Gene editing for inflammatory disorders.

Technology for precise and efficient genetic editing is constantly evolving and is now capable of human clinical applications. Autoimmune and inflammatory diseases are chronic, disabling, sometimes life-threatening, conditions that feature heritable components. Both primary genetic lesions and the inflammatory pathobiology underlying these diseases represent fertile soil for new therapies based on the capabilities of gene editing. The ability to orchestrate precise targeted modifications to the genome will likely enable cell-based therapies for inflammatory diseases such as monogenic autoinflammatory disease, acquired autoimmune disease and for regenerative medicine in the setting of an inflammatory environment. Here, we discuss recent advances in genome editing and their evolving applications in immunoinflammatory diseases. Strengths and limitations of older genetic modification tools are compared with CRISPR/Cas9, base editing, RNA editing, targeted activators and repressors of transcription and targeted epigenetic modifiers. Commonly employed delivery vehicles to target cells or tissues of interest with genetic modification machinery, including viral, non-viral and cellular vectors, are described. Finally, applications in animal and human models of inflammatory diseases are discussed. Use of chimeric autoantigen receptor T cells, correction of monogenic diseases with genetically edited haematopoietic stem and progenitor cells, engineering of induced pluripotent stem cells and ex vivo expansion and modification of regulatory T cells for a range of chronic inflammatory diseases are reviewed.

Feedback regulation of NEUROG2 activity by MTGR1 is required for progression of neurogenesis.

The sequential steps of neurogenesis are characterized by highly choreographed changes in transcription factor activity. In contrast to the well-studied mechanisms of transcription factor activation during neurogenesis, much less is understood regarding how such activity is terminated. We previously showed that MTGR1, a member of the MTG family of transcriptional repressors, is strongly induced by a proneural basic helix-loop-helix transcription factor, NEUROG2 in developing nervous system. In this study, we describe a novel feedback regulation of NEUROG2 activity by MTGR1. We show that MTGR1 physically interacts with NEUROG2 and represses transcriptional activity of NEUROG2. MTGR1 also prevents DNA binding of the NEUROG2/E47 complex. In addition, we provide evidence that proper termination of NEUROG2 activity by MTGR1 is necessary for normal progression of neurogenesis in the developing spinal cord. These results highlight the importance of feedback regulation of proneural gene activity in neurodevelopment.

Interaction of MTG family proteins with NEUROG2 and ASCL1 in the developing nervous system.

During neural development, members of MTG family of transcriptional repressors are induced by proneural basic helix-loop-helix (bHLH) transcription factors and in turn inhibit the activity of the bHLH proteins, forming a negative feedback loop that regulates the normal progression of neurogenesis. Three MTG genes, MTG8, MTG16 and MTGR1, are expressed in distinct patterns in the developing nervous system. Various bHLH proteins are also expressed in distinct patterns. We asked whether there is a functional relationship between specific MTG and bHLH proteins in developing chick spinal cord. First, we examined if each MTG gene is induced by specific bHLH proteins. Although expression of NEUROG2, ASCL1 and MTG genes overlapped, the boundaries of gene expression did not match. Ectopic expression analysis showed that MTGR1 and NEUROD4, which show similar expression patterns, are regulated differently by NEUROG2 and ASCL1. Thus, our results show that expression of MTG genes is not regulated by a single upstream bHLH protein, but represents an integration of the activity of multiple regulators. Next, we asked if each MTG protein inhibits specific bHLH proteins. Transcription assay showed that NEUROG2 and ASCL1 are inhibited by MTGR1 and MTG16, and less efficiently by MTG8. Deletion mapping of MTGR1 showed that MTGR1 binds NEUROG2 and ASCL1 using multiple interaction surfaces, and all conserved domains are required for its repressor activity. These results support the model that MTG proteins form a higher-order repressor complex and modulate transcriptional activity of bHLH proteins during neurogenesis.

Structural properties of the primary medial knee ligaments.

BACKGROUND: The structural properties of the individual components of the superficial medial collateral ligament (MCL), deep MCL, and posterior oblique ligament (POL) have not been studied in isolation. To define the necessary strength requirements for an anatomical medial knee reconstruction, knowledge of these structural properties is necessary. HYPOTHESIS: The components of the superficial MCL, POL, and deep MCL have significantly different structural properties. STUDY DESIGN: Controlled laboratory study. METHODS: This study used 20 fresh-frozen nonpaired cadaveric knee specimens with a mean age of 54 years (range, 27 to 68 years). These knees provided 8 samples for each tested medial knee structure, which was individually isolated and loaded to failure at 20 mm per minute. Specifically tested were the superficial MCL with intact femoral and detached proximal tibial attachments, the superficial MCL with intact femoral and detached distal tibial attachments, the central arm of the POL, and the isolated deep MCL. Load was recorded as a function of displacement. Stiffness of the ligament at failure was calculated from these measurements. RESULTS: The mean load at failure for the superficial MCL with the intact femoral and distal tibial attachments was 557 N. Mean load at failure was 88 N for the intact femoral and proximal tibial divisions of the superficial MCL, 256 N for the POL, and 101 N for the deep MCL. Stiffness of the ligaments just before failure was 63, 17, 38, and 27 N/mm, in the same order as above. CONCLUSION: The proximal and distal tibial divisions of the superficial MCL, POL, and deep MCL produced loads of clinical importance. CLINICAL RELEVANCE: Knowledge of the structural properties of these attachment sites will assist in reconstruction graft choices, fixation method choices, and overall operative treatment of medial knee injury.