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306. A Robust and Efficient Lentiviral Platform for Delivery of MicroRNA and MicroRNA-Based siRNA into Primary Cells: Validation by Targeting FOXP3 in Human Regulatory T Cells
RNA Virus Vectors: Functional Applications 306. A Robust and Efficient Lentiviral Platform for Delivery of MicroRNA and MicroRNA-Based siRNA into Primary Cells: Validation by Targeting FOXP3 in Human Regulatory T Cells
Mario Amendola,1,2 Laura Passerini,1 Grazia Andolfi,1 Lucia Sergi Sergi,1 Rosi Bacchetta,1 Maria Grazia Rondarolo,1,2 Luigi Naldini.1,2 1 Telethon Institute for Gene Therapy (TIGET), San Raffaele Scientific Institute, Milan, Italy; 2San Raffaele Vita-Salute University, Milan, Italy.
The development of efficient platforms to deliver microRNA (miR) and siRNA in primary cells and in vivo will tremendously empower gene function studies and gene therapy applications. Towards this goal, we identified an efficiently processed pri-miR among a panel of highly expressed miR, engineered its backbone for expressing heterologous mature miR or siRNA, and optimized lentiviral vectors (LV) to express the selected pri-miR from a Pol-II promoter together with a marker gene. To evaluate miR activity we generated a panel of reporter cell lines each one expressing GFP mRNA tagged with perfectly complementary sequences to the investigated miR. For each miR we compared exogenous delivery vs. endogenous expression by introducing the reporter construct in cells expressing or not the miR. We then inserted the best performing pri-miR, miR 223, in different positions within LV and selected an intronic placement which enabled efficient segregation between the mutually exclusive miR and mRNA maturation. This design allowed miR expression levels comparable to the highest measured endogenous ones, up to 30-fold GFP repression, and easy detection/selection of the transduced cells by marker gene co-expression. Importantly, miR 223 LV did not alter expression and activity of several tested endogenous miR. We further developed our platform to efficiently co-express two miR and to switch ON and OFF miR expression by tetracycline. Once established miR 223 performance, we replaced its stem loop with that of other miR and showed that the activity of this chimeric miR (miR-sl) was similar to that of either parental miR. This finding indicates that miR 223 flanking sequences can be exploited to conveniently express other miR and test their function in controlled fashion and relevant models. We also generated a miR-sl 30 and compared its activity to that of wildtype pri-miR 30 delivered by the same LV design. Surprisingly, miR 30 activity was 10-fold higher with the miR 223 backbone suggesting that pri-miR 30, although widely used to express siRNA, is not efficiently processed. Finally, we validated our platform for siRNA expression by replacing the miR 223 targeting sequence with a siRNA of interest (miR-si). We generated two miR-si LV against FOXP3 and transduced human CD4+CD25+ regulatory T cells (Treg) to address whether FOXP3 is required for maintenance of suppressor activity. After two weeks of expansion, the phenotype and function of cells transduced by either miR-siFOXP3 or control LV were evaluated. We obtained marked FOXP3 down-regulation that resulted in reduced CTLA4 expression and increased IL-2 production. Moreover, FOXP3 knock-down reverted the anergy and in vitro suppressive capacity of the transduced Treg. These results prove the proficiency of our new platform at delivering RNAi into up to now challenging targets and open new possibilities to RNAi application in gene therapy.
307. Cell – Cell Transmission of HIV-1 Derived Lentivector Particles Involves Their Association with CD63 and CD81 Tetraspanin Proteins
Amy Skinner,1 Lee O’Neill,1 Peter Kurre.1,2 Pediatrics, Oregon Health & Science University, Portland, OR; 2Cell & Developmental Biology, Oregon Health & Science University, Portland, OR. 1
Trans-infection by wild-type HIV-1 virus is a recently described mechanism whereby primary immune cells (dendritic, macrophage, Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Langerhans) capture particles and subsequently transfer them to CD4+ T lymphocytes for productive infection. We have previously reported the protease- and complement- resistant intracellular persistence of replication-deficient HIV-1 derived lentivector particles in hematopoietic cells as well as their resultant infection (in trans) of secondary targets with peak cell- cell transfer occurring between 24 and 48 hours from initial vector exposure. To identify the cellular compartment involved in their persistence and transmission from primary (“carrier”) cells to secondary targets we screened endocytic-, proteasome- and lysosome- pathways - all known to participate in wild-type and vector particle trafficking - by pharmacologic inhibition in carrier cells. Among the reagents and experimental conditions tested, only the endosomal uptake and processing inhibitor ammonium chloride seemed to affect (increase) secondary transduction of 293T cells. We next turned to an immunofluorescent imaging approach to track GFPvpr fusion protein labeled vector particles in carrier cells and determine their potential colocalization with antibodies directed against components of cellular compartments. Results showed that vector particles colocalize with endosomal compartment markers (EEA1, transferrin receptor, and adaptor protein-2) during uptake, within minutes of vector exposure, and to a lesser extent with the lysosomal marker (LAMP1). In contrast, when we examined colocalization of vector particles with the tetraspanin proteins CD63 and -81, we observed a statistically significant, time-dependent increase in colocalization in SupT-1 and Jurkat carriers. In addition, the kinetics of association paralleled those previously noted in our functional co-culture transduction studies. Further, when we prevented intracellular rearrangement of tetraspanins by treating SupT1 and Jurkat cells with the actin remodeling inhibitor Latrunculin A for 1 hour we observed a significant difference in the percentage of particles colocalized with CD63 in untreated versus treated cells. Finally, in live-cell imaging studies we observed carrier cell derived CD81 associated GFPvpr particles colocalizing with DsRed actin in 293T secondary targets. These data show remarkable analogy with reports on the emerging role of tetraspanins (CD9, -63, -81) in wildtype HIV-1 egress as well as recently reported MLV vector particle trafficking in producer cells. Taken together, our observations reported herein corroborate our previous functional studies and suggest a role for tetraspanins in cell-cell particle transmission. Our findings suggest the possibility of an alternate cellular fate for replication-deficient vector particles (in addition to integration of degradation) and have implications for the use of pseudotyped vectors for gene transfer.
308. Effective Chemoprotection Conferred by Allogeneic MGMT Gene-Modified Cells in the Dog Brian C. Beard,1 Christina Gooch,1 Hans-Peter Kiem.1,2 Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Medicine, University of Washington, Seattle, WA.
1
We have previously demonstrated successful in vivo selection and chemoprotection in dogs that received myeloablative allogeneic stem cell transplantation with gene-modified cells. Here we wished to investigate whether in vivo selection, chemoprotection, and modulation of donor chimerism could also be achieved after reduced intensity transplantation which should be less toxic and thus more appropriate for older patients and patients with genetic diseases. We have used a lentivirus vector encoding the P140K mutant of methylguanine-DNA methyltransferase (MGMT-P140K) to transduce AMD3100-mobilized DLA matched littermate CD34+ cells. The use of a lentivirus vector allowed us to keep the ex vivo/transduction time short, less than 24 hours which improves engraftment in the allogeneic setting. Three dogs received reduced intensity conditioning consisting of 300cGy total body irradiation (TBI) before infusion of the genemodified CD34+ cells. All three dogs had stable gene marking (1-3%) and donor chimerism (20-60%), before receiving O6-benzylguanine (O6BG) and temozolomide. Even with relatively low levels of gene S115
Mario Amendola,1,2 Laura Passerini,1 Grazia Andolfi,1 Lucia Sergi Sergi,1 Rosi Bacchetta,1 Maria Grazia Rondarolo,1,2 Luigi Naldini.1,2 1 Telethon Institute for Gene Therapy (TIGET), San Raffaele Scientific Institute, Milan, Italy; 2San Raffaele Vita-Salute University, Milan, Italy.
The development of efficient platforms to deliver microRNA (miR) and siRNA in primary cells and in vivo will tremendously empower gene function studies and gene therapy applications. Towards this goal, we identified an efficiently processed pri-miR among a panel of highly expressed miR, engineered its backbone for expressing heterologous mature miR or siRNA, and optimized lentiviral vectors (LV) to express the selected pri-miR from a Pol-II promoter together with a marker gene. To evaluate miR activity we generated a panel of reporter cell lines each one expressing GFP mRNA tagged with perfectly complementary sequences to the investigated miR. For each miR we compared exogenous delivery vs. endogenous expression by introducing the reporter construct in cells expressing or not the miR. We then inserted the best performing pri-miR, miR 223, in different positions within LV and selected an intronic placement which enabled efficient segregation between the mutually exclusive miR and mRNA maturation. This design allowed miR expression levels comparable to the highest measured endogenous ones, up to 30-fold GFP repression, and easy detection/selection of the transduced cells by marker gene co-expression. Importantly, miR 223 LV did not alter expression and activity of several tested endogenous miR. We further developed our platform to efficiently co-express two miR and to switch ON and OFF miR expression by tetracycline. Once established miR 223 performance, we replaced its stem loop with that of other miR and showed that the activity of this chimeric miR (miR-sl) was similar to that of either parental miR. This finding indicates that miR 223 flanking sequences can be exploited to conveniently express other miR and test their function in controlled fashion and relevant models. We also generated a miR-sl 30 and compared its activity to that of wildtype pri-miR 30 delivered by the same LV design. Surprisingly, miR 30 activity was 10-fold higher with the miR 223 backbone suggesting that pri-miR 30, although widely used to express siRNA, is not efficiently processed. Finally, we validated our platform for siRNA expression by replacing the miR 223 targeting sequence with a siRNA of interest (miR-si). We generated two miR-si LV against FOXP3 and transduced human CD4+CD25+ regulatory T cells (Treg) to address whether FOXP3 is required for maintenance of suppressor activity. After two weeks of expansion, the phenotype and function of cells transduced by either miR-siFOXP3 or control LV were evaluated. We obtained marked FOXP3 down-regulation that resulted in reduced CTLA4 expression and increased IL-2 production. Moreover, FOXP3 knock-down reverted the anergy and in vitro suppressive capacity of the transduced Treg. These results prove the proficiency of our new platform at delivering RNAi into up to now challenging targets and open new possibilities to RNAi application in gene therapy.
307. Cell – Cell Transmission of HIV-1 Derived Lentivector Particles Involves Their Association with CD63 and CD81 Tetraspanin Proteins
Amy Skinner,1 Lee O’Neill,1 Peter Kurre.1,2 Pediatrics, Oregon Health & Science University, Portland, OR; 2Cell & Developmental Biology, Oregon Health & Science University, Portland, OR. 1
Trans-infection by wild-type HIV-1 virus is a recently described mechanism whereby primary immune cells (dendritic, macrophage, Molecular Therapy Volume 16, Supplement 1, May 2008 Copyright © The American Society of Gene Therapy
Langerhans) capture particles and subsequently transfer them to CD4+ T lymphocytes for productive infection. We have previously reported the protease- and complement- resistant intracellular persistence of replication-deficient HIV-1 derived lentivector particles in hematopoietic cells as well as their resultant infection (in trans) of secondary targets with peak cell- cell transfer occurring between 24 and 48 hours from initial vector exposure. To identify the cellular compartment involved in their persistence and transmission from primary (“carrier”) cells to secondary targets we screened endocytic-, proteasome- and lysosome- pathways - all known to participate in wild-type and vector particle trafficking - by pharmacologic inhibition in carrier cells. Among the reagents and experimental conditions tested, only the endosomal uptake and processing inhibitor ammonium chloride seemed to affect (increase) secondary transduction of 293T cells. We next turned to an immunofluorescent imaging approach to track GFPvpr fusion protein labeled vector particles in carrier cells and determine their potential colocalization with antibodies directed against components of cellular compartments. Results showed that vector particles colocalize with endosomal compartment markers (EEA1, transferrin receptor, and adaptor protein-2) during uptake, within minutes of vector exposure, and to a lesser extent with the lysosomal marker (LAMP1). In contrast, when we examined colocalization of vector particles with the tetraspanin proteins CD63 and -81, we observed a statistically significant, time-dependent increase in colocalization in SupT-1 and Jurkat carriers. In addition, the kinetics of association paralleled those previously noted in our functional co-culture transduction studies. Further, when we prevented intracellular rearrangement of tetraspanins by treating SupT1 and Jurkat cells with the actin remodeling inhibitor Latrunculin A for 1 hour we observed a significant difference in the percentage of particles colocalized with CD63 in untreated versus treated cells. Finally, in live-cell imaging studies we observed carrier cell derived CD81 associated GFPvpr particles colocalizing with DsRed actin in 293T secondary targets. These data show remarkable analogy with reports on the emerging role of tetraspanins (CD9, -63, -81) in wildtype HIV-1 egress as well as recently reported MLV vector particle trafficking in producer cells. Taken together, our observations reported herein corroborate our previous functional studies and suggest a role for tetraspanins in cell-cell particle transmission. Our findings suggest the possibility of an alternate cellular fate for replication-deficient vector particles (in addition to integration of degradation) and have implications for the use of pseudotyped vectors for gene transfer.
308. Effective Chemoprotection Conferred by Allogeneic MGMT Gene-Modified Cells in the Dog Brian C. Beard,1 Christina Gooch,1 Hans-Peter Kiem.1,2 Clinical Research, Fred Hutchinson Cancer Research Center, Seattle, WA; 2Medicine, University of Washington, Seattle, WA.
1
We have previously demonstrated successful in vivo selection and chemoprotection in dogs that received myeloablative allogeneic stem cell transplantation with gene-modified cells. Here we wished to investigate whether in vivo selection, chemoprotection, and modulation of donor chimerism could also be achieved after reduced intensity transplantation which should be less toxic and thus more appropriate for older patients and patients with genetic diseases. We have used a lentivirus vector encoding the P140K mutant of methylguanine-DNA methyltransferase (MGMT-P140K) to transduce AMD3100-mobilized DLA matched littermate CD34+ cells. The use of a lentivirus vector allowed us to keep the ex vivo/transduction time short, less than 24 hours which improves engraftment in the allogeneic setting. Three dogs received reduced intensity conditioning consisting of 300cGy total body irradiation (TBI) before infusion of the genemodified CD34+ cells. All three dogs had stable gene marking (1-3%) and donor chimerism (20-60%), before receiving O6-benzylguanine (O6BG) and temozolomide. Even with relatively low levels of gene S115