Spell Checking Nature: Versatility of CRISPR/Cas9 for Developing Treatments for Inherited Disorders.

Clustered regularly interspaced short palindromic repeat (CRISPR) has arisen as a frontrunner for efficient genome engineering. However, the potentially broad therapeutic implications are largely unexplored. Here, to investigate the therapeutic potential of CRISPR/Cas9 in a diverse set of genetic disorders, we establish a pipeline that uses readily obtainable cells from affected individuals. We show that an adapted version of CRISPR/Cas9 increases the amount of utrophin, a known disease modifier in Duchenne muscular dystrophy (DMD). Furthermore, we demonstrate preferential elimination of the dominant-negative FGFR3 c.1138G>A allele in fibroblasts of an individual affected by achondroplasia. Using a previously undescribed approach involving single guide RNA, we successfully removed large genome rearrangement in primary cells of an individual with an X chromosome duplication including MECP2. Moreover, removal of a duplication of DMD exons 18-30 in myotubes of an individual affected by DMD produced full-length dystrophin. Our findings establish the far-reaching therapeutic utility of CRISPR/Cas9, which can be tailored to target numerous inherited disorders.

Lethal Disorder of Mitochondrial Fission Caused by Mutations in DNM1L.

We describe two infants with hypotonia, absent respiratory effort, and giant mitochondria in neurons due to compound heterozygosity for 2 nonsense mutations of DNM1L. DNM1L has a critical role in regulating mitochondrial morphology and function. This observation confirms the central role of mitochondrial fission to normal human development.

Pre-B cell colony-enhancing factor (PBEF/Nampt/visfatin) primes neutrophils for augmented respiratory burst activity through partial assembly of the NADPH oxidase.

Pre-B cell colony-enhancing factor ([PBEF] also known as Nampt/visfatin) is a pleiotropic 52-kDa cytokine-like molecule whose activity has been implicated in multiple inflammatory disease states. PBEF promotes polymorphonuclear neutrophil (PMN) proinflammatory function by inhibiting constitutive PMN apoptosis. We investigated whether PBEF activates or primes for PMN respiratory burst. We found that although PBEF did not activate respiratory burst on its own, it primed for increased reactive oxygen species generation through the NADPH oxidase. PBEF promoted membrane translocation of cytosolic NADPH oxidase subunits p40 and p47, but not p67, induced p40 phosphorylation on Thr(154), and activated the small GTPase Rac. Priming, translocation, and phosphorylation were dependent on activation of p38 and ERK MAPKs, but not of PI3K. Priming by PBEF occurred independent of its NAD-generating capacity because neither nicotinamide mononucleotide or NAD could recapitulate the effects, and a specific inhibitor of PBEF, APO-866, could not inhibit priming. Taken together, these results demonstrate that PBEF can prime for PMN respiratory burst activity by promoting p40 and p47 translocation to the membrane, and this occurs in a MAPK-dependent fashion.

Pre-B cell colony-enhancing factor (PBEF)/visfatin: a novel mediator of innate immunity.

Pre-B cell colony-enhancing factor (PBEF), also known as visfatin, is a highly conserved, 52-kDa protein found in living species from bacteria to humans. Originally a curiosity identified serendipitously in microarray studies but having no obvious functional importance, PBEF has now been shown to exert three distinct activities of central importance to cellular energetics and innate immunity. Within the cell, PBEF functions as a nicotinamide phosphoribosyl transferase, the rate-limiting step in a salvage pathway of nicotinamide adenine dinucleotide (NAD) biosynthesis. By virtue of this role, it can regulate cellular levels of NAD and so impact not only cellular energetics but also NAD-dependent enzymes such as sirtuins. Although it lacks a signal peptide, PBEF is released by a variety of cells, and elevated levels can be found in the systemic circulation of patients with a variety of inflammatory diseases. As an extracellular cytokine, PBEF can induce the cellular expression of inflammatory cytokines such as TNF-alpha, IL-1beta, and IL-6. Finally, PBEF has been shown to be an adipokine expressed by fat cells that exerts a number of insulin mimetic and antagonistic effects. PBEF expression is up-regulated in a variety of acute and chronic inflammatory diseases including sepsis, acute lung injury, rheumatoid arthritis, inflammatory bowel disease, and myocardial infarction and plays a key role in the persistence of inflammation through its capacity to inhibit neutrophil apoptosis. This review summarizes the admittedly incomplete body of emerging knowledge about a remarkable new mediator of innate immunity.

Modulating neutrophil apoptosis.

Polymorphonuclear neutrophils are short-lived phagocytic cells that serve as cardinal early cellular effectors of innate immunity. Both oxidative and non-oxidative mechanisms contribute to microbial killing by the neutrophil. Neutrophil defence mechanisms are potent but non-specific, with the result that inadvertent injury to host tissues commonly accompanies the activation of a neutrophil mediated response; this bystander injury has been implicated in the tissue injury of sepsis. The capacity for neutrophils to cause injury to host tissues is attenuated by the relatively short in vivo lifespan of the neutrophil, a consequence of a constitutively expressed program of apoptosis. That program can be inhibited, and neutrophil survival prolonged, through the interaction of the neutrophil with a variety of mediators of both microbial and host origin. These, in turn, inhibit apoptosis by increasing the expression of anti-apoptotic genes within the neutrophil: interleukin (IL)1beta and a novel cytokine-like molecule pre-B cell colony-enhancing factor (PBEF) are central to this inhibitory influence. Conversely, the phagocytosis of a micro-organism activates the apoptotic program, and so contributes to the resolution of acute inflammation. A complex series of interactions between the neutrophil and microorganisms or their products regulates the duration and intensity of an inflammatory response, and so provides an attractive target for therapeutic manipulation.

Stem cells on alert: priming quiescent stem cells after remote injury.

A recent paper published by Rodgers et al. describes a novel phase of stem cell quiescence, termed GAlert, that serves to prime cells in response to injury-induced signals. These stem cells are located distal to the site of injury and require mTORC1 activity to elicit the alert response.