ACS Publications Division - Journals/Magazines
to Modern Drug Discovery home
FOCUS: Careers/Salary/ Employment
Feature Article
October 2000, Volume 3, No.8, pp, 55–60.

Genetic Transportation

By Mark S. Lesney

Finding the right viral vehicle for gene therapy presents conflicting choices.opening art - signposts

The most expensive gift is a wasted gesture if it gets lost or broken in transit. It is especially problematic if the recipient slashes his or her fingers on cut glass when opening the package. So too in delivering the intended gift of genetic health. Although all the glamour of genetic research rests in finding prospective genetic solutions to this or that disease, the mundane issue of safe and effective transport and delivery of genetic “cures” can make or break a therapeutic protocol. Creating an effective genetic fix for individual diseases depends on finding a reliable workhorse to carry it—the appropriate vector.

The realization is growing that there is (and in some sense can be) no prospect for a universal carrier for all gene products, all tissues, and all diseases. Neither will there ever be a vector that can be guaranteed to be equally safe and effective under all circumstances. Although vectors can be designed to enhance safety and success, vector choice seems destined to remain a biological (rather than an economic) cost–benefit decision, with no universal or multipurpose vector likely to take over.

In the foreseeable future, choosing the appropriate vector system will likely remain heavily dependent on the unique requirements of the particular disease being treated. Among the possible vectors currently available, this article will deal exclusively with viral systems. At present, these are the most widely studied vectors in laboratories and clinical trials. Some of these viral systems hold the promise of being highly versatile or adaptable to different needs; others are more restricted in their potential. (Nonviral vectors are a large and complex topic and will be discussed in a future article.)

Promise and peril
The now infamous case of Jesse Gelsinger, who died in a 1999 gene therapy trial, points out how the demand for gene therapy progress can overwhelm the system. Why is there such a phenomenal demand? It’s not just about money, career advancement, and prestige, although these are compelling incentives. The research is driven by the real hope of ameliorating diseases hitherto unassailable by modern medicine. From cancer to hemophilia, from bubble-boy syndrome to heart disease, gene therapy is one of the most active and heavily touted biomedical research fronts. Ultimately, the utility of the Human Genome Project may be its ability to aid in developing successful and safe gene therapy protocols based on the profusion of genetic information that it will provide. According to Savio L. C. Woo, in his presidential address at the 3rd Annual Meeting of the American Society of Gene Therapy, “We are at the dawn of the postgenome era, and each and every 50 to 100,000 human genes can potentially be used as medicine to treat some form of disease.”

Setting up the problem
For all of the hoopla associated with gene therapy, it is important to remember how truly young a procedure it really is. The first authentic gene therapy took place in 1990 under the auspices of W. French Anderson in an attempt to cure severe combined immunodeficiency (SCID), or bubble-boy syndrome. Although some effect from the inserted gene was shown initially, it was not a cure for SCID. The treated patients continue to require repeated injections of recombinant enzyme rather than relying on the inserted gene. To the embarrassment of many in the field, there has been more hype than success until very recently (see box, “Success at last?”).

Success at last?
The following cases were deemed so remarkable as achievements that they were extolled by Savio L. C. Woo in his presidential address to the American Society of Gene Therapy in June as the promise of the future. In a Phase I gene transfer trial for Hemophilia B by Kathryn High at the Children’s Hospital of Philadelphia and Mark Kay at Stanford University, “significant reduction in whole blood clotting times was observed in patients for several months after receiving the lowest intramuscular administration of the lowest dose of a recombinant AAV vector expressing the human coagulation factor IX gene.” Similarly, in another Phase I trial, patients of Alain Fischer of the Hospital Necker-Enfants Malades in Paris who received autologous transplants of bone marrow stem cells transformed in vitro using a MMLV retrovirus-derived vector containing the complement to the normal gene for X-linked SCID (bubble-boy syndrome) have “reconstituted their immune systems for up to one year and the children are now living normally with their families.” Significantly, this success was with the first disease ever treated by gene therapy—just more than 10 years ago.
There are many reasons why gene therapy is not straightforward. Even before a vector system is chosen, a phenomenal amount of research must go into choosing the appropriate gene. Questions must be answered: Is it the right form of the gene to solve the specific disease problem? Is its product stable enough to act as a replacement for the missing or aberrant “natural” product? What is the most effective promoter to get the new gene to express properly (in time, space, and amount)?

Without an appropriate vector system, these questions are moot. To get the gene product produced in the appropriate cellular location, a vector must breach a variety of barriers—at the cell, tissue, and organ level—that are designed to keep out foreign genetic material. Once such a breach is made, the cellular transcription/translation mechanisms have to be co-opted to produce recombinant product efficiently and for the length of time required for therapeutic purposes. In cases in which a transient gene product can do the job or is the only option available, high efficiency of infection (to effect the greatest number of cells during any one dose) and the ability to repeat dosages are critical for success in ameliorating disease. In cases in which long-term solutions are necessary to achieve “repair” of the genetic problem, the manifold difficulties of integrating foreign genes into host chromosomes in a safe and efficient manner must be considered. Some of these challenges involve the structure of the gene itself, but many are determined by the means of vectoring.

Of equal if not more importance for patient safety than concerns about initial effectiveness, the vector must not trigger disease or a cumulative immune response that could either inactivate continuing therapy or create the risk of anaphylactic shock. These worries have been especially linked to the use of viral vectors, specifically adenovirus vectors, since the Gelsinger case.

Choosing a virus vector
There are at least two distinct kinds of gene therapy. Which to use depends on a complex calculation involving the disease and the particular health needs of the patient. In “traditional” gene therapy, a functional gene copy is added to replace the null or aberrant activity of the defective gene. Pharmacological gene therapy transmits a gene for the production of a particular novel therapeutic product, a vast excess of one of the body’s own functional gene products, or a toxic product to kill cells (such as cancer cells). It essentially turns the patient’s own cells into pharmaceutical manufacturing plants.

The characteristics to be considered in choosing a viral vector are dictated by which of these types of therapy are intended. The available viral vectors differ widely. Chief among these differences are cellular infectivity (which cell types or stages can be targeted) and cellular integration (if and where a gene becomes integrated into the host genome). For vectors that do not integrate, the issue of cytoplasmic stability (how long a nonintegrated gene actively makes product) is critical. Viral vectors also differ in the size of the gene or genes that can be packaged for transmission.

Additional considerations in choosing a vector are its compatibility with the chosen application route. Typically, delivery of the therapy can occur via an aerosol to the lungs (as in cystic fibrosis), by injection or perfusion into particular tissues, via transplantation of cells transformed in vitro (as with bone marrow transformation), or through systemic injection or iv drip. Making the proper choice concerning the mode of physical delivery can be as important as choosing the right biological vector.

Producing the vector
caution sign (with a benzene)All viral vectors used for gene therapy are perforce recombinant. Not only must they have the gene of interest added to them, but deleterious genes (involved in the production of disease by the native virus particles) must be removed. Such viruses are referred to as replication-defective. But removing the viral genes creates a problem—how to generate large numbers of recombinant viral particles for use in gene therapy when the virus is no longer capable of replicating on its own. Virus vectors for gene therapy are bulk-produced in mammalian (usually human) cell cultures containing helpers (modified viruses or, more commonly, plasmids) that provide the necessary genes for viral replication proteins in a manner genetically separated from the actual recombinant virus. Because the helpers do not contain the nucleic acid sequences for the virus coat material to package around them, they are not made into virus particles. The recombinant virus is subsequently purified from the cultures and away from the helpers.

Developing and maintaining optimal cell culture lines is an art form in itself (as well as a major commercial concern). It requires the production of reproducibly high virus titers. At the same time, the cells must be kept free of exogenous pathogens. In addition, unwanted recombinations of the vector virus with helper plasmids also have to be eliminated by genetic design and/or careful monitoring to ensure that the vector does not gain back the ability to replicate and thus, its pathogenicity.

As a side note, the necessary inability of the gene therapy virus to replicate is the reason that such high numbers of particles must be used in clinical protocols—to infect a sufficient number of cells in the patient to achieve therapeutic levels of product. This is the main reason that despite being immunogenic, viruses such as adenovirus are tested in such high titers. Typically, this high dosage caused no real problems. Unfortunately, in at least one case, the titers were high enough to cause a deadly reaction in an unexpectedly sensitive individual. Jesse Gelsinger died from what was essentially a massive and systemic allergic reaction to the adenovirus vector, not from some unexpected regaining of pathogenicity by the replication-defective virus.

According to the Journal of Gene Medicine (Wiley) clinical Web site, 37.6% of gene therapy clinical trials use retroviral vectors, making them the largest vector category. Retroviruses are enveloped single-stranded RNA viruses that produce double-stranded DNA copies of themselves in infected cells and that can integrate into the host cell chromosomes. This provides the profound benefit of stable transformation. The most commonly used retroviral vectors are based on Moloney murine leukemia virus (MMLV), which can infect mouse and human cells. The major obstacle to using retrovirus vectors such as MMLV is that they only infect rapidly dividing cells. An MMLV-based vector was recently responsible for one of gene therapy’s few validated successes.

According to the Journal of Gene Medicine Web site, adenoviruses accounted for some 20% of the gene therapy clinical trials. The suspension of many such protocols pending investigation of Jesse Gelsinger’s death was not listed as a factor in these percentages. Adenoviruses are DNA-containing viruses typical of the common cold. Despite their immunogenic properties, they are considered valuable vectors for several reasons. They have a broad host-cell range, including the important ability to infect nondividing cells; they are very easy to produce in high titer; they are highly infectious; and finally, they do not integrate into the host genome. Lack of integration is a disadvantage for those diseases described above, in which long-term genetic repair is envisioned as the sole solution. In such cases, repeated treatments would be required. But for diseases in which gene therapy is seen as the application of a short-term genetic “drug”, especially in cases in which cell suicide is the desired outcome, the lack of integration can be a positive benefit.

Adeno-associated viruses
These viruses, known as AAVs, are small DNA viruses that cause no known diseases in humans. They have several benefits compared with adenoviruses; most prominently, they stimulate a low immune response (increasing their safety factor). In addition, these viruses can achieve stable integration of the gene of interest into the treated cells and can target nondividing cells. Unfortunately, they have lower levels of infectivity than adenoviruses, and because they are smaller, they cannot incorporate many of the typically large genes required for effective gene therapy. They are also difficult to produce in mass quantities. An AAV vector, however, was responsible for one of the gene therapy field’s few clear successes.

yield sign (with DNA strand)Recombinant vaccinia virus and recombinant herpes simplex virus are being studied for potential use as gene therapy vectors. Research is also being done on the use of modified bacteriophage or phagmid vectors. Lentiviruses (of which HIV is the most prominent member) are increasingly of interest for gene therapy. The chief benefits of lentivirus vectors are stable integration and the ability to transform nondividing cells. Obviously, using a recombinant HIV-based vector poses a significant public perception problem, but the life-threatening nature of the majority of genetic diseases for which gene therapy is being developed may lead to eventual clinical acceptance of these vectors. This would be especially true when attacking the problem of AIDS itself—a possibility given the vector’s ability to target the same T cells involved in the disease.

Various chimeric vectors with cell-specific recognition proteins embedded in their viral coats to trigger attachment and uptake are being investigated in hopes of improving cell targeting without increasing pathogenicity risk. One way that is being studied to accomplish improved therapeutic targeting is the use of antibody-conjugated viruses. The antibodies lead to specific cell recognition and attachment (some cancer cells and most tissues have unique membrane proteins). Because proper attachment usually leads to virus uptake, these techniques have the potential to improve therapeutic efficiency.

No panaceas
As far as the foreseeable future, there is likely to be no universal vector. Different clinical needs will ensure that the choice and development of vector systems will rely heavily on an increasing knowledge of the unique physiology of cell, tissue, and organ systems as much as on the molecular genetics of the particular malady at issue. Although the death of Jesse Gelsinger cast a pall on some viral-vector studies and created a move toward increased regulation, there is too much riding on gene therapy for it to just go away.

Suggested Web sites and reading

  1. Virus databases online, including The Universal Virus Database developed by the International Committee on Taxonomy of Viruses: http://life.anu.
  2. Virtual Lectures from the University of Rochester Department of Microbiology, including in-depth discussions of Herpesviruses and Lentiviruses:
  3. Institute for Human Gene Therapy (University of Pennsylvania). Information on the death of Jesse Gelsinger and extensive resources on gene therapy:
  4. American Society of Gene Therapy:
  5. Gene Therapy Clinical Trials listing:
  6. Adenoviruses: Basic Biology to Gene Therapy; Prem, S., Ed.; Landes Bioscience: New York, 1999.
  7. Gene Therapy: Therapeutic Mechanisms and Strategies; Templeton, N. S., Lasic, D. D., Eds.; Marcel Dekker: New York, 2000.
  8. Abelda, S. M.; Wiewrodt, R.; Zuckerman, J. B. Gene therapy for lung disease: Hype or hope? Ann. Intern. Med. 2000, 132 (8), 649–660.

Return to Top || Table of Contents