In Utero Efficacy of Cystic Fibrosis Gene Therapy: Difficult Studies, Positive or Negative
Authors: Terence R Flotte
Journal: Molecular Therapy (2008)

In the first decade after the discovery of the cystic fibrosis transmembrane conductance regulator (CFTR), there was great expectation that delivery of CFTR with robust viral vector systems might result in clinical efficacy for CF patients in the relatively short term. This view was fueled by numerous demonstrations of robust gene expression in the lungs of a wide range of animal systems, including rodents, rabbits, and nonhuman primates.1Engelhardt JF Simon RH Yang Y Zepeda M Weber-Pendleton S Doranz B et al.Adenovirus-mediated transfer of the CFTR gene to lung of nonhuman primates: biological efficacy study.Hum Gene Ther. 1993; 4: 759-769Crossref PubMed Scopus (205) Google Scholar,2Rosenfeld MA Siegfried W Yoshimura K Yoneyama K Fukayama M Stier LE et al.Adenovirus-mediated transfer of a recombinant alpha 1-antitrypsin gene to the lung epithelium in vivo [see comments].Science. 1991; 252: 431-434Crossref PubMed Scopus (672) Google Scholar The most commonly used vector system, recombinant adenovirus (rAd), demonstrated the highest efficiency and most rapid rate of onset of expression in these early reporter gene studies. Demonstration of biological efficacy of rAd-CFTR gene therapy in animal models was considerably more challenging for several reasons. The Cftr knockout and mutant mouse disease models that were developed did not spontaneously develop lung disease in a manner similar to that in CF patients. Furthermore, the original mouse strains experienced early mortality due to intestinal obstruction, which made it difficult to study the respiratory tract phenotype in more detail.3Snouwaert JN Brigman KK Latour AM Malouf NN Boucher RC Smithies O et al.An animal model for cystic fibrosis made by gene targeting.Science. 1992; 257: 1083-1088Crossref PubMed Scopus (770) Google Scholar Intermediate end points, such as tracheal chloride potential measurements, were also problematic, primarily because alternative cyclic adenosine monophosphate (cAMP)-activated chloride currents were detected in murine trachea. In this context, a study by Larson et al. reported that in utero rAd-CFTR gene transfer by intra-amniotic fluid injection resulted in dramatic improvement in survival of Cftr knockout mice,4Larson JE Morrow SL Happel L Sharp JF Cohen JC Reversal of the cystic fibrosis phenotype in mice by gene therapy in utero.Lancet. 1997; 349: 619-620Abstract Full Text Full Text PDF PubMed Scopus (93) Google Scholar even though CFTR expression was of very limited duration. This publication led to an expectation that even a transient vector-mediated expression of CFTR by a rAd vector, if given at a critical interval of lung development, might have a long-lasting therapeutic effect. That original report has been examined critically for several reasons. First, relatively few data were presented with intermediate end points, such as specific mechanisms of longer-term effects of transient CFTR messenger RNA (mRNA) or protein expression or electrophysiologic correction of cAMP-activated chloride transport, although subsequent work suggested a mechanism based on stretch-induced differentiation. Second, other groups have not yet reported similarly positive results. It was suggested that the findings could be explained by genetic variation among the Cftrtm/Cam mice, which at that time were available only on a hybrid genetic background. However, the highly challenging technical nature of these studies has prevented a clear determination of whether in utero Ad-CFTR is worth pursuing further. The articles by Davies et al.5Davies LA Varathalingam A Painter H Lawton AE Sumner-Jones SG Nunez-Alonso GA et al.Adenovirus-mediated in utero expression of CFTR does not improve survival of CFTR knockout mice.Mol Ther. 2008; 16: 812-818Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar and Buckley et al.6Buckley SMK Waddington SN Jezzard S Bergau A Themis M MacVinish LJ et al.Intra-amniotic delivery of CFTR-expressing adenovirus does not reverse cystic fibrosis phenotype in inbred CFTR-knockout mice.Mol Ther. 2008; 16: 819-824Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar in this issue of Molecular Therapy undertake the very difficult task of attempting to definitively determine the inability to reproduce the work of another group's positive efficacy result. Such studies always present inherent challenges, even though it is absolutely essential to undertake this work. There is a natural tendency toward prejudgment of such studies. Depending on the perspective, one may be predisposed to expect a negative result because of the unexpected nature of the original report. Conversely, one may be predisposed to dismiss any negative result, because there may be countless minor technical details that one could cite as discrepancies. Because of these challenges, it is essential that these confirmatory studies be meticulously controlled. The strength of the new studies reported in this issue is that both include such controls and updated assays. Both groups used the appropriate strains of mice and vector (including C57/BL6 congenic mice and the original Cambridge mice, and first-generation A25 vector) and confirmed robust expression of the CFTR transgene in all groups. Finally, both studies made use of a sufficient number of animals to demonstrate that the untreated controls produced the expected survival curves.5Davies LA Varathalingam A Painter H Lawton AE Sumner-Jones SG Nunez-Alonso GA et al.Adenovirus-mediated in utero expression of CFTR does not improve survival of CFTR knockout mice.Mol Ther. 2008; 16: 812-818Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar,6Buckley SMK Waddington SN Jezzard S Bergau A Themis M MacVinish LJ et al.Intra-amniotic delivery of CFTR-expressing adenovirus does not reverse cystic fibrosis phenotype in inbred CFTR-knockout mice.Mol Ther. 2008; 16: 819-824Abstract Full Text Full Text PDF PubMed Scopus (27) Google Scholar Davies et al. first made use of fluorescent-labeled particles of a diameter matching that of Ad virions (100 nm) to optimize the timing of intra-amniotic injection so as to obtain the most favorable biodistribution of particles to the target organs of interest (lung and intestine).5Davies LA Varathalingam A Painter H Lawton AE Sumner-Jones SG Nunez-Alonso GA et al.Adenovirus-mediated in utero expression of CFTR does not improve survival of CFTR knockout mice.Mol Ther. 2008; 16: 812-818Abstract Full Text Full Text PDF PubMed Scopus (28) Google Scholar They then confirmed the ability to transduce lung and intestines of the mice following intra-amniotic delivery of an rAd-luciferase reporter administered on day 16 of gestation at a dose matching that of the Larson et al. study (1 × 108 plaque-forming units, PFUs) and went on to reproduce the CFTR gene transfer experiment under similar circumstances in the Cftrtm1UNC homozygous mouse strain. Under these carefully matched circumstances, with gene transfer and expression confirmed, no survival advantage was observed. Buckley et al. fully replicated the study design of Larson et al., using intra-amniotic delivery at day 16 of gestation of exactly the same dose (1 × 108 PFUs) as well as a 20-fold higher dose (2 × 109 PFUs) of the rAd-CFTR vector with an rAd-lacZ vector control. They used mice carrying the same Cftrtm/Cam allele, but in this instance on a completely inbred genetic background (129 S6Sv/Ev). They were able to confirm CFTR complementary DNA presence and mRNA expression in these mice, and yet they did not observe any differences in survival between the treatment and control groups. Furthermore, the treated mice that did survive to necropsy displayed the obstructive intestinal pathology that is typical of Cftr-deficient mice. It is important to point out that pure-background strains of Cftr mutant mice and several relevant techniques were not readily available in 1997 when the article by Larson et al. was published. There also still may be some inconsistency between vector titers, which could be resolved by comparing each back to the Ad5 reference standard. Although the lack of “home run” efficacy from in utero rAd-CFTR is inevitably disappointing, the tools to measure efficacy of CF gene therapy in animal models have been substantially improved. This provides significant hope that future versions of CF gene therapy can be tested and refined and retested with much greater certainty and efficiency. Examples of this greater emphasis on detailed interim analysis can be seen in work done by the United Kingdom Cystic Fibrosis Gene Therapy Consortium (http://www.cfgenetherapy.org.uk), which did a highly systematic comparison of potential nonviral agents before entering into an ongoing phase I nasal instillation trial with a cationic lipid-plasmid DNA complex to deliver CFTR, and in a recently presented collaborative study7Flotte TR Fischer A Goetzmann J Mueller C Poirier A Cebotaru L et al.Direct comparative analysis of rAAV1 and rAAV5 pseudotyped vectors using aerosol delivery of Firefly and Renilla luciferase reporters co-delivered to the lungs of chimpanzees [Abstract].In Vivo Barriers to Gene Delivery. 2007; (November 2007; Cold Spring Harbor, NY, p.6, abstract No. 6): 26-29Google Scholar to determine the optimal recombinant adeno-associated virus serotype for the next rAAV-based CFTR aerosol trial. In both instances, detailed studies in CFTR mouse models have been complemented by meticulous studies of the vector constructs in a variety of cell culture and other systems. Only through such careful preparation can experiments be truly informative, regardless of the outcome.