- Email: [email protected]
6 Mice
conventional RY,whereas LV with LTRs containing strong Rv-like enhancer-promoters significantly accelerated tumor onset. These results underline the critical role of LTRs with strong enhancers in oncogenesis and encourage the use of SIN vectors for clinical gene therapy. Intriguingly, LV with the same RV-Iike enhancer-promoter in internal position did not display any obvious genotoxic effect, suggesting that both the position and type of the genetic elements present in the vector and its integration site selection significantly influence the safety profile. We are mapping the vector integration sites in pre-transplant cells and tumors to elucidate the potential mechanism of insertional mutagenesis driven by the different vector types. These new results show the power of our in vivo model to highlight the vector features most critical for genotoxicy and will be instrumental to select the safest vector designs for clinical applications.
1064. HIV-Derived Lentivector Particles Persist in Hematopoietic Target Cells and Exhibit Delayed Release Kinetics
Amy M. Skinner,' Lee O'Neill, I Peter Kurre.P I Pediatrics. Oregon Health & Science University, Portland. OR; leell & Developmental Biology. Oregon Health & Science University, Portland. OR.
HIV-derived lentiviral particles are frequently used in gene transfer protocols. We recently described the prolonged adherence oflentivector particles to hematopoietic cells (murine bone marrow, and T-cell lines) (Pan et. al., 1. Virol. 2006). Specifically, we reported that particles of multiple pseudotypes remained attached to cellular surfaces after routine washes and were protected from complement inactivation in vitro. Transplantation of ex vivo-transduced hematopoietic cells resulted in subsequent systemic dissemination of VSV-G pseudotyped particles to non-target tissues in nonmyeloablated murine recipients. To further characterize apparent particle binding to primary ("carrier) cells and release to secondary cells, we generated vpr-GFP fusion protein tagged VSV-G vector to track particles via deconvolution microscopy. Murine L 1210 cells were exposed to vector, washed in pronase for inactivation of surface-bound particles, and transferred to direct co-culture with fibroblasts. Remarkably, microscopy images revealed that lentivector particles persist in the carrier cell cytoplasm. To evaluate the carrying capacity of primary cells we performed lentivector exposures with escalating doses of GFP tagged particles followed by pronase wash. FACS results demonstrate input vector-dose dependent shifts in fluorescence and further eonfinn the existence of an expandable compartment responsible for vector capture. These data were corroborated in functional studies that show a vector exposure time dependent increase in pronase resistant particle persistence, which is abrogated when particle entry is experimentally prevented. To determine the kinetics of persistence and release, Ll210 cells were transduced with VSV-G/GFP vector particles, washed with pronase and propagated for an extended duration, before transfer to direct co-culture with 293T cells. FACS analysis for GFPevents to measure particle transfer to 293T cells unequivocally supports the release of intact particles as late as five days after the initial vector exposure. The kinetics of release from primary cells show modest release to secondary cells prior to 24 hours, with a peak at 48 hours followed by subsequent decreases. Efforts to improve our understanding of the cellular compartment responsible for vector persistence are underway. In conclusion, we provide data to support a novel intracellular fate for lentiveetor particles. Based on models of HIV transfer from dendritic cells to T-cells, hematopoietic 'carrier' cells may allow systemic delivery of particles to secondary targets in situ. Insight into the mechanistic basis for capture and release may allow the development of novel cellular therapies whereby carrier S406
cells deliver particles with genetic payload and provide targeting function to tissues of interest.
1065. Neonatal Retroviral Vector Gene Therapy Does Not Induce Tumors in Normal C57BLl6 Mice Xiucui Ma,1 Mindy Tittiger,' Rachel Burrows,' Lingfei Xu,' Katherine P. Ponder,' 'Internal Medicine, Washington University School a/Medicine. St. LOllis. Mo. Neonatal IV injection of a gamma retroviral vector (RV) can result in stable expression ofthe therapeutic gene and correction of hemophilia and mueopolysaceharidosis (MPS) in mice and dogs. However, integration into the host cell chromosome could contribute to the development of cancers due to insertional mutagenesis. Indeed, liver tumors have developed in mice after fetal or neonatal systemic injection ofsome, but not other, lentiviral vectors in mice. In addition, leukemias developed in mice and humans that received ex vivo transduction ofhematopoeitic stem cells (HSC), and many ofthese were associated with integration into known oncogene loci. Finally, tumors developed in MPS VII mice that received neonatal IV injection of an AAV vector. The goal of this study was to determine if neonatal IV injection ofagamma RV would induce tumors in mice . Normal C57BLl6 mice were injected at 2 or 3 days after birth with 1010 transducing unitslkg of the RV designated hAATcGUSB-WPRE. This expresses the canine ~-glucuronidase cDNA from the liver-specific human ai-antitrypsin promoter, although the intact long-terminal repeat promoter can also drive expression in non-hepatic cells. Mice achieved 706+1-507 (SD) U/ml of serum GUSB activity in serum that was stable for the duration of evaluation. The percentage ofhepatocytes that were transduced was 23+19% as assessed by histochemical stain at 4 months after birth. Since _107 hepatocytes are present in newborns, this represents 2.3x 106 independent integrations per mouse, or l.Sx l O" total integrations for the 53 mice that were evaluated. Control mice derived from the same breeding colony were housed in the same animal room and did not receive any treatment. Mice were followed for 1.5 to 1.75 years after birth. Ninety-four percent of53 total RV-treated mice survived until the completion ofthe study, which was not statistic ally different from the value of 97% in the control group (N=65). The cause of death was not clear in any of these mice, who had been healthy at the most recent time ofexamination. The mean age of sacrifice was 21.5±1.4 months for the RV-treated mice , which was not statistically different from the value of 21.9± 1.6 months for the control mice. For the RV-treated mice, 12% had enlarged spleens, which were lymphosarcomas on pathological analysis for those that have been analyzed to date. This was similar to the frequency ofenlarged spleens in the control group of20%, which again were lymphosarcomas for those that have been evaluated. Most of the mice with enlarged spleens had enlarged lymph nodes andlor nodules within the liver, which were lymphosarcomas on histological analysis for samples that have been analyzed to date. None of the mice without enlarged spleens had visible nodules in the liver after sectioning at 5 mm intervals through the liver. We conclude that neonatal gene therapy does not increase the frequency ofliver tumors in C57BL/6 mice that were followed for 1.75 years after gene therapy.
Molecular Therapy Volume 15, Supplemen t I. ,\br 2007 Co pyright © Th e American Society o f G ene Thcr.Lp)·
1064. HIV-Derived Lentivector Particles Persist in Hematopoietic Target Cells and Exhibit Delayed Release Kinetics
Amy M. Skinner,' Lee O'Neill, I Peter Kurre.P I Pediatrics. Oregon Health & Science University, Portland. OR; leell & Developmental Biology. Oregon Health & Science University, Portland. OR.
HIV-derived lentiviral particles are frequently used in gene transfer protocols. We recently described the prolonged adherence oflentivector particles to hematopoietic cells (murine bone marrow, and T-cell lines) (Pan et. al., 1. Virol. 2006). Specifically, we reported that particles of multiple pseudotypes remained attached to cellular surfaces after routine washes and were protected from complement inactivation in vitro. Transplantation of ex vivo-transduced hematopoietic cells resulted in subsequent systemic dissemination of VSV-G pseudotyped particles to non-target tissues in nonmyeloablated murine recipients. To further characterize apparent particle binding to primary ("carrier) cells and release to secondary cells, we generated vpr-GFP fusion protein tagged VSV-G vector to track particles via deconvolution microscopy. Murine L 1210 cells were exposed to vector, washed in pronase for inactivation of surface-bound particles, and transferred to direct co-culture with fibroblasts. Remarkably, microscopy images revealed that lentivector particles persist in the carrier cell cytoplasm. To evaluate the carrying capacity of primary cells we performed lentivector exposures with escalating doses of GFP tagged particles followed by pronase wash. FACS results demonstrate input vector-dose dependent shifts in fluorescence and further eonfinn the existence of an expandable compartment responsible for vector capture. These data were corroborated in functional studies that show a vector exposure time dependent increase in pronase resistant particle persistence, which is abrogated when particle entry is experimentally prevented. To determine the kinetics of persistence and release, Ll210 cells were transduced with VSV-G/GFP vector particles, washed with pronase and propagated for an extended duration, before transfer to direct co-culture with 293T cells. FACS analysis for GFPevents to measure particle transfer to 293T cells unequivocally supports the release of intact particles as late as five days after the initial vector exposure. The kinetics of release from primary cells show modest release to secondary cells prior to 24 hours, with a peak at 48 hours followed by subsequent decreases. Efforts to improve our understanding of the cellular compartment responsible for vector persistence are underway. In conclusion, we provide data to support a novel intracellular fate for lentiveetor particles. Based on models of HIV transfer from dendritic cells to T-cells, hematopoietic 'carrier' cells may allow systemic delivery of particles to secondary targets in situ. Insight into the mechanistic basis for capture and release may allow the development of novel cellular therapies whereby carrier S406
cells deliver particles with genetic payload and provide targeting function to tissues of interest.
1065. Neonatal Retroviral Vector Gene Therapy Does Not Induce Tumors in Normal C57BLl6 Mice Xiucui Ma,1 Mindy Tittiger,' Rachel Burrows,' Lingfei Xu,' Katherine P. Ponder,' 'Internal Medicine, Washington University School a/Medicine. St. LOllis. Mo. Neonatal IV injection of a gamma retroviral vector (RV) can result in stable expression ofthe therapeutic gene and correction of hemophilia and mueopolysaceharidosis (MPS) in mice and dogs. However, integration into the host cell chromosome could contribute to the development of cancers due to insertional mutagenesis. Indeed, liver tumors have developed in mice after fetal or neonatal systemic injection ofsome, but not other, lentiviral vectors in mice. In addition, leukemias developed in mice and humans that received ex vivo transduction ofhematopoeitic stem cells (HSC), and many ofthese were associated with integration into known oncogene loci. Finally, tumors developed in MPS VII mice that received neonatal IV injection of an AAV vector. The goal of this study was to determine if neonatal IV injection ofagamma RV would induce tumors in mice . Normal C57BLl6 mice were injected at 2 or 3 days after birth with 1010 transducing unitslkg of the RV designated hAATcGUSB-WPRE. This expresses the canine ~-glucuronidase cDNA from the liver-specific human ai-antitrypsin promoter, although the intact long-terminal repeat promoter can also drive expression in non-hepatic cells. Mice achieved 706+1-507 (SD) U/ml of serum GUSB activity in serum that was stable for the duration of evaluation. The percentage ofhepatocytes that were transduced was 23+19% as assessed by histochemical stain at 4 months after birth. Since _107 hepatocytes are present in newborns, this represents 2.3x 106 independent integrations per mouse, or l.Sx l O" total integrations for the 53 mice that were evaluated. Control mice derived from the same breeding colony were housed in the same animal room and did not receive any treatment. Mice were followed for 1.5 to 1.75 years after birth. Ninety-four percent of53 total RV-treated mice survived until the completion ofthe study, which was not statistic ally different from the value of 97% in the control group (N=65). The cause of death was not clear in any of these mice, who had been healthy at the most recent time ofexamination. The mean age of sacrifice was 21.5±1.4 months for the RV-treated mice , which was not statistically different from the value of 21.9± 1.6 months for the control mice. For the RV-treated mice, 12% had enlarged spleens, which were lymphosarcomas on pathological analysis for those that have been analyzed to date. This was similar to the frequency ofenlarged spleens in the control group of20%, which again were lymphosarcomas for those that have been evaluated. Most of the mice with enlarged spleens had enlarged lymph nodes andlor nodules within the liver, which were lymphosarcomas on histological analysis for samples that have been analyzed to date. None of the mice without enlarged spleens had visible nodules in the liver after sectioning at 5 mm intervals through the liver. We conclude that neonatal gene therapy does not increase the frequency ofliver tumors in C57BL/6 mice that were followed for 1.75 years after gene therapy.
Molecular Therapy Volume 15, Supplemen t I. ,\br 2007 Co pyright © Th e American Society o f G ene Thcr.Lp)·