Leslie Stratford-Perricaudet

In vivo transfer of the human cystic fibrosis transmembrane conductance regulator gene to the airway epithelium

Direct transfer of the normal cystic fibrosis (CF) transmembrane conductance regulator (CFTR) gene to airway epithelium was evaluated using a replication-deficient recombinant adenovirus (Ad) vector containing normal human CFTR cDNA (Ad-CFTR). In vitro Ad-CFTR-infected CFPAC-1 CF epithelial cells expressed human CFTR mRNA and protein and demonstrated correction of defective cAMP-mediated Cl- permeability. Two days after in vivo intratracheal introduction of Ad-CFTR in cotton rats, in situ analysis demonstrated human CFTR gene expression in lung epithelium. PCR amplification of reverse transcribed lung RNA demonstrated human CFTR transcripts derived from Ad-CFTR, and Northern analysis of lung RNA revealed human CFTR transcripts for up to 6 weeks. Human CFTR protein was detected in epithelial cells using anti-human CFTR antibody 11-14 days after infection. While the safety and effectiveness remain to be demonstrated, these observations suggest the feasibility of in vivo CFTR gene transfer as therapy for the pulmonary manifestations of CF.

Widespread long-term gene transfer to mouse skeletal muscles and heart.

Successful treatment of muscular disorders awaits an adapted gene delivery protocol. The clinically applicable technique used for hematopoietic cells which is centered around implantation of retrovirally modified cells may not prove sufficient for a reversal of phenotype when muscle diseases are concerned. We report here efficient, long-term in vivo gene transfer throughout mouse skeletal and cardiac muscles after intravenous administration of a recombinant adenovirus. This simple, direct procedure raises the possibility that muscular degenerative diseases might one day be treatable by gene therapy.

Diversity of airway epithelial cell targets for in vivo recombinant adenovirus-mediated gene transfer.

A variety of pulmonary disorders, including cystic fibrosis, are potentially amenable to treatment in which a therapeutic gene is directly transferred to the bronchial epithelium. This is difficult to accomplish because the majority of airway epithelial cells replicate slowly and/or are terminally differentiated. Adenovirus vectors may circumvent this problem, since they do not require target cell proliferation to express exogenous genes. To evaluate the diversity of airway epithelial cell targets for in vivo adenovirus-directed gene transfer, a replication deficient recombinant adenovirus containing the Escherichia coli lacZ (beta-galactosidase [beta-gal]) gene (Ad.RSV beta gal) was used to infect lungs of cotton rats. In contrast to uninfected animals, intratracheal Ad.RSV beta gal administration resulted in beta-gal activity in lung lysate and cytochemical staining in all cell types forming the airway epithelium. The expression of the exogenous gene was dose-dependent, and the distribution of the beta-gal positive airway epithelial cells in Ad.RSV beta gal-infected animals was similar to the normal cell differential of the control animals. Thus, a replication deficient recombinant adenovirus can transfer an exogenous gene to all major categories of airway epithelial cells in vivo, suggesting that adenovirus vectors may be an efficient strategy for in vivo gene transfer in airway disorders such as cystic fibrosis.

Gene Therapy: The Advent of Adenovirus

Adenovirus has been developed as an alternative gene transfer vehicle which has emerged as a potent vector with a promising future. Infection of cells with replication-incompetent adenoviruses allows the production of foreign proteins encoded by the engineered viruses without otherwise affecting the host cell. Of utmost relevance to gene therapy, postmitotic cells seem particularly adapted to gene transfer by adenovirus, since the viral genome is not integrated into the cell’s chromosome and presumably persists the life of the cell. Administration of recombinant adenoviruses directly in vivo opens the way to new therapeutic strategies. The first successful somatic gene therapy of a hepatic enzyme deficiency in an animal model was achieved using an adenovirus (Stratford-Perricaudet et al., 1990). Since this initial demonstration of the feasibility of correcting a genetic defect with adenovirus, many potential applications have been imagined. The field has aroused much enthousiasm as reflected at the December 1992 RAC meeting where approval was given to carry out clinical trials using three different recombinant adenoviruses capable of expressing the human CFTR, one of which was constructed in our laboratory (Rosenfeld et al., 1992).

Adenovirus-mediated In Vivo Gene Therapy

Adenoviruses are widespread in nature since they have been found in many mammalian and avian species. All members of the adenovirus family have similar chemical and physical properties, but can be distinguished by their individual type-specific antigens. In man, 47 distinct serotypes (Ad1 to Ad47) that form six groups (A to F) have been isolated to date, most of them during the decade following their discovery by Rowe (1953). Host specificity seems to be very stringent inasmuch as no spread from one host species to another has ever been reported. An abortive growth cycle usually results after infection of cells with virus from another species. Clinical illness associated with adenovirus infection depends on the serotype, but is usually mild and rarely life-threatening. The primary target for adenovirus cytopathology is the epithelial cell. Productive infections of human adenoviruses take place in gastrointestinal, respiratory, or ocular epithelial cells, resulting in pathological alterations mediated by direct tissue damage. For example, some serotypes (Ad 3, 20) are associated with “swimming pool conjunctivitis” or gastroenteritis (Ad40), while Ad5 is responsible for respiratory illnesses. The near-terminally differentiated quiescent cells of the upper respiratory tract lining are the natural host cells in which Ad5 replicates. From the primary sites of infection the viral progeny then enter the bloodstream and spread to the other body tissues.