Abstract Purpose Successful in utero or perinatal gene therapy for congenital lung diseases, like cystic fibrosis and surfactant protein deficiency, requires identifying clinically relevant viral ...vectors that efficiently transduce airway epithelial cells. The purpose of the current preclinical large animal study was to evaluate lung epithelium transduction of adeno-associated viral vector (AAV) serotypes following intratracheal delivery. Methods Six different AAV serotypes (AAV1,AAV5,AAV6,AAV8,AAV9, AAVrh10) expressing the green fluorescent protein (GFP) as the transgene were injected into the right upper lobe (RUL) of perinatal sheep via bronchoscopy. At one week samples were harvested, analyzed by fluorescent stereomicroscopy and immunohistochemistry, and quantified using a radial grid and quantitative real time PCR. Results Fluorescent stereomicroscopy demonstrated GFP expression in the RUL following injection of all AAV serotypes assessed except AAV5. Immunohistochemistry analysis confirmed GFP expression in small and medium sized airways following intratracheal injection of AAV1,6,8,9, and rh10. However; only AAV8 and AAVrh10 resulted in transgene expression in large airways. These results were confirmed by qPCR, yet, after 40-cycles AAV1 did not show GFP gene amplification. Conclusion AAV serotypes 6,8,9, and rh10 demonstrated efficient GFP transgene expression at early time points and AAV8 demonstrated efficient transduction of all airway sizes with high pulmonary GFP expression tested using qPCR.
Abstract only
Cystic fibrosis (CF) is a life‐threatening genetic disease caused by mutations in the CF transmembrane conductance regulator gene. It is the most common genetic disease among ...Caucasians. There is an urgent need to create more therapies for CF since current drugs are not effective in treating all the mutations that cause this disease. Furthermore, gene therapy is a potential treatment for CF airway disease. Because of this, various viral‐based gene transfer vectors have been evaluated for their efficiency in correcting the CF airway disease phenotype. This project aimed to compare side‐by‐side the differences in the transduction efficiency and targeting of specific cell types of adenovirus (Ad)‐based and adeno‐associated virus (AAV)‐based vectors in different models of the airway. Different serotypes of AAV, namely 1, 2, 5, 6, and Ad5‐based vectors, expressing green fluorescent protein (GFP), firefly luciferase (ffLuc) or ß‐galactosidase (LacZ) were assessed in
in vitro
and
in vivo
gene transfer studies. Madin‐Darby Canine Kidney (MDCK) cells were grown submerged on plastic, and human airway epithelial (HAE) cells were grown on transwells and fed only from the basolateral side to attempt to model the human conducting airways. The viral vectors expressing GFP were tested at doses of 10
2
–10
3
genome copies (GC) for AAV or plaque‐forming unit (pfu) for Ad, and the level of transduction was assessed at different time points. The data demonstrates that in MDCK and HAE cells, the highest transduction was achieved by the Ad5 vector followed by the AAV2 vector, while AAV6 had the lowest transduction. Additionally, as examined in HAE cells, the age of the culture impacts transduction efficiency. In parallel, AAV vectors 1, 2, 5, 6 (at a dose of 8×10
10
– 3×10
11
GC) and Ad5 vector (at a dose of 1×10
11
pfu) expressing ffLuc or LacZ were intranasally delivered to mice to assess the transduction efficiency in the epithelium of both the nose and lungs. AAV6 was the most efficient at transducing the cells of the nasal epithelium of mice and AAV5, at transducing lung cells. AAV2 did not transduce airway cells in mice. In conclusion, we confirmed that the viral vectors have different transduction profiles in different models of airway epithelium. More airway models should be studied to better understand which are more useful for preclinical assessments of CF gene therapeutics.
Support or Funding Information
This project was supported by the National Heart, Lung, and Blood Institute (R25‐HL084665‐14).
Integrating lentiviral vectors based on the human immunodeficiency virus type-1 (HIV-1) can transduce quiescent cells, which in lung account for almost 95% of the epithelial cell population. ...Pseudotyping lentiviral vectors with the envelope glycoprotein from the Ebola Zaire virus, the lymphocytic choriomeningitis virus (LCMV), the Mokola virus, and the vesicular stomatitis virus (VSV-G) resulted in transduction of mouse alveolar epithelium, but gene expression in the lung of C57BL/6 and BALB/c mice waned within 90 days of vector injection. Intratracheal delivery of the four pseudotyped lentiviral vectors resulted in transgene-specific T-cell activation in both mouse strains, albeit lower than that achieved by intramuscular injection of the vectors. We performed an adoptive transfer of luciferase-specific T cells, isolated from spleen or lung of donor mice injected with VSV-G-pseudotyped lentivirus vector expressing luciferase into the muscle or lung, respectively, into recipient recombination-activating gene (RAG)–deficient mice transduced in lung with adenovirus expressing firefly luciferase (ffluc2). Gene expression declined within 7 days of adoptive transfer approaching background levels by day 36. Taken together, our results suggest that the loss of transduced cells in lung is due to VSV-G.HIV vector–mediated activation of transgene-specific T cells rather than as result of normal turnover of airway cells.
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.
Gene therapy for cystic fibrosis (CF) airway disease has emerged as a potentially successful therapy, because expression of the CF gene would be expected to restore the electrophysiological function ...of the airway epithelium to normalcy. Although, cellular and humoral immune responses to viral gene transfer vectors have been studied extensively, there has been no evaluation of T cell-mediated responses to the therapeutic human CF gene product. Using an adenovirus vector we demonstrated that T cells against human CF gene protein are elicited in CF gene knockout (KO), heterozygote (Het), and wild-type (wt) mice. A dominant CD8 T cell epitope found in CF gene KO, Het, and wt mice was mapped to NTYLRYITV. In CF gene KO mice we also identified (to a conserved region of the CF gene CSQFSWIMPGTIKEN), a minor T cell epitope that did not show any activity in the Het or wt mice.
Airway submucosal glands are sites of high expression of the cystic fibrosis transmembrane conductance regulator (CFTR) Cl â channel and contribute to fluid homeostasis in the lung. However, the ...molecular mechanisms of gland ion and fluid transport
are poorly defined. Here, submucosal gland serous acinar cells were isolated from murine airway, identified by immunofluorescence
and gene expression profiling, and used in physiological studies. Stimulation of isolated acinar cells with carbachol (CCh),
histamine or ATP was associated with marked decreases in cell volume (20 ± 2% within 62 ± 5 s) that were tightly correlated
with increases in cytoplasmic Ca 2+ concentration (Ca 2+ i ) as revealed by simultaneous DIC and fluorescent indicator dye microscopy. Simultaneous imaging of cell volume and the Cl â -sensitive fluorophore SPQ indicated that the 20% shrinkage was associated with a fall of Cl â i from 65 m m to 28 m m , reflecting loss of 67% of cell Cl â content, accompanied by parallel efflux of K + . Upon agonist removal, Ca 2+ i relaxed and the cells swelled back to resting volume via a bumetanide-sensitive Cl â influx pathway, likely to be NKCC1. Accordingly, agonist-induced serous acinar cell shrinkage and swelling are caused by
activation of solute efflux and influx pathways, respectively, and cell volume reflects the secretory state of these cells.
In contrast, elevation of cAMP failed to elicit detectible volume responses, or enhance those induced by submaximal CCh,
because the magnitude of the changes were likely to be below the threshold of detection using optical imaging. Finally, when
stimulated with cholinergic or cAMP agonists, cells from mice that lacked CFTR, as well as wild-type cells treated with a
CFTR inhibitor, exhibited identical rates and magnitudes of shrinkage and Cl â efflux compared with control cells. These results provide insights into the molecular mechanisms of salt and water secretion
by lung submucosal glands, and they suggest that while murine submucosal gland fluid secretion in response to cholinergic
stimulation can originate from CFTR-expressing serous acinar cells, it is not dependent upon CFTR function.
The human severe acute respiratory syndrome coronavirus (SARS-CoV) is a highly infectious virus that causes severe respiratory infections in humans. The spike envelope glycoprotein of SARS-CoV, the ...main determinant of SARS-CoV tropism, was isolated and used to pseudotype a human immunodeficiency virus (HIV)-based vector. Spike-pseudotyped HIV vector was generated and evaluated in vitro on well-differentiated human airway epithelial cells and bronchial explants and in vivo in murine airways. The spike envelope was less efficient at promoting HIV vector transduction of murine airway epithelium than an optimized deletion mutant of the Zaire ebolavirus envelope glycoprotein (NTD6L), which was used as a benchmark. However, spike-pseudotyped HIV vector was substantially more efficient than NTD6L-pseudotyped vector on human airway epithelium as demonstrated by lacZ gene transfer in primary cultures of epithelial cells and bronchial explants. In addition, this study shows that spike-pseudotyped HIV -based vector can efficiently transduce human dendritic cells and epithelial cells of the esophagus, which may have implications in investigating mechanisms of SARS-CoV pathogenesis. Spike-pseudotyped HIV-based vector is a novel lung-directed gene transfer vehicle that holds promise for the treatment of genetic lung diseases such as cystic fibrosis or alpha(1)-antitrypsin deficiency.
Virus vector-mediated gene transfer has been developed as a treatment for cystic fibrosis (CF) airway disease, a lethal inherited disorder caused by somatic mutations in the cystic fibrosis ...transmembrane conductance regulator gene. The pathological proinflammatory environment of CF as well as the naïve and adaptive immunity induced by the virus vector itself limits the effectiveness of gene therapy for CF airway. Here, we report the use of an HDAC inhibitor, valproic acid (VPA), to enhance the activity of the regulatory T cells (T(reg)) and to improve the expression of virus vector-mediated gene transfer to the respiratory epithelium. Our study demonstrates the potential utility of VPA, a drug used for over 50 years in humans as an anticonvulsant and mood-stabilizer, in controlling inflammation and improving the efficacy of gene transfer in CF airway.
Lung delivery of plasmid DNA encoding the CFTR gene complexed with a cationic liposome is a potential treatment option for patients with cystic fibrosis. We aimed to assess the efficacy of non-viral ...CFTR gene therapy in patients with cystic fibrosis.
We did this randomised, double-blind, placebo-controlled, phase 2b trial in two cystic fibrosis centres with patients recruited from 18 sites in the UK. Patients (aged ≥12 years) with a forced expiratory volume in 1 s (FEV1) of 50–90% predicted and any combination of CFTR mutations, were randomly assigned, via a computer-based randomisation system, to receive 5 mL of either nebulised pGM169/GL67A gene–liposome complex or 0·9% saline (placebo) every 28 days (plus or minus 5 days) for 1 year. Randomisation was stratified by % predicted FEV1 (<70 vs ≥70%), age (<18 vs ≥18 years), inclusion in the mechanistic substudy, and dosing site (London or Edinburgh). Participants and investigators were masked to treatment allocation. The primary endpoint was the relative change in % predicted FEV1. The primary analysis was per protocol. This trial is registered with ClinicalTrials.gov, number NCT01621867.
Between June 12, 2012, and June 24, 2013, we randomly assigned 140 patients to receive placebo (n=62) or pGM169/GL67A (n=78), of whom 116 (83%) patients comprised the per-protocol population. We noted a significant, albeit modest, treatment effect in the pGM169/GL67A group versus placebo at 12 months' follow-up (3·7%, 95% CI 0·1–7·3; p=0·046). This outcome was associated with a stabilisation of lung function in the pGM169/GL67A group compared with a decline in the placebo group. We recorded no significant difference in treatment-attributable adverse events between groups.
Monthly application of the pGM169/GL67A gene therapy formulation was associated with a significant, albeit modest, benefit in FEV1 compared with placebo at 1 year, indicating a stabilisation of lung function in the treatment group. Further improvements in efficacy and consistency of response to the current formulation are needed before gene therapy is suitable for clinical care; however, our findings should also encourage the rapid introduction of more potent gene transfer vectors into early phase trials.
Medical Research Council/National Institute for Health Research Efficacy and Mechanism Evaluation Programme.
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