Gene Therapy Vector Contamination Assay

A method for identifying virus able to replicate in normal human cells in a sample comprising viral gene therapy vector unable to replicate in normal human cells, the method comprising:

• a. Obtaining a sample comprising viral gene therapy vector unable to replicate in normal human cells,
• the viral vector comprising a transgene and a viral genome which has been genetically modified from a wild-type viral genome by having a modification or deletion of a region of the wild-type viral genome essential for replication of the virus in a normal human cell, whereby the resulting viral gene therapy vector is intended to be unable to replicate in normal human cells, and then
• b. Mixing the sample with live target cells which can be transduced by the viral gene therapy vector to make a transduction mixture, and then
• c. Maintaining the transduction mixture for a time and under conditions adequate to enable the viral gene therapy vector to transduce the target cells, and then
• d. Separating the target cells from any residual sample, and then
• e. Lysing the target cells to release their intracellular contents, and then
• f. Mixing the intracellular contents of the lysed target cells with live assay cells which can be infected by the virus to make an infection mixture, and then
• g. Maintaining the infection mixture for a time and under conditions adequate to enable the virus (if any) to infect the assay cells, and then
• h. Lysing the assay cells to release their intracellular contents, and then
• i. Isolating nucleic acid from the assay cells' intracellular contents, and then
• j. Evaluating the isolated nucleic acid by digital PCR, using a probe which hybridizes to said region of the viral genome essential for viral replication which has been modified or deleted,
• thereby measuring in the sample the approximate quantity of virus able to replicate in normal human cells.