Gene therapy is a growing field where disease is treated by the addition of genetic material into cells to compensate for missing or abnormal genes. To facilitate the movement of genetic material into cells, viruses such as adeno may be modified into replication-incompetent vectors.
Adenovirus is a commonly used vector due to its broad tropism and ability to infect non-replicating cells.
The adenovirus genome consists of linear double-stranded DNA that is approximately 36 kilobases in length. The adenovirus genome contains early regions (E1-E4), late regions (L1-L5), and short inverted terminal repeats. To render the adenovirus replication-incompetent, vectors contain deletions in the E1 region of the genome.
Webinar Series: Platforms for Gene Therapy Characterization
Watch this educational webinar series to enhance your existing analytical methods and explore innovative, novel tools to support safe and effective gene therapy development.
Historically, adenovirus vectors have been produced using human embryonic kidney cells (HEK293), which were transformed using human adenovirus 5. These cells contain the E1 region of adenovirus and can aid in the replication of replication-incompetent adenovirus vectors. However, due to the homology between the E1 flanking sequences in the HEK293 cells and the vectors, recombination events can occur. A recombination event could lead to the insertion of the cellular E1 gene into the vector product, which would render the vector to be replication-competent adenovirus (RCA).
The contamination of adenoviral vectors with RCA is a major safety concern. Adenovirus typically causes mild respiratory disease in healthy humans; however, it can cause life-threatening infections in immunocompromised patients. To mitigate and control the risk, the FDA has mandated that there must be less than 1 RCA in 3 x 1010 vector particles. To ensure the safety of these life-saving products, assays have been developed to screen adenovirus vectors for RCA.
The RCA assay is performed by inoculating vector material onto human cells, which only support the growth of replication-competent adenovirus. The cells are observed over an incubation period to ensure that the vector product is free of viral contaminants. The usage of a cell-based assay allows for the clear separation between replication competent and replication defective adenovirus particles. The RCA assay can be customized and qualified to ensure the safety profile of specific vectors.
Our team is here to answer any questions you have on this assay and how it can benefit your program.