Genome engineering of stem cells for autonomously regulated, closed-loop delivery of biologic drugs

Chronic inflammatory diseases such as arthritis are characterized by dysregulated responses to pro-inflammatory cytokines such as interleukin-1 (IL-1) and tumor necrosis factor α (TNF-α). Pharmacologic anti-cytokine therapies are often effective at diminishing this inflammatory response but have significant side effects and are used at high, constant doses that do not reflect the dynamic nature of disease activity. Using the CRISPR/Cas9 genome-engineering system, we created stem cells that antagonize IL-1- or TNF-α-mediated inflammation in an autoregulated, feedback-controlled manner. Our results show that genome engineering can be used successfully to rewire endogenous cell circuits to allow for prescribed input/output relationships between inflammatory mediators and their antagonists, providing a foundation for cell-based drug delivery or cell-based vaccines via a rapidly responsive, autoregulated system. The customization of intrinsic cellular signaling pathways in stem cells, as demonstrated here, opens innovative possibilities for safer and more effective therapeutic approaches for a wide variety of diseases

A genome-engineered bioartificial implant for autoregulated anticytokine drug delivery

[Figure: see text]

Towards a Pharmacophore for Amyloid

Diagnosing and treating Alzheimer's and other diseases associated with amyloid fibers remains a great challenge despite intensive research. To aid in this effort, we present atomic structures of fiber-forming segments of proteins involved in Alzheimer's disease in complex with small molecule binders, determined by X-ray microcrystallography. The fiber-like complexes consist of pairs of β-sheets, with small molecules binding between the sheets, roughly parallel to the fiber axis. The structures suggest that apolar molecules drift along the fiber, consistent with the observation of nonspecific binding to a variety of amyloid proteins. In contrast, negatively charged orange-G binds specifically to lysine side chains of adjacent sheets. These structures provide molecular frameworks for the design of diagnostics and drugs for protein aggregation diseases

Vaccinia Virus Proteins A52 and B14 Share a Bcl-2–Like Fold but Have Evolved to Inhibit NF-κB rather than Apoptosis

Vaccinia virus (VACV), the prototype poxvirus, encodes numerous proteins that modulate the host response to infection. Two such proteins, B14 and A52, act inside infected cells to inhibit activation of NF-κB, thereby blocking the production of pro-inflammatory cytokines. We have solved the crystal structures of A52 and B14 at 1.9 Å and 2.7 Å resolution, respectively. Strikingly, both these proteins adopt a Bcl-2–like fold despite sharing no significant sequence similarity with other viral or cellular Bcl-2–like proteins. Unlike cellular and viral Bcl-2–like proteins described previously, A52 and B14 lack a surface groove for binding BH3 peptides from pro-apoptotic Bcl-2–like proteins and they do not modulate apoptosis. Structure-based phylogenetic analysis of 32 cellular and viral Bcl-2–like protein structures reveals that A52 and B14 are more closely related to each other and to VACV N1 and myxoma virus M11 than they are to other viral or cellular Bcl-2–like proteins. This suggests that a progenitor poxvirus acquired a gene encoding a Bcl-2–like protein and, over the course of evolution, gene duplication events have allowed the virus to exploit this Bcl-2 scaffold for interfering with distinct host signalling pathways

Cellular and Biomaterial Engineering for Orthopaedic Regenerative Medicine

<p>The ends of long bones that articulate with respect to one another are lined with a crucial connective tissue called articular cartilage. This tissue plays an essential biomechanical function in synovial joints, as it serves to both dissipate load and lubricate articulating surfaces. Osteoarthritis is a painful and debilitating disease that drives the deterioration of articular cartilage. Like many chronic diseases, pro-inflammatory cytokines feature prominently in the onset and progression of osteoarthritis. Because cartilage lacks physiologic features critical for regeneration and self-repair, the development of effective strategies to create functional cartilage tissue substitutes remains a priority for the fields of tissue engineering and regenerative medicine. The overall objectives of this dissertation are to (1) develop a bioactive scaffold capable of mediating cell differentiation and formation of extracellular matrix that recapitulates native cartilage tissue and (2) to produce stem cells specifically tailored at the scale of the genome with the ability to resist inflammatory cues that normally lead to degeneration and pain. </p><p>Engineered replacements for musculoskeletal tissues generally require extensive ex vivo manipulation of stem cells to achieve controlled differentiation and phenotypic stability. By immobilizing lentivirus driving the expression of transforming growth factor-β3 to a highly structured, three dimensionally woven tissue engineering scaffold, we developed a technique for producing cell-instructive scaffolds that control human mesenchymal stem cell differentiation and possess biomechanical properties approximating those of native tissues. This work represents an important advance, as it establishes a method for generating constructs capable of restoring biological and mechanical function that may circumvent the need for ex vivo conditioning of engineered tissue substitutes.</p><p>Any functional cartilage tissue substitute must tolerate the inflammation intrinsic to an arthritic joint. Recently emerging tools from synthetic biology and genome engineering facilitate an unprecedented ability to modify how cells respond to their microenvironments. We exploited these developments to engineer cells that can evade signaling of the pro-inflammatory cytokine interleukin-1 (IL-1). Our study provides proof-of-principle evidence that cartilage derived from such engineered stem cells are resistant to IL-1-mediated degradation. </p><p>Extending on this work, we developed a synthetic biology strategy to further customize stem cells to combat inflammatory cues. We commandeered the highly responsive endogenous locus of the chemokine (C-C motif) ligand 2 gene in pluripotent stem cells to impart self-regulated, feedback-controlled production of biologic therapy. We demonstrated that repurposing of degradative signaling pathways induced by IL-1 and tumor necrosis factor toward transient production of cytokine antagonists enabled engineered cartilage tissue to withstand the action of inflammatory cytokines and to serve as a cell-based, auto-regulated drug delivery system.</p><p>In this work, we combine principles from synthetic biology, gene therapy, and functional tissue engineering to develop methods for generating constructs with biomimetic molecular and mechanical features of articular cartilage while precisely defining how cells respond to dysfunction in the body’s finely-tuned inflammatory systems. Moreover, our strategy for customizing intrinsic cellular signaling pathways in therapeutic stem cell populations opens innovative possibilities for controlled drug delivery to native tissues, which may provide safer and more effective treatments applicable to a wide variety of chronic diseases and may transform the landscape of regenerative medicine.</p>Dissertatio

Additional file 1 of Nuclear receptor ligand screening in an iPSC-derived in vitro blood–brain barrier model identifies new contributors to leptin transport

Additional file 1. Tables S1-S4 and Figures S1-S3

Additional file 2 of Nuclear receptor ligand screening in an iPSC-derived in vitro blood–brain barrier model identifies new contributors to leptin transport

Additional file 2. Table S5