In the first clinical attempt to transfer genes directly into cells,five patients with advanced, untreatable, disseminatedmelanoma received injections of DNA straight into their skinlesions in hopes of inciting their immune systems to reject thetumors as foreign.

In all five test cases the transferred genes expressed theirantigenic proteins in the tumors. Equally important, none of thepatients produced antibodies against the alien DNA orexperienced any toxic side effects from the experimentaltreatment. And in one of the five trial patients, tumorsregressed not only at the site of DNA injection but in remotelung metastases.

"It was sort of a Phase I trial, so although we had someencouraging responses to treatment, it would be premature toinclude anything about efficacy," said molecular biologist GaryNabel of the University of Michigan Medical Center, the study'sprincipal investigator.

Nabel and his co-workers reported their pioneering genetherapy trial in Wednesday's issue of the Proceedings of theNational Academy of Science (PNAS), in an article titled "Directgene transfer with DNA-liposome complexes in melanoma:Expression, biologic activity, and lack of toxicity in humans."

Until now, human gene therapy efforts PP all requiring pre-approval by the Recombinant-DNA Advisory Committee (RAC)to the National Institutes of Health and the FDA PP have mixedtherapeutic genetic materials with cells removed from a patientin Petri dishes or similar ex vivo vessels. Nabel's favorableresults demonstrated that the direct DNA transfer approachworks, and marks out the path to imminent future in vivo genetransfer trials.

Nabel's team is leading with a second clinical study under thesame RAC review, which on Feb. 10, 1992, authorized him tointroduce DNA directly into a total of 24 test subjects with end-stage melanomas and other cancers.

Beginning usually with a small bluish-reddish wart-like patchor bump on the skin, melanoma swifty strikes inward to spreadmetastases in lungs, liver and other visceral organs. If caughtearly, wide surgical excision can often halt this deadlyprogression. Otherwise, chemotherapy and radiation are oflittle effect, and an estimated 6,000 Americans die ofmelanoma every year.

The three men and two women in the Michigan trial -- four ofthem in their 60s -- were beyond other treatment and had alife expectancy of less than one year. The only weapon left inthe therapeutic arsenal was their own immune response,specifically killer T cells aroused and beefed up to attack theirtumors.

This strategy called for a highly immunogenic trigger, a Class Itransplantation antigen that Nabel's team chose as itsdesignated hitter. This HLA-B7 is the same immune systemprotein that assaults and destroys incompatible organ grafts.By the same token, the researchers reasoned, most peoplewould recognize its gene as foreign and raise their killer T cellhackles against it.

The scientists constructed a cDNA that encoded this B7immunogen and inserted it in a modified Rous sarcoma virusplasmid. Then they wrapped this package in a liposome-likesheath and injected it directly into the melanotic lesions.

"We put the transplantation gene into the tumor," Nabel toldBioWorld. "The immune system sees it as a foreign antigen andresponds to it by elaborating cytokines that stimulate immunedefenses locally. Then these cytolytic T lymphocytes go on torecognize other tumors that we haven't injected, in a so-called'bystander effect.' "

This effect caused metastases to regress in the lungs of onepatient, a 68-year-old man, as well as the lysing of local skinnodules remote from the injection sites. "So presumably theseimmune cytotoxic T cells were free to circulate to those siteswhere they could recognize the tumor," Nabel observed.

"However, the antigen gene's expression was site-specific," headded. "That is, we found the B7 protein in the tumors, but notelsewhere in the body. This is fairly important; we're beginningto find ways to target genes to specific sites."

Another patient experienced a fivefold increase in theprecursors to killer T cells after treatment.

Nabel is now recruiting patients for his second in vivo trial,which will also be Phase I, or rather Phase I/II, but with twodifferences. "We will have a new and improved liposomepreparation and a better vector, both developed by Phil(Felgner) at Vical," Nabel said.

Biochemist Felgner is chief scientist at Vical Inc. of San Diego.The University of Michigan has licensed Nabel's gene therapytechnology to Vical, and Nabel sits on the company's scientificadvisory board.

"The improved vectors we're producing here we refer to as'cytofectins,' " Felgner explained to BioWorld. "These arecationic phospholipid molecules that interact spontaneouslywith DNA. They actually ensheath the DNA in a lipid envelope.Not as a liposome would, but more like a viral envelope, inclose association with DNA strands."

Vical's second improvement, Felgner added, "is to make thosesynthetic gene constructs synthesize more gene product. So weare optimizing the promoter to produce plasmids that expresshigher levels of the transgene." This will enable Nabel to give100-fold to 1,000-fold higher doses of DNA than in his firsttrial.

Vical will use another cytofectin version, dubbed 'allovectin' inin vivo gene therapy trials of its own, for which the company isseeking approval at the RAC meeting this Friday. "The trialsVical sponsors will look similar to the type of protocols thatGary Nabel is engaged in," Felgner said. They, too, will treatmelanomas and other solid tumors PP RAC willing.

Also, if RAC concurs on Friday, John Kovach, who chairs theMayo Clinic's oncology department and directs its NationalCancer Institute-designated Comprehensive Cancer Center, willundertake the third in vivo direct gene transfer trial, inconsultation with Nabel.

"We believe Dr. Nabel's approach should be evaluated in avariety of diseases," Kovach told BioWorld, "particularly thecommon cancers for which we have no effective therapy, oncethey are in a disseminated state. We are asking RAC'spermission to evaluate this in vivo approach involved intransfer of MHC Class I genes into colon carcinomas metastaticto the liver. We will see if it elicits an antibody and/orcytotoxic T cell response, not only to transduced cells, but tocells at a different site that did not receive the vector."

-- David N. Leff Science Editor

(c) 1997 American Health Consultants. All rights reserved.