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

Financial Intermediation, Capital Accumulation and Crisis Recovery

This paper integrates banks into a two-sector neoclassical growth model to account for the fact that a fraction of firms relies on banks to finance their investments. There are four major contributions to the literature: First, although banks’ leverage amplifies shocks, the endogenous response of leverage to shocks is an automatic stabilizer that improves the resilience of the economy. In particular, financial and labor market institutions are essential factors that determine the strength of this automatic stabilization. Second, there is a mix of publicly financed bank re-capitalization, dividend payout restrictions, and consumption taxes that stimulates a Pareto-improving rapid build-up of bank equity and accelerates economic recovery after a slump in the banking sector. Third, the model replicates typical patterns of financing over the business cycle: procyclical bank leverage, procyclical bank lending, and countercyclical bond financing. Fourth, the framework preserves its analytical tractability wherefore it can serve as a macro-banking module that can be easily integrated into more complex economic environments

Runx2-Genetically Engineered Skeletal Myoblasts for Bone Tissue Engineering

Bone tissue engineering is a promising approach to address the limitations of currently used bone tissue substitutes. However, an optimal cell source for the production of osteoblastic matrix proteins and mineral deposition has yet to be defined. In response to this deficiency, ex vivo gene therapy of easily accessible non-osteogenic cells, such as skeletal myoblasts, has become a prevalent strategy for inducing an osteoblastic phenotype. The majority of these approaches focus on constitutive overexpression of soluble osteogenic growth factors such as bone morphogenetic proteins (BMPs). In order to avoid aberrant effects of unregulated growth factor secretion, this work focuses on delivery of the osteoblastic transcription factor Runx2 as an autocrine osteogenic signal under the control of an inducible expression system. The overall objective of this research was to engineer an inducible cell source for bone tissue engineering that addresses the limitations of current cell-based approaches to orthopedic regeneration. Our central hypothesis was that inducible Runx2 overexpression in skeletal myoblasts would stimulate differentiation into a regulated osteoblastic phenotype. We have demonstrated that Runx2 overexpression stimulates transdifferentiation of primary skeletal myoblasts into a mineralizing osteoblastic phenotype. Furthermore, we have established Runx2-engineered skeletal myoblasts as a potent cell source for bone tissue engineering applications in vitro and in vivo, similar to BMP-2-overexpressing controls. Finally, we exogenously regulated osteoblastic differentiation by myoblasts engineered to express a tetracycline-inducible Runx2 transgene. This conversion into an osteoblastic phenotype was inducible, repressible, recoverable after suppression, and dose-dependent with tetracycline concentration. This work is significant because it addresses cell sourcing limitations of bone tissue engineering, develops controlled and effective gene therapy methods for orthopedic regeneration, and establishes a novel strategy for regulating the magnitude and kinetics of osteoblastic differentiation.Ph.D.Committee Chair: Garcia, Andres; Committee Member: Boyan, Barbara; Committee Member: Guldberg, Robert; Committee Member: Le Doux, Joseph; Committee Member: Pavlath, Grace; Committee Member: Sambanis, Anthanassio

Public Debt and the Balance Sheet of the Private Sector

This paper studies the impact of corporate political influence on fiscal policy. We in-troduce different interest groups, firms and households, into a simple growth model with incomplete markets and heterogeneous agents. Firms face non-insurable id-iosyncratic productivity shocks. They finance their productive investments by issu-ing bonds but cannot issue equity. Households’ savings are invested into corporate bonds and public debt. The government selects the levels of taxes and public debt so as to maximize a weighted sum of the welfares of firms’owners and households. More government debt reduces corporate leverage, increases the risk free rate r and decreases the growth rate g. A. The weight of firms in social welfare determines whether r g at the optimum, with different dynamics in both regimes

Gene targeting to the ROSA26 locus directed by engineered zinc finger nucleases

Targeted gene addition to mammalian genomes is central to biotechnology, basic research and gene therapy. For example, gene targeting to the ROSA26 locus by homologous recombination in embryonic stem cells is commonly used for mouse transgenesis to achieve ubiquitous and persistent transgene expression. However, conventional methods are not readily adaptable to gene targeting in other cell types. The emerging zinc finger nuclease (ZFN) technology facilitates gene targeting in diverse species and cell types, but an optimal strategy for engineering highly active ZFNs is still unclear. We used a modular assembly approach to build ZFNs that target the ROSA26 locus. ZFN activity was dependent on the number of modules in each zinc finger array. The ZFNs were active in a variety of cell types in a time- and dose-dependent manner. The ZFNs directed gene addition to the ROSA26 locus, which enhanced the level of sustained gene expression, the uniformity of gene expression within clonal cell populations and the reproducibility of gene expression between clones. These ZFNs are a promising resource for cell engineering, mouse transgenesis and pre-clinical gene therapy studies. Furthermore, this characterization of the modular assembly method provides general insights into the implementation of the ZFN technology

In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy

Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the CRISPR/Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR/Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR/Cas9-based genome editing as a potential therapy to treat DMD

Directed evolution of recombinase specificity by split gene reassembly

The engineering of new enzymes that efficiently and specifically modify DNA sequences is necessary for the development of enhanced gene therapies and genetic studies. To address this need, we developed a robust strategy for evolving site-specific recombinases with novel substrate specificities. In this system, recombinase variants are selected for activity on new substrates based on enzyme-mediated reassembly of the gene encoding β-lactamase that confers ampicillin resistance to Escherichia coli. This stringent evolution method was used to alter the specificities of catalytic domains in the context of a modular zinc finger-recombinase fusion protein. Gene reassembly was detectable over several orders of magnitude, which allowed for tunable selectivity and exceptional sensitivity. Engineered recombinases were evolved to react with sequences from the human genome with only three rounds of selection. Many of the evolved residues, selected from a randomly-mutated library, were conserved among other members of this family of recombinases. This enhanced evolution system will translate recombinase engineering and genome editing into a practical and expedient endeavor for academic, industrial and clinical applications

A Mechanism Design Approach to Climate Agreements *

Abstract: We analyze environmental agreements in contexts with voluntary participation by sovereign countries, incentives problems and possible limits on enforcement and commitment. Taking a mechanism design perspective, we study how countries may agree on effort targets and compensations to take into account multilateral externalities. The optimal mechanism unveils an important trade-off between solving a free riding problem in effort provision at the intensive margin for participating countries and another free riding problem at the extensive margin to ensure that all countries participate. This mechanism can easily be approximated by means of simple menus with attractive implementation and robustness properties. However, limits on enforcement and commitment might hinder its performances making the "business as usual" scenario more attractive. Keywords: public goods, incentive constraints, mechanism design, global warming. JEL Codes: Q54, D82, H23. * We thank workshop participants at Paris School of Economics, the Paris Environmental and Energy Economics Seminar, the CIRPEE (UQAM/HEC Montréal, Laval University)'s annual conference, CREST-LEI Paris, Frankfort, GREQAM-Marseille, the Congress of the Canadian Economic Association in Calgary, the Workshop on the Economics of Climate Change CDC Paris, Collège de France Paris, and ETH Zürich, but also Antoine Bommier Jean-Marc Bourgeon, Renaud Bourlès, Gabrielle Demange, Pierre Fleckinger, Hans Gersbach, Bard Harstad, Jérôme Pouyet, Jean-Charles Rochet, François Salanié and Alain Trannoy for helpful comments on an earlier draft. We also thank Daniel Coublucq and Perrin Lefèbvre for outstanding research assistance. All errors are ours

Chromatin Remodeling of Colorectal Cancer Liver Metastasis is Mediated by an HGF‐PU.1‐DPP4 Axis

Colorectal cancer (CRC) metastasizes mainly to the liver, which accounts for the majority of CRC-related deaths. Here it is shown that metastatic cells undergo specific chromatin remodeling in the liver. Hepatic growth factor (HGF) induces phosphorylation of PU.1, a pioneer factor, which in turn binds and opens chromatin regions of downstream effector genes. PU.1 increases histone acetylation at the DPP4 locus. Precise epigenetic silencing by CRISPR/dCas9KRAB or CRISPR/dCas9HDAC revealed that individual PU.1-remodeled regulatory elements collectively modulate DPP4 expression and liver metastasis growth. Genetic silencing or pharmacological inhibition of each factor along this chromatin remodeling axis strongly suppressed liver metastasis. Therefore, microenvironment-induced epimutation is an important mechanism for metastatic tumor cells to grow in their new niche. This study presents a potential strategy to target chromatin remodeling in metastatic cancer and the promise of repurposing drugs to treat metastasis