Incomplete rescue of cystic fibrosis transmembrane conductance regulator deficient mice by the human CFTR cDNA
Incomplete rescue of cystic fibrosis transmembrane conductance regulator deficient mice by the human CFTR cDNARichard Rozmahel1,4, Katalin Gyömörey2,5, Sue Plyte1, Van Nguyen1, Michael Wilschanski2,3, Peter Durie3,6, Christine E. Bear2,5 and Lap-Chee Tsui1,4,*
1Department of Genetics, 2Division of Cell Biology and 3Division of Gastroenterology, The Hospital for Sick Children, Toronto, Ontario, Canada and 4Department of Molecular and Medical Genetics, 5Department of Physiology and 6Department of Pediatrics, University of Toronto, Toronto, Ontario, Canada
Received February 28, 1997;Revised and Accepted April 30, 1997
We have used a mouse model to study the ability of human CFTR to correct the defect in mice deficient of the endogenous protein. In this model, expression of the endogenous Cftr gene was disrupted and replaced with a human CFTR cDNA by a gene targeted `knock-in' event. Animals homozygous for the gene replacement failed to show neither improved intestinal pathology nor survival when compared to mice completely lacking CFTR. RNA analyses showed that the human CFTR sequence was transcribed from the targeted allele in the respiratory and intestinal epithelial cells. Furthermore, in vivo potential difference measurements showed that basal CFTR chloride channel activity was present in the apical membranes of both nasal and rectal epithelial cells in all homozygous knock-in animals examined. Ussing chamber studies showed, however, that the cAMP-mediated chloride channel function was impaired in the intestinal tract among the majority of homozygous knock-in animals. Hence, failure to correct the intestinal pathology associated with loss of endogenous CFTR was related to inefficient functional expression of the human protein in mice. These results emphasize the need to understand the tissue-specific expression and regulation of CFTR function when animal models are used in gene therapy studies.
Cystic fibrosis (CF) is a common autosomal recessive disease characterized by severe obstructive lung pathology, abnormal intestinal tract function and elevated sweat electrolyte levels (1 ). The clinical manifestations of CF are thought to result from misregulation of a cAMP-regulated chloride channel in the apical membranes of epithelial cells (2 ,3 ). Study of the molecular defect underlying CF has been made possible by cloning of the cystic fibrosis transmembrane conductance regulator (CFTR) gene (4 ,5 ). Subsequent expression studies showed that CFTR could not only perform as a cAMP-activated chloride channel (6 -8 ) but that it may also have additional functions (9 -12 ).
To investigate the role of CFTR in CF pathophysiology, several mouse models have been generated (13 -17 ). These `knock-out' animals were produced by gene targeting to ablate endogenous CFTR function and were found to have electrophysiological abnormalities of the respiratory and intestinal epithelia similar to that observed in CF patients (14 -19 ). Different from the human disease in which respiratory lesions are the primary cause of morbidity, however, the majority of CF mice died of intestinal tract obstruction (13 -15 ,17 ). This unique pathology could be explained by the relatively high levels of Cftr expression which is presumably required for normal function of the mouse intestine (20 ).
Since gene therapy presents a promising treatment strategy for the CF-associated lung disease, CF mice have been used as convenient recipients in preclinical trials. To assess the ability of the human CFTR cDNA to correct the pathological lesions in these mice in vivo, several transgenic constructs have been examined (21 -23 ); however, these attempts have been only marginally successful. In studies where a human CFTR transgene driven by the rat fatty acid binding protein (Fabp) promoter was used, only one of several transgenic mice demonstrated apparent amelioration of intestinal pathology (21 ,23 ). Even in the one `corrected' mouse (21 ), although human CFTR transcript was detected in the colon, no electrophysiological rectification was apparent at this site; there was only minor rectification of small intestinal bioelectric properties. However, since the cell-specific expression did not reflect the endogenous situation and the level of human CFTR mRNA relative to endogenous levels was not quantified, the efficiency of the human CFTR to correct overall epithelial bioelectric properties was not resolved. Since strain variability has been documented for CF disease severity in mice, the pathological correction in the single Fabp-hCFTR CF mouse could be due to mechanism(s) only partially related to the transgene activity (17 ). In another study, expression of human CFTR was placed under the control of the villin promoter which had been shown to express along the intestinal villi epithelia, in a pattern more similar to Cftr than that of the Fabp promoter (24 ). None of the Cftr-/Cftr- transgenic mice carrying the villin-hCFTR transgene showed any significant pathological correction, although CFTR expression was demonstrated (22 ).
RNA analyses in humans (25 ) and mice (26 ) suggested that as little as 10% of endogenous CFTR expression was adequate for providing normal respiratory and intestinal functions. It was argued that 10% endogenous Cftr expression was sufficient to confer survival in a large fraction of mutant mice with an exon 10 insertion (CftrHGU/CftrHGU) (26 ), although an abnormal electrophysiological profile was observed. While the minimal amount of CFTR expression required for normal function is still a subject of investigation, the failure of human CFTR cDNA under the control of heterologous promoters to correct the pathology in the CF mice might be due to incorrect cell-specific expression. It is also possible that since the human and mouse CFTR sequences are not completely identical, the human cDNA does not efficiently substitute for the endogenous mouse gene in terms of alleviating the bioelectrical and pathological deficiencies in the CF mice.
To ensure proper cell-specific expression of human CFTR in the CF mice, we have exploited a `knock-in' strategy to generate a transgene construct expressing the human gene in place of the endogenous Cftr gene (CftrhCFTR). In the design, a full-length human CFTR cDNA/minigene (7 ) is inserted into the 5' untranslated region of mouse Cftr exon 1 (Fig. 1 a) via homologous recombination in embryonic stem cells, such that expression of the human cDNA would be under the transcriptional control of the endogenous mouse gene. The strategy is an extension of our previous study (17 ) in which the mouse gene was disrupted through the insertion of a foreign sequence into its exon 1, resulting in `knock-out' animals (Cftrm1HSC/Cftrm1HSC) with no CFTR function and intestinal lesions similar to other severely-affected CF mouse models. If expression of human CFTR could completely compensate for the loss of endogenous Cftr function, the `knock-in' animals would present a normal phenotype with respect to both electrophysiological and pathophysiological parameters.
The `knock-in' gene-targeting vector was electroporated into ES cells (Fig. 1 b) and chimeric animals were produced according to standard techniques (Materials and Methods). As expected, heterozygous F1 mice (CftrhCFTR/Cftr+) were phenotypically normal, with no reduced survival when compared to their wild-type Cftr+/Cftr+ siblings (data not shown). When the heterozygous animals were crossed to produce homozygous F2 animals, however, the fraction ofCftrhCFTR/CftrhCFTR mice was less than that expected from simple autosomal recessive inheritance (+/+:+/hCFTR:hCFTR/hCFTR = 86:159:66; P < 0.25). It was noted that a proportion of mice were lost in the immediate post-natal stage; although their genotype was not determined, these missing animals were presumed to be primarily CftrhCFTR/ CftrhCFTR homozygotes. Moreover, 14 of the remaining 66 CftrhCFTR/CftrhCFTR mice died within 2 weeks and another 17 between 2 and 6 weeks after birth (Fig. 2 ). Taken together, ~60% of the CftrhCFTR/CftrhCFTR mice did not survive beyond 6 weeks, which was slightly, although not significantly (P < 0.25), better than the mortality observed in knock-out Cftrm1HSC/Cftrm1HSC animals (68%) in the same genetic background. Since no significant improvement in survival was evident by these experiments, the animals viable beyond 6 weeks of age were considered to be equivalent to the `mildly-affected' Class III CF animals previously reported by us (17 ), and thus were not genotyped for the CF modifier locus (17 ).
To compare the level of expression of the CftrhCFTRallele to that of the wildtype endogenous alleletotal RNA was extracted from several representative organs of heterozygous animals of 6-12 weeks old. The transcripts were amplified in the form of cDNA by the reverse transcriptase-polymerase chain reaction (RT-PCR) with primers common to both the mouse and human sequences. To distinguish between the two different CFTR transcripts, a BamHI restriction site present in exon 9 of the human but not the mouse gene (Fig. 3 a) was used. The relative amounts of the human and mouse CFTR transcripts could thereby be estimated from the RT-PCR products after BamHI digestion. In our preliminary analysis (data not shown), the amounts of BamHI-sensitive (human) and BamHI-resistant (mouse) products were roughly equal in all tissues examined (brain, lung, kidney, liver and intestinal tract), verifying expression of the human CFTR cDNA from the knock-in allele and suggesting preservation of transcriptional regulation from the modified allele.
To assess epithelial ion transport in these animals, the nasal and intestinal epithelial bioelectric properties of 6-10 week old CftrhCFTR/CftrhCFTR mice was compared to age-matched wildtype controls, Class III Cftrm1HSC/Cftrm1HSC and Cftrm1UNC/ Cftrm1UNC mice by in vivo and in vitro studies. Measurements of basal potential difference (PD) across nasal and rectal epithelia in vivo should reflect the transepithelial resistance and electrogenic transport across basolateral or apical membranes. As previous reported (27 ), the basal nasal PD and amiloride-sensitive component of the basal PD was higher in CF relative to normal mice, suggesting increased amiloride-sensitive sodium absorption in CF nasal mucosa. Nevertheless, whereas the Class III Cftrm1HSC/Cftrm1HSC mice exhibited nasal basal and amiloride-sensitive PD values intermediate between those of wildtype and Cftrm1UNC/Cftrm1UNC mice (Fig. 4 a), the CftrhCFTR/CftrhCFTR animals demonstrated complete `correction' of both bioelectric parameters (Fig. 4 a).
The in vivo studies showed that CFTR was functionally expressed in the nasal and rectal epithelia of CftrhCFTR/CftrhCFTR animals and thus failed to explain the comparable pathological lesions between the homozygous knock-in and knock-out animals. Hence, we sought to determine whether the human CFTR protein was functionally expressed in the small intestine, specifically in regions most severely affected in the CF knock-out animals. Accordingly, we assessed the electrophysiological properties of the jejunal, ileal and proximal colonic epithelia from the different mice using Ussing chamber analysis in vitro.
The basal potential differences (PDs) measured across the ileum, jejunum and proximal colon segments obtained from the Class III Cftrm1HSC/Cftrm1HSC, Cftrm1UNC/Cftrm1UNC and CftrhCFTR/ CftrhCFTR mice were significantly lower than those measured in control mice (Fig. 5 a). This decrease in basal PD likely reflects a decreased electrogenic ion transport capacity in the Cftr knock-out mice since there was no corresponding decrease in transepithelial resistance, except in the proximal colon of the Cftrm1UNC/Cftrm1UNC mice, coincident with marked damage of the mucosa in at these sites (13 -15 ,17 ). The basal PDs measured in the intestinal segments of CftrhCFTR/CftrhCFTR animals were comparable to those of Class III Cftrm1HSC/Cftrm1HSC mice, indicating no overall correction of this electrophysiological parameter.
Using a knock-in strategy, we have generated transgenic mice with tissue-specific human CFTR expression in place of the endogenous gene. RNA analyses showed that the level of hCFTR expression was equivalent to 30-38% of that of the endogenous gene. Since previous studies suggested that 10% CFTR could be sufficient to confer a normal phenotype (25 ,26 ), we were surprised that we did not observe significant improvement in the intestinal pathology nor survival among the homozygous knock-in mice expressing human CFTR as compared to the Cftr-deficient animals. In a previous investigation, in which hCFTR was expressed in CF mice under the control of the Fabp promoter (21 ), a partial correction of intestinal electrophysiology and an apparently complete amelioration of intestinal disease was demonstrated. Although quantitation of transgene expression relative to endogenous CFTR levels was not performed in that study, it is possible that the one corrected line presented with much elevated hCFTR expression relative to the other lines and our `knock-in' model.
Among the surviving knock-in animals, however, we observed `reconstituted' nasal and rectal epithelial bioelectric properties, improved weight gain and normal female reproductive ability relative to the knock-out mice, indicating adequate compensation of the physiological deficits caused by the loss of the endogenous CFTR activity in these tissues. `Correction' of the various CF-associated lesions in these mice appeared to be tissue dependent. The inability to exhibit a normal phenotype in the small intestines of the knock-in mice could reflect either more stringent requirements for high levels of CFTR, or activity of hitherto unknown tissue-specific factors.
It is possible that the human CFTR protein failed to respond to the endogenous cellular signalling pathways in mice. It is also possible that human CFTR failed to regulate other channel activities in the mouse epithelial cells. In support of both of these assumptions, the primary amino acid sequences of the two proteins are only 78% identical, with the highest conservation (81%) for the two ATP-binding domains and the lowest (69%) between the regulatory (R) domains (28 ). In addition, the two proteins appeared to have distinct chloride channel properties (29 ). First, the unitary conductance of murine CFTR is slightly less than that of human CFTR. Second, although both murine and human CFTR channel activity is stimulated by phosphorylation, once activated, the murine CFTR channel exhibits a reduced open probability that is ~10-20% of the human CFTR channel (29 ). This latter observation suggests inherent differences in the activation of the two proteins. The inability to activate the human CFTR protein in the mouse background could be due to specificity of isoforms of signaling molecules, kinases or phosphatases. Consistent with these explanations, although activation of the human CFTR protein appeared to be impaired in mice, basal activity is expected to remain unaffected, as indicated by our electrophysiological data.
The functional expression of CFTR is also required for the activation of other distinct chloride channels, including the outward rectifying chloride channel (30 ) which is known to be present in the mouse intestinal tract (31 ). Thus, an alternative explanation for the apparent inefficiency of human CFTR to correct the cAMP-mediated chloride conductance in CF mice could relate to its inability to interact with or activate the different chloride transport pathways in mouse intestine.
It is of interest to note that the in vitro Ussing chamber measurements demonstrated a correction of the cAMP-elicited chloride current in at least one intestinal segment of two CftrhCFTR/CftrhCFTR animals. The basis for the differences amongst these animals in regulating cAMP-mediated chloride conductance is currently unknown, but could arise from differences in quantity of apically localized CFTR and/or other chloride transport pathways that are activated by CFTR. Alternatively, variations in the signalling molecules required for the full activation of human CFTR or other chloride channels activated by CFTR may underlie the differences in these mice. Since approximately equal levels of CFTR mRNA were detected in the intestinal tracts of all mice examined, it is unlikely that the observed discrepancy originates from differences in levels of transcription or post-transcriptional processing of human CFTR. Furthermore, inherent differences in post-translational maturation, trafficking or stability of the human CFTR protein does not provide a sufficient explanation because apically localized CFTR was equally detected in the nasal and rectal epithelia of all animals. Thus, the observed variability in CFTR activation may be due to differences in intrinsic or alternative CFTR regulatory pathways. As discussed above, survival of the CftrhCFTR/CftrhCFTR mice used in the electrophysiological studies are likely to be due to secondary genetic factors contributed from the CD1 and 129/Sv background (17 ). The differences observed among these mice may reflect the different mechanisms underlying the prolonged survival of these animals as well as the interactions between these regulatory pathways.
In conclusion, although the homozygous CftrhCFTR knock-in mice have tissue-specific expression of human CFTR at ~30% equivalent of the endogenous transcript, they suffer intestinal obstruction leading to early mortality similar to the knock-out animals. The problem in the CftrhCFTR mice appears to be due to their inability to activate the human CFTR chloride channel via cAMP agonists to a completely open state. Lack of full cAMP-mediated activation of CFTR in the absence of endogenous CFTR function results in abnormal intestinal bioelectric properties and intestinal disease. Understanding the differences in regulation between human and murine CFTR proteins may allow us to dissect the complex signalling pathways. The mouse intestine appears to be a highly sensitive system for such studies in vivo. Consequently, it may be necessary to generate transgenic CF mice properly expressing the murine Cftr cDNA in the intestine in order to provide the required CFTR function to keep the animals alive.
The construction of the targeting vector has been previously described (17 ). A full length human CFTR cDNA (7 ) was inserted into the SalI site engineered into the 5' untranslated region of exon 1. The complete plasmid was ~27 kb in size. Prior to electroporation, the vector was linearized at a unique NotI site present within the plasmid sequence (adjacent to the long arm). The vector was then transfected into the R1 ES cell (32 ) that was subsequently selected for homologous recombination according to the positive-negative selection protocol (33 )for resistance to the drug G418 and sensitivity to the drug gancyclovir. The positive-negative selection procedure resulted in an enrichment factor of ~10-fold. Doubly-resistant colonies were expanded and DNA was isolated. To screen for the presence of a homologous recombination event in the doubly-resistant colonies, their genomic DNA was digested with the restriction enzyme BamHI that would produce a 6.0 kb endogenous fragment and a 5.5 kb novel fragment when hybridized with the probe BP0.6, indicative of a homologous recombination event. The digested DNA was separated by agarose gel electrophoresis, Southern blotted, and hybridized with BP0.6. The targeting frequency in this experiment was ~1/20 clones (3/65 doubly selected clones). Characterization of the targeted ES cell clones by Southern blot analyses confirmed that the inserted CFTR cDNA remained intact. Correctly targeted ES cell clones were expanded and introduced into host blastocysts by aggregation techniques (34 ) with morulae from a CD1 mouse background. One male (A2d) demonstrated germline transmission of the targeted CftrhCFTR allele. F1 heterozygotes (CftrhCFTR/Cftr+) were then generated by breeding the A2d founder with CD1 females. Fidelity of the human CFTR cDNA sequence in these animals was confirmed by its amplification from the mouse genomic DNA by the polymerase chain reaction and sequencing.
The heterozygous F1 mice were intercrossed to generate F2 animals that were homozygous for the exon 1 disruption. The CftrhCFTR/CftrhCFTR, Cftr+/Cftr+ and Cftrm1HSC/Cftrm1HSC mice used in these studies were hybrids of CD1 and 129/Sv genetic backgrounds. The Cftrm1UNC/Cftrm1UNC mice contained a mixed genetic background (as described in ref. 13 ). All mice were genotyped by genomic Southern blot analysis of tail clip DNA digested with the BamHI restriction enzyme and hybridized with the BP0.6 probe either at the time of death or time of weaning. The CD1 animals for this study were purchased from Charles River Laboratories. The CftrhCFTR/CftrhCFTR and Cftrm1HSC/Cftrm1HSC animals were maintained on rodent chow in a sterile pathogen-free environment. The Cftrm1UNC/Cftrm1UNC animals were housed under standard housing conditions and maintained on a liquid diet from time of weaning (3 weeks) (35 ).
Total RNA was isolated from tissues of ten 6-10 week old CftrhCFTR/Cftr+ mice using TrizolTM reagent (Gibco-BRL) according to recommended protocols. The RNA sample was treated with preamplification grade DNAse I (Gibco-BRL) according to protocols provided with the SuperscriptTM Preamplification System (Gibco-BRL). A 100 [mu]g sample of the total RNA was polyA+ purified using the oligotex-dTTM (QIAGEN) method according to the manufacturer's instructions. The polyA+ mRNA was reverse transcribed from random primers using the SuperscriptTM Preamplification System (Gibco-BRL) following the manufacturer's specifications. The products of the reverse transcript reactions were amplified by the PCR using the oligonucleotides MHX8 (5'-AGGAGGGATTTGGGGAATTA-3') and MHX10-rev (5'-CATCATAGGAAACACCAAAG-3') which results in a 338 bp product from mouse Cftr exons 8 to 10. The PCR was performed with Taq DNA polymerase and buffer reagents supplied by Gibco-BRL using the conditions of 15 s at 94oC and 30 s at 60oC for 30 cycles. After 30 cycles of PCR, 10 [mu]l of the product was transferred to a new tube containing fresh reagents and put through four additional cycles of PCR to prevent heteroduplexes in the final product. The products of the second round of PCR were digested with BamHI (Gibco-BRL) and analyzed by electrophoresis on a 2.5% agarose gel. This analysis results in the production of two bands, the mouse Cftr-derived PCR product migrates at a mobility of 338 bp while the human CFTR-derived band results from two overlapping bands at a mobility of 168 and 170 bp. The intensity of the ensuing bands was compared by densitometry analysis of the negative image derived from the ethidium bromide stained agarose gel.
The RNA in situ hybridization was performed according to a previously described protocol (36 ) to duodenal sections from one Cftr+/Cftr+ mouse, two Cftrm1HSC/Cftrm1HSC mouse and two CftrhCFTR/CftrhCFTR mice. All mice for this study were between 12 and 18 weeks of age.
The technique for measuring the transepithelial potential difference was adapted from previously published methods(27 ,37 ). Measurements were taken from 9-18 mice to ascertain a mean value and SEM for each group of animals. The measurement of nasal PD involves insertion of a flowing electrode (PE 10 tubing), through which Ringers Lactate solution (Baxter) is perfused at a rate of 1 ml/h into the nose at a point which is 0.5 cm distal to the nares. Amiloride (100 [mu]M) was perfused via a concentric sleeve over the flowing electrode. The flowing electrode and the reference electrode (placed subcutaneously into the abdomen of the animal) were connected via agar bridges (4% agar in Ringers solution) to a digital voltmeter and the signal recorded on a chart recorder. All animals for this study were between 6 and 10 weeks of age.
Transepithelial ion transport measurements were made using Ussing chambers for 10 CftrhCFTR/CftrhCFTR, 18 Cftr+/Cftr+, 14 Cftrm1HSC/Cftrm1HSC and 10 Cftrm1UNC/Cftrm1UNC mice. Following the PD measurements in vivo, animals were euthanized and jejunal, ileal and colonic segments of intestine were dissected and placed in Ringers solution composed of 140 mM NaCl, 2 mM CaCl2, 25 mM HEPES, 3 mM MgCl2 and 5 mM glucose. The intestinal segments were mounted in Ussing chambers over apertures of 28 mm2 (MRA International, Naples, FL). Both mucosal and serosal surfaces of the epithelia were bathed with Ringers solution (except 5 mM mannitol replaced glucose in the serosal compartment) at 37oC and gassed with 95% O2 and 5% CO2. Transepithelial potential measurements were determined under current clamp conditions. Negative PD values indicate that the mucosal compartment is negative relative to the serosal compartment. Pulses (1 [mu]A/min) were passed continuously through the tissue to determine conductance. Once stable, basal values were determined and the tissues were treated with forskolin (10 [mu]M) serosally and mucosally. All statistical comparisons were made using the Student's t-test; the null hypothesis was rejected at P < 0.05.
The authors would like to thank S. Gyömörey for technical assistance, S. Sauvé for secretarial assistance and C. C. Hui for assistance and advice for the RNA in situ hybridization experiments. The work is supported by grants from the Canadian Cystic Fibrosis Foundation (CCFF) to L.-C.T., C.E.B. and P.D., and Howard Hughes Medical Institute (International Scholarship) and the Canadian Genetic Disease Network to L.-C.T. R.R. and K.G. are supported by CCFF Studentships. L.-C.T. is Sellers Chair in Cystic Fibrosis Research at the Hospital for Sick Children and Senior Scientist of MRC; C.E.B. is MRC Scientist; M.W. is CCFF Research Fellow and is supported by Janssen Pharmaceutica and an American Physicians Fellowship.
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*To whom correspondence should be addressed at: Department of Genetics, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada. Tel: +1 416 813 6015; Fax: +1 416 813 4931; Email: lctsui@genet.sickkids.on.ca
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