Gene therapy involves the transfer of genetic material, encoding one or more therapeutic genes and the sequences necessary for their expression, to target cells to alter their genetic makeup for some desired therapeutic effect. Gene therapy was first used to treat adenosine deaminase (ADA) deficiency, a single-gene genetic disorder, but is now being tested in a wide variety of applications, including complex genetic disorders such as cancer, infectious diseases such as human immunodeficiency virus (HIV) infection, and in tissue engineering.
Genetic material has been successfully delivered to a large number of different human cell types, and their phenotypes have been altered. For example, complementary DNA (cDNA) encoding the gene for ADA has been transferred to blood cells to treat ADA deficient children, cDNA encoding cytokines have been delivered to tumor cells in an attempt to elicit an anti-tumor immune response, and cDNA encoding the receptor for low density lipoprotein (LDL) has been delivered to the hepatocytes of patients suffering from familial hypercholesterolemia.
Most often, the genetic material is transferred ex vivo to tissue that has been removed from the patient . After gene transfer, the tissue is cultured and expanded in vitro, then reimplanted into the patient. If the target tissue cannot be removed or cultured in vitro (e.g., brain, heart, and lungs), the genetic material is injected directly into the patient, in vivo.
Despite several exciting early milestones in gene delivery, to date there are no examples of gene therapy ‘cures’ . One major reason for the lack of a major success is the current inability to efficiently deliver genetic material to target cells. Several gene transfer vector systems have been developed to deliver genetic material more efficiently, but this first generation of systems is somewhat crude and must be significantly improved before the potential of gene therapy can be unlocked. Fundamental engineering principles must be applied to gain a better understanding of the rate-limiting steps of the gene transfer process before the next generation of gene transfer technologies and methodologies can be rationally designed.
We will focus on recombinant retroviruses, although most of the principles discussed are applicable to all gene transfer systems. Recombinant retroviruses are the most common gene transfer vector used in human gene therapy clinical trials, primarily because they can enter most cell types, and they permanently integrate the genetic material into the genome of the target cell . Permanent genetic modification of the target cell is a distinct advantage when a long lasting treatment is desired, as in the treatment of hereditary or chronic disorders.
Unfortunately, recombinant retroviruses, like other gene transfer systems, have several limitations. The major drawback is that transduction efficiencies, defined as the number of gene copies delivered per target cell, are low. Recombinant retroviruses are also unable to: (1) infect nondividing cells, (2) transfer more than 8 kilobases of genetic material, and (3) easily target specific cell types and tissues. A discussion of these latter fundamental biological issues is, however, beyond the scope of this article.
Transduction efficiencies must be increased by improving recombinant retroviruses and methods to deliver them. Higher transduction efficiencies in ex vivo protocols would reduce the number of cells cultured outside the body and increase the expression of the therapeutic gene in each cell, increasing the likelihood of eliciting the desired biological effect. Higher transduction efficiencies in in vivo protocols would minimize the volume of retrovirus stock that must be injected into the body and maximize expression of the therapeutic gene in each cell.
In this article, the major factors that limit the transduction efficiency of retroviral-mediated gene transfer, as well as some of the strategies being used to overcome these limitations, will be discussed. Because the field of gene therapy has only recently been developed, the technology base for its application is also in its infancy. Thus, in anticipation of more comprehensive descriptions of retroviral-mediated gene transfer, which will undoubtedly come in the future as a result of more mature and detailed investigations, we offer a discussion of several exciting avenues of research where biomedical engineers have begun to contribute to the overall process.