Strategies to rescue the consequences of inducible arginase-1 deficiency in mice

Arginase-1 catalyzes the conversion of arginine to ornithine and urea, which is the final step of the urea cycle used to remove excess ammonia from the body. Arginase-1 deficiency leads to hyperargininemia in mice and man with severe lethal consequences in the former and progressive neurological impairment to varying degrees in the latter. In a tamoxifen-induced arginase-1 deficient mouse model, mice succumb to the enzyme deficiency within 2 weeks after inducing the knockout and retain <2 % enzyme in the liver. Standard clinical care regimens for arginase-1 deficiency (low-protein diet, the nitrogen-scavenging drug sodium phenylbutyrate, ornithine supplementation) either failed to extend lifespan (ornithine) or only minimally prolonged lifespan (maximum 8 days with low-protein diet and drug). A conditional, tamoxifen-inducible arginase-1 transgenic mouse strain expressing the enzyme from the Rosa26 locus modestly extended lifespan of neonatal mice, but not that of 4-week old mice, when crossed to the inducible arginase-1 knockout mouse strain. Delivery of an arginase-1/enhanced green fluorescent fusion construct by adeno-associated viral delivery (rh10 serotype with a strong cytomegalovirus-chicken beta-actin hybrid promoter) rescued about 30% of male mice with lifespan prolongation to at least 6 months, extensive hepatic expression and restoration of significant enzyme activity in liver. In contrast, a vector of the AAV8 serotype driven by the thyroxine-binding globulin promoter led to weaker liver expression and did not rescue arginase-1 deficient mice to any great extent. Since the induced arginase-1 deficient mouse model displays a much more severe phenotype when compared to human arginase-1 deficiency, these studies reveal that it may be feasible with gene therapy strategies to correct the various manifestations of the disorder and they provide optimism for future clinical studies

Rehabilitation in patients with radically treated respiratory cancer: A randomised controlled trial comparing two training modalities.

INTRODUCTION: The evidence on the effectiveness of rehabilitation in lung cancer patients is limited. Whole body vibration (WBV) has been proposed as an alternative to conventional resistance training (CRT). METHODS: We investigated the effect of radical treatment (RT) and of two rehabilitation programmes in lung cancer patients. The primary endpoint was a change in 6-min walking distance (6MWD) after rehabilitation. Patients were randomised after RT to either CRT, WBVT or standard follow-up (CON). Patients were evaluated before, after RT and after 12 weeks of intervention. RESULTS: Of 121 included patients, 70 were randomised to either CON (24), CRT (24) or WBVT (22). After RT, 6MWD decreased with a mean of 38m (95% CI 22-54) and increased with a mean of 95m (95% CI 58-132) in CRT (p<0.0001), 37m (95% CI -1-76) in WBVT (p=0.06) and 1m (95% CI -34-36) in CON (p=0.95), respectively. Surgical treatment, magnitude of decrease in 6MWD by RT and allocation to either CRT or WBVT were prognostic for reaching the minimally clinically important difference of 54m increase in 6MWD after intervention. CONCLUSIONS: RT of lung cancer significantly impairs patients' exercise capacity. CRT significantly improves and restores functional exercise capacity, whereas WBVT does not fully substitute for CRT

Conditional Gene Knockout in Human Cells with Inducible CRISPR/Cas9.

The advent of the easily programmable and efficient CRISPR/Cas9 nuclease system has revolutionized genetic engineering. While conventional gene knockout experiments using CRISPR/Cas9 are very valuable, these are not well suited to study stage-specific gene function in dynamic situations such as development or disease. Here we describe a CRISPR/Cas9-based OPTimized inducible gene KnockOut method (OPTiKO) for conditional loss-of-function studies in human cells. This approach relies on an improved tetracycline-inducible system for conditional expression of single guide RNAs (sgRNAs) that drive Cas9 activity. In order to ensure homogeneous and stable expression, the necessary transgenes are expressed following rapid and efficient single-step genetic engineering of the AAVS1 genomic safe harbor. When implemented in human pluripotent stem cells (hPSCs), the approach can be then efficiently applied to virtually any hPSC-derived human cell type at various stages of development or disease

The EMT regulator Zeb2/Sip1 is essential for murine embryonic hematopoietic stem/progenitor cell differentiation and mobilization

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs), and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo, but is essential for normal HSC/HPC differentiation. In addition, Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased beta 1 integrin and Cxcr4 expression. Moreover, deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and, subsequently, improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult. (Blood. 2011; 117(21): 5620-5630

Experiments to test AAV rescue of inducible arginase-1 deficient mice.

<p>Notes:</p><p>*Age of mice is based on the day of the first tamoxifen administration.</p><p>Timing of viral administration is relative to the last dose of tamoxifen (designated 0) and is given in days (-14 refers to two weeks prior to the last dose of tamoxifen, while +7 refers to one week after the last dose. Several extra vehicle-injected control mice were included in the studies (not shown in Table) to exclude viral-mediated toxicity.</p><p>^#Mouse A2 was euthanized at Day +21, even though healthy (within 2% of starting body weight and expressing 75% of control liver arginase activity; see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.g008" target="_blank">Fig 8D</a>.</p><p>^!Mouse died due to non-viral mediated testicular morbidity.</p><p>CB7, hybrid cytomegalovirus enhancer/chicken β-actin promoter; TBG, thyroxine-binding globulin promoter. Mouse designation corresponds with data found in Fig <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.g008" target="_blank">8C</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.g008" target="_blank">8D</a>.</p><p>^♂, male;</p><p>^♀, female.</p><p>Experiments to test AAV rescue of inducible arginase-1 deficient mice.</p

Floxed <i>Arg1-Cre</i> mice (8–12 weeks old) treated with the standard tamoxifen dosing regimen (A) show evidence of reduced liver arginase-1 enzyme activity and protein (n = 3 all points, except Day +12 where n = 4) over time (B).

<p>Day 0 is counted as the last day of tamoxifen administration, while Day +13 is the usual day for humane euthanization. Each Western blot lane (lower blot) corresponds to the enzyme activity bar below, with the exception that there is one extra lane in the blot corresponding to a mouse euthanized on Day +14. Image J quantitation relative to Day -2 is shown below the blot. The upper blot is the loading control for β-actin (β-Act). Enzyme activity was significantly decreased (*, <i>p</i><0.05) compared to the “baseline” measurement (Day -2). (C) Typical phenotypic pattern of weight loss induced by Arg1 deficiency (n = 7 male; n = 5 female). Weight loss at humane endpoint was always significantly lower (<i>p</i><0.05) than at Day +4. (D) Plasma ammonia in the same groups of mice used for protein and enzyme activity analysis. Day +12 levels were elevated but not significantly (<i>p</i> = 0.12).</p

Generation of adeno-associated viral (AAV) vectors used in studies to rescue induced Arg1 deficiency.

<p>(A) Maps (not to scale) of two AAV constructs used and the packaging serotypes (AAV8 and rh10). ITR, inverted terminal repeat; TBG, thyroxine-binding globulin; CB7-CI, hybrid cytomegalovirus enhancer/chicken β-actin promoter (CB7), along with a chicken β-actin intron (CI); WPRE, woodchuck hepatitis virus post-transcriptional regulatory element; bGH-pA, bovine growth hormone polyA adenylation signal sequence; rBG, rabbit beta-globin polyA adenylation signal sequence. (B) Western blot indicating level of expression in livers of 4 separate mice (lanes 1–4) of AAV-expressed Arg1-eGFP relative to WT Arg1 and tubulin loading control post-tamoxifen (Day +13;4-week old mice injected with 2x10^<i>11</i> gc of the AAV8 construct shown in panel A) on Day -4. Lane 5 represents normal level of expression of Arg1 (and tubulin) in a mouse not injected with tamoxifen. In order to observe sufficient signal of the inducible Arg1 knockout band of lanes 1–4, the signal becomes over-saturated when detecting normal endogenous levels of Arg1 (lane 5). (C) Images of liver sections (i-iii) and dissociated hepatocytes (iv, v) from AAV8.TBG.PI.Arg1-eGFP.WPRE.bGH injected 4-week old mice 1 week after virus administration. Strong fluorescence signal from the fusion transgene is observed in the cytoplasm of some periportal hepatocytes (ii,iii) and also in dissociated cells (v) with corresponding phase contrast images (i, iv).</p

Ornithine supplementation does not rescue the induced arginase-1 deficiency phenotype.

<p>(A) Citrulline (Cit), ornithine (Orn), arginine (Arg) and methionine (Met) plasma concentrations are similar in control and ornithine-treated groups of 4 week old mice at baseline (n = 4). (B) Ornithine-treated mice (●, n = 4) indeed show elevations in plasma ornithine, which demonstrates the efficacy of the protocol, and have significant increases in arginine compared to control mice (■,n = 5) *, p<0.05 relative to control. (C) Mice in both groups show similar patterns of weight loss (surrogate marker of health status). Weight loss at humane endpoint was always significantly lower (*, <i>p</i><0.05) than at Day +4.</p

Some male mice are rescued from the lethal consequences of induced Arg1 deletion by AAVrh10.CB7.CI.Arg1-eGFP.WPRE.rBG administration.

<p>(A) Survival of tamoxifen-treated male (n = 24) and female (n = 10) induced Arg1 KO mice injected with AAV vector (Expts 4–7, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.t001" target="_blank">Table 1</a>). Seven “rescued” healthy mice were sacrificed at Day +21 (mouse A2, n = 1), Day +49 (mice S9, S11; n = 2), Day +90 (mice S1, S4; n = 2), and Day +180 (mice A1, A10; n = 2) for analysis of Arg1-eGFP expression. This explains the drop off in male survival beyond Day +21 denoted by asterisks. (B) <i>In situ</i> liver showing extensive green fluorescence expression from the Arg1-eGFP transgene in one rescued male mouse S1 sacrificed at Day +90, viewed under a handheld UV light (purple background). Note absence of expression in the gall bladder. (C) Western blot depicting Arg1-eGFP transgene expression in six of the rescued male mice mentioned above (mouse A2 not tested). The lane corresponding to mouse S6 was non-AAV-treated (Arg activity = 0.24 units; not shown in panel D). Protein standards (std) are not visible on the Western blot. Con, control non-tamoxifen treated mouse injected with an AAV-GFP construct. (D) Liver arginase enzyme activity at endpoint in AAV-injected mice of Expts 4–7 (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0125967#pone.0125967.t001" target="_blank">Table 1</a>). Activity of rescued (A1, A2, A10, S1, S4, S9, S11) mice are labeled, along with three mice not rescued but with substantial enzyme activity (S8, S10, S12).</p

Real time quantitative PCR expression of <i>Arg1</i>.

<p>Brain (A), kidney (B), and liver (C) of control C57BL/6 (n = 3), <i>R26-Arg1</i> transgenic (TG; n = 3–5), and <i>Arg1</i>^Δ knockout (KO; n = 3–5) mice.</p