- Email: [email protected]
447. Packaging and Transfer of Gag-Pol mRNA to Target Cells by EIAV Based Lentiviral Vectors
RNA VIRUS VECTORS II GCV-resistant cells. To overcome the sensitivity limitation, Black et al (Proc. Natl. Acad. Sci USA,93: 3525-3529, 1996; Cancer Res. 61, 3022-3026, 2001) generated mutants of HSV-TK that confer increased GCV sensitivity to to a wide variety of mammalian cells. Two particularly effective mutants, mutant 75 and mutant 39, increased cell killing 43-fold and 249-fold, respectively, compared to wild type HSV-TK. Recently, we developed a novel chimeric suicide gene by fusing the extracellular and transmembrane domains of human CD34 to the entire coding region of wild type HSV-TK. The CD34-TK chimeric suicide gene is advantageous because it simultaneously provides a cell surface marker for efficient tracking and purification of gene-modified cells as well as information about HSV-TK expression at the single cell level. In this study, new CD34TK suicide genes containing the mutant 75 and mutant 39 HSV-TK enzymes were generated and tested for the ability to enhance in vitro cell sensitivity to GCV. Furthermore, since GCV resistance in human T cells has been attributed to altered splicing of the HSV-TK gene in retroviral packaging cells (Garin et al Blood, 97:122-129, 2001), we eliminated the potential for altered splicing in our CD34TK constructs by introducing third base degenerate changes into the CD34-TK75 and CD34-TK39 genes. High efficiency gene transfer of the chimeric CD34-TK variants to NIH 3T3 cells and primary human T cells was accomplished using a VSV-G pseudotyped, Moloney murine leukemia virus-based retrovirus. Following transduction, cells expressing similar levels of the CD34-TK suicide genes were purified to greater than 97% by CD34 immunomagnetic selection and evaluated for the level of sensitivity to GCV. Surprisingly, CD34-TKwt, CD34-TK75 and CD34-TK39 exhibited similar sensitivity to GCV. In 3T3 cells, the IC50 values of CD34TKwt, CD34-TK75, and CD34-TK39 were 53 nM, 68 nM, and 32 nM respectively. A similar trend was observed in human T cells, where CD34-TKwt, CD34-TK75, and CD34-TK39 exhibited IC50 values of 3.1 nM, 3.3 nM, and 2.2 nM respectively. Not surprisingly, introducing third base degenerate changes at the previously characterized cryptic splice donor and acceptor sites of HSV-TK had no effect on the GCV sensitivity of the CD34-TK variants. Taken together, these data demonstrate that the chimeric suicide genes CD34-TK75 and CD34-TK39 expressing the mutant TK enzymes offer no increased cell sensitivity to GCV compared to CD34-TKwt, but significantly lower IC50 values (100-1000X) than that previously reported by others for wild type HSV-TK.
446. Lentiviral Transduction of MonocyteDerived Dendritic Cells for Cancer Immunotherapy Nachimuthu Chinnasamy,1 Dhanalakshmi Chinnasamy.1 1 Immunotherapy Program, St. Luke’s Medical Center, Milwaukee, WI. Dendritic cells (DCs) are the most potent professional antigen presenting cells and play vital role in primary immune response. DCs engineered to express immunomodulators have shown feasibility in eliminating tumor in murine models. DCs instruct and activate a naïve immune system to mount a response toward foreign proteins. Therefore, it has been hypothesized that gene transfer of tumor antigens into DCs would be an ideal strategy for cancer immunotherapy. Genetic engineering of DCs allows constitutive expression of encoded proteins and thus prolong the presentation of tumor-associated antigens (TAA). In addition, multiple and unidentified epitopes encoded by the entire gene may induce strong antitumor immunity by inducing cytotoxic T lymphocyte response against multiple antigenic epitopes. However, methods for safe and efficient gene delivery to DCs, and clinically feasible culture and gene transfer procedures are required to translate this approach to clinical trial. Previously, we and others have shown that a multiply attenuated HIV-1-based lentiviral vector could efficiently transduce S176
monocyte-derived DCs. Recently, a self-inactivating (SIN) lentiviral vector with a 400-nucleotide deletion in the 3’ LTR was shown to be biologically safe and efficient in transducing various cell types. Here we report the efficiency of SIN lentiviral vector in transducing monocyte-derived DCs. We generated vesicular stomatitis virus G glycoprotein (VSV.G) pseudotyped SIN lentiviral vector particles expressing enhanced green fluorescent protein (EGFP) as a marker gene under the control of human phosphoglycerate kinase (PGK) promoter and used them to transduce the human peripheral blood monocyte-derived DCs. Then, we optimized procedures for high titer SIN lentiviral vector production and efficient transduction of peripheral blood monocyte-derived DCs using clinically applicable cell culture reagents. Our results demonstrate that high titer SIN lentivector could be generated in culture media containing 5% human serum, which could transduce more than 80% of the cells at an MOI of 10, resulting high level expression of the transgene. These results were comparable to that obtained with vector produced in medium containing fetal calf serum. Vector production was optimal and comparable in all the three culture media (Opti-MEM, Ex-Vivo-10, and DMEM) tested. DCs transduced under these conditions retain their immature immunophenotype and maintain their ability to respond to maturation signals, and showed enhanced T cell immunostimulatory potential. Further, lentivirally modified EGFP expressing DCs were able to prime and generate antigen-specific cytotoxic T cells capable of killing EGFP positive autologous cells. Overall, these findings show that SIN lentiviral vector could be produced in high titer using clinical grade culture reagents and provide a highly efficient gene delivery system to generate gene-modified DC vaccine for cancer immunotherapy.
447. Packaging and Transfer of Gag-Pol mRNA to Target Cells by EIAV Based Lentiviral Vectors James E. Miskin,1 Daniel D. Chipchase,1 Susan M. Kingsman,2 Kyriacos A. Mitrophanous.1 1 Virology Group, Oxford BioMedica (UK) Ltd., Oxford, United Kingdom; 2Oxford BioMedica (UK) Ltd., Oxford, United Kingdom. Lentiviral vectors hold great promise as tools for gene therapy and there are many examples of therapeutic efficacy in model systems. These vectors are now being considered for clinical evaluation. Vector safety is maximised by eliminating all extraneous vector-derived sequences from the transfer genome and from the production system. Such ‘minimal systems’ should have a restricted opportunity for generating replication competent lentiviruses (RCLs) from homologous recombination between the vector components; suitable RCL assays are being devised to validate this assumption. There are potential issues regarding the vectors’ interaction with endogenous or exogenously acquired retroviral or lentiviral-like sequences in the target cells. We have attempted to model the potential for such interactions by examining the packaging of GagPol sequences into the vector particles and the transfer of these Gag-Pol sequences to target cells. The Gag-Pol proteins are essential for copying and integrating novel DNA sequences into a cell. Thus, theoretically the transfer of an active Gag-Pol gene to a target cell could result in the production of virus-like particles, which, in the presence of a suitable envelope, could be transferred to other cells. We have shown that EIAV and HIV derived vector particles contain Gag-Pol RNA. This occurs because lentiviral vectors are assembled in the cytoplasm of producer cells, an environment that is rich in host-cell and vector derived mRNA species. The degree of packaging depends upon the Gag-Pol expression cassette. By using a codonoptimised Gag-Pol gene with no packaging signals we have reduced the packaging of Gag-Pol mRNA to background levels. However, this still provides the possibility of the transfer of Gag-Pol RNA to target cells, as a result of the unavoidable non-specific association of Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
RNA VIRUS VECTORS II RNAs with vector particles. We now show that there is indeed transfer and integration of Gag-Pol sequences into the genomes of target cells but that this represents a small fraction of the number of transducing units (TU) in a clinical vector dose, which we currently assume to be a maximum of 1010 TU. We have considered the possibility that this transfer results from a recombination event between the vector genome and the packaging construct, but we have found no evidence for this. The frequency of transfer was correlated with the degree of packaging of the Gag-Pol RNA. Therefore, we believe that we are detecting integration of an inefficiently reverse transcribed entity and not DNA contamination. The mechanism of this integration is under investigation. We have failed to detect the production of any Gag-Pol proteins or particles from a transduced cell population using sensitive PERT analysis, suggesting that the transferred sequences are not active. Our data therefore indicate lentiviral vectors potentially transfer Gag-Pol sequences to target cells, and this cannot be eliminated, but using optimised EIAV systems minimises this transfer and no inappropriate particle production or biological effects are observed in target cells. Optimised minimal EIAV based vectors are now being evaluated for the delivery of therapeutic genes for the treatment of late stage Parkinson’s disease.
448. Optimization of Lentiviral Transduction of Primary Murine Splenocytes for Adoptive Immunotherapy Oscar Szentirmai,1 Bob S. Carter,1 Richard C. Mulligan.1 Department of Genetics, Harvard Medical School, Boston, MA, United States. 1
Adoptive immunotherapy protocols may benefit from genetic retargeting of lymphocyte specificity with chimeric T-cell receptors. We have previously shown that a retroviral transduction strategy could be employed to retarget primary murine lymphocytes to angiogenic blood vessels associated with tumor growth. (PNAS 99:7009, 2002) One potential difficulty with these approaches has been the transduction process itself, which has required an extensive ex vivo culturing with exposure to CD3 and CD28 for up to 72 hours prior to retroviral infection, with associated activation induced cell death. We are currently working to develop protocols that employ lentiviral vectors to transduce murine CD8+ cells with minimal ex vivo culturing and minimal activation. Using a strategy of VSV-G pseudotyping and concentration to titers of >10^9/cc, we have compared the efficacy of transduction of primary murine CD8+ cells using a single lentiviral supernatant exposure (marker gene eGFP) comparing the following variables: 1) no preculturing versus preculturing with exposure to soluble CD3/CD28 antibody 2) culture in Il-2 versus Il-7 and 3) use of protamine sulfate versus polybrene versus no cationic supplement. Using an approach that involves no pretransduction stimulation and no use of cationic exposure, with subsequent culturing in IL-7 we found that we were able to achieve ~90% infection of 10^6 CD8+ cells with a single viral supernatant exposure. Application of this strategy may improve in vivo survival of transduced T-cells.
449. Secretion and Uptake of Modified Mouse Galactosylceramidase Delivered by Lentiviral Vectors X.-Y. Zhang,1 S.-C. Li,2 J. Reiser.1 1 Gene Therapy Program, LSUHSC, New Orleans, LA, United States; 2Biochemistry, Tulane University School of Medicine, New Orleans, LA, United States. Krabbe disease or globoid cell leukodystrophy (GLD), is an inherited recessive disorder affecting the central and peripheral nervous systems caused by a genetic deficiency of the lysosomal Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
enzyme, galactosylceramidase (galactocerebroside b-galactosidase, GALC). Like most chronic neurodegenerative disorders, effective treatments for Krabbe disease are impeded by the structural and functional complexity and relative inaccessibility of the CNS. With a view toward designing gene therapy strategies for Krabbe disease, HIV-1-based lentiviral vectors encoding mouse GALC were constructed and the efficiency of expressions of the GALC enzyme was tested in cell lines and in primary cultures. High levels of GALC activity (up to 80-fold above endogenous levels) were detected in monkey COS-7 cells, human 293T cells and in human bone marrow derived mesenchymal stem cells following transduction with GALC lentiviral vectors. Sustained production of GALC activity in the culture medium of virus transduced cells over a period of at least two weeks was observed. Lentiviral vectors pseudotyped with VSVG were able to transduce efficiently skin derived fibroblast cells from twitcher mice (a murine model of GLD) and resulted in cross correction of the enzyme deficiency in these cells. Modified mouse GALC with a 24-amino acid addition at its C-terminus including a myc epitope and 6 His residues (termed GALC-MH) revealed an unaltered specific activity. Likewise, mouse GALC tagged with an 11-amino acid Tat protein transduction domain (PTD) in addition to the myc epitope and the 6 His residues (termed GALC-TMH) was only slightly impaired in terms of its specific activity. Tagged GALC protein could be easily detected by immunocytochemistry in transduced cells and by Western blotting in cell extracts and in the cell culture medium from transduced cells. GALC-MH and GALCTMH secreted from transduced 293T or COS-7 cells were taken up by untransduced 293T, COS-7 and H9 cells by mannose 6-phosphate receptor (M6PR) mediated endocytosis. These results provide evidence that the newly synthesized GALC enzyme in transduced cells maintains typical features of the native lysosomal GALC enzyme. Interestingly, GALC-TMH also revealed M6PRindependent uptake in vitro, which indicates that the PTD motif of the Tat protein facilitates the distribution of GALC protein between cells. Therefore, functional Tat-modified mouse GALC delivered by lentiviral vector-medicated gene transfer in vivo might improve the therapeutic potency in GALC deficient twitcher mice.
450. Virus
RNA-Mediated Inhibition of Hepatitis B
Kathryn L. Nash,1 Graeme J. Alexander,1 Andrew M. Lever.1 Department of Medicine, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom. 1
Chronic hepatitis B virus (HBV) infection is frequently associated with cirrhosis and hepatocellular carcinoma. HBV is a double stranded DNA virus that utilises reverse transcription to replicate via a RNA intermediate. Four major viral RNAs are produced that share a common 3’ terminus. Strategies that target viruses by sequence specific recognition of their RNA have recently received interest as potential therapeutic tools. The aim of this study was to design genes to produce antisense RNA or ribozymes (small catalytic RNA molecules) that target HBV. The ability of these genes to inhibit HBV gene expression was assessed by using lentiviral vectors to deliver the genes to a HBV producing cell line in vitro. Antisense RNA was designed to target the HBV mRNAs encoding the surface antigen, X protein and the viral encapsidation signal. Ribozymes targeting the viral surface and X RNAs were also created. The cDNAs for each of these were cloned into lentiviral vectors. Vectors containing sense RNA and catalytically inactive ribozymes were created as controls. Replication deficient lentiviral vectors containing these genes were produced and applied to HepG2 (2.2.15) cells in vitro. A ribonuclease protection assay was used to determine HBV RNA levels in transduced cells. The ribozyme-containing vectors produced the most dramatic reductions in HBV RNA levels in transduced cells. The X ribozyme S177
446. Lentiviral Transduction of MonocyteDerived Dendritic Cells for Cancer Immunotherapy Nachimuthu Chinnasamy,1 Dhanalakshmi Chinnasamy.1 1 Immunotherapy Program, St. Luke’s Medical Center, Milwaukee, WI. Dendritic cells (DCs) are the most potent professional antigen presenting cells and play vital role in primary immune response. DCs engineered to express immunomodulators have shown feasibility in eliminating tumor in murine models. DCs instruct and activate a naïve immune system to mount a response toward foreign proteins. Therefore, it has been hypothesized that gene transfer of tumor antigens into DCs would be an ideal strategy for cancer immunotherapy. Genetic engineering of DCs allows constitutive expression of encoded proteins and thus prolong the presentation of tumor-associated antigens (TAA). In addition, multiple and unidentified epitopes encoded by the entire gene may induce strong antitumor immunity by inducing cytotoxic T lymphocyte response against multiple antigenic epitopes. However, methods for safe and efficient gene delivery to DCs, and clinically feasible culture and gene transfer procedures are required to translate this approach to clinical trial. Previously, we and others have shown that a multiply attenuated HIV-1-based lentiviral vector could efficiently transduce S176
monocyte-derived DCs. Recently, a self-inactivating (SIN) lentiviral vector with a 400-nucleotide deletion in the 3’ LTR was shown to be biologically safe and efficient in transducing various cell types. Here we report the efficiency of SIN lentiviral vector in transducing monocyte-derived DCs. We generated vesicular stomatitis virus G glycoprotein (VSV.G) pseudotyped SIN lentiviral vector particles expressing enhanced green fluorescent protein (EGFP) as a marker gene under the control of human phosphoglycerate kinase (PGK) promoter and used them to transduce the human peripheral blood monocyte-derived DCs. Then, we optimized procedures for high titer SIN lentiviral vector production and efficient transduction of peripheral blood monocyte-derived DCs using clinically applicable cell culture reagents. Our results demonstrate that high titer SIN lentivector could be generated in culture media containing 5% human serum, which could transduce more than 80% of the cells at an MOI of 10, resulting high level expression of the transgene. These results were comparable to that obtained with vector produced in medium containing fetal calf serum. Vector production was optimal and comparable in all the three culture media (Opti-MEM, Ex-Vivo-10, and DMEM) tested. DCs transduced under these conditions retain their immature immunophenotype and maintain their ability to respond to maturation signals, and showed enhanced T cell immunostimulatory potential. Further, lentivirally modified EGFP expressing DCs were able to prime and generate antigen-specific cytotoxic T cells capable of killing EGFP positive autologous cells. Overall, these findings show that SIN lentiviral vector could be produced in high titer using clinical grade culture reagents and provide a highly efficient gene delivery system to generate gene-modified DC vaccine for cancer immunotherapy.
447. Packaging and Transfer of Gag-Pol mRNA to Target Cells by EIAV Based Lentiviral Vectors James E. Miskin,1 Daniel D. Chipchase,1 Susan M. Kingsman,2 Kyriacos A. Mitrophanous.1 1 Virology Group, Oxford BioMedica (UK) Ltd., Oxford, United Kingdom; 2Oxford BioMedica (UK) Ltd., Oxford, United Kingdom. Lentiviral vectors hold great promise as tools for gene therapy and there are many examples of therapeutic efficacy in model systems. These vectors are now being considered for clinical evaluation. Vector safety is maximised by eliminating all extraneous vector-derived sequences from the transfer genome and from the production system. Such ‘minimal systems’ should have a restricted opportunity for generating replication competent lentiviruses (RCLs) from homologous recombination between the vector components; suitable RCL assays are being devised to validate this assumption. There are potential issues regarding the vectors’ interaction with endogenous or exogenously acquired retroviral or lentiviral-like sequences in the target cells. We have attempted to model the potential for such interactions by examining the packaging of GagPol sequences into the vector particles and the transfer of these Gag-Pol sequences to target cells. The Gag-Pol proteins are essential for copying and integrating novel DNA sequences into a cell. Thus, theoretically the transfer of an active Gag-Pol gene to a target cell could result in the production of virus-like particles, which, in the presence of a suitable envelope, could be transferred to other cells. We have shown that EIAV and HIV derived vector particles contain Gag-Pol RNA. This occurs because lentiviral vectors are assembled in the cytoplasm of producer cells, an environment that is rich in host-cell and vector derived mRNA species. The degree of packaging depends upon the Gag-Pol expression cassette. By using a codonoptimised Gag-Pol gene with no packaging signals we have reduced the packaging of Gag-Pol mRNA to background levels. However, this still provides the possibility of the transfer of Gag-Pol RNA to target cells, as a result of the unavoidable non-specific association of Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
RNA VIRUS VECTORS II RNAs with vector particles. We now show that there is indeed transfer and integration of Gag-Pol sequences into the genomes of target cells but that this represents a small fraction of the number of transducing units (TU) in a clinical vector dose, which we currently assume to be a maximum of 1010 TU. We have considered the possibility that this transfer results from a recombination event between the vector genome and the packaging construct, but we have found no evidence for this. The frequency of transfer was correlated with the degree of packaging of the Gag-Pol RNA. Therefore, we believe that we are detecting integration of an inefficiently reverse transcribed entity and not DNA contamination. The mechanism of this integration is under investigation. We have failed to detect the production of any Gag-Pol proteins or particles from a transduced cell population using sensitive PERT analysis, suggesting that the transferred sequences are not active. Our data therefore indicate lentiviral vectors potentially transfer Gag-Pol sequences to target cells, and this cannot be eliminated, but using optimised EIAV systems minimises this transfer and no inappropriate particle production or biological effects are observed in target cells. Optimised minimal EIAV based vectors are now being evaluated for the delivery of therapeutic genes for the treatment of late stage Parkinson’s disease.
448. Optimization of Lentiviral Transduction of Primary Murine Splenocytes for Adoptive Immunotherapy Oscar Szentirmai,1 Bob S. Carter,1 Richard C. Mulligan.1 Department of Genetics, Harvard Medical School, Boston, MA, United States. 1
Adoptive immunotherapy protocols may benefit from genetic retargeting of lymphocyte specificity with chimeric T-cell receptors. We have previously shown that a retroviral transduction strategy could be employed to retarget primary murine lymphocytes to angiogenic blood vessels associated with tumor growth. (PNAS 99:7009, 2002) One potential difficulty with these approaches has been the transduction process itself, which has required an extensive ex vivo culturing with exposure to CD3 and CD28 for up to 72 hours prior to retroviral infection, with associated activation induced cell death. We are currently working to develop protocols that employ lentiviral vectors to transduce murine CD8+ cells with minimal ex vivo culturing and minimal activation. Using a strategy of VSV-G pseudotyping and concentration to titers of >10^9/cc, we have compared the efficacy of transduction of primary murine CD8+ cells using a single lentiviral supernatant exposure (marker gene eGFP) comparing the following variables: 1) no preculturing versus preculturing with exposure to soluble CD3/CD28 antibody 2) culture in Il-2 versus Il-7 and 3) use of protamine sulfate versus polybrene versus no cationic supplement. Using an approach that involves no pretransduction stimulation and no use of cationic exposure, with subsequent culturing in IL-7 we found that we were able to achieve ~90% infection of 10^6 CD8+ cells with a single viral supernatant exposure. Application of this strategy may improve in vivo survival of transduced T-cells.
449. Secretion and Uptake of Modified Mouse Galactosylceramidase Delivered by Lentiviral Vectors X.-Y. Zhang,1 S.-C. Li,2 J. Reiser.1 1 Gene Therapy Program, LSUHSC, New Orleans, LA, United States; 2Biochemistry, Tulane University School of Medicine, New Orleans, LA, United States. Krabbe disease or globoid cell leukodystrophy (GLD), is an inherited recessive disorder affecting the central and peripheral nervous systems caused by a genetic deficiency of the lysosomal Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright © The American Society of Gene Therapy
enzyme, galactosylceramidase (galactocerebroside b-galactosidase, GALC). Like most chronic neurodegenerative disorders, effective treatments for Krabbe disease are impeded by the structural and functional complexity and relative inaccessibility of the CNS. With a view toward designing gene therapy strategies for Krabbe disease, HIV-1-based lentiviral vectors encoding mouse GALC were constructed and the efficiency of expressions of the GALC enzyme was tested in cell lines and in primary cultures. High levels of GALC activity (up to 80-fold above endogenous levels) were detected in monkey COS-7 cells, human 293T cells and in human bone marrow derived mesenchymal stem cells following transduction with GALC lentiviral vectors. Sustained production of GALC activity in the culture medium of virus transduced cells over a period of at least two weeks was observed. Lentiviral vectors pseudotyped with VSVG were able to transduce efficiently skin derived fibroblast cells from twitcher mice (a murine model of GLD) and resulted in cross correction of the enzyme deficiency in these cells. Modified mouse GALC with a 24-amino acid addition at its C-terminus including a myc epitope and 6 His residues (termed GALC-MH) revealed an unaltered specific activity. Likewise, mouse GALC tagged with an 11-amino acid Tat protein transduction domain (PTD) in addition to the myc epitope and the 6 His residues (termed GALC-TMH) was only slightly impaired in terms of its specific activity. Tagged GALC protein could be easily detected by immunocytochemistry in transduced cells and by Western blotting in cell extracts and in the cell culture medium from transduced cells. GALC-MH and GALCTMH secreted from transduced 293T or COS-7 cells were taken up by untransduced 293T, COS-7 and H9 cells by mannose 6-phosphate receptor (M6PR) mediated endocytosis. These results provide evidence that the newly synthesized GALC enzyme in transduced cells maintains typical features of the native lysosomal GALC enzyme. Interestingly, GALC-TMH also revealed M6PRindependent uptake in vitro, which indicates that the PTD motif of the Tat protein facilitates the distribution of GALC protein between cells. Therefore, functional Tat-modified mouse GALC delivered by lentiviral vector-medicated gene transfer in vivo might improve the therapeutic potency in GALC deficient twitcher mice.
450. Virus
RNA-Mediated Inhibition of Hepatitis B
Kathryn L. Nash,1 Graeme J. Alexander,1 Andrew M. Lever.1 Department of Medicine, University of Cambridge, Cambridge, Cambridgeshire, United Kingdom. 1
Chronic hepatitis B virus (HBV) infection is frequently associated with cirrhosis and hepatocellular carcinoma. HBV is a double stranded DNA virus that utilises reverse transcription to replicate via a RNA intermediate. Four major viral RNAs are produced that share a common 3’ terminus. Strategies that target viruses by sequence specific recognition of their RNA have recently received interest as potential therapeutic tools. The aim of this study was to design genes to produce antisense RNA or ribozymes (small catalytic RNA molecules) that target HBV. The ability of these genes to inhibit HBV gene expression was assessed by using lentiviral vectors to deliver the genes to a HBV producing cell line in vitro. Antisense RNA was designed to target the HBV mRNAs encoding the surface antigen, X protein and the viral encapsidation signal. Ribozymes targeting the viral surface and X RNAs were also created. The cDNAs for each of these were cloned into lentiviral vectors. Vectors containing sense RNA and catalytically inactive ribozymes were created as controls. Replication deficient lentiviral vectors containing these genes were produced and applied to HepG2 (2.2.15) cells in vitro. A ribonuclease protection assay was used to determine HBV RNA levels in transduced cells. The ribozyme-containing vectors produced the most dramatic reductions in HBV RNA levels in transduced cells. The X ribozyme S177