Preclinical studies of ribozyme-mediated gene therapy for HIV-1

Sammanfattning: Approximately 40 million people are infected with HIV-1 and AIDS is the number one cause of death in Africa as well as, the fourth leading cause of death globally. Patients in developed countries generally receive highly active antiretroviral therapy (HAART), even though the drugs are expensive and sometimes cause side-effects and treatment failure. This antiretroviral therapy does not cure or eradicate HIV. Gene therapy using ribozymes could be an alternative approach. Ribozymes are small catalytic RNAs, acting as sequence-specific endoribonucleases. They can be designed to hybridize to and cleave specific RNAs. This thesis focuses on basic cellular and molecular studies to add information necessary for the prospect of bringing ribozymes to the clinic as a functional gene therapy against HIV-1 infection. The ribozyme constructs were all developed with the rationale of using a trans-targeting (antiHIV-1) ribozyme followed by a cis-cleaving ribozyme. The purpose of the cis-cleaving ribozyme is to release the upstream trans-targeting anti-HIV-1 ribozyme (a hammerhead or a hairpin ribozyme) and in this way achieve a short ribozyme with a defined 3´end. The resulting 3´end is stable to exoribonucleases due to a 2'3 cyclic phosphate resulting from the cis-cleavage reaction. The ribozyme transcripts were generated from the CMV promoter, a potent constitutively and ubiquitously expressing RNA polymerase Il promoter. The efficiency of the cis-cleavage reaction was studied by introducing such cassettes with anti-HIV-1 ribozyme into a human cell line or transgenic mice. When expression cassettes were stably transfected into a human T cell line (HUT78), the occurrence of an efficient ciscleavage was found to affect both the steady-state level as well as, the anti-viral efficiency of the trans-cleaving ribozyme. If the cis-cleavage was inhibited, by insertion of a point mutation, higher steady-state levels of the full-length ribozyme transcript were observed however, it also abolished the protection against HIV-1 infection. In transgenic mice, an HIVI specific hairpin ribozyme was found at significantly higher steady-state levels than a hammerhead ribozyme. In addition, an unexpected organ difference; kidney > liver > lung > spleen, regarding steady-state levels of both 5' and 3' cleavage products, was found (Paper I). The relative ability of hammerhead and hairpin ribozymes directed against a well-studied target site in the untranslated 5' region (U5) to inhibit HIV-1 replication was tested. The results revealed a similar antiviral efficacy, despite the fact that the hammerhead showed faster cleavage kinetics in a cell free system. It was also found that ribozymes could limit or prevent the appearance of integrated provirus, indicating cleavage of the infecting viral RNA before reverse transcription (Paper II). The prospect of combining HAART and ribozyme gene therapy was supported by the finding that less amount of antiretroviral drugs were required to reduce HIV-1 p24 levels when administered to HUT78 cells expressing an efficient anti-HIV1 ribozyme (Paper III). An eased drug burden would decrease the common side-effects and reduce the development of drug-resistant virus. Five ribozymes targeting different sites in the HIV-1 SF2 genome were evaluated individually and in combinations for inhibition of HIV-1 replication in cell culture. The best of all combinations was a double ribozyme (hhnef-hpU5), exhibiting an 86% protection against HIV1SF2 (Paper IV). Interactions between hammerhead ribozymes and RNA substrates in a cell free test system could readily be followed in real-time using SPR technology. This could be relevant for evaluating the impact of various factors that may enhance the ribozyme cleavage (Paper V).