Jian Zuo, PhD - US grants

DESCRIPTION (provided by applicant): A large number of genes have recently been identified that are implicated in vestibular disorders and in development of inner ear hair cells. Mouse models in which these genes can be inactivated in a hair cell-specific, temporally controlled manner are crucial to in vivo functional studies of these genes. However, to date there has been no effective method of conditional gene targeting in hair cells in vivo. This proposal seeks to overcome the limitations of traditional transgenic methods by expressing the bacterial recombinase Cre specifically and inducibly in vestibular and cochlear hair cells in transgenic mice. Preliminary studies have succeeded in creating and characterizing transgenic mice in which the reporter gene green fluorescent protein (GFP) is expressed specifically and abundantly in developing and adult hair cells of the vestibular and cochlear sensory epithelia. This success was achieved through the novel approach of modifying a bacterial artificial chromosome (BAC) that contains the alpha-9 AChR gene, which is important to efferent innervation and specific to vestibular and cochlear hair cells. The specific aims of this proposal are to: 1. Create mice in which Cre is expressed specifically in vestibular and cochlear hair cells. 2. Create mice in which Cre is activated specifically in vestibular and cochlear hair cells only when an exogenous ligand is delivered. The creation and characterization of these Cre-hair cell mice will greatly facilitate studies of gene function in sensory-motor systems responsive to gravity. Ultimately, such studies will also contribute to the understanding, prevention, and treatment of vestibular diseases in humans. All mice made in FVB/N strain will be available to the academic community.

DESCRIPTION (Applicant's Description): This application focuses on elucidating the normal functions of a recently cloned human gene, retinitis pigmentosa 1 (RP1), and the mechanisms by which the mutant forms of the RP1 gene cause retinal degeneration. Retinitis pigmentosa (RP) is the most common form of inherited retinopathy, affecting 1 in 3.500 people world wide (1.5 million). RP is characterized by night blindness and progressive degeneration of the peripheral retina, culminating in severe reduction of visual fields and in blindness. We have recently identified a novel retinal photoreceptor- specific gene on chromosome arm 8q in which nine mutations were found to cause the RP1 form of autosomal dominant RP in seventeen unrelated families. It is unclear whether mutations in RP1 represent loss-of-function or dominant-negative alleles of the RP1 gene. The protein encoded by this gene consists of 2,156 amino acids; its function is unknown although its amino terminus has significant homology to that of human doublecortin, and its carboxy terminus contains a nucleoside diphosphate kinase domain and several nuclear localization signal domains. We mapped the mouse homolog of the human RP1 gene to mouse chromosome 1 where there is no existing mouse retinal degenerative mutant. In this application we plan: (1) to generate and characterize transgenic mice that over-express wild-type or mutant Rp1 genes in a pattern that recapitulates the pattern of endogenous Rp1 expression; (2) to generate and characterize mice without the Rp1 gene; and (3) to determine the sub-cellular localization of wild-type and mutant Rp1 proteins. These studies will lead to a better understanding of the normal function of the RP1 gene during retinal development. The characterization of molecular pathways in mouse models of RP1 will facilitate the prevention and treatment of retinal degenerative diseases in humans. Although there have been dramatic successes in recent years in the identification of multiple genes that can cause retinal diseases, very little is known about how mutations in these genes cause diseases. A number of these disease genes display complex patterns of gene expression; some mutations that cause dominant retinal diseases are likely to be gain-of-function mutations. Thus, a combination of the transgenic technology using bacterial artificial chromosome and the knockout technology can be widely applicable for making mouse models of these retinal diseases, and our studies of RP1 will provide an example for studying functions of these genes in vivo.

DESCRIPTION (from applicant's abstract): Although there have been dramatic successes in recent years in identifying multiple genes that can cause deafness in humans, very little is known about how mutations in these genes cause disease. We have recently succeeded in expressing a reporter gene specifically in hair cells in transgenic mice using a modified bacterial artificial chromosome (BAC). We found that in our transgenic mice the expression of the reporter gene recapitulates the pattern of the endogenous gene expression. Thus, this BAC transgenic technology can be widely used for making mouse models of dominant hearing disorders in which the disease genes display complex expression patterns, or in which the mutations are gain-of-function mutations. This application focuses on making mouse models of DFNA15, a progressive, nonsyndromic, autosomal dominant hearing loss in humans. Recently, an 8-bp deletion has been found in the POU4F3 gene in a family with DFNA15. In the inner ear, Pou4f3 is expressed specifically in hair cells from the earliest onset of development through adulthood. Targeted deletion of the Pou4f3 gene in mice suggested that the 8-bp deletion in POU4F3 does not represent a loss-of function allele, but rather a gain-of function allele of the POU4F3 gene. In this application we plan to test this hypothesis by: 1. generating transgenic mice that overexpress the mutant Pou4f3 gene in a pattern that recapitulates the pattern of endogenous Pou4f3 expression; 2. generating transgenic mice that overexpress the wild-type Pou4f3 gene in a pattern that recapitulates the pattern of endogenous Pou4f3 expression. The creation and characterization of mouse models of DFNA15 will facilitate the prevention and treatment of the disease in humans and will also lead to a better understanding of the normal function of the POU4F3 gene during hair cell development.

DESCRIPTION (Applicant's Description): This application focuses on elucidating the normal functions of a recently cloned human gene, retinitis pigmentosa 1 (RP1), and the mechanisms by which the mutant forms of the RP1 gene cause retinal degeneration. Retinitis pigmentosa (RP) is the most common form of inherited retinopathy, affecting 1 in 3.500 people world wide (1.5 million). RP is characterized by night blindness and progressive degeneration of the peripheral retina, culminating in severe reduction of visual fields and in blindness. We have recently identified a novel retinal photoreceptor- specific gene on chromosome arm 8q in which nine mutations were found to cause the RP1 form of autosomal dominant RP in seventeen unrelated families. It is unclear whether mutations in RP1 represent loss-of-function or dominant-negative alleles of the RP1 gene. The protein encoded by this gene consists of 2,156 amino acids; its function is unknown although its amino terminus has significant homology to that of human doublecortin, and its carboxy terminus contains a nucleoside diphosphate kinase domain and several nuclear localization signal domains. We mapped the mouse homolog of the human RP1 gene to mouse chromosome 1 where there is no existing mouse retinal degenerative mutant. In this application we plan: (1) to generate and characterize transgenic mice that over-express wild-type or mutant Rp1 genes in a pattern that recapitulates the pattern of endogenous Rp1 expression; (2) to generate and characterize mice without the Rp1 gene; and (3) to determine the sub-cellular localization of wild-type and mutant Rp1 proteins. These studies will lead to a better understanding of the normal function of the RP1 gene during retinal development. The characterization of molecular pathways in mouse models of RP1 will facilitate the prevention and treatment of retinal degenerative diseases in humans. Although there have been dramatic successes in recent years in the identification of multiple genes that can cause retinal diseases, very little is known about how mutations in these genes cause diseases. A number of these disease genes display complex patterns of gene expression; some mutations that cause dominant retinal diseases are likely to be gain-of-function mutations. Thus, a combination of the transgenic technology using bacterial artificial chromosome and the knockout technology can be widely applicable for making mouse models of these retinal diseases, and our studies of RP1 will provide an example for studying functions of these genes in vivo.

DESCRIPTION (provided by applicant): The mammalian cochlea responds to sound stimuli with remarkable sensitivity by a mechanical amplification process (termed cochlear amplification) that resides in the cochlea's outer hair cells (OHCs). The change of OHC soma length driven by transmembrane voltage (termed OHC electromotility) has been hypothesized to provide mechanical feedback and, thereby, cochlear amplification. A complex of molecules within the lateral wall of OHCs (termed the motor complex) is thought to be responsible for OHC electromotility. In this proposal, we focus on the genetic analysis of the mechanism underlying OHC electromotility and cochlear amplification in mice. Using a knockout mouse, we have provided evidence that prestin, a recently discovered protein in the plasma membrane of the motor complex, is required for OHC electromotility and cochlear amplification. To further elucidate the molecular basis of OHC electromotility and its role in cochlear amplification, we plan to determine: 1. whether prestin-mediated OHC electromotility is the only active mechanism in OHCs to generate cochlear amplification. 2. how prestin-mediated OHC electromotility provides feedback for cochlear amplification; and 3. how other molecules of the motor complex in the OHC's lateral wall contribute to prestin-mediated OHC electromotility and, thereby, cochlear amplification. Biochemical, physiologic, and genetic analyses of mutant mice will enable us to elucidate the molecular pathway that underlies OHC electromotility and cochlear amplification. Our studies may provide insights into the mechanisms by which hearing loss involving deficiencies in OHC electromotility occurs in humans.

[unreadable] DESCRIPTION (provided by applicant): Our ability to manipulate gene expression specifically in the hair cells of the inner ear during development and adulthood in mice is crucial for understanding the physiology of hearing and the pathology of deafness in humans. Recent advances from our laboratory and many others have demonstrated that gene expression can be manipulated in developing mouse hair cells in a spatially and temporally controlled manner. However, a key feature remains elusive -- our ability to manipulate gene expression specifically in mature inner hair cells (IHCs) and outer hair cells (OHCs). Here we propose to develop transgenic or knockin mice that express inducible CreERT2, an effective fusion of Cre and estrogen receptor (CreER), in mature cochlear hair cells (OHCs in Aim 1; IHCs and OHCs in Aim 2). The characterization and availability of these mouse lines will enable auditory researchers to inactivate or activate genes of their interest in mature hair cells. The creation of conditional (loxP) alleles of every mouse gene in their genome, which has been proposed in the NIH's Knockout Mouse Project (KOMP), will provide a complementary resource for the use of the inducible Cre lines that we generate. Finally, the strategies we adopt here can be used to express genes specifically in other mature cell types of the inner ear (i.e., IHCs, spiral ganglia, stria fibrocytes and marginal cells) at any given time. All mouse lines will be genetically engineered in 129S7/C57BL/6J mixed or FVB/NJ strains, transferred to the CBA/CaJ strain, and then deposited in the NIH-sponsored Mutant Mouse Regional Resource Center (MMRRC). Public Health Relevance Statement An estimated 28 million people in the United States are deaf or hard of hearing. Approximately 1.5 million individuals aged 3 years or older are deaf in both ears and 2 to 3 per 1,000 live births suffer congenital hearing loss. More than 40 million persons in the United States suffer various levels of noise induced hearing loss. Despite the significant progress in our understanding of these hearing disorders, very little is known about the disease causes and about the normal hearing processes in adults. Many genetic factors (genes) that, when mutated, cause hearing impairment in people, play critical roles in both infants and adults. We propose here to develop genetic tools that would allow us to investigate the function of hearing related genes in adult animal models. These tools will be able to surgically activate genes in a specific set of cells in the hearing process at any time of the adult life of the animal. This temporal and spatial precision is critical for our understanding of hearing process and developing therapeutic targets for intervention of the hearing disorders. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Hearing is impaired in more than 10% of the human population. Despite significant progress in neonatal regeneration of mammalian cochlear hair cells (HCs), such regeneration in adults has proved extremely difficult. While gamma secretase inhibitors have shown some promise in regenerating noise-damaged auditory HCs in adult mice, no drugs have been proven effective for auditory HC regeneration in adult humans. Interestingly, we recently demonstrated that supporting cells (which surround hair cells) can be converted to HCs in mature cochleae through combined manipulation of two key genes, one of which (p27Kip1 or p27) is inactivated and one of which (Atoh1) is activated. These findings led us to screen for small-molecule inhibitors of p27 and to characterize them in cell lines. We propose to test the inhibitory effects of these lead compounds in cochlear explants and in vivo in adult wild-type and transgenic mice, with or without noise damage. These exploratory studies will provide the key proof of concept for using small-molecule inhibitors of p27, together with small-molecule activators of Atoh1, to regenerate damaged auditory HCs in adult mammals. The final lead compounds identified here will advance to the drug development pipeline for optimization, selection, and preclinical safety analysis, and eventually to clinical trials for HC regeneration in humans. These studies may lead to a breakthrough in the treatment of hearing loss caused by noise, antibiotics, chemotherapy, or age.

PROJECT SUMMARY: To date, there are no drugs approved by the US Food and Drug Administration (FDA) for protection against cisplatin- or noise-induced hearing loss. We have developed an unbiased, phenotypic drug screen for protection against cisplatin ototoxicity, using a cell line derived from neonatal mouse cochleae. From a library of 4,359 unique compounds, including 844 FDA-approved drugs, we have identified a top hit that exerts strong protection against cisplatin ototoxicity in the cochlear cell line and cochlear explant culture, with an excellent therapeutic index (>200) and an IC50 of ~150 nM. We have confirmed that when locally delivered to adult mice via injection through the eardrum, this top hit protects against cisplatin and noise injury. Therefore, we have a validated top hit that protects against cisplatin- and noise-induced hearing loss. Here we propose to optimize our top hit to generate a lead compound by improving local delivery methods and by performing sequential analyses, including medicinal chemistry, structure-activity relationship (SAR), absorption, distribution, metabolism, excretion, safety/toxicology (ADMET), and in vivo pharmacokinetics (PK) and pharmacodynamics (PD) studies. Our top hit and its analogs, after testing in our streamlined assays, will serve as candidate compounds for preclinical and clinical studies of their safety and efficacy when locally delivered for treatment of hearing loss.

? DESCRIPTION (provided by applicant): Regeneration of sensory hair cells in the mature cochlea is a major therapeutic challenge. Atoh1, a transcription factor important for hair cell development, can induce hair cell formation from surrounding supporting cells in postnatal murine cochleae. However, hair cells regenerated by Atoh1 are too few, are incompletely mature, and most importantly, exhibit age-dependent decline in the postnatal cochlea. It remains unclear which molecules and signaling pathways modulate Atoh1-mediated conversion of supporting cells to hair cells in the mature cochlea in vivo. Our preliminary results demonstrate that genetic manipulation of additional factors, in conjunction with Atoh1 activation, in supporting cells can convert them to hair cells in the mature cochlea in vivo. Remarkably, we also showed that one of the three factors by itself can convert supporting cells to hair cells in te mature cochlea in vivo. Moreover, we have established RNA profiles of newly regenerated hair cells that identify transcription factors and signaling molecules that are likely involved in regulation of Atoh1-mediated hair cell regeneration in the mature cochlea. These novel and exciting findings led us to test whether combined genetic manipulation of these molecules would increase the efficiency of conversion of supporting cells to hair cells in the mature cochlea in vivo and determine the molecular mechanisms of such conversion. Direct conversion of supporting cells to hair cells is a key initial step toward hair cell regeneration and functional restoration of hearing. Our approach has been highly fruitful in generating new hair cells in vivo?both previously, in the postnatal cochlea, and now in the mature cochlea. For example, we have achieved a conversion rate of supporting cells to hair cells as high as 20% in vivo, a rate comparable to those in other regenerative systems such as adult pancreas, heart, and CNS. Our proposed studies will reveal 1) novel mechanisms of hair cell regeneration in the mature mammalian cochlea and 2) novel therapeutic targets for future clinical restoration of hearing.

ABSTRACT There is a significant unmet medical need to develop pharmacotherapeutics for auditory disorders. Yet, few academic centers in the world are dedicated to these efforts. With a strong focus on pharmacotherapeutics for such disorders in the Translational Hearing Center (THC), our Drug Discovery & Delivery Core (DDDC) aims to establish a state-of-the-art drug development pipeline to facilitate individual research projects in THC. We will leverage projects developed by Research Project Leaders (RPLs) and allied Center Investigators to significantly advance the field of pharmacotherapeutics for auditory disorders. Our DDDC will mentor and train the next generation of hearing researchers and attract new investigators to contribute to this fast advancing field. During Phase 1, DDDC will: 1) develop in silico, in vitro and in vivo high-throughput screens to discover and validate novel therapeutics for auditory disorders; 2) establish a medicinal chemistry pipeline to optimize chemical entities used in research projects or discovered in 1 or in RPLs); 3) establish drug delivery and pharmacokinetic/pharmacodynamic (PK/PD) methodologies; 4) develop a sustainability plan for DDDC services. With strong commitment from Creighton University and neighboring institutes (Boys Town National Laboratory and University of Nebraska Medical Center), the DDDC has already established nearly all necessary equipment and expert personnel. The Director and Co-Directors of DDDC have extensive experience in drug development for auditory disorders, medicinal chemistry and computational structural studies. The sustained impact of the DDDC will be to build a state-of-the-art and highly collaborative center so that additional extramural grants and sponsored programs will gradually cover the main cost of DDDC. We will also emphasize the educational component so that new investigators will become familiar with this fast-moving field of ototherapeutics and maintain the momentum for years to come.