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Rapid advances in cancer gene therapy are driven by an explosive development of gene transfer technology and a strong demand for effective alternatives to unsatisfactory conventional cancer therapies. Discovery of the genetic basis of cancer has indicated that cancer is a disease of genes. Among a variety of approaches to gene therapy of cancer, anti-oncogene and tumor suppressor gene therapy of cancer are the two strategies that aim at correcting genetic disorders of cancer. The potential effectiveness of these approaches is promised by their precise targeting at the mechanisms of the disease. Successful examples of human lung cancer animal models by applying anti-K-ras retrovirus and recombinant p53 adenovirus are reviewed. Future development of these approaches towards clinical application is also discussed.

Cancer, as a genetic disease, is a logical target for gene-oriented therapy--either by replacing the missing/nonfunctioning gene or by depressing the activity of an unwanted gene. The latter is really the inhibition of gene expression using oligonucleotide-based or "antisense" treatment. There are several strategies to achieve this: anti-gene or anti-code with triplex formation; ribozyme with endogenous catalytic RNase activity; antisense with oligonucleotides through steric inhibition or RNaseH activation; and sense strategy to inhibit or trap proteins by nucleic acids. There are two essential partners of the approach: targeted sequence in the unwanted gene/molecule and the complementary antisense oligo (-ribo- or -deoxyribonucleotide). The antisense sequences require chemical modifications (mostly on the phosphodiester backbone, less in the sugar or in the bases) to avoid nucleases, to form complexes for better delivery (in the organism and also in the cell). The activity of the unwanted target should be non-randomly associated with cancer (e.g. abl/ber). Both aspects of antisense treatment require further improvements to get longer lasting and real sequence-specific antitumor effect which could be competitive with the available therapeutic modalities.

Chronic infection with the hepatitis B virus is a major health problem worldwide. The only established therapy is interferon-alpha, with an efficacy of only 30-40% in highly selected patients. Nucleoside analogues do not show a significant clinical benefit. Molecular therapeutic strategies aimed at blocking gene expression include antisense DNA/RNA and ribozymes acting at the posttranscriptional level and triple helix formation blocking at the transcriptional level. In vitro, antisense oligodeoxynucleotides inhibit viral replication and gene expression in human hepatoma cell lines. In vivo, an antisense oligodeoxynucleotide directed against the 5'-region of the pre-S gene of the duck hepatitis B virus inhibited viral replication and gene expression in ducks. In vitro, ribozymes accurately cleave HBV substrate RNA. Triple helix formation is another very promising molecular approach. Results in hepadnaviral infection are not yet available, however.

Genetic manipulation of somatic cells may be of therapeutic value in a variety of infectious diseases, particularly in human immunodeficiency virus (HIV) infection. Stable insertion of custom-designed 'resistance genes' into cells susceptible to HIV could reduce the viral burden in infected individuals and potentially retard the characteristic progressive immune dysfunction. Alternatively, ectopic expression of genes that encode viral antigens might induce potent antiviral immune responses and form the basis for novel prophylactic and therapeutic vaccines. While laboratory studies have proved that the approach works in principle, preclinical and clinical studies will be necessary to evaluate the therapeutic benefit of such gene-based therapies.

In this chapter I have attempted to outline the rationale that underlies the antisense approach to treatment of virus infection, to catalog the effector molecules that are currently available, and to estimate the relative worth of each. In so doing I have tried to describe the criteria that might be employed in their design and the factors that may determine their efficacy in tissue culture and, perhaps, in vivo. Finally, I have described the few examples presently available that indicate that antisense approaches may one day be therapeutically useful in treatment of disease of viral or nonviral origin.

The retroviral life cycle and genetic plasticity of human immunodeficiency virus 1 (HIV-1) present unprecedented therapeutic challenges. Twelve years into the HIV epidemic, satisfactory treatment remains elusive. Our current understanding of AIDS pathogenesis calls for early intervention with antiviral agents. Although still in its infancy, human gene therapy holds considerable potential for the long-term treatment of genetic disorders, cancer and chronic infectious diseases. Gene therapy for HIV infection is receiving particularly intensive study: approaches that are in development include both immunotherapy (e.g. therapeutic vaccines and adoptive transfer of CD8+ T-cell clones) and direct antiviral therapy (intracellular immunization). The latter strategies include transdominant modifications of HIV proteins, RNA decoys, antisense RNA, ribozymes and modifications of cellular proteins (e.g. intracellular antibodies, soluble CD4). Several of these strategies are now entering clinical trials. While significant conceptual and technical hurdles remain to be overcome before the promise of gene therapy for HIV infection can be fully realized, progress in this field is likely to be rapid and to contribute to the broader applicability of human gene therapy to the treatment of other disorders.

Rat C6 glioma cells express insulin-like growth factor I (IGF-I) and form rapidly growing tumors in syngeneic animals. When transfected with an episome-based vector encoding antisense IGF-I complementary DNA, these cells lost tumorigenicity. Subcutaneous injection of IGF-I antisense-transfected C6 cells into rats prevented formation of both subcutaneous tumors and brain tumors induced by nontransfected C6 cells. The antisense-transfected cells also caused regression of established brain glioblastomas when injected at a point distal to the tumor. These antitumor effects result from a glioma-specific immune response involving CD8+ lymphocytes. Antisense blocking of IGF-I expression may reverse a phenotype that allows C6 glioma cells to evade the immune system.

Antisense catalytic RNAs that specifically base-pair with and cleave target RNA sequences have potential for use as therapeutic agents against viral as well as endogenous gene expression. With the ultimate goal of developing anti-human immunodeficiency virus type 1 (HIV-1) ribozymes for therapeutic use, we have been exploring ways to improve upon the functional activity of ribozymes in living cells. This is being done by the systematic exploration of parameters that affect antisense, and hence ribozyme, function. These include target accessibility, stability of the catalyst, methods for delivery, and intracellular localization of the ribozyme. In addition, we have been examining the kinetic consequences of having extra, nontargeted sequences appended to the ribozyme flanking sequences. Perhaps the single most important consideration for ribozyme effectiveness in an intracellular environment is the accessibility of the target RNA for cleavage. By exploiting the mechanisms by which naturally occurring antisense RNAs interact with their target sequences, we hope to be able to address this problem of targeting and fully capitalize upon the potential of ribozymes as therapeutic agents.

CML, a myeloproliferative clonal disorder of myeloid stem cells, is characterized by the consistent presence of a bcr-c-abl fusion gene which is formed as a result of a translocation of the c-abl gene from chromosome 9 to downstream of the bcr gene on chromosome 22 (ph'). Current approaches to the treatment of CML are chemotherapy (conventional or aggressive with immuno-modulators) and bone marrow transplantation (BMT). Neither of the above treatment modalities results in long-term remission or cure. Hence, an alternative approach which aims at correcting the genetic defect should be considered. Taking advantage of the consistent abnormal presence of the bcr-c-abl gene in the treated and untreated CML patients at all stages, a gene therapy at the level of blocking mRNA might be considered. Such an antisense RNA therapy should include removal of patient's bone marrow, administration of the gene for constitutive expression of an antisense RNA for the bcr-c-abl fusion gene into the myeloid stem cells and reinjecting the engineered marrow into the patient. Such an approach, comparable to autologous BMT, will have the advantages of absence of graft rejection and possibility of 100% remission. The possible nature of the gene construct for such an antisense RNA therapy is discussed.

Two isoforms of the regulatory subunits of cyclic AMP (cAMP)-dependent protein kinase that bind cAMP are inversely expressed during ontogeny and cell differentiation. These cAMP-binding receptor proteins in harmony may regulate the growth of normal cells and their differentiation into nondividing states. Cancer cells can also be made to differentiate and stop growing when the functional balance of these cAMP receptor proteins is restored by treatment with site-selective cAMP analogues or by the use of an antisense oligodeoxynucleotide, suggesting new approaches to cancer treatment.