TMPRSS3 promoter, Myo7a promoter, Human Cytomegalovirus (HCMV) promoter, cytomegalovirus/chicken β -actin (CBA) promoter, and Pou4f3 promoter.
19. The method of claim 15, wherein the expression vector is administered by injection into the inner ear of the subject.
20. The method of claim 19, wherein the method of injection is selected from the group consisting of a cochlear ostomy, a round window membrane, an endolymphatic sac, a cochlear canal, a ductostomy, a cochlear duct through an endolymphatic sac, or any combination thereof.
21. The method of claim 15, wherein the subject has one or more genetic risk factors associated with hearing loss.
22. The method of claim 21, wherein one of the genetic risk factors is selected from the group consisting of a TMPRSS3 gene mutation or a LOXHD1 gene mutation.
23. The method of claim 21, wherein the subject does not exhibit any clinical manifestations of hearing loss.
24. A transgenic mouse comprising a hearing loss causing mutation selected from the group consisting of a mutation in the human LOXHD1 gene or a mutation in the human TMPRSS3 gene.
Technical Field
Compositions and methods for treating and/or preventing hearing loss and deafness caused by genetic mutations in the TMPRSS3 gene or LOXHD1 gene are disclosed.
Background
Hearing loss is the most common sensory defect in humans. According to an estimate of the number of disabling hearing losses released by the World Health Organization (WHO) in 2012, 3.6 million people worldwide have disabling hearing losses (3.28 million adults and 3200 million children), 89% (or 3.22 million) of those people live in low-income countries (WHO's global estimate of prevalence of hearing losses; mortalities and Burden of Diseases and preservation of Blindness and loss, WHO 2012).
There is currently no approved therapeutic agent for the prevention or treatment of hearing loss or deafness. The current treatment option for those patients with disabling hearing loss is cochlear implant. Cochlear implants are a common procedure with significant medical costs, with a lifetime cost of over $ 1,000,000 per patient (Mohr PE et al (2000); the scientific models of surgery to contaminated surgery in the United States; int. JTechnol Association Health Care; 16(4): 1120-35).
The demand for cochlear implant currently exceeds the supply. The productivity of cochlear implant devices is 50,000 per year. According to the current birth rate and the incidence and prevalence of disabling hearing loss in newborns, 134,000 cochlear implants are required each year to provide 1 cochlear implant for each child suffering from the disease. This number increases if patients requiring bilateral (2) cochlear implants are involved.
The lifetime cost of cochlear implants is high for most people, particularly those living outside of the developed countries where most people with disabling hearing loss live. There is a need to provide an economical treatment option to replace cochlear implants, particularly for those living outside of the developed world.
More than 50% of presbycusis is inherited, i.e. hereditary diseases (disease control and prevention center-hearing loss genetics). Hereditary hearing loss and deafness can be conductive, sensorineural, or both; syndromic (with malformation of the outer ear or other organs or medical problems involving other organ systems) or non-syndromic (without obvious abnormalities of the outer ear or any associated medical problems); and pre (pre language development) or post (post language development) (Richard JH Smith, MD, a Eliot Shearer, Michael S Hildebrand, PhD, and Guy VanCamp, PhD, defanessand heredity heart searching love Overview, GeneReviews was originally issued: 14/2/1999, final revision: different gene loci for non-syndromic DeaFNess were designated DFN (DeaFNess), gene loci were named based on genetic means: DFNA (autosomal dominant), DFNB (autosomal recessive) and DFNX (X-linked). This change reflects both genetic and environmental influences and the interaction between environmental triggers and the genetic predisposition of an individual.
Sensorineural hearing loss (SNHL) is the most common neurodegenerative disease in humans, and there is currently no approved pharmaceutical intervention. SNHL may be caused by genetic diseases, but also by, for example, acoustic trauma and ototoxic injury. Genetic diagnosis has demonstrated that at least 100 genes cause non-syndromic SNHL. Recent research advances in genetics and gene therapy techniques have shown that many occult deafness can be rescued by gene therapy (Akil et al, 2012; Askew et al, 2015). Long-term gene delivery to the inner ear has been achieved using adeno-associated viral vectors (AAV) (Shu, Tao, Wang, et al, 2016). Despite these observations were first described several years ago, they have not been transformed into humans for clinical trials. The ideal disease target for transformation studies in this field is recessive inherited hearing loss, which affects a defined set of cells in the inner ear and occurs after language development. The prevalence of the mutation is another consideration.
As described herein, by carefully assessing the incidence of common underlying causes of hearing loss and taking into account the size of the genes, it is possible to develop a gene therapy approach for a realistic and fairly common patient population. For example, although less common than other mutant genes, TMPRSS3 is a fairly common gene that can cause hearing loss with sufficient severity to determine the need for cochlear implants. Furthermore, patients with the TMPRSS3 mutation may not respond to artificial cochlear implants and patients with other mutations (Shearer et al, 2017). This provides a therapeutic opportunity to target the TMPRSS3 gene or other genes such as LOXHD1 as a stand-alone therapeutic agent or in combination with other therapeutic agents and/or cochlear implants to improve the implantation effects of this disorder. Table 1 (adapted from (Miyagawa, Nishio, & Usami,2016)) indicates that TMPRSS3 mutation is probably the most common cause of post-verbal recessive hearing loss, its distribution within the cochlea is rather limited, and due to the size of the genes, it can be attempted to build it into existing AAV vectors.
Table 1: incidence of different mutations in 176 adult cochlear implant patients
The human transmembrane protease, serine 3(TMPRSS 3; also known as DFNB10, DFNB8, ECHOS1, TADG 12; Acc: HGNC:11877) was identified by its association with congenital (present at birth) and childhood onset autosomal recessive deafness. Mutations in the TMPRSS3 gene were associated with autosomal recessive non-syndromic hearing impairment types DFNB8 and 10. TMPRSS3 is a 1646 base pair gene encoding a serine protease and, in association with DFNA8/10, may account for 1-5% of hearing loss patients undergoing cochlear implant implantation (weegernk et al, 2011). The broad spectrum of hearing phenotypes caused by the appearance of this gene loss of function depends on the site of the mutation. Both congenital and adult-onset progressive hearing loss are associated with loss of this gene.
While the onset of hearing loss with DFNB10 was pre-prandial (congenital), the onset of hearing loss with DFNB8 was post-prandial (age 10-12 years). This phenotypic difference reflects a difference in genotype. The variant caused by DFNB8 was a splice site variant, indicating that inefficient splicing is associated with a reduced amount of normal protein that is sufficient to prevent pre-speech deafness but insufficient to prevent eventual hearing loss. (see Richard JH Smith, MD, et al (2014.) Genes Known ToCause automated non-reactive searching impact: afterness and heredity Heart Overview; GeneReviews).
The TMPRSS3 mutation on chromosome 21 known to cause hearing loss is described in table 2.
Lipoxygenase homeodomain 1 genes (LOXHD 1; also known as LH2D1, DFNB77, FLJ 32670; OMIM: 613072; Acc: HGNC: 26521) encode highly conserved proteins consisting entirely of the PLAT (polycystic protein/lipoxygenase/alpha-toxin) domain, thought to be involved in targeting proteins to the plasma membrane. Mouse studies have shown that the gene is expressed in mechanosensory hair cells of the inner ear, and that mutations in the gene lead to hearing defects, indicating that the gene is critical to the function of normal hair cells. Screening for human familial isolated deafness identified a mutation in this gene that resulted in DFNB77, a progressive autosomal-recessive non-syndromic hearing loss (ARNSHL) 77. Alternatively, different isoforms encoded by splice transcript variants of the gene have been noted.
Clinical features of LOXHD 1:
autosomal recessive inheritance
Hearing loss, sensorineural, bilateral (low frequency mild hearing loss)
Congenital attacks leading to cochlear implant between 7-10 years of age in the family of German Uyghur
In the near-relative iran family during adulthood, age episodes of 7-8 years develop to moderate to severe loss with moderate frequency and high frequency
Evidence suggests that autosomal recessive inherited non-syndromic hearing loss-77 (DFNB77) is caused by a homozygous mutation in the LOXHD1 gene (613072) on chromosome 18q 21.
Expression of Loxhd1 was detected by in situ hybridization in the inner ear of mice developing at embryonic stages 13.5 and 16 days, but not in any other tissue. On postnatal day 4, expression was detected in the cochlea and vestibular hair cells, with the highest concentration in the nucleus. Loxhd1 gradually localizes to the cytoplasm, and in adults, Loxhd1 is expressed in hair cells along the length of the cilia.
Using N-ethyl-N-nitrosourea (ENU) mutagenesis screening, Grillet et al (2009) developed a "samba" murine line, hearing impaired at 3 weeks of age and deafness at 8 weeks of age. Homozygous samba mice showed no other neurological or vestibular abnormalities, and heterozygous samba mice appeared completely normal. The stereo cell development of homozygous samba mice was not affected, but hair cell function was disturbed and the hair cells eventually regressed.
Grilett et al (2009) found mutations in samba mouse Loxhd1 gene, destabilizing the β -sandwich structure of PLAT domain 10. The mutation did not alter mRNA or protein stability or localization of Loxhd1 protein along the length of the cilia. However, at postnatal day 21, some hair cells showed morphological defects with fused cilia and membrane folds at the apical cell surface. Significant degenerative changes including hair cell loss and loss of spiral ganglion neurons occurred at day 90 after birth. The degeneration of the helical ganglion neurons hypothesized by Grillet et al (2009) may be secondary to perturbations in the function and maintenance of hair cells.
The LOXHD1 mutation on chromosome 18 known to cause hearing loss is described in table 3.
U.S. publication No.2013/0095071, incorporated herein by reference in its entirety, describes a gene therapy approach to restore age-related hearing loss using a mutant tyrosine adeno-associated viral vector to deliver X-linked apoptosis-inhibiting protein (XIAP) to the round window membrane of the inner ear. However, this disclosure does not contemplate the delivery of a nucleic acid sequence encoding a functional TMPRSS3 or LOXHD1 to prevent or delay the onset or recovery of hearing loss or deafness caused by a genetic mutation in the TMPRSS3 or LOXHD1 genes, as disclosed herein.
Furthermore, one important drawback in the prior art for developing clinical gene therapy for hearing impairment is the lack of animal models reflecting hearing loss in humans. Many mouse models of inherited hearing loss available for adult onset in humans exist that complicate delivery studies with congenital hearing loss. Models with few hereditary hearing loss episodes after hearing development. Delivery of the vector in neonatal mice results in a different transfection pattern than in adult mice (Shu, Tao, Li et al, 2016). There is a need for new animal models that can be used to assess hearing rescue using different vector systems and gene targets.
There is currently no approved treatment for the prevention or treatment of hearing loss or deafness, and there is a lack of effective preclinical animal models for testing such treatments. The present invention describes compositions and methods for delivering viral vector genes of TMPRSS3 or LOXHD1 into the inner ear to restore the activity of mutated TMPRSS3 or LOXHD1 genes, to promote hair cell survival and restore hearing in patients with hearing loss or deafness, and cell-based and animal-based models for testing these compositions and methods.
Disclosure of Invention
Disclosed herein are nucleic acids comprising SEQ ID NO:1 or SEQ ID NO: 2, or an expression vector corresponding to the nucleic acid sequence of SEQ id no:1 or SEQ ID NO: 2, wherein the nucleic acid sequence is operably linked to a promoter. Also disclosed herein are pharmaceutical compositions for use in a method of treating or preventing hearing loss, the pharmaceutical compositions comprising a peptide having SEQ ID NO:1 or SEQ ID NO: 2, or an expression vector corresponding to the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO: 2, wherein the nucleic acid sequence is operably linked to a promoter. In some embodiments, the nucleic acid sequence is identical to SEQ ID NO:1 or SEQ ID NO: 2 has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the expression vector is selected from an adeno-associated viral vector, an adenoviral vector, a herpes simplex viral vector, a vaccinia viral vector, a helper-dependent adenoviral vector, or a lentiviral vector. In some embodiments, the vector is an adeno-associated viral vector selected from AAV2, AAV2/Anc80, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or Anc80. In some embodiments, the adeno-associated viral vector is AAV2 or Anc80. In some embodiments, the promoter is selected from any hair cell promoter that drives expression of an operably linked nucleic acid that develops early and maintains expression throughout life, e.g., TMPRSS3 promoter, Human Cytomegalovirus (HCMV) promoter, cytomegalovirus/chicken β -actin (CBA) promoter, Myo7a promoter, or Pou4f3 promoter.
Disclosed herein are cells having an expression vector comprising SEQ ID NO:1 or SEQ ID NO: 2, or a nucleotide sequence identical to SEQ ID NO:1 or SEQ ID NO: 2, wherein the nucleic acid sequence is operably linked to a promoter. In some embodiments, the nucleic acid sequence is identical to SEQ ID NO:1 or SEQ ID NO: 2 has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the cell is a stem cell. In some embodiments, the stem cell is an induced pluripotent stem cell.
Disclosed herein are methods for treating or preventing hearing loss comprising administering to a subject in need thereof an effective amount of an expression vector comprising SEQ ID NO:1 or SEQ ID NO: 2, or a nucleotide sequence identical to SEQ id no:1 or SEQ ID NO: 2, wherein the nucleic acid sequence is operably linked to a promoter. In some embodiments, the nucleic acid sequence is identical to SEQ ID NO:1 or SEQ ID NO: 2 has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% sequence identity. In some embodiments, the expression vector is selected from an adeno-associated viral vector, an adenoviral vector, a herpes simplex viral vector, a vaccinia viral vector, a helper-dependent adenoviral vector, or a lentiviral vector. In some embodiments, the vector is an adeno-associated viral vector selected from AAV2, AAV2/Anc80, AAV5, AAV6, AAV6.2, AAV7, AAV8, AAV9, AAVrh8, AAVrh10, AAVrh39, AAVrh43, AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, or Anc80. In some embodiments, the adeno-associated viral vector is AAV2 or Anc80. In some embodiments, the promoter is selected from any hair cell promoter that drives expression of an operably linked nucleic acid that develops early and maintains expression throughout life, e.g., TMPRSS3 promoter, Human Cytomegalovirus (HCMV) promoter, cytomegalovirus/chicken β -actin (CBA) promoter, Myo7a promoter, or Pou4f3 promoter. In some embodiments, the expression vector is administered into the inner ear of the subject, e.g., by injection. In some embodiments, the method of Delivery is selected from the group consisting of a cochlear ostomy, a round window membrane, a ductal ostomy, or any combination thereof (see, Erin E. LearySwan, et al (2008) Inner Ear Drug Delivery for Audio applications. adv Drug Delivery Rev.60 (15): 1583-. In some embodiments, the expression vector is delivered into the cochlear duct by an endolymphatic sac (Colletti V, et al (2010) event of gallium distribution from the endogenous pharmaceutical sac to the endogenous pharmaceutical compositions of the human endocrine. Audio neurool.15 (6): 353-63; Marco Mandal, MD, et al (2010) induced endogenous pharmaceutical flow from the endogenous pharmaceutical sac to the cochlea in M ni's disease. aerobic-blood and feed nerve 143, 673-679; Yasoba T, et al (1999) enzyme vector expression vector therapeutic in therapeutic human tissue, 769.10.9). In some embodiments, the subject has one or more genetic risk factors associated with hearing loss. In some embodiments, one of the genetic risk factors is a mutation in the TMPRSS3 gene. In some embodiments, the mutation in the TMPRSS3 gene is selected from any one or more TMPRSS3 mutations known to cause hearing loss (see, e.g., table 2). In some embodiments, one of the genetic risk factors is a mutation in the LOXHD1 gene. In some embodiments, the mutation in the LOXHD1 gene is selected from any one or more LOXHD1 mutations known to cause hearing loss (see, e.g., table 3). In some embodiments, the subject does not exhibit any clinical signs of hearing loss.
In some embodiments, the expression vectors described herein are administered as a combination therapy with one or more expression vectors comprising additional nucleic acid sequences and/or with one or more additional active agents to treat hearing loss. For example, the combination therapy may include a polypeptide having SEQ ID NO:1 and a first expression vector having the nucleic acid sequence of SEQ ID NO: 2, wherein both expression vectors are administered to the subject as part of a combination therapy to treat hearing loss.
Disclosed herein are transgenic mice having a mutant human TMPRSS3 gene selected from any one or more TMPRSS3 mutations known to cause hearing loss (see, e.g., table 2). Disclosed herein are transgenic mice having a human LOXHD1 gene with a mutation selected from any one or more LOXHD1 mutations known to cause hearing loss (see, e.g., table 3).
Drawings
Figure 1 shows a cDNA sequence encoding wild-type human TMPRSS3 (GenBank accession No. BC 074847.2);
FIG. 2 shows the amino acid sequence of wild-type human TMPRSS3 encoded by the cDNA in FIG. 1;
figure 3 shows a cDNA sequence encoding wild-type human LOXHD1 (GenBank accession No. AK 057232.1);
FIG. 4 shows the wild type human LOXHD1 amino acid sequence encoded by the cDNA of FIG. 3;
FIG. 5 shows
Detailed Description
The subject matter which is particularly pointed out and distinctly claimed in the claims at the conclusion of the specification is regarded as the invention. The foregoing and other objects, features, and advantages of the invention will be apparent from the following detailed description of the invention, taken in conjunction with the accompanying drawings.
As used herein, the terms "treatment", "treating" and "treatment" include various activities that are intended to achieve a desired change in clinical outcome. For example, as used herein, the term "treatment" includes any activity intended to achieve or indeed achieve a detectable improvement in one or more clinical indicators or symptoms of hearing loss as described herein.
Hearing loss due to TMPRSS3 mutation or LOXHD1 mutation commonly occurs in two populations: (i) the subjects were born with a congenital population of hearing loss and (ii) the subjects were born with no measurable symptoms of hearing loss, but over a period of time exhibited a progressive population of progressive hearing loss. Thus, in certain instances, a subject may have a mutation in the TMPRSS3 gene or LOXHD1 gene (e.g., as detected in a genetic diagnostic test) but have not yet exhibited a clinical marker or symptom of hearing loss, thus providing a window during which therapeutic intervention may begin. Thus, in some embodiments, the present invention provides methods of therapeutic intervention during gradual hearing loss. The method of the invention may be started before this time period. The methods of treating hearing loss provided by the present invention include, but are not limited to, methods for preventing or delaying the onset of hearing loss or the progression of a clinical indicator or symptom of hearing loss.
As used herein, the term "hearing loss" is used to describe a reduced ability to hear sounds, and includes deafness and complete inability to hear sounds.
As used herein, the term "effective amount" or "therapeutically effective amount" refers to an amount of an active agent as described herein that is sufficient to achieve or contribute to achieving one or more desired clinical results, such as those described in the "treatment" description above. The appropriate "effective" amount in any individual case can be determined using standard techniques known in the art, such as dose escalation studies.
The term "active agent" as used herein refers to a molecule (e.g., an AAV vector described herein) that is intended for use in the compositions and methods described herein, and is intended to be biologically active, e.g., for the purpose of treating hearing loss.
The term "pharmaceutical composition" as used herein refers to a composition comprising at least one active agent or two or more active agents as described herein, and one or more other components suitable for drug delivery, such as carriers, stabilizers, diluents, dispersants, suspending agents, thickeners, excipients, and the like.
The terms "subject" or "patient" as used interchangeably herein include mammals, including, but not limited to, humans, non-human primates, rodents (e.g., rats, mice and guinea pigs), and the like. In some embodiments of the invention, the subject is a human.
The present invention can calculate the dosage of the active agent based on studies in humans or other mammals to determine the efficacy and/or effective amount of the active agent. The dosage and frequency or timing of administration can be determined by methods known in the art and can depend on factors such as the pharmaceutical form of the active agent, the route of administration, whether only one active agent or multiple active agents are used (e.g., when such an agent is used in combination with a second active agent, the required dosage of the first active agent can be lower), and patient characteristics, including age, weight, or the presence of any relevant factors that affect the medical condition of drug metabolism.
In one embodiment, a single dose may be administered. In another embodiment, multiple doses may be administered over a period of time, e.g., at specified intervals, e.g., four times a day, two times a day, once a week, once a month, etc.
Clinical features of hearing loss. Hereditary hearing loss and deafness can be conductive, sensorineural, or a combination of both; syndromic (with malformation of the outer ear or other organs or medical problems involving other organ systems) or non-syndromic (without obvious abnormalities of the outer ear or any associated medical problems); pre (pre language development) or post (post language development) (Richard JH Smith, MD et al (2104) Defafess and heredity HearingLoss Overview, GeneReviews).
Diagnosis/detection. Hereditary hearing loss must be distinguished from the acquired (non-hereditary) cause of hearing loss. Hereditary hearing loss is diagnosed by otoscientific, audiological and physical examination, family history, auxiliary tests (e.g., CT examination of the temporal bone), and molecular genetic tests. Molecular genetic testing may play an important role in the diagnosis and genetic counseling of many types of syndromic and non-syndromic deafness.
Selection test for detecting hearing loss:
1. distortion products otoacoustic emissions (DPOAE). Distortion product otoacoustic emission (DPOAE) refers to the response that occurs when the cochlea is simultaneously stimulated by two pure tone frequencies with a frequency ratio between 1.1 and 1.3. Recent studies on the mechanism of DPOAE generation have emphasized the presence of important components in two DPOAE responses, one generated by intermodulation "distortion" and one generated by "reflections".
The incidence of DPOAE in normal adult ears is 100%. The responses from the left and right ears are usually correlated (i.e., they are very similar). For normal subjects, women have higher DPOAE amplitudes. Aging processes (aging processes) affect the DPOAE response by reducing the DPOAE amplitude and narrowing the DPOAE response spectrum (i.e., the higher frequency response is gradually reduced). DPOAEs can also be recorded using other animal species used in clinical studies, such as lizards, mice, rats, guinea pigs, chinchillas, chickens, dogs, and monkeys. (Otoacoustic emission website)).
2. Auditory Brainstem Response (ABR). The hearing brainstem response (ABR) test provides information about the inner ear (cochlea) and the hearing brain pathways. This test is sometimes also referred to as hearing evoked potential (AEP). The test may be used to detect children or other persons who have had difficulty with routine behavioral methods of hearing screening. ABRs are also useful in humans who indicate signs, symptoms or disease of certain hearing loss in the brain or brain pathways. The test is for humans and animals. ABR is performed by electrodes affixed to the head-similar to electrodes placed around the heart during electrocardiographic operation-recording brain wave activity in response to sound. The test is performed while the tested person is resting or sleeping quietly. No response is necessary. ABRs may also be used as screening tests for neonatal hearing screening programs. When used as a screening test, only one intensity or loudness level is checked, with or without the infant passing the screening. (American Speech Hearing Association Website).
Clinical manifestations of hearing loss. The type and onset of hearing loss are described:
type (B)
Conductive hearing loss is caused by abnormalities in the outer and/or middle ear ossicles.
Sensorineural hearing loss is caused by dysfunction of the inner ear structure (i.e., the cochlea).
Mixed hearing loss is a combination of conductive and sensorineural hearing loss.
Central auditory dysfunction is caused by injury or dysfunction at the level of the eighth cranial nerve, auditory brainstem or cerebral cortex.
Onset of disease
Pre-speech hearing loss is present before language development. All congenital (present at birth) hearing losses are pre-lingual, but not all pre-lingual hearing losses are congenital.
Post-lingual hearing loss occurs after normal language development.
(Richard JH Smith, MD, etc.;Deafness and Hereditary Hearing Loss Overview(ii) a GeneReviews; initially issuing: 14 days 2 month 1999; and finally revising: 2014 1 month 9 days)
Severity of hearing loss. Hearing is measured in decibels (dB). The threshold or 0dB marker for each frequency refers to the level at which a normal young person perceives a tone burst 50% of the time. If one's threshold is within 15dB of the normal threshold, then hearing is considered normal. The severity of hearing loss is shown in table 4.
Percentage of hearing impairment. To calculate the percent hearing impairment, 25dB was subtracted from the pure tone averages at 500Hz, 1000Hz, 2000Hz, 3000 Hz. The result is multiplied by 1.5 to obtain a specific level of the ear. By weighting the better ear five times the worse earTo determine the damage (see table 5). Since spoken speech is about 50-60dB HL (hearing level), calculating functional impairment based on pure tone averages can be misleading. For example, 45-dB hearing loss is far more than 30% in functional significance. Different grade scales are suitable for young children, and even limited hearing loss has a great influence on language developmentNorthern&Downs 2002]。
The frequency of hearing loss. The frequency of hearing loss was specified as:
low (<500Hz)
Middle (501 + 2000Hz)
High (>2000Hz)
And (4) gene therapy. Gene therapy refers to the introduction of DNA into a patient to treat a genetic disease. The new DNA usually contains a functional gene to correct the effects of pathogenic mutations in existing genes. Gene transfer for experimental or therapeutic purposes relies on a vector or vector system to transfer genetic information into a target cell. The vector or vector system is considered to be a major determinant of the efficiency, specificity, host response, pharmacology and longevity of the gene transfer reaction. Currently, the most efficient and effective method of achieving gene transfer is through the use of vectors or vector systems based on viruses that have been made replication-defective (PCT publication No. WO 2015/054653; Methods of differentiating antibiotic Virus Sequences and uses thereof).
And (3) a carrier. To date, adenovirus, adeno-associated virus, herpes simplex virus, vaccinia virus, retrovirus, helper-dependent adenovirus, and lentivirus have all been tested for cochlear gene delivery. Among the most potent are adeno-associated viruses (AAV): it is non-replicating, can efficiently transfer the transgene to the inner ear, and does not cause ototoxicity. In particular, AAV can efficiently transfect inner hair cells, a key feature if one wishes to correct genetic defects by hair cell-specific mutations. To date, many different AAV subtypes have been successfully used for cochlear gene delivery, demonstrating little if any damage to the organ of Corti. Recent reports of
The size of the target gene that can be corrected is limited by the carrying capacity of AAV (Wu, Yang, & Colosi, 2010). For translation purposes, this limits potential targets for those genetic disorders that are recessive hearing loss caused by relatively small genes (<4.6 kB). As an initial approach to developing gene therapy drugs, target gene mutations should be relatively common and hearing loss occurs after language development. Matching these features identifies patients with progressive hearing loss, providing an opportunity for intervention and cessation or possible reversal of their progressive loss. Inner ear gene therapy trials have been initiated in humans with acquired deafness and therefore many safety and delivery issues are being addressed (clinical trial identification number NCT 02132130). Increased availability and accuracy of gene testing will enable identification of patients who would benefit from these types of interventions (prepacido et al, 2005).
Mouse model for gene therapy: several mouse models of different recessive hereditary deafness have been rescued by gene therapy. Examples include correction of hearing loss caused by VGLUT3, TLC1, rotanin,
TMPRSS3 mutation induces hearing loss: hearing loss associated with the TMPRSS3(DFNA8/10) mutation can occur in a variety of different phenotypes. Congenital profound hearing loss has been described as well as adult onset progressive hearing loss (weegernk et al, 2011). At present, the mechanism of Tmprss3 dysfunction is not clear. Two mouse models have been developed to determine hearing loss at birth, the other being the onset of hearing loss at a later time point but still before hearing and mouse maturation. Fasquerel et al developed ethyl-nitrosourea-induced mutant mice carrying a protein-truncated nonsense mutation at
Tmprss3 genotype-phenotype studies indicate a number of different types of hearing loss, ranging from severe congenital to onset progressive hearing loss in adults (Chung et al, 2014; Gao et al, 2017; Weegrink et al, 2011). Studies have shown that hearing loss due to the Tmprss3 mutation may account for 2 to 5% of adult cochlear implant patients (Jolly et al, 2012; Miyagawa, Nishio, & Usami, 2016; Sloan-Heggen et al, 2016). Many patients with these mutations have significant residual hearing. This would make it an attractive target for potential rescue treatment as it would have substrates for cells that could be treated. There are several divergent studies on the success of cochlear implant implantation in patients with mutations. At least some types of hearing loss caused by Tmprss3 loss may be unsuitable for cochlear implant implantation as compared to other types of hereditary hearing loss (Shearer et al, 2017). This may be associated with the ability of this gene to be expressed in hair cells and up to 50% of spiral ganglion cells (see FIG. 5). These differences need to be taken into account when selecting vector system delivery. The vector will be tested by using strong hair cell tropism and binding hair cell and spiral ganglion cell tropism. Inner ear delivery in neonates and adults also showed differences in vector tropism (Shu, Tao, Li et al, 2016; Shu, Tao, Wang et al, 2016 a). Since the target clinical population is humans with a mature hearing system, a mouse model is disclosed herein that has episodes of hearing loss after hearing maturation that can be used as a disease progression model (see example 1) and a delivery model for adult cochlear rescue therapy (see example 2).
AAV serotype for delivery to the inner ear: a number of different AAV serotypes have been demonstrated for transfection of inner ear tissue (Etc., 2017; shu, Tao, Wang, et al, 2016 b; the total of Xia, Yin,&wang, 2012). The distribution of the vector delivered transgenes was significantly different in neonates and adults, and in addition, there was a difference in perilymph versus endolymph delivery when hair cell transfection was evaluated (Kilparick et al, 2011; Wang et al, 2013). In a mouse model, delivery of the vector to the endolymph would result in improved hair cell transfection but would result in residual hearing loss, making it difficult to mimic progressive hearing loss. Possibly, in larger animals or in humans, the vector can be delivered into the endolymphatic sac without causing hearing loss, thereby improving the transfection efficiency (Colletti et al, 2010). For the purposes of the planning study, delivery to perilymph would enable mouse hearingAnd (4) evaluating. Delivery of AAV2 to hair cells and spiral ganglion cells in adult animals has been shown (Tao et al, 2017). Another advantage of AAV2 is that it has retained widely documented and safe data in human gene therapy clinical trials (Santiago-Ortiz)&Schafer, 2016). Recently, various studies have shown that synthetic AAV (AAV2/Anc80) can improve delivery to hair cells, but spiral ganglion delivery comparable to native AAV2 may not be achieved (Landegger et al, 2017; Suzuki, Hashimoto, Xiao, Vandenberghe,&liberman, 2017). The present invention provides a mouse model with adult onset hearing loss and compares whether Tmprss3 gene therapy of AAV2 or AAV2/Anc80 can better salvage the function of hearing and spiral ganglia.
Induced pluripotent stem cells (ipscs). Induced pluripotent stem cells (IPS or IPSC) refer to stem cells produced from adult cells such as skin, liver, stomach or other mature cells, which are transformed into cells with all the characteristics of embryonic stem cells by introducing genes that re-encode the cells. The term pluripotent means the ability of a cell to produce multiple cell types, including all three embryonic lineages that form body organs, nervous system, skin, muscle and bone.
CRISPR/Cas9 gene editing. The methods described herein also contemplate using CRISPR/Cas9 genome editing to remediate hearing by editing mutations in the TMPRSS3 or LOXHD1 genes. This technique has been used in two mouse models of hereditary hearing loss (Tmcl and Pmca2) and successfully rescues hearing (Askew, C et al (2015) Tmc gene therapy hearing aid function in following mice. Sci Transl Med.7(295):295ral 08). Although this technique is primarily used for dominant hearing loss, it can be used to restore hearing against recessive hearing loss in a Tmprss3 knock-in mouse model and ultimately for hearing loss caused by mutations in the Tmprss3 gene or LOXHD1 gene. The use of CRISPR/Cas9 gene editing to repair defective gene sequences is further described in PCT publication No. WO2016/069910, PCT publication No. WO2015/048577, and U.S. application publication No. 2015/0291966, each of which is incorporated herein by reference in its entirety.
- 上一篇：一种医用注射器针头装配设备
- 下一篇：抗沉淀的小分子药物制剂