GENE THERAPY: POTENTIAL, PITFALLS AND PROSPECTS

Inder M. Verma, Ph.D., The Salk Institute, P.O. Box 85800, San Diego, CA 92186

Gene therapy is a form of molecular medicine which will have a

major impact on human health in the coming century. At present, gene

therapy is being contemplated for both genetic and acquired diseases. In

the former case, a wide variety of somatic tissues are being explored for

the introduction of foreign genes with a view towards gene therapy. A

prime requirement for successful gene therapy is the sustained expression

of the therapeutic gene without any adverse effect on the recipient. We,

and others in the past, have used fibroblasts and myoblasts for

introducing genes whose products are secreted into the circulation. In

most cases, however, the expression of the transgene, introduced via

infection with a recombinant retrovirus either under the control of a

viral long terminal repeat (LTR) or heterologous internal promoter, was

"shut-off". Recently we have used primary myoblasts for gene therapy,

partly because of their ready accessibility, ease of in vitro and in vivo

manipulations and that skeletal muscles constitute nearly 40% of the body

weight of an average individual. We have observed that : (i) primary

myoblasts can be efficiently infected with recombinant retroviral vectors;

(ii) the expression of foreign genes is not compromised following fusion

to form myotubes; (iii) recombinant vectors containing muscle specific

(mck) enhancer element linked to CMV manifest the highest expression

following transplantation of myoblasts in vivo; (iv) expression of factor

IX protein can be detected for over two years without any apparent adverse

effect on the recipient mice. However, when similar experiments were

performed in hemophilic dogs using canine factor IX and autologous

myoblasts, little or no expression was observed, presumably because the

dog myoblasts fuse very inefficiently with dog myotubes. This limitation

imposed by the inability of retroviruses to infect dividing cells will

remain a major handicap for efficient gene delivery in most somatic

tissue. Therefore, we switched to the use of adenovirus based vectors to

deliver factor IX gene in vivo. Therapeutic levels (5 µg/ml) of factor IX

are secreted in the plasma of recipient nude mice. However, when the same

recombinant adenovirus was introduced into adult normal mouse muscle, high

levels of expression were observed for only a week. The loss of

expression was due to cytotoxic T lymphocytes (CTL's) to adenoviral and

transgene products. Furthermore, there was extensive humoral response to

viral structural proteins to preclude repeated injection of the

recombinant adenoviral vector. We have also infected the muscles of

hemophilic dogs by direct injection of adenoviruses containing factor IX

cDNA. Transient correction of the clotting defect can be observed. The

advantages and limitations of adenovirus-based delivery system with regard

to gene therapy will be discussed.

A highly desirable vector will be the one which can be generated at high

titers, integrate in non-dividing cells and have little or no associated

immune problems. Therefore, we have generated lentivirus-based vectors

which can integrate in non-dividing cells in vitro and infect tissues in

vivo. We will describe the construction of novel vectors using VSVG

protein instead of viral glycoprotein and show their utility in vivo. We

will discuss the use of lentiviral vectors to infect rat brain in vivo.

Finally, We will discuss the generation of targeted vectors with

particular reference to the use of single chain variable fragment (SCVF)

linked to viral envelope gene. We will elaborate on the use of

retroviruses containing human LDL-receptor SCVF to infect human cells. At

the end of the talk I will summarize the pros and cons of currently used

methods of gene therapy, requirements for future improvements, and the

overall prospects for gene therapy.