Gene Therapy: If at first you don't succeed…

Gene therapy represents the culmination of medical research and application to human health. Less than 60 years ago, Linus Pauling and others described at the molecular and genetic level what was to be the first of many molecular diseases.¹ In the most simple form, a mutation of just one base pair in a gene can alter the production or function of a protein that is vital to health and life. It is estimated that by the end of the year a “rough draft” of the human genome sequence will be completed, and with this, the sequence of every human gene will be available to scientists, physicians, and the general public. Subsequently, the mapping of diseases to individual genes will be only a matter of time and effort. Over the past half century, investigators have teased out the mechanisms of many diseases, both at the physiological and genetic level, and have made great progress developing pharmacological drugs to alleviate these maladies. The next step which has already begun is to use genes themselves as the drugs, replacing or altering the expression of defective or misregulated genes - to treat patients at the molecular level.

Unfortunately, while gene therapy may be to the 21^st century what antibiotics were to the last, we have a long way to go before success is at hand. As can be seen in the headlines above, gene therapy has had multiple “first” successes followed by the realization that much of the enthusiasm for each success has been perhaps premature or overstated. Further, the third headline refers to the recent death of a young man involved in one clinical trial that has dampened much of the early excitement about this approach in the public's view. However, as with all discoveries and new fields, problems do exist, but they are identified, studied, and overcome. Indeed, these are exciting times to practice medicine, but with regard to gene therapy, much of the initial unbridled enthusiasm has worn off, and now the real work has begun.

Gene therapy began in the mid-1970s when it was discovered that DNA could be manipulated in the test tube. Forward thinking investigators quickly realized that the possibility of expressing normal or foreign genes in human cells and ultimately in humans themselves could usher in a new era of medicine. Mutant genes in patients with such diseases as sickle cell disease and cystic fibrosis theoretically could be replaced and the diseases corrected by transferring the wild type, or normal, genes into individuals.² Other diseases such as cancer might be treated with genetic therapy by adding genes to increase anti-tumor immune responses or by inhibiting angiogenesis and cutting off the tumor's blood supply.³ Further, infectious diseases might be combated by altering immune responses.⁴ As more and more of the human genome was discovered, more associations between genes and diseases were identified, and the possibilities for genetic medicines became apparent. All of these treatments require moving desired genes into the appropriate cells in both animals and people. This is where the problems have been encountered over the past 10 years.^{5, 6}

There have been two major approaches taken to deliver genes to cells: viral and nonviral. In the case of viral delivery, the vectors (delivery agents) are modified viruses that have had their genomes largely replaced with therapeutic genes.⁵ This allows the desired genes to be packaged into virus particles that are very effective at entering cells within the body. By removing most of the viral genes, the chance for viral replication and aberrant infection in the host is removed (hopefully). The two families of viruses that have been used in the majority of clinical trials so far are adenoviruses and retroviruses. Adenoviruses are very good at infecting non-dividing or very slowly dividing cells which make up most of our tissues, while retroviruses can infect only dividing cells, thus limiting their applications, but result in life-long expression of the carried gene because it actually incorporates itself into the host's chromosome.⁷ Unfortunately, such effectiveness at gene delivery comes at a cost: viral vectors, especially adenovirus, can induce significant localized and systemic inflammation and a sustained immune response. While each individual will vary in their inflammatory responses to viral administration, the results can range from unnoticeable to severe. Indeed, in the recent case at the University of Pennsylvania, administration of recombinant adenovirus carrying a gene to alleviate ornithine transcarbamylase (OTC) deficiency to a 17 year old resulted in activation of his innate immune system, fever, and ultimately lead to his death.^{8, 9} Although the preceding 17 patients in the same study that had received the same viral vector showed nowhere near these levels of inflammation or response, revealing no indication predictive of the patient's death, this only exemplifies the variations possible.

Another result of an immune response to viral vectors is that it limits the number of times the virus can be administered. Once antibody and T cell responses are developed, as soon as a second dose of the viral vector is given, it and the cells it infects will be targeted for destruction, thereby greatly limiting the expression of the transferred therapeutic gene. Although these drawbacks may seem daunting, investigators are actively engaged in moderating these responses and finding ways to circumvent the problems. To this end, novel and modified viruses are being tested and show great promise in terms of significantly reduced inflammatory responses and the ability to give repeated doses for multiple gene therapies.

The second approach for gene delivery and therapy is to use nonviral systems. These include plasmid DNA that is either delivered directly (termed “Naked DNA”) or complexed with carriers, such as liposomes that encase the DNA, add stability and increase cellular delivery and entry.¹⁰ Plasmids, based on design, have the ability to express in both dividing and non-dividing cells, thus making them appropriate for all cells of the body. Further, the lack of viral proteins and genes that the body sees as foreign and worthy of reaction, means that little inflammation is induced and no immune response is mounted against the DNA, making multiple effective treatments possible. Unfortunately, the level of expression obtained with nonviral methods is usually much lower than that found with their viral counterparts. Other potential delivery vehicles are in development that are producing increased gene expression. These include delivery to the skin by Star Trek-like pneumatic guns and the use of electric fields and sound waves to drive DNA into cells.

Even though the levels of nonviral DNA delivery and gene expression are much lower than those obtained using viruses, the lack of an immune response may lead the field to favor these systems over the potentially hazardous viral methods. However, as time goes on, it will most likely be found that each system has its unique applications and both will ultimately find their way to the clinic.

So, has gene therapy succeeded? In the realm of theory and laboratory experimentation the answer is a resounding yes. Experiments have demonstrated that genes can be transferred to animals and humans and exert therapeutic effects. In animal models, diseases can be corrected transiently and even long term in some cases. In the earliest “first success” of gene therapy, Michael Blaese and colleagues were able to transfer the gene for adenosine deaminase (ADA) using a retrovirus to T cells isolated from two little girls with severe combined immunodeficiency as a result of ADA deficiency.¹¹ When transfused back into the patients, small increases in ADA levels were detected as were minor increases in T cells and in immune responses. However, the benefit was incomplete and did not fully “cure” the patients.¹¹ In a more recent “first success”, researchers in France have taken a similar approach to the earlier work to treat a different form of severe combined immunodeficiency, but rather than target T cells, they targeted stem cells isolated from three different infants' bone marrow also using a retrovirus.¹² In this case, the successfully transduced stem cells when transfused back into the infants were able to propagate and expand, outgrowing and replacing the endogenous defective cells. Indeed, success may be at hand, but as in over 390 other gene therapy clinical trials over the past 10 years, only time will tell.

In conclusion, how close are you to gene therapy in the Family Practice setting? When will you prescribe a shot of recombinant virus or a plasmid infusion to control hypertension or sickle cell disease, or cancer, instead of using the traditional approaches? When will the first success of clinical importance be? Hopefully soon, but to be realistic, definitely not before you have to take the Boards again.