2000 — 2002 |
Brigande, John Vincent |
F32Activity Code Description: To provide postdoctoral research training to individuals to broaden their scientific background and extend their potential for research in specified health-related areas. |
Models of Inner Ear Morphogenesis @ Purdue University West Lafayette
The vertebrate inner ear is a structurally complex organ consisting of fluid-filled ducts and toroidal canals protruding from a central vestibule. Congenital defects of inner ear morphogenesis can lead to deafness and balance disorders. This proposal aims to gain insight into the origins of congenital ear malformations by probing the molecular-genetic basis of otic vesicle morphogenesis. Our hypothesis is that gene expression domains subdivide the vertebrate otocyst into compartments that interact at boundaries to specify cell fate and pattern formation. We will fate map the chick and mouse otic cup by iontophoretic injection of fluorescent tracer dye. The fate maps will provide essential mechanistic information regarding the cellular movements responsible for conversion of the otic cup to an ovoid epithelial vesicle. This information will fill a critical void in the ear development field as it is not currently known how gene expression patterns at the placode stage correlate with those at the vesicle stage or beyond. In addition, the fate maps will provide a blueprint for the focal misexpression of a gene by retrovirus-mediated gene transfer. Our idea is that if precise boundaries of gene expression in the nascent otocyst are required for patterning the inner ear, then creation of temporally or spatially inappropriate foci of gene expression will likely perturb morphogenesis in a predictable way. A corollary goal of this proposal is to establish a mouse whole embryo culture model for studying early otic ontogeny. Experimental access to the ever-expanding list of natural and induced mutant mice would likely further our understanding of the genetic mechanisms that underlie inner ear morphogenesis and provide a basis for eliminating congenital deafness and balance disorders.
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0.934 |
2005 — 2006 |
Brigande, John Vincent |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Lineage Analysis of the Developing Mammalian Inner Ear @ Oregon Health and Science University
DESCRIPTION (provided by applicant): The long term goal of the proposed research is to understand the molecular mechanism underlying pattern formation and cell fate specification in the mammalian inner ear. Virtually nothing is known about the cell fate choices made by otic epithelial progenitors during development. Lineage analysis, however, can teach us about the types and timing of these choices. Defining lineage relationships is essential because mammals possess a unique array of inner ear cell types that are not present in lower vertebrates. Unfortunately, the formative stages of mammalian inner ear development occur when the early postimplantation embryo is encased in a complex arrangement of maternally-derived tissues. Transuterine microinjection techniques useful for introducing bioactive reagents into the otic epithelium at late postimplantation stages are thus ineffective at early stages where the opacity of materal tissues thwarts conventional imaging efforts. This inaccessibility of the developing inner ear in utero must be overcome to address fundamental questions regarding the molecular genetic mechanisrm underlying mammalian inner ear formation and function. This application seeks to establish a model system for the experimental manipulation of the developing mouse inner ear in utero by: 1) applying ultrasound biomicroscopy to image the mouse inner ear in utero; 2) pioneering ultrasound biomicroscopy-guided transuterine microinjection into the otic placode, cup, and vesicle; and 3) establishing the clonal relationships among component cells of the inner ear using retrovirus-mediated lineage analysis. Ultrasound biomicroscopy generates high-resolution images of embryonic tissues in real time. We will microinject into the otic placode, cup, and early vesicle under ultrasotmd guidance a replication-defective retrovirus encoding a lineage label, alkaline phosphatase, as well as an oligonucleotide library that serves as a tag for each integration event. Unambiguous assignment of clonal identity will be conducted by amplification and sequencing of the integration tag from individual alkaline phosphatase-positive cells in the mature, postnatal inner ear. Introduction of lineage virus at the earliest stages of inner ear development should produce a large number of clones of complex composition, which will be ideal for identifying lineage relationships. Defining these relationships may advance our understanding of the underlying mechanisms responsible for patterning the mammalian inner ear and specifying cell fate. Moreover, experimental embryological access to the developing mouse inner ear in utero should assist in the design of rational therapies to ameliorate or eliminate congenital forms of human deafness.
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1 |
2007 — 2018 |
Brigande, John Vincent |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Molecular Embryology of the Mammalian Inner Ear @ Oregon Health & Science University
DESCRIPTION (provided by applicant): The long term goal of our laboratory is to define gene- and cell-based strategies that restore hearing and balance in the dysfunctional inner ear. The short term goals are to advance our understanding of morphogenesis, pattern formation, and cell fate specification in the mouse inner ear. We use the mouse as a model system because an ever-expanding array of natural and induced genetic mutations exist that serve as accurate paradigms for human inner ear dysfunction. Our overarching technical approach relies on experimental embryology, a palette of surgical, imaging, microinjection and molecular techniques that permit access to the developing mouse inner ear in utero and enable genetic manipulation of precursor cells that give rise to the auditory and vestibular sensory structures. I this proposal, we aim: 1) to fate map the mouse otic vesicle in vivo~ 2) to define the types of cel fate choices otic precursors make and the timing of those choices~ and 3) to define the clonal contributions of neuroepithelial progenitors to the differentiated inner ear. A fate map describes what distinct populations of cells become as the inner ear matures. Developmental biologists use fate maps to understand how progenitor gene expression in a developing tissue or organ drives maturation and results in the acquisition of form and function. Intersection of indelibly labeled precursors with known domains of gene expression can teach us the molecular signals required to pattern sensory organs and specify cell fate. Lineage analysis, on the other hand, identifies the differentiated cells an individual otic precursor makes and reveals their location i the mature sensory organ. Clonal relationships will teach us if genetically defined pools of precursors are programmed early on in development to give rise to gross anatomical structures in the inner ear and to the sensory patches. The output of fate mapping and lineage analysis is a deeper understanding of the genetic regulation of progenitor cell identity and behavior. And that regulation promises to be far more complex that originally envisioned. Recent genetic fate mapping data show that neuroepithelial progenitor cells from the neural tube and likely the neural crest contribute precursor cells to the otic epithelium that generate sensory and nonsensory cells upon differentiation. These novel data firmly challenge the precept that the inner ear develops exclusively from placodal ectoderm and imply that neural ectoderm may uniquely participate in patterning and cell fate specification. Clonal analysis of these geneticall separable neuroepithelial precursor populations will advance our core knowledge about the embryonic origins of the mouse inner ear. A more complete understanding of the fate, lineage, and behavior of inner ear progenitor cells will inform the design of gene- and cell-based strategies aimed at the restoration of hearing and balance.
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1 |
2013 — 2014 |
Brigande, John Vincent |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
In Utero Protein Transduction to Interrogate Inner Ear Sensory Patch Formation @ Oregon Health & Science University
DESCRIPTION (provided by applicant): A long term goal of our laboratory is to define gene-based strategies that restore auditory and vestibular function in the diseased or damaged inner ear. Significant barriers to progress in the field of regenerative medicine are the identification f genes that have authentic therapeutic potential and the creation of reliable strategies to efficaciously modulate their expression and function. To begin to address these barriers, we have devised in utero gene transfers techniques that permit gain-of-function studies in the developing mouse inner ear that rely on viral vectors and in vivo electroporation. In the present proposal, we seek to define a rapid, cost effective, and technically simplified experimental paradigm that enables modulation of gene expression in otic precursors by in vivo protein transduction. Virtually all proteins do not spontaneously enter cells which restricts their usefulness as research tools. However, two new technologies have emerged that show enormous potential: surface remodeling of proteins and virus-like particles. Surface remodeling of proteins by replacement of nonconserved residues facilitates endocytosis in part by maximizing productive interactions with sulfated proteoglycans in the glycocalyx. Next generation virus-like particles are derived from an avian viral vector and effectively deliver a protein rather than a nucleic acid payload to the infected cell. In Aim 1, we propose to initiate somatic recombination in otic precursors by transuterine microinjection of bioactive Cre recombinase using the surface remodeling and virus-like particle formats. In subaim A, we test both formats using a floxed allele of a fluorescent reporter to define the time course of recombination, the type and distribution of recombined cells, and the potential impact of these reagents on postnatal acquisition of hearing and balance. In subaim B, we will generate inner ears mosaic for atonal homolog 1 (Atoh1) expression by Cre-mediated recombination of the floxed Atoh1 gene. We predict that abrogation of Atoh1 expression will reduce the number of sensory hair cells formed and allow us to test the hypothesis that Atoh1 positive cells can instruct the formation of Atoh1 negative hair cells. An additional property of surface remodeled proteins is their ability to reversibly complex with nucleic acids while retaining their protein transduction characteristics. In Aim 2, we propose to transfect otic precursors with expression plasmid or small interfering RNA (siRNA) by transuterine microinjection of surface remodeled protein/nucleic acid complexes. In subaim A, we will define the parameters for efficient expression plasmid transfection and test the bioactivity of an Atoh1 construct which is predicted to induce the formation of extra hair cells. In subaim B, we will define the parameters for efficiet siRNA transfection and test the bioactivity of siRNAs directed against Atoh1 to knock down gene expression and perturb hair cell fate specification. Successful completion of the proposed studies will establish a gain- and loss-of-function experimental platform to discern genes that have therapeutic potential and will introduce in vivo protein transduction as a potential therapeutic strategy for regenerative interventions in the diseased inner ear.
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1 |
2014 — 2018 |
Brigande, John Vincent |
R01Activity Code Description: To support a discrete, specified, circumscribed project to be performed by the named investigator(s) in an area representing his or her specific interest and competencies. |
Fetal Pharmacotherapy For Congenital Deafness @ Oregon Health & Science University
DESCRIPTION (provided by applicant): Bilateral permanent congenital hearing loss is estimated to affect 1-3 neonates per 1,000 live births worldwide. A genetic basis is causal in approximately 50% of the individuals. Treatment options for congenitally deaf or hearing impaired children are limited in scope and efficacy and typically involve hearing aids or cochlear implants. Neither intervention fully restores the richness of native hearing. The conceptual basis of this proposal is to define fetal therapies that correct defects in gene expression or function prior to the onset of overt pathogenic changes in the sensory epithelia of the developing inner ear. We use the mouse as a model system because an ever-expanding array of natural and induced genetic mutations exists that serve as accurate paradigms for human inner ear dysfunction. Our technical approach relies on experimental embryology, a palette of surgical, imaging, microinjection and molecular techniques that permit access to the developing mouse inner ear in utero and enable genetic manipulation of precursor cells that give rise to the auditory and vestibular sensory structures. In this proposal, we aim: 1) to restore auditory function in a mouse model defective in synaptic transmission~ and 2) to restore auditory and vestibular function in a mouse model of Usher syndrome. A missense mutation in the human SLC17A8 gene that encodes vesicular glutamate transporter-3 (VGLUT3) has been associated with an autosomal-dominant form of progressive, high-frequency nonsyndromic deafness. VGLUT3 is selectively localized to inner hair cells in the cochlea and loads the excitatory amino acid neurotransmitter glutamate into synaptic vesicles. The VGLUT3 knockout mouse is born deaf due to the inability of inner hair cells to release glutamate at the afferent nerve terminals. We hypothesize that virus-mediated gene transfer of VGLUT3 to otic epithelial precursors will force VGLUT3 expression in inner hair cells and restore synaptic transmission and hearing in the VGLUT3 knockout mouse. Usher syndrome is the leading genetic cause of combined deafness and blindness and is associated with 13 loci and 10 genes in humans. Three distinct clinical forms are differentiated by the time of onset and severity of auditory, vestibular, and visual dysfunction. Usher syndrome type 1 is the most severe with bilateral profound hearing loss and balance difficulty at birth and retinitis pigmentosa present by early adolescence. The USH1 gene encodes the protein harmonin that is expressed in the sensory hair cell bundle and the ribbon synapse. A single base pair mutation in the harmonin gene creates a cryptic splice site resulting in a frameshift and truncated harmonin protein. The USH1C mouse mutant is born deaf, uncoordinated, and develops vision impairment. We hypothesize that fetal administration of an antisense oligonucleotide will correct harmonin messenger RNA splicing and restore auditory and vestibular function in the USH1C mouse mutant. We are optimistic that the proposed studies will establish the first fetal therapies for congenital deafness and vestibular dysfunction and will help inform translation of these approaches to the clinic.
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1 |
2019 — 2021 |
Brigande, John Vincent Hennebold, Jon D |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Fetal Gene Therapy For Congenital Deafness and Imbalance @ Oregon Health & Science University
PROJECT SUMMARY/ABSTRACT Extensive knowledge of the genetic mutations responsible for congenital hearing loss and imbalance has led to gene-based therapeutic strategies aimed at rescuing sensory function. The mouse is the dominant model system because of the availability of natural and induced mutations, the accessibility of the neonatal inner ear, and its responsiveness to genetic manipulation. A striking observation from these studies is that virus-mediated gene therapies and pharmacotherapies targeted to the postnatal day 0 (P0) through P5 mouse inner ear yield optimal rescue of hearing and balance. Intervention thereafter dramatically lessens or entirely eliminates therapeutic benefits. Critically, the P0-P5 mouse inner ear is functionally immature with hearing onset at P12 consonant with the emergence of the acoustic startle reflex. In humans, acoustic startle arises at gestational week 19 during the second trimester of pregnancy, suggesting that the window of therapeutic efficacy from P0-P5 in the mouse may predicate a prenatal window of efficacy in the human fetus. The conceptual basis of this proposal is that the early neonatal mouse inner ear functionally models the prenatal human inner ear. To discern if gene therapies defined in the early neonatal mouse inner ear may safely and effectively translate to the clinic, a higher vertebrate model system characterized by the precocious emergence of fetal hearing is needed. Our long-term goal is to establish a rhesus macaque model system to test fetal versus neonatal gene therapy to treat congenital deafness and imbalance. In Aim 1, we will define the onset of fetal hearing in the rhesus macaque. Pure tones at 100, 250, 500, 1000, or 3,000 Hz will be transmitted across the maternal abdomen with increasing intensities. Ultrasonic assessment of acute head, arm, or torso movements will indicate startle. We predict that startle to lower frequency stimuli will emerge first during development as they do in the human fetus. We further hypothesize that the optimal time to intervene therapeutically will precede the age of hearing onset. In Aim 2, we will define a fetal survival surgery to access the inner ear. An adeno-associated viral vector encoding green fluorescent protein (GFP) will be microinjected into membranous labyrinth. The viral transduction efficiency will be estimated by whole mount immunofluorescence to detect GFP. We hypothesize that an AAV2-based vector pseudotyped with a synthetic or naturally occurring capsid will robustly transduce the majority of immature hair cells in the fetal inner ear. In Aim 3, a CRISPR/Cas9-based genome editing technology will be deployed to create rhesus embryos with bi-allelic mutations in harmonin. We hypothesize that correct targeting will produce a model of Usher syndrome type 1C characterized by congenital deafness and profound vestibular dysfunction. Successful completion of the proposed studies will define the optimal gestational age to initiate fetal gene therapy in rhesus; identify an AAV vector capable of delivering harmonin to fetal sensory hair cells; and create a primate model of congenital inner ear disease. These resources will be deployed in future studies to test the safety and efficacy of fetal versus neonatal gene therapy to rescue hearing and balance.
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1 |
2020 |
Brigande, John Vincent Hennebold, Jon D |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Fetal Gene Therapy For Congenital Deafness and Imbalance (Administrative Supplement) @ Oregon Health & Science University
PROJECT SUMMARY/ABSTRACT Extensive knowledge of the genetic mutations responsible for congenital hearing loss and imbalance has led to gene-based therapeutic strategies aimed at rescuing sensory function. The mouse is the dominant model system because of the availability of natural and induced mutations, the accessibility of the neonatal inner ear, and its responsiveness to genetic manipulation. A striking observation from these studies is that virus-mediated gene therapies and pharmacotherapies targeted to the postnatal day 0 (P0) through P5 mouse inner ear yield optimal rescue of hearing and balance. Intervention thereafter dramatically lessens or entirely eliminates therapeutic benefits. Critically, the P0-P5 mouse inner ear is functionally immature with hearing onset at P12 consonant with the emergence of the acoustic startle reflex. In humans, acoustic startle arises at gestational week 19 during the second trimester of pregnancy, suggesting that the window of therapeutic efficacy from P0-P5 in the mouse may predicate a prenatal window of efficacy in the human fetus. The conceptual basis of this proposal is that the early neonatal mouse inner ear functionally models the prenatal human inner ear. To discern if gene therapies defined in the early neonatal mouse inner ear may safely and effectively translate to the clinic, a higher vertebrate model system characterized by the precocious emergence of fetal hearing is needed. Our long-term goal is to establish a rhesus macaque model system to test fetal versus neonatal gene therapy to treat congenital deafness and imbalance. In Aim 1, we will define the onset of fetal hearing in the rhesus macaque. Pure tones at 100, 250, 500, 1000, or 3,000 Hz will be transmitted across the maternal abdomen with increasing intensities. Ultrasonic assessment of acute head, arm, or torso movements will indicate startle. We predict that startle to lower frequency stimuli will emerge first during development as they do in the human fetus. We further hypothesize that the optimal time to intervene therapeutically will precede the age of hearing onset. In Aim 2, we will define a fetal survival surgery to access the inner ear. An adeno-associated viral vector encoding green fluorescent protein (GFP) will be microinjected into membranous labyrinth. The viral transduction efficiency will be estimated by whole mount immunofluorescence to detect GFP. We hypothesize that an AAV2-based vector pseudotyped with a synthetic or naturally occurring capsid will robustly transduce the majority of immature hair cells in the fetal inner ear. In Aim 3, a CRISPR/Cas9-based genome editing technology will be deployed to create rhesus embryos with bi-allelic mutations in harmonin. We hypothesize that correct targeting will produce a model of Usher syndrome type 1C characterized by congenital deafness and profound vestibular dysfunction. Successful completion of the proposed studies will define the optimal gestational age to initiate fetal gene therapy in rhesus; identify an AAV vector capable of delivering harmonin to fetal sensory hair cells; and create a primate model of congenital inner ear disease. These resources will be deployed in future studies to test the safety and efficacy of fetal versus neonatal gene therapy to rescue hearing and balance.
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1 |
2020 — 2021 |
Brigande, John Vincent |
R21Activity Code Description: To encourage the development of new research activities in categorical program areas. (Support generally is restricted in level of support and in time.) |
Therapeutic Cellular Reprogramming in the Adult Mammalian Inner Ear by Fetal Gene Transfer @ Oregon Health & Science University
PROJECT SUMMARY/ABSTRACT Hearing loss is the most common sensory deficit worldwide. Disabling hearing loss will affect an estimated 900 million individuals globally by 2050 at an annual cost of US$ 750 billion. There is compelling socioeconomic rationale to devise novel therapeutic strategies to treat hereditary and non-hereditary forms of inner ear disease. Mouse models of deafness and vestibular dysfunction are most commonly exploited to test gene and pharmacotherapeutics designed to rescue sensory function. A widespread experimental approach is to deliver genes or drugs to the functionally immature neonatal inner ears of mice that model human deafness and then assess structural and functional recovery at mature stages. This work takes advantage of the plasticity of the pre-hearing mammalian inner ear to accommodate microinjection of aqueous reagents without significantly affecting acquisition of auditory or vestibular function. However, a pressing need is to define experimental systems that model the responsivity of the adult inner ear to therapeutic genetic manipulation. The conceptual basis of this proposal is that delivery of functionally silent genetic constructs to the fetal inner ear will enable atraumatic activation in differentiated cell types of mature inner ear. We hypothesize that transuterine microinjection of Cre recombinase-responsive genetic elements into the otic vesicle of mice harboring tamoxifen- inducible alleles will permit control of the timing and cell type-specificity of therapeutic gene delivery without compromising inner ear structure or function. Our long term goal is to verify where in the inner ear and when specific genes must be modulated to restore or protect auditory function in models of hereditary and non- hereditary hearing loss. In Aim 1, we will atraumatically deliver a chemically inducible genetic switch flanking green fluorescent protein (GFP) to the fetal inner ear using a recombinant adeno-associated viral vector (rAAV) and then pharmacologically trigger expression in the adult inner ear. We hypothesize that GFP expression will be constrained to inner or outer hair cells, subsets of supporting cells in the organ of Corti, to vestibular supporting cells in the cristae and maculae, and spiral ganglion neurons as predicated by relevant Cre driver alleles. In Aim 2, we will deploy the genetic switch system to reprogram adult mouse supporting cells into hair cells by conditional expression of the Pou4f3, Gfi1, and Atoh1 transcription factors. We hypothesize that exogenous bioactive signals will be efficiently transmitted to supporting cells in the adult mouse inner ear. In Aim 3, we will use an inducible hybrid transcriptional activation system to reprogram supporting cells into hair cells. We hypothesize that forced transcriptional activation of endogenous Pou4f3, Gfi1, and Atoh1 in adult mouse supporting cells will induce a hair cell fate. Successful completion of our aims may establish a mouse model system that enables in vivo validation of druggable genetic targets that can preserve hearing and balance in the mature inner ear.
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1 |