Bulletin of Experimental Treatments for AIDS, No. 28; March, 1996
Henry E. Chang and Mark Bowers

During the last few years, considerable progress has been made in gene therapy technology for HIV infection and AIDS. Reports of extremely rapid progress in this high-tech arena have distorted public opinion by catapulting expectations, while at the same time some researchers have called for a renewed emphasis on basic research. This article will review some of the key issues in gene therapy as it relates to HIV disease, and will report early results from some of the more than 100 studies currently approved by the Recombinant DNA Advisory Committee (RAC) at the National Institutes of Health (NIH).

What is Gene Therapy?

Gene therapy is the descriptive term for a method which seeks to correct a disease by genetic manipulation. Genes, the basic units of heredity, consist of a long stretch of deoxyribonucleic acid (DNA), which forms a part of the chromosome, a body in the nucleus that is the bearer of genetic information. Human DNA contains all of the information that the body needs to form new cells and tissues, to communicate with other cells, and to regulate the continued existence and biological evolution of humankind. Human beings have between 50,000-100,000 genes (the collection is called a genome). The Human Genome Project is working now to characterize all human genes; several thousand are known so far.

Each gene contains the information required to make a protein. Human cells contain an abundance of different proteins, each with a different job to do. If even a single gene is damaged in some way, the production of the protein that is made by that gene will be changed. The changed or missing protein could then change the way the cell functions, ultimately leading to genetic disease. Many diseases are possible when proteins are not produced in the right amounts, are produced at the wrong time or are not produced at all.

One goal of gene therapy is to replace a gene that has become defective. Another goal is to put new working genes into the human body that do specific useful things, such as increase the immune system's ability to find and neutralize HIV. In order to accomplish either of these goals, the gene must first be delivered to the right cell by a process called gene transfer. There are many technical difficulties in accomplishing gene transfer, and much basic research focuses on safe methods to put genes into human cells. Once the gene has been successfully inserted in the right place in the target cell, the gene must produce enough protein to bring about some therapeutic effect. Again, protein production presents many technical challenges. Finally, the whole procedure must result in clinical benefit to patients.

Gene Transfer Methods

Once an appropriate gene has been identified, it needs to be put into the right cell. Viruses have a natural ability to insert their own genes into the chromosomes of cells. The virus takes over the cellular machinery, inducing the cell to make the proteins the virus needs to survive and reproduce. Gene therapy researchers have genetically engineered several different kinds of modified viruses to carry desired genes into target cells. This involves removing some of the virus's genes and replacing them with genes that make proteins that may provide therapeutic benefit.

So far, researchers have identified 3 broad problems with using viruses as transferring vehicles, or "vectors." First, the virus that has had modifications in its DNA must effectively and reliably put the target gene into a host cell. Once that happens, the cell is permanently modified, as are all cells produced by the subsequent division of that cell. The second problem is that host cells need to be infected outside the body with the virus that transfers the desired genes. Once the DNA is incorporated into the host cell's genetic material, the cells are encouraged to divide many times (a process referred to as "cell expansion"). Finally, cells are tested for contaminants and viruses that could reproduce and cause disease. Only after all the steps have succeeded are the modified cells put back into the body.

The Recombinant DNA Advisory Committee (RAC) of the Food and Drug Administration (FDA) has approved studies of viral gene transfer approaches. Practical difficulties and high costs have spurred researchers to look for alternative ways to move desirable genes into target cells. One strategy is to deliver the genes of interest directly into a living person, using either modified viruses or other transfer technologies. Mouse retroviruses are considered safe for moving genes into the cells of living humans. The use of mouse retroviruses also results in stable long-term expression of proteins that have a therapeutic effect. Direct gene transfer of pure DNA without the intermediary use of a virus has also been attempted. The arguments for this kind of transfer are that it is safer, more convenient and cheaper than using viruses.

Antiviral Gene Therapies

Antiviral gene therapy is a strategy that is being developed to interfere with the reproduction of HIV. Some gene therapy techniques are designed to enhance or modify the human immune response to HIV infection. The information that will come from early studies of immune enhancement will yield important information about how to restore lost immune functions that result from advanced HIV disease. Recent experience with gene therapy approaches to HIV disease has also taught researchers about immune responses to gene therapy itself, and has given some clues about how to best use these immune responses.

CD8 HIV-Specific Cytotoxic T-Cells

Stanley Riddell, MD, and colleagues at the Fred Hutchinson Cancer Research Center in Seattle reported the results of a small gene therapy study in the February issue of Nature Medicine. These researchers infused 6 HIV positive individuals 4 times with genetically altered cytotoxic T-cells (CTL), immune cells with CD8 markers that can destroy the virally infected cells that they find. The new cells were specifically targeted at gag, a gene of HIV, and also contained a "suicide gene" that produces an enzyme for which a powerful drug already exists. If the new cells were to prove to be a problem for any reason, the drug could be administered to effectively destroy them. Each time the volunteers received an infusion of altered cells, their bodies mounted a defensive immune response that eliminated the altered cells. In 5 of the 6 participants, the cells either disappeared rapidly or were found in much decreased numbers after the third and fourth infusions.

Was the study a failure or a qualified success? Certainly, new and important questions arise because of these study results. There must be a way around the generation of a strong anti-CTL response in almost all of the HIV positive volunteers. Researchers are now considering using drugs or antibodies to block the immune response that destroyed most of the infused cells. Also under consideration is the use of certain cytokines (chemical messengers) such as interleukin 10 (IL-10) to dampen the vigorous immune response to the transplanted cells. Researchers have also begun to wonder if adding CTL to existing antiretroviral therapies (such as nucleoside analogs and protease inhibitors) will be enough to halt disease progression.

Ribozymes

Flossie Wong-Stahl, MD, and her colleagues at the University of California in San Diego have developed an RNA molecule called a ribozyme that can bring about certain chemical reactions; it can cut HIV's genetic material at a certain point and prevent the production of new HIV in treated cells. This is an interesting avenue of research, because the creation of ribozymes, or "molecular scissors," is not limited to a single strain of HIV, and because ribozymes can successfully be put into human T-cells from HIV negative as well as HIV positive individuals. A Phase I clinical study was developed and approved by the RAC in 1993. The study is designed to find out if a double ribozyme can safely and effectively be introduced into human volunteers. The study has not yet opened. Nonetheless, researchers are optimistic that they will be able to justify the clinical use of ribozymes in treating HIV disease.

Transdominant Negative Mutants

As in the previous 2 examples of gene therapy, transdominant negative mutants are introduced to target cells by viral vectors, or delivery systems. This particular approach takes advantage of a phenomenon that has been frequently observed in nature: mutant proteins can interfere with a large number of wild-type proteins. Wild-type means the kind of protein that HIV normally produces. Transdominant negative mutants include altered forms of the HIV genes gag, rev, tat and env.

Gary Nabel, MD, and his colleagues at the University of Michigan at Ann Arbor have chosen to develop one transdominant negative mutant called Rev M10. This mutant has been able to suppress HIV replication in chronically infected cells. Rev binds to an area of genetic material called the Rev Response Element (RRE). Mutant rev binds to the same place and creates a backlog of HIV RNA in the nucleus of the cell. The RNA cannot leave the nucleus, and thus no new HIV proteins can be made. Nabel's group has begun a Phase I study of Rev M10. Two different delivery systems are being used: viral and microparticle. The study is expected to provide data about the safety of the 2 procedures and some hints as to the effectiveness of the overall strategy.

Conclusion

Gene therapy, an exciting and promising field of research, has not yet produced any striking therapeutic benefits. Both researchers and the public have been enthusiastic about the possibilities of correcting or compensating for disease at the genetic level, but some of the more fundamental problems -- delivery and continued existence of altered cells -- remain incompletely solved. Gene therapy is an infant science, and like an infant, the first steps are halting. Gene therapy will one day be used with currently approved drugs -- nucleoside analogs and non-nucleoside reverse transcriptase inhibitors, protease inhibitors, and others not yet imagined -- to control HIV disease and restore immunity, but the process will take time. In 3-5 years, the tools should be available, and several years after that, widespread use of gene therapy may be a reality. For now, gene therapy research is progressing no faster than vaccine research. Answering key questions must precede widespread clinical adoption of any specific gene therapy strategy.

References

Afton E. Gene therapy. New England Journal of Medicine 334(5):332. February 1, 1996.

Fox J. One panel tells NIH not so fast on gene therapy; another says streamline RAC. ASM News 62(2):66-67. February 1996.

Friedman T. Human gene therapy:an immature genie, but certainly out of the bottle. Nature Medicine 2(2):144-147. February 1996.

Koenig S. A lesson from the HIV patient: the immune response is still the bane (or promise) of gene therapy. Nature Medicine 2(2):165-167. February 1996.

Leiden J. Gene therapy: promise, pitfalls, and prognosis. New England Journal of Medicine 333(13):871-873. September 28, 1995.

Riddell S and others. T-cell mediated rejection of gene-modified HIV-specific cytotoxic T lymphocytes in HIV-infected patients. Nature Medicine 2(2):216-223. February 1996.

Touchette N. Gene therapy: not ready for prime time. Nature Medicine 2(1):7-8. January 1996.

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