Browsing by Subject "Gene Targeting"
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Item CRISPR and gene editing: one tool to rule them all(2017-05-09) Wolinetz, Carrie D.Advances in gene editing, particularly the development of CRISPR-cas9, have allowed for new applications of this technology, ranging from gene drives to development of new animal models for research. This emerging biotechnology is pushing the boundaries of science, even as it provides new and evolving challenges to our policy framework and oversight mechanisms. How do we ensure responsible and feasible oversight while not constraining scientific progress that expands our knowledge base and improves human health? What are the intersection points between new gene editing applications and the current policy landscape?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 Generation of HIV-Resistant T-Cells and Correction of the Sickle Cell Mutation by Targeted Genome Engineering(2013-07-23) Voit, Richard Alexander 1983-; Goodman, Joel M.; Porteus, Matthew H.; Sternweis, Paul C.; Graff, Jonathan M.Targeted genome engineering is a powerful method to create specific modifications at chromosomal loci. This technique makes it feasible to precisely alter DNA sequences by introducing a specific DNA double-strand break, which is repaired by the natural cellular machinery. These double strand breaks are induced by engineered chimeric nucleases – either zinc finger nucleases (ZFNs) or Tal effector nucleases (TALENs) – and depending on the experimental design, can result in gene disruption, gene correction or targeted transgene integration. In this thesis, I present two applications of this approach in the context of two prevalent human diseases, HIV infection and sickle cell disease. HIV infects CD4+ T-cells by binding to the CD4 receptor and either the CCR5 or CXCR4 co-receptor on the surface of those cells. Previously, ZFNs were described that create gene specific knockouts of CCR5, protecting cells against CCR5-tropic (R5) HIV, but not against CXCR4-tropic (X4) HIV. I hypothesized that combining ZFN-mediated CCR5 disruption with targeted integration of a cassette of anti-HIV genes would confer higher levels of resistance against R5-tropic virus and also be protective against X4-tropic virus. In a T-cell reporter line, I showed that CCR5 disruption alone conferred 16-fold protection against R5-tropic virus but had no effect against X4-tropic HIV. In contrast, CCR5 disruption, combined with targeted gene integration into that locus, of the anti-HIV restriction factors human-rhesus hybrid TRIM5α, APOBEC3G D128K and Rev M10 was completely protective against both viral tropisms. Sickle cell disease is caused by a point mutation in the β-globin gene, and I sought to correct this mutation by synthesizing TALENs specific for that site. The β-globin TALENs stimulated integration of therapeutic β-globin cDNA in approximately 20% of cells prior to selection. Using FDA-approved drugs to select for modified cells, I showed virtually complete enrichment of targeted cells. Furthermore, I used the β-globin TALENs to target GFP to the β-globin start codon and designed TALENs to target tdTomato to the start codon of the γ-globin gene, upregulation of which is a goal of sickle cell disease pharmacotherapy. In this way, I developed an endogenous dual promoter reporter system and screened for drugs that preferentially upregulated γ-globin.Item Identifying Context-Specific Synthetic Lethal miRNA Inhibition in Non-Small Cell Lung Cancer(2013-07-24) Borkowski, Robert John; Scaglioni, Pier Paolo; Pertsemlidis, Alexander; White, Michael A.; Cobb, Melanie H.; Corey, David R.Non-small cell lung cancer (NSCLC) is the leading cause of cancer-related fatalities in the US. This is due in part to a lack of highly effective therapies for advanced cases, and this is of special concern as most NSCLC cases are not diagnosed until they are in an advanced, later stage. Recent successes in developing genotypically-targeted therapies with potency only in a well-defined subpopulation of tumors suggests that identifying targeted therapies for additional common NSCLC genotypes will improve patient survival. In this study I utilized a library of inhibitors to microRNAs, a class of post-transcriptional gene regulators, to identify novel synthetic lethal miRNA inhibition:molecular mechanism interactions in NSCLC. I accomplished this by screening a panel of 13 NSCLC and immortalized normal lung epithelium (HBEC) cell lines in two phases to identify miRNA inhibitors with selective toxicity in the NSCLC cell lines that were also benign in an HBEC cell line. Two inhibitors, the miR-92a and miR-1226* inhibitors, met these criteria. I then collected toxicity data in an expanded panel of 29 total cell lines. This expanded toxicity data was used to identify p53 loss as a molecular mechanism correlated with sensitivity to the miR-92a and miR-1226* inhibitors in NSCLC cell lines. This was recapitulated by demonstrating sensitivity after knockdown of p53 in the previously resistant HBEC30KT cell line. I determined that the inhibitors were toxic in a very sequence-specific manner and that they down-regulated the miR-17~92 polycistron. Down-regulation of the polycistron was toxic in a context-specific manner, and the down-regulation of the miR-17~92 cluster in sensitive cell lines mimicked activation of a 1α, 25-dihydroxyvitamin D3 response in NSCLC cell lines in a manner consistent with sensitivity to the miR-92a inhibitor. The results of this investigation demonstrate that the screening approach utilized in this study was capable of identifying a synthetic lethal miRNA inhibition:molecular mechanism interaction, and that I was then able to use a genetically defined model of the mechanism to identify a relevant mechanism of action for the toxic inhibitors.Item Improving Viral Vectors for Gene Targeting in Gene Therapy(2011-02-01) Ellis, Brian Lee; Porteus, Matthew H.Over 10,000 monogenic diseases in the world affect one out of every hundred live births (WHO). Gene targeting is a term that is used describe the manipulation of genetic material, either by adding a gene in a specific locus, creating a mutation at a specific locus, or correcting a gene at a specific locus. Here, unless otherwise noted, we will use the term to describe the correction of a gene with a homologous piece of donor genetic material whereby a mutant gene that causes monogenic disease is essentially replaced by a wild type copy through homologous recombination. Thus, gene targeting is inherently safer than classic gene therapy, where a gene is randomly introduced into the genome and can cause insertional mutagenesis. Although the rates of homologous recombination are low when simply delivering a donor substrate (1 in a million), creating a deoxyribonucleic acid (DNA) double-stranded break in or around the gene of interest using a nuclease, increases the rate of gene targeting 30,000-50,000 fold. The delivery of the nuclease and donor substrate to these cells is one of the major hurdles in achieving this type of therapy. However, for classic gene therapy there have already been many clinical trials using viral vehicles for gene delivery. One problem with using a virus for gene therapy is the low titer associated with some types of virus, in particular, lentivirus. In the first part of this dissertation, this problem is addressed by showing that the addition of caffeine during viral production can increase titer up to 8-fold. Besides lentivirus, other viruses, like Adeno-associated virus (AAV) have been used in clinical trials. There are nine AAV serotypes, but the most-well characterized is AAV2. Because there are situations where AAV is to be used in cells that cannot be transduced with AAV2, it is essential to know which serotype best infects the desired cell type. The second part of the dissertation describes a comprehensive survey of the ability of AAV1-9 and one engineered serotype to transduce primary and immortalized cells from human, mouse, hamster, and monkey origin. Overall, the results show that AAV1 and AAV6 transduce the most cell types at the highest efficiencies. Though gene targeting has been achieved using the homing endonuclease I-Sce in AAV2, targeting has never been achieved using two zinc-finger nucleases (ZFNs) in any AAV serotype. This is significant because the recognition site for I-Sce is not found in the human genome, while ZFNs are designed to specifically bind in or around a gene of interest. Based on the results from the AAV survey and the advantage of ZFNs, we created an AAV6 virus that carried the genetic information for both ZFNs and donor substrate for gene targeting in cells containing a GFP gene targeting system. We also created an AAV6 virus that carried the donor substrate alone. The third part of this dissertation reveals that dual infection at the optimal multiplicities of infection for both AAV viruses can achieve targeting efficiencies of ~3%, which is ~3-fold higher than by lipofection. Furthermore, we show that the addition of the proteasome inhibitor, MG132, increases the gene targeting level an additional 2-fold. This data suggests that AAV is a great choice for gene therapy by gene targeting. Chapters 3-5 within this body of work make significant contributions to the gene therapy field. The work and the contributions will be described in each section respectively as well as summarized in the last chapter.Item Nuclease-Mediated Targeted Gene Insertion at the Adenosine Deaminase Locus in Primary Cells(2013-07-24) Checketts, Joshua Allen; Albanesi, Joseph P.; Porteus, Matthew H.; Burma, Sandeep; Abrams, John M.; Sternweis, Paul C.Gene therapy is the ability to correct diseases at the DNA level and has long been a goal of science and medicine. The earliest gene therapy clinical trial was for a patient with severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) deficiency. Initial trials looked promising and the technique was extended to other forms of primary immunodeficiency. Unfortunately, some of the patients enrolled in these trials using retroviral vectors to carry replacement genes resulted in insertional oncogenesis. To avoid the insertional oncogenesis caused by random integration into the genome, we postulated that targeted insertion of the gene of interest through homologous recombination would prove to be a safer alternative to random viral insertion of a gene. To this end, we developed several pairs of TAL effector nucleases (TALENs) designed to target exon 1 of ADA. These TALENs function as dimers, and each pair creates a different targeted double strand break near the start site of the ADA gene. The most effective pair induces a DNA double strand break immediately preceding the ADA start codon. Targeted activity of these TALENs was measured through determining the percent of alleles that undergo mutagenic non-homologous end joining upon exposure to the TALENs, with up to 14% of alleles undergoing such mutations. In order to stimulate gene targeting at the ADA locus in human cells, these TALENs were nucleofected into the cells as plasmid DNA, along with a donor plasmid that contains the DNA to be inserted flanked by 800bp arms of homology to the cut site. These TALENs were able to stimulate site-specific integration of the desired fragment at rates of up to 10% in human cell lines. Successful targeted gene insertion was verified through maintained fluorescence, western blots, and sequencing of the targeted alleles through PCR amplification. We demonstrated the ability to enrich for targeted cells through the expression of a selectable marker within the DNA cassette integrated at the ADA locus. In addition to the editing of cell lines, we showed successful stimulation of gene targeting in patient-derived fibroblasts in 1.5% of cells. We demonstrated the feasibility of using the ADA locus as a safe harbor through the targeted insertion of three therapeutically interesting genes. Finally, we demonstrated the successful targeted gene insertion in human CD34+ in up to 0.5% of cells treated. The successful targeting of human CD34+ is especially relevant, as these cells will need to undergo gene targeting in order to be therapeutically relevant as a curative therapy for SCID due to ADA deficiency.Item Prevention of Muscular Dystrophy in Mice by Gene Editing(2014-11-20) Long, Chengzu; Wang, Zhigao; Olson, Eric N.; Cobb, Melanie H.; Chen, Zhijian J.Duchenne muscular dystrophy (DMD) is an inherited X-linked disease caused by mutations in the gene encoding dystrophin, a protein required for muscle fiber integrity. DMD is characterized by progressive muscle weakness and a shortened lifespan, often along with breathing and heart complications. There is no effective treatment. RNA-guided nucleases-mediated genome editing, based on Type II CRISPR/Cas systems, offers a new approach to alter the genome. It can precisely remove a mutation in DNA, allowing the DNA repair mechanisms to replace it with a normal copy of the gene. The benefit of this over other gene therapy techniques is that it can permanently correct the 'defect' in a gene rather than just transiently adding a 'functional' one. We used CRISPR/Cas9-mediated genome editing to correct the dystrophin gene (Dmd) mutation in the germline of mdx mice, a model for DMD, and then monitored skeletal muscle and heart structure and function. Genome editing produced genetically mosaic animals containing 2 to 100% correction of the Dmd gene. Histological analysis of skeletal muscle and heart from these corrected mice showed absence of the dystrophic muscle phenotype and restoration of dystrophin expression. In addition, the degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction, likely reflecting an advantage of the corrected stem cells and their contribution to regenerating muscle. Our experiments provide proof-of-concept that CRISPR/Cas9-mediated genomic editing can correct a causative germline mutation causing muscular dystrophy in a mouse model and prevent development of several characteristic features of the disease. With rapid technological advances of gene delivery systems and improvements to the CRISPR/Cas9 editing system, this strategy may allow correction of disease-causing mutations in the muscle tissue or iPSCs (induced pluripotent stem cells) from patients with genetic diseases.Item [Southwestern News](1996-11-12) Stieglitz, Heather