Progress on Application and Maintenance of Nonviral Vectors

T he concept of gene therapy entered clinical trials in the late 1980s. Initially designed as a means to treat monogenetic disorders, gene therapy has become a treatment strategy for a plethora of diseases, including cancer and infectious disorders. Approximately 20 years later, an increasing number of reports on therapeutic efficacy indicates the successful translation of the concept into clinical practice. In particular, advancements in vector design have critically contributed to this success. Nevertheless, a number of challenges—faced by both classes of gene delivery tools, that is, nonviral as well as viral vectors—still have to be overcome. Problems include low transduction efficacy, lack of cell-type specificity, shutdown of transgene expression, and host immune responses.

The efficacies with which target as well as off-target cells are transduced are influenced by the specific and nonspecific interactions of vectors with surface molecules on cellular membranes. These interactions may also affect induction of immune responses that hamper vector reapplication and/or duration of cell modification. Consequently, efforts in vector development focus on the specific modification of vector–cell interactions, in particular cell binding and uptake of vectors.

Even in the absence of immune responses toward the vector or transgene product, therapeutic efficacy may be lost when transgene cassettes are delivered by standard nonintegrating vector systems to proliferating cells or when the promoters driving transgene expression are silenced. These challenges provide the rationale for developing strategies for targeted integration or episomal maintenance of the delivered vector genomes. Besides promoting stable gene transfer, such strategies help to lower the risk of insertional mutagenesis and the likelihood of promoter silencing. The latter, however, also critically depends on the choice of the promoter. In the case of episomal maintenance, transcriptional activity may even be a prerequisite for replication and chromosomal attachment, further underlining the complexity and interconnectedness of the mechanisms underlying safe and efficient transgenesis.

In a series of five reviews published in three consecutive issues of Human Gene Therapy, with the first two included in this issue, technical improvements in nonviral vector-based gene therapy, in particular those tackling the problems of efficacy and maintenance, will be presented.

Briefly, A. Rochard, D. Scherman and P. Bigey report on the use of plasmid DNA for genetic immunization. Besides its use as a means of producing antibodies for diagnostic and experimental purposes, DNA immunization is exploited for the treatment of autoimmune diseases, allergy, and cancer. Another interesting application would be the tolerization, rather than immunization, of the host toward a specific gene product. These applications have benefited from improvements in vector design and gene delivery methods. The review by Bigey and colleagues summarizes the current status, in particular emphasizing the advantages of electrotransfer. In the second review contained in this issue, M. Ogris and E. Wagner discuss advancements in receptor targeting of DNA- and RNA-based nonviral vectors. As ligands, proteins, peptides, or small molecules are coupled to the outer surface of vectors to (re)direct vector tropism in order to improve the efficacy and specificity of gene transfer.

Subsequent issues of Human Gene Therapy will review three strategies to achieve maintenance or stable cell modification. Stable gene transfer using randomly integrating systems bears the risk of insertional mutagenesis. Although still significantly less efficient than retro- and lentiviral vector systems in establishing stable gene transfer, nonviral vectors bearing S/MAR (scaffold/matrix attachment region) elements are a valid alternative as these elements promote replication and maintenance of plasmid DNA or minicircles as episomes in mammalian cells. C. Hagedorn, S.-P. Wong, R. Harbottle, and H. Lipps report in their review about the discovery of S/MAR elements, on the refinements leading to improved gene expression and size reduction as well as on ex vivo and in vivo applications of S/MAR-based nonviral vectors. A different strategy for lowering the risk of insertional mutagenesis and simultaneously achieving stable gene transfer is targeted integration. The review by S.H. Rahman, M.L. Maeder, J.K. Joung, and T. Cathomen summarizes the current status of the use of zinc-finger nucleases in somatic gene therapy. Zinc-finger nucleases are designed to target specific sequences in the genome in order to increase the efficiency and specificity of homologous recombination. Depending on the experimental setup, zinc-finger nucleases can either be employed to knock out genes, to target integration of a therapeutic expression cassette to a safe harbor, or to modify the sequence of a gene of interest by homologous recombination with a donor DNA. Clinical applications of this technology as well as chances and risks are discussed. Similarly, transposons (mobile genetic elements that can be harnessed to serve as non-viral gene vectors) are exploited as tools for achieving transgene integration at sites in the genome that promote long-term gene expression without disturbing the expression or regulation of neighboring genes. Z. Ivics and Z. Izsvák will introduce this technology and discuss advancements in a third issue of Human Gene Therapy.