Charles A. Gersbach, Ph.D. - US grants

[unreadable] DESCRIPTION (provided by applicant): Deregulation of the apoptotic pathway is responsible for resistance to cancer therapeutics designed to induce cell death. Although there have been many advances in understanding apoptosis, the molecular characteristics involved in resistance or sensitivity to specific apoptotic stimuli are unclear. The identification of genes, which influence the cellular response to apoptosis-based therapeutics will significantly advance the field of cancer cell biology and the rational design of effective anti-cancer strategies. In particular, this research will focus on the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), which selectively stimulates cell death in cancer cells but not normal cells. However the mechanisms by which normal cells and particular transformed cells obtain resistance to TRAIL remain poorly understood. Our long-term goal is to identify novel gene targets for the design of effective anti-cancer therapeutics. The rationale for the proposed work is that libraries of engineered transcription factors are a unique and powerful tool for identifying genes involved in regulating sensitization or resistance to apoptosis-inducing cancer therapies. The overall objective of this proposal is to identify genes to be targeted for improved apoptosis-based therapeutics. Our hypothesis is that genes identified by libraries of engineered transcription factors (ETFs) will modulate sensitivity and resistance to TRAIL-mediated apoptosis. The objective will be accomplished by testing our hypothesis with the following specific aims: (1) Identify genes that regulate sensitivity to TRAIL-induced apoptosis; (2) Identify genes that regulate TRAIL-mediated suppression of tumor development; (3) Evaluate the role of TRAIL receptor and decoy receptor expression in regulating TRAIL sensitivity. Libraries of ETFs have been recently developed by the sponsor's laboratory to activate or repress the expression of genes associated with complex cellular phenotypes. We will use this tool to identify genes that confer resistance or enhance sensitivity to TRAIL in cancerous and normal cells using an apoptosis-based selection strategy. Additionally, we will use these libraries in combination with a human xenograft tumor model to identify genes that regulate TRAIL activity in vivo. Finally, ETFs will be used to regulate TRAIL receptor expression and elucidate the role of these receptors in selective TRAIL activity. The genes identified in this work will serve as the basis for designing cancer therapies that effectively eliminate cancer cells with minimal effects on normal tissues. These treatments will dramatically enhance the quality of life of cancer patients by maximizing the health of non-cancerous organs during treatment, limiting the need for repeated treatments, and decreasing mortality resulting from ineffective cancer therapy. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The regeneration of damaged or diseased skeletal tissues remains a significant clinical challenge and cause for human disability and discomfort. Tissue engineering and regenerative medicine are emerging as promising strategies for treating these conditions by creating living tissue substitutes composed of cells, bioactive factors, and a biodegradable scaffold. Tissue engineering has been particularly successful in creating uniform tissues, such as skin and cartilage. However, many injuries and diseases affect the interfaces between tissues, such as the transition between bone and cartilage. New tissue engineering methods need to be developed to recapitulate the structure and function of these biphasic tissue interfaces. This project develops a method to deliver bioactive factors to cells in a precise spatial pattern within a three dimensional scaffold. As a result, stem cells seeded onto these scaffolds will be stimulated to produce different tissue types in predefined patterns. In particular, we will generate osteochondral tissues with human mesenchymal stem cells through spatially controlled gene transfer of differentiation factors. Precise spatial control will be enabled by innovative methods of biomaterial-mediated gene delivery and a microscale weaving technique for creating 3D polymer scaffolds. This work is significant to developing tissue engineering strategies for creating complex structures that recapitulate the heterogeneity and function of native tissue interfaces.

DESCRIPTION (Provided by the applicant) Abstract: Recent advances in the genetic reprogramming of mammalian cells are challenging the traditional concepts of cell differentiation and providing new avenues for regenerative medicine. These studies have shown that cellular transcription machinery can be manipulated to reprogram gene expression profiles and redirect cell behavior. Highlighted by recent progress in the development of induced pluripotent stem cells (iPSCs), the principles of genetic reprogramming are also generally applicable to controlling a wide variety of cellular phenotypes. In fact, many cell lineages are defined by a single master regulatory transcription factor, several of which are currently being used to control cell fate for gene- and cell-based therapies and other biotechnological applications. However, current methods for genetic reprogramming are limited by several inefficiencies, including a low frequency of reprogrammed cells, long times to achieve full reprogramming, and insufficient robustness of new cell phenotypes. To address these limitations, we propose to enhance the intrinsic properties of the reprogramming factors through directed molecular evolution. This represents a new conceptual direction in the area of genetic reprogramming and capitalizes on the recent progress in the area of directed evolution for enhancing protein function. Importantly, this approach is broadly applicable to improving the efficacy of any transcriptional regulation machinery and therefore will be beneficial to numerous applications and fields of biomedical research. The outputs of this work will include the discovery of transcription factor variants with enhanced reprogramming capabilities, insights into the biochemistry of morphogenetic transcription factors, and the development of new methods for controlling cell behavior. Additionally, this study will propel the field of directed evolution into the new areas of genetic reprogramming and regenerative medicine. This synergistic incorporation of technologies represents the innovation that will be necessary to translate the transformative advances of biomedical research into real benefits for human health. Public Health Relevance: Scientists are investigating methods to use naturally occurring proteins to coordinate complex cell behaviors, including tissue repair. Although this approach shows tremendous promise, these natural proteins are often insufficient for effectively directing cell activity. This proposal outlines a new and unique technology for evolving engineered versions of these proteins that will be more effective in generating robust cell sources for a variety of applications, including the treatment of cardiovascular disease, neurodegenerative conditions, and musculoskeletal disorders.

CBET 1151035/ Gersbach

This NSF CAREER award by the Biotechnology, Biochemical and Biomass Engineering program supports the development of a new technology for controlling the expression of genes in mammalian cells with light. Systems for controlling the expression of genes in mammalian cells have diverse applications in medicine, biopharmaceutical production, biotechnology, and basic science. However, current systems for gene regulation are not able to effectively control the spatial organization of gene expression within a population of cells or tissue. The positional control of gene expression is critical to many processes such as the engineering of complex tissues made of multiple cell types. Additionally, current systems only control genes that have been artificially added to cells, rather than controlling genes in their natural chromosomal context. To address these limitations, light-sensitive proteins from plants will be reengineered so that they control specific target genes in mammalian cells. The ability of these engineered proteins to control gene expression and catalyze specific changes to the genome sequence in response to light will be assessed in human cells. Patterns of light exposure will be used to engineer architecturally complex tissues in which the spatial organization of tissue development can be arbitrarily programmed. This work will develop new tools for engineers and scientists to design and study cellular systems with enhanced precision for applications in tissue engineering and studies of tissue development and cell-cell interactions. The theme of this research project will also be transformed into educational programs that foster enthusiasm and creativity in engineering and science education and are vertically integrated across elementary, middle and high school, undergraduate, and graduate levels. The program will be implemented in collaboration with the National Academy of Engineering Grand Challenges K12 Partners Program. This program will also be complemented by research education experiences in the PI's laboratory by high school and undergraduate students, curriculum development at the undergraduate and graduate level, and the inclusion of underrepresented groups across all levels of research and education.

DESCRIPTION (provided by applicant): Genome sequencing and the identification of epigenetic marks by projects such as ENCODE and the Epigenomics Roadmap Project have transformed biomedical research. Technologies for targeted manipulation of these epigenetic properties are necessary to transform the knowledge gained from these projects into tangible scientific advances and benefits for human health, such as gene therapies that modify the epigenetic code at targeted regions of the genome and the engineering of epigenome-specific drug screening platforms. To address this technology gap, we are developing a suite of well-characterized tools for custom locus- and cell type-specific modification of any epigenomic property with precise spatiotemporal control. These tools consist of fusion proteins of programmable DNA-binding proteins and enzymes that control genome structure and function. These epigenetic modifiers (EGEMs) can be specifically targeted to nearly any site in the genome. Optimized EGEM designs will be tested on both proximal and distal regulatory elements that represent diverse chromatin states, including active, repressive, bivalent, and imprinted marks. The generality of EGEMs will be shown on additional high-value targets that have broad relevance to disease. Importantly, all of these tools function independent of cell- and species-type, and therefore are useful to all fields of biologic research. Comprehensive characterization of EGEM activity in human cells will be provided by targeted and genome-wide analysis of DNA-binding, chromatin structure, and gene regulation. A validated optogenetic approach for controlling protein localization with blue light will be used to achieve precise spatiotemporal control of EGEM activity. The utility of the tool set of epigenetic modifiers will b demonstrated by impacting gene regulation in a manner that is robust, specific, and heritable. We will test the working hypothesis that different genes will require a customized set of epigenetic modification(s) to achieve efficient changes in gene expression. The specificity and stability of epigenetic modifications will be of broad utility to the fields of genomics, epigenomis, imprinting, gene therapy, developmental biology, regenerative medicine, and drug development.

DESCRIPTION (provided by applicant): Summary Osteoarthritis (OA) affects an estimated 27 million people in the United States and is characterized by joint pain and cartilage loss. One of the risk factors for developing OA is having particular genetic variations at certain locations in the genome, such as a specific single nucleotide polymorphism in the regulatory region of the growth and differentiation factor 5 (GDF5) gene. The mechanism by which a causal variant increases the risk for OA is unclear, partly because there is substantial genetic variation among OA patients and lifestyle differences also affect the development of OA. The production of cartilage tissue that is perfectly matched except for the causal variant in question would allow for insight into how a specific genetic variant affects the response of cartilage to OA stimuli such as inflammatory cytokines. Additionally, it would provide an important tool for in vitro drug screens that seek to identify candidate drugs optimized for patients with particular genetic profiles. We propose to develop a novel in vitro system for studying the functional effect of identified OA causal variants on the biochemical and mechanical properties of articular cartilage using genome editing of induced pluripotent stem cells (iPSCs) and cartilage tissue engineering. Our hypothesis is that user-defined precise genetic changes in iPSCs before subsequent chondrogenic differentiation will alter the cartilage production and render the tissue more susceptible to the pro-inflammatory cytokine interleukin-1 alpha (IL-1¿), which has been implicated in the pathogenesis of OA. Genome editing will be performed with engineered nucleases targeted to specific loci to stimulate gene targeting by homologous recombination. We will analyze the engineered cartilage for both biochemical composition and mechanical properties. Furthermore, we will also use RNA-Seq to detect how the genetic changes affect global gene expression. Our first aim will be to pursue the T/C single nucleotide polymorphism rs143383 of GDF5 as an initial example of this generally applicable technique. Our second aim will be to make cartilage that represents five different variants in a region of the genome that has been associated with an increased risk for OA by a genome-wide association study. One goal of this aim will be to determine which of the genetic changes is responsible for the increased OA risk. Together, this work will establish a rigorous in vitro system for testing the effect of particular genetic variants on OA development. This knowledge has the potential to provide a platform technology for discovering OA therapeutics that are matched to patients with particular genetic risk factors.

Project Summary/Abstract The current proposal seeks renewal of the biotechnology training program in Biomolecular and Tissue Engineering (BTE) at Duke University. The objective of the biotechnology training program in BTE is to provide enhanced classroom, laboratory, and research predoctoral training in the design, manipulation, and quantitative characterization of biomolecules, cells and tissues. The BTE training program involves 38 faculty, including 21 engineering faculty and 17 faculty from chemistry and biomedical sciences. To date, a total of 93 students have been supported by interdisciplinary predoctoral traineeships in BTE. The BTE training experience is enriched by (1) performing research that is interdisciplinary in nature and is central to the development of medical biotechnology, (2) including at least two BTE faculty on their doctoral dissertation committee to specifically provide mentorship in the area of medical biotechnology, (3) enrolling in a laboratory- based engineering course in modern biotechnology, engineering electives that provide breadth in BTE, and two advanced courses in the basic biomedical sciences relevant to BTE, (4) participating in four semesters of the interdisciplinary bioengineering seminar series and related journal club for credit, (5) participating in a special seminar series on career choices in the biotechnology industry, (6) engaging in a three-month industrial biotechnology internship, (7) presenting in the annual BTE poster session, and (8) undergoing training in responsible conduct in research. The value of this enriched training program has been demonstrated through student productivity and diverse career outcomes in the biotechnology industry. Moreover, the impact of the training grant has exceeded far beyond the individual trainees through student enrollment in our BTE Certificate Program and the leveraging of institutional resources for training activities for the broader Duke community. Our training program has also increased diversity and the participation of underrepresented groups. In our renewal, we have improved the program and its relevance to the modern biotechnology industry by adding faculty in molecular engineering and biomedical sciences, particularly faculty with a history of translational research. We have also added programmatic activities to enhance student interactions with visiting faculty and outreach opportunities. In this proposal we request the extension of our program to continue to integrate engineering, science, and medicine at Duke and create a unique training environment for our students.

DESCRIPTION: The regeneration of damaged or diseased skeletal tissues remains a significant clinical challenge and cause for human disability and discomfort. Tissue engineering and regenerative medicine are emerging as promising strategies for treating these conditions by creating living tissue substitutes composed of cells, bioactive factors, and a biodegradable scaffold. Tissue engineering has been particularly successful in creating uniform tissues, such as skin and cartilage. However, many injuries and diseases affect the interfaces between tissues, such as the transition between bone and cartilage. Many regenerative medicine studies that address these more complex tissues have been successful in engineering tissues in vitro with combinations of progenitor cells and biomaterials. However, these approaches typically require elaborate, laborious, and expensive procedures for cell isolation, expansion, and in vitro cell conditioning for proper cell differentiation and tissue formation. A primary challenge has been the translation of these promising preclinical studies into methods that are not only straightforward to practice but also effectively direct cell differentiation and tissue formation tat recapitulates the complexity of natural tissues. Technologies that capitalize on advances in cell engineering but minimize in vitro cell manipulation can greatly enhance the promise of regenerative medicine. This project develops a method to deliver bioactive factors to cells in a precise spatial pattern within a three dimensional scaffold. As a result, stem cells seeded onto these scaffolds or endogenous progenitor cells that infiltrate the scaffold will be stimulated to produce different tissue types in predefined patterns. In this study, we will first generate osteochondral tissues with human mesenchymal stem cells through spatially controlled gene transfer of differentiation factors. Precise spatial control will be enabled by innovative methods of biomaterial-mediated gene delivery and a microscale weaving technique for creating 3D polymer scaffolds. We will then expand this in vitro approach to a rabbit model of microfracture surgery, where the scaffold is populated by endogenous progenitor cells from the bone marrow that are instructed to form tissues in situ based on viral transgene delivery from the scaffold. This work is significant to developing tissue engineering strategies for creating complex structures that recapitulate the heterogeneity and function of native tissue interfaces and minimize in vitro cell manipulations.

? DESCRIPTION: Chronic cocaine abuse arises as a result of persistent cocaine-induced adaptations in the function of the neurons that comprise mesolimbocortical brain reward circuits. Cocaine-induced changes in gene transcription contribute to many of these alterations in neuronal function. Furthermore cocaine exposure has been shown to dynamically alter the epigenome by regulating the expression and/or function of histone and DNA modifying enzymes. Taken together, these data have led to the hypothesis that long-lasting changes in the epigenome may underlie the persistence of cocaine-induced addictive-like behaviors. However whether specific changes in chromatin regulation are truly causative for drug-induced behavioral plasticity has remained a challenging hypothesis to test due to the lack of high-throughput in vivo methods for site-specific experimental manipulation of the epigenome. To overcome this limitation we will generate two novel Cre/loxP-conditional CRISPR/Cas9- based transgenic mouse strains in which an enzymatically dead Cas9 protein fused either to the core histone acetyltransferase domain of p300 (dCas-p300) or the KRAB repressor domain (dCas9-KRAB) is knocked into the Rosa26 locus. We have shown that gRNA-mediated recruitment of dCas9 fusions with chromatin regulators is sufficient to induce targeted histone modifications and highly specific corresponding changes in gene transcription. Now by expressing these fusion proteins in transgenic mice, we will achieve regional and temporal control of site-specific epigenome editing in the brain in vivo by intersecting Cre-dependent induction of dCas9-fusion protein expression with stereotaxic viral delivery of validated gRNAs targeting cocaine- regulated enhancers. In the R21 phase of this proposal we will first generate and characterize the conditional dCas9-p300 and dCas9-KRAB mouse strains and then conduct a proof-of-principle experiment to validate whether dCas9-mediated regulation of Fosb in neurons of the nucleus accumbens is sufficient to alter cocaine- induced locomotor sensitization and conditioned place preference. In the R33 phase we will use the dCas9- p300 and dCas9-KRAB mouse strains to test the hypothesis that epigenetic sensitization of Bdnf transcription in dopaminergic neurons of the ventral tegmental area promotes incubation of cocaine craving, an important rodent model for relapse. Finally to accelerate future epigenome editing studies we will first use a novel method to capture nuclei of cocaine-activated neuronal ensembles in the nucleus accumbens for chromatin profiling and then develop and functionally validate gRNAs targeting cocaine-regulated enhancers for use by the broader scientific community. Our studies will provide a novel toolbox for functional epigenomic studies of the molecular mechanisms underlying substance use disorders.

? DESCRIPTION (provided by applicant) Gene therapy is a promising approach to treating Duchenne Muscular Dystrophy (DMD). However, current methods typically require the addition of extra dystrophin genes to the genome or the lifelong re- administration of foreign genetic material that works transiently to restore dystrophin expression, both of which have significant safety and practical concerns. Furthermore, these strategies have been limited by an inability to deliver the large and complex dystrophin gene sequence. An appealing alternative to these gene replacement approaches is the targeted repair of the endogenous mutant dystrophin gene. This concept, known as genome editing, represents a potential cure to DMD without the need for permanent integration of or repeated exposure to foreign biological material. Furthermore, it corrects the problem at the source by correcting the mutation to the naturally occurring dystrophin gene. Genome editing has been made a reality for human gene therapy by the recent development of transformative technologies that use engineered enzymes to cut and paste DNA sequences at specific sites in the genome. In fact, genome editing is now in clinical trials for treating cancer and HIV. The most recently developed genome editing technology, known as CRISPR, is much more robust than previous technologies and has rapidly transformed all areas of biomedical research and biotechnology in less than two years. Several efforts are underway to use CRISPR to correct genetic diseases, and we have demonstrated that it is possible to restore dystrophin expression in muscle cells from DMD patients. However, for this to be viable for clinical translation, we must demonstrate successful genome editing in skeletal and cardiac muscle tissue in animal models of the disease. In this study, we will use adeno- associated virus to delivery CRISPR to skeletal and cardiac muscles of a mouse model of DMD and a mouse model carrying the human dystrophin gene. The overall objective of this research proposal is to develop methods to restore dystrophin expression via targeted genome editing in vivo. The central hypothesis is that nuclease-mediated gene correction will lead proper dystrophin expression and function in mouse models of DMD. This research plan is innovative because it capitalizes on the unfulfilled potential of the CRISPR genome editing technology to address the fundamental limitations of conventional gene therapies and the unmet need for a safe and effective permanent cure to DMD. Importantly, this approach is also broadly applicable to numerous genetic diseases in addition to DMD. Thus in addition to identifying a lead candidate nuclease and delivery method for treatment of DMD, this work will also lead to additional development and refinements of the CRISPR technology to broadly benefit patients affected by hereditary disorders. Finally, the development of technologies for in vivo genome editing in skeletal and cardiac muscle will be broadly useful for biotechnology and basic scientific research.

Project Summary/Abstract The goal of this proposal is to develop a novel high-throughput platform for understanding gene regulatory elements in order to identify new drug targets for common diseases. The human genome encodes approximately 50,000 genes. Understanding how those genes are regulated and how this correlates to complex cell phenotypes has long been a major focus of our team. Follow-up projects to the Human Genome Project, such as the NIH-funded Encyclopedia of DNA Elements (ENCODE) and the Roadmap Epigenomics Project, have identified millions of putative regulatory elements across the human genome for many human cell types and tissues. Importantly, genome wide association (GWA) studies have strongly indicated that non- coding regulatory elements determine the gene expression patterns responsible for most complex diseases including cancer, cardiovascular disease, diabetes, and neurological disorders. However, the function of these regulatory elements and their relationships to these disease phenotype are largely unknown. Additionally, conventional screening technologies for perturbing cellular processes, such as small molecules and RNA interference, cannot directly target genomic regulatory elements. To address this critical limitation and illuminate the fundamental genomic basis of these cell phenotypes, we have recently developed epigenome- editing technologies for directly and precisely activating and repressing genomic regulatory elements in their natural chromosomal location. More recently, we have developed a novel and robust method for using these tools for high-throughput identification and quantification of gene regulatory element activity. Here, we propose to apply these methods to the discovery and validation of regulatory elements associated with cancer and cardiovascular disease as demonstration of this novel platform technology for understanding the genetic basis of complex disease. This technology will be critical to translating modern advances in genetics and genomics into new drug targets, diagnostics, and personalized medicine catered to each patient genome.   

There is a fundamental gap in understanding how the millions of known regulatory elements functionally contribute to gene regulation and phenotypes. Continued existence of that gap is an important problem because, until it is filled, it will remain extremely difficult to identify the genetic mechanisms underlying the thousands of observed genetic associations with disease phenotypes. Our long-term goal is to understand how and to what extent gene regulatory elements alter target gene expression and impact phenotypes. The objectives of this particular proposal are to functionally characterize all regulatory elements contributing to the differentiation of CD4+ T cells. In doing so, we will identify the causal regulatory mechanisms that modulate the immune system. The rationale for this work is that understanding those mechanisms will be the foundation for future efforts to therapeutically modulate the immune system, and will establish a discovery platform for determining the mechanisms underlying countless other model systems. Specifically, we will characterize three complementary components of regulatory element activity: (i) the capacity of regulatory elements to drive expression of a reporter gene, (ii) the effect of each regulatory element on the expression of one or more target genes, and (iii) the contributions of regulatory elements to phenotypic function, namely differentiation. We will accomplish those goals across three specific aims. In Aim 1, we will quantify the activity of all regulatory elements that have evidence of differential activity between subtypes of mouse CD4 T cells. We will do so using a capture-based high-throughput reporter assay that allows us to assay larger (>500 bp) fragments from specific genomic regions of interest. In Aim 2, we will quantify the effects of regulatory elements on target genes using a novel strategy that combines high-throughput CRISPR/ Cas9-based epigenome editing screens and targeted high-throughput single-cell RNA-sequencing. In Aim 3, we will determine which regulatory elements are necessary or sufficient for CD4 T cell differentiation using high-throughput CRISPR/Cas9-based epigenome-editing screens combined differentiation into particular CD4 T cell subtypes. Each aim will provide functional characterization of all of the regulatory elements implicated in CD4 T cell differentiation. Together, the aims will provide a comprehensive, multi-layered, and systematic understanding of the ways that gene regulatory elements modulate the immune system. The result will be an actionable set of targets for designing strategies to modulate immune system activity for therapeutic benefit. Because the approach is general to any model system, the same strategy can be readily transferred to diverse systems including differentiation and disease models. Therefore, we expect that this project will have both immediate and long-term benefit for determining the ways that regulatory elements contribute to health and disease.

PART 1: NON-TECHNICAL SUMMARY

Even with very advanced lithographic techniques, it is difficult or impossible for human engineering to reach down and control matter in the low nanometer length-scale. However, biological systems utilize the ability of molecules to recognize and bind to one another for self-assembling very complex structures with feature sizes down in the nanometer range. Taking inspiration from the rich diversity of functional architectures observed in biology and making use of new discoveries in engineered biomaterials, this project will expand basic understanding of biomaterials and their programmable assembly. Successful completion of the research will, in the short-term, enable significant progress in nano-scale self-assembly using proteins and nucleic acids. In the long-term, this will provide the needed technology for implementation of ever-smaller devices for computing, communications, and sensing applications; implantable medical devices and biosensors; and programmable, artificial molecular machines. This project will provide training opportunities for postgraduate and postdoctoral students in cutting-edge molecular engineering and bionanotechnology. Using NSF?s supplemental funding mechanisms, high school and undergraduate students will be attracted to the project to participate research and to train in the emerging field of nanoscience. This project will expand our scientific understanding of nanometer-scale phenomena and materials as well as to improve our ability to design and engineer new functional materials on this length-scale. The results of this project could impact future application areas in the sustainable fabrication of electronic devices and new approaches to medical diagnostics and therapeutics.


PART 2: TECHNICAL SUMMARY

DNA?s capacity for highly reliable and programmable molecular recognition has led to the field of DNA-based nanotechnology, also known as structural DNA nanotechnology. Researchers in this field develop materials and techniques for DNA-guided molecular and nano-scale self-assembly and have made remarkable recent progress, including the ordering of matter with unprecedented precision and parallelism, nano-scale organization of proteins and metal particles, as well as fascinating demonstrations of artificial molecular machines. One problem that has limited DNA nanotech?s translation from prototype demonstrations to commercially viable applications has been the lack of a general purpose, rapidly-reprogrammable method for functionalizing DNA structures with polypeptides. The Cas9 protein from the CRISPR RNA-directed bacterial immune system is used here as a novel solution to this problem. Cas9 is a programmable protein that acts as an endonuclease for cleaving non-self nucleic acid targets. In an engineered form, dCas9 has been mutated to bind a DNA sequence (specified by an RNA molecule) without cleavage of the targeted DNA. The dCas9 protein, guide RNA, and its target DNA sequences are well understood, modular, and programmable. Consequently, dCas9 is perfect for use in the development of a new family of molecular assembly tools with designed sequence specificities and the ability to act as a new smart glue for the programmed assembly of other nanomaterials including protein enzymes, affinity peptides, inorganic nanoparticles, and carbon nanotubes. The overarching goal of the project is to add the programmable recognition and binding functions of dCas9 to the growing toolbox of materials and methods available to DNA-based nanotech. Specifically, the project seeks to develop fusion proteins bearing mutant Cas9 domains to organize other polypeptides (including ligands, in vitro selected affinity peptides, enzymes, and inhibitors) on DNA nanostructures in programmed patterns for biomedical applications as well as in the bionanofabrication of electronic and photonic devices.

Recent strategies to reprogram cell lineage have the potential to transform in vitro disease modeling, drug screening, and gene and cell therapies for regenerative medicine. There has been a recent expansion in cell reprogramming methods following the discovery of reprogramming of somatic cells to pluripotency by overexpression of master transcription factors. However, it still remains a pertinent challenge to generate cell types with functionally mature phenotypes at high efficiency. To address this bottleneck, we are developing new strategies to identify and relieve barriers to cell reprogramming. We propose to utilize next-generation epigenome editing tools based on the programmable CRISPR/Cas9 system to modulate the endogenous epigenome, discover barriers to reprogramming and phenotypic maturation, and facilitate reprogramming outcomes. In this project, we will use CRISPR/Cas9-based epigenetic modifiers to remodel endogenous chromatin at genes encoding master regulatory factors to direct differentiation of human pluripotent cells and reprogram human fibroblasts to induced neurons. We will use this strategy to induce reprogramming with transient delivery of the synthetic epigenetic modifiers, thus avoiding any genetic manipulation of the starting cell population. We will then exploit the high-throughput capacity of the CRISPR/Cas9 system for (1) the unbiased identification of transcription factor combinations that maximize production of induced neurons and (2) the identification of key regulatory elements that govern neuronal cell fate specification. The insights gained from these studies will have broad relevance to improving cell reprogramming strategies and enhancing our understanding of cell differentiation and plasticity.

? DESCRIPTION: Chronic cocaine abuse arises as a result of persistent cocaine-induced adaptations in the function of the neurons that comprise mesolimbocortical brain reward circuits. Cocaine-induced changes in gene transcription contribute to many of these alterations in neuronal function. Furthermore cocaine exposure has been shown to dynamically alter the epigenome by regulating the expression and/or function of histone and DNA modifying enzymes. Taken together, these data have led to the hypothesis that long-lasting changes in the epigenome may underlie the persistence of cocaine-induced addictive-like behaviors. However whether specific changes in chromatin regulation are truly causative for drug-induced behavioral plasticity has remained a challenging hypothesis to test due to the lack of high-throughput in vivo methods for site-specific experimental manipulation of the epigenome. To overcome this limitation we will generate two novel Cre/loxP-conditional CRISPR/Cas9- based transgenic mouse strains in which an enzymatically dead Cas9 protein fused either to the core histone acetyltransferase domain of p300 (dCas-p300) or the KRAB repressor domain (dCas9-KRAB) is knocked into the Rosa26 locus. We have shown that gRNA-mediated recruitment of dCas9 fusions with chromatin regulators is sufficient to induce targeted histone modifications and highly specific corresponding changes in gene transcription. Now by expressing these fusion proteins in transgenic mice, we will achieve regional and temporal control of site-specific epigenome editing in the brain in vivo by intersecting Cre-dependent induction of dCas9-fusion protein expression with stereotaxic viral delivery of validated gRNAs targeting cocaine- regulated enhancers. In the R21 phase of this proposal we will first generate and characterize the conditional dCas9-p300 and dCas9-KRAB mouse strains and then conduct a proof-of-principle experiment to validate whether dCas9-mediated regulation of Fosb in neurons of the nucleus accumbens is sufficient to alter cocaine- induced locomotor sensitization and conditioned place preference. In the R33 phase we will use the dCas9- p300 and dCas9-KRAB mouse strains to test the hypothesis that epigenetic sensitization of Bdnf transcription in dopaminergic neurons of the ventral tegmental area promotes incubation of cocaine craving, an important rodent model for relapse. Finally to accelerate future epigenome editing studies we will first use a novel method to capture nuclei of cocaine-activated neuronal ensembles in the nucleus accumbens for chromatin profiling and then develop and functionally validate gRNAs targeting cocaine-regulated enhancers for use by the broader scientific community. Our studies will provide a novel toolbox for functional epigenomic studies of the molecular mechanisms underlying substance use disorders.

The structure of the human genome inside of a living cell is precisely and dynamically controlled to determine the level of each gene in different cell types, in response to environmental stimuli, and in various disease conditions. The chemical modification and three-dimensional folding of our DNA plays an even greater role than our inherited genetics in human development, disease progression, and drug response. There have been tremendous advances in mapping genome sequence and structure but our understanding of the relationship between genome structure and function is relatively poor. The objective of this proposal is to cross interdisciplinary boundaries to develop the necessary technologies to accurately predict, quantitatively monitor, and deterministically program genome structure to generate improved disease models that will catalyze transformative drug development. The team will also develop educational programs to inspire the next generation of scientists in this emerging discipline.

Innovative new technologies, including reporters of dynamic molecular structure and CRISPR/Cas9-based epigenome editing, provide a unique opportunity to monitor and perturb epigenetic states that govern chromatin structure. The team will generate new molecular tools for monitoring changes to epigenetic states in live cells using FRET-based nanosensors that report on structural changes to chromatin structure and mechanics in response to targeted perturbations by CRISPR/Cas9-based epigenome editing. This data will inform molecular models that predict the effect of epigenetic changes on chromatin structure and consequent changes in gene expression. These technologies and models will be validated in the context of human tumor models to illuminate the relationship between chromatin structure and cancer progression. Collectively, this will enable the development of new technologies and models that are broadly useful for disease modeling and drug screening.

This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.

The recent revolution in novel nucleic acid-targeting systems has generated incredible opportunities for treating disease by targeted manipulation of DNA and RNA. While the emphasis thus far has largely been on genome editing to treat rare, inherited disorders, this represents only one mechanism by which these DNA-targeting tools can be applied to improve human health. In fact, a significantly broader set of pathologies can be addressed by modulating gene regulation and epigenetic states, in contrast to altering underlying DNA sequences. Moreover, this approach has a number of advantages with respect to efficiency, safety, and reversibility. While several studies have demonstrated proof-of-principle that in vivo somatic cell epigenome editing can be used to program cell phentoypes and modulate therapeutic targets, there are a number of challenges that must be overcome to prepare this technology for treatment of human disease. First, an ideal DNA-targeting system that is facile and broadly applicable has yet to be developed. While CRISPR-Cas9 systems have dramatically transformed genome engineering, their application for human epigenome editing is limited by specificity, incompatibility with size-restricted viral vectors, and pre-existing immunity in the human population. Therefore, we will mine bacterial genomes for novel small CRISPR-Cas9/Cas12 systems that meet these criteria for in vivo epigenome editing. We will examine genome-wide specificity of epigenomic modifications with unbiased assays and assess both induced immunity in mouse models and pre-existing immunity in human samples. Second, it remains unclear in the field of epigenome editing which epigenetic modifications are necessary and sufficient to achieve desired outcomes in gene expression and genome structure. We will complete a comprehensive analysis of the relationship between epigenetic states and epigenome editing activity to develop a set of rules for achieving corresponding changes in gene expression. Finally, we will validate these epigenome editing tools in vivo in a set of pilot experiments in mouse models of neuromuscular disease encompassing a representative set of epigenomic states. In close collaboration with the Somatic Cell Genome Editing Consortium, this work will prepare epigenome editing technology for human clinical translation in which it may have a transformative effect on a broad array of both rare and common disease.

Abstract: Genome editing technologies have significant potential to treat a variety of devastating human diseases and disorders. However, there are a number of challenges that genome editing therapies must overcome to reach their full promise. Specifically, there are many possible adverse consequences that are unique to genome editing tools, such as genome integrity, immune responses, and loss of therapeutic efficacy due to cell turnover, for which there are currently are no optimal systems for rigorous assessment. Moreover, these consequences are unique to human physiology, genome sequence, and immune systems, and therefore typical animal models are not completely informative. To address this unmet need, we have assembled a team of collaborative investigators that have developed advanced genome editing strategies and methods for engineering human microphysiological tissue systems that recapitulate human physiology and function, with an emphasis on skeletal and cardiac muscle. We will combine these technologies in this proposal to systemically evaluate tissue physiology, genomic alterations, tissue regeneration, and immune response in response to various genome editing strategies and delivery methods. Specifically, this will include comprehensive and unbiased mapping of unintended modifications to human genome sequences, including at on-target and off-target sites. We will also determine the role of resident tissue stem cells, cell turnover, and tissue injury and regeneration in the stability of genome editing. Finally, we can incorporate immune cells into these microphysiological tissues to understand the consequences of immunity to bacteria-derived genome editing components. Collectively, this proposal will develop a platform to systematically address the most significant challenges to realizing the transformative potential of genome editing therapies in human tissues.

Understanding the genetic causes of human disease has immense potential to benefit human health. The human genetics community has devoted tremendous resources to identifying those causes, including, most recently, whole genome sequencing of patient cohorts. Those studies have found genetic variation in non-coding regions of the genome to be most often associated with diseases and drug responses. Unfortunately, since the effects of genetic variation on gene regulation remain poorly understood and difficult to study at the genome-wide scale, the full benefit of most of those studies has yet to be realized. Our long-term goal is to understand how non-coding genetic variants act through gene regulatory elements to influence phenotypes. The objective of this proposal, a step towards that long-term goal, is to develop a platform of empirical and statistical methods to reliably and systematically determine the regulatory mechanisms underlying human traits and diseases. Specifically, in Aim 1, we will use high- throughput reporter assays to quantify the effects of millions of human genetic variants on regulatory element activity. Those variants will represent diverse human ancestries, and will cover over 60% of all regions associated with a trait or disease via GWAS. The outcome will be the most extensive catalog of human regulatory variation every created. In Aim 2, we will develop new technologies to systematically relate those changes in regulatory element activity to changes in gene expression. That technology will combine our previous work developing CRISPR-Cas9- based epigenome editing screens with targeted single-cell RNA-seq. In Aim 3 we will develop statistical analyses to integrate the effects of regulatory variants to infer changes in gene expression and differences in phenotypes between individuals. The resulting method will be analogous to gene based association tests, but for the noncoding genome. The expected outcomes of this project are (i) dramatically improved ability to establish mechanisms underlying non-coding associations with human traits and diseases; (ii) better understanding of the genetic architecture of regulatory element activity and gene regulation that will guide the design and interpretation of future genetic association studies; and (iii) novel reagents, protocols, and software that other labs can use to complete similar investigations of their own model systems of interest. Taken together, we expect that this project will be a major step towards fully realizing the potential of genome wide and whole genome association studies.

ABSTRACT Large scale genome annotation consortia such as ENCODE, Epigenomics Roadmap, and others have identified millions of putative regulatory elements. We now need to focus efforts on comprehensively characterizing and quantifying the function of those elements, and noncoding variants that map within these regions, on gene expression and cell phenotypes. Our long-term goal is to assign function to every regulatory element and noncoding variant in the human genome, understand how that function changes in different contexts, and use that information to better understand cell fitness, disease mechanisms, cell lineage specification, and tissue homeostasis. To accomplish this goal, we have developed multiple novel high-throughput CRISPR-based technologies for characterizing the function of putative gene regulatory elements by perturbing their activity in their endogenous, native context. We have coupled these methods with single-cell RNA-seq to identify the target gene(s) for each regulatory element. We have also developed dCas9 effector mice to characterize elements in their natural in vivo context. In addition, we have developed population-based high-throughput reporter assays (POP-STARR) to characterize the impact of noncoding genetic variation across the entire genome. The objective of this proposal is to apply and share our compendium of complementary, robust, scaleable, and well-characterized methods by working collaboratively to support the IGVF Consortium goals of understanding how genomes and genomic variation function and orchestrate complex phenotypes. Our track record in developing, applying, and sharing these high-throughput characterization methods, as well as providing access to all data, supports that we will be successful in accomplishing our objective via the following specific aims: Aim 1. Characterize all gene regulatory elements essential for cell survival. Aim 2. Characterize all gene regulatory elements essential to cell lineage specification. Aim 3. Characterize all gene regulatory elements in select eQTL regions. Aim 4. Characterize all non- coding elements essential to tissue homeostasis in a mouse model. We will make all data immediately available, as well as share comprehensive protocols, reagents, and analysis tools to the scientific community. Together, the diverse approaches of this Characterization Center will lead to transformative progress in understanding the role of regulatory elements and noncoding variants across many diverse phenotypes.