Browsing by Subject "Genetic Therapy"
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Item Biologic effects of nuclear weapons(1961-10-19) Sanford, Jay P.Item Correction of Hot Spot Mutations in Duchenne Muscular Dystrophy by CRISPR/Cas9 Gene Editing(2018-11-26) Min, Yi-Li; Schneider, Jay W.; Sadek, Hesham A.; Hill, Joseph A.; Olson, Eric N.The ability to efficiently modify the genome using CRISPR technology has rapidly revolutionized biology and genetics and will soon transform medicine. Duchenne muscular dystrophy (DMD) represents one of the first monogenic disorders that has been investigated with respect to CRISPR-mediated correction of causal genetic mutations. DMD results from mutations in the gene encoding dystrophin, a scaffolding protein that maintains the integrity of striated muscles. Thousands of different dystrophin mutations have been identified in DMD patients, who suffer from a loss of ambulation followed by respiratory insufficiency, heart failure, and death by the third decade of life. Most DMD patients have an inherited or spontaneous deletion in the dystrophin gene that disrupts the reading frame resulting in an unstable truncated product. The major DMD mutational hotspots are found between exons 6 to 8, and exons 45 to 53. Mutations that delete exon 44 of the dystrophin gene represent one of the most common causes of DMD and can be corrected in ~12% of patients by editing surrounding exons, which restores the dystrophin open reading frame. In this study, a new DMD mouse model was generated by deleting exon 44, thereby creating a human DMD hotspot mutation in a mouse animal model. Using CRISPR/Cas9-mediated genomic editing, the reading frame of the exon 44 DMD mouse model was restored and dystrophin expression was rescued. Furthermore, I present an efficient strategy for correction of exon 44 deletion mutations by CRISPR/Cas9 gene editing in cardiomyocytes obtained from patient-derived induced pluripotent stem cells and in a new mouse model harboring the same deletion mutation. Using AAV9 encoding Cas9 and single guide RNAs, I also demonstrate the importance of the dosages of these gene editing components for optimal gene correction in vivo. Our findings provide therapeutic insight to develop possible CRISPR therapies for DMD.Item Development of a Novel Gene Therapy Strategy for SCID-X1 and a Method for Measuring Gene Targeting Outcomes at Endogenous Loci(2016-04-05) Kildebeck, Eric James; Brown, Kathlynn C.; Porteus, Matthew H.; Wright, Woodring E.; Zinn, Andrew R.Two decades of gene therapy trials for primary immunodeficiencies have seen tremendous clinical success with a significant majority of patients developing functional immune systems. The development of leukemia in some patients has led to the development of precise gene targeting tools to correct genetic deficits without inducing genomic instability. In this thesis I report the development of a novel gene therapy strategy for SCID-X1 and the development of a useful method for measuring gene editing outcomes at endogenous loci in any cell type. TALENs designed to target IL2RG exon 1 are shown to be highly active and stimulate precise integration of IL2RG cDNA under the control of the endogenous IL2RG promoter. Activity levels of IL2Rγ in cells targeted with a codon-optimized cDNA and an artificial intron are also shown to be as high or higher than WT levels, demonstrating the potential for this approach to correct the functional deficit seen in SCID-X1. Furthermore, these TALENs successfully stimulate gene targeting in CD34+ hematopoietic stem and progenitor cells at frequencies 10-fold higher than the highest levels previously reported, while displaying less toxicity than ZFNs already in use in clinical trials. The high activity and low toxicity of these TALENs in combination with the potential for gene targeting at exon 1 to correct more than 98% of SCID-X1-causing mutations make this a promising strategy for gene therapy, which could one day form the basis for a safe and effective cure for SCID-X1.Item Hemophilia: past, present, and future(2018-09-28) Rambally, SiayarehItem Prevention of Duchenne Muscular Dystrophy by CRISPR/Cas Therapeutic Genome Editing(2020-01-13) Zhang, Yu; Sadek, Hesham A.; Olson, Eric N.; Mendell, Joshua T.; Chen, ElizabethSkeletal muscle is one of the largest tissues in the human body and hence muscle diseases caused by genetic mutations have a profound and systemic impact on human health. Duchenne muscular dystrophy (DMD) is a lethal neuromuscular disorder, caused by mutations in the DMD gene on the X chromosome, which consists of 79 exons encoding dystrophin protein. Patients with DMD develop progressive muscle weakness and cardiomyopathy, and ultimately succumb to respiratory and cardiac failure in their mid-20s. The dystrophin gene was identified three decades ago and mutations in the DMD gene are well-characterized. However, there is no effective treatment for this debilitating disease. The CRISPR/Cas (clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins) was first discovered as an adaptive immune system in bacteria and archaea for defending against phage infection. Recently, the CRISPR/Cas system has been applied for mammalian genome editing because it provides site-specific DNA double-stranded breaks with simplicity and precision. In this study, I demonstrate the feasibility of using CRISPR/Cpf1 to correct a DMD exon 48-50 out-of-frame deletion mutation in cardiomyocytes derived from patient induced pluripotent stem cells by exon skipping and exon reframing strategies. Next, I precisely correct a Dmd exon 23 nonsense mutation in mdx mouse by CRISPR/Cpf1-mediated germline editing. Furthermore, I apply CRISPR/Cas9-mediated post-natal genome editing to correct a Dmd exon 44 out-of-frame deletion mutation in a DMD mouse model. Finally, I develop an effective strategy to improve CRISPR/Cas9-mediated in vivo genome editing by packaging Cas9 nuclease in conventional single-stranded AAV and CRISPR single guide RNAs in double-stranded self-complementary AAV. This strategy significantly reduces the amount of AAV vector needed for therapeutic genome editing and enhances dystrophin restoration after delivery into a mouse model of DMD harboring an exon 44 deletion. These findings represent an important advancement toward therapeutic translation of genome editing technology for permeant correction of Duchenne muscular dystrophy.Item Prospects for gene therapy of ischemic heart disease(1993-02-18) Williams, R. SandersItem Site-Specific Genome Engineering in Mouse Primary Fibroblasts(2014-01-07) Barker, Jenny; Amatruda, James F.; Porteus, Matthew H.; Huang, Lily; Castrillon, Diego H.Site-specific genome engineering is a powerful tool for medical therapeutics and basic scientific research. As the name implies, site-specific genome engineering describes the ability to add or subtract nucleic acid information in a precise, controlled manner within the genome. This technology has developed as a result of two key discoveries. The first is that a cell’s double strand break machinery can be highjacked to affect a desired change in the genome. Double stranded breaks are typically repaired by a pathway called non-homologous end joining (NHEJ) which can allow for disruption of an endogenous locus, or the pathway of homologous recombination (HR) which can allow for insertion of sequences if a homologous donor is supplied. The second major discovery is that chimeric enzymes can be engineered to create site-specific double stranded breaks, and that these enzymes can dramatically stimulate the frequency of gene targeting at a given locus. Recently, there has been an outpouring of studies performing site-specific genome engineering in human cell lines and primary cells, including embryonic stem cells, induced pluripotent stem cells and CD34+ hematopoietic stem cells. However, very little has been accomplished in terms of animal modeling of these principles. The work described in this thesis seeks to address some of these issues in a reporter mouse model that we have developed to study genome engineering. With this model we have demonstrated gene correction of an endogenous locus and also site-specific gene addition through a novel strategy which does not require disruption of the gene product at the site of the insertion. We have accomplished gene correction in several cell types including embryonic and adult fibroblasts, astrocytes and embryonic stem cells. For gene addition, we have demonstrated site-specific insertion of multiple transgenes including human growth hormone and human platelet derived growth factor-B, as well as a surface selectable marker (the truncated nerve growth factor receptor) and drug selectable marker. The tantamount goal for ex vivo genome engineering is to be able to modify a patient’s cells and then re-transplant them into the patient for a therapeutic outcome. In our mouse model, we have described a strategy where primary fibroblasts can be modified and then transplanted back into a recipient mouse. We are currently investigating the ability of these fibroblasts to serve as vehicles for local protein delivery to augment biological processes, such as tissue repair and wound healing. This work contributes to the body of knowledge that will enable translation of genome engineering from a scientific endeavor into a clinical reality.Item [Southwestern News](2004-12-10) Satyanarayana, MeghaItem [Southwestern News](1997-10-23) Stieglitz, HeatherItem [Southwestern News](1998-10-28) Mullen, KrisItem [Southwestern News](2005-04-04) Satyanarayana, MeghaItem [Southwestern News](1995-05-16) Martinez, EmilyItem [Southwestern News](1996-12-10) Steeves, Susan A.Item [Southwestern News](1994-09-13) McNeill, Bridgette RoseItem [Southwestern News](1995-07-05) Lyons, Morgan