Browsing by Subject "Gene editing"
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Item Open Access Gene Editing for Duchenne Muscular Dystrophy(2018) Robinson-Hamm, JacquelineDuchenne muscular dystrophy (DMD) is a muscle wasting disease that results from a lack of dystrophin protein, which is an essential musculoskeletal protein. Patients are typically non-ambulatory by their teenage years and suffer prematurely fatal respiratory and/or cardiac complications by the third decade of life. DMD is caused by deleterious mutations in the dystrophin gene, which creates an out-of-frame shift leading to a lack of dystrophin protein and manifestation of DMD. Although scientists have had an understanding of the genetic basis of DMD for decades there has been only modest advancement in improving quality of life for these patients.
Becker muscular dystrophy (BMD) is an allelic disease; BMD is also caused by mutations in the dystrophin gene, although these mutations maintain the translational reading frame and thus a truncated, partially functional dystrophin protein is created. BMD patients have a wide range of symptoms, but BMD typically has much less severe symptoms than DMD. Thus, a common approach to creating a therapy for DMD is to shift the DMD genotype to a BMD genotype. One therapy targeting the genetic cause of the DMD by shifting the messenger RNA (mRNA) and thus protein product to one of BMD has been conditionally approved by the US Food and Drug Administration (FDA), but the treatment is transient and thus far has not demonstrated reliable clinical benefit. DMD presents some unique challenges for developing gene therapies. First, the full-length gene is so large that exogenous delivery in size restricted viral vectors is not an option. Second, popular strategies being explored are transient and would require lifelong administration. The work presented in this dissertation utilized gene editing technology. Building on prior proof-of-concept studies, we show a CRISPR-Cas9 system utilizing Staphylococcus aureus Cas9 (SaCas9) can be used to create permanent changes to the dystrophin gene. This technique overcomes the main challenges presented, as editing the native locus does not require delivering the gene exogenously, and CRISPR-Cas9 mediated DNA double stranded breaks result in permanent changes of the genome. Here we further the proof-of-concept body of work for utilizing CRISPR-Cas9 to treat DMD by targeting exon 51 for excision in a humanized mouse model.
Initially, we recognized the need for a relevant small animal model. A majority of DMD in vivo work is done in the mdx mouse or variants of the mdx mouse, which contains a mutated mouse dystrophin gene such that it does not produce dystrophin protein and displays a mild dystrophic phenotype. While this is a useful research tool, in order to move genome editing closer to the clinic we need to be able to test guide RNAs (gRNAs) that target the human dystrophin gene in a small animal model. As the gRNAs target exact sequences of the genome they must be designed to the human DMD gene. These human DMD targeting gRNAs would not match the mouse Dmd gene, and thus there was a clear need for a preclinical humanized small animal model of DMD. We obtained an hDMD/mdx mouse that contains the full-length, healthy, wild type human DMD gene on mouse chromosome 5. Although this mouse has the human DMD gene, it is ultimately a healthy mouse. Thus, we utilized Streptococcus pyogenes CRISPR-Cas9 (SpCas9) to excise exon 52 of the human DMD gene in the mouse zygotes. We identified a founder mouse that lacked exon 52 in the genomic DNA (gDNA) and bred that mouse with the mdx mouse line. Thus, using genome editing, we created the hDMDΔ52/mdx mouse, which lacks both human and mouse dystrophin protein expression. We confirmed this biochemically by sequencing the gDNA to ensure lack of exon 52 between the gRNA targeted sites, lack of exon 52 in the cDNA, and lack of dystrophin protein by both immunohistochemistry (IHC) staining and Western blot. The hDMDΔ52/mdx mouse also displayed a mild dystrophic phenotype compared to its healthy counterpart, the hDMD/mdx mouse. We have characterized this hDMDΔ52/mdx mouse and shown it lacks dystrophin and has a mild dystrophic phenotype, and this mouse will be a meaningful tool for testing potential DMD therapies.
Next, we created a CRISPR-SaCas9 system that would target the human DMD gene for exon 51 excision. While our lab has previously shown efficacy of this method utilizing SpCas9, we switched to the smaller SaCas9 in order to better accommodate the small packaging limit of adeno-associated virus (AAV). gRNAs were designed to target conserved regions in the intronic area flanking exon 51 of dystrophin in both humans and rhesus macaques. gRNAs were tested individually for on-target activity in HEK293T cells and those with on-target activity were assessed for off-target activity in silico. One gRNA upstream of human dystrophin exon 51 and one gRNA downstream of exon 51 were selected based on distance from the exon, percent modification measured by the Surveyor nuclease assay, and potential for off-target activity in humans and rhesus macaques. Those chosen two gRNAs were tested as a deletion pair in both HEK293T cells and immortalized myoblasts from a DMD patient, lacking exons 48 through exon 50 that is correctable by removing exon 51, and shown to create the desired deletion. Currently there is a lack of rules about what makes an effective gRNA, and in particular even the length of the gRNA protospacer sequence for SaCas9 can have effects on on-target activity. Thus, the two chosen gRNAs were tested with protospacer lengths varying from 19 to 23 base pairs (bp) both individually and as deletion pairs in HEK293T cells. The most effective on-target pair was with both gRNA protospacer sequences at 23 bp long. These 23 bp length gRNAs were re-tested in HEK293T cells and DMD patient immortalized myoblasts and shown to be effective at creating deletions in the genome, having that edit carry over in the mRNA of differentiated myoblasts resulting in the loss of exon 51 and the junction of exon 47 to exon 52 when Sanger sequenced, and restored dystrophin protein expression in the differentiated myoblasts by Western blot. Off-target sequences of these 23 bp length protospacers were assessed in silico and ten of the predicted off-target sites for each gRNA were tested in vitro in HEK293T cells by deep sequencing. Although the upstream gRNA did have two off-target sites that had notable small insertion or deletion (indel) rates measured by treated gDNA/untreated gDNA, ultimately all measurable off-target activity was at least two orders of magnitude lower than the on-target rate of indel formation.
Finally, we created a CRISPR-SaCas9 system with gRNAs that target human DMD for exon 51 removal, and these exact gRNAs tested in vitro were tested in vivo in our previously characterized hDMDΔ52/mdx mouse. Initially we did a small proof-of-concept study by packaging our system in AAV8 and performed local injections into the tibialis anterior (TA) muscle of adult hDMDΔ52/mdx mice. 8 weeks after treatment the TA was analyzed. We noted deletion of exon 51 between the gRNA targeted sites in the gDNA, as well as dystrophin protein restoration by IHC and Western blot. While promising, DMD is a systemic disease that affects all skeletal and cardiac muscles. Thus, we next delivered our CRISPR-SaCas9 system using AAV9 systemically by tail vein injections in adult hDMDΔ52/mdx mice or temporal vein injections in neonatal hDMDΔ52/mdx mice. At 16 weeks of age mice were sacrificed for biochemical analysis. Deep sequencing of gDNA at each gRNA target site showed measurable indel formation above the limit of detection in all tissues assayed in mice treated as both adults and neonates. There were a few trends that emerged in this data and hold true throughout analysis of on-target editing: the upstream gRNA is generally more effective at on-target activity than the downstream gRNA, the mice treated as neonates show more on-target activity than mice treated as adults, and there is much more on-target activity in the heart than in the skeletal muscles. Indels are a measure of on-target activity, but we delivered a system to create a deletion and not just individual cuts. Thus, gDNA from the heart and TA of mice treated as adults was assayed by linear amplification sequencing, which revealed approximately 4% deletions of exon 51 in gDNA from the heart and about 1% deletions of exon 51 in gDNA from the TA. Through this method we are also investigated inversions of the targeted sequence and AAV integrations into the targeted cut site, both of which were much more prominently present in the heart gDNA than the TA gDNA. Confident we were able to edit the genome at low, although measurable, levels, we examined changes in mRNA. In the mRNA from hearts of both mice treated as adults and neonates we see clear deletions of exon 51 by endpoint polymerase chain reaction (PCR). Sanger sequencing the deletion band revealed the exact junction of exon 50 to exon 53 as expected. We performed quantitative droplet digital PCR (ddPCR) on cDNA from the heart, TA, diaphragm, and gastrocnemius, and similar to the indel formation we saw the highest amount of exon 51 deletions in the heart cDNA at about 20% in both mice treated as adults and neonates. The deletions in skeletal muscles varied from about 0.15% to about 1.5% and were all measurable above the limit of detection as defined by the average of samples from untreated mice. Lastly, we examined dystrophin protein expression. By Western blot we saw mouse to mouse variability in intensity, but largely some degree of dystrophin protein expression restoration in protein extracted from hearts and gastrocnemius muscles from mice treated as both adults and neonates, although qualitatively the mice treated as neonates have more dystrophin protein expression than those treated as adults. IHC on hearts and TA muscle sections similarly showed variable but nonetheless present dystrophin protein expression restoration in both mice treated as adults and neonates. Consistent with prior data, we saw more dystrophin expression in the heart than in the TA, and this difference is exacerbated in the mice treated as adults.
In sum, the objective of this dissertation was to create a clinically relevant CRISPR-SaCas9 system and test it in vitro and in vivo in a diseased humanized mouse model. This work is an incremental step to propel forward methods to permanently correct the dystrophin gene by gene editing technology to treat DMD. We created a useful mouse model for the field to test preclinical therapies in vivo and make the most of the rapidly advancing gene editing tools. Collectively this work is significant in extending early proof-of-principle studies to a translational strategy for gene editing as a potential treatment for DMD.
Item Open Access Genome Engineering Tools to Dissect Gene Regulation(2019) Kocak, Daniel DewranOver 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.
Item Open Access Knock-out mutagenesis of zebrafish genes using a CRISPR/Cas9 approach(2019-05) Hwang, JamesDetermining effective methods of shutting down genes or inserting a specific gene into the genome can provide insight about gene functionality and mechanisms for disease. My project specifically investigates methods of CRISPR/Cas9-mediated gene knock-out. I targeted two zebrafish genes, tram1 and clta. For tram1, I used one CRISPR/Cas9 mutagenesis site to generate loss-of-function alleles. For clta, I targeted two genomic sites around 140-bp apart to excise a portion of the chromosome. After raising several generations of fish, successful mutagenesis was confirmed. Analysis of genomic DNA showed tram1 mutant alleles with various insertions and deletions. Analysis of clta fish showed insertions and deletions as well as an allele with a 136-bp deletion. Results showed successful mutagenesis using both one- and two-target site approaches. The one-site approach proved to be an effective way of generating random mutations. The two-site approach proved to be an effective method of excising a portion of the genome.Item Open Access The Interrogation of Cas9 Aptamers and sgRNA Structures Through SELEX(2022) Bush, Korie BWhile much of the current focus on advancing CRISPR-Cas9 editing revolves around the engineering of Cas9, the interrogation and evolution of sgRNA scaffold, in addition to novel Cas9 binding RNAs, represent another echelon of development and therapeutic potential. Currently, the majority of research utilizes a singular guide RNA scaffold sequence (the sgRNA) for a given Cas protein (e.g., the Streptococcus pyogenes Cas9 and associated guide RNA). This sequence inflexibility makes many sites within the genome intractable to CRISPR/Cas editing, often due to undesirable intramolecular interactions that result in undesirable secondary structures. Additionally, given the electrostatic potential of Cas9, it may be possible to generate additional Cas9 binding RNA molecules.Here, we use utilize SELEX to both identify novel Cas9 binding RNAs and interrogate the sequence depth of the sgRNA scaffold. First, a SELEX scheme utilizing a nitrocellulose filter binding assay was utilized to identify modified RNA aptamers that bind to Cas9 with specificity and an affinity rivaling that of the sgRNA. The aptamer was shown to tolerate truncations and sequence additions, demonstrating an ability to localize oligonucleotide-based therapeutics to the Cas9 protein. We believe that this aptamer can be expanded upon to incorporate novel functions instead of altering the sgRNA . Second, we use a novel combinatorial approach that utilizes a functional SELEX (Systematic Evolution of Ligands by Exponential Enrichment) to identify numerous, diverse sgRNA variants that bind S. pyogenes Cas9 and support DNA cleavage. These variants demonstrate surprising malleability in the sgRNA sequence and are utilized in a combinatorial approach to identify scaffolds that enhance editing efficiencies when paired DNA-binding antisense domains. Using molecular evolution, guide RNA scaffolds can be generated for specific targets and optimized to ensure that secondary structure is maintained. This selection approach should be valuable for generating gRNAs with a range of new activities.
Item Open Access Tissue Engineered Blood Vessels to Study Endothelial Dysfunction in Hutchinson-Gilford Progeria Syndrome(2022) Abutaleb, Nadia OsamaHutchinson-Gilford Progeria Syndrome (HGPS) is a rare, fatal genetic disease that causes progressive atherosclerosis and accelerated aging in children resulting in fatality at an average of 14.6 years of age. With a limited pool of HGPS patients, clinical trials face unique challenges and require reliable preclinical testing. Preclinical studies to date have relied on 2D cell culture using HGPS fibroblasts which does not accurately model the 3D physiological microenvironment and limits the scope many studies to only the fibroblast response, which may not translate to a vascular benefit. Further, only two HGPS murine models develop atherosclerosis and these exhibit symptoms that are not present in HGPS patients. An ideal model would incorporate human cells in a microenvironment mimicking in vivo conditions and replicate key aspects HGPS vascular pathology. Such a model would contribute significantly to the ability to study HGPS mechanisms and test therapies. One significant area of research that requires further study is the contribution of endothelial cells to HGPS pathology. The endothelium plays a critical atheroprotective role in maintaining vascular homeostasis. When it is damaged, dysfunctional endothelium becomes inflammatory and proatherogenic, contributing significantly to atherosclerosis and cardiovascular disease. Progressive atherosclerosis is the most severe symptom of HGPS and is the common underlying cause of mortality in HGPS patients, yet only a few studies have investigated the HGPS endothelial phenotype. The goal of our work was to develop and characterize a tissue engineered model of HGPS vasculature that could be used to study endothelial dysfunction in HGPS and test therapies to alleviate HGPS vascular pathology.In Aim 1, we developed and characterized a tissue engineered blood vessel (TEBV) model of HGPS using vascular smooth muscle cells (viSMCs) and endothelial cells (viECs) differentiated from induced pluripotent stem cells (iPSCs) from two HGPS patients. HGPS viSMCs and viECs exhibited manifestations consistent with typical symptoms of HGPS including progerin expression, abnormal nuclear morphology, and premature senescence. HGPS viECs exhibited cell responses consistent with endothelial dysfunction including impaired tube formation, elevated reactive oxygen species (ROS) levels, reduced proliferation, and increased levels of DNA damage compared with healthy viECs. HGPS viECs also displayed a blunted response to shear stress including limited flow-sensitive gene expression and reduced nitric oxide production. TEBVs fabricated with HGPS cells exhibited reduced vasoactivity compared with healthy TEBVs and replicated HGPS vascular pathology including SMC loss, excess extracellular matrix (ECM) protein deposition, inflammation, and vascular calcification. In Aim 2, we tested the effects of HGPS therapeutics the farnesyltransferase inhibitor Lonafarnib and mTOR inhibitor Everolimus, currently in Phase I/II clinical trial, on endothelial dysfunction and HGPS TEBVs phenotype. Everolimus decreased reactive oxygen species levels, increased proliferation, and reduced DNA damage in HGPS vascular cells. Lonafarnib improved flow-sensitive gene expression of HGPS viECs exposed to physiological shear stress. Both Lonafarnib and Everolimus were able to restore nitric oxide production to healthy levels in HGPS viECs exposed to physiological shear stress. While Everolimus improved vasoconstriction, Lonafarnib increased vasodilation, ECM deposition, inflammation, and calcification in HGPS TEBVs. Combination treatment with Lonafarnib and Everolimus maintained the benefits of each monotherapy and also resulted in additional benefits such as improved endothelial marker expression and reduced apoptosis. The 3D TEBV model was critical to reveal the benefit of Lonafarnib and Everolimus combination treatment which was more limited in 2D studies but became clear in the TEBVs. In Aim 3, we tested a recently developed targeted gene therapy for HGPS known as adenine base editing (ABE). ABE corrects the genetic heterozygous point mutation that causes HGPS with high efficiency and minimal off-target effects. ABE ameliorated all the dysfunctional endothelial phenotypes that we tested in HGPS viECs including elevated ROS levels, reduced proliferation, and increased DNA damage. Most critically for endothelial function, ABE restored nitric oxide production and flow-sensitive gene expression to healthy levels in HGPS viECs. In TEBVs, ABE restored healthy levels of vasoconstriction and vasodilation, improved SMC retention, increased proliferation, and inhibited excess ECM protein expression. In summary, this work contributes data supporting the hypothesis that progerin induces endothelial dysfunction in HGPS endothelial cells which could contribute to the vascular pathology observed in HGPS. This work also tests current and novel therapies in the first 3D tissue engineered model of HGPS, validating the model as a valuable platform for preclinical testing that can supplement and improve information gathering from current 2D and animal models.