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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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Integrative Analysis

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Integrative Analysis
Seth Ament, Ph.D. (co-chair), University of Maryland
Ronna Hertzano, M.D., Ph.D. (co-chair), National Institute on Deafness and Other Communication Disorders
Albert Edge, Ph.D., Mass Eye & Ear
Stefan Heller, Ph.D., Stanford University
David Raible, Ph.D., University of Washington
Jennifer Stone, Ph.D., University of Washington

This group will take the lead on data curation and analysis. A dedicated full-time HRP analyst is working across groups to help collect and process data, thereby facilitating a broader analysis of cell states and trajectories across species. The group will start by annotating hair cell types from all species so that anyone in the field can assess what kind of hair cell their regeneration approaches may produce, while also easing identification of common hair cell genes, which will help the Cross-Species Epigenetics group. Analysis of the hair cells produced in mouse organoids will be performed as an example. The Ament lab will leverage their expertise in bioinformatics, while the Hertzano lab will continue to oversee upkeep of gEAR, with a goal of making it even easier for HRP members to post their new data and for others in the community to analyze those data. The Edge lab will take the lead on the development of organoids as a screening platform for the future. The Heller, Hertzano, Raible, and Stone labs will validate markers by in situ hybridization across species, and all working group members will help direct the analysis.

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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.

Integrative Systems Biology of Hearing Restoration

Integrative Systems Biology of Hearing Restoration
Seth Ament, Ph.D., University of Maryland School 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

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.

Detection of Transcriptome Changes in Single Cells After Aminoglycoside-Induced Hair Cell Loss in the Chicken Basilar Papilla

Detection of Transcriptome Changes in Single Cells After Aminoglycoside-Induced Hair Cell Loss in the Chicken Basilar Papilla
Stefan Heller, Ph.D., Stanford University

Birds robustly regenerate their cochlear hair cells through the conversion of dormant supporting cells into new hair cells. Our project uses selective, high-sensitivity methods to reveal the molecular changes in supporting cells after their activation by, for example, ototoxic drugs that cause hair cell death. By examining the responses of many single cells, we have begun to identify triggers that initiate, execute, sustain, and ultimately terminate the regenerative process. Recent experiments have confirmed what we already knew, that the two halves of the chicken cochlea (neural and abneural) predominantly use different regeneration mechanisms. Using bioinformatics methods to process the resulting data, this year we are focusing on analyzing chicken cochlea cell populations isolated at various time points during hair cell regeneration and specifically characterizing the two distinct responses. By examining regeneration in an animal that replaces hair cells after damage, we will be able to find triggers that may be activated in mammals to reverse hair cell loss.

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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.

Implementing the gEAR for Data Sharing Within the HRP

Implementing the gEAR for Data Sharing Within the HRP
Ronna Hertzano, M.D., Ph.D., University of Maryland School of Medicine

One of the successes of the HRP has been the development of the gEAR portal (gene Expression Analysis Resource, umgear.org). The gEAR has many public and private datasets, and these complex datasets can be compared by scientists without the need for sophisticated programming expertise. The gEAR is also the primary data sharing, visualization, and analysis tool for auditory researchers outside of the HRP, becoming a platform that supports the hearing research community at large. This year we will build on past successes, continuing to support data upload, develop new visualization tools, and further enable the greater research community to exploit this resource.

Mouse Model Systems to Interrogate Candidate Genes for Sensory Hair Cell Regeneration

Mouse Model Systems to Interrogate Candidate Genes for Sensory Hair Cell Regeneration
John Brigande, Ph.D., Oregon Health & Science University

As HRP scientists detect and characterize genes that are hypothesized to participate in the activation or inhibition of hair cell regeneration, methods for altering or disrupting those genes are critical to the demonstration of their importance. This project aims to couple together two sophisticated methods for manipulating genes in mice, the so-called CRISPR-READI and i-GONAD methods. CRISPR-READI enables efficient, large gene edits, and i-GONAD simplifies the delivery of reagents for gene editing. Together, the proposed CRISPR-READI-GO method should allow for rapid and efficient gene editing to be put to use by HRP investigators.

Integrative Systems Biology of Hearing Restoration

Integrative Systems Biology of Hearing Restoration
Seth Ament, Ph.D., University of Maryland School of Medicine

This project will focus on integrating multiple datasets from the HRP to gain insight into hair cell development and regeneration and prioritize specific "driver" genes that can be targeted to induce regeneration. The main premise is that we will be able to regenerate hair cells if we activate the correct set of hair cell–promoting genes in supporting cells. This process is called transdifferentiation, and it occurs naturally in species such as birds and fish, but not in the inner ear of adult mammals. HRP researchers have generated numerous genomic datasets that describe cochlear development and transdifferentiation in multiple species. By analyzing all of these data together using sophisticated network analysis tools, we aim to identify which genes are involved in these processes as well as key differences that may explain the inability of human and mouse cells to transdifferentiate. Finally, this will enable the identification of genes that can be targeted to enable transdifferentiation.

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.

Detection of Transcriptome Changes in Single Cells After Aminoglycoside-Induced Hair Cell Loss in the Chicken Basilar Papilla

Detection of Transcriptome Changes in Single Cells After Aminoglycoside-Induced Hair Cell Loss in the Chicken Basilar Papilla
Stefan Heller, Ph.D., Stanford University

Birds regenerate cochlear hair cells by activating dormant supporting cells. This project builds on innovative methods and findings to study how supporting cells are activated when ototoxic drugs cause hair cell death. The project uses single cell analysis (during which we study the complement of genes that are active in many individual cells) to identify triggers that initiate, execute, sustain, and ultimately terminate the regenerative process. Using bioinformatics methods to process the resulting data, we will focus this year on the analysis of the chicken cochlea cell populations isolated at various time points during hair cell regeneration, which will reveal the molecular steps that occur during hair cell regeneration. We have already identified a first candidate gene signaling pathway that may regulate the regenerative process in the chicken cochlea, and we will be confirming that this pathway plays a role in hair cell regeneration. Interestingly, this pathway is not active during inner ear development, which sets it apart from other pathways linked to chicken (and zebrafish) hair cell regeneration; the other pathways are involved in cell development and may not represent a unique regenerative trigger. Finding triggers that specifically control regeneration may be an important stepping stone on the path to developing cures for hair cell loss in mice and, eventually, humans.

Epigenetics Analysis of Maturation and Regenerative Responses in the Mouse Organ of Corti and Utricle (2019)

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

Although hair cell regeneration does not occur in mammals, newborn mice harbor a latent capacity for some regenerative responses. However, this capability disappears within the first few weeks of life. This observation provides an experimental window that this proposal exploits to address fundamental questions about the failure of hair cell regeneration in mammals. Specifically, we propose experiments to identify those changes in the genetic material, the chromatin, that are responsible for orchestrating the differentiation of new hair cells within the newborn organ of Corti in the inner ear, and to investigate the changes in the chromatin that lead to the failure of regeneration in the adult mammalian inner ear.

Implementing the gEAR for Data Sharing Within the HRP

Implementing the gEAR for Data Sharing Within the HRP
Ronna Hertzano, M.D., Ph.D., University of Maryland School of Medicine

When a group of geographically dispersed scientists collaborate on hair cell regeneration in three different animal models—chicken, zebrafish, and mouse—and use multiple methods to track how genes “instruct” cells (multi-omics), an enormous amount of data results. The work of visualizing, conceptualizing, and analyzing these data presents a considerable challenge, and as technology has advanced, much of the multi-omic data is generated at the single cell level, resulting in datasets and files that are too big to process with traditional tools, such as Excel worksheets. The gEAR portal (gene Expression Analysis Resource, umgear.org) responds to this need by enabling meaningful visualization and analysis of these complex datasets in the public or private domain—no advanced programming skills required. It has also evolved to become a primary data sharing, visualization, and analysis tool for auditory researchers outside of the HRP to become a platform that supports the hearing research community at large.

Mouse Model Systems to Interrogate Candidate Genes for Sensory Hair Cell Regeneration

Mouse Model Systems to Interrogate Candidate Genes for Sensory Hair Cell Regeneration
John Brigande, Ph.D., Oregon Health & Science University

Given what the HRP has discovered thus far, this project proceeds from the assumption that manipulation of multiple genes that regulate diverse signaling pathways may be required to reprogram supporting cells to become functional hair cells. We must first verify the expression pattern of candidate genes in the inner ear, then develop methods that allow us to modulate multiple candidate genes in each supporting cell in order to transmit the genetic instructions that trigger regeneration. This proposal’s first goal is to establish improved genome editing via oviductal nucleic acids delivery (iGONAD) technology, allowing us to identify as well as eliminate candidate genes. Our second goal is to turn on multiple genes simultaneously in the same cell using a mouse model. The overall objective is to establish a rapid method to describe candidate gene expression and function in the inner ear at any stage, and to define an approach to determine which genes are critical to modulate signaling pathways for hair cell regeneration.

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.

Transcriptome Changes in Single Chick Cells

Transcriptome changes in single chick cells
Stefan Heller, Ph.D., Stanford University

Chicks regenerate hair cells in auditory and vestibular organs after damage, making them a valuable animal model to study the signals controlling hair cell regeneration. This project aims to identify changes in gene expression after hair cell loss in the chick cochlea and vestibular system. The collected data will be comprehensive because it will cover all detectable expressed genes. Subsequent data analysis will focus on establishing a sequence of gene expression changes that we hypothesize will correlate with important steps of the hair cell regeneration process. These steps include the signals that initiate, execute, sustain, and ultimately terminate the regenerative process. Comparison among the different organs and across species through collaborations with other HRP investigators will allow us to draw conclusions about species-specific specialized mechanisms as well as more general processes that control hair cell regeneration.

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Epigenetics of the Mouse Inner Ear

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Epigenetics of the mouse inner ear
Michael Lovett, Ph.D., Imperial College London
David Raible, Ph.D., University of Washington
Neil Segil, Ph.D., University of Southern California
Jennifer Stone, Ph.D., University of Washington

Inner ear supporting cells from newborn mice harbor a latent capacity for some regenerative responses, but these disappear within the first few weeks of life. This observation provides an experimental window this proposal will exploit to address fundamental questions about the failure of hair cell regeneration in mammals. Specifically, we propose experiments to identify those changes in the genetic material, the chromatin, that are responsible for orchestrating the differentiation of new hair cells within the perinatal organ of Corti; and investigating the changes in the chromatin, the epigenome, that lead to the failure of regeneration in the adult inner ear.

Signaling Molecules Controlling Avian Hair Cell Regeneration

Signaling molecules controlling avian hair cell regeneration
Jennifer Stone, Ph.D., University of Washington

HRP members have spent the past three years gathering information about genes that are turned on or off after inner ear hair cell damage in the chick, fish, and mouse. Some of these genes may encode therapeutic agents that can be applied to stimulate hair cell regeneration in humans. Our HRP studies and others have found that five signaling pathways (Wnt, VEGF, BMP4, Notch, and FGF) are important regulators of hair cell regeneration in the chick cochlea (the basilar papilla). The expression and activity of these pathways change significantly after hair cell damage, and the experimental manipulation of activity in each pathway either boosts or dampens hair cell regeneration. Furthermore, each pathway shows distinct regional expression patterns in the basilar papilla, which implicates it in either mitotic regeneration or non-mitotic regeneration—two distinct ways in which hair cells are replaced after damage. Studies in other growing tissues demonstrate that these five pathways regulate one another in temporally and spatially restricted patterns, in order to coordinate cell growth, differentiation, and patterning. Thus, it is likely that any therapy leading to safe and stable hair cell regeneration will require coordinated manipulation of more than one gene or pathway in the cochlea. In this study, we propose to begin to determine how these five powerful pathways interact to enable and control hair cell regeneration in the chick basilar papilla after hair cell damage.

Integrated Systems Biology of Hearing Restoration

Integrated systems biology of hearing restoration
Seth Ament, Ph.D., University of Maryland School of Medicine

The goal of this proposal is to support the HRP through data integration and systems biology. We propose two related goals for 2018, based on our preliminary network modeling results and discussions with other HRP investigators. We will (a) predict regulatory genes driving cell fate decisions in the developing mouse cochlea using refined transcriptional regulatory networks, gene co-expression networks, protein-protein interaction networks, and related methods. We will (b) extend these analyses to the zebrafish and chick models by projecting networks from the mouse cochlea onto data from these other species. Our goal is to generate testable predictions about driver genes and perturbations (deviations) that could influence hearing restoration.

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