Last week the first cystic fibrosis patient entered the firstin vivo clinical trial of an untried gene therapy deliveryvehicle _ adeno-associated virus (AAV).
The year-long, Phase I study, at Johns HopkinsChildren's Center in Baltimore, will test the safety anddosage of the novel vector, developed by parvovirologistBarrie Carter and his team at Targeted Genetics Corp., inSeattle. The trial is under the direction of Hopkinspediatric pulmonologist Terence Flotte.
The genetically engineered AAV sequence will transfer agene encoding CFTR _ cystic fibrosis transmembraneconductance regulator _ which is missing or mutated incystic fibrosis sufferers. A flexible fiberopticbronchoscope, under direct vision, instilled the geneconstruct into the superior segment of the right lower lobeof the patient's lung, Flotte told BioWorld Today.
The day before, he and his team had dripped thetransgene material into and onto airway surface epithelialcells in one of her nostrils.
Flotte said, "We were blinded as to which nasal sidecontained the vector, and which one only a controlsolution. As the patient didn't know either, it was double-blinded."
This first patient, he said, "is a 45-year-old woman with afamily history of cystic fibrosis, but relatively mildsymptoms of lung disease."
Carter told BioWorld Today that the patient will befollowed by 11 others, all adults with mild but clear-cutcystic fibrosis, treated one month apart to escalating nasaland pulmonary doses of the transgene package.
"The reason for that," Carter explained, "is that whilewe've done extensive safety studies in rabbits andprimates, we've never used AAV vectors before inpatients.
"This Phase I trial," he added, "is designed to be done byanalogy with the first cystic fibrosis gene therapy studies,by Ron Crystal, of New York Hospital/Cornell MedicalCenter, and Jim Wilson, at the University of Pennsylvaniain Philadelphia.
"Our patients," Flotte said, "at least initially, will beisolated for two weeks following administration of therecombinant virus. We'll monitor viral shedding, a riskperiod we think will be much shorter than two weeks.Then we'll be able to be much nicer to our patients."
Introducing AAV, Which Leans On AV for Support
The earlier cystic fibrosis gene therapy trials in New Yorkand Philadelphia, now being reevaluated, employedadenovirus (AV) rather than AAV as their gene transfervector.
"AV," Carter pointed out, "is a human pathogen thatcauses respiratory disease, such as common-coldsymptoms. As a transgene vector, it has a lot of viralgenes, which aren't especially necessary for thetherapeutic application, and may or may not cause avariety of complications, including adverse cellularimmune responses. AV has to be re-engineered so as notto be pathogenic any longer."
AAV is called "associated" with AV, because it can'treplicate at all, except in the presence of the latter virus.
"AAV is a human virus, which has never been reported tocause any disease," Carter observed. "It's a parvovirus,one of many, some of which are disease-causing. ButAAV is defective, and can't replicate by itself."
This has made AAV a tempting candidate vector toreplace AV in gene therapy experiments. Carter, then atthe National Institute for Diabetes, Digestive and KidneyDiseases, pioneered development of the first AAVvectors in 1984, five years before CFTR was cloned in1989. That year marked the founding of TargetedGenetics, which Carter joined in 1992.
He and Flotte have since collaborated on working up tothe Phase I clinical trial, which Flotte launched lastMonday at Hopkins. He is co-director of the NationalInstitutes of Health-supported Cystic Fibrosis GeneTherapy Center there.
"The AAV virion has two genes," Carter explained, "repand cap. Cap codes for the capsid, or protein shell, ofAAV. Rep is required for replication, in addition to thehelper function provided by AV. In order to make ourAAV vector," he continued, "we've deleted both of thesegenes. In fact, the only gene present in our construct is theone that encodes CFTR. No viral genes at all, just twosmall, 145-nucleotide, inverted terminal repeats hitchedonto the CFTR's cDNA. You need them to package thevector."
"So now AAV is doubly defective: It can't replicatewithout AV, and also because we've removed its repgene."
Targeted Genetics holds an exclusive, worldwide licenseto the NIH patent covering the construct.
Rabbits that in 1992 and 1993 received the AAV-transferred CFTR gene instillation, targeted to onepulmonary lobe, continued to show gene expression forup to six months after treatment, Carter reported at thetime. Since then, he said, "We have done analogousstudies in 20 or so rhesus monkeys, with the clinicalAAV vector. Again we can see dose-dependent deliveryof vector, with persistence and expression out through sixmonths."
He pointed out that safety and dosage data from the just-begun Phase I trial, programmed to dose 12 subjectsstepwise at one-month intervals, need not await the fullyear. "We should begin to get numbers from some of theearlier patients before that, so it may be speeded uptoward the end," he said.
After Safety And Dosage Comes Efficacy
To confirm gene transfer, Flotte said, "we will employcytologic brushing to get small samples of cells fromnasal orifices and lung segments, for DNA, RNA andprotein analyses. For this Phase I study," he pointed out,"the main outcome that we can evaluate rigorously issafety. Patient numbers are too small to test for any kindof efficacy."
That will involve a subsequent, larger, clinical trial,Carter observed, "Call it a follow-up Phase I, Phase II orI/II."
After the ongoing study ends, he added, "The obviousplace to go next, is to deliver vector to the whole lung,since this is the organ we ultimately want to treat in cysticfibrosis, not just one lobe of it.
"By actually getting it into patients, we have hopefullyopened the door for other people to study several of theissues that are particular to AAV in a better modelsystem _ which is Homosapiens, if you will." n
-- David N. Leff Science Editor
(c) 1997 American Health Consultants. All rights reserved.