A common cause of the common cold is the adenovirus. Thispathogenic perpetrator of acute respiratory infection is now performingcommunity service as a DNA-delivery vehicle for gene therapy ofcystic fibrosis (CF).Gene therapists at four institutions are conducting Phase I clinical trialsof the adenovirus vector for ferrying the CF transmembraneconductance regulator gene to the airways of patients. The preliminarystudies, to evaluate toxicity rather than efficacy, are taking place at theUniversity of Iowa and Genzyme Corp; University of North Carolina;Cornell University and the National Institutes of Health; and theUniversity of Pennsylvania.A fifth entry, Jeffrey Whitsett, at the University of Cincinnati, is poisedto join this line-up, said cell biologist John F. Engelhardt, JamesWilson's collaborator at the University of Pennsylvania.Engelhardt is lead author of a paper published Tuesday in theProceedings of the National Academy of Sciences (PNAS), whichreports efforts to overcome adenovirus' shortcomings as a vector. Itstitle: "Ablation of E2A in recombinant adenovirus improves transgenepersistence and decreases inflammatory response in mouse liver.""First-generation" adenoviruses currently conscripted as gene therapyvectors have been stripped of genes enabling them to replicate in cellsthey infect with DNA payloads. "Current E1-deleted recombinantadenoviruses," reads the PNAS paper, "have shown tremendouspromise for the treatment of inherited and acquired diseases by genetherapy."Nevertheless, Engelhardt told BioWorld Today, "Most of the animalmodels tested to date, leading to these Phase I trials, "have generatedtwo severe limitations with the adenovirus vectors, although these arethe best available DNA delivery systems." For one thing, he explained,their useful life within the target organ is very short; for another,inflammations flare up at the organ site.The Goal: To Match Nude-Mouse PersistenceThe most prominent drawback, he said, is the lack of persistence invivo. "In all immune-competent animal models we've studied to date,transgene expression in liver or lung did not persist past 14 days." Innude mice _ which lack T cell immunity _ the vector-deliveredgenes stayed on the job for 100 days or longer, without diminishedexpression of their therapeutic product."Our hypothesis for generating vectors with increased persistence,"Engelhardt and Wilson wrote in PNAS, "is that the presentrecombinant adenoviruses express viral proteins that stimulate cellularimmune responses leading to destruction of the infected cells [bycytotoxic T cells] and repopulation of the organ with non-transgene-containing cells."This observation set the stage for Engelhardt and Wilson to explorewhat additional viral genes they might remove, to render their vectorialadenovirus "invisible to the recipient's immune system," and soachieve persistence on a par with nude-mouse performance.Such a result would make gene therapy much more practical,Engelhardt foresees, with repeat DNA transfers perhaps at annualintervals rather than monthly, as now envisaged.Their first sequence alteration in this direction was to introduce asingle-base-pair mutation into a viral gene within the b-galactosidase-expressing adenovirus viral genome. This encoded a temperature-sensitive DNA-binding protein called E2A. This mutation enhancedpersistence in a temperature-related manner.The mutant strikingly lengthened the shelf-life of the adenovirus-delivered DNA in mice made transgenic. Animals that received thesecond-generation vector expressed the transgene for at least 70 days_ a five-fold improvement over the first-generation's 14 days."E2A," Engelhardt explained, "is involved in viral DNA replication, aswell as transcriptional regulation of other viral genes. Either of thesemechanisms could be involved in the increased persistence, anddecreased inflammatory immune response." But he hastened to add,"We don't invoke elevating the temperature of patients to make thisvector more effective. Rather, we use this second-generationadenovirus to prove the principle that if we go in and alter the viralsequences, and further handicap these vectors, then we can change thetherapeutic outcome."So the next key step in Wilson and Engelhardt's ongoing effort "is togo back and completely get rid of that mutant sequence."He went on, "We were very excited to see this type of response bymodifying these vectors. It opens up a whole new field for vectoringwith these particular viruses, increasing their efficacy and safety forgene therapy."Asked about recent proposals to do away with vectors altogether, andinject transgenes directly into their target organs, Engelhardt said,"Direct DNA injection has worked well in muscle, but to myknowledge is not successful in airways. So for our gene therapydirected at CF, injection doesn't seem to be an efficacious method."Cornell's Ronald Crystal, who pioneered development of adenovirusvectors, wonders whether Wilson's temperature-sensitive mutantapproach will be useful in humans."I think it's an academic question," he told BioWorld Today. "From acommercial point of view, we've passed on that strategy. I don't thinkit would be enormously useful in terms of final products." Crystalfounded GenVec, Inc., of Rockville, Md., last year to pursue genetherapy commercially. (See BioWorld Today, Oct. 20, 1993, p. 2.)"But from an academic point of view," he added, "I think it's a usefulidea, and should be tried. I'm sure Jim [Wilson] is going to come to theRAC and the FDA with it, probably at the next meeting or the one afterthat."For CF gene therapy, Crystal thinks the first-generation adenovirusvector "is not going to solve the problem. We'll have to have second-,third- and fourth-generation vectors _ which we're working very hardon."He predicts that adenovirus' cytotoxic T cell problem "will be solvedwithin a year," but emphasizes, "It's the humoral immunity that's aworry. Because to antibodies, adenoviruses are just a bag of proteinswith some nucleic acid. If antibodies go to the site where you'redelivering the transgene, they may prevent you from giving it a secondtime."One strategy around that potential pitfall, he suggests, is to getconsiderably longer persistence of expression. But, he noted, "In ourfirst four patients, we've seen no neutralizing antibodies at all."For short-term therapy," Crystal declared, "as in cancer orcardiovascular disease, where you only need expression for days orweeks, I think adenoviruses are going to be terrific. There's noquestion they're the gold standard for in vivo delivery." n

-- David N. Leff Science Editor

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