A lung targeted miR-29 Mimic as a Therapy for Pulmonary Fibrosis

AbstractmicroRNAs are non-coding RNAs that negatively regulate gene networks. Previously, we reported a systemically delivered miR-29 mimic MRG-201 that reduced fibrosis in animal models, but at doses prohibiting clinical translation. Here, we generated MRG-229, a next-gen miR-29 mimic with improved chemical stability, conjugated with the internalization moiety BiPPB (PDGFbetaR-specific bicyclic peptide). In TGF-b-treated human lung fibroblasts and precision cut lung slices, MRG-229 decreased COL1A1 and ACTA2 gene expression and reduced collagen production. In bleomycin-treated mice, intravenous or subcutaneous delivery of MRG-229 downregulated profibrotic gene programs at doses more than ten-fold lower than the original compound. In rats and non-human primates, and at clinically relevant doses, MRG-229 was well tolerated, with no adverse findings observed. In human peripheral blood decreased mir-29 concentrations were associated with increased mortality in two cohorts potentially identified as a target population for treatment. Collectively, our results provide support for the development of MRG-229 as a potential therapy in humans with IPF.One Sentence SummaryOne Sentence Summary: A stabilized, next-generation miR-29 mimic has been developed that demonstrates efficacy at commercially viable doses with a robust safety margin in non-human primates.

Comparison of Long-term Human Precision-cut Lung Slice Culture Methodology and Response to Challenge: An Argument for Standardisation

As non-animal alternatives gain acceptance, a need for harmonised testing strategies has emerged. Arguably the most physiologically-relevant model for assessing potential respiratory toxicants, that based on human precision-cut lung slices (hPCLS) has been utilised in many laboratories, but a variety of culture methodologies are employed. In this pilot study, combinations of three different hPCLS culture methods (dynamic organ roller culture (DOC), air–liquid interface (ALI) and submersion) and various media (based on E-199, DMEM/F12 and RPMI-1640) were compared. The hPCLS were assessed in terms of their viability and responsiveness to challenge. The endpoints selected to compare the medium–method (M–M) combinations, which included histological features and viability, were evaluated at day 14 (D14) and day 28 (D28); protein and adenylate kinase (AK) content, and cytokine response to immunostimulants (lipopolysaccharide (LPS) at 5 μg/ml; polyinosinic:polycytidylic acid (Poly I:C) at 15 μg/ml) were evaluated at D28 only. Based on the set of endpoints assessed at D28, it was clear that certain culture conditions significantly affected the hPCLS, with the tissue retaining more of its native features and functionality (in terms of cytokine response) in some of the M–M combinations tested more than others. This pilot study indicates that the use of appropriate M–M combinations can help maintain the health and functional responses of hPCLS, and highlights the need for the standardisation of culture conditions in order to facilitate effective inter-laboratory comparisons and encourage greater acceptance by the regulatory community.

Organophosphorus pesticides exhibit compound specific effects in rat precision-cut lung slices (PCLS): mechanisms involved in airway response, cytotoxicity, inflammatory activation and antioxidative defense

Organophosphorus compound pesticides (OP) are widely used in pest control and might be misused for terrorist attacks. Although acetylcholinesterase (AChE) inhibition is the predominant toxic mechanism, OP may induce pneumonia and formation of lung edema after poisoning and during clinical treatment as life-threatening complication. To investigate the underlying mechanisms, rat precision-cut lung slices (PCLS) were exposed to the OP parathion, malathion and their biotransformation products paraoxon and malaoxon (100–2000 µmol/L). Airway response, metabolic activity, release of LDH, cytokine expression and oxidative stress response were analyzed. A concentration-dependent inhibition of airway relaxation was observed after exposure with the oxon but not with the thion-OP. In contrast, cytotoxic effects were observed for both forms in higher concentrations. Increased cytokine expression was observed after exposure to parathion and paraoxon (IL-6, GM-CSF, MIP-1α) and IL-6 expression was dependent on NFκB activation. Intracellular GSH levels were significantly reduced by all four tested OP but an increase in GSSG and HO-1 expression was predominantly observed after malaoxon exposure. Pretreatment with the antioxidant N-acetylcysteine reduced malaoxon but not paraoxon-induced cytotoxicity. PCLS as a 3D lung model system revealed OP-induced effects depending on the particular OP. The experimental data of this study contribute to a better understanding of OP toxicity on cellular targets and may be a possible explanation for the variety of clinical outcomes induced by different OP.

Optimization of long-term cold storage of rat precision cut lung slices (PCLS) with a tissue preservation solution

Precision cut lung slices (PCLS) are used as ex vivo model of the lung to fill the gap between in vitro and in vivo experiments. To allow optimal utilization of PCLS, possibilities to prolong slice viability via cold storage using optimized storage solutions were evaluated. Rat PCLS were cold stored in DMEM/F-12 or two different preservation solutions for up to 28 days at 4 °C. After rewarming in DMEM/F-12, metabolic activity, live/dead staining and mitochondrial membrane potential was assessed to analyze overall tissue viability. Single cell suspensions were prepared and proportions of CD45+, EpCAM+, CD31+ and CD90+ cells analyzed. As functional parameters, TNF-α expression was analyzed to detect inflammatory activity and bronchoconstriction after acetylcholine stimulus. After 14 days cold storage, viability and mitochondrial membrane potential were significantly better preserved after storage in solution 1 (potassium chloride rich) and solution 2 (potassium- and lactobionate-rich analog) compared to DMEM/F-12. Analysis of cell populations revealed efficient preservation of EpCAM+, CD31+ and CD90+ cells. Proportion of CD45+ cells decreased during cold storage but was better preserved by both modified solutions than by DMEM/F-12. PCLS stored in solution 1 responded substantially longer to inflammatory stimulation than those stored in DMEM/F-12 or solution 2. Analysis of bronchoconstriction revealed total loss of function after 14 days storage in DMEM/F-12 but in contrast, a good response in PCLS stored in the optimized solutions. An improved base solution with a high potassium chloride concentration optimizes cold storage of PCLS and allows shipment between laboratories and stockpiling of tissue samples.

Using a micro-physiological system to prolong the preservation of ex vivo lung tissue

Current in vitro and in vivo disease models have been reported to lack sufficient translation to human. Precision-Cut Lung Slices (PCLS) are viable sections of lung tissue and have been described to be a translational model for the ex vivo assessment of pharmacological and toxicological compounds. In most studies PCLS were cultured under static conditions. These lung sections, however, suffer from the limited viability. Here we present a novel modular microphysiological system (MPS) to prolong the cultivation of ex vivo lung tissue. A tailored MPS setup was designed using the PDMS free modular plug&play MPS construction kit. PCLS from mice were cultivated for up to one week under static versus perfused conditions. Using the MPS technology enabled a prolonged culture period with improved viability as shown by lowered lactate dehydrogenase release and improved membrane integrity. Using this technology might allow us to use PCLS for longer culture periods such as e.g. repeated dose toxicity or pharmacology studies.