Gene therapy vector contamination assay

Abstract

Certain viral gene therapy vectors are by design unable to replicate in a patient. Nonetheless, during manufacture of viral gene therapy vector, undesirable replication-competent virus ('RCV') may form due to random mutation or other events. Viral gene therapy vector manufacturers thus assay for the presence of contaminating RCV. Regulatory agencies require this to be done by assaying for serial infection, i.e., transducing target cells with the viral vector, and then lysing the transduced cells, and then mixing the lysate with live assay cells, and then microscopically observing the assay cells to visually determine whether they have been infected with virus. We have tested various alternative approaches, and surprisingly found that digital PCR is not only faster than the prior art approach, but is also over an order of magnitude more sensitive, able to detect, for example, in 3×10assay cells, as few as seven (7) replication competent adenoviruses (“RCA”).