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1. Garcia, C. et al. Nat. Biotechnol. 25, 107–116 (2007)

2. Casadevall, A., Dadachova, E. & Pirofski, L. Nat. Rev. Microbiol. 2, 695–703 (2004).

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Lentiviral vectors ready forprime-timeDonald B Kohn

The first clinical trial of a lentiviral vector highlights the promise of this new class of gene-therapy vector.

Research on gene therapy for HIV-1 infection has spawned a profusion of synthetic ‘anti-HIV’ genes, including antisense, ribozymes, RNA decoys, dominant-negative HIV-1 genes, chimeric T-cell receptors, virion fusion inhibitors and short hairpin RNA (shRNA). Although these genes are generally effective in vitro, results from at least ten clinical tri-als have failed to demonstrate clear-cut evi-dence of therapeutic benefits1. The major limitation of these studies, in which T cells or hematopoietic stem/progenitor cells from HIV-1–infected subjects were modified ex vivo using plasmids or gamma-retroviral vectors, has been inefficient gene delivery to clinically relevant numbers of primary cells that per-sist after reinfusion. In a recent issue of the Proceedings of the National Academy of Science, June and coworkers2 describe a clinical trial in which an anti-HIV-1 gene is transferred and expressed using a lentiviral vector. The first reported clinical trial to use lentiviral vectors, this phase 1 study demonstrates efficient and safe gene delivery to the patients’ T cells with good persistence in vivo. Moreover, it suggests that lentiviral vectors hold promise for more effective gene delivery to treat a large number of disorders.

Lentiviral vectors were developed by inves-tigators at the Salk Institute in the mid-1990s, using design strategies defined over the prior decade to optimize gamma-retroviral vectors

for safety and effectiveness (such as minimal viral sequences in the transfer vector, splitting genes for packaging functions over multiple plasmids, pseudotyping with alternate enve-lope proteins, and efficient production by transient transfection)3,4. Like the lentiviral subclass of retroviridae from which they are derived, lentiviral vectors can integrate in nondividing cells, making them more effec-tive than gamma-retroviral vectors for gene transfer to postmitotic or slowly dividing cells, which may include hematopoietic stem cells and T cells.

In addition, researchers have developed len-tiviral vectors that retain the lentiviral rev/RRE elements, which facilitate nuclear-to-cytoplas-mic transport of large, incompletely spliced transcripts. This property may be highly advantageous for transfer of intact, complex transgene cassettes with multiple transcrip-tional control elements, as has been demon-strated with the human beta globin gene and associated locus control regions5.

The occurrence of insertional oncogen-esis in patients with severe combined immune deficiency, who received hematopoietic stem cells transduced by a gamma-retroviral vector, has focused attention on the integration-site patterns of different gene delivery vectors6,7. Characteristically, gamma-retroviral vectors have a bias toward integration near the 5′ ends of genes, a site that may be favorable for the viral genome to remain transcriptionally active. But this pattern may also increase the potential for strong enhancer elements in the retroviral long terminal repeats to transactivate the adja-cent cellular gene promoter—the underlying mechanism of insertional oncogenesis.

Lentiviral vectors have a different integra-tion-site pattern, targeting gene-rich regions at even higher frequency than gamma-retroviruses

but without the predilection for the 5′ ends of genes. It is not known whether these dif-ferences make lentiviral vectors intrinsically less likely to transactivate cellular genes than gamma-retroviral vectors. However, a par-ticular design modification commonly used for lentiviral vectors may have this effect. Lentiviral vectors with so-called ‘self-inacti-vating’ (SIN) long terminal repeats lack strong viral enhancers and instead may use weaker transcriptional control elements coupled to elements that improve post-transcriptional steps to yield clinically relevant levels of trans-gene expression. Such SIN vectors have been shown to have a lower propensity to transac-tivate adjacent genes than gamma-retroviral vectors, but the vector configurations were dif-ferent and thus further studies are needed8.

Initial concerns about the safety of lentiviral vectors were, quite naturally, based on their derivation from pathogenic HIV-1. Multiple iterations of lentiviral vectors and their pack-aging components were derived to delete as much of the HIV-1 genome as possible and to eliminate overlap of the sequences contained in different components, to minimize the risks of generating recombinants with the ability to replicate autonomously. Current lentivi-ral vectors do not contain any open reading frames from HIV-1 and, when integrated into chromosomes, should serve to express only the transgene. Nevertheless, the prospect of inducing clinical problems, however remote, remains a theoretical possibility. Thus, study-ing patients already infected with wild-type HIV-1 and evaluating a potential anti-HIV-1 gene therapy may offer a favorable risk-to-benefit ratio for the first clinical trials with these vectors.

But the use of a lentiviral vector in patients infected with wild-type HIV-1 presents a new problem: the potential for HIV-1 to infect a cell containing the lentiviral vector and to ‘mobilize’ it by packaging the vector genomic transcript and transferring it to another cell. For patients infected with HIV-1, it may actu-ally be beneficial to transfer an anti-HIV-1 gene into as many cells as possible by in vivo spread. However, transfer of the recombinant vector genome among individuals would pose complex biosafety and ethical problems and must be specifically avoided.

June and coworkers performed their clini-cal trial in a productive collaboration between industry and academia. VIRxSYS Corp. (Gaithersburg, MD, USA) developed the len-tiviral vector, which expresses antisense RNA against the HIV-1 envelope transcript, as a con-ditionally replicative vector that is activated by accessory proteins provided only when wild-type HIV-1 coinfects a cell bearing the vector

Donald B. Kohn is at the University of Southern California Keck School of Medicine, Division Research Immunology/Bone Marrow Transplantation, The Saban Research Institute of Childrens Hospital Los Angeles, 4650 Sunset Blvd., Mail Stop #62, Los Angeles, California, 90027-6016, USA.e-mail: dkohn@chla.usc.edu

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66 VOLUME 25 NUMBER 1 JANUARY 2007 NATURE BIOTECHNOLOGY

(Fig. 1a). They also established methods to produce the lentiviral vector at clinical grade and scale, with appropriate qualified assays to certify the preparations.

June and his colleagues at the University of Pennsylvania have been leaders in establish-ing optimal procedures to genetically modify human T lymphocytes under current Good Manufacturing Practice conditions9. In this study, they treated five patients with stable but fairly advanced HIV-1 infection. The patients’ peripheral blood T lymphocytes were isolated by leukopheresis, cultured and transduced with the lentiviral vector, and ~1010 cells were later reinfused (Fig. 1b).

The lentiviral vector expressed an antisense molecule to HIV-1 envelope sequences. As the grand-daddy of anti-HIV genes, antisense is seemingly ‘old school’, yet may be suffi-ciently suppressive of HIV-1 gene expression to decrease HIV-1 growth and pathogenicity in cells that become infected. The relatively long length of the antisense RNA produced by the vector (937 bases) may lessen sensi-tivity to small nucleotide differences in the HIV-1 transcript target, differences that may be problematic for shRNA approaches using short-sequence targets.

Of note, the VIRxSYS vector is capable of being mobilized by incoming HIV-1, as it does not have the enhancer-deleted SIN long termi-nal repeats that are used in most lentiviral vec-tors and thus produces full-length transcripts from the long terminal repeats competent for packaging into the HIV-1 virion10. This prop-erty of the vector to be mobilized could lead to protection of other susceptible target cells

by in vivo spread, but could be problematic if it were prolonged.

The primary endpoints of this phase 1 study were chosen to assess the safety of the approach. Follow-up over two years has not detected any adverse clinical effects. Some mobilization of vector sequences was observed by identifying viral particles in the circulation that contained the vector transcript, presum-ably packaged by pseudotyping by wild-type HIV-1 coinfecting vector-transduced cells. This mobilization was a transient event, seen only over the first one to two months.

Analyses of vector integration sites in blood cells showed the preference for gene-rich regions typical of lentiviral vectors, with no evidence of clonal proliferation. There was relatively effective gene delivery to the T cells, with ongoing detection of gene-modified T cells for more than one year in two of the sub-jects. No data were provided on gene expres-sion by the vector in vivo. Improvements in CD4 cell levels and HIV-1 viral loads in one of the subjects must be written off as anec-dotal and potentially attributable to previ-ous changes in the antiretroviral medication regimen. It is too early to conclude whether lentiviral vectors yield improved gene trans-fer to T cells compared to gamma-retroviral vectors, although the long-term persistence of transduced cells seen in this study appears promising. The greater advantage of lentiviral vectors may be for the transduction of hema-topoietic stem cells.

Several prior studies of gene therapy for HIV-1 have shown glimmers of efficacy, with modest evidence for protection of T cells from

HIV-1–induced cytopathicity, but these have been isolated and anecdotal in Phase 1 safety studies that were not statistically powered to detect efficacy. Short-term effects may be subtle in patients on effective antiretroviral therapy or with advanced disease, with ben-efits realized only over long time periods if gene therapy leads to some protection of T-cell survival and function.

Gene modification of human lymphocytes and hematopoietic stem cells holds the prom-ise of providing improved therapies not only for infection by HIV-1 but also for blood-cell disorders, such as hemoglobinopathies, stem cell defects, and storage and metabolic diseases, as well as for cancer. Lentiviral vec-tors may be a key element in advancing gene therapy to expand the range of conditions that may respond, beyond the primary immune deficiencies that have constituted the sole suc-cesses to date using gamma-retroviral vectors. The findings of June and coworkers provide initial positive data on the clinical suitability of lentiviral vectors.

1. Podsakoff, G.M., Engel, B. & Kohn, D.B. Biol. Blood Marrow Transplant. 11, 972–976 (2005).

2. Levine, B.L. et al. Proc. Natl. Acad. Sci. USA 103, 17372–17377 (2006).

3. Naldini, L. et al. Science 272, 263–267 (1996).4. Zufferey, R. et al. Nat. Biotechnol. 15, 871–875

(1997).5. May, C. et al. Nature 406, 82–86 (2000).6. Hacein-Bey-Abina, S. et al. Science 302, 415–419

(2003).7. Bushman, F. et al. Nat. Rev. Microbiol. 3, 848–858

(2005).8. Montini, E. et al. Nat. Biotechnol. 24, 687–696

(2006).9. Humeau, L.M. et al. Mol. Ther. 9, 902–913 (2004).10. Dropulic, B., Hermankova, M. & Pitha, P.M. Proc. Natl.

Acad. Sci. USA 93, 11103–11108 (1996).

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Figure 1 Lentiviral vectors make their first prime-time appearance in the clinic. (a) Maps of the wild-type HIV-1 genome and the VRX495 lentiviral vector are shown. The transcript from the VRX495 lentiviral vector contains sequences complementary (antisense) to a portion of the env gene transcript from the wild-type HIV-1. Potentially, base pairing between the antisense RNA from the vector and the HIV-1 transcript may inhibit expression of the HIV-1 genes, impairing viral replication, as shown in preclinical in vitro studies10. (b) The experimental schema for the clinical trial is depicted. HIV-1+ subjects enrolled in the study underwent leukopheresis. CD4+ T lymphocytes were enriched, stimulated with antibodies to CD3 and CD28 bound to beads, and transduced in vitro by addition of the VRX495 lentiviral vector. The transduced cells were expanded and then cryopreserved, while samples were tested for safety parameters, to meet the certificate of analysis (C.O.A.). Cells were then thawed and given as an intravenous infusion back to the donors. Subjects were followed over one year, during which trial endpoints for safety and efficacy were measured.

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