HIV Meets Its Maker

The killer will be turned against itself, if a human trial of gene therapy for AIDS goes forward in a few months, using a vector derived from HIV-1. Biotech startup VIRxSYS Corp. of Gaithersburg, Md., is complying with requests for more information concerning its clinical protocol that were made in October by the National Institutes of Health Recombinant DNA Advisory Committee and the Food and Drug Administration. The candidate drug's inventor, Boro Dropulic, says VIRxSYS has received about $18 million in investment funds and expects to begin Phase 1 trials in the second quarter of this year. The treatment capitalizes on improvements made over recent years in the use of vectors derived from lentiviruses, a retroviral subfamily that includes HIV. Only lentiviral and oncoretroviral vectors can permanently integrate into the genomes of blood stem cells, which are mostly nondividing in their primitive state and are central to treating diseases of the blood and immune system. But when oncoretroviral vectors are used, the transgene fails to express sufficiently, for unknown reasons. That leaves the field to lentiviral vectors, and the leading such vehicle is based on HIV-1. Inder Verma, whose lab at the Salk Institute for Biological Sciences first developed an HIV-based lentiviral vector for transduction of nondividing cells,1 was at the October FDA meeting and says the questions asked of VIRxSYS seemed "completely answerable." He thinks the trial will go forward and believes it's "not a bad idea." Noting that vectors based on equine and feline lentiviruses are also being studied, he says, "I think they're equally useful, but it's not clear that they have any special advantages. We chose HIV because we know all about it." Dropulic developed his method from 1994 to 1999 in the Johns Hopkins University School of Medicine's molecular biology and genetics department. The university patented the invention, which uses conditionally replicating HIV-1 (crHIV) vectors that interfere with replication and spread of wild-type HIV-1. It works "like a molecular scissors," he says. "It cuts and destroys wild-type virus." The vector cannot code for HIV proteins, and contains ribozymes that cleave wild-type HIV RNA but not crHIV RNA.2 In the ex vivo process, about 90% of T cells purified from the patient's blood and cultured with the vector containing the transgene. After freezing, the cells are administered intravenously. Dropulic acknowledges the concerns that even a disabled HIV-1 vector can engender for human use. But he notes, "These will be patients who have no options left. They've already failed two drugs, their CD4 counts are low and their viral loads are high." Researcher Would Volunteer Oncology assistant professor Linzhao Cheng of the Johns Hopkins Kimmel Cancer Center also works with a vector based on HIV-1. He cites safety and efficiency improvements such as minimizing HIV sequences, eliminating viral accessory proteins, and using a self-inactivating modification developed in Verma's lab that permanently turns off the viral promoter gene after the vector integrates into the host genome. "Personally, I believe it's safe," he says. "If they're going to start clinical trials and want a normal, healthy person, I'd be more than happy to volunteer." Last month, Cheng and colleagues demonstrated the ability to turn on a fluorescent jellyfish transgene only after the vector had integrated into human hematopoietic stem progenitor cells that had differentiated into antigen-presenting cells. The transgene was highly expressed in dendritic cells, which are critical in regulating immunity.3 Such targeted expression has long been sought in gene therapy because it increases effectiveness and reduces the potential for adverse reactions. Two recent examples of targeted treatment of blood-borne genetic diseases in mice models are reversal of skin photosensitivity from protoporphyria, and correction of sickle cell disease.4,5 New Animal Models Another stumbling block that now looks surmountable is the unavailability of higher animal models for diseases such as AIDS and neurodegenerative conditions. A paper from the lab of California Institute of Technology president David Baltimore describes two new ways to create transgenic animals using lentiviral vectors.6 Verma's lab will announce similar results in a paper currently in press. The conventional method of injecting the vector into the pronucleus or nuclear material of a fertilized ovum is practical only for special strains of mice. Because pronuclei of most mammals cannot be seen under a microscope, other transgene delivery methods were needed. "We were using lentiviral vectors for modifying cells and realized they might have this potential, so we tried it," Baltimore recalls. Vectors carrying the fluorescent jellyfish gene were injected into a space between the cytoplasmic membrane and an outer covering called the zona pellucida that protects newly fertilized eggs. In an alternative procedure, the zona pellucida was removed by bathing the eggs in a hydrochloric solution, then the denuded embryo was incubated with a lentiviral suspension. Carlos Lois, lead author of the Caltech paper, describes embryo stripping as a "trivial" procedure. "This allows you to do transgenics on the bench of the lab," he says. It should prove popular with researchers willing to accept slightly reduced effectiveness compared to injection of cells with intact zona pellucidae. The trade-off is production of numerous animals without the expense of sophisticated microinjection technology. Even more important, the study shows that the new methods work with rats, suggesting that they can be extended to all mammals. "We've already started working with monkeys," Lois reveals. "We already know that the gene expresses very well in their embryos." However, Lois warns that because offspring will carry the transgene, the new technique should not be applied to people. "That's something I think would be better not to think about too much," he says. Steve Bunk (sbunk@the-scientist.com) is a contributing editor. References 1. L. Naldini et al., "In vivo gene delivery and stable transduction of nondividing cells by a lentiviral vector," Science, 272:263-7, 1996. 2. B. Dropulic et al., "A conditionally replicating HIV-1 vector interferes with wild-type HIV-1 replication and spread," Proceedings of the National Academy of Sciences, 93:11103-8, 1996. 3. Y. Cui et al., "Targeting transgene expression to antigen-presenting cells derived from lentivirus-transduced engrafting human hematopoietic stem/progenitor cells," Blood, 99:399-408, Jan. 15, 2002. 4. E. Richard et al., "Gene therapy of a mouse model of protoporphyria with a self-inactivating erythroid-specific lentiviral vector without preselection," Molecular Therapy, 4:331-8, October 2001. 5. R. Pawliuk et al., "Correction of sickle cell disease in transgenic mouse models by gene therapy," Science, 294:2368-71, Dec. 14, 2001. 6. C. Lois et al., "Germline transmission and tissue-specific expression of transgenes delivered by lentiviral vectors," Science, published online Jan. 10, 2002, www.sciencemag.org/sciencexpress/recent.shtml.

HIV Meets Its Maker

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