Nenad Bursac - US grants

[unreadable] DESCRIPTION (provided by applicant): Implantation of exogenous donor cells into the heart (cellular cardiomyoplasty) is an emerging methodology for the treatment of post-ischemic ventricular remodeling. While initial clinical implantations of autologous skeletal myoblasts and bone marrow cells have been promising, the intrinsic potential of these cells to affect electro-mechanical function of surrounding heart tissue has still not been elucidated. Therefore, the main goal of this proposal is to systematically study the ability of different donor cells (i.e. skeletal myoblasts and mesenchymal stem cells, as compared to control cardiac myofibroblasts) to propagate electro-mechanical activity through a host cardiac network in vitro. We hypothesize that: 1) propagation of electrical activity through donor cells depends on their type and stage of differentiation, as well as the presence of not only electrical but also mechanical junctions between the donor-donor and host-donor cell pairs, and 2) electro- mechanical propagation in the donor cell implant can be improved through upregulation of the intercellular communication by specific growth factors. To test these hypotheses we propose to study electrical conduction through donor cells using a geometrically simplified, reproducible one-dimensional setting, i.e. the micropatterned cardiomyocyte strands with inserts made of donor cells. The propagation of cell membrane potentials and intracellular calcium transients in donor cells will be optically mapped in the presence of various differentiation agents and growth factors. Obtained results will be correlated with those from immunohistochemical and molecular analyses. The findings from this proposal are expected to elucidate potential of different donor cells to functionally integrate in the heart. Eventually, the proposed experimental framework will allow us to perform high throughput in vitro analysis of the factors that can improve electromechanical propagation of different donor cells. The final aim is to aid in the development of efficient and safe cell-based approaches for the treatment of regional heart injury due to ischemia, infarction, or congenital defects. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Implantation of exogenous donor cells into the heart (cellular cardiomyoplasty) is an emerging methodology for the treatment of post-ischemic ventricular remodeling. While initial clinical implantations of autologous skeletal myoblasts and bone marrow cells have been promising, the intrinsic potential of these cells to affect electro-mechanical function of surrounding heart tissue has still not been elucidated. Therefore, the main goal of this proposal is to systematically study the ability of different donor cells (i.e. skeletal myoblasts and mesenchymal stem cells, as compared to control cardiac myofibroblasts) to propagate electro-mechanical activity through a host cardiac network in vitro. We hypothesize that: 1) propagation of electrical activity through donor cells depends on their type and stage of differentiation, as well as the presence of not only electrical but also mechanical junctions between the donor-donor and host-donor cell pairs, and 2) electro- mechanical propagation in the donor cell implant can be improved through upregulation of the intercellular communication by specific growth factors. To test these hypotheses we propose to study electrical conduction through donor cells using a geometrically simplified, reproducible one-dimensional setting, i.e. the micropatterned cardiomyocyte strands with inserts made of donor cells. The propagation of cell membrane potentials and intracellular calcium transients in donor cells will be optically mapped in the presence of various differentiation agents and growth factors. Obtained results will be correlated with those from immunohistochemical and molecular analyses. The findings from this proposal are expected to elucidate potential of different donor cells to functionally integrate in the heart. Eventually, the proposed experimental framework will allow us to perform high throughput in vitro analysis of the factors that can improve electromechanical propagation of different donor cells. The final aim is to aid in the development of efficient and safe cell-based approaches for the treatment of regional heart injury due to ischemia, infarction, or congenital defects. [unreadable] [unreadable] [unreadable]

[unreadable] DESCRIPTION (provided by applicant): Cardiac tissue injury during myocardial infarction often leads to congestive heart failure. The field of tissue engineering offers the promise of generating a muscle patch that would structurally and functionally repair tissue damage resulting from infarction or congenital heart defects. However, current cardiac tissue engineering techniques suffer from a number of limitations that preclude their use in clinical applications. In particular, safe and efficient repair of myocardial infarction requires that engineered cardiac tissue patch: 1) mimics the anisotropic (aligned) architecture of native cardiac muscle and 2) exhibits sufficient thickness (multiple muscle layers) in order to prevent dilation of the heart and improve its contractile function. Nevertheless, the method to engineer a 3D tissue patch with a cm2 area and uniform cell alignment throughout its volume is still non-existent, even for patches as thin as 50 [unreadable]m. This proposal will test the hypothesis that cultivation of cardiac cells within microfabricated porous hydrogel networks will improve the diffusion of nutrients and oxygen to embedded cells while simultaneously enabling control over local cell alignment. The specific aims of this project are: 1) to develop methods to micropattern and stack thin porous cell/hydrogel networks into a relatively thick anisotropic cardiac tissue patch that will be cultured in a rotating bioreactor and 2) to assess the electrical and mechanical function of the resulting cardiac patch as a function of its thickness and micropatterned pore geometry. In the future, the methods developed in this study will be applied to clinically relevant cell types (e.g. embryonic stem cell-derived cardiomyocytes, skeletal myoblasts, mesenchymal stem cells, resident cardiac progenitor cells), and the resultant tissue patches will be assessed in animal studies for their ability to repair cardiac tissue damage and prevent the onset of heart failure. [unreadable] [unreadable] [unreadable]

DESCRIPTION (provided by applicant): The engineering of three-dimensional (3D) bioartificial skeletal muscle holds promise for the treatment of a variety of muscle diseases and injuries, including muscular dystrophy, traumatic muscle damage, prolonged denervation, and cardiac infarction. To improve impaired muscle function, bioartificial skeletal muscle should survive and rapidly vascularize and innervate in vivo while containing sufficient numbers of aligned muscle fibers to generate the necessary contractile force. However, state-of-the-art engineered skeletal muscle tissues consist of only a few hundred ¿m thick sheets or muscle bundles that generate active forces too small to be clinically used for direct repair of muscle damage. Therefore, we propose to develop a novel, reproducible tissue engineering approach to fabricate relatively large skeletal muscle tissues made of aligned and differentiated muscle fibers that generate forces comparable to those of native muscle. To achieve this goal, we will integrate expertise in 3D tissue microfabrication and muscle mechanotransduction with non-invasive imaging of tissue growth and function in vitro and vascularization in vivo. Specifically, we will: 1) fabricate porous aligned skeletal myoblast networks using a cell/hydrogel micromolding approach and by stacking multiple networks create thicker skeletal muscle constructs, 2) enhance the functional properties of the muscle constructs using optimized regimens of electromechanical stimulation, and 3) endothelialize the muscle constructs with different pore sizes to optimize for construct survival and force production after implantation in a rat dorsal skinfold chamber. The obtained knowledge and technologies developed in this proposal can be applied in the future to create other tissues with complex architecture. PUBLIC HEALTH RELEVANCE: A variety of muscular diseases and injuries, including muscular dystrophy, craniofacial defects, traumatic injury and cardiac infarction would benefit from the implantation of a functional bioartificial muscle. This proposal describes a novel tissue engineering approach to fabricate relatively large bioartificial muscle tissues made of aligned and differentiated muscle fibers with potential to be used for experimental studies and tissue replacement therapies.

DESCRIPTION (provided by applicant): Irreversible tissue damage during myocardial infarction often leads to congestive heart failure. Implantation of exogenous cells in the heart, either as a cell suspension or tissue patch, is proposed for treatment of post-infarction disease. Although promising, initial clinical trials with injection of skeletal myoblasts and bone marrow derived stem cells have yielded only marginal improvements in cardiac function, thus prompting the quest for a better cell source. Novel, actively pursued cells are cardiogenic in nature and include adult resident cardiac stem cells (CSCs), embryonic stem cell derived cardiomyocytes (ESC-CMs) or cardiovascular progenitor cells (ESC-CPCs), and recently, cardiac progenitors isolated from induced pluripotent stem cells (iPSCs). Of the considered cell types, CSCs and ESC- or iPSC-derived cardiovascular progenitors can differentiate into cardiomyocytes, vascular smooth muscle, and endothelial cells and, at least in theory, may reconstitute both cardiac muscle tissue and its vasculature. Therefore, in this proof-of-concept study we propose to test the ability of mouse ESC-CPCs to form a functional cardiac tissue patch when placed in a 3-dimensional cardio-mimetic environment. Our main hypothesis is that, owing to the presence of supporting endothelial and smooth muscle cells, the ESC-CPC patches will exhibit superior structure and function compared to patches made of pure cardiomyocytes (ESC-CMs). Specific aims of this project are to: 1) develop a tissue culture bioreactor for electro-mechanical stimulation of engineered patches that mimics the contraction phases of the native cardiac cycle, 2) apply this cardio- mimetic stimulation to ESC-CPCs embedded in hydrogel/nanofiber biomaterials to create aligned and differentiated cardiac tissue patches, and 3) systematically assess electrical propagation and generated contractile forces in the obtained ESC-CPC patches in comparison to patches made of pure ESC-CMs. Towards the end of the project, the optimized design rules for engineering the ESC-derived cardiac tissue patch will be tested with mouse iPSC-CMs. In the future, this 3-dimensional cardiomimetic culture system will be applied as a reproducible test bed for studying the potential of different stem cells to undergo cardiogenesis in vitro. The obtained knowledge will be finally applied to constructing a functional cardiac tissue patch made of human cells and to testing its potential to restore cardiac function after infarction. PUBLIC HEALTH RELEVANCE: Heart failure is one of the most prominent cardiac diseases in USA that develops due to irreversible damage of heart tissue following a heart attack. This proposal describes a proof-of-concept study to engineer a living heart tissue substitute (cardiac patch) starting from novel cardiogenic stem cells. Engineered cardiac patch has a potential to be used in the future as a replacement for damaged heart tissue and to prevent the occurrence or progression of heart failure.

The moderate clinical success of stem cell injections for the treatment of myocardial infarction has been mainly attributed to the low retention and survival of injected cells. Implantation of the engineered cardiac tissue patch is expected to yield improved survival of delivered cells, and potentially, a more efficient structural and functional tissue reconstruction at the infarct site. While in the past 15 years the field of cardiac tissue engineering has benefited from the use of neonatal rat cardiomyocytes, it is well recognized that these cells will remain limited to in vitro model systems and proof-of-concept in vivo studies. On the other hand, cardiac tissue patches made of stem cells offer a potential for translation to clinical practice. In particular, large quantities of cardiogenic cells can be obtained from pluripotent stem cell sources (embryonic or induced pluripotent stem cells), which offers an exciting opportunity to develop and utilize a relatively large, functional cardiac tissue patch for the treatment of myocardial injury. Unfortunately, the clear design rules to engineer a highly functional, stem cell- derived cardiac tissue patch are currently non-existent. Therefore, in order to significantly promote the field of cardiac tissue engineering, we propose to combine our novel tissue engineering approach with tools from developmental and cancer biology to design an electromechanically functional, stem cell-derived cardiac tissue patch that can rapidly vascularize and functionally integrate with host tissue and yield the repair of myocardial injury. Specifically, we propose to: 1) systematically study different mouse embryonic stem cell-derived cardiogenic populations for their ability to functionally integrate with neonatal rat myocytes and assemble into a highly functional cardiac tissue patch in vitro, 2) explore different structural and biochemical factors to enhance vascularization, survival, and functionality of these tissue patches upon implantation in mouse dorsal skin flap chamber model, and 3) investigate implantation conditions in the setting of mouse myocardial infarction to yield safe and efficient functional integration of the patch and host tissue, and consequently, a significantly improved cardiac function. The knowledge obtained in this project will allow us to pursue in the future engineering of a functional cardiac tissue patch made of human stem cells for potential clinical applications.

DESCRIPTION (provided by applicant): Although electronic pacemakers represent standard of care for treatment of symptomatic bradyarrhythmias and heart failure, they have a number of limitations including no neurohumoral responsiveness, a relatively short battery life, potential for primary and secondary infections, electromagnetic inference from other devices, and no adaptation to growth of pediatric patients. This has stimulated development of novel gene and cell-based biopacemaker therapies with the goal to eventually supplement or replace the use of electronic pacemakers. Thus far, cell-based biopacemaker therapies have involved intramyocardial injection of spontaneously active embryonic stem cell derived cardiomyocytes or bone marrow derived stem cells genetically engineered to express pacemaking HCN gene. However, since the proliferation and differentiation of injected stem cells is difficult to control, stem cell-based biopacemakers may have heterogeneous and unstable phenotype, and potentially induce immune rejection, tumors or arrhythmias. Ideally, cell-based biological pacemaker therapies should involve the implantation of patient's own cells engineered to autonomously generate stable pacemaking rhythm. Therefore, we propose to develop a novel approach to cell-based biopacemaker therapy wherein tissue-engineered grafts made of terminally differentiated somatic cells with stable, genetically engineered pacemaking properties are used to normalize rhythm of diseased hearts. Specifically, in this exploratory proposal we will: 1) identify sets of genes that enable conversion of unexcitable cells into stable autonomous pacemakers and 2) study how geometry of engineered pacemaker tissues affects their ability to pace 2D and 3D cardiac networks in vitro. The knowledge obtained in this project will allow us to pursue in the future clinically relevant procedures for the successful application of tissue-engineered pacemaking patch in the treatment of heart rhythm abnormalities. PUBLIC HEALTH RELEVANCE: Use of electronic pacemakers to restore normal heart rhythm is associated with a number of limitations including high cost, bacterial infections, lead or battery failure, and inability to adapt pacing rate to physiological needs. Biological pacemakers hold potential to replace electronic pacemakers and resolve the above limitations. The goal of this exploratory proposal is to establish tissue and genetic engineering rules that would enable generation of stable autonomous biopacemaking tissues starting from electrically unexcitable cells.

DESCRIPTION (provided by applicant): Glycogen storage disease type II (Pompe disease) is a fatal degenerative disease caused by the deficiency of acid-alpha glucosidase (GAA) or acid maltase. This disease is characterized by progressive myopathy resulting from the accumulation of lysosomal glycogen in skeletal and cardiac muscle cells. Enzyme replacement therapy (ERT) with recombinant human GAA is the only FDA-approved treatment for Pompe disease, which despite being beneficial, is highly expensive and inefficient, requiring enzyme doses 100-fold greater than those used for other lysosomal disorders. Furthermore, the ability of ERT to correct important aspects of the disease including autophagy, glycogen accumulation, and low exercise capacity, remains questionable. Therefore, the need for the development of alternative or adjuvant therapies to ERT is obvious, and although the mouse GAA knockout (GAA-KO) model is often utilized for this purpose, the differences in size and physiology of mice and humans and less severe disease phenotype in mice limit the translational utility of these studies. Human cells isolated from patients' muscle biopsies offer an alternative system to study muscle disease in vitro, however, no methods exist to generate functional contractile muscle fibers starting from human muscle cells. In this project we for the first time describe engineering of contractile, electrically responsive human muscle tissues (bioartificial muscle) made of primary myogenic cells obtained using standard muscle biopsies from normal individuals and Pompe disease patients. We propose to utilize these 3D cell cultures as a predictive in vitro screen for candidat drug and gene therapeutics for human muscle disease. By combining bioengineering and clinical expertise of the two principal investigators, we will carry out a set of translational in itro and in vivo studies in order to screen and validate alternative and adjuvant drug and gene therapies for Pompe disease. In particular, we will: 1) Optimize functional properties of healthy and Pompe disease human bioartifical muscle tissues and systematically characterize their molecular, metabolic and functional properties, 2) Mechanistically study novel candidate drug and AAV therapies for Pompe disease using GAA-KO mice, and 3) Screen the efficacy of these candidate approaches in vitro using engineered human Pompe disease muscle and further validate the most promising therapies in vivo using a novel humanized mouse model of Pompe disease. In the future, the experimental framework established in this project will allow us to undertake similar translational studies to aid treatment of other skeletal and cardiac muscle disorders.

? DESCRIPTION (provided by applicant): Although highly promising, the use of stem cell-derived cardiomyocytes for treatment of heart disease faces a number of challenges. In particular, therapies involving pluripotent stem cells suffer from the lack of methods to select a desired cardiac electrical phenotype and generate mature cardiomyocytes in vitro, as well as control differentiation fate of transplanted cells and their interactions with host cardiomyocytes n vivo. Together, these limitations can render stem cell-based cardiac therapies not only inefficient, but also tumorigenic and arrhythmogenic. Similarly, recently developed reprogramming techniques to directly convert cardiac fibroblasts to cardiomyocytes suffer from low efficiency and reproducibility, as well as the inability to obtain functional phenotype with human cells even after several weeks of culture. Ideally, a safe and efficient cardiac cell-based therapy should involve the implantation of homogeneous cells or engineered tissue grafts with the functional properties that are stable and similar to those of the surrounding adult cardiomyocytes. Thus, we propose to develop a new bioengineering strategy for cell-based cardiac repair that does not rely on the use of stem cells or direct reprogramming of fibroblasts to cardiomyocytes. Rather, building on our proof-of-concept studies with immortalized human cell lines, we propose to rapidly and efficiently convert primary human dermal fibroblasts into electrically active cells capable of action potential conduction and functional coupling with cardiomyocytes. Specifically, we propose to: 1) develop genetic engineering algorithm to stably convert adult human fibroblasts into electrically conducting cells with tailored electrophysiological phenotype resembling that of neonatal or adult rat cardiomyocytes, and 2) evaluate the therapeutic potential of injected electrically active fibroblasts and tissue patches made of these cells in a rat model of myocardial infarction. Successful competition of the proposed studies will allow us to evaluate the potential of engineered excitable somatic cells for future use in experimental studies in vitro and cell-based cardiac therapies in vivo. The outcomes of this project may also promote the development of new gene therapies for heart disease where selective in situ conversion of endogenous cardiac fibroblasts into electrically excitable and conducting cells could significantly improve compromised heart function.

? DESCRIPTION (provided by applicant): Stem cell injections into the heart are actively being pursued as a potential therapy for myocardial infarction and heart failure. While the ongoing trials with adult-derived stem cells show moderate clinical benefits, significant progress in the field is expected to arise from the use of cardiomyocytes derived from induced pluripotent stem cells. Despite great promise, eventual clinical use of pluripotent stem cell-derived cardiomyocytes faces a number of challenges that need to be resolved including key issues with inadequate cell maturation, phenotypic heterogeneity, arrhythmogenesis, low viability after implantation, and scale-up. Therefore, in this project we aim to establish a novel approach for cardiac cell and gene therapy that does not rely on the use of stem cells. Instead we propose to employ in vitro or in situ genetic engineering of fibroblasts into electrically active cells with customizable electrical phenotype that can couple with surrounding cardiomyocytes and improve their electrical and contractile function. Specifically, in Aim 1 we will utilize minimum st of genetic manipulations to rapidly and efficiently convert adult human fibroblasts into a readily expandable and homogeneous source of excitable cells that autonomously fire and conduct action potentials. In Aim 2, engineered fibroblasts with select electrophysiological phenotypes will be characterized for their functional interactions with neonatal rat cardiomyocytes in well-controlled in vitro co- culture systems. In Aim 3, we will establish if contractile function of infarcted rat hearts can be improved by implantation of engineered excitable fibroblasts or retroviral conversion of endogenous fibroblasts into electrically active cells. In addition to abov experimental studies, we will utilize computer simulations to facilitate genetic engineering of excitable cells and enhance mechanistic understanding of their functional interactions with native cardiomyocytes in vitro and in vivo. We believe that the proposed genetic and tissue engineering approach will provide strong foundation for the future experimental and clinical use of engineered fibroblasts in cell- and gene-based therapies for cardiac infarction and arrhythmias.

The derivation of functional cardiomyocytes from human embryonic stem cells (hESCs) fifteen years ago, as well as the discovery of iPSCs, has opened doors to the engineering of human cardiac tissue surrogates for use in drug discovery, disease modeling, and regenerative medicine. Still, translating human iPSC technology to clinical therapy for heart disease has been slow due to a number of challenges including immature and heterogeneous cardiomyocyte phenotype, their low expansion capacity, high metabolic demand and low viability after implantation, potential for tumor and arrhythmia induction, and high costs. To address these limitations, we propose to explore a novel strategy for cell- and gene-based cardiac repair that does not rely on the use of stem cells. Instead, we will develop methods for engineering of terminally differentiated human fibroblasts into cells capable of action potential conduction. These cells will be generated rapidly, at low cost, have stable, homogeneous, and customizable electrical phenotype, be readily expandable in vitro and available off-the-shelf, and be able to electrically couple with cardiomyocytes and significantly improve electrical and contractile function of the infarcted heart. Specifically, in Aim 1 we propose to utilize prokaryotic ion channels to engineer human fibroblasts into a readily expandable and homogeneous source of electrically excitable cells that autonomously fire and conduct action potentials. In Aim 2, we will utilize well-controlled in vitro co-culture systems to explore how engineered fibroblasts with specific electrophysiological properties affect electrical and mechanical function of native cardiomyocytes. In Aim 3, we propose to directly compare actively conducting fibroblasts and PSC-derived cardiomyocytes for their antiarrhythmic action and ability to improve contractile and hemodynamic function of infarcted rat hearts. In addition, we will utilize computer simulations to facilitate genetic engineering of actively conducting fibroblasts and enhance mechanistic understanding of their functional interactions with native cardiomyocytes in vitro and in vivo. We expect that successful completion of this project will enable future applications of engineered fibroblasts in cell-based therapies for myocardial infarction and arrhythmias.

ABSTRACT Ischemic heart disease continues to have a tremendous impact on public health, shortening lifespan and impairing the quality of life. The inability of the adult human myocardium to undergo regeneration after a myocardial infarction has inspired research using cell therapy for myocardial repair. However, clinical trials to date have shown modest or no benefit, suggesting the need to consider other cell sources and approaches. In large animal models, derivatives of human pluripotent stem cells have provided promising results, but the grafts have generally been small, transient, and of limited functional benefit. In addition, there remain important questions regarding cardiac cells derived from iPSCs, including the optimal delivery strategy, immunogenicity, maturity, and the ability to couple effectively to the native myocardium without causing arrhythmias. In this proposal, three integrated projects will address these challenges and advance toward the long-term goal of utilizing a functional human cardiac tissue patch (hCTP) for repair of ischemic myocardium. The first project aims to generate novel cell populations, including induced cardiac progenitor cells and genetically engineered cell lines that will be evaluated for their immunogenicity in a novel humanized mouse model. These and other cell products, including commercially available sources, will be utilized to generate large vascularized hCTPs in the second project. The third project will utilize a porcine post-infarction model to test hCTPs and optimize electrical and vascular integration as assessed by optical mapping technology and MRI/NMR spectroscopy, respectively. These studies will overcome critical barriers to generating large, fully functional human cardiac tissues that can be integrated safely into the native myocardium to provide a powerful new approach for treatment of advanced ischemic heart disease.

Successful engineering of biomimetic skeletal muscle tissues could allow creation of accurate models of muscle physiology and disease and aid treatment of various muscle disorders. This project is based on our recently developed methods to utilize adult rat myogenic cells for engineering of 3D skeletal muscle tissues with structural and functional properties comparable to those of native muscle. Specifically, we have established conditions for robust in vitro expansion of adult rat myogenic cells and have successfully utilized them to engineer skeletal muscle tissues with contractile capacity 10- 100 fold higher than previously reported. Importantly, in a comprehensive set of preliminary studies we for the first time show that self-regenerative capacity of adult-derived engineered muscle in vitro and survival in vivo can be significantly enhanced by a 3-D co-culture of skeletal muscle progenitors with non-polarized macrophages derived from bone marrow. We propose to build on these exciting results and systematically explore the use of 3D muscle-macrophage co-culture system to create highly contractile and regenerative muscle tissues with the capacity for rapid vascular and neuronal integration and successful repair of skeletal muscle injury in vivo. We will study: (1) the cellular and molecular mechanisms of macrophage mediated self-repair of tissue-engineered muscle in vitro, (2) the combined effects of macrophages, vascular cells, and biophysical cues on the ability of in vitro formed pre- vascularized engineered muscle to undergo rapid blood perfusion and functional maturation in vivo, and (3) the roles of macrophage supplementation and synaptogenic stimulation in vitro upon the ability of muscle-macrophage implants to functionally integrate with and repair damaged skeletal muscle in vivo. Successful completion of the proposed studies will establish foundation for the future applications of tissue engineering methodologies to human muscle repair. Furthermore, our novel strategy to utilize immune system cells as pro-regenerative adjuvants inside tissue-engineered implants may find broad applications in the field of regenerative medicine.

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.