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