Adriana Heguy

Gene expression profiling of human alveolar macrophages of phenotypically normal smokers and nonsmokers reveals a previously unrecognized subset of genes modulated by cigarette smoking

Cigarette smoking is the leading cause of the respiratory diseases collectively known as chronic obstructive pulmonary disease (COPD). While the pathogenesis of COPD is complex, there is abundant evidence that alveolar macrophages (AM) play an important role. Based on the concept that COPD is a slow-progressing disorder likely involving multiple mediators released by AM activated by cigarette smoke, the present study focuses on the identification of previously unrecognized genes that may be linked to early events in the molecular pathogenesis of COPD, as opposed to factors associated with the presence of disease. To accomplish this, microarray analysis using Affymetrix microarrays was used to carry out an unbiased survey of the differences in gene expression profiles in the AM of phenotypically normal, ∼20 pack-year smokers compared to healthy nonsmokers. Although smoking did not alter the global gene expression pattern of AM, 75 genes were modulated by smoking, with 40 genes up-regulated and 35 down-regulated in the AM of smokers compared to nonsmokers. Most of these genes belong to the functional categories of immune/inflammatory response, cell adhesion and extracellular matrix, proteolysis and antiproteolysis, lysosomal function, antioxidant-related function, signal transduction, and regulation of transcription. Of these 75 genes, 69 have not been previously recognized to be up- or down-regulated in AM in association with smoking or COPD, including genes coding for proteins belonging to all of the above categories, and others belonging to various functional categories or of unknown function. These observations suggest that gene expression responses of AM associated with the stress of cigarette smoking are more complex than previously thought, and offer a variety of new insights into the complex pathogenesis of smoking-induced lung diseases.

Upregulation of pirin expression by chronic cigarette smoking is associated with bronchial epithelial cell apoptosis

BackgroundCigarette smoke disrupts the protective barrier established by the airway epithelium through direct damage to the epithelial cells, leading to cell death. Since the morphology of the airway epithelium of smokers does not typically demonstrate necrosis, the most likely mechanism for epithelial cell death in response to cigarette smoke is apoptosis. We hypothesized that cigarette smoke directly up-regulates expression of apoptotic genes, which could play a role in airway epithelial apoptosis.MethodsMicroarray analysis of airway epithelium obtained by bronchoscopy on matched cohorts of 13 phenotypically normal smokers and 9 non-smokers was used to identify specific genes modulated by smoking that were associated with apoptosis. Among the up-regulated apoptotic genes was pirin (3.1-fold, p < 0.002), an iron-binding nuclear protein and transcription cofactor. In vitro studies using human bronchial cells exposed to cigarette smoke extract (CSE) and an adenovirus vector encoding the pirin cDNA (AdPirin) were performed to test the direct effect of cigarette smoke on pirin expression and the effect of pirin expression on apoptosis.ResultsQuantitative TaqMan RT-PCR confirmed a 2-fold increase in pirin expression in the airway epithelium of smokers compared to non-smokers (p < 0.02). CSE applied to primary human bronchial epithelial cell cultures demonstrated that pirin mRNA levels increase in a time-and concentration-dependent manner (p < 0.03, all conditions compared to controls).Overexpression of pirin, using the vector AdPirin, in human bronchial epithelial cells was associated with an increase in the number of apoptotic cells assessed by both TUNEL assay (5-fold, p < 0.01) and ELISA for cytoplasmic nucleosomes (19.3-fold, p < 0.01) compared to control adenovirus vector.ConclusionThese observations suggest that up-regulation of pirin may represent one mechanism by which cigarette smoke induces apoptosis in the airway epithelium, an observation that has implications for the pathogenesis of cigarette smoke-induced diseases.

367. Safety and Immunological Responses Following Intrapleural Administration of an AAV5 Vector Encoding Human |[alpha]|1-Antitrypsin to Non-Human Primates

|[alpha]| 1-antitrypsin (|[alpha]|1AT), a 52 kDa serine proteinase inhibitor secreted by the liver, provides >95% of the functional anti-protease protection of the lower respiratory tract. |[alpha]|1AT deficiency is an autosomal recessive disorder associated with early-onset emphysema if serum |[alpha]|1AT levels are 1.6-fold the accepted therapeutic dose in humans. Importantly, the |[alpha]|1AT level in the bronchoalveolar lavage fluid (normalized to total protein) resulting from delivery of the AAV5CUh|[alpha]|1AT to the pleura was comparable to that found in the serum. In preparation for a clinical trial, the present study addresses the safety of administration of AAV5CUh|[alpha]|1AT to the pleural space of non-human primates at doses scalable to humans (evaluation of human |[alpha]|1AT levels in these animals is not technically feasible due to the 94% homology between African green monkey and human |[alpha]|1AT). The AAV5CUh|[alpha]|1AT vector (1013 gc/animal, n=3) and control (AAV5-luciferase, 1013 gc, n=1) were administered under fluoroscopy guidance in 5 ml of PBS into the right pleural space of juvenile African green monkeys, using an 22 gauge angiocatheter in the 5th or 6th intercostal space. Chest X-rays carried out immediately after vector administration, and at days 3,14 and 56 post-vector administration demonstrated the absence of pneumothorax, pleural effusion, pneumonia or any other pulmonary complications. Serum was collected at 3 time points (days-30,-15 and 0) prior to vector administration, to obtain baseline blood count and chemistry values, and at days 3, 14, 28, 56 and 91 post-vector administration. There were no significant differences (p>0.05 all parameters) between baseline (pre-vector administration) blood counts at any time point, except for transient increases in white blood cell count at days 3 and 14 post-vector administration (p=0.007), likely reflecting a physiological response to the procedure and/or vector administration. There were no significant differences in blood chemistry values at any time point, with the exception of serum Ca+ levels that decreased from initially high values into the range for adult African green monkeys. The anti-AAV5 neutralizing antibody titers post-vector administration ranged from 250 to 640 above background. Together with the long term gene expression following gene transfer to mice, these findings support the concept of a clinical trials to assess the safety of intrapleural administration of AAV5CUh|[alpha]|1AT to individuals with |[alpha]|1AT deficiency.

508. Impact of Sex on Serum |[alpha]|1-Antitrypsin Levels Following Intrapleural and Intravenous Administration of AAVrh.10 Vectors to Mice

The level of transgene expression following intravenous administration of AAV2 vectors to mice varies with sex, with males expressing 5- to 13-fold higher levels than females at the same dose of vector (Davidoff et al, Blood 2003, 102:480-488). This effect is independent of the AAV capsid, the promoter/transgene expression cassette and is postulated to be associated with androgen-enhanced transition from single stranded to double stranded DNA following gene transfer. We have extended this concept by testing the hypothesis that there is a difference in serum |[alpha]|1AT levels between male and female mice following intrapleural gene transfer with AAVrh.10, a highly effective AAV derived from rhesus macaque. Male or female C57Bl/6 mice were injected by intrapleural or intravenous route with 5|[times]|1010 gc of AAVrh.10|[alpha]|1AT and serum |[alpha]|1AT levels were assessed at 0, 4, 14 and 28 days. Consistent with our prior studies in males only using the intravenous route (De et al, Mol Ther 2004, 9:S128), the serum |[alpha]|1AT level at 28 days was 1520|[plusmn]|380 |[mu]|g/ml in male mice. Strikingly, however, the |[alpha]|1AT levels at 28 days were 300|[plusmn]|89 |[mu]|g/ml in females (p<0.05), a 5.1-fold difference. Sex differences were also observed with intrapleural administration: the serum |[alpha]|1AT level at 28 days was 1360|[plusmn]|250 |[mu]|g/ml in males and 480|[plusmn]|58 |[mu]|g/ml in females, a 2.8-fold difference (p<0.05 male vs female). By both routes, human |[alpha]|1AT was found in serum 4 days following gene transfer; and even at that time point, the serum |[alpha]|1AT levels were higher in males than in females (p<0.05). The sex difference in |[alpha]|1AT levels following intrapleural administration of AAVrh.10|[alpha]|1AT may be due to leakage of vector to the systemic circulation and thereby to liver where males expressed higher |[alpha]|1AT levels. Therefore, an AAVrh.10 vector expressing a non-secreted transgene, luciferase, was injected intrapleurally into male and female mice and the luciferase activity of various organs assessed after 1 wk. As expected, there was a 4.0-fold higher luciferase activity in the livers of male mice. But there was also a striking sex difference in other organs with diaphragm and chest wall, the major sites of transgene expression following intrapleural administration, showing a 6.5- and 4.0-fold higher expression level respectively in males. Similarly, the left lung showed a 9.6-fold higher expression level in males. By contrast two organs where luciferase levels were low, the quadriceps and kidney showed no significant difference in expression levels between males and females. We conclude that preferential expression of transgene from AAV vectors in male mice is not confined to intravenous administration and may occur in multiple organs. Although the responsible mechanism is not entirely clear, it is striking how an AAVrh.10 vector that rapidly yields over 100 times the expression level that is derived from AAV2 vectors shows a similar pattern of sex-specific transgene expression. The data suggest that sex is a significant parameter that needs to be considered in both toxicology studies and human clinical trials of AAV derived vectors.

812. Non-Human Primate Serotype rh.10 AAV Directed Therapeutic Serum Levels of |[alpha]|1-antitrypsin Following Intrapleural Administration in Mice with Pre-Existing Immunity to AAV

Alpha 1-antitrypsin (|[alpha]|1AT), a serine protease inhibitor secreted by the liver, protects the lung from degradation by neutrophil proteases. |[alpha]|1AT deficiency is an autosomal recessive disorder associated with the accelerated development of emphysema if serum |[alpha]|1AT levels are 99% for AAV2- or AAV5-mediated |[alpha]|1AT gene transfer (p 0.4) in previously AAV2+AAV5 immune mice (830|[plusmn]|150 |[mu]|g/ml) and previously naive mice (1100|[plusmn]|140 |[mu]|g/ml). We have also demonstrated the concept that |[alpha]|1AT expression levels can be boosted by the sequential use of two different serotypes. In mice that initially received intrapleural injection of 1011 gc AAV5CUh|[alpha]|1AT, the serum |[alpha]|1AT levels after 6 wk were 752|[plusmn]|274 |[mu]|g/ml. When these mice were then treated with AAV5CUh|[alpha]|1AT, there was no increase in serum |[alpha]|1AT levels at 2 wk (p>0.5). However, when these mice were treated with AAVrh.10h|[alpha]|1AT, a 164|[plusmn]|21% increase in serum |[alpha]|1AT levels was observed (p<0.01 compared to AAV5CUh|[alpha]|1AT boosted animals). These data indicate that intrapleural administration of an AAVrh.10h|[alpha]|1AT vector can provide therapeutic levels of |[alpha]|1AT and overcome high level immunity to human AAV serotypes.