Andy Groves Ph.D.

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Reprogramming and Gene Delivery

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Reprogramming and Gene Delivery
Andy Groves, Ph.D. (chair), Baylor College of Medicine
John Brigande, Ph.D., Oregon Health & Science University
Yehoash Raphael, Ph.D., University of Michigan
Segil Lab, University of Southern California

This group will take the lead on transitioning to Phase II, testing candidate genes. They will study the effects of current transcription factor reprogramming cocktails on supporting cell behavior, including the collection of additional transcriptomic and epigenetic data that will be shared with the CSE group. These experiments will be performed in a variety of systems, including a flattened epithelium guinea pig model that shares features with chronic human deafness. The Groves lab will use transgenic mice to detail the effects of the reprogramming cocktails in the organ of Corti. The Brigande lab will continue its work on creating efficient mouse model systems to interrogate candidate genes for sensory hair cell regeneration, as pioneered by testing whether viral delivery of these same reprogramming factors is also efficacious. The Raphael lab will take a similar approach in the guinea pig model, in this case using a virus designed and generated by the Groves and Segil labs. These experiments are part of the group’s broader efforts to develop new methods to deliver molecules and/or genes, for example by endogenous activation of reprogramming factors via CRISPR/Cas-9.

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Cross-Species Epigenetics

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Cross-Species Epigenetics
Tatjana Piotrowski, Ph.D. (chair), Stowers Institute for Medical Research
Alain Dabdoub, Ph.D., Sunnybrook Research Institute
Andy Groves, Ph.D., Baylor College of Medicine
Stefan Heller, Ph.D., Stanford University
The Lab of Neil Segil, Ph.D., University of Southern California
Litao Tao, Ph.D., Creighton University

This group will complete the collection of transcriptomic and epigenetic data from systems that regenerate (neonatal mouse, zebrafish, chick) and those that do not (mature mouse and human). In addition, they will begin to perform cross-species comparisons of the behavior of a shared set of hair cell loci across species. Collection of chick data is spearheaded by the Heller lab; the Groves, Segil, and Tao labs are responsible for mouse data; the Piotrowski lab leads work on zebrafish; and the Dabdoub lab will add data from humans. High-quality single-cell RNA-seq, ATAC-seq, and SHARE-seq data from multiple timepoints and conditions will be generated by all member labs.

Comparison of Three Reprogramming Cocktails in the Organ of Corti: Cells, Transcriptomes, and Epigenomes

Comparison of Three Reprogramming Cocktails in the Organ of Corti: Cells, Transcriptomes, and Epigenomes
Andy Groves, Ph.D., Baylor College of Medicine

Each cell type in the human body is defined by its activation of a unique combination of genes that endow each cell type with unique properties. The activation of these genes is achieved by special proteins known as transcription factors, or “switches,” responsible for switching on appropriate genes in one cell type and preventing inappropriate genes from being activated. In recent years, investigators have identified a number of these transcription factors that cause the formation of hair cells. The goal this year is to examine why these cocktails of transcription factors are able to “reprogram” nonsensory cells, but not supporting cells of the inner ear, to become hair cells.

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Epigenetics Analysis of Maturation and Regenerative Responses in the Mouse Organ of Corti and Utricle

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Epigenetics Analysis of Maturation and Regenerative Responses in the Mouse Organ of Corti and Utricle
Neil Segil, Ph.D., University of Southern California
Andy Groves, Ph.D., Baylor College of Medicine

The broad premise of the HRP is to identify molecules that could control hair cell regeneration. To do this, we are studying cell types and regenerative processes in multiple contexts and species, then integrating these data together to identify mechanisms that could potentially be turned on in the mouse cochlea to drive transdifferentiation (activating the correct set of hair cell–promoting genes in supporting cells). The role of this systems biology project is to provide the necessary data integration “glue,” binding together the results from the data generation projects. We will combine much of the data that is being generated by the HRP to advance our knowledge of hair cells, supporting cells, conversion of one cell type to another, and the potential for regeneration. By modeling all of the available HRP data, we will identify regulatory molecules that may contribute to regeneration.

Comparison of Three Reprogramming Cocktails in the Organ of Corti: Cells, Transcriptomes, and Epigenomes

Comparison of Three Reprogramming Cocktails in the Organ of Corti: Cells, Transcriptomes, and Epigenomes
Andy Groves, Ph.D., Baylor College of Medicine

In the past year we have been investigating whether we are able to use genetic reprogramming techniques to generate new hair cells in the mouse cochlea. The results of this work are extremely promising: We were able to turn nonsensory cells of the mouse cochlea into hair cells. However, we consistently find that supporting cells in our mouse models do not respond to reprogramming. This is curious, as supporting cells are the cells responsible for producing new sensory hair cells in birds and fish. In the coming year, we will first examine supporting cells after our reprogramming attempts to see whether they have been able to activate any aspects of a hair cell program. Second, we will test whether hair cell death alters the adjacent supporting cells so that they become more responsive to reprogramming.

Comparison of Three Reprogramming Cocktails

Comparison of three reprogramming cocktails
Andy Groves, Ph.D., Baylor College of Medicine

Each cell type in the human body is defined by its activation of a unique combination of genes that endow each cell type with specific properties. The activation of these genes is achieved by special proteins known as transcription factors. These “switches” are responsible for turning on appropriate genes in one cell type and preventing inappropriate genes from being activated. In recent years, investigators have identified a number of these transcription factors that lead to the formation of hair cells in the inner ear. The goal of this project is to rigorously test the extent to which a cocktail of transcription factors is able to reprogram supporting cells of the inner ear to turn into hair cells.

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Growing Supporting Cells in Culture: Toward High-Throughput Screens

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Growing supporting cells in culture: toward high-throughput screens
Alain Dabdoub, Ph.D. University of Toronto
Andy Groves, Ph.D. Baylor College of Medicine
Neil Segil, Ph.D. University of Southern California

There is now a need for experimental systems to quickly test the candidates identified in Phase I experiments before moving into animal models and ultimately clinical trials. These researchers will develop a supporting cell culture system that can be used to quickly screen for factors that promote supporting cell growth or that promote supporting cells to generate hair cells. They will then test if this system can be used to grow cells at low density (allowing fewer cells to be used), to grow cells from older animals (necessary for a real drug), and to grow cells from the adult mouse utricle (which already shows limited regeneration). This project may give us the assay system needed to identify small molecule or gene therapeutics.

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A Cross-Species Approach Toward Functional Testing for Hair Cell Regeneration

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A cross-species approach toward functional testing for hair cell regeneration
Andy Groves, Ph.D. Baylor College of Medicine
David Raible, Ph.D. University of Washington
Jennifer S. Stone, Ph.D. University of Washington

This multi-modal, cross-species analysis project allows the HRP to test the role of molecules identified in our previous and ongoing experiments. Experience gained in the first year has led to first screening pathway inhibitors in chicks, then validating these initial hits by generating gene knockouts in zebrafish using CRISPR technology. Targets that pass both screens will then be assessed in the mouse. This project exemplifies the consortium’s shift toward Phase II of the HRP’s strategic research plan.

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A Cross-Species Approach to Functional Testing of Hair Cell Regeneration Pathways

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A cross-species approach to functional testing of hair cell regeneration pathways
David Raible, Ph.D. University of Washington
Andy Groves, Ph.D. Baylor College of Medicine
Jennifer S. Stone, Ph.D. University of Washington

This group has developed a multi-modal, cross-species analysis platform that will allow the HRP to test the role of molecules identified in our previous and ongoing experiments. Coupling gene knockout in zebrafish using the newly emerging CRISPR technology, which allows rapid testing, with pathway manipulation and gene knockouts in chicks and mice, we will be able to rapidly interrogate the many molecules the group wishes to examine. This project exemplifies the consortium’s shift towards phase II of the project.

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RNAseq Analysis of Mouse Auditory and Vestibular Supporting Cells Following Hair Cell Killing In Vitro in DTR Mice

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RNAseq analysis of mouse auditory and vestibular supporting cells following hair cell killing in vitro in DTR mice
Neil Segil, Ph.D. University of Southern California
Jennifer S. Stone, Ph.D. University of Washington
Andy Groves, Ph.D. Baylor College of Medicine

At the fall 2014 meeting, the HRP consortium realized that we lacked an important genomics dataset, which would characterize at a molecular level how supporting cells respond to immediate death of hair cells. Using a mouse model developed by Ed Rubel, an HRP investigator, this project will examine how RNA expression changes in mice whose hair cells are killed with an application of diphtheria toxin. These “DTR mice” respond to the diphtheria toxin because all of their hair cells are uniquely sensitive to the toxin because of expression of a specific receptor. We will be able to compare the responses of supporting cells to the toxin with responses to hair cell death from aminoglycosides, already carried out by this group.

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