Gene therapy, the correcting of a disease by addition of exogenous genetic material (transgene), was envisioned in the early 1970s. These early pioneers faced the challenge of how to introduce a transgene adequately and safely into human cells to cure or reduce disease severity. Although several methods for introducing genetic material into a cell have been developed, viral vectors have proven the most efficient; taking advantage of a virus's natural ability to invade and introduce their genetic material into human cells. Several different viruses have been used as vectors for gene therapy including adenoviruses (AV), adeno-associated viruses (AAV), retroviruses, lentiviruses (LV) and herpes simplex viruses (HSV) with each virus having its advantages and limitations. The genetic material contained in DNA viruses like AV, AAV and HSV remain largely episomal and get diluted as cells divide while the transgenes in RNA viruses like retroviruses and LVs are integrated into the host cell's chromosome and provide long term transgene expression. Viral vectors have been used to treat a variety of diseases including monogenic syndromes and cancers and can be delivered both in vivo, in situ and ex vivo (in vitro). While in vivo delivery has proven successful in mitigating some diseases, it is not without risks including causing severe immunogenic responses, insertional mutagenesis resulting in oncogenesis, and cytotoxicity. Safety issues that arose in several gene therapy clinical trials in the 1990s including a death and several instances of viral mediated leukemia cast doubt on the ability of gene therapy to safely cure or mitigate disease. These safety issues, however, prompted further research into understanding the virus-host interaction that resulted in new generations of viral vectors that abrogated many of these issues and sparked a renaissance in gene therapy.
Viral vectors for gene therapy, like the viruses they are derived from, consists of genetic material (double stranded DNA, single stranded DNA, or RNA) encased in a protective protein shell or capsid. Retroviral and LV vectors are also encased in lipid bilayer or envelope that can contain proteins necessary for cell invasion. The size of the capsid largely dictates the amount of genetic material that can be contained within a virus and correspondingly the size of the transgene. Larger viruses like AVs being able to carry transgenes of up to 36 kb while smaller viruses like AAVs only being able to accommodate a transgene of 3-4 kb. Regardless of the size of the viral particle or the nature of its genetic material, all vectors have some common basic characteristics.
- Viral vectors are infectious but replication defective. In order to deliver a transgene into a cell, the viral particles need to be infectious. However, to prevent potential cytopathic effects (i.e. cell lysis), the viral particles are rendered replication deficient meaning that following infection and introduction of the transgene no more virus is produced. This is done by deleting genetic sequences necessary for viral replication and/or production of a capsid which also makes room in the viral genome for the transgene. Due to safety concerns with a replicating virus, supporting assay data to confirm the vector is replication defective is required. The viral genome for most current gene therapies essentially consists of the transgene, viral sequences necessary for packaging the viral genome in the capsid during manufacture, a promoter for transgene expression and genetic elements necessary for integration of the transgene into the host chromosome in the case of retroviruses and LVs.
- Transgene expression is controlled. Whether the transgene remains episomal or is integrated into the host chromosome, to get expression of the transgene it must have a promoter. Early LV-based gene therapies used strong viral promoters that were associated with serious adverse events in clinical trials, however, most current LV-based therapies use human housekeeping promoters (constitutive or regulated) and regulatory elements encoded in the viral vector genome to better control transgene expression. In addition, tissue specific and inducible promoters have been used to allow expression of the transgene in particular tissue cell types and at particular times during treatment.
- Cell tropism can be controlled. While tissue specific promoters provide some cell tropism, the type of host cell targeted by a gene therapy is often dictated by proteins in the viral capsid or envelope. Many AAV serotypes have been identified that have subtle differences in the capsid proteins that bind to different cell surface structures on host cells and provide tissue preferences for virus invasion. This allows for design of gene therapies that target particular cell types by using specific AAV serotypes. Furthermore, advances in understanding viral biology has allowed for directed evolution of capsid sequences to provide greater control of cell tropism.
- Immunogenicity is still an issue. While great strides have been made in the design of viral vectors to reduce cytopathic effects, control transgene expression and provide tissue specific targeting, immunogenicity remains problematic both in terms of reactogenicity during delivery but also in reducing effectiveness due to immune-mediated inactivation. This is particularly a problem for large viruses like AVs and HSVs which tend to be more immunogenic.
Overall, viral vector design for gene therapy has come a long way since its inception and continues to evolve as knowledge of viral biology grows. While in vivo use of viral vectors remains challenging, continuing advances in vector design are expected to lead to improvements in the efficacy of gene therapies and a reduction in safety concerns.