Alan W. Cuthbert

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.

Repeat administration of DNA/liposomes to the nasal epithelium of patients with cystic fibrosis.

The major cause of mortality in patients with cystic fibrosis (CF) is lung disease. Expression of the cystic fibrosis transmembrane conductance regulator (CFTR) gene product in the airways is a potential treatment. Clinical studies in which the CFTR cDNA was delivered to the respiratory epithelia of CF patients have resulted in modest, transient gene expression. It seems likely that repeated administration of the gene transfer vector will be required for long-term gene expression. We have undertaken a double-blinded study in which multiple doses of a DNA/liposome formulation were delivered to the nasal epithelium of CF patients. Ten subjects received plasmid DNA expressing the CFTR cDNA complexed with DC-Chol/DOPE cationic liposomes, whilst two subjects received placebo. Each subject received three doses, administered 4 weeks apart. There was no evidence of inflammation, toxicity or an immune response towards the DNA/liposomes or the expressed CFTR. Nasal epithelial cells were collected 4 days after each dose for a series of efficacy assays including quantitation of vector-specific DNA and mRNA, immunohistochemistry of CFTR protein, bacterial adherence, and detection of halide efflux ex vivo. Airway ion transport was also assessed in vivo by repeated nasal potential difference (PD) measurements. On average, six of the treated subjects were positive for CFTR gene transfer after each dose. All subjects positive for CFTR function were also positive for plasmid DNA, plasmid-derived mRNA and CFTR protein. The efficacy measures suggest that unlike high doses of recombinant adenoviral vectors, DNA/liposomes can be successfully re-administered without apparent loss of efficacy.

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.

KININS STIMULATE NET CHLORIDE SECRETION BY THE RAT COLON

1 Short circuit current (SCC), transepithelial conductance and ion fluxes were measured across the isolated descending colon of the rat in response to bradykinin or kallidin. 2 Kinins added to the serosal bath caused immediate increases in SCC but were ineffective when added to the mucosal bath. Increases in SCC were accompanied by significant increases in transepithelial conductance. Threshold kinin concentration was 0.5 nm and maximal increases were seen at 50–100 nm. 3 A rat glandular kallikrein (7 nm) or mellitin (2 μm) also increased SCC if added to the serosal bath. 4 Responses to kinins were unaffected by mucosal amiloride (100 μm) but attenuated or blocked by serosal frusemide (100 μm), indomethacin (1 μm) or mepacrine (50 μm). 5 Replacement of chloride ion in the serosal bath by gluconate and sulphate ions abolished responses to kinins which reappeared after chloride re‐addition. 6 Measurement of 36Cl, 22Na and 86Rb fluxes across the tissue showed that the kinin‐induced increase in SCC resulted principally from increased net chloride secretion. Effects upon 22Na or 86Rb flux were minimal and made no contribution to the current responses observed in this tissue. 7 The results prove that kinins stimulate net chloride secretion in the rat colon, most probably via a prostaglandin‐dependent pathway.

The effect of neuropeptide Y and peptide YY on electrogenic ion transport in rat intestinal epithelia.

1. Neuropeptide Y (NPY), peptide YY (PYY) and, to a lesser extent, human pancreatic polypeptide (HPP) reduced short‐circuit current (SCC) in a concentration‐dependent manner in epithelial preparations of rat jejunum and descending colon. 2. From concentration‐response curves in the jejunum EC50 values of 3 nM for PYY and 10 nM for NPY were obtained. HPP was much less potent, the threshold concentration being around 100 nM, and NPY 13‐36 was inactive. 3. Repeated exposure of jejunal preparations to either NPY or PYY led to a rapid desensitization. Cross‐desensitization to the actions of these two peptides was also observed. Neither tetrodotoxin (TTX) nor phentolamine affected responses to either NPY or PYY on the jejunum. 4. Responses to both peptides were inhibited by the presence of transport inhibitors, particularly diphenylamine‐2‐carboxylate (DPC, a chloride channel blocker) and piretanide (Na+‐K+‐2Cl‐ co‐transport inhibitor). These results may indicate that the reduction in SCC caused by the neuropeptides is due to a net increase in chloride movement in the apical to basolateral direction. 5. 36Cl‐flux studies identified an inhibition of chloride secretion as the predominant mechanism of action of NPY and PYY, together with a smaller stimulation of chloride absorption. No significant changes in the movement of 22Na were seen in either direction. 6. The cyclo‐oxygenase inhibitors piroxicam (5 microM) and indomethacin (5 microM) significantly reduced the responses to both NPY and PYY in rat jejunum. From this and other evidence it was concluded that the peptides depended for their effect on the endogenous formation of eicosanoids, the prevention of which attenuated the SCC reduction due to the peptides.

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.

WNK1 affects surface expression of the ROMK potassium channel independent of WNK4.

The WNK (with no lysine kinase) kinases are a novel class of serine/threonine kinases that lack a characteristic lysine residue for ATP docking. Both WNK1 and WNK4 are expressed in the mammalian kidney, and mutations in either can cause the rare familial syndrome of hypertension and hyperkalemia (Gordon syndrome, or pseudohypoaldosteronism type 2). The molecular basis for the action of WNK4 is through alteration in the membrane expression of the NaCl co-transporter (NCCT) and the renal outer-medullary K channel KCNJ1 (ROMK). The actions of WNK1 are less well defined, and evidence to date suggests that it can affect NCCT expression but only in the presence of WNK4. The results of co-expressing WNK1 with ROMK in Xenopus oocytes are reported for the first time. These studies show that WNK1 is able to suppress total current directly through ROMK by causing a marked reduction in its surface expression. The effect is mimicked by a kinase-dead mutant of WNK1 (368D > A), suggesting that it is not dependent on its catalytic activity. Study of the time course of ROMK expression further suggests that WNK1 accelerates trafficking of ROMK from the membrane, and this effect seems to be dynamin dependent. Using fragments of full-length WNK1, it also is shown that the effect depends on residues in the middle section of the protein (502 to 1100 WNK1) that contains the acidic motif. Together, these findings emphasize that the molecular mechanisms that underpin WNK1 regulation of ROMK expression are distinct from those that affect NCCT expression.

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.

Effects of some pyrazinecarboxamides on sodium transport in frog skin.

1 The inhibitory effect of amiloride (N‐amidino‐3,5‐diammo‐6‐chloropyrazinecarboxamide) on sodium transport in isolated skin of frog has been compared with 17 of its analogues. The dissociation constant of amiloride for passive sodium channels was 181.9 ± 8.9 nm, and the maximal percentage inhibition of sodium transport was 101.3 ± 0.4% (means of 123 measurements) when measured at a sodium concentration of 111 mm. 2 The N‐benzylamidino and N‐o‐chlorobenzylamidino compounds had affinities approximately 20 times larger than those for amiloride, and produced maximal inhibition of transport. 3 Substitution of chlorine in the 6‐position by other halogens showed that the bromo‐compound was equally active to amiloride, whereas the iodo derivative had an affnity equal to 15% of that for amiloride. 4 Substitution in the 5‐amino group in 10 compounds reduced the affinities to less than 1% of that of amiloride, without affecting their ability to produce complete inhibition of transport. 5 N‐Amidino‐3,5‐diaminopyrazinecarboxamide was unique in that it produced an unusual concentration‐response relationship.