Repeat or single-dose lentiviral vector administration to mouse lungs? It's all about the timing.

Lentiviral vectors are attractive delivery vehicles for cystic fibrosis gene therapy owing to their low immunogenicity and ability to integrate into the host cell genome, thereby producing long-term, stable gene expression. Nonetheless, repeat dosing may be required to increase initial expression levels, and/or boost levels when they wane. The primary aim of this study was to determine if repeat dosing of a VSV-G pseudotyped LV vector delivered into mouse lungs is more effective than a single dose. C57Bl/6 mouse lungs were conditioned with lysophosphatidylcholine, followed one-hour later by a LV vector carrying the luciferase reporter gene, using six different short-term (≤1 wk) and long-term (>1 wk) dosing schedules. Luciferase expression was quantified using bioluminescence imaging over 12 months. Most dosing schedules produced detectable bioluminescence over the 12-month period, but the shorter intervals (≤1 wk) produced higher levels of flux than the longest interval (five doses at least 1-month apart). Ex vivo lung analysis at 12 months showed that the estimated mean flux for the group that received two doses 1-week apart was significantly greater than the single dose group and the two groups that received doses over a period greater than 1-week. These results suggest that early consecutive multiple doses are more effective at improving gene expression in mouse lungs at 12 months, than longer repeat dosing intervals.

Changes in Essential Fatty Acids and Ileal Genes Associated with Metabolizing Enzymes and Fatty Acid Transporters in Rodent Models of Cystic Fibrosis.

Cystic fibrosis (CF), the result of mutations in the CF transmembrane conductance regulator (CFTR), causes essential fatty acid deficiency. The aim of this study was to characterize fatty acid handling in two rodent models of CF; one strain which harbors the loss of phenylalanine at position 508 (Phe508del) in CFTR and the other lacks functional CFTR (510X). Fatty acid concentrations were determined using gas chromatography in serum from Phe508del and 510X rats. The relative expression of genes responsible for fatty acid transport and metabolism were quantified using real-time PCR. Ileal tissue morphology was assessed histologically. There was an age-dependent decrease in eicosapentaenoic acid and the linoleic acid:α-linolenic acid ratio, a genotype-dependent decrease in docosapentaenoic acid (n-3) and an increase in the arachidonic acid:docosahexaenoic acid ratio in Phe508del rat serum, which was not observed in 510X rats. In the ileum, Cftr mRNA was increased in Phe508del rats but decreased in 510X rats. Further, Elvol2, Slc27a1, Slc27a2 and Got2 mRNA were increased in Phe508del rats only. As assessed by Sirius Red staining, collagen was increased in Phe508del and 510X ileum. Thus, CF rat models exhibit alterations in the concentration of circulating fatty acids, which may be due to altered transport and metabolism, in addition to fibrosis and microscopic structural changes in the ileum.

Essential Fatty Acid Deficiency in Cystic Fibrosis Disease Progression: Role of Genotype and Sex.

Adequate intake of nutrients such as essential fatty acids (EFA) are critical in cystic fibrosis (CF). The clinical course of deterioration of lung function in people with CF has been shown to relate to nutrition. Independent of the higher energy consumption and malabsorption due to pancreatic insufficiency, EFA deficiency is closely associated with the risk of pulmonary infection, the most significant pathology in CF. This review will focus on the EFA deficiency identified in people with CF, as well as the limited progress made in deciphering the exact metabolic pathways that are dysfunctional in CF. Specifically, people with CF are deficient in linoleic acid, an omega 6 fatty acid, and the ratio of arachidonic acid (omega 6 metabolite) and docosahexaenoic acid (omega 3 metabolite) is increased. Analysis of the molecular pathways in bronchial cells has identified changes in the enzymes that metabolise EFA. However, fatty acid metabolism primarily occurs in the liver, with EFA metabolism in CF liver not yet investigated, indicating that further research is required. Despite limited understanding in this area, it is well known that adequate EFA concentrations are critical to normal membrane structure and function, and thus are important to consider in disease processes. Novel insights into the relationship between CF genotype and EFA phenotype will be discussed, in addition to sex differences in EFA concentrations in people with CF. Collectively, investigating the specific effects of genotype and sex on fatty acid metabolism may provide support for the management of people with CF via personalised genotype- and sex-specific nutritional therapies.

Improved in-vivo airway gene transfer via magnetic-guidance, with protocol development informed by synchrotron imaging.

Gene vectors to treat cystic fibrosis lung disease should be targeted to the conducting airways, as peripheral lung transduction does not offer therapeutic benefit. Viral transduction efficiency is directly related to the vector residence time. However, delivered fluids such as gene vectors naturally spread to the alveoli during inspiration, and therapeutic particles of any form are rapidly cleared via mucociliary transit. Extending gene vector residence time within the conducting airways is important, but hard to achieve. Gene vector conjugated magnetic particles that can be guided to the conducting airway surfaces could improve regional targeting. Due to the challenges of in-vivo visualisation, the behaviour of such small magnetic particles on the airway surface in the presence of an applied magnetic field is poorly understood. The aim of this study was to use synchrotron imaging to visualise the in-vivo motion of a range of magnetic particles in the trachea of anaesthetised rats to examine the dynamics and patterns of individual and bulk particle behaviour in-vivo. We also then assessed whether lentiviral-magnetic particle delivery in the presence of a magnetic field increases transduction efficiency in the rat trachea. Synchrotron X-ray imaging revealed the behaviour of magnetic particles in stationary and moving magnetic fields, both in-vitro and in-vivo. Particles could not easily be dragged along the live airway surface with the magnet, but during delivery deposition was focussed within the field of view where the magnetic field was the strongest. Transduction efficiency was also improved six-fold when the lentiviral-magnetic particles were delivered in the presence of a magnetic field. Together these results show that lentiviral-magnetic particles and magnetic fields may be a valuable approach for improving gene vector targeting and increasing transduction levels in the conducting airways in-vivo.

In vitro optimization of miniature bronchoscope lentiviral vector delivery for the small animal lung.

Current gene therapy delivery protocols for small animal lungs typically utilize indirect dose delivery via the nasal airways, or bolus delivery directly into the trachea. Both methods can result in variable transduction throughout the lung, as well as between animals, and cannot be applied in a targeted manner. To minimize variability and improve lung coverage we previously developed and validated a method to visualize and dose gene vectors into pre-selected lobes of rat lungs using a mini-bronchoscope. Lentiviral (LV) vectors are known to be fragile and can be inactivated easily by temperature or the application of shear stresses. There are several ways that the bronchoscope could be configured to deliver the LV vector, and these could result in different amounts of functional LV vector being delivered to the lung. This study evaluated several methods of LV vector delivery through the bronchoscope, and how flow rates and LV vector stabilizing diluents impact LV vector delivery. NIH-3T3 cells were exposed to LV vector containing the green fluorescent protein (GFP) reporter gene using various bronchoscopic delivery techniques and the number of GFP-positive cells produced by each was quantified by flow cytometry. The results showed that directly drawing the LV vector into the bronchoscope tip resulted in 80-90% recovery of viable vector, and was also the simplest method of delivery. The fluid delivery rate and the use of stabilizing serum in the vector diluent had no effect on the viability of the LV vector delivered. These findings can be used to optimize LV vector dose delivery into individual lung lobes of small animal models.

The Effects of Conditioning and Lentiviral Vector Pseudotype on Short- and Long-Term Airway Reporter Gene Expression in Mice.

A gene addition therapy into the conducting airway epithelium is a potential cure for cystic fibrosis lung disease. Achieving sustained lung gene expression has proven difficult due to the natural barriers of the lung. The development of lentiviral (LV) vectors pseudotyped with viral envelopes that have a natural tropism to the airway has enabled persistent gene expression to be achieved in vivo. The aims of this study were to compare the yields of hemagglutinin (HA) and vesicular stomatitis virus-glycoprotein (VSV-G) pseudotyped HIV-1 vectors produced under the same conditions by our standard LV vector production method. We then sought to measure gene expression in mouse airways and to determine whether lysophosphatidylcholine (LPC) conditioning enhances short- and long-term gene expression. C57Bl/6 mouse airways were conditioned with 10 µL of 0.1% LPC or saline control, followed 1 h later by a 30 µL dose of an HA or VSV-G pseudotyped vector carrying either the LacZ or luciferase reporter genes. LacZ expression was assessed by X-gal staining after 7 days, while lung luminescence was quantified regularly for up to 18 months by bioluminescent imaging. The HA pseudotyped vectors had functional titers 25 to 60 times lower than the VSV-G pseudotyped vectors. Conditioning the lung with LPC significantly increased the total number of LacZ-transduced cells for both pseudotypes compared to saline control. Regardless of LPC conditioning, the VSV-G pseudotype produced higher initial levels of gene expression compared to HA. LPC conditioning did not increase the number of transduced basal cells for either pseudotype compared to saline, and was not required for long-term gene expression. Both pseudotyped vectors effectively transduced the upper conducting airways of wild-type mice. The use of LPC conditioning before vector delivery was not required in mouse lungs to produce long-term gene expression, but did improve short-term gene expression.

Towards Human Translation of Lentiviral Airway Gene Delivery for Cystic Fibrosis: A One-Month CFTR and Reporter Gene Study in Marmosets.

Gene therapy continues to be a promising contender for the treatment of cystic fibrosis (CF) airway disease. We have previously demonstrated that airway conditioning with lysophosphatidylcholine (LPC) followed by delivery of a HIV-1-based lentiviral (LV) vector functionally corrects the CF transmembrane conductance regulator (CFTR) defect in the nasal airways of CF mice. In our earlier pilot study we showed that our technique can transduce marmoset lungs acutely; this study extends that work to examine gene expression in this nonhuman primate (NHP) 1 month after gene vector treatment. A mixture of three separate HIV-1 vesicular stomatitis virus G (VSV-G)-pseudotyped LV vectors containing the luciferase (Luc), LacZ, and hCFTR transgenes was delivered into the trachea through a miniature bronchoscope. We examined whether a single-dose delivery of LV vector after LPC conditioning could increase levels of transgene expression in the trachea and lungs compared with control (phosphate-buffered saline [PBS]) conditioning. At 1 month, bioluminescence was detected in vivo in the trachea of three of the six animals within the PBS control group, compared with five of the six LPC-treated animals. When examined ex vivo there was weak evidence that LPC improves tracheal Luc expression levels. In the lungs, bioluminescence was detected in vivo in four of the six PBS-treated animals, compared with five of the six LPC-treated animals; however, bioluminescence was present in all lungs when imaged ex vivo. LacZ expression was predominantly observed in the alveolar regions of the lung. hCFTR was detected by qPCR in the lungs of five animals. Basal cells were successfully isolated and expanded from marmoset tracheas, but no LacZ-positive colonies were detected. There was no evidence of an inflammatory response toward the LV vector at 1 month postdelivery, with cytokines remaining at baseline levels. In conclusion, we found weak evidence that LPC conditioning improved gene transduction in the trachea, but not in the marmoset lungs. We also highlight some of the challenges associated with translational lung gene therapy studies in NHPs.

Phenotypic Characterization and Comparison of Cystic Fibrosis Rat Models Generated Using CRISPR/Cas9 Gene Editing.

Animal models of cystic fibrosis (CF) are essential for investigating disease mechanisms and trialing potential therapeutics. This study generated two CF rat models using clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeats-associated protein 9 gene editing. One rat model carries the common human Phe508del (ΔF508) CF transmembrane conductance regulator (CFTR) mutation, whereas the second is a CFTR knockout model. Phenotype was characterized using a range of functional and histologic assessments, including nasal potential difference to measure electrophysiological function in the upper airways, RNAscope in situ hybridization and quantitative PCR to assess CFTR mRNA expression in the lungs, immunohistochemistry to localize CFTR protein in the airways, and histopathologic assessments in a range of tissues. Both rat models revealed a range of CF manifestations, including reduced survival, intestinal obstruction, bioelectric defects in the nasal epithelium, histopathologic changes in the trachea, large intestine, and pancreas, and abnormalities in the development of the male reproductive tract. The CF rat models presented herein will prove useful for longitudinal assessments of pathophysiology and therapeutics.

Epithelial mesenchymal transition (EMT): a universal process in lung diseases with implications for cystic fibrosis pathophysiology.

Cystic Fibrosis (CF) is a genetic disorder that arises due to mutations in the Cystic Fibrosis Transmembrane Conductance Regulator gene, which encodes for a protein responsible for ion transport out of epithelial cells. This leads to a disruption in transepithelial Cl-, Na + and HCO3- ion transport and the subsequent dehydration of the airway epithelium, resulting in infection, inflammation and development of fibrotic tissue. Unlike in CF, fibrosis in other lung diseases including asthma, chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis has been well characterised. One of the driving forces behind fibrosis is Epithelial Mesenchymal Transition (EMT), a process where epithelial cells lose epithelial proteins including E-Cadherin, which is responsible for tight junctions. The cell moves to a more mesenchymal phenotype as it gains mesenchymal markers such as N-Cadherin (providing the cells with migration potential), Vimentin and Fibronectin (proteins excreted to help form the extracellular matrix), and the fibroblast proliferation transcription factors Snail, Slug and Twist. This review paper explores the EMT process in a range of lung diseases, details the common links that these have to cystic fibrosis, and explores how understanding EMT in cystic fibrosis may open up novel methods of treating patients with cystic fibrosis.

Lobe-Specific Gene Vector Delivery to Rat Lungs Using a Miniature Bronchoscope.

For respiratory research utilizing gene vector delivery to the lung, the size of rodent models has typically necessitated relatively "blind" dosing via the nose, via an endotracheal tube, or through a surgical incision into the trachea. This commonly results in a limited ability to dose specific small regions of the lung reliably, and contributes to high levels of transduction variability between animals. The resultant poor reliability, reproducibility, and high variability compromises statistical capability, and so demands greater animal sample sizes than should be feasible. The first reliable targeted gene vector dosing of small regions in rat lungs has been designed and successfully implemented using a miniature rigid bronchoscope containing a working channel. Using this setup, this technique can currently access airway branches down to at least the fourth generation in the lungs of rats >200 g in body weight, allowing dosing and re-dosing of specific lobes via airway branch points in the lung tree. Here, the protocol for performing this minimally invasive technique is reported, along with the effect of delivering vesicular stomatitis virus G pseudotyped lentivirus to selected lung lobes. Examples of other applications, such as delivery of agar beads, are also shown. It is expected that the availability of this technique will substantially enhance gene vector studies in rat models for a range of lung diseases.

Epithelial disruption: a new paradigm enabling human airway stem cell transplantation.

BACKGROUND: Airway disease is a primary cause of morbidity and early mortality for patients with cystic fibrosis (CF). Cell transplantation therapy has proven successful for treating immune disorders and may have the potential to correct the airway disease phenotype associated with CF. Since in vivo cell delivery into unconditioned mouse airways leads to inefficient engraftment, we hypothesised that disrupting the epithelial cell layer using the agent polidocanol (PDOC) would facilitate effective transplantation of cultured stem cells in mouse nasal airways. METHODS: In this study, 4 µL of 2% PDOC in phosphate-buffered saline was administered to the nasal airway of mice to disrupt the epithelium. At 2 or 24 h after PDOC treatment, two types of reporter gene-expressing cells were transplanted into the animals: luciferase-transduced human airway basal cells (hABC-Luc) or luciferase-transduced human amnion epithelial cells (hAEC-Luc). Bioluminescence imaging was used to assess the presence of transplanted luciferase-expressing cells over time. Data were evaluated by using two-way analysis of variance with Sidak's multiple comparison. RESULTS: Successful transplantation was observed when hABCs were delivered 2 h after PDOC but was absent when transplantation was performed 24 h after PDOC, suggesting that a greater competitive advantage for the donor cells is present at the earlier time point. The lack of transplantation of hAECs 24 h after PDOC supports the importance of choosing the correct timing and cell type to facilitate transplantation. CONCLUSIONS: These studies into factors that may enable successful airway transplantation of human stem cells showed that extended functioning cell presence is feasible and further supports the development of methods that alter normal epithelial layer integrity. With improvements in efficacy, manipulating the airway epithelium to make it permissive towards cell transplantation may provide another option for safe and effective correction of CF transmembrane conductance regulator function in CF airways.

Large-scale production of lentiviral vectors using multilayer cell factories.

Lentiviral-mediated gene therapy has been proposed for the treatment of a range of diseases, and due to its genome integration properties, it offers the potential for long-lasting benefit from a once-off treatment. Production methods for pre-clinical studies in animal models, and ultimately for human clinical trials, must be capable of producing large quantities of high-quality lentiviral vector in an efficient and cost-effective manner. We report here a medium-scale method (from 1.5 L to 6 L of vector supernatant) for lentiviral vector production in adherent cell cultures using the NUNC™ EasyFill™ Cell Factory™ from Thermo Fisher Scientific. Downstream purification uses a Mustang Q XT5 anion exchange capsule from Pall, and an ultracentrifugation step to concentrate the vector. This method is capable of producing lentiviral vector with concentrated titres of 108-109 TU/ml, with reduced manual handling compared to single monolayer flask methods.

Role of Basal Cells in Producing Persistent Lentivirus-Mediated Airway Gene Expression.

Cystic fibrosis (CF) lung disease is an ideal candidate for a genetic therapy. It has been shown previously that preconditioning with lysophosphatidylcholine (LPC) prior to lentiviral (LV) vector delivery results in long-term in vivo gene expression in the airway epithelium of CF mice. It was hypothesized that this outcome is largely due to transduction of airway basal cells that in turn pass the transgene onto their progeny. The aim of these studies was to confirm if the in vivo delivery of a human immunodeficiency virus type 1 (HIV-1) vesicular stomatitis virus envelope glycoprotein (VSV-G) pseudotyped LV vector following LPC airway conditioning results in transduction of mouse airway basal cells in situ and if the transgene is passed onto their progeny. Additionally, the study sought to determine the efficiency of in vitro transduction of human airway basal cells. First, normal mouse nasal airways were pretreated with LPC prior to delivery of a HIV-1 VSV-G pseudotyped LV vector carrying a LacZ marker gene (LV-LacZ). An epithelial ablation model utilizing polidocanol was then used to demonstrate that clonal outgrowth of linear and spotted clusters of transgene expressing ciliated, basal, and goblet cells occurs following transduction of basal cells. Second, human basal cells were cultured from primary bronchial epithelial cells, with identity confirmed by keratin 5 staining. High levels of transgene expression were found following LV-LacZ transduction. This study demonstrates the ability of the vector delivery protocol to transduce mouse airway basal cells, the LV vector to transduce human basal cells, and the likely role of these cells in maintaining long-term gene expression. These findings support and further develop the potential of LV gene transfer for persistent correction of CF airway disease.

Mucopolysaccharidosis enzyme production by bone marrow and dental pulp derived human mesenchymal stem cells.

Mucopolysaccharidoses (MPS) are inherited metabolic disorders that arise from a complete loss or a reduction in one of eleven specific lysosomal enzymes. MPS children display pathology in multiple cell types leading to tissue and organ failure and early death. Mesenchymal stem cells (MSCs) give rise to many of the cell types affected in MPS, including those that are refractory to current treatment protocols such as hematopoietic stem cell (HSC) based therapy. In this study we compared multiple MPS enzyme production by bone marrow derived (hBM) and dental pulp derived (hDP) MSCs to enzyme production by HSCs. hBM MSCs produce significantly higher levels of MPS I, II, IIIA, IVA, VI and VII enzyme than HSCs, while hDP MSCs produce significantly higher levels of MPS I, IIIA, IVA, VI and VII enzymes. Higher transfection efficiency was observed in MSCs (89%) compared to HSCs (23%) using a lentiviral vector. Over-expression of four different lysosomal enzymes resulted in up to 9303-fold and up to 5559-fold greater levels in MSC cell layer and media respectively. Stable, persistent transduction of MSCs and sustained over-expression of MPS VII enzyme was observed in vitro. Transduction of MSCs did not affect the ability of the cells to differentiate down osteogenic, adipogenic or chondrogenic lineages, but did partially delay differentiation down the non-mesodermal neurogenic lineage.

Dietary fish oil replacement with canola oil up-regulates glutathione peroxidase 1 gene expression in yellowtail kingfish (Seriola lalandi).

The marine carnivore yellowtail kingfish (YTK, Seriola lalandi) was fed diets containing 5% residual fish oil (from the dietary fish meal) plus either 20% fish oil (FO), 20% canola oil (CO), 20% poultry oil (PO), 10% fish oil plus 10% canola oil (FO/CO) or 10% fish oil plus 10% poultry oil (FO/PO) and the effects on fish growth and hepatic expression of two glutathione peroxidase (GPx 1 and GPx 4) and two peroxiredoxin (Prx 1 and Prx 4) antioxidant genes were investigated. Partial (50%) replacement of the added dietary fish oil with poultry oil significantly improved fish growth whereas 100% replacement with canola oil significantly depressed fish growth. The fatty acid profiles of the fish fillets generally reflected those of the dietary oils except that there was apparent selective utilization of palmitic acid (16:0) and oleic acid (18:1n-9) and apparent selective retention of eicospentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). The Prx 1 and 4 genes were expressed at 10- and 100-fold the level of the GPx 4 and 1 genes, respectively, and at one-tenth the level of the highly expressed ß-actin reference gene. Dietary fish oil replacement with canola oil significantly up-regulated GPx 1 gene expression and there was a non-significant tendency towards down-regulation of Prx 1 and Prx 4. The results are discussed in terms of the effects of fish oil replacement on the peroxidation index of the diets and the resulting effects on the target antioxidant enzymes.