AAV Vector Development Milestones
The Vector Core at Nationwide Children’s has historically demonstrated a commitment to constantly improving its services and providing gene transfer vectors of extremely high quality. The following list of Vector Core milestones documents Nationwide Children’s contributions to the field of AAV vector development.
Nationwide Children’s develops the first stable rAAV producer cell lines and Ad hybrid production methods in 1996 3,5,6,9.
1998 - 2006
In 1998, the research Viral Vector Core was established to support pre-clinical SIV/HIV genetic vaccine studies using stable producer cell line technology.
Targeted Genetics Inc. licenses the technology and uses the HeLa cell platform to safely dose more than 400 patients without serious adverse events related to the vector.
Nationwide Children’s is the first to report the use of Heparin-based FPLC for AAV2 vector purification 2.
Nationwide Children’s develops Rep+ cell lines (C12) to titer rAAV infectious units 4.
Nationwide Children’s develops Taqman qPCR technique to titer rAAV vector containing particles 2.
Nationwide Children’s defines the molecular form and frequency of persistence of rAAV and wt AAV in mouse and human tissue and demonstrates that the vast majority of AAV (wild-type or recombinant) persists as episomes 1,8,10,11.
The Vector Core develops novel AAV helper plasmids based on in vivo molecular isolates. A novel rh.74 serotype is isolated from rhesus macaque LN and shown to efficiently traverse the vascular endothelium for systemic delivery applications 7
Using plasmid DNA transfection methodology, the Vector Core successfully produces of pre-clinical vector to support a National Institute of Neurological Disorders and Stroke sponsored U54 Project for development of two AAV-based biologics towards IND application. The Vector Core meets all associated milestones.
A Nationwide Children’s Hospital internal initiative to fund a cGMP clinical manufacturing facility is approved. The five-year, multi-million dollar plan provides for facility, equipment and full-time employee infrastructure.
The CMF successfully produces an AAV gene therapy tox product for testing in a GLP tox study to support an IND application.
The facility successfully produces a qualified mammalian MCB for use in cGMP clinical vector manufacturing.
In 2009-2010 the facility procures a second qualified HeLa MCB and human Ad5 Master Viral Bank for use in cGMP clinical vector manufacturing.
The CMF initiated clinical production of its first drug substance in support of an IND application.
The CMF produced a tox lot for an AAV1 gene therapy product that was used to support a recently approved IND.
The CMF produced two phase I clinical AAV products, the first was an AAV2 product approved in Great Britain by the MHRA (FDA equivalent) to treat a congenital blindness disorder called choroideremia.12 The product has successfully entered human testing. The second was an AAV1 product approved by the FDA in October 2011, with the ongoing phase I trial initiated at Nationwide Children’s Hospital in February 2012.
1. Chen, C-L., R. L. Jensen, B. C. Schnepp, M. J. Connell, R. Shell, T. J. Sferra, J. S. Bartlett, K. R. Clark, and P. R. Johnson. 2005. Molecular Characterization of Adeno-Associated Viruses in Children. J Virol 79: 14781–14792.
2. Clark, K. R., X. Liu, J. P. McGrath, and P. R. Johnson. 1999. Highly purified recombinant adeno-associated virus vectors are biologically active and free of detectable helper and wild-type viruses. Hum Gene Ther 10:1031-1039.
3. Clark, K. R., F. Voulgaropoulou, D. M. Fraley, and P. R. Johnson. 1995. Cell lines for the production of recombinant adeno-associated virus. Hum Gene Ther 6:1329-1341.
4. Clark, K. R., F. Voulgaropoulou, and P. R. Johnson. 1996. A stable cell line carrying adenovirus-inducible rep and cap genes allows for infectivity titration of adeno-associated virus vectors. Gene Ther 3:1124-1132.
5. Liu, X., F. Voulgaropoulou, R. Chen, P. R. Johnson, and K. R. Clark. 2000. Selective Rep-Cap gene amplification as a mechanism for high-titer recombinant AAV production from stable cell lines. Mol Ther 2:394-403.
6. Liu, X. L., K. R. Clark, and P. R. Johnson. 1999. Production of recombinant adeno-associated virus vectors using a packaging cell line and a hybrid recombinant adenovirus. Gene Ther 6:293-299.
7. Rodino-Klapac LR, Janssen PM, Montgomery CL, Coley BD, Chicoine LG, Clark KR, Mendell JR. 2007. A translational approach for limb vascular delivery of the micro-dystrophin gene without high volume or high pressure for treatment of Duchenne muscular dystrophy. J of Transl Med, 5:45.
8. Schnepp, B., K. Clark, D. Klemanski, C. A. Pacak, and P. R. Johnson. 2003. Genetic Fate of Recombinant Adeno-Associated Virus Vector Genomes in Muscle. J Virol 77:3495-3504.
9. Schnepp, B., and K. R. Clark. 2002.Highly purified recombinant adeno-associated virus vectors. Preparation and quantitation. Methods Mol Med 69:427-443.
10. Schnepp, B., R. L. Jensen, C-L Chen, P. R. Johnson and K. R. Clark. 2005. Characterization of Adeno-Associated Virus Genomes Isolated from Human Tissues. J Virol 79: 14793–14803.
11. Schnepp BC, Jensen RL, Clark KR, and Johnson PR. 2009. Infectious molecular clones of adeno-associated virus isolated directly from human tissues. J Virol 83:1456-1464.
12. TouchOncology.com "Viral Vector Holds Key in Treating Genetic Blindness Disorder"