By Frances Bishopp

Gene therapy company, Targeted Genetics Corp., and the Medical College of Ohio received notice of allowance of a broad U.S. patent that covers technology for production of adeno-associated viral (AAV) vectors using stable cell lines capable of expressing the AAV replication gene using any promoters other than the native AAV promoter.

A formidable barrier in the development of AAV vectors for gene therapy has been the difficulty of generating large quantities of pharmaceutical-grade product.

The components required to manufacture an AAV vector include a permissive cell in which to grow the vector; a replication (rep) gene, which allows the vector to be produced in quantity; a capsid (cap) gene, which is needed to form the three-dimensional viral vector particles containing the therapeutic gene to be delivered; promoter sequences required to turn on the rep and cap genes; and a helper virus, such as adenovirus, which facilitates production of the vector.

Jon Case, director of corporate development, explained there are two ways to make AAV vectors: a transient approach or a stable packaging cell line.

In order to manufacture AAV vectors, most groups have used the transient production method. That involves the stepwise introduction into cells (by transfection) of one or more DNA plasmids containing the rep gene, the cap gene and the vector and then requires exposing the cells to an adenovirus, which activates the replication and assembly of the vector.

Plasmid transfection methods have disadvantages, in that there is a need to produce multiple bulk raw materials, purification of the product is difficult, and the process is cumbersome and less amenable to scale-up.

A more commercially acceptable approach and one used to manufacture most recombinant biological products, Case said, involves the establishment of stable mammalian cell lines which, in the case of AAV vector product, already contain the necessary rep and cap genes and the vector critical to the production process.

A cell line-based manufacturing method reduces the number of steps necessary in production, provides a more scaleable system and appears to result in a more easily purified product.

"The transient approach requires so many run-throughs of the process and so much labor and material, it is likely to be impractical to do it on a large scale," said Stephen Lupton, associate director of corporate development at Targeted Genetics.

The patent covers the use of stable cell lines that use many non-native promoter sequences to turn on the rep gene.

Using any promoter (DNA sequences that control expression of the gene) other than the native AAV promoter could allow the achievement of higher levels of expression and higher levels of vector protection with the protein than is possible using the native AAV promoter, Lupton said.

Also, Lupton said, a major problem with generating the cell lines is the rep protein either stops cells from growing or it actually kills growing cells.

Regulation is an issue, Case added, because if too much of the rep protein is produced at the wrong time, it can kill the cells.

"Using other promoters gives us the opportunity to regulate expression of the rep gene in ways that it wouldn't be possible using the native AAV promoter," Lupton said.

Cell Line Approach More Flexible

Instead of starting with a cell line that has nothing in it, Case said, "you create a cell line that has whatever gene you are trying to package and you eventually have an entity that includes the gene of interest and rep and cap inside the cell line. Once you do this, you never have to do it again," Case said.

"This method gives us great flexibility," he added.

Targeted Genetics is the exclusive licensee of this patent, which is based on the work of James Trempe, at the Medical College of Ohio at Toledo.

The company also has patents pending which would provide coverage of stable cell lines using a native AAV promoter sequence to activate the rep gene.

Seattle-based Targeted Genetics' lead product, tgAAV-CFTR, currently in Phase II clinical trials, is designed to correct the genetic defects responsible for cystic fibrosis.

Barbara Hoffman, an analyst with Vector Securities International Inc., of Deerfield, Ill., said Targeted Genetics, which is trading at a market cap of $60.6 million and a technology value of $45 million could be a candidate for acquisition in that the valuations compare favorably to other publicly traded gene and cellular therapy companies and are dramatically below valuations historically paid for such companies.

"This company has 'know-how' as well as patents," Hoffman said. "I think their cystic fibrosis gene therapy is the leader in that field."

Targeted Genetics had $15.6 million in cash as of March 31, 1997. Its stock (NASDAQ:TGEN) closed Monday at $3.125, up $0.125. *