Despite some early setbacks involving toxicological effects and poor efficacy, the gene therapy market in the United States is expected to reach approximately $125 million in 2006 and possibly even surpass $6.5 billion by 2011, according to recent reports by Frost & Sullivan. Several technological advances in the design, development, and production of viral vectors, however, are beginning to generate renewed interest in the sector.
One success story is gendicine, produced by China's Shenzhen SiBiono Gene Technologies, an adenoviral vector that targets largely head and neck squamous cell cancers by introducing the p53 tumor suppressor gene. A related technology that is also showing increased attention is lentiviral vectors (LVs).
Looking at lentiviruses
LVs are derived from special group of viruses, of which HIV is a member. HIV has evolved and adapted itself to enter human cells in a very effective manner. While this is one of the reasons that the disease is so difficult to eradicate, these very same qualities also enable the virus to be engineered into a very effective gene delivery system once it is "gutted" of its harmful elements.
Gene delivery is accomplished by the binding and fusion of the LV pseudotyped envelope protein to the target cell membrane. This complex is then released into the cell and an enzyme called reverse transcriptase converts the RNA into DNA by a process called reverse transcription. The DNA complex then enters the nucleus of the cell integrating into the target cell's chromosomal DNA.
Gene delivery is stable because the target gene (or gene silencing sequence) is integrated in the chromosome and is copied along with the DNA of the cell every time the cell divides. In addition to stability, LVs can deliver genes with up to 100 percent efficiency. Other viral vector systems such as adenoviral and adeno-associated viral vectors can achieve high, but not stable, gene delivery only in dividing cells, such as blood cells. In addition, vectors such as murine retroviral vectors can deliver stable genes, but cannot do so efficiently. For these reasons, LVs may provide the best solution.
Given this multifaceted profile, a significant number of applications have emerged for LVs. LVs have potential uses as: a drug discovery tool; an efficient production method for small molecules and vaccines; and ultimately, as a human therapeutic.
LVs have vast potential as drug discovery tool, including use in target validation and the generation of engineered cell lines and transgenic animals. Medical researchers need a robust gene delivery system to unlock the mystery of gene function. Researchers generally discover gene mechanisms by either "knocking in" or "knocking out" the gene of interest and then using sensitive "biochip" technology for analysis.
One such application would be the use of LV technology to validate drug targets by "knocking out" the target gene in cells and then showing lack of effect of the drug in those cells. Presently, there is no robust and reliable commercially available product to obtain stable long-term over-expression or "knock down" of genes without a labor-intensive process for researchers. Researchers have several options, but all of them suffer serious limitations.
For example, plasmids have low delivery efficiency, which requires selection of a few cells while killing the majority of cells to generate stable cell lines expressing the transgene. This approach is not suitable for high-throughput or sensitive cell analysis. On the other hand, LVs offer significant benefits because they are capable of efficiently generating cell lines that stably express the transgene of interest without need for selection.
LVs can also facilitate drug discovery by genetically engineering primary cells to closely mimic the normal and abnormal cells in the body through the introduction of genes or inhibitors of gene expression (such as RNAi), that generate novel cell lines of the desired phenotype. In this way, the time to identify a lead drug candidate that has high potency and low toxicity will be dramatically reduced.
LVs can similarly be used in target validation to knock out the drug target gene and then validate that the lead drug candidate does not produce any adverse "off-target" effects. LVs add value to this process over other delivery systems by efficiently delivering the gene or RNAi into cells with high efficiency into difficult-to-engineer primary cells. This is important because it is known that multiple copies are required for efficient RNAi-mediated inhibition of gene expression in cells.
LVs could also be used for a variety of other applications for example, the production of transgenic animals for use in drug validation and toxicity studies. By introducing specific genes or gene combinations, researchers can better mimic various human disease states within the model animals, increasing the effectiveness of their preclinical screening efforts.
Beyond discovery, LVs may also offer substantial improvements in both protein and vaccine production. They can be designed to more efficiently express proteins, monoclonal antibodies (MAbs) and vaccines from producer cell lines. They will cut down the time and increase yields by using the highly efficient delivery properties LVs offer.
Although chemical-based drugs have been successfully used for the treatment of various diseases, many diseases remain without current treatment options or what is available is toxic. Medicine is rapidly moving toward biological based therapies that are more targeted and promise better outcomes for patients, without significant side effects. On the leading edge of this revolution in medicine is the treatment of disease by transfer of therapeutic genes into diseased cells of the body.
In contrast to vector systems used in the past, LVs can deliver genes into cells with high efficiency and stability. Although there have been some concerns in using such a novel vector system, LVs have now been evaluated in human clinical trials. Recently, a Phase I clinical trial using a LV for the treatment of patients with HIV/AIDS was successfully completed at the University of Pennsylvania, showing excellent safety profiles.
Presently, there are several companies that are in Phase III clinical trials for gene therapy products, some look very promising for licensure. For example, Cell Genesys is in the midst of a Phase III clinical trial with its GVAX product for the treatment of various forms of cancer. Similarly, Oxford BioMedica has several lentivirus-based therapeutics in preclinical and Phase I clinical development, including RetinoStat for age-related macular degeneration and ProSavin for late-stage Parkinson's disease. Many clinical investigators at academic centers and biotechnology companies are now looking to LVs for the treatment such human disorders.
Providing lentiviral vectors
To supply the burgeoning pharmaceutical and biotechnology industry with viral vectors, a number of companies exist that focus on providing adenoviral and adeno-associated viral vectors. A short list would include the likes of Clontech, Invitrogen and Stratagene.
Some companies, such as Invitrogen, have also provided LV plasmids that the researcher then uses to subclone the sequence of interest. The researcher then uses a packaging mix to produce the LV particles in-house. The basic materials for the production of LVs results in the researcher spending several months cloning, producing and titering their LV, with varying results. Lentigen, however, is the only company supplying custom LVs, where the researcher simply designs and orders a LV over the Web and Lentigen, in turn, manufactures the custom LV particles for the customer to high titer and quality. Lentigen believes that researchers would prefer to focus their efforts upon understanding their gene-of-interest, rather than spending months constructing and producing a LV lot.
Lentigen is positioning itself to become the leading product and service provider of LVs to academic, pharmaceutical, biotechnology and other commercial organizations. The added value that Lentigen provides is a proprietary highly concentrated, pure and quality-controlled LV preparations—LentiMax, a synthetically optimized HIV-1 based lentiviral vector, and the LentiMax Production System—that provide efficient gene delivery in cells, providing a reliable result when coupled with sensitive assays for analysis. In addition to research-grade LVs, Lentigen provides large scale LV manufacturing services including GLP scale for animal toxicity experiments and cGMP clinical-grade vector material for clinical trials.
Dr. Boro Dropulic is founder and CEO of Lentigen Corp. He is also a member of the Infectious Diseases and Industrial Liaisons Committees for the American Society of Gene Therapy. Prior to founding Lentigen, he was founder and CSO of VIRxSYS, where he led the first ever lentiviral vector clinical trial in humans. Dr. Dropulic was also an adjunct assistant professor at Johns Hopkins University School of Medicine.