- Email: [email protected]
597. Challenges in the Production of Large and Complex Plasmid Vectors for Gene Therapy
CELL PROCESSING AND VECTOR PRODUCTION 595. Measurement of Viral Titer by Fluorescence Nanoparticle Tracking Analysis Andrew Malloy,1 Duncan A. Griffiths,2 Patrick Hole,1 Bob Carr.1 1 NanoSight Ltd., Amesbury, Wiltshire, United Kingdom; 2 NanoSight USA, Costa Mesa, CA.
Measurement of viral titer and sample aggregation is a ubiquitous requirement in viral vector development. Nanoparticle Tracking Analysis (NTA) is a new methodology which provides total viral titer in minutes and real time measurement of sample aggregation. The ability to measure these parameters at key points throughout the downstream purification process allows manufacturers to monitor and optimize the sample purification. The technique images viruses individually in liquid suspension and then calculates size from their Brownian motion on a virus-by-virus basis. By individually counting and sizing the viruses, a high resolution number vs size distribution is generated which relates the number of virus monomer to aggregates. Total virus count or concentration is simultaneously derived from this measurement. Fluorescence based measurements allow speciation of appropriately labeled sub-populations within the sample. Fluorescent labeling of the virus capsids, envelope, or DNA allows distinction of virus from cell debris making the technique suitable for working in crude harvest materials. Operating under light scatter the technique is inherently non-specific and hence suited to working in purified samples. As all particles, virus or otherwise, are measured, this can be combined with the fluorescence measurements to provide a measure of contaminant or empty capsid concentrations. The technique is designed to work alongside traditional technologies such as infectivity assays, as these assays provide valuable, yet limited information. Infectivity assays have no ability to pick up aggregation within a sample and do not give a measure of total viruses within a sample. When this data set is merged with the NTA data, the user can monitor infective vs non-infective viruses vs sample aggregation to better understand the quality of a viral preparation.
596. Where Human Gene Transfer Is Illegal: Local Regulation of rDNA Clinical Research
Jan P. Vleck,1 Gary M. Johnson,2 Ethan Mascoop.3 IBC Services, Western Institutional Review Board, Olympia, WA; 2 MetroWest Medical Center, Framingham, MA; 3Framingham Board of Health, Framingham, MA.
1
“The use of humans as experimental subjects in recombinant DNA research, as defined and regulated by the NIH Guidelines, shall not be permitted in the Town of Framingham.” [Board of Health, Rules and Regulations Relative to the Use of Recombinant DNA Technology within the Town of Framingham]1. A local regulation outlawing the use of human subjects in recombinant DNA (rDNA) research was discovered during preparation for a commerciallysponsored, multicenter, Phase II human gene transfer trial at a hospital in Framingham, Massachusetts. Penalties for violation including fines and closure of the “laboratory”. The hospital succeeded in obtaining a variance allowing the research. Selected Requirements of the Framingham BOH Regulation conform to NIH Guidelines intestinal flora surveillance BOH-approved procedure manual investigate and report all worker illness emergency response plan ban on P3, P4 research at least 2 BOH-appointed IBC members ban on use of human subjects IBC minutes to BOH institutional registration with BOH local screening for purity and antibiotic fine $200/day and lab closure resistance
Local Context. A controversial 1976 Harvard University plan for a P3 research laboratory in downtown Cambridge prompted various local actions to regulate use of rDNA. In Framingham, the Town Board of Health is charged with “registering recombinant DNA technologies.”2 MetroWest Medical Center (MWMC) is an independent regional health care system serving the Framingham area. Opening the trial. On September 14, 2010 MWMC registered an S228
IBC with NIH OBA in preparation for opening its first gene transfer trial. IBC review was scheduled for October 19. On October 7, a local ban on human gene transfer, probably from the early 1980s, was discovered. MWMC considered several options. Abandon the trial. This was not acceptable. Move the trial. Logistical and public relations considerations made it untenable to relocate to a MWMC hospital in neighboring Natick. Modify the regulatory status. This could both address public health concerns, and increase the feasibility of future HGT research in Framingham. This option was chosen. On October 8, the IBC roster was re-registered to add two Town residents unaffiliated with MWMC (one the Director of Public Health). MWMC submitted background safety information to the BOH and requested a permanent variance allowing FDA regulated, industry sponsored rDNA clinical trials. On October 28, the BOH approved a single-study variance as a test of procedural competence and safety. Legal notice of the BOH decision and amendments to the regulation was printed in the local paper. The BOH filed its actions with the Massachusetts Department of Environmental Protection. On November 10, the IBC approved the research. The local newspaper ran two stories.3,4 The first screening visit occurred January 11, 2011. Discussion: A local regulation outlawing human gene transfer research caused a 3-week delay in IBC approval for this regional community hospital site in a multi-center trial. The strategy of seeking a regulatory variance, going public in the local newspaper, and expanding public participation in the IBC was successful. 1. http://www.framinghamma.gov/DocumentView.aspx?DID=2812, accessed 01-11-2011[Home/Government/Departments/Board of Health/Rules and Regulations/Recombinant DNA Technologies Regulation] 2. http://www.framinghamma.gov/index.aspx?NID=852, accessed 01-11-2011 [Home/Government/Departments/Board of Selectmen/ Selectmen Appointed Committees/Board of Health] 3. Morton, M. MetroWest Medical seeks update to Framingham’s DNA regulation. The MetroWest Daily News. http://www. metrowestdailynews.com/lifestyle/health/x370073169/MetroWestMedical-seeks-update-to-Framinghams-DNA-regulation, posted 10-29-2010, accessed 01-11-2011 4. Morton, M. MetroWest Medical Center approved to for [sic] HPV treatment trial. The MetroWest Daily News. http://www. metrowestdailynews.com/lifestyle/health/x600431471/MetroWestMedical-Center-approved-to-for-HPV-treatment-trial, posted 11-142010, accessed 01-11-2011
597. Challenges in the Production of Large and Complex Plasmid Vectors for Gene Therapy Ying Cai,1 Stephen Rodriguez,1 Luke Clifford,1 Henry L. Hebel.1 VGXI, Inc, The Woodlands, TX.
1
Regulatable gene therapy appears as a promising approach by adjusting or switching on/off gene expression on demand. However, along with advantages of safety and flexibility, the size and complexity of the vector are increased to accommodate various control elements. A number of obstacles arise during production of such plasmids at pre-clinical or clinical grade, such as: low yield, instability, shearsensitive, and abundance of impurities. To address these challenges, VGXI devised process development programs for each stage of plasmid production. Extensive screening and bioreactor simulation ensured the selection of a high quality and high yield clone for seed banks. Large plasmids with aberrant sequences typically have high recombination ratios, and the quality of the desired plasmid form is associated with growth temperature. We have found the normal growth temperature of 37°C led to an unacceptable high level of recombinants. Reducing grow temperature improved product quality, but resulted in several fold reduction of initially low copy number plasmids. To accommodate both quality and yield, a novel fed-batch strategy was developed, which not only minimized undesirable forms Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
CLINICAL GENE & CELL THERAPY ORAL ABSTRACT SESSION but also increased yield titer up to 4-fold. Downstream processing also faced unusual barriers because large plasmids share similar physical/ chemical characteristics contaminating genomic DNA. Alkaline lysis is of particular concern as large plasmids are inefficient at renaturing. Special attention to process shear is required to maintain structural integrity. We have optimized downstream process conditions at every step. For a plasmid of size greater than 12 kb, the product demonstrated high purity suitable for pre-clinical or clinical applications.
Clinical Gene & Cell Therapy Oral Abstract Session 598. A Gene Therapy Approach to HIV: Adoptive Transfer of Zinc Finger Nuclease (ZFN) Modified Autologous CD4 T-Cells to Aviremic HIV-Infected Subjects with Suboptimal CD4 Counts (200-500 Cells/mm3) Shelley Wang,1 Jay Lalezari,2 Ronald Mitsuyasu,3 Steven Deeks,4 Winson Tang,1 Gary Lee,1 Michael Holmes,1 Phillip Gregory,1 Marty Giedlin,1 Dale Ando.1 1 Sangamo Biosciences, Richmond, CA; 2Quest Clin Research, San Francisco, CA; 3UCLA, Los Angeles, CA; 4UCSF, San Francisco, CA.
Background: A significant number of HIV+ patients on HAART are aviremic but continue to have CD4+ T-cells <500 cells/mm3. Previous attempts to use adoptive transfer to protect CD4+ T-cells from HIV infection have shown limited cell persistence. ZFN mediated disruption of CCR5 in CD4+ T-cells and selective expansion has been shown to protect against R5 HIV infection in both in vitro and in vivo T-cell and stem cell models. This study assesses the following effects of a single infusion of autologous CCR5-disrupted CD4+ T-cells administered intravenously to HAART-treated aviremic HIV subjects: 1) safety and tolerability, 2) cell persistence, 3) increases in CD4+ cell count, and 4) homing to gut mucosa. Methods: Nine aviremic HIV infected subjects with CD4+ counts between 200 and 500 cells/mm3 were enrolled, three each at dose levels of 1X1010, 2X1010 and 3X1010 total cells. Autologous CCR5 disrupted CD4+ T-cells were successfully expanded ex vivo with a mean CCR5 disruption efficiency of 26% (range 14-36%). After infusion, subjects were followed weekly for one month and then monthly for 11 months. Results: The median follow-up to date for cohort 1 is 9 months (range 7-11). CCR5-disrupted CD4+ T-cell infusions were safe and well tolerated with only mild and reversible infusion related adverse effects such as fever and chills. CD4 T-cell counts increased at all time points post infusion in all subjects. The average CD4+ T-cell counts at Day 28 increased by 155 cells/mm3 (range 86-211). The number of CCR5-disrupted T-cells in the peripheral blood as measured by PCR was 3.9-, 0.3- and 4.6-fold of the predicted number (2.5% total CD4 in 4.7L of blood) at Day 14 and persisted for the duration of followup (7 to 11 months). The percentage of CCR5 disrupted CD4 cells detected in the peripheral blood was as high as 19% in one subject at Day 28. CCR5 disrupted cells were detected in the rectal mucosa of all cohort 1 subjects at Day 14 and at 3 months. Conclusions: CCR5 disrupted CD4+ T-cells can be consistently manufactured from patients with suboptimal CD4+ counts to generate amounts greater than the intended 10-30 billion cell dose. Reinfusion to HIVinfected subjects is safe and well tolerated with disrupted cells being detected at frequencies up to 4.6-fold higher than the predicted input 14 days after infusion. This level of gene marking is approximately 1-log greater than has been observed previously in adoptive transfer studies with co-stimulated CD4+ T cells. Improvements in CD4+ T-cell counts were seen in all 3 subjects as was homing of these modified cells to the gut mucosa, suggesting that the modified CD4+ T-cells may distribute normally. These preliminary data suggest that Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
the CCR5 disrupted CD4 T cells persist at significant levels post infusion and can bolster CD4 cell counts even in HIV patients with undetectable viral load.
599. Clinical Trial Results: Intralesional Injection of a Tumor Selective Oncolytic Vaccinia Virus
Herbert J. Zeh,1 Mark O’Malley,1 Heather Jones,1 David H. Kirn,2 Moon Anne,2 Hwang H. Tae,3 David L. Bartlett.1 1 Surgery, University of Pittsburgh, Pittsburgh, PA; 2Jennerex Biotherapeutics, Inc., San Francisco, CA; 3Dong-A University, Busan, Korea.
Introduction: The WR strain of vaccinia virus (VV) has numerous potential advantages as an oncolytic virus. We have created a tumor selective mutant of VV (vvDD) by deleting the thymidine kinase (TK) gene and the vaccinia growth factor (VGF) gene. These gene products are non-essential in tumor cells with E2F and/or EGFR pathway activation mutations (including ras), but essential for replication in normal, non-dividing cells. After extensive pre-clinical work, we embarked on a phase I trial of vvDD as an intralesional injection in patients with accessible tumors. Methods: Patients with metastatic breast cancer (n=4), colorectal cancer (n=10), and pancreatic cancer (n=2) were treated with a single percutaneous injection of vvDD. A standard phase I dose escalation was performed with a starting dose of 3X107 plaque forming units (pfu), and a maximum feasible dose of 3X109 pfu. Up to 3 lesions were injected. Patients were observed in the hospital for 24 hours, then followed closely with serial samples for viral shedding analysis and analysis of response. Cutaneous tumors allowed the direct observation of the response and biopsies for viral recovery, while CT scans were used for internal lesions. Patients were stratified for prior vaccinia exposure, but only one patient had not been previously vaccinated. Results: There were no dose limiting toxicities. Grade 3 toxicities possibly related to the treatment, included pain after injection. This was predominately abdominal pain after deep injection of hepatic metastases. Grade 1 and 2 adverse events included fever, chills, swelling and erythema at the injection site, diffuse rash (not systemic vaccinia), and thrombocytopenia. We did not see evidence of viral shedding by vaccinia titers. 4 patients underwent biopsies of their tumors on day 8, and 2 patients had recoverable virus. These 2 patients also had evidence of viral spread from the injected lesions to other metastatic deposits. 2 patients had complete resolution of their injected lesions, and 1 patient had evidence of a distant lesion responding. The virus did not spread to normal skin. Conclusions: We have demonstrated the safety of vvDD as a single injection into tumors, with evidence of replication, tumor response, and recoverable virus in distant tumors. The vaccinia replication was specific for tumor, with complete sparing of the normal skin. Future plans include systemic delivery of this tumor selective virus.
600. Safety and Efficacy of Re-Administration of AAV2.hRPE65v2 in Subjects with Leber Congenital Blindness Due to RPE65 Mutations
Jean Bennett,1,2 Albert M. Maguire,1,2 Federico Mingozzi,2 Eric A. Pierce,1,2 Daniel C. Chung,1,2 Jeannette Bennicelli,1 Junwei Sun,2 J. Fraser Wright,1,2 Kathleen Marshall,2 Jennifer M. Wellman,2 Katherine A. High.1,2 1 Ophthalmology, University of Pennsylvania, Philadelphia, PA; 2 Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA.
Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We and others previously demonstrated that adenoassociated virus (AAV)-mediated delivery of RPE65 via subretinal injection results in improved vision/retinal function in animal models of and in humans with Leber Congenital Amaurosis due to RPE65 S229
Measurement of viral titer and sample aggregation is a ubiquitous requirement in viral vector development. Nanoparticle Tracking Analysis (NTA) is a new methodology which provides total viral titer in minutes and real time measurement of sample aggregation. The ability to measure these parameters at key points throughout the downstream purification process allows manufacturers to monitor and optimize the sample purification. The technique images viruses individually in liquid suspension and then calculates size from their Brownian motion on a virus-by-virus basis. By individually counting and sizing the viruses, a high resolution number vs size distribution is generated which relates the number of virus monomer to aggregates. Total virus count or concentration is simultaneously derived from this measurement. Fluorescence based measurements allow speciation of appropriately labeled sub-populations within the sample. Fluorescent labeling of the virus capsids, envelope, or DNA allows distinction of virus from cell debris making the technique suitable for working in crude harvest materials. Operating under light scatter the technique is inherently non-specific and hence suited to working in purified samples. As all particles, virus or otherwise, are measured, this can be combined with the fluorescence measurements to provide a measure of contaminant or empty capsid concentrations. The technique is designed to work alongside traditional technologies such as infectivity assays, as these assays provide valuable, yet limited information. Infectivity assays have no ability to pick up aggregation within a sample and do not give a measure of total viruses within a sample. When this data set is merged with the NTA data, the user can monitor infective vs non-infective viruses vs sample aggregation to better understand the quality of a viral preparation.
596. Where Human Gene Transfer Is Illegal: Local Regulation of rDNA Clinical Research
Jan P. Vleck,1 Gary M. Johnson,2 Ethan Mascoop.3 IBC Services, Western Institutional Review Board, Olympia, WA; 2 MetroWest Medical Center, Framingham, MA; 3Framingham Board of Health, Framingham, MA.
1
“The use of humans as experimental subjects in recombinant DNA research, as defined and regulated by the NIH Guidelines, shall not be permitted in the Town of Framingham.” [Board of Health, Rules and Regulations Relative to the Use of Recombinant DNA Technology within the Town of Framingham]1. A local regulation outlawing the use of human subjects in recombinant DNA (rDNA) research was discovered during preparation for a commerciallysponsored, multicenter, Phase II human gene transfer trial at a hospital in Framingham, Massachusetts. Penalties for violation including fines and closure of the “laboratory”. The hospital succeeded in obtaining a variance allowing the research. Selected Requirements of the Framingham BOH Regulation conform to NIH Guidelines intestinal flora surveillance BOH-approved procedure manual investigate and report all worker illness emergency response plan ban on P3, P4 research at least 2 BOH-appointed IBC members ban on use of human subjects IBC minutes to BOH institutional registration with BOH local screening for purity and antibiotic fine $200/day and lab closure resistance
Local Context. A controversial 1976 Harvard University plan for a P3 research laboratory in downtown Cambridge prompted various local actions to regulate use of rDNA. In Framingham, the Town Board of Health is charged with “registering recombinant DNA technologies.”2 MetroWest Medical Center (MWMC) is an independent regional health care system serving the Framingham area. Opening the trial. On September 14, 2010 MWMC registered an S228
IBC with NIH OBA in preparation for opening its first gene transfer trial. IBC review was scheduled for October 19. On October 7, a local ban on human gene transfer, probably from the early 1980s, was discovered. MWMC considered several options. Abandon the trial. This was not acceptable. Move the trial. Logistical and public relations considerations made it untenable to relocate to a MWMC hospital in neighboring Natick. Modify the regulatory status. This could both address public health concerns, and increase the feasibility of future HGT research in Framingham. This option was chosen. On October 8, the IBC roster was re-registered to add two Town residents unaffiliated with MWMC (one the Director of Public Health). MWMC submitted background safety information to the BOH and requested a permanent variance allowing FDA regulated, industry sponsored rDNA clinical trials. On October 28, the BOH approved a single-study variance as a test of procedural competence and safety. Legal notice of the BOH decision and amendments to the regulation was printed in the local paper. The BOH filed its actions with the Massachusetts Department of Environmental Protection. On November 10, the IBC approved the research. The local newspaper ran two stories.3,4 The first screening visit occurred January 11, 2011. Discussion: A local regulation outlawing human gene transfer research caused a 3-week delay in IBC approval for this regional community hospital site in a multi-center trial. The strategy of seeking a regulatory variance, going public in the local newspaper, and expanding public participation in the IBC was successful. 1. http://www.framinghamma.gov/DocumentView.aspx?DID=2812, accessed 01-11-2011[Home/Government/Departments/Board of Health/Rules and Regulations/Recombinant DNA Technologies Regulation] 2. http://www.framinghamma.gov/index.aspx?NID=852, accessed 01-11-2011 [Home/Government/Departments/Board of Selectmen/ Selectmen Appointed Committees/Board of Health] 3. Morton, M. MetroWest Medical seeks update to Framingham’s DNA regulation. The MetroWest Daily News. http://www. metrowestdailynews.com/lifestyle/health/x370073169/MetroWestMedical-seeks-update-to-Framinghams-DNA-regulation, posted 10-29-2010, accessed 01-11-2011 4. Morton, M. MetroWest Medical Center approved to for [sic] HPV treatment trial. The MetroWest Daily News. http://www. metrowestdailynews.com/lifestyle/health/x600431471/MetroWestMedical-Center-approved-to-for-HPV-treatment-trial, posted 11-142010, accessed 01-11-2011
597. Challenges in the Production of Large and Complex Plasmid Vectors for Gene Therapy Ying Cai,1 Stephen Rodriguez,1 Luke Clifford,1 Henry L. Hebel.1 VGXI, Inc, The Woodlands, TX.
1
Regulatable gene therapy appears as a promising approach by adjusting or switching on/off gene expression on demand. However, along with advantages of safety and flexibility, the size and complexity of the vector are increased to accommodate various control elements. A number of obstacles arise during production of such plasmids at pre-clinical or clinical grade, such as: low yield, instability, shearsensitive, and abundance of impurities. To address these challenges, VGXI devised process development programs for each stage of plasmid production. Extensive screening and bioreactor simulation ensured the selection of a high quality and high yield clone for seed banks. Large plasmids with aberrant sequences typically have high recombination ratios, and the quality of the desired plasmid form is associated with growth temperature. We have found the normal growth temperature of 37°C led to an unacceptable high level of recombinants. Reducing grow temperature improved product quality, but resulted in several fold reduction of initially low copy number plasmids. To accommodate both quality and yield, a novel fed-batch strategy was developed, which not only minimized undesirable forms Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
CLINICAL GENE & CELL THERAPY ORAL ABSTRACT SESSION but also increased yield titer up to 4-fold. Downstream processing also faced unusual barriers because large plasmids share similar physical/ chemical characteristics contaminating genomic DNA. Alkaline lysis is of particular concern as large plasmids are inefficient at renaturing. Special attention to process shear is required to maintain structural integrity. We have optimized downstream process conditions at every step. For a plasmid of size greater than 12 kb, the product demonstrated high purity suitable for pre-clinical or clinical applications.
Clinical Gene & Cell Therapy Oral Abstract Session 598. A Gene Therapy Approach to HIV: Adoptive Transfer of Zinc Finger Nuclease (ZFN) Modified Autologous CD4 T-Cells to Aviremic HIV-Infected Subjects with Suboptimal CD4 Counts (200-500 Cells/mm3) Shelley Wang,1 Jay Lalezari,2 Ronald Mitsuyasu,3 Steven Deeks,4 Winson Tang,1 Gary Lee,1 Michael Holmes,1 Phillip Gregory,1 Marty Giedlin,1 Dale Ando.1 1 Sangamo Biosciences, Richmond, CA; 2Quest Clin Research, San Francisco, CA; 3UCLA, Los Angeles, CA; 4UCSF, San Francisco, CA.
Background: A significant number of HIV+ patients on HAART are aviremic but continue to have CD4+ T-cells <500 cells/mm3. Previous attempts to use adoptive transfer to protect CD4+ T-cells from HIV infection have shown limited cell persistence. ZFN mediated disruption of CCR5 in CD4+ T-cells and selective expansion has been shown to protect against R5 HIV infection in both in vitro and in vivo T-cell and stem cell models. This study assesses the following effects of a single infusion of autologous CCR5-disrupted CD4+ T-cells administered intravenously to HAART-treated aviremic HIV subjects: 1) safety and tolerability, 2) cell persistence, 3) increases in CD4+ cell count, and 4) homing to gut mucosa. Methods: Nine aviremic HIV infected subjects with CD4+ counts between 200 and 500 cells/mm3 were enrolled, three each at dose levels of 1X1010, 2X1010 and 3X1010 total cells. Autologous CCR5 disrupted CD4+ T-cells were successfully expanded ex vivo with a mean CCR5 disruption efficiency of 26% (range 14-36%). After infusion, subjects were followed weekly for one month and then monthly for 11 months. Results: The median follow-up to date for cohort 1 is 9 months (range 7-11). CCR5-disrupted CD4+ T-cell infusions were safe and well tolerated with only mild and reversible infusion related adverse effects such as fever and chills. CD4 T-cell counts increased at all time points post infusion in all subjects. The average CD4+ T-cell counts at Day 28 increased by 155 cells/mm3 (range 86-211). The number of CCR5-disrupted T-cells in the peripheral blood as measured by PCR was 3.9-, 0.3- and 4.6-fold of the predicted number (2.5% total CD4 in 4.7L of blood) at Day 14 and persisted for the duration of followup (7 to 11 months). The percentage of CCR5 disrupted CD4 cells detected in the peripheral blood was as high as 19% in one subject at Day 28. CCR5 disrupted cells were detected in the rectal mucosa of all cohort 1 subjects at Day 14 and at 3 months. Conclusions: CCR5 disrupted CD4+ T-cells can be consistently manufactured from patients with suboptimal CD4+ counts to generate amounts greater than the intended 10-30 billion cell dose. Reinfusion to HIVinfected subjects is safe and well tolerated with disrupted cells being detected at frequencies up to 4.6-fold higher than the predicted input 14 days after infusion. This level of gene marking is approximately 1-log greater than has been observed previously in adoptive transfer studies with co-stimulated CD4+ T cells. Improvements in CD4+ T-cell counts were seen in all 3 subjects as was homing of these modified cells to the gut mucosa, suggesting that the modified CD4+ T-cells may distribute normally. These preliminary data suggest that Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy
the CCR5 disrupted CD4 T cells persist at significant levels post infusion and can bolster CD4 cell counts even in HIV patients with undetectable viral load.
599. Clinical Trial Results: Intralesional Injection of a Tumor Selective Oncolytic Vaccinia Virus
Herbert J. Zeh,1 Mark O’Malley,1 Heather Jones,1 David H. Kirn,2 Moon Anne,2 Hwang H. Tae,3 David L. Bartlett.1 1 Surgery, University of Pittsburgh, Pittsburgh, PA; 2Jennerex Biotherapeutics, Inc., San Francisco, CA; 3Dong-A University, Busan, Korea.
Introduction: The WR strain of vaccinia virus (VV) has numerous potential advantages as an oncolytic virus. We have created a tumor selective mutant of VV (vvDD) by deleting the thymidine kinase (TK) gene and the vaccinia growth factor (VGF) gene. These gene products are non-essential in tumor cells with E2F and/or EGFR pathway activation mutations (including ras), but essential for replication in normal, non-dividing cells. After extensive pre-clinical work, we embarked on a phase I trial of vvDD as an intralesional injection in patients with accessible tumors. Methods: Patients with metastatic breast cancer (n=4), colorectal cancer (n=10), and pancreatic cancer (n=2) were treated with a single percutaneous injection of vvDD. A standard phase I dose escalation was performed with a starting dose of 3X107 plaque forming units (pfu), and a maximum feasible dose of 3X109 pfu. Up to 3 lesions were injected. Patients were observed in the hospital for 24 hours, then followed closely with serial samples for viral shedding analysis and analysis of response. Cutaneous tumors allowed the direct observation of the response and biopsies for viral recovery, while CT scans were used for internal lesions. Patients were stratified for prior vaccinia exposure, but only one patient had not been previously vaccinated. Results: There were no dose limiting toxicities. Grade 3 toxicities possibly related to the treatment, included pain after injection. This was predominately abdominal pain after deep injection of hepatic metastases. Grade 1 and 2 adverse events included fever, chills, swelling and erythema at the injection site, diffuse rash (not systemic vaccinia), and thrombocytopenia. We did not see evidence of viral shedding by vaccinia titers. 4 patients underwent biopsies of their tumors on day 8, and 2 patients had recoverable virus. These 2 patients also had evidence of viral spread from the injected lesions to other metastatic deposits. 2 patients had complete resolution of their injected lesions, and 1 patient had evidence of a distant lesion responding. The virus did not spread to normal skin. Conclusions: We have demonstrated the safety of vvDD as a single injection into tumors, with evidence of replication, tumor response, and recoverable virus in distant tumors. The vaccinia replication was specific for tumor, with complete sparing of the normal skin. Future plans include systemic delivery of this tumor selective virus.
600. Safety and Efficacy of Re-Administration of AAV2.hRPE65v2 in Subjects with Leber Congenital Blindness Due to RPE65 Mutations
Jean Bennett,1,2 Albert M. Maguire,1,2 Federico Mingozzi,2 Eric A. Pierce,1,2 Daniel C. Chung,1,2 Jeannette Bennicelli,1 Junwei Sun,2 J. Fraser Wright,1,2 Kathleen Marshall,2 Jennifer M. Wellman,2 Katherine A. High.1,2 1 Ophthalmology, University of Pennsylvania, Philadelphia, PA; 2 Center for Cellular and Molecular Therapeutics, The Children’s Hospital of Philadelphia, Philadelphia, PA.
Gene therapy has the potential to reverse disease or prevent further deterioration of vision in patients with incurable inherited retinal degeneration. We and others previously demonstrated that adenoassociated virus (AAV)-mediated delivery of RPE65 via subretinal injection results in improved vision/retinal function in animal models of and in humans with Leber Congenital Amaurosis due to RPE65 S229