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597 challenges in the production of large and complex plasmid vectors for gene therapy

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597 Challenges in the Production of Large and Complex Plasmid Vectors for Gene Therapy Molecular Therapy Volume 19, Supplement 1, May 2011 Copyright © The American Society of Gene & Cell Therapy S228[.]

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 NanoSight Ltd., Amesbury, Wiltshire, United Kingdom; 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 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; MetroWest Medical Center, Framingham, MA; 3Framingham Board of Health, Framingham, MA “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 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 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] http://www.framinghamma.gov/index.aspx?NID=852, accessed 01-11-2011 [Home/Government/Departments/Board of Selectmen/ Selectmen Appointed Committees/Board of Health] 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 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 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 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

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