Browsing by Subject "Gene Therapy"

Now showing 1 - 5 of 5
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    Alleviation of Chronic Neuropathic Pain by Agmatine Requires the GluN2B Subunit of the NMDA Receptor
    (2017-11) Peterson, Cristina
    Effective treatment for chronic pain patients remains an area of largely unmet need. However, chronic pain patients receiving traditional opioid therapy are consistently surrounded by the potential risks and social stigmas of opioid dependence, misuse, and addiction. These concerns are heightened in the face of the expanding opioid epidemic. The need for new, non-opioidergic therapeutics for management of the large population of chronic patients is widely recognized. Agmatine, also known as decarboxylated arginine, is an endogenous small molecule that has been shown to modulate maladaptive neuroplasticity that underlies the experience of chronic pain. Agmatine has been established to meet the criteria of acting as a neurotransmitter including synthesis in neurons, release from nerve terminals, and binding to post-synaptic receptors. We have previously demonstrated the efficacy of exogenously delivered agmatine in reversing chronic pain behaviors in models of neuropathic pain. Targeting primary sensory neurons through gene vectors such as serotypes of the adeno-associated virus has recently been identified as a powerful emerging strategy to treat chronic, intractable pain. Gene therapy has been approved for market use in Europe and the United States, making it a viable tool for translation from bench side to clinic. To this end, a viral vector encoding the synthetic enzyme for agmatine, namely arginine decarboxylase was developed. It has been shown that intrathecally injected viral vector particles distribute to sites of interest for chronic pain. The primary objective of my thesis work has been to expand both the application and mechanistic understanding of agmatine as a non-opioidergic therapeutic in the treatment of chronic pain. The central hypothesis of this work is that enhanced expression of arginine decarboxylase in nociceptive pathways results in long-term reduction of neuropathic pain due to agmatine production and agmatine’s antagonism of the NMDA receptor. The rationale for this research was that delivery of a gene therapy to enhance agmatine’s inhibition of NMDA signaling would be a viable, long term solution for management of chronic pain. In this thesis, I will expand upon the dual public health crises of chronic pain and prescription opioid abuse. These call for new, non-opioid therapeutic approaches for chronic pain, leading to the therapeutic development of agmatine as an NMDA receptor antagonist.
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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.
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    A synthetic approach to produce recombinant adeno-associated virus (rAAV) in Chinese hamster ovary (CHO) cells
    (2023-09) Cai, Wen
    Recombinant adeno-associated virus (rAAV) is one of the most promising gene delivery vectors for somatic gene therapy. Currently, its prevailing manufacturing technologies are relying on transiently transfecting host cells with three plasmids or infection of producer cell lines with helper viruses. Both methods pose real issues in process development, such as difficulty to scale-up or cleaning up helper virus from final products. Commonly used host cell lines for rAAV manufacturing are HEK293, and Sf9. Our lab has previously designed a helper virus-free, plasmid-free, stable cell line production system for rAAV2 via synthetic biology approach. The stable cell line was constructed by integrating multiple copies of rAAV2 genome, AAV2 Cap, AAV2 Rep and Ad5 helper genes which are under inducible promoter control and organized as three separate segments in Genome module, Replication module and Packaging module, into HEK293 cells genome. The stable cell line produced infectious rAAV2 particles upon induction. In this study, we aimed to explore the possibility of using Chinese hamster ovary (CHO) cell as the host cell and creating a stable producer cell line for rAAV2 production. Compared to HEK293 and Sf9, CHO cell holds many advantages. As the most commonly used industrial cell line for therapeutic protein production, it could reach high density in suspension cell culture using serum-free media and is resilient and robust in manufacturing conditions. This study showed that CHO cells were capable of translating AAV2 viral proteins, replicating rAAV genomes, and packaging them into rAAV vector in transient transfection using the Genome, Replication and Packaging modules. Expression of two Ad5 gene, E1A and E1B, could further enhance rAAV titer. E1A and E1B could be stably integrated into the CHO-K1 host cell genome along with the three modules under inducible promoter control. Current producer cell lines had a low productivity and their productivity appeared unstable. Nevertheless, our study demonstrated the potential of CHO cell lines as a novel production platform for rAAV manufacturing.
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    Use of Oncolytic Adenovirus expressing Interferon (IFN) Alpha as a tool to improve IFN-based chemoradiation regimen to treat pancreatic cancer
    (2018-02) Salzwedel, Amanda
    Pancreatic Ductal Adenocarcinoma (PDAC) may soon become the third-leading cause of cancer-related death in the United States. Aside from curative resection of tumors, there is no highly effective therapy to treat PDAC. Because patients are usually diagnosed in the late stage of the disease, more than 80% of them are not eligible to undergo surgery, which results in a post-diagnosis survival rate of three to four months, and an extremely low five-year survival rate of 6%. Despite discouraging current clinical outcomes, clinical trials treating PDAC patients with adjuvant therapy combined with IFN- (IFN), Fluorouracil (5-FU), Cisplatin (CDDP), and radiation have shown to improve patients' two-year survival rates by 20-41%, and improve their five-year survival rates by 35%. These clinical trials show that IFN-therapy is now one of the few therapeutic regimens that can significantly improve the short- and long-term survival of PDAC patients. Despite its high efficacy, the drawbacks of IFN therapy included high IFN systemic toxicity, which resulted in increased patient drop-out rates in clinical trials, and low levels of the cytokine in tumors, which hampered the chemo-radio-sensitization capability of IFN during therapy. To attempt to improve the efficacy and overcome the drawbacks of IFN therapy, we studied the use of IFN-expressing replication competent oncolytic adenovirus (OAd) vectors in combination with chemoradiation mimicking the aforementioned chemoradiation and IFN-based clinical trials. Because IFN therapy can stimulate a tumor-specific immune response, we first evaluated the anti-tumor effect of oncolytic adenovirus vector expressing hamster IFN (OAd-hamIFN) in a syngeneic immunocompetent PDAC hamster model. To further understand the interaction between IFN-expressing OAd and chemotherapy, radiation, and chemoradiation, and to evaluate the therapeutic effect of the virus used in treatments mimicking the IFN therapy, we also tested the effect of a human IFN-expressing oncolytic adenovirus (OAd-IFN) in immunodeficient mice bearing human PDAC xenografts. To increase infectivity of OAd-hamIFN in hamster pancreatic cancer cell lines, we included the RGD-4C (Arginive-Glycine-Aspartic) motif in the HI loop of OAd-hamIFN fiber. This modification is known to shift viral tropism from the Coxsackie and Adenovirus Receptor (CAR) to integrins αvβ3 and αvβ5, which are widely expressed in hamster pancreatic cancer cell lines. To increase infectivity of OAd-IFN in human pancreatic cancer cells lines, we replaced the adenovirus type 5 fiber with the chimeric adenovirus 5/3 fiber, which consisted of the fiber of adenovirus type 5 with the knob of adenovirus type 3. This modification has been proven to shift viral tropism from CAR receptor, which shows low expression in human pancreatic cancer cells, to Desmoglein type-2 and CD46 proteins, which are widely expressed in human pancreatic cancer cell lines. To improve spreading in tumors and oncolytic effect of both OAd-hamIFN and OAd-IFN in PDAC cells, and to achieve replication-dependent expression of IFN, we included the Adenovirus Death Protein (ADP) and respective IFN genes in the adenovirus (Ad) E3 region. To restrict the replication of human IFN expressing OAd vector (OAd-IFN) in human PDAC cells, we added the cyclooxygenase-2 (Cox-2) promoter upstream of the Ad E1 region, which is the region responsible for initiating viral replication. Combinations of 5-FU, radiation, and 5-FU + Radiation with OAd-hamIFN in hamster PDAC cells, or with OAd-IFN in human PDAC cells, resulted in highly synergistic and cytotoxic combinations in vitro. Studies in the syngeneic hamster PDAC model showed that including OAd-hamIFN in combination with therapeutics used in IFN therapy improved treatment efficacy, and that using the virus in treatment mimicking the IFN therapy (OAd-hamIFN + 5-FU + Radiation) was the most effective treatment strategy in the study. When we analyzed the effect of OAd-IFN in combination with 5-FU + CDDP in human PDAC cells, the combinations were antagonistic and weakly cytotoxic. But adding radiation to the treatment (OAd-IFN + (5-FU + CDDP) + Radiation) overcame the chemotherapy antagonism, which resulted in highly synergistic and extremely potent treatments in vitro and in mice bearing PDAC xenografts. Because radiation eliminated the antagonism of chemotherapy to the virus in vivo, we tested different radiation protocols in combination with OAd-IFN in mice bearing PDAC tumors. We concluded that administering radiation before infecting tumors with OAd-IFN improved treatment efficacy and that using radiation before OAd-IFN infection should potentiate the effect of Cox-2 controlled IFN-expressing OAds in combination with chemoradiation. In summary, our data strongly support including an IFN expressing OAd in treatments mimicking IFN therapy. As this therapy is one of the few therapeutic regimens shown to improve patients' short- and long-term survival rates, developing a virus-based IFN treatment protocol may result in a highly effective means to treat PDAC.