of the head and neck. However, despite more than 1,300 clinical trials to date, no products have been FDA-approved.
At the same time, tragic setbacks, including the deaths of patients in two trials, have hindered progress. A severe inflammatory response caused by the adenovirus used in a 1999 trial for the treatment of ornithine transcarbamylase deficiency was proved to be the cause of death and resulted in a temporary halt to all gene therapy trials. In addition, at least 2 of the 11 children in the Cavazzana-Calvo γc-SCID trial developed leukemia as a result of retroviral insertion of the therapeutic sequence in or near a gene associated with childhood leukemias. Thus a key limitation to the development of human gene therapy remains the lack of safe and efficient methods of gene delivery (Verma and Somia, 1997).
Current gene-delivery methods comprise recombinant viruses, which are used in the majority of clinical trials, and synthetic materials, including peptides, polymers, and liposomes. Although viruses are the most efficient vectors, they often initiate immune responses, are limited in the size of genetic material they can carry, are difficult to produce and purify, and exhibit limited target-cell specificity (or often nonspecificity). Cationic polymers (Felgner and Rolland, 1998; Pack et al., 2005; Smith et al., 1997) have the potential to be nontoxic and nonimmunogenic, are chemically and physically stable, are relatively easy to produce in large quantities, and can be targeted to desired cell types; but in general, they are not efficient enough for clinical use. Even the most efficient polymers are orders of magnitude less efficient than viruses (micrograms of DNA are required to achieve transgene expression comparable to that resulting from a virus suspension containing about 10 picograms of genetic material).
To escort genes from a solution (e.g., in a vial) to the cell nucleus, gene-delivery vectors must navigate a series of obstacles, both extracellular and intracellular. Viruses have evolved functions to address each of these challenges, but synthetic vectors generally lack one or several of these functions. These obstacles must all be taken into consideration for the rational design of new materials.
The first set of barriers facing gene-delivery vectors appears in transporting genes from the test tube to the membrane of a target cell. First, the vector must bind and condense plasmid DNA to a sufficiently small size to allow efficient cellular internalization and protect the genes from nuclease degradation. Polycations and DNA spontaneously form tight complexes (polyplexes) through entropically driven electrostatic interactions. The resulting particles typically comprise several DNA molecules and hundreds of polymer chains and range in size from a few tens to several hundred nanometers in diameter. Second, the polyplexes must form a stable solution under physiological conditions, which can often be achieved by coating them with a hydrophilic polymer, such as polyethylene glycol. Third, for