Trinucleotide mRNA Cap Analogue N6-Benzylated at the Site of Posttranscriptional m6Am Mark Facilitates mRNA Purification and Confers Superior Translational Properties In Vitro and In Vivo.

Eukaryotic mRNAs undergo cotranscriptional 5'-end modification with a 7-methylguanosine cap. In higher eukaryotes, the cap carries additional methylations, such as m6Am─a common epitranscriptomic mark unique to the mRNA 5'-end. This modification is regulated by the Pcif1 methyltransferase and the FTO demethylase, but its biological function is still unknown. Here, we designed and synthesized a trinucleotide FTO-resistant N6-benzyl analogue of the m6Am-cap-m7GpppBn6AmpG (termed AvantCap) and incorporated it into mRNA using T7 polymerase. mRNAs carrying Bn6Am showed several advantages over typical capped transcripts. The Bn6Am moiety was shown to act as a reversed-phase high-performance liquid chromatography (RP-HPLC) purification handle, allowing the separation of capped and uncapped RNA species, and to produce transcripts with lower dsRNA content than reference caps. In some cultured cells, Bn6Am mRNAs provided higher protein yields than mRNAs carrying Am or m6Am, although the effect was cell-line-dependent. m7GpppBn6AmpG-capped mRNAs encoding reporter proteins administered intravenously to mice provided up to 6-fold higher protein outputs than reference mRNAs, while mRNAs encoding tumor antigens showed superior activity in therapeutic settings as anticancer vaccines. The biochemical characterization suggests several phenomena potentially underlying the biological properties of AvantCap: (i) reduced propensity for unspecific interactions, (ii) involvement in alternative translation initiation, and (iii) subtle differences in mRNA impurity profiles or a combination of these effects. AvantCapped-mRNAs bearing the Bn6Am may pave the way for more potent mRNA-based vaccines and therapeutics and serve as molecular tools to unravel the role of m6Am in mRNA.

Inhibition of Macrophage-Specific CHIT1 as an Approach to Treat Airway Remodeling in Severe Asthma.

Chitotriosidase (CHIT1) is an enzyme produced by macrophages that regulates their differentiation and polarization. Lung macrophages have been implicated in asthma development; therefore, we asked whether pharmacological inhibition of macrophage-specific CHIT1 would have beneficial effects in asthma, as it has been shown previously in other lung disorders. CHIT1 expression was evaluated in the lung tissues of deceased individuals with severe, uncontrolled, steroid-naïve asthma. OATD-01, a chitinase inhibitor, was tested in a 7-week-long house dust mite (HDM) murine model of chronic asthma characterized by accumulation of CHIT1-expressing macrophages. CHIT1 is a dominant chitinase activated in fibrotic areas of the lungs of individuals with fatal asthma. OATD-01 given in a therapeutic treatment regimen inhibited both inflammatory and airway remodeling features of asthma in the HDM model. These changes were accompanied by a significant and dose-dependent decrease in chitinolytic activity in BAL fluid and plasma, confirming in vivo target engagement. Both IL-13 expression and TGFß1 levels in BAL fluid were decreased and a significant reduction in subepithelial airway fibrosis and airway wall thickness was observed. These results suggest that pharmacological chitinase inhibition offers protection against the development of fibrotic airway remodeling in severe asthma.

Targeting arginase-1 exerts antitumor effects in multiple myeloma and mitigates bortezomib-induced cardiotoxicity.

Multiple myeloma (MM) remains an incurable malignancy of plasma cells despite constantly evolving therapeutic approaches including various types of immunotherapy. Increased arginase activity has been associated with potent suppression of T-cell immune responses in different types of cancer. Here, we investigated the role of arginase 1 (ARG1) in Vκ*MYC model of MM in mice. ARG1 expression in myeloid cells correlated with tumor progression and was accompanied by a systemic drop in ʟ-arginine levels. In MM-bearing mice antigen-induced proliferation of adoptively transferred T-cells was strongly suppressed and T-cell proliferation was restored by pharmacological arginase inhibition. Progression of Vκ*MYC tumors was significantly delayed in mice with myeloid-specific ARG1 deletion. Arginase inhibition effectively inhibited tumor progression although it failed to augment anti-myeloma effects of bortezomib. However, arginase inhibitor completely prevented development of bortezomib-induced cardiotoxicity in mice. Altogether, these findings indicate that arginase inhibitors could be further tested as a complementary strategy in multiple myeloma to mitigate adverse cardiac events without compromising antitumor efficacy of proteasome inhibitors.

Pharmacological Inhibition of Chitotriosidase (CHIT1) as a Novel Therapeutic Approach for Sarcoidosis.

Introduction: Sarcoidosis is a systemic disease of unknown etiology characterized by granuloma formation in the affected tissues. The pathologically activated macrophages are causatively implicated in disease pathogenesis and play important role in granuloma formation. Chitotriosidase (CHIT1), macrophage-derived protein, is upregulated in sarcoidosis and its levels correlate with disease severity implicating CHIT1 in pathology. Methods: CHIT1 was evaluated in serum and bronchial mucosa and mediastinal lymph nodes specimens from sarcoidosis patients. The therapeutic efficacy of OATD-01 was assessed ex vivo on human bronchoalveolar lavage fluid (BALF) macrophages and in vivo in the murine models of granulomatous inflammation. Results: CHIT1 activity was significantly upregulated in serum from sarcoidosis patients. CHIT1 expression was restricted to granulomas and localized in macrophages. Ex vivo OATD-01 inhibited pro-inflammatory mediators' production (CCL4, IL-15) by lung macrophages. In the acute model of granulomatous inflammation in mice, OATD-01 showed anti-inflammatory effects reducing the percentage of neutrophils and CCL4 concentration in BALF. In the chronic model, inhibition of CHIT1 led to a decrease in the number of organized lung granulomas and the expression of sarcoidosis-associated genes. Conclusion: In summary, CHIT1 activity was increased in sarcoidosis patients and OATD-01, a first-in-class CHIT1 inhibitor, demonstrated efficacy in murine models of granulomatous inflammation providing a proof-of-concept for its clinical evaluation in sarcoidosis.

Inhibition of CHIT1 as a novel therapeutic approach in idiopathic pulmonary fibrosis.

Idiopathic pulmonary fibrosis (IPF) is a progressive and eventually fatal lung disease with a complex etiology. Approved drugs, nintedanib and pirfenidone, modify disease progression, but IPF remains incurable and there is an urgent need for new therapies. We identified chitotriosidase (CHIT1) as new driver of fibrosis in IPF and a novel therapeutic target. We demonstrate that CHIT1 activity and expression are significantly increased in serum (3-fold) and induced sputum (4-fold) from IPF patients. In the lungs CHIT1 is expressed in a distinct subpopulation of profibrotic, disease-specific macrophages, which are only present in patients with ILDs and CHIT1 is one of the defining markers of this fibrosis-associated gene cluster. To define CHIT1 role in fibrosis, we used the therapeutic protocol of the bleomycin-induced pulmonary fibrosis mouse model. We demonstrate that in the context of chitinase induction and the macrophage-specific expression of CHIT1, this model recapitulates lung fibrosis in ILDs. Genetic inactivation of Chit1 attenuated bleomycin-induced fibrosis (decreasing the Ashcroft scoring by 28%) and decreased expression of profibrotic factors in lung tissues. Pharmacological inhibition of chitinases by OATD-01 reduced fibrosis and soluble collagen concentration. OATD-01 exhibited anti-fibrotic activity comparable to pirfenidone resulting in the reduction of the Ashcroft score by 32% and 31%, respectively. These studies provide a preclinical proof-of-concept for the antifibrotic effects of OATD-01 and establish CHIT1 as a potential new therapeutic target for IPF.

Discovery of OATD-01, a First-in-Class Chitinase Inhibitor as Potential New Therapeutics for Idiopathic Pulmonary Fibrosis.

Chitotriosidase (CHIT1) and acidic mammalian chitinase (AMCase) are the enzymatically active chitinases that have been implicated in the pathology of chronic lung diseases such as asthma and interstitial lung diseases (ILDs), including idiopathic pulmonary fibrosis (IPF) and sarcoidosis. The clinical and preclinical data suggest that pharmacological inhibition of CHIT1 might represent a novel therapeutic approach in IPF. Structural modification of an advanced lead molecule 3 led to the identification of compound 9 (OATD-01), a highly active CHIT1 inhibitor with both an excellent PK profile in multiple species and selectivity against a panel of other off-targets. OATD-01 given orally once daily in a range of doses between 30 and 100 mg/kg showed significant antifibrotic efficacy in an animal model of bleomycin-induced pulmonary fibrosis. OATD-01 is the first-in-class CHIT1 inhibitor, currently completed phase 1b of clinical trials, to be a potential treatment for IPF.

A critical role for ABC transporters in persistent lung inflammation in the development of emphysema after smoke exposure.

Macrophage infiltration is common to both emphysema and atherosclerosis, and cigarette smoke down-regulates the macrophage cholesterol efflux transporter ATP binding cassette (ABC)A1. This decreased cholesterol efflux results in lipid-laden macrophages. We hypothesize that cigarette smoke adversely affects cholesterol transport via an ABCA1-dependent mechanism in macrophages, enhancing TLR4/myeloid differentiation primary response gene 88 (Myd88) signaling and resulting in matrix metalloproteinase (MMP) up-regulation and exacerbation of pulmonary inflammation. ABCA1 is significantly down-regulated in the lung upon smoke exposure conditions. Macrophages exposed to cigarette smoke in vivo and in vitro exhibit impaired cholesterol efflux correlating with significantly decreased ABCA1 expression, up-regulation of the TLR4/Myd88 pathway, and downstream MMP-9 and MMP-13 expression. Treatment with liver X receptor (LXR) agonist restores ABCA1 expression after short-term smoke exposure and attenuates the inflammatory response; after long-term smoke exposure, there is also attenuated physiologic and morphologic changes of emphysema. In vitro, treatment with LXR agonist decreases macrophage inflammatory activation in wild-type but not ABCA1 knockout mice, suggesting an ABCA1-dependent mechanism of action. These studies demonstrate an important association between cigarette smoke exposure and cholesterol-mediated pathways in the macrophage inflammatory response. Modulation of these pathways through manipulation of ABCA1 activity effectively blocks cigarette smoke-induced inflammation and provides a potential novel therapeutic approach for the treatment of chronic obstructive pulmonary disease.-Sonett, J., Goldklang, M., Sklepkiewicz, P., Gerber, A., Trischler, J., Zelonina, T., Westerterp, M., Lemaître, V., Okada, V., D'Armiento, J. A critical role for ABC transporters in persistent lung inflammation in the development of emphysema after smoke exposure.

The divergent roles of secreted frizzled related protein-1 (SFRP1) in lung morphogenesis and emphysema.

Developmentally expressed genes are believed to play a central role in tissue repair after injury; however, in lung disease their role has not been established. This study demonstrates that SFRP1, an inhibitor of Wnt signaling normally expressed during lung embryogenesis, is induced in the lungs of emphysema patients and in two murine models of the disease. SFRP1 was found to be essential for alveolar formation as Sfrp1(-/-) mice exhibited aberrant Wnt signaling, mesenchymal proliferation, and impaired alveoli formation. In contrast, SFRP1 activated ERK and up-regulated MMP1 and MMP9 without altering TIMP1 production when expressed in human lung epithelial cells. These findings demonstrate that SFRP1 promotes normal alveolar formation in lung development, although its expression in the adult up-regulates proteins that can cause tissue destruction. Thus, SFRP1 induction during tissue injury is unlikely to contribute to the repair response but rather is a participatory factor in the pathogenesis of emphysema and tissue destruction.