AAV Vectors

Adeno-Associated Viruses are small non-enveloped single-stranded DNA viruses. They are non-pathogenic human parvoviruses and are dependent on helper viruses, including adenovirus, herpes simplex virus, vaccinia virus and CMV, for replication. To date, 11 serotypes of AAV have been isolated.

AAV vectors are currently one of the most promising systems for human gene therapy. Using recombinant AAV (rAAV) vectors, genes can be delivered into a wide range of host cells including many different human and non-human cell lines or tissues. Because AAV is non-pathogenic and does not illicit an immune response, a multitude of pre-clinical studies have reported excellent safety profiles. rAAVs are capable of transducing a broad range of cell types and transduction is not dependent on active host cell division. High titers, > 10⁸ vp/ml, are easily obtained in the supernatant and ³ 10¹¹ -10¹² vp/ml with further concentration. The transgene is integrated into the host genome so expression is long term and stable.

AAV2 is the best characterized serotype and the serotype for which most gene transfer studies have been based upon. The design of AAV-based vectors in pretty straight-forward. The ITRs flank the transgene and the AAV Rep and Cap and helper virus genes are provided in trans. Several systems have been developed to produce rAAV with each system having its own advantages and disadvantages. With each system, the disadvangates vary, however one disadvantage remains the same, the small packing capacity of the rAAV vectors. rAAV cannot package genomes greater than 4 kb.

All AAV vectors produced in the VDL are produced in compliance with GLP. We produce rAAV using a 2 transfection method. One plasmid contains the AAV2 rep and cap genes and the adenovirus helper genes (E2, E4, VA1/II) while the other plasmid contains an expression cassette (CMV promoter, multiple cloning site and SV40 polyA) flanked by AAV2 ITRs. The two plasmids are transfected into 50, 150mm dishes of 293 cells. At 48 hours post-transfection, the cells are harvested and virus is purified by cesium chloride gradient ultracentrifugation. The virus is dialyzed, diluted and stored in a specific storage buffer. Using this method, we usually get 1x10^12 particles.

Our quality assurance/ quality control testing includes, quantification of AAV genomes, determination of infectivity titer, and tests for replication competent adeno-associated virus (RCAAV). Check out our Catalog to get more information on these services.

To address some of the disadvantages of current rAAV production methods, we developed a New rAAV Production System that can be easily scaled up for production of large quantities of highly purified rAAV.

To request an AAV service, visit our Initate AAV Service page or fill out and fax your Service Request Form to us at 713-798-1230 or e-mail to vector@bcm.tmc.edu.