Gene therapy has the potential to cure any disease caused by genetic mutations. For gene therapy to work, normal copies of genetic DNA, replacement messenger RNA or gene-editing constructs must be delivered to affected cells. Nucleic acids are rapidly degraded in the bloodstream so any gene therapy must be delivered to cells using protective vectors.
The vector options currently available are viral vectors and lipid nanoparticles, although naturally occurring vault nanoprotein vectors are also under development. Retroviruses are an attractive option as gene therapy vectors because they naturally contain the enzymes needed to insert DNA into the host genome. However, this ability is also one of their biggest threats to human patients as replication-competent retroviruses can replicate inside host cells and insert viral DNA in multiple places in the host genome. This increases the risk of insertional mutagenesis (1).
Minimizing Risk in Gene Therapy Vectors
Insertional mutagenesis occurs when a retrovirus inserts DNA in the middle of a host gene, thus mutating that gene (2). This can cause cancer if the mutated gene happens to be a protooncogene, or a gene that controls cell division. Retroviruses use the integrase enzyme to insert viral DNA into the host genome. This insertion is arbitrary and can occur anywhere in the host genome. Scientists can minimize the risk of insertional mutagenesis by using gene therapy vectors that do not contain replication-competent retroviruses.
Stable packaging cell lines are used to produce viral vectors for gene therapy. These cell lines are designed to minimize the risk of the viral vectors becoming replication-competent. The resulting viral vectors should not be able to replicate and should transfer only the gene of interest and not any viral genes into patient cells (2).
FDA Requirements for Gene Therapy Vector Testing
In 2006, the FDA issued guidelines on testing for replication-competent retroviruses in gene therapy products that contain retroviral vectors (3). The FDA recommends that researchers use a cell bank system to ensure their vector packaging cell lines are consistent. They recommend developing a Master Cell Bank with a supply of cells derived from a single cell or tissue, and a Working Cell Bank that comes from the Master Cell Bank.
The FDA guidelines recommend that all Master Cell Bank cell lines, all Working Cell Bank cell lines and all final products are tested for replication-competent retroviruses.
The FDA has since mandated that all vectors be tested before they can be used in clinical trials, and re-tested if they are cultured for more than 4 days (4). All patients who receive retroviral vectors in clinical trials must also be tested for replication-competent retroviruses. Retroviral vectors and patient blood samples must be cultured for at least 5 passages, or around 3 weeks, to rule out the presence of replication-competent retroviruses (4). This means that testing is expensive, time-consuming and must be conducted in specialized labs.
National Gene Vector Biorepository
The National Gene Vector Biorepository in the US is funded by the National Institutes of Health (NIH) and the National Heart, Lung and Blood Institute (NHLBI). The National Gene Vector Biorepository provides services, databases and educational resources to non-profit organizations, academic institutions and companies with NIH small-business grants. Services include archiving clinical samples, GMP material and pharmacology/toxicology studies, and FDA-required testing of vector products.
The National Gene Vector Biorepository recently published data on replication-competent vector testing (4). They tested 286 retroviral vector gene therapy products from 14 clinical trials. They also tested blood from 241 clinical trial participants who received gene therapy products with retroviral vectors. The PG13 packaging cell line was used to generate most of the vector products tested in this study.
No RCR was found in any gene therapy products or in patient blood.
The data from this study suggest that the PG13 packaging line is safe. This supports previous data published by the National Gene Vector Biorepository showing that 460 cell products used in cancer immunotherapy trials were free from replication-competent retroviruses. Therefore, the authors propose that perhaps the FDA guidelines could be revised so that less testing of PG13-derived vector products is required.
- Bear et al. Replication-Competent Retroviruses in Gene-Modified T Cells Used in Clinical Trials: Is It Time to Revise the Testing Requirements? Mol Ther. 2012
- The use of retroviral vectors for gene therapy – what are the risks? A review of retroviral pathogenesis and its relevance to retroviral vector-mediated gene delivery. Genet Vaccines Ther. 2018.
- Guidance for Industry: Supplemental Guidance on Testing for Replication Competent Retrovirus in Retroviral Vector Based Gene Therapy Products and During Follow-up of Patients in Clinical Trials Using Retroviral Vectors. U.S Department of Health and Human
- Cornetta et al. Screening Clinical Cell Products for Replication Competent Retrovirus: The National Gene Vector Biorepository Experience. Mol Ther Methods Clin Dev. 2018