Browsing by Subject "Lentivirus"

Now showing 1 - 3 of 3
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    Dynamics of the mammalian APOBEC3 locus and the relationship between mammalian APOBEC3 and Lentiviral Vif proteins.
    (2010-08) LaRue, Rebecca St. Claire
    The mammalian immune system must be dynamic in the face of a diverse and ever-changing array of pathogens. Successful pathogens have evolved to overcome host immunity. One of the most successful pathogens in the last century is the human immunodeficiency virus (HIV), which is the etiological agent responsible for the global acquired immune deficiency syndrome (AIDS) epidemic. Currently, one of the most studied host-pathogen interactions is between the cellular anti-retroviral APOBEC3 (A3) proteins and HIV viral infectivity factor (Vif) protein. A3 proteins are cytosine deaminases that primarily inhibit retroviruses and retrotransposons by mutating cytosines to uracils in retroviral DNA during reverse transcription. When HIV Vif is present, it can bind to certain A3 proteins and recruit cellular degradation complexes to target these anti-retroviral proteins for proteasomal degradation. HIV-like viruses (lentiviruses) infect other mammals and are the inspiration for studying A3 proteins in other mammals. The ultimate goal of this comparative study was to gain a better understanding of how the lentiviral Vif protein counteracts host A3 proteins. The first component of this dissertation is dedicated to the process of determining the complete A3 protein repertoires of cow and sheep (representatives of the artiodactyl lineage), which are both infected with a lentiviruses. The second component of this dissertation was to test if these artiodactyl A3 proteins were functional. Cow and sheep A3 proteins demonstrate intrinsic DNA cytosine deaminase activity and localize in cells similar to human A3 proteins. Furthermore, certain cow A3 proteins are capable of restricting HIV and are neutralized in the presence of cattle-specific lentiviral Vif (bovine immunodeficiency virus). The last component of this dissertation, a panel of conserved mammalian A3 proteins were tested to determine if each A3 protein interacts specifically with its species lentiviral Vif protein. It was shown that each representative host A3 protein is degraded in the presence of its species specific lentiviral Vif, suggesting a conserved interaction between mammalian A3 proteins and their species specific lentiviral Vif protein.
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    Gene therapy for Athabascan SCID
    (2010-09) Multhaup, Megan Marie
    Artemis is an endonuclease characterized as a key factor involved in both nonhomologous end joining (NHEJ) and variable (diversity) joining (V(D)J) recombination. Mutations in the gene encoding Artemis result in a radiationsensitive form of severe combined immunodeficiency (SCID) found at a high incidence in Athabascan-speaking Native Americans (SCID-A) and characterized by the absence of mature B and T lymphocytes. Early treatment is critical since otherwise the disease results in severe infections that ultimately lead to fatality at a young age. The current therapy for SCID-A is allogeneic hematopoeitic cell transplantation (HCT); however, HCT often results in incomplete reconstitution of B lymphocytes and may lead to complications such as graft versus host disease. Transplantation with genetically corrected autologous cells is an alternative approach that may provide improved treatment of SCID-A. Lentiviral vectors pseudotyped with VSV-G are compelling candidate vectors for gene transfer considering their high transduction efficiency and capability to mediate gene transfer in non-dividing cells populations, such as quiescent hematopoietic stem cells. Accordingly, I developed several lentiviral vectors for the transduction of human Artemis cDNA into hematopoeitic cells for the correction of a murine model of SCID-A. Upon characterization of these vectors I found that Artemis over-expression results in a decrease in cell survival due to genomic DNA fragmentation, cell cycle arrest, and ultimately apoptosis. These data emphasize the importance of transgene regulation and demonstrate the necessity of establishing conditions that provide Artemis expression at a level iv that is non-toxic yet sufficient to complement Artemis deficiency. To this end, I subsequently recovered and characterized the endogenous human Artemis promoter (APro) as a one-kilobase region located directly upstream of the human Artemis translational start site. APro conferred a moderate level of reporter gene expression in vitro and in vivo, including secondary mouse transplant recipients, thus demonstrating reliable expression after lentiviral gene transfer into hematopoeitic stem cells. Subsequently, I compared innate regulation of the human Artemis cDNA using its own endogenous promoter sequence to that of the strong EF1α and more moderate PGK promoter for the capacity to mediate correction of a murine model of Artemis deficiency presenting a B- T- phenotype and exhibiting no leakiness (mArt -/-). Transplantation with both APro-hArtemis and PGK-hArtemis transduced mArt -/- marrow led to complete reconstitution of the immune compartment in the recipient animals. Beginning at 8 weeks posttransplant, the recipient animals had wild-type levels of CD3+CD4+ and CD3+CD8+ T lymphocytes and B220+NK1.1- B lymphocytes, cell populations that are absent in mArt -/- immunodeficient mice. However, transplantation with EF1α-hArtemis transduced marrow did not support immune reconstitution, suggestive of cytotoxic effects caused by Artemis over-expression. AProhArtemis treated mice exhibited restored IgM and IgG responses against 4- hydroxyl-3-nitrophenylacetyl hapten conjugated-keyhole limpet hemocyanin as well as restored cellular immune function, as assessed by in vitro stimulation of isolated splenocytes with anti-CD3 or concanavalin A. These results demonstrate that the naturally regulated Artemis lentiviral vector effectively complemented murine SCID-A, contributing to the development and advancement of gene transfer as a clinically relevant and feasible approach for treatment of SCID-A in humans.
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    Methotrexate resistance gene transfer in stem cells
    (2008-11) Gori, Jennifer Leah
    Gene modification of hematopoietic stem cells (HSCs) has the potential to cure genetic, malignant and acquired diseases. Despite success in pre-clinical gene therapy studies, achieving genetic correction or a therapeutic response in humans has been challenging. HIV-1-based lentivirus vectors have come to the forefront of pre-clinical studies due to their ability to more effectively transduce quiescent HSCs. Drug resistance gene expression coupled to chemotherapy after HSC transplantation may support in vivo selection of gene-modified cells while protecting the patient from chemotoxicity. We hypothesized that lentivirus-mediated transfer of a methotrexate (MTX) resistance gene, Tyr22-dihydrofolate reductase (Tyr22-DHFR), into stem cells would support long-term stable gene expression in vivo and protect hematopoietic daughter cells from MTX toxicity. To test our hypothesis, we first generated high-titer lentivirus vectors expressing Tyr22-DHFR and green fluorescent protein (GFP) in different genetic configurations, and then compared MTX resistance, enzyme activity and GFP fluorescence in mouse and human cell lines including human embryonic stem cells (hESCs). Tyr22-DHFR-HSCs protected transplanted mice from MTX myelotoxicity, and conferred a significant survival advantage compared to MTX treated GFP-HSC transplanted mice. To assess the feasibility of a physiologic scale-up in a large animal model, we demonstrated DHFR-GFP expression in canine CD34+ cells and long-term engraftment of gene-modified cells in vivo. MTX administration increased gene-marking in the peripheral blood of one dog, without causing cytopenia. We also defined the optimal priming of HSCs (c-G-CSF/c-SCF BM), transduction conditions and MTX tolerated doses in dogs. Finally, we present a novel application of selective expansion of hESCs-derived cells in mouse xenografts. Methotrexate-resistant (MTXr)-DHFR hESCs gave rise to MTXr-GFP+ teratomas, indicating that that gene-modified cells retain their pluripotency during MTX treatment. MTXr-hESCs placed in stromal cell co-culture differentiated into GFP+ hemato-endothelial cells, including CD34+CD45+ subsets, which subsequently gave rise to MTXr-hematopoietic colony forming cells (CFCs). Finally, we showed that MTX administration of mice bearing hESC xenografts supported in vivo selection of Tyr22-DHFR-hESC-hematopoietic cells and increased engraftment of gene-modified cells in the bone marrow of treated mice. Taken together, these results show that lentivirus vectors effectively transduce MTXr-DHFR into HSCs, thereby preventing life-threatening myelotoxicity (as observed in our mouse studies), and supporting long-term engraftment of gene-modified cells in vivo. These studies mark significant progress of MTX resistance gene therapy toward clinical trials in humans.