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- Hotchkiss, Graham
(författare)
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Towards ribozyme-mediated gene therapy of HIV-1 infections
- 2000
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Doktorsavhandling (övrigt vetenskapligt/konstnärligt)abstract
- HIV-1 is the causative agent in AIDS, the world's fourth most frequent cause of death. HIV is a retrovirus that primarily infects macrophages and T helper cells - required for a proper immune response. An infection is characterised by a lengthy period of clinical latency, during which a slow decline in the number of T helper cells eventually leads to immunodeficiency. However, the virus continues to replicate during this period with disease progression driven by viral load. Despite effective control of viral replication by combinations of antiviral drugs, the disease remains incurable and its high mutation rate can lead to selection of mutants resistant to these therapeutics. Over 95% of those infected live in the developing world, where the application of these expensive drugs is extremely limited. Gene therapy using sequence-specific designer ribozymes (catalytic RNA) is an alternative approach against HIV. Ribozymes can be designed to specifically bind to and cleave the virus RNA, allowing flexibility of drug design to match virus evolution. We demonstrated that catalytic RNA could be tolerated and function in higher eukaryotes by generating transgenic mice expressing ribozymes against a model gene, beta-2-microglubulin (ß2m). Steady state levels of ß2M mRNA were reduced by up to 90% in these mice (main reference 1). Oligonucleotides complementary to a conserved region in the HIV-1 Nef gene reduced virus production from infected primary lymphocytes demonstrating accessibility of this portion of the viral RNA. Ribozymes against this site conferred strong inhibition to productive viral infection when expressed in a human T cell line (main reference II). Ribozymes against several different sites were subsequently found to have comparable antiviral efficacy in this cell line system. Furthermore, we determined that certain of these ribozymes had the ability to block infection, presumably by cleavage of the infecting RNA-a property that may give cells expressing such agents a selective advantage in vivo (main reference III). In order to allow meaningful reconstitution of immunity any protective agent must be produced in cells that retain sufficient replication capacity. Telomeres, special structures at the ends of eukaryotic chromosomes shorten during mitosis and can be used as a molecular marker of replication potential. We demonstrated that the telomere lengths in peripheral blood mononuclear cells from HIV-1 patients were indistinguishable from healthy controls. However, treatment with highly active drug combinations lead to a moderately accelerated rate of telomere loss that may be of concern both to the patient and for the application of gene therapy in such individuals (main reference IV).
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