Meeting FDA Guidance recommendations for replication-competent virus and insertional oncogenesis testing

Figure 1. Vector design and manufacture methods (A) Top: a schematic of a gammaretroviruses shows the gag/pol gene region that encodes the structural proteins and enzymes required for virion formation, reverse transcription, and integration. These membrane-bound viruses also contain a viral envelope glycoprotein, which facilitates entry into the target cell. Bottom: almost all the gag/pol region and envelope sequence can be replaced by the transgene of interest, making the vector replication defective. The long-terminal repeats (LTR) are retained, as they contain the viral promoter and enhancer that drive transgene expression and are also required for integration of the vector into the target-cell DNA. (B) Generation of retroviral particles requires expression of vector and packaging DNA. Packaging cell lines are created by transfecting viral genes into immortalized cell lines and selecting clones that integrate and stably express the DNA. The line may also contain stable integration of the transgene (gene of interest, GOI), or the transgene DNA can be introduced by transient transfection. An increasingly common method of viral vector production is to use transient transfection of all…

Figure 2. Replication-competent retrovirus (RCR) testing requirements The current recommendations by the US FDA for testing vector products and ex vivo-transduced cells and for monitoring patients. EOP cells, end-of-production cells; ∗, whichever is less.

Figure 3. Biologic assays for replication-competent retrovirus detection The diagram represents an analysis of vector supernatant, which typically contains replication-defective vector particles in excess of 109 particles per milliliter. The challenge is to detect an RCR present at very low numbers within the test material. This is facilitated by exposing vector products to permissive cells, which can amplify the RCR to high levels that are easily detectable, while the replication-incompetent vector particles dissipate as the cells expand.

Figure 4. Mechanisms for insertional alteration of gene expression The most common mechanism for insertional oncogenesis has been oncogene promoter upregulation by the enhancer element within gammaretroviral vectors. While vector promoters driving oncogene transcription have been reported in murine cancers, this has not been noted in human cancers arising after vector insertion. Activation by truncation has been shown after insertion led to the truncation of mRNA, removing a let-7 site that normally regulated gene expression (noted by asterisk). This led to transient clonal expansion without cancer formation. Integration and disruption of gene expression has also inactivated a tumor-suppressor gene, which led to clonal expansion without malignancy in a patient with an inherited hypomorphic mutation in the non-transduced allele. Green, promotor region; red, exon.

Figure 5. Long-term follow-up (LTFU) monitoring plan An example algorithm for monitoring patients for insertional oncogenesis.