Browsing by Subject "Gene Therapy"
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Item Epistatic Interactions in the Suppression of Autoimmunity(2005-08-11) Subramanian, Srividya; Wakeland, Edward K.Sle1 is a susceptibility locus for autoimmunity derived from the lupus-prone NZM2410 mouse. The NZW-derived suppressive modifier Sles1 was identified as a specific modifier of Sle1 and prevents the development of anti-chromatin autoantibodies mediated by Sle1 on the B6 background. Fine-mapping of Sles1 with truncated congenic intervals localized it to a ~956 KB segment on mouse chromosome 17. Sles1 completely abrogated the development of activated lymphocyte populations in B6.Sle1, but splenic B cells from B6.Sle1|Sles1 still exhibited intrinsic ERK phosphorylation. Classic genetic complementation tests using the non-autoimmmune 129/SvJ mouse, suggests that this strain possesses a Sles1 allele complementary to NZW, as evidenced by the lack of autoimmunity in [129 XB6.Sle1|Sles1]F1s. These findings localized and characterized the suppressive properties of Sles1 and implicated 129 as a useful strain for aiding in the identification of this elusive epistatic modifier gene. In contrast, introduction of the suppressive modifiers Sles2 and Sles3 onto B6.Sle1 led to a decrease in the penetrance and mean titres of autoantibody production, but not complete suppression. These results suggested that genes, such as Sles1, which can specifically modify the effects of susceptibility alleles causing a breach in tolerance, could prevent systemic autoimmunity, even in the presence of additional susceptibility genes. To test this hypothesis we introduced the Sle1-specific suppressive locus Sles1 onto two different lupus-prone models: B6.Sle1|yaa and B6.Sle1|Sle2|Sle3/5, to determine whether and how Sles1 mediated suppression of Sle1 impacts the highly penetrant systemic autoimmunity characteristic of these two mouse strains. Comparing the development of a variety of pathological, immunological, functional and molecular phenotypes between the B6.Sle1|yaa and 6.Sle1|Sles1|yaa strains at different ages, revealed that Sles1 mediated a profound and complete suppression of systemic autoimmunity in this model. In contrast, the introduction of Sles1 onto the B6.Sle1|Sle2|Sle3/5 strain improved survival, and altered the kinetics of disease, but did not completely suppress disease in this model. These data suggest that the nature of the additional susceptibility loci interacting with Sle1 influences the degree of Sles1-mediated epistatic suppression of Sle1. Further characterization of these different models will help identify specific pathways Sles1 impacts, and provide insight into potential therapeutic strategies.Item Gene therapy 2000: the end of the beginning or the beginning of the end?(2000-06-29) Hofmann, Sandra L.Item Gene therapy of cancer(1993-08-05) Carbone, DavidItem Human gene therapy(1990-10-25) Fleischman, Roger A.Item Human gene therapy: from promise to practice(1993-06-17) Moreadith, Randall W.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 The Role of Tumor Necrosis Factor (TNF) in Microglial Activation and Progressive Degeneration of Dopaminergic Neurons(2010-05-14) Harms, Ashley Nicole Simpson; Tansey, Malú G.Parkinson’s disease (PD) is a progressive neurodegenerative disorder characterized by a loss of dopaminergic (DA) neurons in the substantia nigra pars compacta (SNpc). A number of studies have implicated chronic inflammation in the pathophysiology of PD; however, it is unclear which inflammatory mechanisms directly contribute to neuronal loss in PD. A number of cytokines, including TNF, are elevated in post-mortem brain and cerebrospinal fluid of patients with PD. Previous studies from our group have shown that blocking solTNF signaling at the time of a unilateral 6-OHDA striatal lesion attenuated behavioral deficits and the acute loss of dopaminergic neuron loss by 50%. However, a critical question of clinical relevance is whether delayed solTNF signaling inhibition can prevent the progressive loss of DA neurons that occurs after a CNS insult. I report here that a single intranigral injection of a lentivirus encoding a dominant negative TNF inhibitor delivered 2 weeks after an intrastriatal 6-OHDA lesion attenuated microgliosis in SNpc and halted the progressive loss of nigral DA neurons and the associated locomotor deficits. Given the potential contribution of microglial activation to PD and the suggestion that anti-TNF therapies in the CNS may exert neuroprotective effects on vulnerable dopaminergic neuron populations, I also investigated the role of TNF in regulating microglia effector functions to gauge the potential detrimental effects of anti-TNF therapies on the microglia functional response. I found that microglia from TNF-null mice produced reduced protein levels of cytokines and chemokines in response to LPS stimulation and they displayed no cytotoxic effects on dopaminergic neuroblastoma MN9D cells when activated in vitro. I also demonstrated that microglia isolated from TNF-null mice failed to display the expected morphological changes in response to LPS stimulation, including enhanced perinuclear expression of the activation marker CD45. My results suggest that TNF plays a critical role in microglia activation, regulation of several microglia effector functions and is the primary microglia-derived inflammatory factor that compromises survival of dopaminergic neurons. Furthermore, these studies suggest that TNF-dependent neuroinflammation directly contributes to the delayed and progressive degeneration of nigral DA neurons after neurotoxic injury and further validates solTNF as a potential therapeutic target in PD.Item Targeted Molecular Imaging: A Guide to Combination Therapy(2006-12-20) Ren, Gang; Öz, Orhan K.Recombinant adenovirus is widely used to deliver genes for cancer gene therapy. Coxsackie and Adenovirus' Receptor (CAR) is the primary receptor for recombinant adenovirus. Little attention has been paid to determine CAR protein expression and promoter activity in vivo. This study tested the hypothesis that targeted molecular imaging of CAR could predict the tissue receptivity to viral infection and the response to treatment with histone deacetylases inhibitor (HDACi). To image CAR protein expression, human prostate cancer xenografts were established in nu/nu mice. The ability of iodinated anti-CAR intact, F(ab')2 fragments and control F(ab')2 fragments to distinguish CAR (+) tumors was tested by biodistribution, gamma camera scintigraphy and validated by western blot. Tumor susceptibility to infection was tested with adenoviruses carrying the reporter β-galactosidase. To assess CAR promoter activity, a sodium iodide symporter (NIS) reporter construct containing the NIS open reading frame driven by the CAR promoter (CAR-NIS), was constructed by directional cloning. Tumor cell lines stably expressing CAR-NIS or empty vector were established. NIS protein function was assessed by intracellular accumulation of 99mTcO4- and mRNA level was tested by RT-PCR. The inductivity of the CAR promoter by HDACi in vivo was tested by imaging CAR-NIS tumors after administration of 99mTcO4- using a gamma camera. A replication-deficient recombinant adenovirus coding CAR-NIS was constructed to deliver the reporter construct to cells and tumors to permit radionuclide imaging. Radiolabeled anti-CAR F(ab')2 fragments more effectively distinguished CAR(+) from CAR(-) tumors at early time points. Tumors with greater retention of radiolabeled anti-CAR showed higher levels of CAR protein expression by western blot and ß-galactosidase activity after adenoviral infection. Stable CAR-NIS transfectants showed 16-fold to180-fold increase of 99mTcO4- accumulation after HDACi treatment in PC3 prostate cancer cells or TCC bladder cancer cells, respectively (p<0.001). Ad-CAR-NIS effectively delivered the HDACi inducible CAR-NIS into target cells in a dose dependent manner. The transgene CAR-NIS expression was imaged in vivo using gamma camera scintigraphy. Molecular imaging approaches to image CAR protein expression and assess CAR promoter activity in vivo can predict tissue receptivity to adenoviral infection and have potential to direct combination of gene delivery and chemotherapy.