2019 |
Geleoc, Gwenaelle S Naash, Muna I. |
R56Activity Code Description: To provide limited interim research support based on the merit of a pending R01 application while applicant gathers additional data to revise a new or competing renewal application. This grant will underwrite highly meritorious applications that if given the opportunity to revise their application could meet IC recommended standards and would be missed opportunities if not funded. Interim funded ends when the applicant succeeds in obtaining an R01 or other competing award built on the R56 grant. These awards are not renewable. |
Gene Therapy of Usher Syndrome @ Boston Children's Hospital
Abstract: Usher syndrome is the most frequent cause of deafness and blindness in humans. It is classified under three clinical subtypes (USH-1, -2 and -3) according to the severity of the symptoms. Approximately 2/3 of patients with Usher syndrome suffer from USH2, 85% of whom have mutations in the USH2A gene. USH2 is clinically characterized by moderate to severe congenital, progressive hearing impairment and retinitis pigmentosa leading to blindness in early childhood to late adulthood. While there is no cure for Usher syndrome, early gene therapy treatment may be viable option to halt the progression of the disease. We have recently demonstrated successful restoration of auditory and vestibular function in a mouse model of USH1C using adeno-associated viral vectors (AAVs) injected through the round window membrane (RWM) at early postnatal age (Pan et al., 2017). This work offers proof-of-principal that the approach is viable for treatment of inherited hearing loss. In this proposal, we take a multi-angle, team lead approach to use stat-of-the-art technologies to develop therapeutic strategies and test their efficacy in halting the progression of hearing loss in several animal models of the disease associated with the most common mutation in the USH2A gene. This mutation, a single base-pair deletion in exon 13 (c.2299delG: p.Glu767Serfs*21) leads to a premature termination codon. Because the USH2A coding sequence extends beyond the capacity for AAV vectors, we propose alternative approaches to develop gene therapy treatments for USH2A. We will assess these therapies in mouse models for this mutation including the c.2299delG (Ush2adelG/delG) which we have partially characterized to show progressive deafness and late-onset visual loss associated with structural defects in the cochlea along with retinal degeneration. We are also in the process of generating a delG swine model to perform pre-clinical studies. In aim1, we will assess gene supplementation strategies using high capacity adenoviral vectors (AdV) or DNA nanoparticles (NP) to mediate full phenotypic rescue when injected into the inner ear of mutant mice. Multiply-deleted (-E1a/b, -E3, -pol, -pTP) AdV are well suited for this approach due to their high capacity (>15kb) and ability to transduce hair cells successfully both in vitro and in vivo. NP are ideal complement to AdV especially for delivery of large gene as they have been demonstrated to successfully transduce photoreceptor cells of the retina. These vectors will be injected into the inner ear of newborn Ush2adelG/delG mice and longevity of the functional/structural rescue will be evaluated at different times post treatment. Aim2 will assess the efficacy of gene silencing strategies to correct the c.2299delG mutation. These will include exon skipping strategies using CRISPR/Cas9 constructs as well as antisense oligonucleotides (ASO). To assess and validate gene therapy strategies for future treatment of USH2A, Aim 3 will generate and characterize the auditory phenotype in miniature pig model with the c.2299delG mutation and test the efficacy of viral and non-viral vectors to deliver usherin gene to hair cells and the preservation of auditory sensitivity. In summary, this proposal will provide a solid foundation for development of gene therapy to treat genetic deafness in human.
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0.936 |
2021 |
Geleoc, Gwenaelle S |
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. |
Functional Development of Hair Cells and Neurons in the Inner Ear @ Boston Children's Hospital
Project summary Hair cells of the inner ear are the primary receptors of the auditory system. They transduce mechanical information, associated with sound waves, into electro-mechanical (outer hair cells) and electro-chemical (Inner hair cells) signals, which lead to amplification of the initial signal and activation of afferent neuronal fibers, respectively. While hair cells and neuronal fibers appear before birth in mice, development and maturation of the hair cells, neurons and synapses proceeds until hearing onset, ~postnatal day 12. This process is believed to be dynamic and modulated by hair cell activity. In particular, recent work has shown that lack of hair cell transmission, due to absence of functional synapses or defective mechanosensation, leads to altered neuronal maturation and specification. Successful outcomes for new therapies, including gene therapy, aimed at restoring hair cell function after birth, may depend on restoration of auditory circuits, including mature and functional hair cell synapses and neuronal fibers. Here we propose to assess how disruption or loss of sensory transduction in several mouse models affects hair cell function, synaptic maturation and spiral ganglion specification. Furthermore, we will determine if inner ear gene therapy is capable of reversing any of these observed changes and identify the conditions for optimal recovery of auditory function. We will combine state-of-the-art technologies to address these important questions, including high-resolution imaging, electrophysiology, single cell RNA sequencing and localization of RNA transcripts.
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0.936 |