Charleen Hollmann

Safety of Local Delivery of Low- and Intermediate-Dose Adenovirus Gene Transfer Vectors to Individuals with a Spectrum of Morbid Conditions

To help define the safety profile of the use of adenovirus (Ad) gene transfer vectors in humans, this report summarizes our experience since April 1993 of the local administration of E1(-)/E3(-) Ad vectors to humans using low (<10(9) particle units) or intermediate (10(9)-10(11) particle units) doses. Included in the study are 90 individuals and 12 controls, with diverse comorbid conditions, including cystic fibrosis, colon cancer metastatic to liver, severe coronary artery disease, and peripheral vascular disease, as well as normals. These individuals received 140 different administrations of vector, with up to seven administrations to a single individual. The vectors used include three different transgenes (human cystic fibrosis transmembrane conductance regulator cDNA, E. coli cytosine deaminase gene, and the human vascular endothelial growth factor 121 cDNA) administered by six different routes (nasal epithelium, bronchial epithelium, percutaneous to solid tumor, intradermal, epicardial injection of the myocardium, and skeletal muscle). The total population was followed for 130.4 patient-years. The study assesses adverse events, common laboratory tests, and long-term follow-up, including incidence of death or development of malignancy. The total group incidence of major adverse events linked to an Ad vector was 0.7%. There were no deaths attributable to the Ad vectors per se, and the incidence of malignancy was within that expected for the population. Overall, the observations are consistent with the concept that local administration of low and intermediate doses of Ad vectors appears to be well tolerated.

Neurological deterioration in late infantile neuronal ceroid lipofuscinosis

Background: Late infantile neuronal ceroid lipofuscinosis (LINCL) is associated with progressive degeneration of the brain and retina starting in early childhood. Methods: Thirty-two individual neurologic, ophthalmologic, and CNS imaging (MRI and MRS) assessments of 18 children with LINCL were analyzed. Disease severity was followed by two rating scales, one previously established but modified to solely assess the brain and exclude the retinal disease (modified Hamburg LINCL scale), and a newly developed scale, with expanded evaluation of the CNS impairment (Weill Cornell LINCL scale). Results: For the 18 children, the Weill Cornell scale yielded a closer correlation with both age and time since initial clinical manifestation of the disease than did the modified Hamburg scale. There were no significant differences as a function of age or time since initial manifestation of the disease in the rating scales among the most frequent CLN2 mutations (G3556C, 56% of all alleles or C3670T, 22% of all alleles). Measurements of cortical MRS N-acetyl-aspartate content, MRI ventricular, gray matter and white matter volume, and cortical apparent diffusion coefficient correlated to a variable degree with the age of the children and the time since initial clinical manifestation of the disease. All imaging measurements correlated better with the Weill Cornell CNS scale compared to the modified Hamburg LINCL scale. Conclusion: The data suggest that the Weill Cornell late infantile neuronal ceroid lipofuscinosis (LINCL) scale, together with several of the MRI measurements, may be useful in the assessment of severity and progression of LINCL and for the evaluation of novel therapeutic strategies.

Effects of university of wisconsin solution on endothelium-dependent coronary artery relaxation in the rat

University of Wisconsin (UW) solution has been reported to enhance myocardial preservation in heart transplantation. To evaluate the effects of UW solution on coronary artery endothelial function, we designed experiments to compare UW solution with a standard crystalloid hyperkalemic cardioplegic solution (CHCS). Isolated rat hearts were studied in a modified Langendorff apparatus for coronary endothelial function. Groups 1 and 2 were perfused with 4 degrees C CHCS (24 mmol/L of KCl) and UW solution, respectively, for 10 minutes at a pressure of 80 cm H2O, whereas group 3 underwent warm ischemia for 10 minutes. Groups 4 and 5 were perfused with and stored for 4 hours in cold (4 degrees C) CHCS and UW solution, respectively. Group 6 underwent 4 hours of topical cooling (4 degrees C) without any cardioplegic perfusion. All groups had 6 hearts each. Endothelium-dependent relaxation and endothelium-independent relaxation of the coronary arteries were tested by infusing 5-hydroxytryptamine (5HT) (10(-6) mol/L) and sodium nitroprusside (10(-5) mol/L), respectively, before and after perfusion with and storage in one of the two cardioplegic solutions. The coronary vasodilatation induced by 5HT and sodium nitroprusside was not altered in hearts perfused with (group 1) or perfused with and stored in CHCS (group 4). Coronary flow increase after 5HT infusion was significantly decreased in hearts perfused with (group 2) (before, 35% +/- 10%; after, 13% +/- 10%; p < 0.01) or perfused with and stored in UW solution (group 5) (before, 34% +/- 5%; after, -5% +/- 12%), indicating severe endothelial dysfunction.(ABSTRACT TRUNCATED AT 250 WORDS)

Persistence of Smoking-Induced Dysregulation of MiRNA Expression in the Small Airway Epithelium Despite Smoking Cessation

Even after quitting smoking, the risk of the development of chronic obstructive pulmonary disease (COPD) and lung cancer remains significantly higher compared to healthy nonsmokers. Based on the knowledge that COPD and most lung cancers start in the small airway epithelium (SAE), we hypothesized that smoking modulates miRNA expression in the SAE linked to the pathogenesis of smoking-induced airway disease, and that some of these changes persist after smoking cessation. SAE was collected from 10th to 12th order bronchi using fiberoptic bronchoscopy. Affymetrix miRNA 2.0 arrays were used to assess miRNA expression in the SAE from 9 healthy nonsmokers and 10 healthy smokers, before and after they quit smoking for 3 months. Smoking status was determined by urine nicotine and cotinine measurement. There were significant differences in the expression of 34 miRNAs between healthy smokers and healthy nonsmokers (p<0.01, fold-change >1.5), with functions associated with lung development, airway epithelium differentiation, inflammation and cancer. After quitting smoking for 3 months, 12 out of the 34 miRNAs did not return to normal levels, with Wnt/β-catenin signaling pathway being the top identified enriched pathway of the target genes of the persistent dysregulated miRNAs. In the context that many of these persistent smoking-dependent miRNAs are associated with differentiation, inflammatory diseases or lung cancer, it is likely that persistent smoking-related changes in SAE miRNAs play a role in the subsequent development of these disorders.

Intraflagellar transport gene expression associated with short cilia in smoking and COPD.

Smoking and COPD are associated with decreased mucociliary clearance, and healthy smokers have shorter cilia in the large airway than nonsmokers. We hypothesized that changes in cilia length are consistent throughout the airway, and we further hypothesized that smokers with COPD have shorter cilia than healthy smokers. Because intraflagellar transport (IFT) is the process by which cilia of normal length are produced and maintained, and alterations in IFT lead to short cilia in model organisms, we also hypothesized that smoking induces changes in the expression of IFT-related genes in the airway epithelium of smokers and smokers with COPD. To assess these hypotheses, airway epithelium was obtained via bronchoscopic brushing. Cilia length was assessed by measuring 100 cilia (10 cilia on each of 10 cells) per subject and Affymetrix microarrays were used to evaluate IFT gene expression in nonsmokers and healthy smokers in 2 independent data sets from large and small airway as well as in COPD smokers in a data set from the small airway. In the large and small airway epithelium, cilia were significantly shorter in healthy smokers than nonsmokers, and significantly shorter in COPD smokers than in both healthy smokers and nonsmokers. The gene expression data confirmed that a set of 8 IFT genes were down-regulated in smokers in both data sets; however, no differences were seen in COPD smokers compared to healthy smokers. These results support the concept that loss of cilia length contributes to defective mucociliary clearance in COPD, and that smoking-induced changes in expression of IFT genes may be one mechanism of abnormally short cilia in smokers. Strategies to normalize cilia length may be an important avenue for novel COPD therapies.

The role of preservation solutions in coronary endothelial damage during cold storage.

The effects of cold storage and type of preservation solution on coronary endothelial function are not well understood. Experiments were designed to evaluate coronary endothelial-dependent relaxation after a 4-hr cold (4°C) storage in different preservation solutions. Isolated rat hearts were studied in the Langendorff aprparatus for coronary endothelial function. After 30 min of stabilization, hearts were arrested with a 10-min perfusion of 4°C crystalloid hyperkalemic cardioplegic solution (CHCS) containing 24 mmol/L of KC1 and stored for 4 hr in the following preservation solutions: CHCS, Krebs-Ringers solution (KR), 0.9% NaCl (NS), and University of Wisconsin solution (UW). A fifth group was perfused and stored in UW solution. Endothelial-dependent and independent coronary artery vasorelaxation were tested, respectively, by infusing 5-hydroxy-tryptamine (5-HT) (1×10-6 mol/L) and sodium nitroprusside (SNP) (1×10-5 mol/L) before and 30 min after the storage period. In hearts stored in CHCS and KR, the coronary artery flow increase to 5-HT and SNP infusion were not significantly affected. However, in hearts preserved with NS and UW solutions, 5-HT coronary response was significantly decreased, indicating endothelial dysfunction. In addition to these findings, coronary flow increase to SNP infusion was decreased in the group perfused and stored with UW, suggesting smooth muscle damage. These experiments suggest that 4-hr cold storage in NS or UW impairs endothelial-dependent coronary relaxation in the isolated rat heart model.

Long-Term Follow-Up Assessment of a Phase 1 Trial of Angiogenic Gene Therapy Using Direct Intramyocardial Administration of an Adenoviral Vector Expressing the VEGF121 cDNA for the Treatment of Diffuse Coronary Artery Disease

On the basis of studies in experimental animals demonstrating that AdVEGF121, an E1(-)E3(-) serotype 5 adenovirus coding the 121 isoform of vascular endothelial growth factor (VEGF), could mediate the generation of new blood vessels and reverse coronary ischemia, a clinical study of direct myocardial administration of AdVEGF121 was initiated in patients with late-stage, diffuse coronary artery disease. This study provides long-term (median, 11.8 years) follow-up on these patients. From 1997 to 1999, AdVEGF121 was administered by direct myocardial injection to an area of reversible ischemia in 31 patients with severe coronary disease, either as an adjunct to conventional coronary artery bypass grafting (group A) or as minimally invasive sole (MIS) therapy, using a minithoracotomy (group B). There was no control group; the study participants served as the control subjects. The 5- and 10-year survival was 10 of 15 (67%) and 6 of 15 (40%) for the group A patients, and 11 of 16 (69%) and 5 of 16 (31%) for group B sole therapy patients, respectively. In comparison, maximal medical therapy in comparable groups in the literature have a 3- to 5-year survival rate of 52 to 59%. For the survivors, the angina score for group A was 3.4±0.5 at time 0 and 1.9±1.0 at last follow-up, and for group B it was 3.4±0.6 and 2.0±1.1, respectively. The incidences of malignancy and retinopathy were no greater than that expected for the age-matched general population. We conclude that adenovirus-mediated VEGF direct myocardial administration to patients with severe coronary artery disease is safe, and future larger trials are warranted to assess efficacy.

Persistence of circulating endothelial microparticles in COPD despite smoking cessation

Introduction Increasing evidence links COPD pathogenesis with pulmonary capillary apoptosis. We previously demonstrated that plasma levels of circulating microparticles released from endothelial cells (EMPs) due to apoptosis are elevated in smokers with normal spirometry but low diffusion capacity, that is, with early evidence of lung destruction. We hypothesised that pulmonary capillary apoptosis persists with the development of COPD and assessed its reversibility in healthy smokers and COPD smokers following smoking cessation. Methods Pulmonary function and high-resolution CT (HRCT) were assessed in 28 non-smokers, 61 healthy smokers and 49 COPD smokers; 17 healthy smokers and 18 COPD smokers quit smoking for 12 months following the baseline visit. Total EMP (CD42b−CD31+), pulmonary capillary EMP (CD42b−CD31+ACE+) and apoptotic EMP (CD42b−CD62E+/CD42b−CD31+) levels were quantified by flow cytometry. Results Compared with non-smokers, healthy smokers and COPD smokers had elevated levels of circulating EMPs due to active pulmonary capillary endothelial apoptosis. Levels remained elevated over 12 months in healthy smokers and COPD smokers who continued smoking, but returned to non-smoker levels in healthy smokers who quit. In contrast, levels remained significantly abnormal in COPD smokers who quit. Conclusions Pulmonary capillary apoptosis is reversible in healthy smokers who quit, but continues to play a role in COPD pathogenesis in smokers who progressed to airflow obstruction despite smoking cessation. Trial registration number NCT00974064; NCT01776398.

Pulmonary Abnormalities in Young, Light-Use Waterpipe (Hookah) Smokers

RATIONALE Waterpipes, also called hookahs, are currently used by millions of people worldwide. Despite the increasing use of waterpipe smoking, there is limited data on the health effects of waterpipe smoking and there are no federal regulations regarding its use. OBJECTIVES To assess the effects of waterpipe smoking on the human lung using clinical and biological parameters in young, light-use waterpipe smokers. METHODS We assessed young, light-use, waterpipe-only smokers in comparison with lifelong nonsmokers using clinical parameters of cough and sputum scores, lung function, and chest high-resolution computed tomography as well as biological parameters of lung epithelial lining fluid metabolome, small airway epithelial (SAE) cell differential and transcriptome, alveolar macrophage transcriptome, and plasma apoptotic endothelial cell microparticles. MEASUREMENTS AND MAIN RESULTS Compared with nonsmokers, waterpipe smokers had more cough and sputum as well as a lower lung diffusing capacity, abnormal epithelial lining fluid metabolome profile, increased proportions of SAE secretory and intermediate cells, reduced proportions of SAE ciliated and basal cells, markedly abnormal SAE and alveolar macrophage transcriptomes, and elevated levels of apoptotic endothelial cell microparticles. CONCLUSIONS Young, light-use, waterpipe-only smokers have a variety of abnormalities in multiple lung-related biological and clinical parameters, suggesting that even limited waterpipe use has broad consequences on human lung biology and health. We suggest that large epidemiological studies should be initiated to investigate the harmful effects of waterpipe smoking.

969. Development of Non-Invasive Imaging Strategies to Assess the Impact of Gene Therapy: Phenotypic Assessment of the Neurodegenerative CNS Disorder Late Infantile Neuronal Ceroid Lipofuscinosis Using Magnetic Resonance Spectroscopy

Several animal models of neurodegenerative lysosomal storage disease have been successfully treated by intracranial gene transfer. Among the most challenging aspects of moving these strategies to the clinic is to identify objective outcome parameters to measure the success of gene therapy. Classical phenotyping with measurement of neurological functional parameters and/or survival will take years of observation, it is not ethical to obtain brain biopsies following treatment for histological analysis, and there are limited numbers of children with these disorders to develop comparable treated and control groups. We have commenced a program of AAV-mediated gene therapy for the CNS manifestations of late infantile neuronal ceroid lipofuscinosis (LINCL), a fatal childhood neurodegenerative lysosomal storage disease with no known therapy resulting from mutations in the CLN2 gene and a deficiency of the lysosomal protease tripeptidyl peptidase I. As part of this program, we have assessed whether magnetic resonance spectroscopy (MRS) is sufficiently sensitive and reproducible to assess the impact of intracranial gene transfer in humans with neurodegenerative diseases. To date, 8 children with LINCL (ages 6.25 to 13.75 yr old) have been successfully scanned and four of these children were scanned twice at intervals from 17 to 126 days. MRS scans were performed in a 3.0 Tesla GE Signa LX scanner generating 1H images of 4 brain sections of 15 mm thickness separated by 3.5 mm. Strong MRS signals from pericranial lipid resonances and water were suppressed. The raw data were processed to give the concentrations of four metabolites, lactate, choline, creatine (Cr) and N-acetylacetate (NAA) in multiple voxels (8 |[times]| 8 |[times]| 15 mm). Analysis was confined to those regions of the cortex that were accessible for surgical administration of gene transfer vectors. Initially we assessed the NAA/Cr ratio, an index of neuronal function. The NAA/Cr ratio in the target area was 1.60 |[plusmn]| 0.16 in the youngest LINCL patient and 0.58 |[plusmn]|0.18 in the oldest. The NAA/Cr ratio decreased with a slope of |[minus]|0.10/yr (r2=0.43; 95% confidence intervals |[minus]|0.186 to |[minus]|0.019/yr). The difference in NAA/Cr ratio between the first and second scans from the same subjects was |[minus]|0.52|[plusmn]|0.53/yr suggesting that the NAA/Cr ratio is poorly reproducible in duplicate scans of similar brains. Assuming a low estimate of the overall coefficient of variation in measurement of NAA/Cr ratio of 20% (from spatial variability and variability among scans), we calculated that if the NAA/Cr ratio was stabilized by gene therapy in one third of the voxels in the target area, this would be evident by statistical analysis at 18 months post-surgery. We conclude that MRS may provide objective evidence of local changes in metabolite concentration following gene transfer, but to accomplish this will require repeat scans over greater than 1 yr following gene transfer.