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

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

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

N-cadherin is key to expression of the nucleus pulposus cell phenotype under selective substrate culture conditions

published_or_final_versio

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

Translating the genomics revolution: the need for an international gene therapy consortium for monogenic diseases

Letter to the Edito

Pulling the genome in opposite directions to dissect gene networks

Abstract Orthogonal CRISPR-Cas systems have been integrated into combinatorial screens to decipher complex genetic relationships in two recent studies