CF Airway Epithelial Cells Research Articles

Gene expression responses of CF airway epithelial cells exposed to elexacaftor/tezacaftor/ivacaftor (ETI) suggest benefits beyond improved CFTR channel function.

Gene expression responses by CF AEC exposed to ETI suggest that in addition to improving CFTR channel function, ETI is likely to increase resistance to bacterial infection by increasing levels of beta defensin 1 (hBD-1). ETI may also reduce lung damage by suppressing MMP10, and reduce airway inflammation by repressing proinflammatory cytokine secretion by AEC cells.

400: Metformin improves CFTR response to modulators in hyperglycemia-exposed CF airway epithelial cells

400: Metformin improves CFTR response to modulators in hyperglycemia-exposed CF airway epithelial cells

Chemotaxis and Binding of Pseudomonas aeruginosa to Scratch-Wounded Human Cystic Fibrosis Airway Epithelial Cells.

Confocal imaging was used to characterize interactions of Pseudomonas aeruginosa (PA, expressing GFP or labeled with Syto 11) with CF airway epithelial cells (CFBE41o-, grown as confluent monolayers with unknown polarity on coverglasses) in control conditions and following scratch wounding. Epithelia and PAO1-GFP or PAK-GFP (2 MOI) were incubated with Ringer containing typical extracellular salts, pH and glucose and propidium iodide (PI, to identify dead cells). PAO1 and PAK swam randomly over and did not bind to nonwounded CFBE41o- cells. PA migrated rapidly (began within 20 sec, maximum by 5 mins) and massively (10–80 fold increase, termed “swarming”), but transiently (random swimming after 15 mins), to wounds, particularly near cells that took up PI. Some PA remained immobilized on cells near the wound. PA swam randomly over intact CFBE41o- monolayers and wounded monolayers that had been incubated with medium for 1 hr. Expression of CFTR and altered pH of the media did not affect PA interactions with CFBE41o- wounds. In contrast, PAO1 swarming and immobilization along wounds was abolished in PAO1 (PAO1ΔcheYZABW, no expression of chemotaxis regulatory components cheY, cheZ, cheA, cheB and cheW) and greatly reduced in PAO1 that did not express amino acid receptors pctA, B and C (PAO1ΔpctABC) and in PAO1 incubated in Ringer containing a high concentration of mixed amino acids. Non-piliated PAKΔpilA swarmed normally towards wounded areas but bound infrequently to CFBE41o- cells. In contrast, both swarming and binding of PA to CFBE41o- cells near wounds were prevented in non-flagellated PAKΔfliC. Data are consistent with the idea that (i) PA use amino acid sensor-driven chemotaxis and flagella-driven swimming to swarm to CF airway epithelial cells near wounds and (ii) PA use pili to bind to epithelial cells near wounds.

142 Development of an S/MAR based episomal vector for the CFTR gene delivery

Objectives The recent use of mutation-specific correctors in Cystic Fibrosis, reinforces the concept of gene therapy as a therapeutic treatment for all CF patients. Non-viral vectors are preferable to viral ones, particularly in treatment of chronic diseases, for their simpler composition, lack of immunogenicity and genotoxicity. Episomal vectors based on the S/MAR (scaffold/matrix attachment region) sequence have self-replicating capability and episomal persistence in different cell types, both in vitro and in vivo. To obtain long-term and episomal maintenance of the CFTR gene in target cells, we constructed a vector containing the S/MAR, GFP and the CFTR cDNA (pBQ-S/MAR). Methods pBQ-S/MAR was transfected in CF(DF508/DF508) airway epithelial cells and the cells were grown in non-selective conditions up to 14 days after transfection. The cells were evaluated for the episome maintenance and persistence as well as for CFTR gene expression (RT-PCR, immunofluorescence, western blot). Results The resulting data demonstrate that pBQ-S/MAR is maintained as an episome, with unchanged structure respect to the input vector, for at least 14 days after transfection, corresponding to about six cell duplications. CFTR expression analyses confirmed the presence of the wt transcript and the mature form of the protein. Additionally, the expression of a functional CFTR in pBQ-S/MAR-transfected cells was associated with an increase of Trans Epithelial Resistance respect to parental cells. Conclusion Our results suggest that the pBQ-S/MAR is stably maintained as an episome in airway epithelial cells and support expression of a functional CFTR.

Deleterious impact of hyperglycemia on cystic fibrosis airway ion transport and epithelial repair

BackgroundCystic fibrosis (CF)-related diabetes (CFRD) is associated with faster pulmonary function decline. Thus, we evaluated the impact of hyperglycemia on airway epithelial repair and transepithelial ion transport, which are critical in maintaining lung integrity and function. MethodsNon-CF and CF airway epithelial cells were exposed to low (LG) or high (HG) glucose before ion current and wound repair rate measurements. ResultsCFTR and K+ currents decreased after HG treatments. HG also reduced the wound healing rates of non-CF and CF cell monolayers. Although CFTR correction with VRT-325 accelerated the healing rates of CF cells monolayers under LG conditions, this improvement was significantly abrogated under HG conditions. ConclusionsOur data highlights a deleterious impact of hyperglycemia on ion transport and epithelial repair functions, which could contribute to the deterioration in lung function in CFRD patients. HG may also interfere with the beneficial effects of CFTR rescue on airway epithelial repair.

241 : Increased IL-17C production by the TLR3 ligand poly (I:C) in primary cystic fibrosis airway epithelial cells

Cystic fibrosis is characterized by chronic inflammation that ultimately lead to death from respiratory failure. Overproduction of IL-8, a key chemokine that is crucial for an induction of neutrophil-dominated inflammation, has been considered as one of the important hallmarks of CF airway. Thus, identification of novel pathways underlying IL-8 induction could provide novel drug targets for prevention and treatment of CF. Importantly, recent studies suggest that airway epithelial cells produce IL-17C, an IL-17 member that is produced by epithelial but not Th17 cells, which contributes to production of proinflammatory cytokines such as IL-8 in an autocrine manner. However, the regulatory mechanism responsible for IL-17C expression in both non-CF and CF airway epithelial cells is still largely unexplored. The present study sought to determine how IL-17C expression is regulated during pathogenic infection in CF airway. Among the tested ligands of toll-like receptors (TLRs) that mimic infection signaling, poly (I:C), a synthetic analog of viral double-stranded RNA that works as a ligand for TLR3, strongly induced IL-17C gene expression and secretion in primary airway epithelial cells derived from non-CF (NHBE) and CF (DHBE-CF) subjects. Maximum induction of IL-17C gene expression and secretion was observed at 24 h post-treatment in both cells, and the induction was mainly through NF-kB and partially through MAPKs (ERK, JNK, p38). Importantly, the poly (I:C)-induced IL-17C up-regulation was strongly enhanced in DHBE-CF cells. Moreover, TLR3 expression and poly (I:C)-induced up-regulation of IL-8 and IFNb were also higher in DHBE-CF cells, suggesting that the enhancement of poly (I:C)-dependent IL-17C induction appears to be the result of increased TLR3 expression in CF airway epithelial cells. Thus, these findings firstly provide the idea that poly (I:C) signal is a critical pathway in accelerating IL-17C expression in CF airway, which may imply that the TLR3-IL-17C axis is crucial for exacerbating viral infection-associated inflammation in CF lung disease.

152 Estrogen-regulated MicroRNAs control the expression of secretory leukoprotease inhibitor in monocytes

Apoptosis is a physiological process essential for homeostasis of epithelial organisation and function. CF lung disease is characterised by chronic infection and inflammation and previous work suggests that apoptosis is dysfunctional in the CF airways with conflicting results. In addition, controversy exists regarding how CFTR misfolding contributes to apoptosis. In this study, we evaluated the relationship between CFTR mutation and apoptosis in DF508-CFTR CF airway epithelial cells. Basal activity of the executioner caspase, caspase-3, was significantly increased in CF tracheal and bronchial epithelial cell lines and primary bronchial epithelial cells compared to non-CF controls. In addition, activity of the upstream initiator caspase, caspase-8, was significantly increased in CF epithelial cells compared to controls, suggesting involvement of extrinsic apoptosis signalling, which is mediated by the activation of death receptors, such as Fas (CD95). Increased levels of Fas were observed in CF epithelial cells, and neutralization of Fas significantly inhibited caspase-3 activity in CF epithelial cells compared to untreated cells. Furthermore, activation of Fas significantly increased caspase-3 activity and apoptosis in CF epithelial cells compared to control cells. Overall, these results suggest that CF airway epithelial cells are more sensitive to apoptosis via increased levels of Fas and subsequent activation of the Fas death receptor pathway.

150 CF epithelial cells are primed for apoptosis as a result of increased Fas

Apoptosis is a physiological process essential for homeostasis of epithelial organisation and function. CF lung disease is characterised by chronic infection and inflammation and previous work suggests that apoptosis is dysfunctional in the CF airways with conflicting results. In addition, controversy exists regarding how CFTR misfolding contributes to apoptosis. In this study, we evaluated the relationship between CFTR mutation and apoptosis in DF508-CFTR CF airway epithelial cells. Basal activity of the executioner caspase, caspase-3, was significantly increased in CF tracheal and bronchial epithelial cell lines and primary bronchial epithelial cells compared to non-CF controls. In addition, activity of the upstream initiator caspase, caspase-8, was significantly increased in CF epithelial cells compared to controls, suggesting involvement of extrinsic apoptosis signalling, which is mediated by the activation of death receptors, such as Fas (CD95). Increased levels of Fas were observed in CF epithelial cells, and neutralization of Fas significantly inhibited caspase-3 activity in CF epithelial cells compared to untreated cells. Furthermore, activation of Fas significantly increased caspase-3 activity and apoptosis in CF epithelial cells compared to control cells. Overall, these results suggest that CF airway epithelial cells are more sensitive to apoptosis via increased levels of Fas and subsequent activation of the Fas death receptor pathway.

151 Investigation of MicroRNA regulation of interleukin-8 production in bronchial epithelial cells

Apoptosis is a physiological process essential for homeostasis of epithelial organisation and function. CF lung disease is characterised by chronic infection and inflammation and previous work suggests that apoptosis is dysfunctional in the CF airways with conflicting results. In addition, controversy exists regarding how CFTR misfolding contributes to apoptosis. In this study, we evaluated the relationship between CFTR mutation and apoptosis in DF508-CFTR CF airway epithelial cells. Basal activity of the executioner caspase, caspase-3, was significantly increased in CF tracheal and bronchial epithelial cell lines and primary bronchial epithelial cells compared to non-CF controls. In addition, activity of the upstream initiator caspase, caspase-8, was significantly increased in CF epithelial cells compared to controls, suggesting involvement of extrinsic apoptosis signalling, which is mediated by the activation of death receptors, such as Fas (CD95). Increased levels of Fas were observed in CF epithelial cells, and neutralization of Fas significantly inhibited caspase-3 activity in CF epithelial cells compared to untreated cells. Furthermore, activation of Fas significantly increased caspase-3 activity and apoptosis in CF epithelial cells compared to control cells. Overall, these results suggest that CF airway epithelial cells are more sensitive to apoptosis via increased levels of Fas and subsequent activation of the Fas death receptor pathway.

Interaction and Localization of Synthetic Nanoparticles in Healthy and Cystic Fibrosis Airway Epithelial Cells: Effect of Ozone Exposure

Nanoparticles (NPs) produced by nanotechnology processes have taken the field of medicine by storm. Concerns about safety of these NPs in humans, however, have recently been raised. Although studies of NP toxicity have focused on lung disease the mechanistic link between NP exposure and lung injury remained unclear. This is primarily due to a lack of availability of appropriate airway disease models and sophisticated microscopic techniques to study nano-sized particulate delivery and resulting responses. Air-liquid interface (ALI) cultures of non-cystic fibrosis (CF) and CF airway epithelial cells were exposed to the FITC-labeled NPs using a PennCentury microsprayer™. Uptake of NPs was assessed by FACS. Laser scanning microscopy (LSM) was performed and the images were analyzed by an advanced imaging software to study particle deposition and uptake. Flow cytometry data revealed that CF cells accumulated increased amounts of NPs. The increased NP uptake could be attributed to the reduced CF transmembrane conductance regulator (CFTR) function as a similar increased retention/uptake was observed in cells whose CFTR expression was downregulated by antisense oligonucleotide. NPs alone did not induce pro-inflammatory cytokine release or cell death. The cell culture system was sensitive to ozone but exposure to the uncoated synthetic NPs used in this study, did not cause any synergistic or suppressive effects. LSM imaging and subsequent image restoration further indicated particle uptake and intracellular localization. Exposure to ozone increased nuclear uptake in both non-CF and CF cells. Our findings demonstrate the uptake of NPs using ALI cultures of non-CF and CF airway epithelial cells. The NPs used here were useful in demonstrating uptake by airway epithelial cells without causing adverse effects in presence or absence of ozone. However, to totally exclude toxic effects, chronic studies under in vivo conditions using coated particulates are required.

The effect of S-nitrosoglutathione and L-cysteine on chloride efflux from cystic fibrosis airway epithelial cells

The endogenous bronchodilator, S-nitrosoglutathione (GSNO), has been proposed as a possible pharmacological remedy that reverses the ΔF508-CFTR (cystic fibrosis transmembrane conductance regulator) maturation defect and increases CFTR-mediated chloride efflux in cultured cystic fibrosis airway epithelial cells (CFBE41o -). It has also been reported that L-cysteine enhanced S-nitrosothiol uptake and increased the intracellular S-nitrosothiol levels, likely through transnitrosation chemistry. The present study investigated whether L-cysteine augmented the effect of GSNO on chloride efflux from CF airway epithelial cells. Treatment with 10 μM GSNO combined with 20 μM L-cysteine resulted in increased chloride efflux from CFBE41o - cells after 5 minutes exposure compared to the control efflux rate and to the efflux rate in the presence of L-cysteine alone as measured using the fluorescent dye N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE). Chloride efflux rates from these cells after 4 h exposure to GSNO and L-cysteine were not different from control. Treatment with 10 μM GSNO alone increased chloride efflux from CFBE41o − cells after 4 h but not at shorter incubation times. GSNO with or without L-cysteine did not alter epithelial tight junction integrity. In conclusion, a combination of GSNO with L-cysteine led to significant increase in chloride efflux in CFBE41o - cells but the effect was transient and not sustained beyond minutes.

Identification of TRPC6 as a novel potential target to activate CaCC in human CF airway epithelial cells

Identification of TRPC6 as a novel potential target to activate CaCC in human CF airway epithelial cells

MiR-126 Is Downregulated in Cystic Fibrosis Airway Epithelial Cells and Regulates TOM1 Expression

Cystic fibrosis (CF) is one of the most common lethal genetic diseases in which the role of microRNAs has yet to be explored. Predicted to be regulated by miR-126, TOM1 (target of Myb1) has been shown to interact with Toll-interacting protein, forming a complex to regulate endosomal trafficking of ubiquitinated proteins. TOM1 has also been proposed as a negative regulator of IL-1beta and TNF-alpha-induced signaling pathways. MiR-126 is highly expressed in the lung, and we now show for the first time differential expression of miR-126 in CF versus non-CF airway epithelial cells both in vitro and in vivo. MiR-126 downregulation in CF bronchial epithelial cells correlated with a significant upregulation of TOM1 mRNA, both in vitro and in vivo when compared with their non-CF counterparts. Introduction of synthetic pre-miR-126 inhibited luciferase activity in a reporter system containing the full length 3'-untranslated region of TOM1 and resulted in decreased TOM1 protein production in CF bronchial epithelial cells. Following stimulation with LPS or IL-1beta, overexpression of TOM1 was found to downregulate NF-kappaB luciferase activity. Conversely, TOM1 knockdown resulted in a significant increase in NF-kappaB regulated IL-8 secretion. These data show that miR-126 is differentially regulated in CF versus non-CF airway epithelial cells and that TOM1 is a miR-126 target that may have an important role in regulating innate immune responses in the CF lung. To our knowledge, this study is the first to report of a role for TOM1 in the TLR2/4 signaling pathways and the first to describe microRNA involvement in CF.

Functional cystic fibrosis transmembrane conductance regulator expression in cystic fibrosis airway epithelial cells by AAV6.2-mediated segmental trans-splicing.

Cystic fibrosis is characterized by deficiency of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) transporter. The packaging constraints of adeno-associated viral (AAV) vectors preclude delivery of both an active promoter and CFTR cDNA to target cells. We hypothesized that segmental trans-splicing, in which two AAV vectors deliver the 5' and 3' halves of the CFTR cDNA, could mediate splicing of two pre-mRNAs into a full-length, functional CFTR mRNA. Using a segmental trans-splicing 5' donor-3' acceptor pair that split the CFTR cDNA between exons 14a and 14b, cotransfection of donor and acceptor plasmids into CFTR(-) cells resulted in full-length CFTR message and protein. Microinjection of plasmids into CFTR(-) cells produced cAMP-activated Cl(-) conductance. Vectors created with an engineered human serotype, AAV6.2, were used to deliver CFTR donor and acceptor constructs, resulting in full-length CFTR mRNA and protein as well as cAMP-activated Cl(-) conductance in CFTR(-) cells, including human CF airway epithelial IB3-1 cells. Thus, segmental trans-splicing can be used with AAV vectors to mediate expression of CFTR, a strategy potentially applicable to individuals with CF.

Tissue Transglutaminase Activation Modulates Inflammation in Cystic Fibrosis via PPARγ Down-Regulation

Cystic fibrosis (CF), the most common life-threatening inherited disease in Caucasians, is due to mutations in the CF transmembrane conductance regulator (CFTR) gene and is characterized by airways chronic inflammation and pulmonary infections. The inflammatory response is not secondary to the pulmonary infections. Indeed, several studies have shown an increased proinflammatory activity in the CF tissues, regardless of bacterial infections, because inflammation is similarly observed in CFTR-defective cell lines kept in sterile conditions. Despite recent studies that have indicated that CF airway epithelial cells can spontaneously initiate the inflammatory cascade, we still do not have a clear insight of the molecular mechanisms involved in this increased inflammatory response. In this study, to understand these mechanisms, we investigated ex vivo cultures of nasal polyp mucosal explants of CF patients and controls, CFTR-defective IB3-1 bronchial epithelial cells, C38 isogenic CFTR corrected, and 16HBE normal bronchial epithelial cell lines. We have shown that a defective CFTR induces a remarkable up-regulation of tissue transglutaminase (TG2) in both tissues and cell lines. The increased TG2 activity leads to functional sequestration of the anti-inflammatory peroxisome proliferator-activated receptor gamma and increase of the classic parameters of inflammation, such as TNF-alpha, tyrosine phosphorylation, and MAPKs. Specific inhibition of TG2 was able to reinstate normal levels of peroxisome proliferator-activated receptor-gamma and dampen down inflammation both in CF tissues and CFTR-defective cells. Our results highlight an unpredicted central role of TG2 in the mechanistic pathway of CF inflammation, also opening a possible new wave of therapies for sufferers of chronic inflammatory diseases.

The GCC repeat length in the 5'UTR of MRP1 gene is polymorphic: a functional characterization of its relevance for cystic fibrosis

BackgroundAmong the members of the ATP binding cassette transporter superfamily, MRPs share the closest homology with the CFTR protein, which is defective in CF disease. MRP1 has been proposed as a potential modifier gene and/or as novel target for pharmacotherapy of CF to explain the clinical benefits observed in some CF patients treated with the macrolide AZM. The 5'UTR of the MRP1 gene contains a GCC triplet repeat that could represent a polymorphic site and affect the activity of the promoter.MethodsThe MRP1 5' flanking region was amplified by PCR from 36 CF patients and 100 non-CF subjects and the number of GCC triplets of each allele was determined by sequence and electrophoretic analysis. We performed gene reporter studies in CF airway epithelial cells 16HBE14o-AS3, in basal conditions and in the presence of AZM.ResultsWe found that the GCC repeat is polymorphic, ranging from 7 to 14 triplets either in CF or in non-CF subjects. Our data are preliminary and have to be confirmed on a larger population of CF subjects. The transcriptional activity of the proximal MRP1 5' regulatory region revealed no statistically significant correlations between the number of repeats and treatment with AZM.ConclusionWe identified a novel polymorphism in the 5'UTR of MRP1 gene that provides multiple alleles in a gene relevant for multidrug resistance as well as for CF, determining that this region is transcriptionally active and that this activity does not appear to be influenced by AZM treatment.

49 Reduction of tumor necrosis factor alpha (TNFα) expression by azithromycin (AZM) in CF airway epithelial cells

AZM has been described to ameliorate airway inflammation in CF patients. We aimed to study properties of this macrolide relevant for therapy, in particular antiinflammatory effects. Since TNFo. plays a relevant role in pathogenesis o f CF as pro-inflammatory molecule, we compared its levels of mRNA and protein expression, by QPCR and ELISA, in two CF airway epithelial cell lines. Cells were incubated with AZM or josamycin (JM), a macrolide lacking clinical anti-inflammatory effects. Activation o f pro-inflammatory transcription factors (TF) involved in the regulation of TNFo. transcription as Nuclear Factor-kB (NF-kB) and Specificity protein 1 (Spl) was determined by immunofluorescent staining in untreated cells and in the presence of AZM. We found that AZM reduces the levels of TNFo. mRNA in both cell lines (about 30%, p < 0.01) as well as TNFo. protein levels (about 55%, p < 0.01) compared to untreated cells, while JM was not significantly effective. Nuclear staining of NFkB and Spl was reduced in the presence of AZM in comparison with untreated cells. In an isogenic non-CF cell line we detected statistically significantly lower levels of TNFo. compared to the CF line, specifically supporting the therapeutic relevance of our data. Our study demonstrates that AZM reduces the expression of TNFo. mRNA and protein and suggests the inhibition of nuclear localization of NF-kB and Spl as a possible mechanism. Consistent with our previous detection of inhibition of interleukin 8 expression by AZM, the results of the present study further support the proposed anti-inflammatory effects of this macrolide relevant for therapy o f CE Supported by Italian Cystic Fibrosis Research Foundation (FFC10/05) and Legge 548/93 Finanziamento Ricerca Fibrosi Cistica 2004. 3. New therapies'

Spliceosome-Mediated RNATrans-Splicing with Recombinant Adeno-Associated Virus Partially Restores Cystic Fibrosis Transmembrane Conductance Regulator Function to Polarized Human Cystic Fibrosis Airway Epithelial Cells

We previously reported that spliceosome-mediated RNA trans-splicing (SMaRT), using recombinant adenoviral vectors expressing pre-trans-splicing molecules (PTMs), could partially restore cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel activity to polarized human DeltaF508 CF airway epithelia. Although these studies proved that SMaRT could correct CFTR mRNA defects, recombinant adenoviral infection from the basolateral surface was required because of inefficient infection from the apical membrane. Hence, applications of SMaRT technology for CF gene therapy require further testing with alternative, more clinically viable, vector systems. Furthermore, because recombinant adeno-associated virus (rAAV) vectors have packing limitations with respect to the size of the CFTR transgene insert, SMaRT correction of CFTR has the added attraction of a smaller transgene cassette. In the present study, we investigated whether rAAV vectors could effectively rescue CFTR chloride conductance in polarized human CF airway epithelial cells, using a SMaRT approach. AAV vectors were generated to carry a PTM engineered to bind intron 9 of CFTR pre-mRNA and then trans-splice the normal sequence for human CFTR exons 10-24 into the endogenous pre-mRNA. Human CF polarized airway epithelia were infected from the apical membrane with rAAV2 or rAAV5 CFTR-PTM vectors in the presence of proteasome-modulating agents (doxorubicin and N-acetyl-L-leucinyl-L-leucinyl-L-norleucinal) to enhance transduction. Epithelia were then evaluated for cAMP-sensitive short-circuit currents 2 weeks postinfection. Levels of CFTR correction seen with rAAV2 (1.07 +/- 0.24 microA) and rAAV5 (0.90 +/- 0.20 microA) CFTR-PTM vectors were similar, representing conductance equivalent to 14.2 and 13.6% of that observed in non-CF human polarized epithelia, respectively. RT-PCR analysis demonstrated the existence of wild-type CFTR transcript in CFTR-PTM-corrected epithelia, whereas only DeltaF508 mRNA was detected in polarized cells infected with control rAAV LacZ-PTM vectors. These results provide evidence that rAAV vectors are capable of using SMaRT to correct CFTR function after apical infection of human CF airway epithelia. The ability of CFTR-PTM-mediated correction to maintain endogenous CFTR regulation of the transgene product may further improve the efficacy of gene therapy for CF.

521. Spliceosome-Mediated RNA Trans-Splicing with rAAV Partially Restores CFTR Function to Polarized Human CF Airway Epithelial Cells

Top of pageAbstract We previously reported that Spliceosome-Mediated RNA Trans-splicing (SMaRT) using recombinant adenoviral vectors expressing pre-trans-splicing molecules (PTMs) could partially restore CFTR chloride channel activity to polarized human |[Auml]|F508 CF airway epithelia. Although these studies proved that SMaRT could correct CFTR mRNA defects, recombinant adenoviral infection from the basolateral surface was required due to inefficient infection from the apical membrane. Hence, applications of SMaRT technology for CF gene therapy require further testing with alternative, more clinically viable, vector systems. Furthermore, since rAAV vectors have packing limitations with respect to the size of the CFTR transgene insert, SMaRT correction of CFTR has the added attraction of a smaller transgene cassette. In the present study, we investigated whether rAAV vectors could effectively rescue CFTR chloride conductance in polarized human CF airway epithelial cells using a SMaRT approach. AAV vectors were generated to contain a PTM engineered to bind intron 9 of CFTR pre-mRNA and then trans-splice the normal sequence for human CFTR exons 10-24 into the endogenous pre-mRNA. Human CF polarized airway epithelia were infected from the apical membrane with rAAV2 or rAAV5 CFTR-PTM vectors in the presence of proteasome-modulating agents (Doxorubicin and LLnL) to enhance transduction. Epithelia were then evaluated for cAMP-sensitive short circuit currents at 2 weeks post-infection. Levels of CFTR correction seen with rAAV2 (1.07 |[plusmn]| 0.24 micro-Amps) and rAAV5 (0.90 |[plusmn]| 0.20 micro-Amps) CFTR-PTM vectors were similar, representing a conductance equivalent to 14.2% and 13.6% of that observed in non-CF human polarized epithelia, respectively. RT-PCR analysis demonstrated the existence of wild type CFTR transcript in CFTR-PTM correct epithelia, while only the |[Delta]|F508 mRNA was detected in polarized cells infected with control rAAV LacZ-PTM vectors. These results provided evidence that rAAV vectors are capable of utilizing SMaRT for correction of CFTR function following apical infection of human CF airway epithelia. The ability of CFTR-PTM-mediated correction to maintain endogenous CFTR regulation of the transgene product may further improve the efficacy of gene therapy for CF.

315. Targeted Gene Repair Exhibits a Polar Bias in the Directionality of Correction

The restoration of gene function through targeted correction of specific mutations guarantees that the correction is physiologically regulated and is expressed in “cell-appropriate” fashion. Such an approach has significant potential as a gene therapy strategy. Previous in vitro studies carried out in human airway epithelial cells showed both genomic and phenotypic correction of the most common cystic fibrosis transmembrane conductance regulator (CFTR) mutation, a 3-bp deletion in exon 10 (ΔF508), following introduction of small fragment homologous replacement (SFHR) comprised of WT CFTR sequences into transformed CF airway epithelial cells (Kunzelmann. et al 1996, Goncz, et al 1998, 2001, 2002)1,2,3,4. To enhance the efficacy of gene correction for therapeutic application, the mode of DNA delivery witch is an actual limit of gene correction was evaluated in terms of transfection parameters. We compared both physical and chemical DNA delivery approaches as measured by Enhance Green Fluorescent Protein (eGFP) gene expression. Chemically, we compared lipofectamine (Invitrogen), Superfect (Qiagen), Effectene (Qiagen) and a newly developed lipid, KLM47. For physical delivery, we used the AMAXA Nucleofector System. Our studies demonstrated that the nature (i.e., chemical composition) of lipids as well as the lipid/DNA (i.e., +/−) ratio modulate the transfection efficacy for each cell line. We also demonstrated that these cell lines derived from the airway epithelium have different lipid/DNA ratio requirements for optimal transfection. When compared to the AMAXA system, we observed a much higher efficiency of transfection (50–60%) than was observed with the chemical cocktails (5–10%). These studies are intriguing and suggest a physical delivery system may provide an effective means of delivering DNA to the nucleus and that ex vivo DNA delivery to pluripotent stem cells as a therapeutic modality.