Browsing by Subject "Genome engineering"

The ends of long bones that articulate with respect to one another are lined with a crucial connective tissue called articular cartilage. This tissue plays an essential biomechanical function in synovial joints, as it serves to both dissipate load and lubricate articulating surfaces. Osteoarthritis is a painful and debilitating disease that drives the deterioration of articular cartilage. Like many chronic diseases, pro-inflammatory cytokines feature prominently in the onset and progression of osteoarthritis. Because cartilage lacks physiologic features critical for regeneration and self-repair, the development of effective strategies to create functional cartilage tissue substitutes remains a priority for the fields of tissue engineering and regenerative medicine. The overall objectives of this dissertation are to (1) develop a bioactive scaffold capable of mediating cell differentiation and formation of extracellular matrix that recapitulates native cartilage tissue and (2) to produce stem cells specifically tailored at the scale of the genome with the ability to resist inflammatory cues that normally lead to degeneration and pain.

Engineered replacements for musculoskeletal tissues generally require extensive ex vivo manipulation of stem cells to achieve controlled differentiation and phenotypic stability. By immobilizing lentivirus driving the expression of transforming growth factor-β3 to a highly structured, three dimensionally woven tissue engineering scaffold, we developed a technique for producing cell-instructive scaffolds that control human mesenchymal stem cell differentiation and possess biomechanical properties approximating those of native tissues. This work represents an important advance, as it establishes a method for generating constructs capable of restoring biological and mechanical function that may circumvent the need for ex vivo conditioning of engineered tissue substitutes.

Any functional cartilage tissue substitute must tolerate the inflammation intrinsic to an arthritic joint. Recently emerging tools from synthetic biology and genome engineering facilitate an unprecedented ability to modify how cells respond to their microenvironments. We exploited these developments to engineer cells that can evade signaling of the pro-inflammatory cytokine interleukin-1 (IL-1). Our study provides proof-of-principle evidence that cartilage derived from such engineered stem cells are resistant to IL-1-mediated degradation.

Extending on this work, we developed a synthetic biology strategy to further customize stem cells to combat inflammatory cues. We commandeered the highly responsive endogenous locus of the chemokine (C-C motif) ligand 2 gene in pluripotent stem cells to impart self-regulated, feedback-controlled production of biologic therapy. We demonstrated that repurposing of degradative signaling pathways induced by IL-1 and tumor necrosis factor toward transient production of cytokine antagonists enabled engineered cartilage tissue to withstand the action of inflammatory cytokines and to serve as a cell-based, auto-regulated drug delivery system.

In this work, we combine principles from synthetic biology, gene therapy, and functional tissue engineering to develop methods for generating constructs with biomimetic molecular and mechanical features of articular cartilage while precisely defining how cells respond to dysfunction in the body’s finely-tuned inflammatory systems. Moreover, our strategy for customizing intrinsic cellular signaling pathways in therapeutic stem cell populations opens innovative possibilities for controlled drug delivery to native tissues, which may provide safer and more effective treatments applicable to a wide variety of chronic diseases and may transform the landscape of regenerative medicine.

Skeletal muscle has the innate ability to robustly regenerate in a highly orchestrated fashion that is initiated by satellite cells, the resident stem cell population. These cells are defined by their uniform expression of the transcription factor, PAX7, which plays a key role in myogenesis through specification and maintenance of satellite cells, as well as regulation of myogenic differentiation. In conditions of skeletal muscle wasting such as cachexia, sarcopenia, and muscular dystrophies, the deterioration of muscle overwhelms the regenerative capabilities of satellite cells, which are believed to undergo early senescence due to exhaustive proliferation. There is significant potential for harnessing satellite cells for gene and cell therapies for such diseases; however, satellite cell specification and regulation is still poorly understood.

The CRISPR/Cas9 system has been established as a multifaceted tool that can be used as a platform for a variety of applications, including sequence-specific genome and epigenome editing for cell differentiation and treatment of genetic diseases. The objective of my research proposal was to use CRISPR/Cas9-based genome engineering technologies toward applications for skeletal muscle regeneration. First, I used a CRISPR/Cas9-based transcriptional activator to direct differentiation of human pluripotent stem cells into functional skeletal muscle progenitor cells. Next, I conducted a high-throughput CRISPR activation screen to identify novel upstream regulators of myogenic progenitor cell differentiation. Lastly, I demonstrated that satellite cells can be targeted in vivo with AAV and subsequently gene-edited to correct the dystrophin reading frame in a mouse model for Duchenne muscular dystrophy. Together, this work provides novel contributions to the field of satellite cell biology and highlights the utility of CRISPR/Cas9 genome engineering in stem cells for skeletal muscle regeneration.

Over the past several years genome and epigenome engineering has been propelled forward by CRISPR-Cas technologies. These prokaryotic defense systems work well in mammalian cells in a manner that is remarkably robust: they are non-toxic, fold into a catalytically active state, localize to targeted cellular compartments, and act on the eukaryotic genome, which is heavily compacted in chromatin. While all these are true, CRISPR-cas nucleases did not evolve to function as highly specific genome engineering tools. Thus, the major goals of the work presented herein are to i) refine the specificity of CRISPR-Cas enzymes, ii) develop methods that facilitate genome engineering in human cells, and iii) apply these technologies toward outstanding problems in human gene regulation. With regard to the first goal, we set out to develop a method that could be easily applied to increase the specificity of diverse CRISPR systems. Adopting RNA-engineering to achieve this goal, we modulate the kinetics of DNA strand invasion to increase the specificity of Cas enzymes. Since the guide RNA is a feature that is common across all CRISPR systems, we expect that this new method to tune the activity and specificity of Cas enzymes will be broadly useful. To address the second goal, we set out to develop an experimental pipeline for the high throughput, precise modification of mammalian genomes. Specifically, we modify the C-termini of genes to include an epitope tag for the genome-wide profiling of transcription factor binding sites. We apply this method to over 30 genes, encoding a variety of transcription factors, chromatin modifying enzymes, and gene regulatory proteins. Out of the large number of genes we focus particularly on members of the AP-1 transcription factor family and nuclear receptor co-activator and co-repressor families. Using this ChIP-seq data, which profiles genome wide binding, and integrating a variety of other genomic information, including chromatin modifications, chromatin accessibility, other TF binding, and inherent regulatory activity, we investigate the dimerization preferences of AP-1 subunits, their genomic binding patterns, and the regulatory potential of theses subunits. Toward addressing the third goal, we decided to focus on the glucocorticoid receptor (GR). The dual activating and repressive function of the GR is incompletely understood, and this duality is a property of many other stimuli responsive transcriptional responses (e.g. NFKB signaling). Thus, how one transcription factor is biochemically endowed with the ability to both activate and repress gene expression is an outstanding problem in gene regulation. It is hypothesized that the GR recruits a variety of distinct protein complexes in order to mediate its diverse function. We used CRISPR based loss of function screening in order to discover new GR cofactors. Using this method, we find a number of cofactors, both canonical and novel, that regulate this response in A549 cells. Ongoing work investigates how general these cofactors are across the transcriptome and whether they provide an avenue to decouple GR’s dual function, which has been a major goal in drug development. Through these studies we have found a way to make CRISPR systems more specific, developed and applied CRISPR based method to define AP-1 binding and function, and used unbiased CRISPR based screens to discover novel regulators of the glucocorticoid drug response.

Chapter 1 broadly introduces this work, its motivations, and aims of research presented herein.

Chapter 2 provides an introduction to both genome engineering and gene regulation. Specifically, it describes the development and application of CRISPR-cas tools and details outstanding problems in gene regulation through the lens of nuclear receptors.

Chapter 3 describes the purification of Cas9 protein and its characterization biochemically. Specifically, we use AFM to determine the DNA binding properties of Cas9 in vitro.

Chapter 4 introduces a new method to modulate the specificity of CRISPR systems in human cells. Therein we show that RNA secondary structure can be applied to diverse CRISPR systems to tune their activity.

Chapter 5 details a method for the high throughput tagging of transcription factors. It specifically investigates members of the AP-1 transcription factor complex.

Chapter 6 is an investigation of the glucocorticoid receptor and its cofactors. We apply a variety of genome engineering and genomic methods to characterize known co-factors and discover new ones.

Chapter 7 is an outlook on the fields of genome-engineering and gene regulation. It describes key questions that are still unanswered and possible lines of attack to address them.

Electrowetting-on-dielectric (EWD) digital microfluidics is a droplet-based fluid handling technology capable of radically accelerating the pace of genome engineering research. EWD-based laboratory-on-chip (LoC) platforms demonstrate excellent performance in automating labor-intensive laboratory protocols at ever smaller scales. Until now, there has not been an effective means of gene transfer demonstrated in EWD microfluidic platforms. This thesis describes the theoretical and experimental approaches developed in the demonstration of an EWD-enabled electrotransfer device. Standard microfabrication methods were employed in the integration of electroporation (EP) and EWD device architectures. These devices enabled the droplet-based bulk transformation of E. coli with plasmid and oligo DNA. Peak on-chip transformation efficiencies for the EP/EWD device rivaled that of comparable benchtop protocols. Additionally, ultrasound induced in-droplet microstreaming was developed as a means of improving on-chip electroporation. The advent of electroporation in an EWD platform offers synthetic biologists a reconfigurable, programmable, and scalable fluid handling platform capable of automating next-generation genome engineering methods. This capability will drive the discovery and production of exotic biomaterials by providing the instrumentation necessary for rapidly generating ultra-rich genomic diversity at arbitrary volumetric scales.

Gene regulation is a complex and tightly controlled process that defines cell function in physiological and abnormal states. Programmable gene repression technologies enable loss-of-function studies for dissecting gene regulation mechanisms and represent an exciting avenue for gene therapy. Established and recently developed methods now exist to modulate gene sequence, epigenetic marks, transcriptional activity, and post-transcriptional processes, providing unprecedented genetic control over cell phenotype. Our objective was to apply and develop targeted repression technologies for regenerative medicine, genomics, and gene therapy applications. We used RNA interference to control cell cycle regulation in myogenic differentiation and enhance the proliferative capacity of tissue engineered cartilage constructs. These studies demonstrate how modulation of a single gene can be used to guide cell differentiation for regenerative medicine strategies. RNA-guided gene regulation with the CRISPR/Cas9 system has rapidly expanded the targeted repression repertoire from silencing single protein-coding genes to modulation of genes, promoters, and other distal regulatory elements. In order to facilitate its adaptation for basic research and translational applications, we demonstrated the high degree of specificity for gene targeting, gene silencing, and chromatin modification possible with Cas9 repressors. The specificity and effectiveness of RNA-guided transcriptional repressors for silencing endogenous genes are promising characteristics for mechanistic studies of gene regulation and cell phenotype. Furthermore, our results support the use of Cas9-based repressors as a platform for novel gene therapy strategies. We developed an in vivo AAV-based gene repression system for silencing endogenous genes in a mouse model. Together, these studies demonstrate the utility of gene repression tools for guiding cell phenotype and the potential of the RNA-guided CRISPR/Cas9 platform for applications such as causal studies of gene regulatory mechanisms and gene therapy.