Purdue researchers develop new delivery system
for gene therapy
WEST LAFAYETTE, Ind. For scientists working to develop gene transfer and gene therapies, finding an ideal carrier system is half the battle. Current systems are often difficult to produce, and may target only a few cells or offer merely a temporary effect.
Purdue University researchers have combined the traits of two types of viruses to be used as a new delivery system that can carry genes into a wider range of cell types and provide a more stable transfer of genetic material.
The result is a delivery system with the potential for broad applications that may someday be used to treat cancer, diabetes and other diseases caused by an underlying genetic deficiency, says David Sanders, assistant professor of biological sciences at Purdue and the study's principal investigator.
"This new system allows us to infiltrate different types of cells than is currently possible, thus increasing the types of tissues and organisms that we can treat using genetic therapies," Sanders says.
The new virus delivery system also can be used by scientists to enhance studies on a group of viruses known as alphaviruses. Alphaviruses, including the Sindbis virus and Semliki Forest virus, are a family of viruses often transmitted to humans by insects and can cause neurological disease and arthritis.
"These viruses are fairly difficult to study because they pose some health risks," Sanders says. "Also, because they kill cells, scientists were previously unable to study the process of how the virus enters the cell. This new delivery system has allowed us to separate the entry step from other steps of alphavirus replication."
Details of the study were published in the March issue of the Journal of Virology.
Sanders and his Purdue colleague, Associate Professor Richard Kuhn, developed the new gene delivery system by replacing the outer protein shell of a virus, known as retrovirus, with the coat of an alphavirus. This combination virus is called a pseudotyped retrovirus because it infects cell types associated with its protein coat, but acts like a retrovirus once inside the cell.
"This allows us to expand the types of cells that can be treated with genetic therapy, because we get a virus that can infect those cells that the alphavirus would infect rather than the ones that the native retrovirus would infect," he says. "Retroviruses, in their natural state, infect only a few specific types of cells. The HIV virus, for example, will infect essentially only macrophages and T-cells."
Retroviruses are often used in gene transfer because once inside a cell, they can copy part of their DNA into the genetic code of the host cell. For gene therapy, the harmful parts of the virus' genetic code are replaced with genes intended to treat disease, so patients can be treated without suffering any harmful effects from the virus. The cell will then produce new cells that contain the therapeutic gene.
The first commonly used pseudotyped retrovirus system called VSV-G because it was developed using the glycoprotein coat of a vesicular stomatis virus currently is used in some gene therapy processes.
"Both systems can infect mammalian and insect cells, but our new system presents several advantages over VSV-G in gene therapy and gene transfer," Sanders says. "For example, VSV-G is toxic, both to cells that are being infected and to cells that are producing the virus. We have seen no signs of toxicity using our system. That should allow these particular viruses to be the virus of choice for gene transfer mediated by retroviruses."
Experiments in cell cultures also indicate the new system can transfer genes in a permanent, dependable fashion, Sanders says. "One problem with some of the current carrier systems, including VSV-G, is that gene transfer with them can be transient."
Another advantage is that the cell lines developed to produce these carrier viruses can make the viruses indefinitely, Sanders says.
"You cannot do that with the VSV-G," he says. "Once you turn on virus production in those cells, the cells die, which makes it difficult to produce large numbers of the VSV-G carrier."
The study was funded by the Purdue Research Foundation and the National Institutes of Health. Doctoral students C. Matthew Sharkey and Cynthia North also participated in the research. Purdue has filed a patent for the new virus.
Sources: David Sanders, (765) 494-6453, firstname.lastname@example.org
Richard Kuhn, (765) 494-1164, email@example.com
Writer: Susan Gaidos, (765) 494-2081; firstname.lastname@example.org
Purdue News Service: (765) 494-2096; email@example.com
Ross River Virus Glycoprotein-Pseudotyped Retroviruses and Stable Cell Lines for Their Production
C. Matthew Sharkey, Cynthia L. North, Richard J. Kuhn and David Avram Sanders
Pseudotyped retroviruses have important applications as vectors for gene transfer and gene therapy and as tools for the study of viral glycoprotein function. Recombinant Moloney murine leukemia virus (Mo-MuLV)based retrovirus particles efficiently incorporate the glycoproteins of the alphavirus Ross River virus (RRV) and utilize them for entry into cells. Stable cell lines that produce the RRV glycoprotein-pseudotyped retroviruses for prolonged periods of time have been constructed. The pseudotyped viruses have a broadened host range, can be concentrated to high titer, and mediate stable transduction of genes into cells. The RRV glycoprotein-pseudotyped retroviruses and the cells that produce them have been employed to demonstrate that RRV glycoprotein-mediated viral entry occurs through endocytosis and that membrane fusion requires acidic pH. Alphavirus glycoprotein-pseudotyped retroviruses have significant advantages as reagents for the study of the biochemistry and prevention of alphavirus entry and as preferred vectors for stable gene transfer and gene therapy protocols.
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