Gene Therapy: Delivery Systems

DELIVERY SYSTEMS

In many cell types it is feasible to deliver nucleic acids and genes by a variety of methods when the cells are grown in tissue culture (Table 58.1). Nonetheless, some cells, such as pneumocytes and neurons, are not readily isolated from humans and do not grow well in vitro. Furthermore, for many diseases it is essential to alter the phenotype of a significant proportion of the total cell population, making ex vivo gene therapy of limited use.

There is general agreement that no ideal delivery system is available for in vivo gene therapy. Direct or in-tratumoral injection of plasmid DNA or antisense oligomers without a viral vector has been attempted. Expression of genes using traditional nonviral vectors has been low compared to viral strategies. Nonetheless, recent breakthroughs in nonviral delivery systems, in-cluding the gene gun, electroporation and naked DNA, suggest that nonviral gene therapy can achieve local ex-pression of therapeutic genes at levels equivalent to those of viral vectors.

Although the mechanism remains undetermined, the injection of naked DNA into skeletal muscle has demon-strated relatively high transfection efficiency. In this set-ting, DNA is precipitated onto the surface of microscopic metal beads (e.g., gold) and the microprojectiles are ac-celerated and penetrate intact tissue to several cell layers.

In preclinical trials, efficiency remains low, but expression has been noted to last for several weeks, and there has been no significant inflammatory response.

Some investigators have used electrical current (electroporation) to improve DNA (or drug) entry into tumor cells with some preliminary success. Liposomes are attractive vehicles for gene delivery, since they can carry plasmid, antisense, or viral DNA. Compared with viral approaches, however, liposomes remain relatively inefficient at facilitating gene transfer, although their safety profile remains more desirable. Some of the at-tributes and limitations of the nonviral methods are listed in Table 58.1.

Because viruses can efficiently integrate into the genome, many clinical trials are exploring the use of replication-defective recombinant viral vectors and de-livery systems. Retroviruses contain their genetic infor-mation as a double-strand DNA genome that is tran-scribed, and the single-strand proviral DNA product is stably integrated into the host genome. Recombinant DNA technology has been used to remove deleterious viral genes involved in replication, and the resulting vector is replication defective, nonpathogenic, and un-able to produce infectious particles. Ideally, with a retro-viral vector, only a single administration should be re-quired because the gene should be permanently retained and expressed. No clinical evidence of mutage-nesis has emerged from the clinical trials performed to date, but the number of patients treated and the time of exposure has been limited.

Adenoviral vectors have also been used in human trials. These vectors enter cells by either an adenovirus fiber–specific receptor or a surface integrin receptor. They efficiently transfer genes in nonreplicating and replicating cells. Nonetheless, immunological responses to viruses have been noted with adenoviral vectors. Replication-selective adenovirus vectors have been in-troduced to optimize infection of target cells and mini-mize infection of normal cells. Over 200 cancer patients have been treated to date in more than 10 clinical trials with little evidence of toxicity reported. Replication, however, has generally been transient ( 10 days), with limited efficacy observed when the gene therapy was administered as a single agent. More encouraging anti-tumor effects have been observed when the gene ther-apy was combined with cytotoxic chemotherapy. Further modifications are likely to be required before there can be general application of adenoviral vectors for cancer therapy.