Lentivirus in the Clinic

To the Editor Greg Podsakoff's commentary [1Podsakoff G. 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.Mol. Ther. 2001; 4: 282-283Abstract Full Text Full Text PDF PubMed Scopus (22) Google Scholar] on the review of our clinical protocol (protocol #0107-488, "A Phase I, Open-Label Clinical Trial of the Safety and Tolerability of Single Escalating Doses of Autologous CD4 T-Cells Transduced with VRX496 in HIV-Positive Subjects") at the National Institutes of Health Recombinant DNA Advisory Committee (RAC) meeting on September 6, 2001, requires clarification on several points. First, Podsakoff describes our VSV-G pseudotyped vector as being a "first-generation" construct. The first-generation HIV vector system initially described by Verma and colleagues consists of a three-plasmid system with HIV accessory genes present in one of the two helper plasmid constructs [2Naldini L. In vivo gene delivery and stable transduction of non-dividing cells by a lentiviral vector.Science. 1996; 272: 195-199Crossref PubMed Scopus (4039) Google Scholar]. In contrast, our single helper plasmid construct does not contain any accessory genes such as Vif, Vpr, Vpu, or Nef. We engineered our helper plasmid construct to encode for both HIV structural and VSV-G envelope proteins, with the open reading frames transcriptionally partitioned by strong poly(A) signals and synthetic pause sites [3Enriquez-Harris P. Levitt N. Briggs D. Proudfoot N.J. A pause site for RNA polymerase II is associated with termination of transcription.EMBO J. 1991; 10: 1833-1842PubMed Google Scholar] to thwart transcriptional read-through. The two-plasmid packaging system is useful for scale-up of vector manufacturing because significantly more HIV vector can be produced from 293 cells using the two-plasmid system rather than the three-plasmid system. Additional refining features of our packaging system include degeneration of certain regions in the helper that are common to the vector so as to decrease the likelihood of recombination, and insertion of a stop codon in the vector to prevent gag-pol translation if recombination between the vector and helper (or wild-type HIV) should occur. The commentary notes correctly that our fully gutted vectors transduced primary CD4 T lymphocytes to greater than 93% and inhibited wild-type HIV (wt-HIV) replication by 3 logarithmic units of p24 at a multiplicity of infection (MOI) of 0.001. However, it was incorrectly reported that the inhibitory effects may be abrogated by higher MOIs. At the meeting, we stated that we have seen similar inhibition of wt-HIV replication at higher MOIs, up to an MOI of 0.1. The commentary also incorrectly reports that our therapeutic strategy relies on mobilization of the vector. Our therapeutic approach uses an HIV vector to inhibit wt-HIV [4Dropulic B. Hermankova M. Pitha P.M. A conditionally replicating HIV-1 vector interferes with wild-type HIV-1 replication and spread.Proc. Natl. Acad. Sci. USA. 1996; 93: 11103-11108Crossref PubMed Scopus (81) Google Scholar], and can optimally use vector mobilization to increase anti-HIV efficacy. In fact, we have chosen VRX496, a vector with poor mobilization characteristics, because of the perceived safety risks of using a mobilizable vector for the first lentiviral vector clinical trial. Even though we have vectors that mobilize more efficiently and display greater anti-HIV efficacy, we purposely chose VRX496 for greater safety. Podsakoff noted correctly that one of the reviewers raised concerns about a 28-day follow-up period before dose escalation, but failed to report our response. CD4 T cells consist of two cell types. Short-lived cells are generally activated cells and have a short lifespan of less than 2 weeks. Long-lived cells are generally quiescent and can survive for years. Our patient monitoring scheme addresses both types of cells: short-term monitoring at 24, 48, and 72 hours and 7, 14, and 28 days; and long-term monitoring at 3 and 6 months postinfusion and then yearly for the life of the patient. During the meeting we explained that a 28-day follow-up period before dose escalation is appropriate for this study because most of the infused CD4 T cells will be activated cells, capable of supporting wt-HIV replication or presumably the replication of a putative HIV recombinant that may produce an adverse event. In contrast, long-lived cells would be incapable of supporting productive HIV replication while in their quiescent state and would support HIV replication only upon their sporadic activation by specific antigen over the long-term. Because the number of activated cells containing VRX496 is highest immediately after infusion, the greatest risk for an adverse event is short-term rather than long-term. Nevertheless, our patient follow-up scheme will include long-term follow-up at 3 and 6 months and then yearly for life to look for any long-term adverse events that may arise. As it is widely recognized, the major concern for the use of a VSV-G pseudotyped HIV vector is the formation of a replication-competent recombinant virus, particularly with VSV-G envelope sequences. To address this safety issue, we have proposed to the RAC and the FDA the use of sensitive molecular assays for detection of functional VSV-G DNA molecules in the final T-cell product. These assays would be used in addition to and in conjunction with biological assays analogous to those used for replication-competent retrovirus detection in the murine retroviral vector system. We concur that these assays are critical for the use of HIV vectors in the clinic. Finally, it was noted that one of the reviewers stated that the clinical endpoints of increasing CD4 levels and decreasing HIV-1 viral loads seem unrealistic. It is important to note that the proposed trial is a phase I safety trial and not an efficacy trial, so safety is the singularly important goal of the trial. We recognize that a successful T-cell therapeutic approach would require sufficient numbers of VRX496-modified T cells to survive long-term in vivo so as to decrease viral loads and promote the anti-HIV immune response. Several reports indicate that ex vivo expanded T cells can indeed survive long-term in vivo [5Mitsuyasu R.T. Prolonged survival and tissue trafficking following adoptive transfer of CD4z gene-modified autologous CD4(+) and CD8(+) T cells in human immunodeficiency virus-infected subjects.Blood. 2000; 96: 785-793PubMed Google Scholar, 6Walker R.E. Long-term in vivo survival of receptor-modified syngeneic T cells in patients with human immunodeficiency virus infection.Blood. 2000; 96: 467-474PubMed Google Scholar]. Several clinical options are available to further address efficacy issues. A future efficacy trial could look at infusion of multiple doses of VRX496-modified T cells or use vectors that mobilize more efficiently. Other approaches could include conditioning regimens for patients that would endow the modified cells with an advantage over endogenous unmodified cells. Moreover, our target cell population need not be restricted to CD4 T cells—VRX496 could also be delivered into hematopoietic stem cells by bone marrow transplantation, where the modified stem cells could then differentiate into mature T cells. However, no matter the approach, we believe that our preclinical data presented at the meeting demonstrate that HIV-derived vectors offer a unique opportunity for the treatment and management of HIV infection. As with any new class of vector, safety issues are paramount. We are working to address these issues to the satisfaction of the RAC and the Food and Drug Administration. We are optimistic that once safety is established, this new class of vectors will make a significant therapeutic impact in the treatment of AIDS and other diseases. Lentivirus in the Clinic: In ReplyGREG M. PODSAKOFFMolecular TherapyDecember, 2001In BriefThe key issue for the proposed first lentiviral vector clinical study is whether this construct is a safe vector to be applied in the clinic at this time, or whether more thorough characterization of potential outcomes is necessary. The study involves treatment of a lethal disease with a novel vector, which, as Boro Dropulic points out, is "second generation" due to the deletion of the accessory proteins. For this study using the proposed vector, the safety issues are difficult to test, as the in vitro results in cells and mice are limited in their translation to the actual in vivo milieu in human subjects. Full-Text PDF Open Archive

Topic: Virus-based gene therapy research
Subfield: Genetics
Field: Biochemistry, Genetics and Molecular Biology
Domain: Life Sciences