Characterization of a mobilization-competent simian immunodeficiency virus (SIV) vector containing a ribozyme against SIV polymerase Kevin V. Morris, 1 Robert A. Grahn, 2 David J. Looney 1 and Niels C. Pedersen 3 Correspondence Kevin V. Morris kvmorris@ucsd.edu 1 Department of Medicine, Stein Clinical Research Building Room 402, University of California San Diego, La Jolla, CA 92093-0665, USA 2 Department of Population Health and Reproduction, Tupper Hall Room 1114, University of California Davis, Davis, CA 95616, USA 3 Department of Veterinary Medicine and Epidemiology, Tupper Hall Room 2108, University of California Davis, Davis, CA 95616, USA Received 17 January 2003 Accepted 12 January 2004 Exploitation of the intracellular virus machinery within infected cells to drive an anti-viral gene therapy vector may prove to be a feasible alternative to reducing viral loads or overall virus infectivity while propagating the spread of a therapeutic vector. Using a simian immunodeficiency virus (SIV)-based system, it was shown that the pre-existing retroviral biological machinery within SIV-infected cells can drive the expression of an anti-SIV pol ribozyme and mobilize the vector to transduce neighbouring cells. The anti-SIV pol ribozyme vector was derived from the SIV backbone and contained the 59- and 39LTR including transactivation-response, Y and Rev-responsive elements, thus requiring Tat and Rev and therefore limiting expression to SIV-infected cells. The data presented here show an early reduction in SIV p27 levels in the presence of the anti-SIV pol ribozyme, as well as successful mobilization (vector RNA constituted ~17 % of the total virus pool) and spread of the vector containing this ribozyme. These findings provide direct evidence that mobilization of an anti-retroviral SIV gene therapy vector is feasible in the SIV/macaque model. INTRODUCTION Current anti-retroviral combination drug therapy (highly active anti-retroviral therapy, HAART) reduces morbidity and mortality in human immunodeficiency virus type 1 (HIV-1)-infected individuals (reviewed by Gazzard, 1999; Shafer & Vuitton, 1999). However, the toxicity of anti- retroviral drugs, compliance with the life-long regimen and the evolution of antiretroviral resistance in the face of drug pressure illustrate the limitations of this approach (Shafer & Vuitton, 1999). Alternative strategies to inhibit virus replication, either alone or in combination with those currently practised, are desirable (Mautino & Morgan, 2002). One adjunctive strategy explored here involves the use of a ribozyme. Ribozymes are catalytic RNA molecules that can be engineered to cleave specifically and effectively destroy a given target RNA (Cech, 1987), presenting an attractive method for reducing viral load in HIV-1 infection (reviewed by Rossi, 2000). A limiting step in the current use of ribozymes, however, is their delivery to virus-infected cells. Due to safety concerns, emphasis on therapeutic gene delivery has relied on the development of replication-defective, recombinant retroviral vectors (Barinaga, 1994; Miller & Wolgamot, 1997). Successful gene transfer to human T cells, stem cells, dendritic cells and bone marrow has been achieved, with expression of the marker gene ranging from weeks to 36 months (Bauer et al., 1997; Leavitt et al., 1994; Mangeot et al., 2002; Yu et al., 1995). The delivery of gene therapy vectors and subsequent gene transfer have involved direct transduction of the target cells with the desired vector (Buchschacher & Wong-Staal, 2000). This method has not proved practical as it involves ex vivo transductions, with the infusion of the transduced cells back into the infected individual. An alternative strategy for a gene therapy vector delivery system involves revising the current paradigm and using conditional-replicating or mobilizable vectors, with the cells already infected for vector propagation. However, the use of such vectors could be expected to be limited by any anti-viral genes within the vector (Klimatcheva et al., 2001). None the less, it has previously been shown that the packaging of vector RNA creates competition for encapsidation of viral RNA, reducing the amount of wild- type virions roughly sixfold, leading to reduced particle 0001-9106 G 2004 SGM Printed in Great Britain 1489 Journal of General Virology (2004), 85, 1489–1496 DOI 10.1099/vir.0.19106-0