513. A Simple Method To Ensure Titer Conservation of AAV2 Vector Preparations Delivered with Stainless Steel Needles

AAV VECTORS To this end, a real-time quantitative polymerase chain reaction (qPCR) assay was developed to establish the amount of rcAAV or wtAAV present in rAAV stocks. An intact left AAV ITR-rep gene junction is a requisite feature for AAV replication to occur in vivo in the presence of a helper virus, but does not ensure replication competency. Therefore, the assay provides a maximum potential contamination level. The target DNA sequence selected for qPCR amplification spans the 3’ end of the left AAV2 ITR D-sequence and the 5’ end of the AAV2 rep sequence. The assay can therefore be applied to any pseudo-typed rAAV vector which utilizes AAV2 ITRs and rep2 gene. The assay employs sequence-specific PCR primers and a dual-labeled hybridizable probe for detection and quantification of the amplified DNA junction sequence. Linearized plasmid DNA was used to generate standard curves while Hirtextracted wtAAV2 DNA and Hirt-extracted adenovirus-infected 293 cell DNA were used for positive and negative controls, respectively. Assay replicates revealed a coefficient of variation (CV) of 3% for CT (cycle of threshold) values. Furthermore, we were able to establish a limit of quantification (LOQ) of 2.56x10-7 ng linear plasmid DNA standard, or 56 single-stranded rcAAV copies, in a background of 1 x 109 rAAV vector genomes (vg). Analysis of a human clinical trial rAAV-1 lot produced by co-transfection of 293 cells with plasmid DNA and purified by column chromatography revealed rcAAV contamination at a level of 1.36 x106 rAAV vg/1 rcAAV vg (σ = 4.65 x 105). Similarly designed assays specific for other ITR-rep combinations may be applicable to a variety of isotype or pseudotype AAV vectors. R.S., D.K., and T.M. are shareholders or hold stock options in a gene therapy company that is commercializing AAV for clinical gene therapy applications.

511. An Enzyme Linked Immunosorbent Assay for the Detection of Herpes Simplex Virus Type 1 Proteins in a Novel rAAV Production and Purification Process Guo-jie Ye,1 Tammy Mandell.2 Research & Preclinical Development, AGTC, Alachua, FL; 2 Process Development, AGTC, Alachua, FL.

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AGTC has developed a recombinant herpes simplex virus (rHSV1)-based adeno-associated virus (AAV) production process. This method requires two rHSV-1 viruses; rHSV-1-rep/cap which expresses the AAV2 rep and cap proteins, and a second rHSV-1 that contains the therapeutic gene of interest flanked by AAV2 inverted terminal repeats (ITRs) necessary for rAAV production. Following co-infection of these two helper viruses onto HEK-293 cells, the rAAV DNA is replicated and packaged into rAAV capsids. rAAV is then liberated from the cells and purified. Our proprietary rAAV purification scheme removes all contaminants including HSV-1 proteins; however, it is necessary to develop a sensitive and quantitative method to confirm the absence of HSV-1 proteins within a level of detection, in the final rAAV preparation. Here we report the development of an enzyme-linked Immunosorbent assay (ELISA) for quantitative determination of whole rHSV-1 proteins in purified rAAV preparations derived from rHSV-1 viruses. To facilitate the assay development, we prepared HSV-1 proteins standard from HSV-1 infected cell lysate by immunoaffinity column conjugated with polyclonal antibody to HSV-1 proteins. The fully developed assay enables us to quantitatively determine residual HSV-1 proteins at a level as low as 0.1 ng. Within the detection range of 0.1 ng to 5 ng of HSV proteins, the assay is reproducible with high linearity (R2>0.99). Background levels of rAAV as high as 5E+11 DNase resistant particles (DRP) do not interfere with the assay. Moreover, in contrast to other HSV protein ELISA assays targeted to a single protein such as HSV-1 glycoprotein D, this assay is capable of detecting a full range of HSV-1 proteins. Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy

512. Characterization of Adsorption of AdenoAssociated Virus to Commonly Used Catheter Materials: AAV2 vs. AAV1/2 Daisy P. Cross,1 Marty Morris.2 Corporate Science and Technology, Medtronic, Inc., Minneapolis, MN; 2CRM Therapy Delivery, Medtronic, Inc., Minneapolis, MN.

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Previous studies have suggested that some materials that are commonly used in injection catheters (e.g., stainless steel, nitinol) inactivate adenovirus (Adv), a viral vector that is widely used in the field of gene therapy. We have previously reported that the activity of recombinant adeno-associated virus serotype 2 (rAAV2) is also decreased after exposure to stainless steel and nitinol. However, unlike findings with Adv, the decreased rAAV2-mediated gene expression following contact with these materials is due to adsorption of the rAAV2 protein capsids to the materials, not inactivation of the rAAV2 itself. In our current study, we investigated the stability of a different AAV serotype, rAAV1/2, when exposed to metals, ceramics, and polymers. Solutions of rAAV1/2 encoding for enhanced green fluorescent protein (eGFP) were aspirated into small diameter tubing of several different catheter materials. At 75 min after aspiration, virus solution was ejected out of the catheter and applied to cultured cells. Incubation with polypropylene was used as the negative control. EGFP expression was assessed via flow cytometry, and viral titers were assessed via ELISA. Characterization of the material surfaces was performed via scanning electron microscope (SEM) imaging. Our results indicate that 1) AAV1/2-mediated expression of eGFP decreased after exposure to catheter materials and 2) this decrease—similar to the behavior of rAAV2—is primarily due to adsorption rather than inactivation of the virus.

513. A Simple Method To Ensure Titer Conservation of AAV2 Vector Preparations Delivered with Stainless Steel Needles Elias T. Ketchum,1 Kevin Kwok,1 Anthony Ramirez,1 Junshan Hao,1 Brian Kruegel,1 Dominick Vacante,1 Raymond Bartus,1 Mehdi Gasmi.1 1 Product Development, Ceregene, Inc., San Diego, CA. Ceregene is developing AAV2-based gene therapy vectors encoding nerve growth factor (NGF) (CERE-110) and neurturin (NTN) (CERE-120) for the treatment of Alzheimer’s and Parkinson’s diseases respectively. Targeted delivery of therapeutic vectors to the central nervous system for these applications requires the use of low volumes, low vector titers, and especially low flow rates. Under these conditions, interactions between the delivery hardware materials and the vector particles in suspension can have a profound impact on the characteristics of the vector preparation. For instance, we found that when AAV2 vectors are delivered by way of a stainless steel needle, vector titer measured by QPCR can drop up to 95% from its initial concentration. Reduction in titer is maximal when low concentration preparations (2x1010vg/mL) are delivered at a low flow rate (0.5µL/min). Since stainless steel is the most common material used to manufacture surgical needles, we developed a technique to prevent vector titer reduction without the need for chemicals or protein additives in the vector formulation. This technique consists of pre-treating the stainless steel needles with an identical vector suspension at a higher concentration (≥4x1012vg/ mL) followed by a rinsing step using the vector at the desired concentration. This pretreatment of the delivery hardware results in the saturation of vector binding sites in the lumen of the needle and prevents further vector loss when the desired dose is delivered. Our data show that AAV2 vector binds surgical grade stainless steel with high affinity and does not leach back into suspension in physiological conditions of temperature and salt concentrations. This priming S197

DNA VECTOROLOGY technique was applied in our pre-clinical in vivo experiments and it is currently used in our phase I clinical trials evaluating the safety and tolerability of our CERE-110 and CERE-120 vectors.

DNA VECTOROLOGY 514. mRNA Transfection Results in Efficient Overexpression of Transgenes in Leukemic Cell Lines and Hematopoietic Stem Cells Martin F. Ryser,1 Marcus Gentsch,1 Fernando Ugarte,1 Harry L. Malech,2 Sebastian Brenner.1 1 Department of Pediatrics, University Clinic Dresden, Dresden, Germany; 2Laboratory of Host Defenses, NIAID, NIH, Bethesda, MD. DNA-Vector transfection of eukaryotic cells needs transport of the DNA to the nucleus for efficient transcription and subsequent translation. In suspension cells most DNA-transfection methods are either inefficient (e.g. lipofection) or stressful for the cells (electroporation or nucleofection), which leads to high cell losses during the transfection process. We report the use of in vitro transcribed, capped and polyadenylated mRNA for the transfection of leukemic cell lines in comparison to DNA delivery. Templates for in vitro transcription were generated by PCR. The forward primer included the T7-promoter and the reverse primer a fragment of the beta-globin 3’UTR to increase the in vivo half life of the mRNA. Electroporation of GFP-mRNA into Jurkat, K562 or Kg1a lead to a GFP overexpression in 95 % of the transfected cells, with survival rates of 80 to 90%. Electroporation with pEGFP-N1 using the same pulse conditions resulted in GFP-overexpression in 62% of Jurkat or 9% of K562, with survival rates of 50% and 40 %, respectively. Our results suggest that electroporation of DNA is more toxic than mRNA transfection. GFP-expression in mRNA transfected cells was visible two hours after the transfection and peaked at about 12h. In contrast to DNA-transfection, mRNA mediated GFP expression levels were uniform in transfected cells. To investigate whether mRNA transfection results in functional transgene expression, mRNA coding the chemokine receptor CXCR4 was generated and electroporated into CXCR4 negative K562 cells. Flow cytometric analyses showed CXCR4 overexpression in more than 90% of transfected cells. Calcium flux measurements displayed a robust increase of intracellular free calcium in CXCR4-mRNA transfected K562 upon binding of the CXCR4 ligand SDF-1, while no calcium flux was visible in GFP-mRNA transfected or untransfected K562. Electroporation of GFP-mRNA into CD34+ hematopoietic progenitor cells or mesenchymal stem cells resulted in GFP overexpression in > 50% of transfected cells, respectively. Experiments are under way to further improve the mRNA transfection for primary cells. mRNA transfection is a highly efficient means for transient overexpression of transgenes in various cell lines and primary cells.

515. Sequence Requirements for Alveolar Epithelial Cell-Specific Plasmid Nuclear Import James V. DeGiulio,1 David A. Dean.1 1 Pulmonary and Critical Care Medicine, Northwestern University Medical School, Chicago, IL. Non-viral DNA gene delivery has many advantages over viral vectors, including potential for repeated administration and minimal immunological response. However, to date, the gene transfer efficiency remains low with this type of vector. This low efficiency is due to several barriers non-viral vectors must overcome prior to transgene expression. Perhaps the most important barrier in nonviral gene transfer is the nuclear envelope. In non-dividing cells, the S198

plasmid DNA has no access to the nucleus where the transcriptional machinery resides, and thus transgenes are not expressed. However, inclusion of specific sequences termed DNA targeting sequences (DTS) on plasmids will mediate nuclear import via the nuclear pore complex and enhance non-viral DNA gene transfer. These sequences, when included on a plasmid, will bind newly-synthesized transcription factors in the cytoplasm and the plasmid DNA will translocate into the nucleus via the nuclear localization signals on the bound transcription factors. Additionally, we can use cell-specific DTSs to mediate nuclear import only in specific cell types, thus limiting transgene expression to the cell type of choice. Using this novel strategy, we can improve both safety and efficiency of nonviral gene therapy by targeting the nucleus of only specific cell types. We have demonstrated previously that the 365 bp fragment of the human SP-C promoter (-318 to +47) will mediate nuclear import of plasmid DNA in alveolar epithelial cells but in no other cell types tested to date. Within this 365 nucleotide fragment, there exist several known binding sites for transcription factors. Several truncations of the SP-C promoter have been constructed, as well as mutations that will abrogate transcription factor binding to the SPC promoter DNA. Using microinjection and in situ hybridization strategy in both A549 and MLE-12 cell lines, we have determined which sequences within the SP-C promoter are required for nuclear import these alveolar epithelial cell lines. These data gives us a further understanding as to the mechanism of DTS function, knowledge that will allow us in the future to predict or create new DTSs for targeted plasmid DNA transfer to many other cell types.

516. Development of a Minicircle Vector Free of Plasmid Bacterial DNA Sequences and Capable of øC31-Mediated Site-Specific Integration Zhi-Ying Chen,1 Cheng-Yi He,1 Anja Ehrhardt,1 Hui Xu,1 Mark A. Kay.1 1 Departments of Pediatrics & Genetics, Stanford Univrsity School of Medicine, Stanford, CA. It has been well documented that plasmid bacterial DNA sequences (plasmid BB) can silence the expression of an episomal or integrated transgene. Current site-specific integrating technologies are hampered in part by the fact that the integrated transgene expression cassette is covalently linked with the plasmid BB. As a result, a substantial proportion of integrated transgenes are expressed at reduced levels or silenced. We have previously reported the development of a robust method for producing episomal minicircle vectors that are devoid of plasmid BB and capable of persistent and high level transgene expression in quiescent tissues in vivo. The minicircle was generated from a parental plasmid, which contained an expression cassette flanked with the short form recombination sites, sattB and sattP, two copies of the recombinase øC31 and one copy of the endonuclease I-Sce I genes under the control of the inducible promoter BAD. An I-Sce I site was engineered into the plasmid BB. Minicircle containing the therapeutic transgene was generated by the intramolecular recombination between the sattB and sattP sites mediated by the øC31 recombinase after the addition of the inducer L-Arabinose to the bacterial culture. A second DNA circle composed of plasmid BB sequences together with the I-Sce I site was also formed but was degraded after linearization by the I-Sce I endonuclease. This resulted in milligram yields of minicircle DNA from a liter of culture that was then purified by simple column chromatography. To extend the application of the minicircle to rapidly replicating cells, we elected to make a site-selective integrating form of this vector. To do this, we devised a strategy to include a functional sattB site into the minicircle. We inserted an additional sattB site between the sattP and the expression cassette, and replaced the original upstream sattB with a full length attB (LattB), resulting in Molecular Therapy Volume 13, Supplement 1, May 2006 Copyright  The American Society of Gene Therapy