2018 |
Gamm, David M Mullins, Robert Foster (co-PI) [⬀] Stone, Edwin M [⬀] Tucker, Budd A |
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. |
Disease Mechanisms in Best Disease
? DESCRIPTION (provided by applicant): Best disease, or vitelliform macular dystrophy, is a human macular degenerative disorder characterized by progressive and irreversible central vision loss. The disease is caused by a host of different (largely missense) mutations in the gene BEST1. Currently there is no treatment or cure for this condition. In this multicenter, multi-PI proposal, we will evaluate the pathophysiology of Best disease using RPE cells derived from induced pluripotent stem cells (iPSCs) from patients with known BEST1 mutations. Preliminary data show that these cells recapitulate at least some the phenotypes of human RPE cells with Best disease. We propose multidisciplinary experiments that will lead to a better understanding of this blinding disease, and that include: determining the pathology of Best disease patient-derived RPE cell chloride conductance and cellular electrophysiology; determining the pathological relationship between different mutations, and between dominant and recessive forms of Best disease; and evaluating gene therapy/genome editing as a potential treatment for Best disease. These important studies will pave the way for new treatments for this disorder.
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0.934 |
2018 |
Gamm, David M Zack, Donald J. [⬀] |
U01Activity 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. |
Screening For Molecules That Promote Photoreceptor Synaptogenesis @ Johns Hopkins University
PROJECT SUMMARY The NEI Audacious Goals Initiative is a bold effort to ?to restore vision through regeneration of neurons and neural connections in the eye and visual system.? One of the major roadblocks in mammalian photoreceptor transplantation experiments has been, and continues to be, the low efficiency of integration and synapse formation following transplantation of photoreceptor populations. In order to address this roadblock, and in response to RFA-EY-15-002 (which is directed at ?discovery-based approaches to identify unknown factors critical to the regeneration of neurons, guiding their axons to targets, and making new functional connections?), we propose to combine state-of-the-art stem cell methodologies with high content screening approaches to identify novel small molecules and molecular pathways that promote human photoreceptor axonal outgrowth and synapse formation. To accomplish this ambitious goal, we have assembled a multidisciplinary group of investigators who have years of experience in human retinal stem cell biology, retinal cell and molecular biology, high content screening (HCS) assay development and drug screening, axonal guidance, synaptic biology, and microfluidics. Members of the research team have already carried out screens that have successfully identified molecules that promote neurite outgrowth of murine retinal ganglion cells (RGCs) and other retinal neurons, and that increase synapse formation in cultures of human stem cell-derived neurons. For this project, we propose to extend this prior work and develop robust and reproducible in vitro neurite outgrowth and synaptogenesis assays using photoreceptors (PRs) obtained from human pluripotent stem cell (hPSCs) derived 3-dimensional optic vesicle-like structures, and then to use these assays to identify and characterize biologically and clinically relevant molecules. More specifically, SA1 will focus on the development and execution of a two-tiered in vitro screen designed to identify molecules that influence hPSC-PRs axon outgrowth and/or guidance; SA2 will focus on a screen to identify molecules that enhance hPSC-PR synaptic marker expression; and SA3 will focus on the development of assays to confirm functional PR synapse formation in culture. Successful completion of these aims and milestones will yield the first in vitro human assay system designed to rapidly screen and rigorously test molecules for their ability to promote hPSC-PR connectivity. This platform should not only accelerate efforts to achieve functional PR replacement in patients, but could also serve as a valuable human preclinical model system.
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0.901 |
2018 — 2021 |
Gamm, David M Roy, Sushmita (co-PI) [⬀] Saha, Krishanu [⬀] Skala, Melissa Caroline (co-PI) [⬀] |
U01Activity 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. |
Single Cell Profiling to Define Biomarkers of Photoreceptor Dysfunction After Gene Editing Within Psc-Derived Organoids @ University of Wisconsin-Madison
PROJECT SUMMARY Genome editors make targeted changes in the genome and hold great promise in both basic and translational research. Unfortunately, they often produce unwanted adverse effects, including genotoxicity, immune response, and reductions in cellular function. Therefore, screening for adverse events is essential for the development of safe genome editing therapies. Here we propose to develop a generalizable and scalable approach to define biomarkers for adverse events after delivery of a genome editor. Our strategy combines state-of-the-art, label-free optical metabolic imaging (OMI) to measure the physiological, functional, and high-content morphological status, with single cell transcriptomic profiling (scRNA-seq) and regulatory network-based methods to analyze single cell data. The inferred gene regulatory networks can be used to develop a small (~50) set of biomarkers for adverse events within functional cells. Proof-of-concept studies will focus on the retina, specifically on rod and cone photoreceptors (PR) within 3D optic vesicle (OV) organoids derived from human pluripotent stem cells (PSCs). Creation of this dataset and validation of this approach will leverage these bioengineering technologies toward the development of safer genome editing therapeutics. In Aim 1, we will adapt an existing imaging and culture platform to administer Cas9 genome editors into OVs. Cells will be edited with important PR master regulators and challenged with light and chemical perturbations to test functional phototransduction post genome editing. In Aim 2, we will discover gene regulatory networks and biomarkers associated with abnormal metabolism within normal and dysfunctional gene-edited OVs. We will perform scRNA-seq and OMI on metabolically-distinct, gene-edited OVs, and then map the gene regulatory network associated with adverse events within PRs. We plan to validate the biomarker panel with qPCR/immunocytochemistry (ICC) and electrophysiology. In Aim 3, we will test and refine the platform with novel sgRNAs and genome editors within the SCGE toolkit. And finally, in Aim 4, we will expand the platform to detect adverse events that occur only in cone PRs, which constitute a minority of PRs within the retina, yet are critical for human vision. By tackling a 3D, heterogeneous organoid culture, our approach will extend to more complex cultures. Thus, the impact of this work could be broad, with the potential to advance the development of genome editors administered to any tissue.
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0.958 |
2021 |
Ahern, Christopher A (co-PI) [⬀] Gamm, David M Gong, Shaoqin Pattnaik, Bikash Ranjan |
R24Activity Code Description: Undocumented code - click on the grant title for more information. |
Restoring Vision With High-Fidelity Nonsense Codon Correction @ University of Wisconsin-Madison
PROJECT SUMMARY/ABSTRACT Nonsense mutations cause approximately 15% of genetically inherited retinopathies and inherited human diseases in general, accounting for 2.5 to 3 million patients in the U.S. For certain specific genes, nonsense mutation incidences can be as high as 40%. Because nonsense mutations cause premature termination (PTC) of protein translation, the disease phenotype is often severe. Currently, there are only a limited number of therapies for nonsense mutations being tested in human clinical trials, including gene therapy, small molecule read-through drugs, or genome editing. Associated challenges equal the promises of each of these therapeutic options. Looking forward, newer technologies may address these hurdles and provide more safe and efficacious treatments for patients. During protein translation, tRNA functions at the ribosomal site to incorporate a specific amino acid into the polypeptide sequence. We aim to develop the next generation of nucleic acid therapy based on anticodon encoding transfer RNA (ace-tRNA) that incorporates the correct wild type amino acid at the site of a disease-causing nonsense mutation. Because of the many anatomical advantages afforded by the eye, we seek to test the broad applicability of ace-tRNA therapeutics for nonsense mutations that cause retinopathies and related blindness due to defects in a variety of genes, including those encoding ion channel proteins. Specifically we will focus on nonsense mutation in ion channels expressed in photoreceptors (PR) which convert retinal light inputs and retinal pigment epithelium (RPE), which provide support for PR. These two cell types are primarily the site of blindness pathogenesis. In this project, we will: 1) Develop ace-tRNA therapeutics that target specific nonsense mutations across several PR and RPE ion channels. 2) Engineer both viral and non-viral ace-tRNA delivery systems for long-term editing. Using these we will determine the functional outcome of ace-tRNA treatment using cultured cells and human iPSC-derived RPE and iPSC-PR retinal organoids. 3) Test both our viral and non-viral ace-tRNA in vivo using mice harboring genetic defects that cause blindness in humans; and 4) Assess the safety and bioavailability of ace-tRNA therapeutics in our preclinical NHP model systems. There are no FDA-approved therapeutic drugs that target channelopathies because of the complexities associated with precise post-translational modifications, carefully regulated expression, and assembly. Our team?s combined expertise in ace-tRNA development, nanomaterial synthesis, human pluripotent stem cell biology, ion-channel physiology, and pathophysiological model systems is unique and ideally suited to advance ace-tRNA technology toward clinical trials for a wide range of genetic diseases that cause blindness.
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0.958 |