VIRAL PRODUCTION AND NOVEL VECTOR STRATEGIES gradients, either alone or in combination with heparin sulfate chromatography. Although these methods offer significant improvement over earlier methods they still require gradient centrifugation, which is not a feasible approach for large-scale commercial production. In addition, due to the differing receptor specificities of the various rAAV serotypes, heparin sulfate chromatography is useful only for the purification of rAAV2; rAAV2 is to date the only serotype that uses cellular heparin sulfate receptors for cell entry. Here we describe chromatographic methods to reproducibly purify various rAAV serotypes, including serotypes 1, 2 and 5. The procedure is rapid, yielding purified rAAV vector which is greater than 95% pure, and is useful for rAAV made either by the triple transfection or baculovirus production methods. A significant advantage of this method is that it is scaleable for commercial purposes, being designed to process large volumes of cell lysate. In conclusion advancements in the purification of various rAAV serotypes makes the production of biologically active rAAV stocks for gene transfer studies in both preclinical and clinical studies a more achievable goal.
910. A Novel Recombinant Herpes Simplex Virus Used as Helper Virus for Producing AAV1 Pseudotype Vectors Hui Cao,1 Suyue Pan,1 Ming Peng,1 Wenjun Wu,1 Xiaobing Wu.1 AGTC Gene Technology Company Ltd, Beijing, China.
AAV1 vectors were reported more efficient than AAV2 vectors for transducing muscle and liver. The efficient and large-scale production of recombinant AAV1 is a key step to successful use of AAV1 vectors for human gene therapy. We have successfully established a two-component packaging system for AAV2 by using a recombinant herpes simplex virus type 1 carrying AAV2 rep and cap genes to infect a cell lines stably holding AAV2 vector. And here we used the same strategy to construct a new helper virus for AAV1 vector packaging. It was described the hybrid plasmids containing AAV2 rep and AAV1 cap gene can help to package AAV2 genome into AAV1 capsid, resulting in an AAV1 pseudotype virus,ie,AAV2 ITRs in AAV1 capsids. generally pseudotype AAV1 vectors production was carried out by calcium phosphate-mediated cotransfection of AAV2 vector plasmid, rep2/cap1 hybrid plasmid, and adenoviral helper plasmid into HEK 293 cells. The rep2/cap1 hybrid plasmid is composed of AAV2 rep gene and AAV1 cap gene. This method is relatively low efficient and difficult to scale up to obtain enough AAV1 vectors for gene therapy studies. Here we report a new method for AAV1 vectors production by simply transfecting a package cell line with a helper virus. We constructed a novel recombinant herpes simplex virus type 1(HSV1) as helper virus for the production of AAV1 vectors. This recombinant HSV1, designated as rHSV1/r2c1, contains AAV2 rep ORFs and AAV1 cap ORFs in the unique XbaI site of UL2 gene. A set of cosmids, Set C(a kindly gift from Dr. Davision AJ) , was made use of to generate HSV1/r2c1. Set C is composed of 5 cosmids, called cos6, cos14, cos28, cos48, cos56 respectively. Each of the five cosmids contains a fragment of HSV1 strain 17 genome, and has overlapping ends to one another (Conningham C, Davision AJ. Virology, 1993, 197: 116-124). At first, r2c1 fragment was constructed by replace AAV2 cap with AAV1 cap on KpnI site,and then inserted this r2c1 fragment into XbaI site of UL2 gene on cos6 of Set C, obtained cos6/r2c1. 5 to 6 days after cotransfecting cos6/ r2c1 with other four cosmids (cos14, cos28, cos48, cos56) into BHK-21 cells, cytopathologic effect (CPE) became obvious, the supernatant was harvested and three rounds of plaque purification were done to obtain rHSV1/r2c1. To detect the helper function of rHSV1/r2c1 in AAV1 vector packaging, a BHK-21 cell line stably carrying AAV2 vector DNA Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts Copyright ® The American Society of Gene Therapy
containing two AAV2 ITRs and eGFP expression cassette was generated(BHK/AAV-GFP). The cells were infected with rHSV1/ r2c1 at MOI=1. 2 days p.i., the cell was freeze-thawed three times and treated at 56 ° for 30 min to inactivate HSV/r2c1, then centrifuged at 12,000 g for 10 min, removed the pellet, the suspension was the AAV1/eGFP vector stock. The titer of the AAV1/eGFP vector was 1~3x1010 v.g./ml measured by dot blot. There was no detectable HSV in AAV1/eGFP vector stock by plaque assay after 5 passages on BHK-21. Comparing AAV1/eGFP with AAV2/eGFP, we found that AAV1/ eGFP could transfected BHK-21, HEK 293 as efficiently as AAV2/ eGFP, but could not be captured by heparan affinity chromatography as AAV2/eGFP does. Using ion-exchange chromatography to purify the AAV1 vector and in vitro and in vivo comparisons of AAV1 and AAV2 vectors are currently underway.
911. Strategy for the Development of Neurotropic AAV: Biopanning for Neurotropic Peptides James K. Liu,1 Teng Qinshan,1 Mary E. Garrity-Moses,1 Diana Tanase,1 Michael J. Imperiale,2 Nicholas M. Boulis.1 1 Department of Neurosurgery, Lerner Research Institute, the Cleveland Clinic Foundation, Cleveland, OH; 2Department of Microbiology and Immunology, University of Michigan Medical School, Ann Arbor, MI. Adeno-associated virus is a popular vector for the delivery of neurotrophic factors due to its efficiency and low toxicity. Several investigators have shown that small peptide epitopes may be inserted into the capsid without hindering the infectivity of AAV. In order to create a novel neurotropic AAV vector, we plan to insert peptides with high-affinity motor neuron binding into the VP1 coat protein. In order to isolate such neurotropic peptides, we have utilized a biopanning process using phage display technology. We have developed a biopanning strategy that involves screening random phage libraries for peptide binding to the clostridial neurotoxin receptor, GT1b ganglioside. In order to guarantee effective immobilization and ensure proper exposure of the desired binding carbohydrate, we have selectively labeled GT1b with putrescine via a reductive amination reaction. This process allows for a covalent interaction when reacted onto maleic anhydride activated plates (Pierce Biotechnology, Inc., Rockford, IL). The putrescine labeling was performed to ensure proper exposure of the binding site and also effective immobilization of the target glycolipid even during rigorous wash steps. ELISA analysis showed that putrescine labeling was an effective means of immobilization with enhanced carbohydrate receptor presentation. Elution during the first round of panning was performed using a general elution factor in order to gather the small percentage of peptides that exhibit extremely strong binding to our target protein. Three successive rounds of panning were performed using recombinant tetanus toxin C fragment (Roche Diagnostics Corp., Indianapolis, IN) as a specific elution factor in order to obtain phage peptides that specifically mimic binding of C fragment to GT1b. Biopanning using streptavidin as the target protein showed that such biopanning is an effective method for obtaining a consensus sequence that binds to the target. 100 clones chosen from each peptide library following four rounds of panning to GT1b were sequenced. Peptides that were believed to represent a consensus sequence for binding were selected and verified for binding via ELISA analysis.