ABCMdb

PMID: 18388934

Davies LA, Varathalingam A, Painter H, Lawton AE, Sumner-Jones SG, Nunez-Alonso GA, Chan M, Munkonge F, Alton EW, Hyde SC, Gill DR
Adenovirus-mediated in utero expression of CFTR does not improve survival of CFTR knockout mice.
Mol Ther. 2008 May;16(5):812-8. Epub 2008 Mar 11., [PubMed]

Sentences

No. Mutations Sentence Comment

11 ABCC7 p.Ser489* ABCC7 p.Ser489* After vector delivery at 16 days of gestation (equivalent to a 15-to 20-week gestation period in humans), transgene expression is observed in the lung, stomach and gut and is highly correlated with increased fetal breathing movements.4 Several CF transgenic mouse strains have been generated in an attempt to model and evaluate potential treatment strategies, but although these mouse models display many of the -CF-related ion-transport bioelectric defects found in human CF tissues, lung pathology is not recapitulated.5 However, some abnormalities of the CF gastrointestinal tract such as intestinal obstruction are observed in mouse models, where they can be very severe, leading to poor survival.6 The CF knockout mouse strain S489X carrying the Cftrtm1Unc mutation develops intestinal obstruction leading to intestinal perforation, peritonitis and death, resulting in only 5% survival to adulthood,7 although these mice can survive given the appropriate dietary intervention.8 Survival of these mice was also reported to increase dramatically when recombinant adenovirus expressing human CF transmembrane conductance regulator (hCFTR) was delivered via intra-amniotic injection to fetal mice in utero.9 The improved survival was observed even though the adenovirus-mediated CFTR expression was transient, implying that continuous expression of CFTR was not required, therefore challenging the widely held view that it is the absence of the CFTR chloride channel that leads to CF disease in the adult. Login to comment 52 ABCC7 p.Ser489* Effect of hCFTR expression on survival of cftr -/- mice Any conclusion regarding the effectiveness of in utero hCFTR expression in correcting the lethal intestinal defect in S489X Cftrtm1Unc /Cftrtm1Unc mice will be wholly dependent on the number of Cftr -/- mice surviving after injection of AdCFTR compared with injection of control AdLacZ vector. Login to comment 57 ABCC7 p.Ser489* ABCC7 p.Ser489* Finally, to assess the impact of in utero delivery of AdCFTR on the survival of Cftr -/- mice, S489X litters were injected at E16 a pCIKCFTR 195 kd 112 kd Band C Band B AdGFP AdCFTR b 0 -0.1 0.0 Rateof 125 |efflux/min 0.1 0.2 0.3 0.4 0.5 1 2 Time (min) 3 4 Untreated AdLacZ AdCFTR T84 cells c 0 0.01 Untreated AdCFTR 0.1 1 10 100 1,000 hCFTRmRNAcopies/ngRNA (3) (6) (7) (4) (13) (4) (+/+) (+/-) (-/-) d Untreated AdCFTR 0 0.01 0.1 1 10 100 hCFTRmRNAcopies/ngRNA (3) (6) (7) (5) (15) (5) (+/+) (+/-) (-/-) Figure 3 Human cystic fibrosis transmembrane conductance regulator (hCFTR) can be detected in lungs and intestines of S489X Cftrtm1Unc mice following intra-amniotic injection of AdCFTR. Login to comment 63 ABCC7 p.Ser489* Expression of hCFTR mRNA was confirmed in vivo following intra-amniotic injection of 108 plaque forming units (pfu)/fetus of AdCFTR at E16 to litters resulting from heterozygote matings of S489X Cftrtm1Unc mice. Login to comment 68 ABCC7 p.Ser489* Table 1 In utero injection and survival of S489X Cftrtm1Unc /Cftrtm1Unc mice Untreated AdLacZ 107 pfu AdCFTR 107 pfu AdCFTR 108 pfu Litters 21 17 15 13 Injected 0 142 114 102 Lost NA 11 14 6 Born 156 131 100 96 Day 0 -/- 25 20 20 22 +/- 87 54 45 48 +/+ 42 48 30 25 Unknown 2 9 5 1 Day 100 -/- 4 2 2 3 +/- 70 28 39 29 +/+ 33 28 26 13 Unknown 0 0 0 0 Abbreviations: CFTR, cystic fibrosis transmembrane conductance regulator; pfu, plaque forming units; NA, not applicable. Login to comment 69 ABCC7 p.Ser489* Litters from heterozygote S489X matings were injected with AdLacZ or AdCFTR at 16 days of gestation. Login to comment 97 ABCC7 p.Ser489* 0 0 20 40 60 Days after birth 80 100 20 40 60 80 100 Percentsurvival (+/+) (+/-) (-/-) a 0 0 20 40 60 Days after birth 80 100 20 40 60 80 100 Percentsurvival c 0 0 20 40 60 Days after birth 80 100 20 40 60 80 100 Percentsurvival Untreated AdLacZ 107 AdCFTR 107 AdCFTR 108 b 0 0 20 40 60 Days after birth 80 100 20 40 60 80 100 Percentsurvival d Figure 4 Survival of S489X Cftrtm1Unc mice following expression of human cystic fibrosis transmembrane conductance regulator (hCFTR) from AdCFTR. Login to comment 98 ABCC7 p.Ser489* Kaplan-Meier plots of S489X Cftrtm1Unc survival following intra-amniotic injections. Login to comment 105 ABCC7 p.Ser489* It was unfortunate that the S489X Cftrtm1Unc mouse strain dem- onstratedsuchhighlevelsofmaternalcannibalismandneglectlead- ing to a high mortality of newborn pups of all genotypes, especially following surgical interventions. Login to comment 112 ABCC7 p.Ser489* An important difference is the ~20% survival of the Cftr -/- mice observed in our facility compared with a previously reported 5%.14 The intestinal pathology and mortality in CF transgenic mouse models can vary dependent on the precise Cftr knockout mutation, environmental influences and the independent segregation of modifier genes.6,17 The survival chances of one CF mouse model Cftrtm1G551D /Cftrtm1G551D were shown to vary between 27 and 67% depending on undetermined environmental factors in the animal breeding facility.18 Interestingly, when we transferred a subset of our S489X Cftr -/- mice to an alternative animal facility they died of intestinal obstruction within a few days (data not shown). Login to comment 115 ABCC7 p.Ser489* Unavoidably, our studies used S489X Cftrtm1Unc CF knockout mice as a 10th generation backcross onto to C57BL/6 background, as opposed to a 5th generation backcross.10 In the accompanying paper, Buckley et al.16 attempted independent verification of the original study, using the same adenoviral vector, but using an alternative knockout CF mouse allele (Cftrtm1Cam /Cftrtm1Cam ) on an inbred genetic background (129 S6Sv/Ev) to minimize genetic heterogeneity and the effects of potential modifier genes. Login to comment 124 ABCC7 p.Ser489* The Cftr-mutant UNC mouse strain, S489X (Cftrtm1UNC / Cftrtm1UNC ) was obtained as a 10th generation backcross on a C57BL/6 background (Dr Julia Dorin, MRC Human Genetics Unit, Edinburgh, UK) and was maintained via heterozygote matings in open cages with hardwood sawdust and R&M3 breeders` diet (Special Diet Services, Witham, UK) with weaning at 21 days. Login to comment 142 ABCC7 p.Ser489* To generate time-mated pregnancies, heterozygote S489X Cftrtm1Unc females were introduced to cages containing heterozygote males overnight with the following day regarded as day 1 of gestation (E1) in resulting pregnancies. Login to comment