Caitlin Smith has a B.A. in biology from Reed College, a Ph.D. in neuroscience from Yale University, and completed postdoctoral work at the Vollum Institute.
Despite the obvious reason to be wary of viruses, these genetic marvels can be invaluable tools in the lab. Scientists use replication-deficient forms of viruses as vectors to introduce genetic material into cells. Viral vectors have a gained a strong adoption for inserting genes into cell types (e.g., primary or stem cells) that may have been recalcitrant to other methods, such as lipid-based transfection or electroporation. Today there is considerable interest in using viral vectors, combined with gene-editing technologies, to alter and monitor gene expression. Here we discuss the advantages and disadvantages of the lentivirus, adenovirus and adeno-associated virus (AAV) expression systems, along with examples of recent therapeutic applications.
Lentiviral vectors
Lentiviruses are used for either transient or stable gene expression, infecting both dividing and nondividing cells and integrating into the host cell’s genome (however, lentiviruses deficient in integrase are available if needed). Insert capacity for lentiviruses (i.e., the size of the gene to be inserted) is up to about 5 kb, depending on the engineered constructs—overall, lower than other vectors (see below).
Lentiviral vector tools are available for a variety of experimental conditions. For example, Dharmacon (part of GE Healthcare’s Life Sciences business) provides a range of lentiviral products for gene knockdown or knockout. These include the Dharmacon™ Edit-R™ CRISPR-Cas9 platform (for gene editing) and the Precision LentiORFs (human open-reading frames in lentiviral expression vectors), says Melissa Kelley, R&D manager for Dharmacon, GE Healthcare.
Transduction with lentivirus tends to be very efficient (usually near 100%), so Kelley says sometimes you need to be more concerned with your multiplicity of infection (MOI) level, depending on your experiment. A lower MOI (less than 1) is important when you want cells to be infected by only one lentiviral particle (for example, in pooled screening applications or in the creation of stable cell lines). “In this case, you don’t necessarily want a high transduction efficiency—you want to have only single integrations into cells,” says Kelley. Subsequent selection of transduced cells by antibiotic resistance or fluorescent reporters can yield a population of 100% cells transduced with single integrations. Conversely, other situations may call for a high MOI, such as a short-term gene knockdown experiment.
Recent studies using lentiviral vector-based hematopoietic stem cell gene therapy have shown progress in treating X-linked severe combined immunodeficiency (a genetic disorder popularly known as “bubble boy disease”), as well as Wiskott-Aldrich Syndrome (a genetic disease of the immune system). In addition, lentiviral gene therapy for the blood disease beta-thalassemia resulted in patients being able to stop regular blood transfusions.
Adenoviral vectors
Adenoviral vectors infect both dividing and nondividing cells, and they don’t integrate into host cells, so expression is transient. The packaging capacity of adenovirus vectors typically accommodates larger inserts (from 8 kb to 36 kb, when using helper-dependent vectors). Adenoviruses also tend to have high levels of protein expression, which can be valuable depending on the selected application.
For viral vector production and purification, Sartorius Stedim Biotech offers a range of bioprocess tools, from low-volume production (with the ambr® 15 fermentation microscale bioreactor system) to large-scale production (with the BIOSTAT® STR bioreactor). The company’s Vivapure® AdenoPACK™ and LentiSELECT virus purification kits are used in basic research, and many of the tools are used by “companies producing commercial products that are in clinical trials,” says Kim Bure, director of regenerative medicine at Sartorius Stedim Biotech.
AMS Biotechnology also offers many viral vector tools, including lentivirus, adenovirus, and AAV — and a large collection of human open-reading frame cDNA clones in adenovirus and lentivirus vectors. Although many of their tools are used in basic research, “we always work together with researchers to help them move the projects into applied studies, if this is their intention,” says technical sales representative Maja Petkovic, who notes that the persistence of basic research is helping to fuel the progress of therapeutic viral vectors. “Continuing development in changing virus envelope and tissue targeting specificity can help further increase the efficacy of viral gene therapy and reduce the side effects,” says Petkovic.
Adenoviral vectors have recently been used as a delivery vehicle to fight prostate cancer. Tumor cells are transduced with a gene that prompts the patient’s immune system to attack the tumor cells. Because the tumor cells are essentially led to self-destruction, the technique is known as “suicide gene therapy.”
AAV vectors
AAV vectors infect both dividing and nondividing cells for stable expression, are nonintegrating and nonpathogenic to humans (which is convenient for therapeutic applications). AAV vectors don’t insert randomly but rather at a specific site in the human genome. A potential disadvantage, depending on your application, is that AAV vectors have a smaller insert capacity (less than 3 kb) compared with either lenti- or adenoviruses.
Cell Biolabs offers AAV (and other viral) vector tools for research use, such as the ViraDuctin™ AAV Transduction Kit, which uses proprietary reagents that increase AAV transduction efficiency. As viral vectors emerge as a clinical tool, Ken Rosser, director of marketing and sales at Cell Biolabs, notes that it’s important to consider the effects that different viral vectors can have on patients’ immune systems. “The lowest possible immune response is desirable, both to ensure proper delivery of the gene and to minimize complications,” says Rosser. “This gives AAV vectors an advantage, since they have been demonstrated to elicit lower immune responses compared to both lentivirus and adenovirus.”
Being less immunogenic makes AAV vectors good candidates for therapeutic applications, such as the introduction of a normal gene into the eyes of patients with retinal pigmentosa, which gradually leads to blindness, if untreated. Recent clinical trials using AAV vectors have improved or restored patients’ vision. At the University of Pittsburgh School of Medicine, a clinical trial is also underway using AAV to introduce the gene for a key enzyme into patients with Parkinson’s Disease. The enzyme is responsible for converting the drug levodopa into the neurotransmitter dopamine, which prolongs the positive effects of the drug as neurons deteriorate.
The future with viruses
One of the challenges of using viral vectors to treat human diseases is the danger of off-target, or unintended, effects of viral infection.
But new technologies may hold solutions. “The advent of gene-editing technologies such as ZFN, TALEN and CRISPR, and their focus on targeted effects, will have considerable impact on the gene-therapy sector if they increasingly become vehicles for vector delivery,” says Bure. Indeed, the combination of tried-and-true viral vectors plus new gene-editing tools is likely to yield some powerful clinical applications in the near future.
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