Adventitious Mutations in Clinical Grade Vectors: An Issue to Consider?
Authors: David A. Williams, Timothy P. Cripe
Journal: Molecular Therapy (2006)

New viral vectors in development for clinical trials typically undergo extensive preclinical testing of their identity, efficacy and safety. For large viruses, such as those derived from Herpes simplex virus (HSV)-1, sequencing of the entire genome is thought to be somewhat prohibitive. Instead, restriction mapping combined with sequencing of the intentionally manipulated regions has usually been considered sufficient to confirm identity of the recombinant virus. Formal toxicology studies of the final GMP product are also required to support an application for an Investigational New Drug. In this issue, an important paper by Dambach et al. [1Dambach M.J. et al.Oncolytic viruses derived from the g34.5-deleted Herpes simplex virus R3616 encode a truncated UL3 protein.Mol. Ther. 2006; 13 (this issue.)Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar] raises additional questions about how best to characterize the safety of viral vectors being utilized in clinical trials. Replication competent variants of HSV-1 have been developed as oncolytic vectors for cancer therapies. These vectors contain deletions of regions essential for growth in normal cells and specifically show reduced neurovirulence. However, these vectors retain the ability to grow in an attenuated fashion in some cells, a characteristic being exploited for growth in and lysis of cancer cells. Several of these vectors have been tested in human trials. In the manuscript by Dambach et al., the authors demonstrate a nonsense mutation in a region of the HSV-1 genome that encodes a protein of unknown function called UL3. The mutation led to a truncated protein, although the effect of the truncation on protein function has not yet been determined. The mutation occurred during the construction of a virus that has served as the parent virus for one vector already in clinical trials (G207) and at least two others under development for clinical evaluation (GΔ47, MGH1). Although the possibility of generation of mutations during vector construction has been well described and is appreciated by virologists, the gene therapy community at large is probably less aware of this issue. The increasing prevalence of more rapid and “sure-fire” techniques for vector construction using bacterial artificial chromosomes (BACs), such as recently described by Saeki's group [2Terada K. et al.Development of a rapid method to generate multiple oncolytic HSV vectors and their in vivo evaluation using syngeneic mouse tumor models.Gene Ther. 2006; (in the press).Crossref PubMed Scopus (87) Google Scholar], rather than techniques that utilize native recombination events may exacerbate this problem, because these BAC-based methods are likely to be associated with a higher error rate. In addition, mutations that were not present on the input vector and which did not arise during small-scale production could arise during large-scale production of vectors. Mutations that yield a growth advantage are a particular concern due to the increased selective pressure of the additional rounds of infection that occur during large-scale expansion of vectors in culture. The new manuscript by Dambach et al. brings this issue into greater focus and begs the question as to how concerned we should be about the potential effects of these mutations on the function and safety of the vector and how careful we should be in monitoring these mutations. Taken to its logical conclusion, this report might suggest that the entire recombinant virus might need to be sequenced following large-scale production. Such an approach could cause additional delays in clinical trials (a problem we addressed in the last issue of Molecular Therapy [3Williams D.A. An Emerging Consensus on Recommendations to Facilitate Clinical Gene Transfer.Mol. Ther. 2006; 13: 637-638Abstract Full Text Full Text PDF PubMed Scopus (3) Google Scholar]) if workers are compelled to “fix” mutated base pairs that do not actually affect the behavior of the vector. Alternatively, some selected mutations might improve the performance of the vector and for this reason an argument could be made not to require that the full vector sequence be determined or that any adventitious mutations be corrected following large-scale production. It is clear, however, that such mutations might adversely affect vector efficacy and safety. It is in part for this reason that the Food and Drug Administration requires full toxicology studies and confirmatory efficacy studies on the final GMP product, and not just on preclinical grade vector. Thus, one could argue that this is an effective safeguard against adventitious and deleterious mutations introduced during amplification and manufacture. However, we should consider whether these safeguards are sufficient. We believe that we should proceed cautiously, by examining and discussing further the phenomenon described in this new study. Genome sequencing is rather easy and inexpensive these days, and it may be worthwhile to assess the full vector sequence to determine the frequency of the occurrence of mutations before and after vector construction, before and after large-scale production, and possibly even before and after injection into patients. In the latter case, one might culture a virus following injection into a patient's tumor, for example. By collecting these types of data, we could begin to grasp how widespread adventitious mutations are, and whether there is likely to be any discernable impact on the safety or efficacy of vectors used in clinical trials. The field might even undertake to create a reference database of such changes in order to determine which newly identified mutations are likely to be deleterious and which ones are likely to be innocuous. However, even this approach may be further complicated by differences in the behavior of a particular mutation in different vector backgrounds (strain) or in the context of other vector mutations. Nevertheless, the results of Dambach et al. raise an important issue that deserves additional analysis and discussion.