Lesley J. MacVinish

A placebo-controlled study of liposome-mediated gene transfer to the nasal epithelium of patients with cystic fibrosis

Cystic fibrosis (CF) is a common, serious, inherited disease. The major cause of mortality in CF is lung disease, due to the failure of airway epithelial cells to express a functional product of the cystic fibrosis transmembrane conductance regulator (CFTR) gene. A potential treatment for CF lung disease is the expression of CFTR in the airways following gene transfer. We have undertaken a double-blinded, placebo-controlled, clinical study of the transfer of the CFTR cDNA to the nasal epithelium of 12 CF patients. Cationic liposomes complexed with plasmid containing the human CFTR cDNA were administered to eight patients, whilst four patients received placebo. Biopsies of the nasal epithelium taken 7 days after dosing were normal. No significant changes in clinical parameters were observed. Functional expression of CFTR assessed by in vivo nasal potential difference measurements showed transient correction of the CF chloride transport abnormality in two patients (15 days after dosing in one patient). Fluorescence microscopy demonstrated CFTR function ex vivo in cells from nasal brushings. In total, evidence of functional CFTR gene transfer was obtained in six out of the eight treated patients. These results provide proof of concept for liposome-mediated CF gene transfer.

Generation and characterization of a ΔF508 cystic fibrosis mouse model

We have generated mice carrying the most common mutation in cystic fibrosis (CF), ΔF508, within the cystic fibrosis (Cftr) gene. Mutant animals show pathological and electrophysiological changes consistent with a CF phenotype. ΔF508−/− mice die from peritonitis and show deficiencies in cAMP–activated electrogenic Cl− transport. These mice produce ΔF508 transcripts and show the temperature–dependent trafficking defect first described for the human ΔF508 CFTR protein. A functional CFTR Cl− channel not demonstrated by null CF mice or present at 37 °C was detected following incubation of epithelial cells at 27 °C. Thus, these mice are an accurate ΔF508 model and will be valuable for testing drugs aimed at overcoming the ΔF508 trafficking defect.

Chloride secretion in response to guanylin in colonic epithelial from normal and transgenic cystic fibrosis mice.

1 Guanylin, a 15 amino acid endogenous gut peptide, increased the short circuit current (SCC) in the epithelium of the mouse colon, but only when applied to the apical and not the basolateral surface. 2 By use of selective blockers of epithelial ion transport and modification of the bathing solution, it was concluded that guanylin increased electrogenic chloride secretion but also had a minor effect on electrogenic sodium absorption. In addition there were small residual currents which remained unresolved. 3 The threshold concentration of guanylin causing a SCC increase was less than 50 nm, but at concentrations 40 times greater no indication of a maximally effective concentration was found. 4 Two guanylin isomers with the same amino acid sequence but with the disulphide bridges joined in an alternate fashion showed no activity. Thus only guanylin with the greatest structural homology to heat stable enterotoxin (STa) showed biological activity. 5 The action of guanylin was virtually eliminated in colonic epithelia from transgenic cystic fibrosis (CF) mice. As these animals lack the chloride channel coded by the CF gene sequence, it is likely that the final effector process in murine colonic epithelia involves the CFTR (cystic fibrosis transmembrane conductance regulator) chloride channel. 6 Opportunistic infections of the gut generating STa lead to diarrhoeal conditions via an action of the toxin on apical guanylin receptors. Thus, as discussed, the CF heterozygote may have a genetic advantage in this circumstance.

Importance of basolateral K+ conductance in maintaining Cl− secretion in murine nasal and colonic epithelia

1 Epithelia lining the nasal passages and descending colon of wild‐type and cystic fibrosis (CF) mice were examined by the short‐circuit current technique. Additionally, intracellular Ca2+ ion determinations were made in nasal epithelial cells. Forskolin produced anion secretory currents in wild‐type and CF nasal epithelia. It produced similar effects in wild‐type colonic epithelia, but not in colonic epithelia from CF mice. 2 After electrogenic Na+ transport was blocked with amiloride and electrogenic Cl− secretion was stimulated with forskolin, the ability of K+ channel blockers to inhibit the forskolin‐induced Cl− current was determined. The order of efficiency for nasal epithelium was: Ba2+ > clofilium >>> TEA = azimilide >>> trans‐6‐cyano‐4‐(N‐ethylsulphonyl‐N‐methylamino)‐3‐hydroxy‐2,2‐dimethyl‐chromane (293B) = charybdotoxin, whereas for the colonic epithelium the order was: Ba2+= 293B >>> azimilide = TEA >>> clofilium = charybdotoxin. 3 1‐Ethyl‐2‐benzimdazolinone (1‐EBIO) was able to generate large Cl−‐secretory currents in colonic epithelia which were partially sensitive to charybdotoxin, with the remaining current being inhibited by 293B. In nasal epithelia 1‐EBIO produced only a small transient effect on current. 4 Forskolin released intracellular Ca2+ in nasal epithelial cells; this activity was attenuated when more powerful Ca2+‐releasing agents were applied first. 5 It is concluded that an action on basolateral cAMP‐sensitive K+ channels is an important determinant of the maintained responses to forskolin in nasal and colonic epithelia, in addition to the effects on the cystic fibrosis transmembrane conductance regulator (CFTR) in the apical membrane. In CF nasal epithelia the activation of calcium‐activated chloride channels (CACs) substitutes for the effect on CFTR. On the basis of the different orders of potency of the blocking agents and the differential response to 1‐EBIO it is concluded that the cAMP‐sensitive K+ channels are different in the airways and the gut.

Complementation of null CF mice with a human CFTR YAC transgene

We have made transgenic mice carrying a 320 kb YAC with the intact human cystic fibrosis transmembrane regulator (CFTR) gene. Mice that only express the human transgene were obtained by breeding with Cambridge null CF mice. One line has approximately two copies of the intact YAC. Mice carrying this transgene and expressing no mouse cftr appear normal and breed well, in marked contrast to the null mice, where 50% die by ∼5 days after birth. The chloride secretory responses in these mice are as large or larger than in wild‐type tissues. Expression of the transgene is highly cell type specific and matches that of the endogenous mouse gene in the crypt epithelia throughout the gut and in salivary gland tissue. However, there is no transgene expression in some tissues, such as the Brunners glands, where it would be expected. Where there are differences between the mouse and human pattern of expression, the transgene follows the mouse pattern. We have thus defined a cloned fragment of DNA which directs physiological levels of expression in many of the specific cells where CFTR is normally expressed.

Ion-transporting activity in the murine colonic epithelium of normal animals and animals with cystic fibrosis.

Electrogenic ion transport in the isolated co-Ionic epithelium from normal and transgenic mice with cystic fibrosis (CF mice) has been investigated under short-circuit current (Isc) conditions. Normal tissues showed chloride secretion in response to carbachol or forskolin, which was sensitive to the Na-K-2Cl cotransport inhibitor, frusemide. Responses to both agents were maintained for at least 12 h in vitro, but the responses to carbachol changed in format throughout this period. By contrast CF colons failed to show the normal secretory responses to carbachol and forskolin, most preparations showing a decrease in Isc that was immediately reversed by frusemide. In CF colons addition of Ba2+ ions or tetraethylammonium (TEA+) to the apical bathing solution antagonised the reduction in Isc caused by the secretagogues. It is concluded that the reduction in Isc in CF colons is due to electrogenic K+ secretion and this was confirmed by flux studies using rubidium-86. In normal colons exposed to TEA+ the responses to for-skolin were greater, but not significantly so, presumably because the minor K+-secretory responses are dominated by major chloride-secretory responses. Again rubidium-86 fluxes showed an increase of K+ secretion in normal colons receiving forskolin. Since the amiloride-sensitive current was not different in CF and normal colons there was no evidence that the CF mice were stressed in a way that increased mineralocorticoid levels and hence K+ secretion. Knowledge of the phenotype of the colonic epithelium of the CF mouse sets the baseline from which attempts at gene therapy for the gut must be judged.

Activation of Ca2+- and cAMP-sensitive K+ channels in murine colonic epithelia by 1-ethyl-2-benzimidazolone

1-Ethyl-2-benzimidazolone (EBIO) caused a sustained increase in electrogenic Cl- secretion in isolated mouse colon mucosae, an effect reduced by blocking basolateral K+ channels. The Ca2+-sensitive K+ channel blocker charybdotoxin (ChTX) and the cAMP-sensitive K+channel blocker 293B were more effective when the other had been added first, suggesting that both types of K+ channel were activated. EBIO did not cause Cl- secretion in cystic fibrosis (CF) colonic epithelia. In apically permeabilized colonic mucosae, EBIO increased the K+ current when a concentration gradient was imposed, an effect that was completely sensitive to ChTX. No current sensitive to trans-6-cyano-4-( N-ethylsulfonyl- N-methylamino)-3-hydroxy-2,2-dimethylchromane (293B) was found in this condition. However, the presence of basolateral cAMP-sensitive K+channels was demonstrated by the development of a 293B-sensitive K+ current after cAMP application in permeabilized mucosae. In isolated colonic crypts EBIO increased cAMP content but had no effect on intracellular Ca2+. It is concluded that EBIO stimulates Cl-secretion by activating Ca2+-sensitive and cAMP-sensitive K+ channels, thereby hyperpolarizing the apical membrane, which increases the electrical gradient for Cl- efflux through the CF transmembrane conductance regulator (CFTR). CFTR is also activated by the accumulation of cAMP as well as by direct activation.

Calcium‐and cyclic AMP‐dependent chloride secretion in human colonic epithelia

1 Three stable epithelial cell lines (HCA‐7, HCA‐7‐Col 1 and HCA‐7‐Col 3) all derived from the same human adenocarcinoma have been cultured on collagen‐coated Millipore filters. These epithelial monolayers have been used to record short circuit current (SCC) in response to a variety of secretagogues. Similar monolayers, but grown on plastic dishes, were used for measurements of tissue cyclic AMP. 2 Lysylbradykinin, applied to either side of the monolayers, increased SCC in HCA‐7 cells but had little effect on the other two lines. The responses showed rapid desensitization, which could be prevented by cooling to 4°C. Responses to kinin were not significantly attenuated by piroxicam, an inhibitor of cyclo‐oxygenase. 3 Other secretagogues, vasoactive intestinal polypeptide (VIP) and carbachol also increased SCC in monolayers. The responses to VIP were greatest in HCA‐7‐Col 1 monolayers while responses were virtually absent in HCA‐7‐Col 3. A similar profile was seen with carbachol except that responses of HCA‐7 and HCA‐7‐Col 1 monolayers were more equal. With one exception the responses to VIP and carbachol showed sidedness, acting only from the basolateral side. 4 The effects of the secretagogues were inhibited by piretanide, a loop diuretic, applied basolaterally. It is presumed that SCC responses represent electrogenic chloride secretion. 5 Treatment with forskolin increased SCC in HCA‐7 and HCA‐7‐Col 1 monolayers with little effect in HCA‐7‐Col 3. Nevertheless cyclic AMP levels were elevated most in HCA‐7‐Col 3 and least in HCA‐7‐Col 1 monolayers, in reciprocal relationship to the functional response. 6 A23187 increased SCC when applied to HCA‐7 and HCA‐7‐Col 3 monolayers with little effect on HCA‐7‐Col 1. 7 The differential responses of the three human cell lines provide unique opportunities to discover the functional responsibilities of entities involved in the chloride secretory process. HCA‐7‐Col 3 cells which generate high levels of cyclic AMP in response to forskolin but which fail to show a substantial chloride secretory response may be a useful model of some disease conditions.

Kinin effects on ion transport in monolayers of HCA-7 cells, a line from a human colonic adenocarcinoma

Using epithelial monolayers of HCA‐7 cells, derived from a primary human colonic adenocarcinoma and grown on pervious supports, it is shown that responses to lysylbradykinin can be elicited from either side. It is proposed that kinin receptors are inserted into both apical and basolateral membrane domains.

A second dose of a CFTR cDNA–liposome complex is as effective as the first dose in restoring cAMP-dependent chloride secretion to null CF mice trachea

Phase I clinical trials have provided encouraging data suggesting that gene transfer could provide a treatment for cystic fibrosis (CF). However, for all the current viral and nonviral vectors used to deliver the cystic fibrosis transmembrane conductance regulator (CFTR) gene, the duration of CFTR expression is limited, necessitating a repeat dosing regimen to provide a long-term treatment. This study was performed to determine whether a second delivery of a CFTR cDNA–liposome complex could result in a similar level of functional CFTR expression observed after a single delivery and to assess whether the deliveries produced adverse inflammatory responses. CFTR functional expression was assessed by short circuit current measurements of tracheas taken from CF null mice (Cftrtm1Cam) treated with a CFTR cDNA–liposome complex in the upper airways. Mice receiving two deliveries of this complex, the second after the response to the first had declined, showed cAMP-stimulated chloride currents which were not significantly different from normal tracheas or tissues assayed after a single dose of the complex. This double treatment was well tolerated with no discernible inflammation of lung tissue.