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Hearing Restoration Project: Consortium Projects
Hearing Health Foundation (HHF) funds the Hearing Restoration Project (HRP) with the goal to determine how to regenerate human inner ear sensory hair cells and to eventually restore hearing in the millions of people with hearing loss. The HRP consists of leading hearing researchers who have dedicated their careers to understanding hearing and hearing loss.
Hearing works when sensory hair cells detect sound waves, then turn those waves into electrical impulses that are sent to the brain for decoding. In humans and mammals, once these sensitive hair cells are damaged or die, hearing is impaired. However, most species, including birds and fish, are able to spontaneously regrow hair cells and restore their hearing. HRP scientists are tasked with uncovering how to replicate this regeneration process in humans.
We have recently changed how we develop and fund research projects. At the HRP’s annual meeting in Seattle in November 2016, the HRP consortium decided to focus on multi-year projects, which permits researchers to commit to funding people in their labs for two to three years. This approach allows for continuity of the research projects, essential for their success, and was endorsed by the HRP’s Scientific Advisory Board (SAB) and HHF’s Board of Directors. (See Hearing Health Magazine, Winter 2017’s “The HRP Shifts Gears for Greater Impact”)

The outcome of the Seattle meeting was that HRP researchers agreed upon four major multi-year projects (numbers 1 through 4), which focus on single-cell sequencing, epigenetic DNA modification, bioinformatics, and gene testing. We also approved five projects (numbers 5 through 9) that fit into the larger HRP strategic plan outside the four major investigations. We are confident in this strategy shift and look forward to giving updates as the science progresses.
Project 1
Transcriptomics-based analysis of hair cell regeneration in 
Stefan Heller, Ph.D., Stanford University
Tatjana Piotrowski, Ph.D., Stowers Institute for Medical Research
Jennifer Stone, Ph.D., University of Washington
Mark Warchol, Ph.D., Washington University in St. Louis
Michael Lovett, Ph.D., Imperial College London
In this third-year project, we will disrupt hair cells, then analyze how gene expression changes in single cells from species that show robust hair cell regeneration. Heller’s lab will examine the consequences of aminoglycoside damage at the single-cell level in the chick utricle, while the Piotrowski lab will examine fish lateral-line cells. In another component, the project will add 
two time points to the chick cochlea and utricle bulk RNA-seq datasets that were generated by Lovett 
and Warchol and which are 
extremely valuable datasets for 
the HRP consortium.

Project 2
Epigenetics of mouse 
inner ear maturation 
and transdifferentiation
Neil Segil, Ph.D., University of Southern California; Jennifer Stone, Ph.D., University of Washington; Michael Lovett, Ph.D., Imperial 
College London
Many genes are turned off by chemical modifications (epigenetic marks) that prevent activation of the gene. One hypothesis is that mammals cannot activate a hair cell regeneration program after the first few postnatal days because the responsible genes have been epigenetically silenced. This second-year project looks at these epigenetic marks in the ear for every gene, during both early and late development. The investigators will use this analysis to find candidate promoter regions, which control gene activity. Preliminary data support the idea that supporting cells turn off expression of key hair cell genes 
(e.g., Atoh1), and so a plausible approach to triggering regeneration in the mammalian ear is to reverse such changes.

Project 3
Mouse functional testing
John Brigande, Ph.D., Oregon Health & Science University
This Phase II HRP project is entering into its third year. The research involves delivery of genes and reagents that could regulate hair cell regeneration into the embryonic inner ear of mice. Two approaches will be taken. In the first, transcription-factor genes that may activate regeneration pathways will be delivered, and we will then assess their ability to trigger hair cell regeneration after those cells are killed. In the second approach, CRISPR/Cas9 reagents will be used to turn off genes that may be preventing hair cell regeneration. Using experimental approaches, the project will allow us to determine whether any specific molecules are capable of triggering hair cell regeneration in the mouse that may lead to regeneration therapies.

Project 4
Implementing the gEAR for discretionary data sharing within the HRP
Ronna Hertzano, M.D., Ph.D., University of Maryland
Here the gEAR (gene expression for auditory research) portal will be further developed for a second year to perform key gene comparison tasks for the HRP. The gEAR allows for the graphic visualization of gene expression data in an intuitive way: Multiple datasets can be displayed on a single page, all represented by cartoons and dynamically colored based on levels of gene expression. Additional features will be added through dedicated developer time to specifically serve the needs of the consortium, including additional dataset-specific graphics, tools 
for cross-dataset and cross-species comparisons, and intuitive 
integration of DNA-structure and gene expression.

Project 5
Identification of candidate regulators of hair cell regeneration in the chick cochlea and utricle
Jennifer Stone, Ph.D., University of Washington
Mark Warchol, Ph.D., Washington University in St. Louis
This project has two components. In the first, the investigators will focus on validation of miRNA-seq data recently acquired by the HRP consortium; these new experiments will determine whether the genomics experiments accurately reflected miRNA fluctuations in the tissue, and may suggest candidate miRNA modulators of hair cell regeneration. In the second component, this project will test which specific signaling pathways are important for the proliferation of supporting cells and the regeneration of hair cells using pharmacological approaches.
Project 6
Fish epigenomics and enhancer screening
Tatjana Piotrowski, Ph.D., Stowers Institute for Medical Research
As explained in Project #2, many genes are turned off by chemical modifications (epigenetic marks) that silence genes and prevent their activation. This inactivation often occurs at enhancers, which are regions of DNA that control the activation of genes. In this project, Piotrowski will use the ATAC-seq and H3K27ac ChIP-seq methods, which were successful for the mouse inner ear, to find enhancers that are active during hair cell regeneration in the fish. Identification of regeneration enhancers will enable the HRP to examine epigenetic marks comparatively—to determine whether regenerating species, such as the zebrafish and chick, utilize different enhancers than non-regenerating species like the mouse, or whether these enhancers are inactive 
in mammals. 

Project 7
Fish CRISPR/Cas9 screen, enhancer screen
David Raible, Ph.D., University of Washington
The functional testing of candidate genes is essential for us to be able to wade through the dozens or hundreds of candidates that have been put forward from the Lovett-Warchol RNA-seq experiments. Raible has successfully developed a zebrafish CRISPR screen approach that allows for the rapid testing of candidate genes for their role in hair cell development and regeneration. 
Raible will continue in his characterization of genes that affect hair cell regeneration. In addition, the project includes a second aim that proposes to test candidate enhancers in the zebrafish.
Project 8
Integrative systems biology of hearing restoration
Ronna Hertzano, M.D., Ph.D., University of Maryland
The HRP consortium aims to identify factors that can either block or promote regeneration and has generated multiple genomics datasets from inner ears of regenerating and non-regenerating model organisms. This proposal aims to predict these causal factors by a detailed analysis of those datasets. The project’s two investigators—Hertzano, who is well versed in inner ear development and genomics, and Seth Ament, Ph.D., an expert in systems biology and neurobiology—will develop a quantitative model for the gene regulatory networks in hair cells and hair cell precursors that will allow them to predict key genes (e.g., master regulator transcription factors) that are associated with the ability to regenerate hair cells. They will use cutting-edge network biology approaches that integrate information about the preservation and divergence of gene co-expression across species and conditions, with information about the targets of hundreds of transcription factors. 

Project 9
Establishing the human utricle from surgical patients as a translational in vitro model for hair cell regeneration
Alain Dabdoub, Ph.D., University of Toronto
Stefan Heller, Ph.D., Stanford University
Michael Lovett, Ph.D., Imperial College London
These investigators will work with human utricles harvested during surgery to examine whether the response of human inner ear tissue to damage is similar to that of our mammalian model, the mouse. They will culture human utricles for extended lengths of time following damage with aminoglycoside drugs and determine whether the utricles show any proliferation of their supporting cells (making new supporting cells) or regeneration of their hair cells (making new hair cells). Mouse utricles have a limited ability to show proliferation and regeneration, and it is important to determine whether they are a good model for humans. The investigators also intend to examine gene expression in these human utricles to determine the molecular similarity of the cells of interest to their human counterpart cells.
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