Strategies for Gene Therapy

For some diseases the pathology affects the function of a particular organ which must be directly treated, e.g. cystic fibrosis in the lungs (Wheeler et al l996), inflammatory bowel disease (Jobin et al, 1998), Parkinsons disease in the corpus striatum region of the brain (During et al, 1994) & retinitis pigmentosa (Miyoshi et al, 1997). Other diseases are systemic in their effects, e.g. haemophilia & metabolic diseases such as adenosine deaminase deficiency. For these diseases the common sites for therapy are the liver (Numes & Rapier, 1996), gut (Lozier et al, 1997) & muscle (Herzog et al, 1997). These sites are chosen for their ease of access, bulk & metabolic activity. Monogenic recessive diseases only require the functional gene to be expressed, often therapeutically useful levels being much lower than that those found in normal individuals. Monogenic dominant diseases require that the aberrant gene is silenced, usually by means of an anti-sense DNA which is complementary to the aberrant gene (Gerad & Collen, 1997).

Gene Therapy for Cancer:

The aim of immunopotentiation is to enhance the response of the immune system to cancers, thereby leading to their destruction. Passive immunotherapy aims to increase the pre-existing immune response to the cancer whilst active immunotherapy initiates an immune response against an unrecognised or poorly antigenic tumour (Barnes et al l997). Passive immunotherapy usually involves harvesting tumour infiltrating lymphocytes & treating them to express increased cytokines e.g. IL-2 & TNF-alpha (Yanelli et al, 1993). The cell population is then expanded in vitro & returned to the patient. Tumour cells are used for active immunotherapy, genetically modifying them to increase expression of antigen presenting molecules/costimulatory molecules (e.g. B7; Chen et al, 1992), local concentrations of cytokines (e.g. IL-2; Lemig et al, 1996) or tumour antigens (erbB2 oncoprotein; Disis et al, 1994). The cells are then irradiated prior to being returned to the patient, preventing the reintroduction of replication competent tumour cells. These approaches have been termed cancer vaccines.

Oncogene inactivation uses the same techniques employed for dominantly inherited monogenic diseases. The oncogene may be targeted at the level of the DNA, RNA transcription or protein product. Oligodeoxynucleotides are short single stranded pyridimine rich DNA sequences that form a triple helix with purine rich double stranded DNA sequence. Oligodeoxynucleotides are designed in a sequence specific manner to target the promoter regions of oncogenes, (e.g. erb2; Ebbinghouse et al, 1993). At the RNA level antisene techniques prevent transport & translation of the oncogene mRNA by providing a complementary RNA molecule (e.g. c-myc; Collins et al, 1992). Ribozymes, antisense oligoribonucleotides with a cleavage action, will also reduced the stability of oncogene mRNA (e.g. brc, abl; Snyder et al, 1993). Transport of the oncogene product to the cell surface can be prevented by a single chain antibody with specificity for the oncogene product & a localisation signal for the endoplasmic reticulum.(e.g. erb2; Beerli et al, 1994).

Despite multiple genetic abnormalities, restoration of the tumour suppressor gene, such as p53, can be sufficient to cause cellular apoptosis & arrest tumour growth. Moreover, expression of p53 is synergistic with chemotherapeutic drugs such as cisplastin (Ogawa et al, 1997) & adjacent tumour cells that have not been transduced are killed, in what is termed the bystander effect (Roth et al, 1996). The p53 gene has been identified as important in a range of cancers & though less elegant than oncogene inactivation techniques, this method has proved robust enough to be included in a number of clinical trials. Other tumour suppressor genes may also prove useful e.g. BRCA1sv (Tait et al, 1997).

An alternative means of killing a tumour cell is to transduce a gene coding for a toxic product, known as molecular chemotherapy. The gene of choice is usually herpes simplex virus thymidine kinase (HSV/TK) which converts the prodrug ganciclovir into toxic metabolites. The effected cell is supported via the gap junctions of adjacent cells (Wygoden et al, 1997), until the toxin burden is too great killing both the affected cell & its neighbours. An advantage to this system is that all the transduced cells will be killed, allowing allogenic tumour cells to be prepared in advance (Paillard, 1998, Oldfield & Ram, 1995). HSV/TK is also used in other gene therapy protocols, allowing the treatment to be aborted at any time (Contassot et al, 1998).

Many cancer chemotherapy regimes are limited by their low therapeutic index, as determined by heamopoetic cells. By harvesting precursor bone marrow cells & transducing them with the gene MDR1, coding for the drug efflux protein p170, a population of resistant cells can be cultured. When returned to the patient, much higher doses of chemotherapy may be used (Champlin et al, 1994).

Clinical Trials:

Clinically, viral gene transfer to cancer cells has proved more efficient than expected from normal organ gene transfer & viral vectors can spread through three dimensional cell matrices (Fujiwara et al, 1994). Perhaps due to the heterogeneous nature of many malignant tumours, the most successful approaches do not rely on manipulating the immune system, rather act as adjuncts to conventional therapies (drug resistance genes) or use established agents (molecular chemotherapy). The morbidity associated with cancer gene therapies is significantly lower than of conventional treatments, therefore more aggressive application of gene therapies may be beneficial (Roth et al, 1996). Development of therapies for non malignant diseases have been hampered by the short duration of transgene expression. Recent advances however, make it likely that monogenic recessive diseases, such as haemophilia, will be treatable by administering a single dose of the gene vector

Current objectives include improvement of cell targeting through vector & promoter specificity (discussed above) & reducing the immune response to the current vectors. As the understanding of the molecular biology, immunology & virology underlying vector technology & oncology improves, so will our ability to treat cancer. Many of the developments reviewed above are present only in isolated systems, therefore consolidation of existing technology may yield significant improvements. Public expectation, though understandable & used by scientists to generate funds, should be tempered as gene therapy will not be the cure all in the near future. However, in conjunction with existing therapies, gene therapy will substantially reduced the burden of morbidity experienced by an ageing population.

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