ER procollagen storage defect without coupled unfolded protein response drives precocious arthritis.

Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II, the primary collagen in cartilage, can lead to diverse chondrodysplasias. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis. Here, we biochemically and mechanistically characterize an induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. We show that Gly1170Ser procollagen-II is notably slow to fold and secrete. Instead, procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Likely owing to the unique features of the collagen triple helix, this accumulation is not recognized by the unfolded protein response. Gly1170Ser procollagen-II interacts to a greater extent than wild-type with specific ER proteostasis network components, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the easily expandable cartilage model will enable rapid testing of therapeutic strategies to restore proteostasis in the collagenopathies.

ER procollagen storage defect without coupled unfolded protein response drives precocious arthritis.

Collagenopathies are a group of clinically diverse disorders caused by defects in collagen folding and secretion. For example, mutations in the gene encoding collagen type-II, the primary collagen in cartilage, can lead to diverse chondrodysplasias. One example is the Gly1170Ser substitution in procollagen-II, which causes precocious osteoarthritis. Here, we biochemically and mechanistically characterize an induced pluripotent stem cell-based cartilage model of this disease, including both hetero- and homozygous genotypes. We show that Gly1170Ser procollagen-II is notably slow to fold and secrete. Instead, procollagen-II accumulates intracellularly, consistent with an endoplasmic reticulum (ER) storage disorder. Owing to unique features of the collagen triple helix, this accumulation is not recognized by the unfolded protein response. Gly1170Ser procollagen-II interacts to a greater extent than wild-type with specific proteostasis network components, consistent with its slow folding. These findings provide mechanistic elucidation into the etiology of this disease. Moreover, the cartilage model will enable rapid testing of therapeutic strategies to restore proteostasis in the collagenopathies.

Using CRISPR/Cas9 to generate a heterozygous COL2A1 p.R719C iPSC line (MCRIi019-A-6) model of human precocious osteoarthritis.

The human iPSC line MCRIi019-A-6 was generated using CRISPR/Cas9-mediated gene editing to introduce a heterozygous COL2A1 exon 33 c.2155C>T (p.R719C) mutation into the control human iPSC line MCRIi019-A. Both the edited and parental lines display typical iPSC characteristics, including the expression of pluripotency markers, the ability to be differentiated into the three germ lines, and a normal karyotype. This cell line, along with the isogenic control line, can be used to study the molecular pathology of precocious osteoarthritis in a human model, more broadly understand type II collagenopathies, and explore novel therapeutic targets for this class of diseases.

CRISPR/Cas9 editing to generate a heterozygous COL2A1 p.G1170S human chondrodysplasia iPSC line, MCRIi019-A-2, in a control iPSC line, MCRIi019-A.

To develop an in vitro disease model of a human chondrodysplasia, we used CRISPR/Cas9 gene editing to generate a heterozygous COL2A1 exon 50 c.3508 GGT > TCA (p.G1170S) mutation in a control human iPSC line. Both the control and COL2A1 mutant lines displayed typical iPSC characteristics, including normal cell morphology, expression of pluripotency markers, the ability to differentiate into endoderm, ectoderm and mesoderm lineages and normal karyotype. These chondrodysplasia mutant and isogenic control cell lines can be used to explore disease mechanisms underlying type II collagenopathies and aid in the discovery of new therapeutic strategies.

High Throughput Experimentation Using DESI-MS to Guide Continuous-Flow Synthesis.

We demonstrate the use of accelerated reactions with desorption electrospray ionization mass spectrometry (DESI-MS) as a tool for predicting the outcome of microfluidic reactions. DESI-MS was employed as a high throughput experimentation tool to provide qualitative predictions of reaction outcomes, so that vast regions of chemical reactivity space may be more rapidly explored and areas of optimal efficiency identified. This work is part of a larger effort to accelerate reaction optimization to enable the rapid development of continuous-flow syntheses of small molecules in high yield. In order to build confidence in this approach, however, it is necessary to establish a robust predictive connection between reactions performed under analogous DESI-MS, batch, and microfluidic reaction conditions. In the present work, we explore the potential of high throughput DESI-MS experiments to identify trends in reactivity based on chemical structure, solvent, temperature, and stoichiometry that are consistent across these platforms. N-alkylation reactions were used as the test case due to their ease of reactant and product detection by electrospray ionization mass spectrometry (ESI-MS) and their great importance in API synthesis. While DESI-MS narrowed the scope of possibilities for reaction selection among some parameters such as solvent, others like stoichiometry and temperature still required further optimization under continuous synthesis conditions. DESI-MS high throughput experimentation (HTE) reaction evaluation significantly reduced the search space for flow chemistry optimization, thus representing a significant savings in time and materials to achieve a desired transformation with high efficiency.

Mass spectrometric directed system for the continuous-flow synthesis and purification of diphenhydramine.

A highly integrated approach to the development of a process for the continuous synthesis and purification of diphenhydramine is reported. Mass spectrometry (MS) is utilized throughout the system for on-line reaction monitoring, off-line yield quantitation, and as a reaction screening module that exploits reaction acceleration in charged microdroplets for high throughput route screening. This effort has enabled the discovery and optimization of multiple routes to diphenhydramine in glass microreactors using MS as a process analytical tool (PAT). The ability to rapidly screen conditions in charged microdroplets was used to guide optimization of the process in a microfluidic reactor. A quantitative MS method was developed and used to measure the reaction kinetics. Integration of the continuous-flow reactor/on-line MS methodology with a miniaturized crystallization platform for continuous reaction monitoring and controlled crystallization of diphenhydramine was also achieved. Our findings suggest a robust approach for the continuous manufacture of pharmaceutical drug products, exemplified in the particular case of diphenhydramine, and optimized for efficiency and crystal size, and guided by real-time analytics to produce the agent in a form that is readily adapted to continuous synthesis.