Prolonged expression of the CRISPR-Cas9 nuclease and gRNA from viral vectors may cause off-target mutagenesis and immunogenicity. Thus, a transient delivery system is needed for therapeutic genome editing applications. Here, we develop an extracellular nanovesicle-based ribonucleoprotein delivery system named NanoMEDIC by utilizing two distinct homing mechanisms. Chemical induced dimerization recruits Cas9 protein into extracellular nanovesicles, and then a viral RNA packaging signal and two self-cleaving riboswitches tether and release sgRNA into nanovesicles. We demonstrate efficient genome editing in various hardto-transfect cell types, including human induced pluripotent stem (iPS) cells, neurons, and myoblasts. NanoMEDIC also achieves over 90% exon skipping efficiencies in skeletal muscle cells derived from Duchenne muscular dystrophy (DMD) patient iPS cells. Finally, single intramuscular injection of NanoMEDIC induces permanent genomic exon skipping in a luciferase reporter mouse and in mdx mice, indicating its utility for in vivo genome editing therapy of DMD and beyond.

Genome editing therapy for Duchenne muscular dystrophy (DMD) holds great promise, however, one major obstacle is delivery of the CRISPR-Cas9/sgRNA system to skeletal muscle tissues. In general, AAV vectors are used for in vivo delivery, but AAV injections cannot be repeated because of neutralization antibodies. Here we report a chemically defined lipid nanoparticle (LNP) system which is able to deliver Cas9 mRNA and sgRNA into skeletal muscle by repeated intramuscular injections. Although the expressions of Cas9 protein and sgRNA were transient, our LNP system could induce stable genomic exon skipping and restore dystrophin protein in a DMD mouse model that harbors a humanized exon sequence. Furthermore, administration of our LNP via limb perfusion method enables to target multiple muscle groups. The repeated administration and low immunogenicity of our LNP system are promising features for a delivery vehicle of CRISPR-Cas9 to treat skeletal muscle disorders.

Inositol hexakisphosphate kinase 3 (IP6K3) generates inositol pyrophosphates, which regulate diverse cellular functions. However, little is known about its own physiological role. Here, we show the roles of IP6K3 in metabolic regulation. We detected high levels of both mouse and human IP6K3 mRNA in myotubes and muscle tissues. In human myotubes, IP6K3 was upregulated by dexamethasone treatment, which is known to inhibit glucose metabolism. Furthermore, Ip6k3 expression was elevated under diabetic, fasting, and disuse conditions in mouse skeletal muscles. Ip6k3−/− mice demonstrated lower blood glucose, reduced circulating insulin, deceased fat mass, lower body weight, increased plasma lactate, enhanced glucose tolerance, lower glucose during an insulin tolerance test, and reduced muscle Pdk4 expression under normal diet conditions. Notably, Ip6k3 deletion extended animal lifespan with concomitant reduced phosphorylation of S6 ribosomal protein in the heart. In contrast, Ip6k3−/− mice showed unchanged skeletal muscle mass and no resistance to the effects of high fat diet. The current observations suggest novel roles of IP6K3 in cellular regulation, which impact metabolic control and lifespan.

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ABSTRACT. We have previously produced human growth hormone (hGH) transgenic (TG) rats that show low circulating levels of both hGH and endogenous rat GH. Although body length of the TG rats is normal, they develop hyperphagia and severe obesity. The present study was undertaken to elucidate the causes of hyperphagia in the TG rats by focusing on temporal changes in plasma ghrelin levels and hypothalamic neuropeptide Y (NPY) contents. In both wild-type (WT) and TG rats, the highest value of plasma ghrelin levels was observed just before the dark phase, and thereafter plasma ghrelin levels were maintained higher in the TG than WT rats. Although NPY contents also showed the peak level just before the dark phase in both the arcuate (ARC) and paraventricular nuclei (PVN) of the hypothalamus, the values in the ARC, but not the PVN, of the TG rats was always lower than those of the WT rats, suggesting increased transport of NPY from the ARC to PVN in the TG rats. In addition, treatment with antagonists for Y1 and Y5 receptors for NPY reduced food intake much more effectively in the TG than WT rats. Intermittent treatment with recombinant hGH for a week significantly decreased food consumption, adipose tissue weight and plasma triglyceride concentrations in the TG rats. These results suggest that, in the TG rats, insufficiency in circulating GH stimulates the ghrelin-NPY system with a resultant increase in food intake. KEY WORDS: ghrelin, growth hormone, hyperphagia, neuropeptide Y, transgenic rat.J. Vet. Med. Sci. 68(9): 959-965, 2006 Growth hormone (GH) is secreted from the pituitary gland in a pulsatile manner and plays an important role in regulating metabolism as well as somatic growth [8,10]. The actions of GH on metabolism involve both anabolism, such as protein synthesis in the muscle, and catabolism, such as lipolysis in the adipose tissue. Therefore, the role of GH is very important in terms of maintaining the balance of body composition. Indeed, GH-deficient (GHD) subjects become obesity with short stature and this obesity in GHD subjects is improved by GH-replacement therapy [9,29]. Since the subjects of Prader-Willi syndrome (PWS), a type of GHD, typically represent hyperphagia [14], GH may also be involved in maintaining normal feeding.We have previously generated transgenic (TG) rats expressing human GH (hGH) gene under the control of mouse whey acidic protein promoter [18]. Contrary to our expectation, serum hGH levels in the TG rats were relatively low and endogenous pulsatile secretion of rat GH was eliminated. While their body length was similar to that of the wild-type (WT) rats, the TG rats developed severe obesity [15] and hyperphagia [12], suggesting that they suffered from GHD. Additionally, the TG rats showed several symptoms that are linked to non-insulin dependent diabetes mellitus, e.g. hyperglycemia, hyperinsulinemia, and hypertriglyceridemia [16]. The TG rats, therefore, appear to be a good experimental model for the study of hyperphagia and resulting obesity and metabolic diseases under...