SBIR-STTR Award

The purpose of this application is to develop a process for improved purification of the non-enveloped viral vector - Adeno-associated virus. This vector is a vehicle for ex vivo and in vivo gene therapy. Starting from crude material, the resulting product must be of high purity and produced in high yields. In addition, this vector must be free from contaminating cellular components such as chromosomal DNA and RNA, protein, membrane components and endotoxins. Requirements for large-scale production of this vector is creating a demand for improved purification processes. Current processes using three (3) consecutive Cesium chloride gradients are cumbersome, inefficient and difficult to scale-up. Alternative technologies based on column chromatography are often inefficient, resulting in low purity and low yields with limited ability to remove contaminants. Column fouling is a particular problem. A new approach involving an innovative technology is proposed. The technology is based on an inert, non-porous, irregularly-shaped polymer using principles of ion-pair reverse-phase chromatography. The unique properties of this resin provide for higher purity and yield and lower residual contaminants. Also, it is reproducible and scalable and will improve economics of vector purification. PROPOSED COMMERCIAL APPLICATIONS: The PolyFlo resin is manufactured by Puresyn, Inc. and is to be used as part of the purification process in the development of nucleic acid-based products. The results of the proposed research will offer a process to a new technology area to Academic and Industrial Healthcare Markets. High purity and yield as well as a reduction in process time will lower production expenses from analytical through large-scale development

Puresyn, Inc. has developed purification processes for recombinant adeno-associated virus gene therapy vectors utilizing its proprietary chromatographic resin, PolyFlo. In Phase I, two tandem chromatography processes were developed: heparin affinity and PolyFlo and tandem PolyFlo in two different modes. Each process resulted in product of high purity and recovery that exceeds gradient centrifugation processes. We demonstrated each process removes significant amounts of host and viral contaminants. We are seeking Phase II funding to concentrate on AAV production to include large-scale purification up to 5 x 10e14 particles per run without compromising purity, yield or biological activity. We will refine and expand our current processes to assure they are applicable to AAV serotypes 1, 2 and 5. We will also address the significant problem of AAV aggregation. Finally, we will conduct an in vivo mouse study to determine the correlation between purity, biological activity and safety through the evaluation of immune and histopathological responses to the vector. Results of this research will have immediate impact for those engaged in the use of recombinant AAV vectors for gene delivery because it will allow the large-scale development of AAV vectors, It will have positive commercialization implications for PoIyFlo and Puresyn, Inc.

Thesaurus Terms:
adeno associated virus group, chromatography, ion exchange chromatography, protein purification, recombinant virus, resin, technology /technique development, transfection /expression vector gene therapy, microorganism population study, serotyping high performance liquid chromatography, histopathology, laboratory mouse, western blotting