Using a modified virus as a Trojan horse, a team led by Purdue University's David Sanders has found a promising system to deliver genes to diseased liver and brain cells. By placing helpful genetic material within the outer protein shell of Ross River Virus (RRV), Sanders' team was able to alter the mice's liver cells without producing the harmful side effects that have accompanied the use of other retroviruses.
"This represents a major advance in that we have used retroviruses for gene therapy, not just in tissue samples, but in living mice," said Sanders, associate professor of biological sciences at Purdue. "This brings us a giant step closer to treating human diseases."
The research, which is a collaboration between Purdue and the University of Iowa, appeared in the September issue of the Journal of Virology.
Gene therapy is the introduction of new genetic material into an organism for medical benefit, such as correcting the genetic defect responsible for cystic fibrosis. It also can be used to alter or destroy defective cells, which makes gene therapy a possible treatment method for cancer. Viruses play a key role in this fledgling field because of their natural ability to transport and transfer genetic material.
While viruses are often looked upon as harmful, their ability to introduce genes into cells gives them great potential as delivery vehicles for therapeutic genes. Ordinarily, a virus injects its own genetic material into a cell, but viral researchers have learned how to "borrow" the outer shell from a harmful virus and fill it up with other, beneficial genetic material.
The chimeric viruses that Sanders' group constructed consist of an outer shell taken from the RRV alphavirus, which typically infects Australian marsupials. The RRV shell allowed the group to solve two problems that have plagued viral researchers for some time: how to treat living organisms (rather than merely samples of tissue in a test tube) and how to avoid causing damage to those organisms while rebuilding their cells.
"Up until this point, a lot of gene therapy research was being done with a retrovirus coated with a protein called vesicular stomatitis virus G," Sanders said. "It has a protein shell that binds to just about any kind of cell, which is terrific if you want lots of options for gene therapy. The trouble is, the proteins are toxic to most cells as well, which is, of course, not so good."
When the team of Beverly Davidson and Paul McCray at the University of Iowa injected its homemade retrovirus into healthy mice, it proved highly effective at introducing new genes into livers. Just as encouraging was the discovery that during the DNA modification process, the retroviruses did not damage the liver cells.
"Not only were the genes successfully transferred, but the RRV envelope proteins did not damage the cells," Sanders said. "We succeeded on both fronts."
Because RRV can be injected intravenously and can bind to such a large number of cells, Sanders said he believes the technique could be useful for a range of illnesses. One promising target is glial cells in the brain, which provide structural support for neurons. Most brain tumors occur in glial cells, which form most of the brain's mass.
"This research shows that RRV has tremendous utility, especially for treating the liver," Sanders said. "But because of its ability to target glial cells, RRV can also potentially be used for a number of muscular and neurodegenerative diseases such as Parkinson's disease, multiple sclerosis and brain tumors."
Another potential application is delivering protein products directly into the bloodstream, which could lead to treatments for blood disorders.
"This is the direction we need to explore next," Sanders said. "If we can use retroviruses to carry therapeutic proteins directly to the bloodstream, it could provide treatments for hemophilia."
Sanders emphasizes that while the work is a leap forward for gene therapy, it will be several years before the technique is ready for human testing.
"I don't imagine having clinical trials on human diseases for at least five years -- there's still a lot to be done," he said. "What we have done is found a great stepping stone. It should encourage other researchers to search for alternative virus shells for gene delivery."
This work is supported by the National Institutes of Health, the Indiana Elks Charities Inc. and the Cystic Fibrosis Foundation.
Sanders conducts research, in part, at the Purdue Cancer Research Center, which coordinates interdisciplinary cancer-related research in the basic biomedical and life sciences. The center, established in 1976, provides shared resources for nearly 70 research groups on the West Lafayette and other Purdue campuses.
The Purdue Cancer Center is supported by the National Cancer Institute (NCI), the American Cancer Society, the Indiana Elks, the Indiana Lions Clubs and several local county cancer societies. The Purdue Cancer Research Center is a NCI designated basic laboratory research center.
Writer: Chad Boutin, (765) 494-2081, firstname.lastname@example.org
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David Sanders' homepage: http://www.
In vivo gene transfer using a nonprimate lentiviral pseudotyped with Ross River Virus glycoproteins
Yubin Kang, Colleen S. Stein, Jason A. Heth, Patrick L. Sinn, Andrea K. Penisten, Patrick D. Staber, Kenneth R. Ratliff, Hong Shen, Carrie K. Barker, Ine S. Martins, C. Matthew Sharkey, David Avram Sanders, Paul B. McCray, Jr., and Beverly L. Davidson.
Vectors derived from lentiviruses provide a promising gene delivery system. We examined the in vivo gene transfer efficiency and tissue or cell tropism of a feline immunodeficiency virus (FIV)-based lentiviral vector pseudotyped with the glycoproteins from Ross River Virus (RRV). RRV glycoproteins were efficiently incorporated into FIV virions, generating preparations of FIV vector, which after concentration attain titers up to 1.5x10^8 TU/ml. After systemic administration, RRV-pseudotyped FIV vectors (RRV/FIV) predominantly transduced the liver of recipient mice. Transduction efficiency in the liver with the RRV/FIV was ca. 20-fold higher than that achieved with the vesicular stomatitis virus G protein (VSV-G) pseudotype. Moreover, in comparison to VSV-G, the RRV glycoproteins caused less cytotoxicity, as determined from the levels of glutamic pyruvic transaminase and glutamic oxalacetic transaminase in serum. Although hepatocytes were the main liver cell type transduced, nonhepatocytes (mainly Kupffer cells) were also transduced. The percentages of the transduced nonhepatocytes were comparable between RRV and VSV-G pseudotypes and did not correlate with the production of antibody against the transgene product. After injection into brain, RRV/FIV preferentially transduced neuroglial cells (astrocytes and oligodendrocytes). In contrast to the VSV-G protein that targets predominantly neurons, <10% of the brain cells transduced with the RRV pseudotyped vector were neurons. Finally, the gene transfer efficiencies of RRV/FIV after direct application to skeletal muscle or airway were also examined and, although transgene-expressing cells were detected, their proportions were low. Our data support the utility of RRV glycoprotein-pseudotyped FIV lentiviral vectors for hepatocyte- and neuroglia-related disease applications.
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