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754. Volumetry of Mouse Liver during and after Hydrodynamic Injection
GENE THERAPY FOR HIV & AIDS 754. Volumetry of Mouse Liver during and after Hydrodynamic Injection
GENE THERAPY FOR HIV & AIDS
Takeshi Suda, Dexi Liu. 1 Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA.
755. Phase II Clinical Trial Demonstrates the Safety and Tolerability of Multiple Doses of Autologous CD4+ T Cells Transduced with VRX496, a Lentiviral Vector Delivering Anti-HIV Antisense in Patients Failing 1 or More HAART Treatment
Backgrounds & Aims: A prerequisite of a large volume is a major obstacle toward clinical application of hydrodynamic gene delivery (HGD). In this study we measured liver volume and intravascular pressure during and after HGD, and correlated them with gene delivery efficiency. The study aimed to determine minimum volume for efficient gene delivery. Materials & Methods: CMV-driven firefly luciferase and βgalactosidase genes were used as a reporter and mice (16-18 g) as an animal model. Plasmids were injected through a 22G catheter, which was placed into inferior vena cava together with a miniature pressure transducer. A local HGD was performed with temporary clamps near catheter-insertion site and beneath the diaphragm. Pressure at inferior vena cava was continuously monitored during and after HGD. Digital image sequences were recorded before, during and after injection. Coordinates of 8 target points marked on liver surface were calculated by a bundle adjustment method using a 3D_photo software. The volumes of tetrahedrons defined by the target points were calculated based on Euler’s tetrahedron formula. Results: When plasmids were injected in saline of 10% of body weight in 5 sec, the pressure peaked at 27 ± 1 mmHg and dropped by 10 mmHg within a few seconds followed by a slower decline. The pressure changes were coupled with volume expansion of the liver reaching peak size at 296 ± 58% of an initial volume and gradually return into its original size (99 ± 12%) after 90 min. On the other hand, when the injection was performed under the same condition but with injection time of 60 sec, the average peak pressure reached 11 ± 2 mmHg, and the liver volume increased up to 152 ± 18% and returned to 95 ± 13% in 30 min. Although robust Lac-Z signal was detected in 5-sec injection, 60-sec injection did not result in Lac Z gene expression. When plasmids were locally injected into liver in saline of 200, 400, 600, or 800-µl in 5 sec, the average luciferase activity obtained 8 hours post injection was approximately 2x105, 8x106, 8x107 and 2x108 RLU/mg, respectively, and significantly different from each other with the exception between 600 and 800µl injections. Furthermore, 600-µl injection showed a similar level of Lac-Z expression with tail vein injection using 10% of body weight. Conclusions: Our results suggest that around twice volume of liver is minimum requirement for maximum gene delivery efficiency in liver. Clinic application of HGD would benefit from effort to target part of the liver, not the whole organ.
S292
Tessio Rebello,1 Cathy Afable,2 Sherri Callahan,1 Laurent Humeau,3 Arvind Chopra,4 Xiaobin Lu,5 Vladimir Slepushkin.6, 4 1 Clinical Affairs, VIRxSYS, Gaithersburg, MD; 2Quality Assurance, VIRxSYS, Gaithersburg, MD; 3Product Development, VIRxSYS, Gaithersburg, MD; 4Quality Control, VIRxSYS, Gaithersburg, MD; 5Vector Development, VIRxSYS, Gaithersburg, MD; 6Production, VIRxSYS, Gaithersburg, MD. Gene therapy for HIV-1 infection has been proposed as an alternative to antiretroviral drug regimens due to emerging drug resistance and toxicity that raises concerns about HAART as a long term therapy. We have previously reported the successful completion of our Phase I clinical trial testing the safety and tolerability of a single dose of autologous HIV infected CD4+ T cells transduced with a lentiviral vector delivery system expressing a 937-base antisense gene against the HIV envelope (VRX496) for use in T cell therapy for HIV/AIDS. These results have lead us to initiate a Phase II clinical trial to evaluate the safety, tolerability, and biological activity of repeated infusions (4 or 8 doses) of autologous VRX496 transduced T cells. The study will enroll up to 40 male and female HIV-positive subjects in up to 8 centers. Subjects will be 18 years of age and over who have failed or are intolerant to at least one triple combination of antiretroviral drugs. Subjects will have, a viral load between 5,000 and 200,000 copies/ml and a CD4+ count of ≥150. Additionally, subjects must have a Karnofsky Performance score of 80 or higher, have no evidence of active opportunistic infection, congestive heart failure, hemodynamic instability, bleeding diathesis and no contraindications for leukapheresis. Patients will receive repeated doses by i.v. of ∼1*1010 genetically modified autologous T-cells, every two weeks. The primary safety objectives will include incidence of adverse events, changes in viral load, changes in CD4+ T cells, changes in TCR v beta repertoire, and tests for replication competent lentivirus (RCL). Tissue trafficking of gene modified cells will also be monitored in GALT tissue. The first part of the study is evaluating the safety and tolerability of multiple dosing using a repeat dosing design. All patients in the 4 dose cohort have received their infusions as scheduled and have reached the 3 month post-infusion visit, and one patient in the 8 dose cohort has received all of the doses as scheduled. To date, all of the doses have been well tolerated and preliminary results suggest multiple infusions are safe, with no SAE’s due to the product, no changes in hematology or chemistry laboratory evaluations, and no detection of VSVG DNA or RCL. In the second part, we are determining an optimal dosing regimen for future confirmatory trials. To date 18 patients have been enrolled and 11 have started receiving infusions. The data generated from the current clinical trial demonstrates the clinical utility of lentiviral vector technology as an alternative for treatment of HIV infection.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy
GENE THERAPY FOR HIV & AIDS
Takeshi Suda, Dexi Liu. 1 Pharmaceutical Sciences, University of Pittsburgh School of Pharmacy, Pittsburgh, PA.
755. Phase II Clinical Trial Demonstrates the Safety and Tolerability of Multiple Doses of Autologous CD4+ T Cells Transduced with VRX496, a Lentiviral Vector Delivering Anti-HIV Antisense in Patients Failing 1 or More HAART Treatment
Backgrounds & Aims: A prerequisite of a large volume is a major obstacle toward clinical application of hydrodynamic gene delivery (HGD). In this study we measured liver volume and intravascular pressure during and after HGD, and correlated them with gene delivery efficiency. The study aimed to determine minimum volume for efficient gene delivery. Materials & Methods: CMV-driven firefly luciferase and βgalactosidase genes were used as a reporter and mice (16-18 g) as an animal model. Plasmids were injected through a 22G catheter, which was placed into inferior vena cava together with a miniature pressure transducer. A local HGD was performed with temporary clamps near catheter-insertion site and beneath the diaphragm. Pressure at inferior vena cava was continuously monitored during and after HGD. Digital image sequences were recorded before, during and after injection. Coordinates of 8 target points marked on liver surface were calculated by a bundle adjustment method using a 3D_photo software. The volumes of tetrahedrons defined by the target points were calculated based on Euler’s tetrahedron formula. Results: When plasmids were injected in saline of 10% of body weight in 5 sec, the pressure peaked at 27 ± 1 mmHg and dropped by 10 mmHg within a few seconds followed by a slower decline. The pressure changes were coupled with volume expansion of the liver reaching peak size at 296 ± 58% of an initial volume and gradually return into its original size (99 ± 12%) after 90 min. On the other hand, when the injection was performed under the same condition but with injection time of 60 sec, the average peak pressure reached 11 ± 2 mmHg, and the liver volume increased up to 152 ± 18% and returned to 95 ± 13% in 30 min. Although robust Lac-Z signal was detected in 5-sec injection, 60-sec injection did not result in Lac Z gene expression. When plasmids were locally injected into liver in saline of 200, 400, 600, or 800-µl in 5 sec, the average luciferase activity obtained 8 hours post injection was approximately 2x105, 8x106, 8x107 and 2x108 RLU/mg, respectively, and significantly different from each other with the exception between 600 and 800µl injections. Furthermore, 600-µl injection showed a similar level of Lac-Z expression with tail vein injection using 10% of body weight. Conclusions: Our results suggest that around twice volume of liver is minimum requirement for maximum gene delivery efficiency in liver. Clinic application of HGD would benefit from effort to target part of the liver, not the whole organ.
S292
Tessio Rebello,1 Cathy Afable,2 Sherri Callahan,1 Laurent Humeau,3 Arvind Chopra,4 Xiaobin Lu,5 Vladimir Slepushkin.6, 4 1 Clinical Affairs, VIRxSYS, Gaithersburg, MD; 2Quality Assurance, VIRxSYS, Gaithersburg, MD; 3Product Development, VIRxSYS, Gaithersburg, MD; 4Quality Control, VIRxSYS, Gaithersburg, MD; 5Vector Development, VIRxSYS, Gaithersburg, MD; 6Production, VIRxSYS, Gaithersburg, MD. Gene therapy for HIV-1 infection has been proposed as an alternative to antiretroviral drug regimens due to emerging drug resistance and toxicity that raises concerns about HAART as a long term therapy. We have previously reported the successful completion of our Phase I clinical trial testing the safety and tolerability of a single dose of autologous HIV infected CD4+ T cells transduced with a lentiviral vector delivery system expressing a 937-base antisense gene against the HIV envelope (VRX496) for use in T cell therapy for HIV/AIDS. These results have lead us to initiate a Phase II clinical trial to evaluate the safety, tolerability, and biological activity of repeated infusions (4 or 8 doses) of autologous VRX496 transduced T cells. The study will enroll up to 40 male and female HIV-positive subjects in up to 8 centers. Subjects will be 18 years of age and over who have failed or are intolerant to at least one triple combination of antiretroviral drugs. Subjects will have, a viral load between 5,000 and 200,000 copies/ml and a CD4+ count of ≥150. Additionally, subjects must have a Karnofsky Performance score of 80 or higher, have no evidence of active opportunistic infection, congestive heart failure, hemodynamic instability, bleeding diathesis and no contraindications for leukapheresis. Patients will receive repeated doses by i.v. of ∼1*1010 genetically modified autologous T-cells, every two weeks. The primary safety objectives will include incidence of adverse events, changes in viral load, changes in CD4+ T cells, changes in TCR v beta repertoire, and tests for replication competent lentivirus (RCL). Tissue trafficking of gene modified cells will also be monitored in GALT tissue. The first part of the study is evaluating the safety and tolerability of multiple dosing using a repeat dosing design. All patients in the 4 dose cohort have received their infusions as scheduled and have reached the 3 month post-infusion visit, and one patient in the 8 dose cohort has received all of the doses as scheduled. To date, all of the doses have been well tolerated and preliminary results suggest multiple infusions are safe, with no SAE’s due to the product, no changes in hematology or chemistry laboratory evaluations, and no detection of VSVG DNA or RCL. In the second part, we are determining an optimal dosing regimen for future confirmatory trials. To date 18 patients have been enrolled and 11 have started receiving infusions. The data generated from the current clinical trial demonstrates the clinical utility of lentiviral vector technology as an alternative for treatment of HIV infection.
Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright The American Society of Gene Therapy