PI Perspective #14

June 1994     View PDF

Gene Therapy

The field of gene therapy has progressed rapidly over the past three years. The first human gene therapy protocol was started in May 1989 and now several dozen protocols are in human studies for a variety of diseases, including HIV. Gene therapy, sometimes referred to recombinant DNA technology, is a broad term referring to the use of genetic materials. As this technology moves into clinical trials, it is important to understand the goals of each approach and how they differ.

Gene therapy may be used to alter immune system cells to make them resistant to HIV infection and perhaps be useful for immune reconstitution. Other gene therapy approaches are antiviral approaches, which disable the virus during its life cycle in order to inhibit viral replication. Gene therapy can be used as a vehicle to deliver drugs, such as interleukin-2, a chemical which has shown encouraging results in studies being conducted through the National Institutes of Health. Gene therapy approaches may also prove useful in preventing or treating opportunistic infections associated with HIV. While gene therapy research is still in its infancy, this technology is quickly moving toward the clinic, with eight human studies in HIV currently being developed, four of which are already underway.

Immunogenetics
Immunogenetics is an effort to insert or modify genes in an effort to stimulate natural immune defenses. Viagene is testing an approach which delivers a new gene to cells which causes them to produce proteins similar to HIV itself, making them act like a vaccine in hopes of strengthening immune response against HIV. Preliminary results demonstrate the therapy to be safe, and further studies are enrolling volunteers in both Northern and Southern California. Perhaps the most important aspect of the Viagene study is that it is testing a method which directly delivers the new gene into body cells and tissue. Most other gene therapy experiments use indirect, or vector-based approaches to delivery, which is more complex.

Immune Reconstitution
A very small pilot study is about to begin at the Howard Hughes Medical Center, University of Michigan, wherein cells are manipulated outside the body with REVM10, a gene which may render them resistant to HIV infection. If successful, this approach opens new doors to immune reconstitution. The initial study will look at cells drawn from the peripheral blood, to examine the effect of genetic manipulation on cell function and the ability of the cells to express the gene. While the preliminary study will probably not result in clinical benefit, it is groundbreaking work and will lead to valuable insights into direction for this approach to therapy for HIV disease. Eventually this research will lead to inserting this gene into a cell which is typically found in the bone marrow, called a stem cell. Stem cells are the ‘mother of all cells’ and can literally mature and differentiate into all of the cells of the immune system. By inserting a gene which renders a cell resistant to HIV infection into a stem cell, it may be possible to repopulate the immune system with an entire repertoire of cells which cannot be infected by the virus. Stem cells are an attractive target for gene therapy as they may provide the key to true immune reconstitution. Systemix, of Palo Alto, California, is developing stem cell technology as well as conducting research into gene constructs which may be useful in HIV disease.

Drug Delivery
A unique approach to gene therapy, which combines cell therapy with immunogenetics, is being developed at the University of Washington, in Seattle. Phil Greenberg is developing a method to genetically alter cells to produce interleukin-2 (IL-2). IL-2, also known as T-Cell Growth Factor, is an important naturally occurring chemical, which is necessary for T-cell differentiation and development. Preliminary studies of IL-2 in HIV disease are encouraging and suggest that as we better learn how to use the drug, it may become an important part of the armature to fight HIV. By combining cell therapy with genetic manipulation, Dr. Greenberg hopes to boost HIV immune response as well as repair some of the immune dysfunction.

Dr. Greenberg is looking at manipulating CD8+ cells, which are believed to be very important in controlling HIV. Preliminary studies involve inserting a ganciclovir-sensitive gene, often referred to as a ‘suicide gene’. If CD8+ cells, expanded or manipulated outside the body, produced a potent antiviral response when reinfused, the amount of inflammation created by this response could prove dangerous, possibly causing pneumonia or serious swelling of the brain. By inserting a ganciclovir-sensitive gene into these cells, it is possible to administer ganciclovir to destroy the cells if they create too potent of an immune response. A study of the safety and effectiveness of ganciclovir-sensitive gene insertion has enrolled its first patient in Seattle. Because CD8+ cells rely on the presence of IL-2, which is deficient in HIV disease, the possibility of delivering a gene which will make these cells produce IL-2 is attractive. Dr. Greenberg has been working on developing IL-2 receptors which could help cells produce this chemical despite whatever immune dysfunction, caused by HIV, is creating an IL-2 deficiency.

Antiviral Approaches
Technologies which are related to gene therapy include antisense and ribozyme technologies. Both antisense and ribozymes target specific viral RNA sequences. However, antisense is being developed as a drug whereas ribozymes require a gene transfer approach. They also ‘attack’ the viral RNA sequences differently. Antisense drugs bind to the viral RNA whereas ribozymes chop up the viral RNA.

Antisense technology involves arranging short strands of genetic material that are targeted to bind to specific viral RNA sequences. When HIV incorporates into the machinery of a cell, it does so by binding its RNA with the immune cell’s DNA, in a fashion that can be likened to a zipper. When the teeth of the zipper come together and close, the viral RNA being one set of teeth, and the cell’s DNA being another, the virus is initiating the process of ‘transforming’ the cell into a factory for new virus. Antisense acts like a piece of gum in that zipper. By binding to one side of the ‘teeth’, when the zipper comes to close, the antisense has gummed up the binding area and the virus cannot attach, thereby disabling its activity. The first antisense for HIV, being developed by Hybridon, went into humans in Europe last year. Human studies of the Hybridon antisense, called GEM 91, began in spring at University of Alabama, Birmingham. This strategy hold potential for controlling many viral diseases, including those associated with HIV disease. ISIS Pharmaceuticals recently announced that it is about to begin a Phase I/II study of an antisense for CMV. Unfortunately this entire field of research has been slowed down due to restrictions placed on its development by NIH patenting and licensing agreements.

Ribozymes, which are sometimes referred to as molecular scissors, cut viral RNA strands at selected sites. Ribozymes seek out viral particles and chop them up into bits, hopefully rendering them non-infectious and incapable of replicating. One of the problems with ribozymes is that they lose their specificity in chopping up the RNA. In test tube studies, ribozymes can get sloppy and chop up unspecified RNA, which could result in toxicities and may be dangerous. There are different kinds of ribozymes, the two most common forms being hammerhead and hairpin ribozymes. The names simply refer to the similarity of the structure of these ribozymes to a hammerhead shark and a hairpin. The first human trial of a ribozyme was approved last year, and will be a trial of a hairpin ribozyme, which was developed by Flossie Wong-Staal at the University of California, San Diego. Problems with manufacturing have delayed this study, which is now expected to begin sometime this year in Southern California.

Conclusion
Recombinant DNA technology, or gene therapy, holds great potential as therapy for HIV disease. The field of recombinant DNA technology is still in its infancy, however, and there are many unanswered questions about its usefulness. Finding a gene construct which efficiently inhibits HIV replication or protects a cell from HIV infection is only half the battle. Getting that gene into human cells, either by injecting the genetic material directly or by packaging the gene in a virus and delivering it to the cells by injection, or infecting cells outside the body, is another challenge to optimizing gene therapy approaches. A number of non-viral delivery mechanisms are being explored in an attempt to decrease the risk of causing harm in delivering the genes to the appropriate cells in the body. How genetically manipulating cells will affect cell function is a concern which only further human experimentation will clarify. There are many unknowns and uncertainties. Despite these uncertainties, success with gene therapy for other diseases provides reason for cautious optimism.

Early Intervention

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