Jan. 5, 1999 CHAPEL HILL, N.C. -- Molecular doorways on the surface of cells may be important factors in the success or failure of human gene therapy using altered viruses, according to researchers at the University of North Carolina at Chapel Hill.
A report of their study published Jan. 4 in the journal Nature Medicine applies directly to a promising gene therapy method in which a harmless, genetically engineered virus called AAV (adeno-associated virus) is used to deliver genes into the body. The new findings tell us that the entry of AAV into the cell is facilitated by surface receptor molecules called alpha V beta 5-integrins. Without the presence of these integrins, the ability of AAV to "infect" the cell and deliver its genetic payload is diminished, say study authors led by Candace Summerford, doctoral student in pharmacology at the UNC-CH School of Medicine.
"We have been able to show that the virus interacts with two receptors: a primary receptor, which serves as an attachment molecule, and a secondary receptor -- the integrin co-factor -- that facilitates virus internalization," Summerford says. She notes that AAV is yet another example of a virus that uses multiple cell surface receptors to mediate its entry into cells. HIV, herpes simplex, and adenovirus, a cold virus, also employ multiple receptors.
The UNC-CH researchers tested hamster cell lines lacking the alpha-v beta5 integrin. Attempts at getting AAV into these cells proved far more difficult than those on cells that were genetically altered to express the integrin. "In the absence of integrin, the virus sits on the outside of the cell," says Dr. R. Jude Samulski, study senior author and director of the UNC-CH Gene Therapy Center. He points to micro-photographs of the virus glowing fluorescent red on the cell's surface. "This is the same cell where the integrin has been put in," Samulski says. He then refers to the next image in which the fluorescence is gone from the cell surface. "Within 10 minutes, the virus is taking a path to the cell nucleus."
Earlier work by Summerford identified the receptor for AAV that mediates attachment to the cell surface. This was the first step towards understanding how the virus infects the cell. Work done by her gene therapy colleagues has demonstrated that neurons carry this primary receptor for AAV, which probably explains why the AAV delivery of genes to the brain has proven very successful in recent experiments at the center. The new findings will help researchers better understand at the molecular level how the virus enters the cell.
The UNC-CH study not only helps clarify how AAV gets into cells, but also carries important implications for deciding which cells to target for human gene therapy using the virus.
"We will only have a high probability of successfully delivering genes to patient cells if the cells carry virus receptors," Samulski says. "The integrin story tells us that binding is the first step, internalization the second. Now that we know what's involved in bringing the virus into the cell, we can potentially exploit it."
Samulski refers to the next round of research on AAV as a viral vector -- the term used for a virus that carries a healthy gene into the body. "We want to change the virus so that it does something it's not evolved to do -- enter a cell that doesn't have the integrin receptor," he says.
The researcher and his colleagues are also gearing up to find ways to stimulate cells to produce more integrins on their surfaces, thus enhancing the chances of successful gene delivery. Summerford notes that white blood cells called monocytes can be removed from an individual and genetically induced to "express" a greater number of integrins on their surface. These cells may then be infected with AAV and re-infused into that individual.
"Before we can maximize the potential of any viral system for gene therapy, we have to understand the basic virology," she says. "How does the virus initiate infection, how does it attach to and enter the cell? And after it's in the cell, how does it get to the nucleus, and what regulates gene expression? These are very basic questions that need to be answered."
"We just figured out the first two steps of the journey: opening the door and stepping into the passageway," Samulski adds.
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