Allen Lapey

Generation of Multipotent Lung and Airway Progenitors from Mouse ESCs and Patient-Specific Cystic Fibrosis iPSCs

Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development, we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm, then into replicating Nkx2.1+ lung endoderm, and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP, FGF, and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs), creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice.

Steatorrhea and azotorrhea and their relation to growth and nutrition in adolescents and young adults with cystic fibrosis

Fecal fat and nitrogen excretion were determined in 20 patients, 12 to 17 years of age, with cystic fibrosis (CF); all had pancreatic deficiency. Current clinical and nutritional status and growth achievement in relation to fecal nutrient loss, age at diagnosis, and adequacy of treatment were evaluated. Steatorrhea and azotorrhea were present to a variable extent (at times massive) in all patients studied, but bore little relation to existing intestinal symptoms, state of nutrition, growth achieved, or age at diagnosis. Many of the patients with better growth, nutrition, and prognostic scores had been diagnosed relatively late in life and presumably had not received adequate or optimal treatment. Addition of pancreatic supplements resulted in significant, but not dramatic decreases in fecal losses of fat and nitrogen. It was concluded that despite pancreatic deficiency most older patients with CF need little dietary restriction, although treatment should be individualized. Growth failure and state of nutrition seem to be correlated more closely with the pulmonary state than with pancreatic deficiency. For ultimate prognosis, natural variation in the severity of lung involvement is at least as important as early diagnosis and adequate or optimal treatment.

Aerosol and Lobar Administration of a Recombinant Adenovirus to Individuals with Cystic Fibrosis. II. Transfection Efficiency in Airway Epithelium

A phase I clinical trial was conducted in which recombinant adenovirus containing the cystic fibrosis trans-membrane regulator (CFTR) (Ad2/CFTR) was administered by bronchoscopic instillation or aerosolization to the lungs of cystic fibrosis (CF) patients. In this paper, we evaluate the efficiency of Ad2/CFTR-mediated transduction of bronchial airway cells. The ability of an Ad2/CFTR vector to transduce airway cells was first evaluated in patients to whom the vector was administered by bronchoscopic instillation. Cells at the administration site were collected 2 days after treatment by bronchoscopic brushing. Ad2-specific CFTR DNA was detected in four of five individuals by PCR, and Ad2-specific CFTR RNA was detected in three of five individuals by RT-PCR. Ad2/CFTR-mediated transduction of airway epithelial cells was then determined in CF individuals receiving this vector by aerosol inhalation. Ad2-specific CFTR DNA was detected in 13 of 13 individuals 2 days after aerosolization, and in 3 of 5 individuals 7 days after aerosolization. Ad2-specific RNA was detected in 4 of 13 individuals on day 2, but was not detected in the 5 individuals tested on day 7. The percentage of airway epithelial cells containing nuclear-localized vector DNA was < or =2.4% as determined by fluorescence in situ hybridization (FISH). However, in some cases, a high percentage of nonepithelial mononuclear cells or squamous metaplastic epithelial cells was infected with the adenoviral vector. In conclusion, aerosol administration is a feasible means to distribute adenoviral vectors throughout the conducting airways, but improvements in adenovirus-mediated transduction of airway epithelial cells are necessary before gene therapy for CF will be effective.

Aerosol and lobar administration of a recombinant adenovirus to individuals with cystic fibrosis. I. Methods, safety, and clinical implications

Cystic fibrosis (CF), an autosomal recessive disorder resulting from mutations in the cystic fibrosis trans-membrane conductance regulator (CFTR) gene, is the most common lethal genetic illness in the Caucasian population. Gene transfer to airway epithelium, using adenoviruses containing normal CFTR cDNA, leads to transient production of CFTR mRNA and, in some studies, to correction of the airway epithelial ion transport defect caused by dysfunctional CFTR. Inflammatory responses to the adenoviral vector have been reported, particularly at high viral titers. We evaluated the effects of adenovirus-mediated CFTR gene transfer to airway epithelium in 36 subjects with CF (34 individuals, 2 of whom received two separate doses of vector), 20 by lobar instillation and 16 by aerosol administration. Doses ranged from 8 x 10(6) to 2.5 x 10(10) infective units (IU), in 0.5-log increments. After lobar administration of low doses there were occasional reports of cough, low-grade temperature, and myalgias. At the highest lobar dose (2.5 x 10(9) IU) two of three patients had transient myalgias, fever, and increased sputum production with obvious infiltrates on CT scan. After aerosol administration there were no significant systemic symptoms until the 2.5 x 10(10) IU dose, when both patients experienced myalgias and fever that resolved within 24 hr. There were no infiltrates seen on chest CT scans in any of the patients in the aerosol administration group. There were no consistent changes in pulmonary function tests or any significant rise in serum IgG or neutralizing antibodies in patients from either group. Serum, sputum, and nasal cytokines, measured before and after vector administration, showed no correlation with adenoviral dose. Gene transfer to lung cells was inefficient and expression was transient. Cells infected with the vector included mononuclear inflammatory cells as well as cuboidal and columnar epithelial cells. In summary, we found no consistent immune response, no evidence of viral shedding, and no consistent change in pulmonary function in response to adenovirus-mediated CFTR gene transfer. At higher doses there was a mild, nonspecific inflammatory response, as evidenced by fevers and myalgias. Overall, vector administration was tolerated but transfer of CFTR cDNA was inefficient and transgene expression was transient for the doses and method of administration used here.

Safety, pharmacokinetics, and pharmacodynamics of ivacaftor in patients aged 2–5 years with cystic fibrosis and a CFTR gating mutation (KIWI): an open-label, single-arm study

BACKGROUND Ivacaftor has been shown to be a safe, effective treatment for cystic fibrosis in patients aged 6 years or older with a CFTR gating mutation. We aimed to assess the safety, pharmacokinetics, and pharmacodynamics of ivacaftor in children aged 2-5 years. METHODS In the two-part KIWI study, we enrolled children aged 2-5 years weighing 8 kg or more with a confirmed diagnosis of cystic fibrosis and a CFTR gating mutation on at least one allele from 15 hospitals in the USA, UK, and Canada. Participants received oral ivacaftor 50 mg (if bodyweight <14 kg) or 75 mg (if bodyweight ≥14 kg) every 12 h for 4 days in part A (to establish the short-term safety of doses for subsequent assessment in part B), and then for 24 weeks in part B (to assess safety and longer-term pharmacodynamics). Children could participate in both or just one part of the study. Primary outcomes were pharmacokinetics and safety, analysed in all patients who received at least one dose of ivacaftor. Secondary outcomes were absolute change from baseline in sweat chloride concentrations and bodyweight, body-mass index (BMI), and height Z scores, and pharmacokinetic parameter estimation of ivacaftor. This study is registered with ClinicalTrials.gov, number NCT01705145. FINDINGS Between Jan 8, 2013, and March 1, 2013, nine patients were enrolled onto part A of the study, all of whom completed the 4 day treatment period, and eight of whom took part in part B. Between June 28, 2013, and Sept 26, 2013, 34 patients were enrolled in part B, 33 of whom completed the 24 week treatment period. All patients received at least one dose of ivacaftor. Results of ivacaftor pharmacokinetics suggested that exposure was similar to that reported in adults (median Cmin were 536 ng/mL for the 50 mg dose; 580 ng/mL for the 75 mg dose; median ivacaftor AUC values were 9840 ng × h/mL and 10 200 ng × h/mL, respectively). Common adverse events in part B included cough (in 19 [56%] of 34 patients) and vomiting (in ten [29%]). Five (15%) patients had liver function test (LFT) results that were more than eight times higher than the upper limit of normal, four of whom had study drug interrupted, and one of whom had study drug discontinued. Six (18%) of 34 patients had seven serious adverse events; a raised concentration of transaminases was the only serious adverse event regarded as related to ivacaftor and the only adverse event that resulted in study treatment discontinuation. At week 24, in patients for whom we had data, sweat chloride had changed from baseline by a mean of -46·9 mmol/L (SD 26·2, p<0·0001), weight Z score by 0·2 (0·3; p<0·0001), BMI Z score by 0·4 (0·4, p<0·0001), and height Z score by -0·01 (0·3; p=0·84). INTERPRETATION Ivacaftor at doses of 50 mg and 75 mg seems to be safe in children aged 2-5 years with cystic fibrosis with a gating mutation followed up for 24 weeks, although the frequency of elevated LFTs suggests that monitoring should be frequent in young children, particularly those with a history of elevated LFTs. Results of an ongoing extension study assessing durability of these effects and longer-term safety are warranted. FUNDING Vertex Pharmaceuticals Incorporated.

Pooled analysis of two large randomised phase III inhaled mannitol studies in cystic fibrosis

BACKGROUND To evaluate safety and efficacy of inhaled mannitol treatment in subgroups of a large global CF population. METHODS Data were pooled from two multicentre, double-blind, randomised, controlled, parallel group phase III studies in which 600 patients inhaled either mannitol (400 mg) or control (mannitol 50 mg) twice a day for 26 weeks. RESULTS Both the mean absolute change in FEV(1) (mL) and relative change in FEV(1) by % predicted from baseline for mannitol (400 mg) versus control were statistically significant (73.42 mL, 3.56%, both p<0.001). Increases in FEV(1) were observed irrespective of rhDNase use. Significant improvements in FEV1 occurred in adults but not children (6-11) or adolescents (aged 12-17). Pulmonary exacerbation incidence was reduced by 29% (p=0.039) in the mannitol (400 mg) group. CONCLUSIONS Sustained six-month improvements in lung function and decreased pulmonary exacerbation incidence indicate that inhaled mannitol is an important additional drug in the treatment of CF.

Altered membrane sodium transport in Bartter's syndrome

To explore the possibility that Bartters syndrome is the manifestation of an inherited abnormality of sodium transport, we have measured various parameters of sodium transport in erythrocytes from patients with Bartters syndrome, their siblings, and their parents. Sodium transport in six of the eight patients with Bartters syndrome differed significantly from that in the other two patients. On the basis of this difference, the patients were divided into two groups (type I and type II). In the six type I patients, fractional sodium outflux (0.38+/-0.05/hr [SD]) was significantly less than normal (0.50+/-0.07) and erythrocyte sodium concentration (9.48+/-0.84 mmoles/liter cells per hr) was significantly greater than normal (5.24+/-0.66). In the two type II patients, none of the measured parameters of sodium transport differed significantly from normal. Erythrocyte sodium transport in the relatives of three type I patients was altered in a way similar to that in the type I patients and was significantly different from that in the relatives of a type II patient. These findings indicate the presence of inherited alterations of erythrocyte sodium transport in certain patients with Bartters syndrome.

Abnormal membrane sodium transport in Liddle's syndrome

We have documented the presence of abnormal sodium transport in Liddles syndrome by measuring sodium concentration, sodium influx, and fractional sodium outflux in vitro in erythrocytes from normal subjects, two patients with Liddles syndrome, and one patient with primary hyperaldosteronism. Sodium influx and fractional sodium outflux, but not sodium concentration, were significantly increased in patients with Liddles syndrome. Sodium outflux in a patient with primary hyperaldosteronism did not differ significantly from normal. These alterations of sodium transport in erythrocytes from patients with Liddles syndrome were not attributable to circulating levels of aldosterone, renin, angiotensin, or serum potassium. Furthermore, changes in aldosterone secretory rate and levels of circulating renin produced by varying dietary sodium intake, did not alter sodium influx or fractional sodium outflux in either patients with Liddles syndrome or normal subjects. The response of fractional sodium outflux and sodium influx to ouabain, ethacrynic acid, and to changes in the cation composition of the incubation medium suggests that the increased sodium fluxes in Liddles syndrome do not result solely from a quantitative increase in those components of sodium transport which occur in normal human erythrocytes. Instead, at least a portion of the increased erythrocyte sodium transport in Liddles syndrome represents a component of sodium transport which does not occur in normal human erythrocytes.

The Renin-Angiotensin-Aldosterone System in Patients with Cystic Fibrosis of the Pancreas

Extract: Patients with cystic fibrosis of the pancreas (CFP) have elevated plasma renin activity, supine renin 497–595 compared with a normal value of 228 ± 133 ng/100 ml plasma on 109 mEq sodium intake/24 hr, but have normal renin release mechanisms as far as postural changes are concerned, since the renin activity increases normally with the upright posture; upright renin, 594–875 compared with a normal value of 359 ± 210 ng/100 ml plasma on the same sodium intake. The high aldosterone secretion rates (ASR), 161–445 compared with a normal value of 90 ± 31 μg/24 hr, seen on 109 mEq sodium intake were probably secondary to the abnormally high renin release. The same can be said for the lack of adequate suppression to normal of both renin and ASR on 249 mEq sodium intake/24 hr, supine renin 205–544 compared with a normal value of 97 ± 71 ng/100 ml plasma; upright renin 845–893 compared with a normal value of 212 ±61 ng/100 ml plasma; ASR on the same intake, 93–333 compared with a normal value of 62.15 ± 27.8 μg/24 hr. The low metabolic clearance rates and the high calculated plasma aldosterone concentrations on the 109 mEq sodium intake indicate that a state of secondary hypcraldosteronism exists in patients with CFP, probably as an adaptation to frequent, excessive sodium losses via the sweat and consequent contraction of intravascular volume.Speculation: In all the patients that we studied with cystic fibrosis of the pancreas a state of secondary hyperaldosteronism appears to exist. This state of hyperaldosteronism, secondary to increased renin release, probably results from adaptation to frequent, excessive sodium losses via the sweat, leading to depletion of extracellular fluid volume.

Compromised Bone Microarchitecture and Estimated Bone Strength in Young Adults With Cystic Fibrosis

CONTEXT Young adults with cystic fibrosis (CF) are at risk for low bone density and fractures, but the underlying alterations in bone microarchitecture that may contribute to their increased fracture risk are currently unknown. OBJECTIVE The main goal of this study was to use high-resolution peripheral quantitative computed tomography (HR-pQCT) to characterize the bone microarchitecture, volumetric bone mineral density (vBMD), and estimated strength of the radius and tibia in young adults with CF compared with healthy volunteers. DESIGN AND SETTING This was a cross-sectional study at an outpatient clinical research center within a tertiary academic medical center. PARTICIPANTS Thirty young adults with CF, 18 to 40 years of age, were evaluated and compared with 60 healthy volunteers matched by age (±2 years), gender, and race. MAIN OUTCOME MEASURES The primary outcomes were HR-pQCT-derived cortical and trabecular vBMD, bone microarchitecture, and estimates of bone strength. RESULTS At the radius and tibia, young adults with CF had smaller bone cross-sectional area and lower vBMD. Cortical and trabecular microarchitecture were compromised at both sites, most notably involving the trabecular bone of the tibia. These differences translated into lower estimated bone strength both at the radius and tibia. After accounting for body mass index differences, young adults with CF had lower bone area and estimated bone strength at the radius and had compromised trabecular microarchitecture and lower total and trabecular vBMD and estimated bone strength at the tibia. Alterations in trabecular bone density and microarchitecture and estimated strength measures of the tibia were also greater than expected based on dual-energy x-ray absorptiometry-derived areal BMD differences. CONCLUSIONS Young adults with CF have compromised bone microarchitecture and lower estimated bone strength at both the radius and tibia, even after accounting for their smaller body size. These skeletal deficits likely explain the higher fracture risk observed in young adults with CF.