CORE--IMMUNE

Wistar Institute, Philadelphia PA

Investigators

Linked publications & trials

Abstract

Most experiments proposed in the 4 projects of this application require production of AAV vector from plasmids that the individual researchers design using standard recombinant DNA techniques. The goal of the Vector Core Facility is therefore the production of research grade AAV vector for pre-clinical studies in hemostatically normal and hemophilic mice and in non-human primates. This function will ensure the availability of recombinant AAV for the different projects that involve experiments with AAV vectors for expression of therapeutic transgenes. AAV vector is produced in a helper virus-free system based on largescale plasmid transfection of HEK-293 cells using two helper plasmids that supply AAV rep/cap and adenoviral helper functions and a third plasmid encoding the recombinant vector. This will allow production of a variety of different vectors for the investigators. The use of a Core Facility will provide reproducible yield and purity of vector, and will be more cost-effective than vector production in individual laboratories, in particular for experiments that involve large animal models. The service of the Core will include large-scale preparation of helper and vector plasmids, large-scale transfection of HEK-293 cells using calcium phosphate precipitation, recovery and purification of recombinant AAV by cell lysis followed by d fferential precipitation and gradient centrifugation, dialysis, sterile filtration, and storage of purified vector, and quantitative slot blot hybridization. The Core will vector preparations for quality control and assist the different projects with functional assays. Standard vector preparations are expected to yield approximately 10(13) vector genomes, while scale up will result in production of approximately 10(14) vector genomes per preparation. Scale-up of vector production using a roller bottle method has been optimized and will be applied to production of vector for non-human primate studies. The Core will also expand and purify recombinant adenoviral vectors and store and distribute peptide libraries for immunological assays.