REHOVOT, Israel — Gene therapy has taken one more step forward as Israeli scientists have harnessed a second member of the parvovirus family to direct transfected genes to a specific location on the host chromosome.

This discovery promises to significantly affect longevity and intended functioning of introduced genes, as it could allow researchers to insert them at specified positions where their expression will be maximized.

"One of the big questions remaining in genetic engineering is how to ensure that a foreign gene introduced into a nucleus will find its way to that site on the chromosome where it will be integrated, and function as intended," said Ernest Winocour, professor in the molecular genetics department at the Weizmann Institute of Science. "The critical characteristics for the success of gene therapy include exact placement on the chromosome, but at present, ways of delivering a new gene to a predetermined location on a cell's chromosomes are limited."

Winocour and colleagues Jacov Tal, professor at Ben-Gurion University of the Negev School of Medicine, and postdoctoral fellow Joe Corsini published their new method in the December issue of the Journal of Virology.

"The striking feature of parvoviruses is their single-mindedness. They are the only animal viruses that integrate themselves into the cell's chromosomes at unique sites," Tal said.

Knowing in advance where the virus vector is directing the introduced gene gives greater control over what the gene will do once inserted into the organism's genome; location means sequence and sequence influences expression.

"If a healthy replacement copy for a faulty, disease-causing gene will survive and replicate every time the cell divides, that is what will enable gene therapy with a long-lasting effect," Winocour said.

It was previously known that one member of the parvovirus family, called adeno-associated virus (AAV), could deliver a gene to a specific site on human chromosome 19.

The new member of the parvovirus family, called minute virus of mice, or MVM, targets a specific site on an episome, using DNA-encoded signals that differ from those used by AAV.

This biomolecular tool will help define the genetic sequences signaling and directing the insertion of therapeutic genes into a number of different sites. The scientists revealed the mechanism that allows MVM to zero in on a particular target site. They were then able to replicate this mechanism, which consists of signals exchanged by the virus and the chromosomes, in a model system.

"Further research needs to be done to see if our method works in laboratory animals," Winocour said. "If successful, it can have far-reaching applications in medicine and animal breeding."

Yeda Research and Development Co., the Weizmann Institute's technology transfer arm, and Ben-Gurion University of the Negev have filed a patent application for the new method.

Recent studies with a collaborating group at Cornell Medical College, in New York, showed site-specific AAV integration is directed by DNA sequence motifs that are present in both the viral replication origin and the chromosomal pre-integration DNA.

Repairing The Linkage

A sequence of nucleotides in the target and in the viral DNA puts the introduced gene into register with the appropriate chromosomal site, nicks the DNA strand and sets up a local DNA replicase to repair the gap and form a covalent linkage.

These sequences specify binding and nicking sites for the viral regulatory protein, and differ from parvovirus to parvovirus.

Other parvoviruses under investigation have been isolated from a wide variety of animals, from humans to crustacea. They differ in strategies of gene expression, but have in common the activation of the origin by the binding and nicking processes mediated by nonstructural regulatory proteins.

The AAV and MVM belong to distant parvovirus genera, but both succeed in targeting. If the entire parvovirus family — with 50 known members, each of which might be capable of inserting itself at a specific location — is harnessed, it will provide scientists with an extremely useful and versatile array of gene vectors.

These may provide a repertoire of targeting signals for the design of parvovirus vectors intended to integrate at defined genetic loci.

"The implication is that different members of the large parvovirus family may be able to direct therapeutic genes to a number of chosen chromosomal sites, which could then be assessed for maximum expression and minimum disturbance to the resident chromosomal genes," said Winocour.

With support by the German-Israeli Foundation for Scientific Research and Development, studies are continuing with other parvoviruses.

No Comments