Lentiviral Vectors Approach the Clinic but Fall Back: National Institutes of Health Recombinant DNA Advisory Committee Review of a First Clinical Protocol for Use of a Lentiviral Vector
Authors: Greg M. Podsakoff
Journal: Molecular Therapy (2001)

Recent preclinical studies have demonstrated some potential advantages for lentiviral vectors. The development of this vector system has led to the first proposed clinical study in patients with human immunodeficiency virus-1 (HIV-1) infection. The study was presented for the second time by Boro Dropulic from VIRxSYS, Gaithersberg, MD, and Rob Roy MacGregor, Principal Investigator at the University of Pennsylvania, at the September 6, 2001, meeting of the National Institutes of Health Recombinant DNA Advisory Committee (see RAC minutes of March 2001, http://www4.od.nih.gov/oba.RDNA.htm). The study design involves enrollment of HIV-1 infected subjects for whom highly active antiretroviral therapy (HAART) has failed and delivery to lymphocytes of an antisense gene directed against the HIV-1 envelope (env). The proposed target cells are autologous T lymphocytes activated ex vivo with antibodies specific for CD3 and CD28. The VSV-G-pseudotyped lentiviral vector is a “first-generation” construct produced by two-plasmid transfection, with a stop codon in gag and a unique sequence tag for PCR identification. The rationale is that the vector-containing T lymphocytes would encode antisense transcripts against HIV-1 env, following infection by HIV-1 and transactivation of the integrated vector's intact HIV-1 long terminal repeat (LTR) by viral Rev and Tat proteins. The trial design includes dose escalation with four different cell dosages separated by one-half log each, with 6-week intervals between groups. Dropulic presented data that showed if sufficient anti-sense env vector was added to activated T lymphocytes, resulting in > 93% transduction of CD4+ T cells, an approximately 3-log suppression of HIV-1 replication was seen. The suppressive activities of the vector were demonstrated with low multiplicity-of-infection (MOI) HIV-1 challenges (0.001, with strains NL4-3, BK-132 (a primary isolate), Ba-L, and US1) and may be abrogated by higher MOI. Interestingly, the breakthrough of virus typically seen in tissue culture was not observed in HIV-1 challenges if the vector copy numbers per cell were high enough (that is, exceeding six). Although the therapeutic strategy involves mobilization of the vector by rescue with HIV-1, preclinical data showed relatively poor mobilization of approximately 0.5–1.8% double-positive-staining cells by FACS analysis of tissue culture experiments. Both the reviewers and RAC members raised some concerns. John Zaia (City of Hope, Duarte, CA) suggested that the clinical design of cell dose escalation may be problematic, and the follow-up of only 28 days after infusion was deemed insufficient. He noted that the clinical end points of increasing CD4 levels and decreasing HIV-1 viral loads seemed unrealistic and, although problematic, two vectors (one active, one inactive) would more likely address any selective survival advantage. One concern that goes to the issue of proper consent is designating a subject as “HAART failure,” as the current standard of care generally calls for administration of a new HAART regimen, not gene transfer. There is some difficulty in defining failure, as different clinicians and subjects may have different concepts of treatment failure, and in consistently conveying this idea in the consent document to clearly identify those who are eligible. Consequently, Nancy King (University of North Carolina, Chapel Hill, NC) suggested that the “Alternatives” section of the consent may be modified to consider a more balanced and complete view of the therapeutic options. One study design question regarded the chance of adverse events, as the doses of cells activated by the anti-CD3/CD28 beads may lead to post-infusional toxicity. Louise Markert (Duke University, Durham, NC) wondered if T-cell diversity would be limited from transduced lymphocytes, but Carl June (University of Pennsylvania, Philadelphia, PA) suggested that this should not be an exclusion criteria, as it is not known what diversity would be protective in these subjects. Nevertheless, Markert stated that it may be useful to at least measure and follow the extent of T-cell diversity of the subjects pre- and post-infusion. The mechanism of action of suppression was questioned by John Coffin (National Cancer Institute, NCI-Frederick, Frederick, MD), particularly the role of “defective interfering virus” activity in light of suppression at low MOI. Coffin also expressed concern about the wild-type LTR in the vector, which may actually repair some mutant HIV-1 infecting a patient, converting the endogenous virus into a more virulent variety. The major concern of the reviewers was the possibility of the formation of a replication-competent recombinant between the vector and packaging sequences (such as the VSV-G protein) in the vector supernatant and/or transduced CD4+ cells (Estuardo Aguilar-Cordova, Harvard University, Cambridge, MA; John Zaia and Jiing-Kuan Yee, City of Hope). An important issue for VIRxSYS is to validate a reproducible, sensitive method to detect one VSV-env genome in the final product. The plan to infuse approximately 1 × 1010 cells, some with multiple copies of the antisense env gene per cell (approximately 1011 total copies of vector), requires a sensitive, validated detection method. Perhaps combined methods (such as biological amplification followed by PCR) may optimize detection of low levels of VSV-G contamination. This challenge is critical, because validation that a single copy of VSV-G is detectable in the final cell product will be required to support the absence of RCL and to complete product characterization before infusion. The possibility of inadvertent transfer of a mobilized vector to a partner was suggested as a potential serious event, although that would be under circumstances in which the subject's endogenous wild-type HIV-1 could also be passed on. Aguilar-Cordova also suggested that if any degree of vector mobilization may occur, in vivo monitoring studies to define vector rearrangements should be carried out. The outcome of the review of this protocol for the use of lentiviral vectors is not final. As the issues and assays needed to ensure patient safety with this new vector system are still being defined, the public review of the protocol provided an open forum to propose hypothetical risks. It is left to the sponsors to respond to all of the questions that were raised to the satisfaction of the RAC and then the FDA before this application of lentiviral vectors can begin. Of course, the vector system will also need acceptance by the potential subjects of this study. If this and other studies involving “first” and later-generation lentiviral vectors demonstrate safety, ultimately the medical community's perception of the long-term safety of this approach will help determine the fate of this gene delivery system.