Progress in Lentiviral Vector Technologies

R etroviridae is a large family of RNA-based infectious agents that share both virion structure and mode of replication. To those of us working in the field of gene therapy, we have come relatively recently to these agents for transfer of genetic material into cells, and as effective therapeutics. However, we sometimes forget that they have a long history in causation of infectious disease and cancer in animals and humans. Although we are all familiar with the lentivirus human immunodeficiency virus as the cause of acquired immunodeficiency syndrome (AIDS), retroviruses were actually among the first viruses to be identified in nature. For example, equine infectious anemia virus (EIAV), which we also now recognize as a lentivirus, was in fact isolated as a filterable agent as far back as 1904. The first oncoretroviruses were identified soon after, and murine leukemia virus (MLV), which has been extensively adapted for gene transfer, was isolated from a leukemia-prone strain of mice in 1951. There was therefore a large background of basic virological and oncological studies that provided a platform for molecular biologists to design highly efficient gene transfer reagents. The ability of retroviruses to become latent through the formation of a DNA-integrated provirus in particular provided a unique strategy to confer stability of gene transfer through host cell divisions.

The last decade has seen remarkable progress in the application of retroviruses as therapeutic agents, and in the realization that they have significant potency in humans. For patients with inherited immune-hematological and metabolic disease, they have shown clear efficacy in situations where more conventional approaches (usually HLA-mismatched allogeneic hematopoietic stem cell transplantation) were judged to be difficult. However, this success has come at the price of some toxicity. Although to many of us the frequency of leukemia in patients with various diseases arising through insertional mutagenesis may have been somewhat surprising, I also remember being quizzed in the late 1990s at an ethics review of one of our own early protocols for treatment of severe combined immunodeficiency (SCID), in which I was asked whether we had tried to eliminate one of the powerful duplicated enhancer elements in the retroviral long term repeat (LTR). The reasoning was based on preexisting knowledge of virus-induced cancer, and although we felt at the time that the risks would be minimal, in fact this concern was well founded. We are also now accustomed to the idea that retroviruses have different integration profiles in terms of preference for certain locations within the genome, and that this may have significant influence on safety and biological activity of the transgene. However, we should not forget that the notion that retroviral integration may not necessarily be random preceded any human studies. So the point is that we can learn much through understanding natural viral biology, in terms of adapting this family of viruses to safety, but also to ensure that the therapeutic effect is both reliable and sustained where necessary.

Lentiviruses have emerged from the shadow of oncoretroviral vectors (from the genus Gammaretrovirus) because of a superior ability to transduce nondividing or slowly dividing cells and also because they can incorporate relatively sophisticated payloads. They can also be easily adapted to be nonintegrating through specific mutations in the integrase gene, which for some applications will provide enhanced biosafety. Even so, there remain considerable constraints to their wider application because manufacture of clinical-grade stocks in large quantities remains both difficult and expensive. This remains one of the major biotechnological challenges to the field today, but if solved would rapidly broaden the clinical applicability of lentiviral vector gene transfer.

An upcoming series of three reviews will address the basic mechanisms of retroviral integration, and ways in which lentiviral technology is developing to enhance both safety and effectiveness of gene expression. The first, appearing in this issue of Human Gene Therapy, “Mechanisms of Retroviral Integration and Mutagenesis” by A. Cavazza, A. Moiani, and F. Mavilio, brings us up to date with the biology of retroviral integration in terms of site selection within the genome, and explores the unique mechanisms by which retroviruses interact with host DNA-binding factors. An interesting corollary is that transcriptional activity of the cell to some extent determines specific integration patterns, which may be important when targeting different cell lineages, and is also influenced by the transduction environment. Access to high-throughput analysis of integration sites in different cell populations has been the prime driver behind these rapid advances in our knowledge. In the second article appearing in this issue, “Biosafety Features of Lentiviral Vectors,” A. Schambach, D. Zychlinski, B. Ehrnstroem, and C. Baum review the technological advances in replication-competent lentivirus-free production, and strategies to limit insertional mutagenesis, that are already entering the clinical arena. In the third article, “Optimizing Retroviral Gene Expression for Effective Therapies,” which will appear in the April issue of Human Gene Therapy, M. Antoniou discusses the use of various strategies to direct optimal gene expression given that it is usually impossible to incorporate gene regulatory elements in a natural context, and also to prevent undesirable effects such as gene silencing.

These timely reviews will highlight the major advances that have been made in retroviral vector design over the last 10 years, and also provide considerable in-depth information pertaining to the biology of the wild-type virus from which they have been derived.