Tal-effector nucleases (TALEN) and clustered regularly interspaced short palindromic repeats (CRISPR) with CRISPR-associated (Cas) proteins are genome editing tools with unprecedented potential. However, the ability to deliver optimal amounts of these nucleases into mammalian cells with minimal toxicity poses a major challenge. Common delivery approaches are transfection- and viral-based methods; each associated with significant drawbacks. An alternative method for directly delivering genome-editing reagents into single living cells with high efficiency and controlled volume is microinjection. Here, we characterize a glass microcapillary-based injection system and demonstrate controlled co-injection of TALENs or CRISPR/Cas9 together with donor template into single K562 cells for targeting the human β-globin gene. We quantified nuclease induced insertions and deletions (indels) and found that, with β-globin-targeting TALENs, similar levels of on- and off-target activity in cells could be achieved by microinjection compared with nucleofection. Furthermore, we observed 11% and 2% homology directed repair in single K562 cells co-injected with a donor template along with CRISPR/Cas9 and TALENs respectively. These results demonstrate that a high level of targeted gene modification can be achieved in human cells using glass-needle microinjection of genome editing reagents.

Objective-The adaptive response to vascular injury is the formation of functional collateral vessels to maintain organ integrity. Many of the clinical complications associated with sickle cell disease can be attributed to repeated bouts of vascular insufficiency, yet the detailed mechanisms of collateral vessel formation after injury are largely unknown in sickle cell disease. Here, we characterize postischemic neovascularization in sickle cell disease and the role of neutrophils in the production of reactive oxygen species. Approach and Results-We induced hindlimb ischemia by ligation of the femoral artery in Townes SS (sickle cell) mice compared with AA (wild type) mice. Perfusion recovery, ascertained using LASER (light amplification by stimulated emission of radiation) Doppler perfusion imaging, showed significant diminution in collateral vessel formation in SS mice after hindlimb ischemia (76±13% AA versus 34±10% in SS by day 28; P<0.001; n=10 per group). The incidence of amputation (25% versus 5%) and foot necrosis (80% versus 15%) after hindlimb ischemia was significantly increased in the SS mice. Motor function recovery evaluation by the running wheel assay was also impaired in SS mice (36% versus 97% at 28 days post-hindlimb ischemia; P<0.001). This phenotype was associated with persistent and excessive production of reactive oxygen species by neutrophils. Importantly, neutrophil depletion or treatment with the antioxidant N-acetylcysteine reduced oxidative stress and improved functional collateral formation in the SS mice. Conclusions-Our data suggest dysfunctional collateral vessel formation in SS mice after vascular injury and provide a mechanistic basis for the multiple vascular complications of sickle cell disease. Visual Overview-An online visual overview is available for this article.

Whole transcriptome RNA-sequencing was performed to quantify RNA expression changes in whole blood samples collected from steady state sickle cell anemia (SCA) and control subjects. Pediatric SCA and control subjects were recruited from Atlanta (GA)—based hospital(s) systems and consented for RNA sequencing. RNA sequencing was performed on an Ion Torrent S5 sequencer, using the Ion Total RNA-seq v2 protocol. Data were aligned to the hg19 reference genome and analyzed in the Partek Genomics studio package (v7.0). 223 genes were differentially expressed between SCA and controls (± 1.5 fold change FDR p < 0.001) and 441 genes show differential transcript expression (± 1.5 fold FDR p < 0.001). Differentially expressed RNA are enriched for hemoglobin associated genes and ubiquitin-proteasome pathway genes. Further analysis shows higher gamma globin gene expression in SCA (33-fold HBG1 and 49-fold HBG2, both FDR p < 0.05), which did not correlate with hemoglobin F protein levels. eQTL analysis identified SNPs in novel non-coding RNA RYR2 gene as having a potential regulatory role in HBG1 and HBG2 expression levels. Gene expression correlation identified JHDM1D-AS1(KDM7A-DT), a non-coding RNA associated with angiogenesis, enhanced GATA1 and decreased JAK-STAT signaling to correlate with HBG1 and HBG2 mRNA levels. These data suggest novel regulatory mechanisms for fetal hemoglobin regulation, which may offer innovative therapeutic approaches for SCA.

Sickle cell disease (SCD) produces many structural and functional abnormalities in the kidney, including glomerular abnormalities. Albuminuria is the most common manifestation of glomerular damage, with a prevalence between 26 and 68% in adult patients. The pathophysiology of albuminuria in SCD is likely multifactorial, with contributions from hyperfiltration, glomerular hypertension, ischemia-reperfusion injury, oxidative stress, decreased nitric oxide (NO) bioavailability, and endothelial dysfunction. Although its natural history in SCD remains inadequately defined, albuminuria is associated with increased echocardiography-derived tricuspid regurgitant jet velocity, systemic blood pressure, and hypertension, as well as history of stroke, suggesting a shared vasculopathic pathophysiology. While most patients with albuminuria are treated with angiotensin converting enzyme inhibitors/angiotensin receptor blockers, there are no published long-term data on the efficacy of these agents. With the improved patient survival following kidney transplantation, SCD patients with end-stage renal disease should be considered for this treatment modality. Given the high prevalence of albuminuria and its association with multiple SCD-related clinical complications, additional studies are needed to answer several clinically important questions in a bid to adequately elucidate its pathophysiology, natural history, and treatment.

Recent studies have demonstrated pleiotropic effects of statins in various mouse models of kidney disease. In this study, Townes humanized sickle cell mice were treated for 8 weeks with atorvastatin at a dose of 10 mg/kg/day starting at 10 weeks of age. Treatment with atorvastatin significantly reduced albuminuria, and improved both urine concentrating ability and glomerular filtration rate. Atorvastatin also decreased markers of kidney injury and endothelial activation, and ameliorated oxidant stress in renal tissues and peripheral macrophages. Atorvastatin downregulated the expression of mRNA levels of the NADPH oxidases, Cybb (also termed Nox2) and Nox4, which are major sources of oxidant stress in the kidney. These findings highlight the pleiotropic effects of atorvastatin and suggest that it may provide beneficial effects in sickle cell nephropathy.

The prevention of organ damage and early death in young adults is a major clinical concern in sickle cell disease (SCD). However, mechanisms that control adult progression of SCD during the transition from adolescence are poorly defined with no cognate prophylaxis. Here, we demonstrate in a longitudinal cohort of homozygous SCD (SS) mice a link between intravascular hemolysis, vascular inflammation, lung injury, and early death. Prophylactic Nrf2 activation in young SS mice stabilized intravascular hemolysis, reversed vascular inflammation, and attenuated lung edema in adulthood. Enhanced Nrf2 activation in endothelial cells in vitro concurred with the dramatic effect on vascular inflammation in the mice. BM chimeric SS mice lacking Nrf2 expression in nonhematopoietic tissues were created to dissect the role of nonerythroid Nrf2 in SCD progression. The SS chimeras developed severe intravascular hemolysis despite having erythroid Nrf2. In addition, they developed premature vascular inflammation and pulmonary edema and died younger than donor littermates with intact nonhematopoietic Nrf2. Our results reveal a dominant protective role for nonhematopoietic Nrf2 against tissue damage in both erythroid and nonerythroid tissues in SCD. Furthermore, we show that prophylactic augmentation of Nrf2-coordinated cytoprotection effectively impedes onset of the severe adult phenotype of SCD in mice.

Sickle cell disease is associated with hypermetabolism and a consequent shortage of substrates for normal growth and healthy immune response. The protein:energy ratio is a major determinant of dietary adequacy; the requirement for optimal growth of control mice is 20% of energy from dietary protein. This study investigated the efficacy of increased dietary protein for improving weight gain and reducing inflammation in the Berkeley sickle cell mouse model (S). The study examined the effect of diet on weight gain and circulating levels of 2 inflammatory proteins, C-reactive protein (CRP), and cytokine interleukin-6 (IL-6). Male C57BL/6 (C) control (n = 8) and S mice (n = 8) were randomized at weaning to 40 d of isoenergetic diets containing 20% (normal) and 35% (high) of energy from protein (C20, C35, S20, S35), replacing dextrin. Rate of weight gain was calculated and plasma CRP and IL-6 concentrations determined by ELISA. Liver mRNA expression of these proteins was measured by real-time PCR and L-arginase by colorimetric assay. S35 mice tended to gain weight more rapidly than S20 mice (P = 0.06) and more rapidly than C35 mice (P < 0.01). Circulating CRP and IL-6 levels were also lower in S35 mice than in S20 mice (P < 0.05), as was liver CRP mRNA expression (P < 0.01). These results demonstrate that introducing a high protein diet at weaning attenuates the steady-state inflammation in this S mouse model. Dietary L-arginine availability was investigated as a possible mechanism for increased nitric oxide production and consequent reduced inflammation. © 2008 American Society for Nutrition.

Chronic kidney disease (CKD) is common and appears to be progressive in sickle cell disease (SCD) and is also associated with increased mortality in this condition (Ataga, et al 2014). Albuminuria is a risk factor for worsening kidney function (Ataga, et al 2014). Albuminuria and changes in glomerular filtration rate occur well after substantial structural and functional tissue damage have ensued (Guasch, et al 1996). Available biomarkers of CKD fail to provide information on underlying causal biochemical pathways, limiting the discovery of novel therapeutic or preventive measures. Metabolite profiling and the bioinformatics tools to study them may aid in the discovery of novel associations with disease markers that uncover pathophysiological pathways in SCD. This exploratory study evaluated plasma metabolomics profiles in SCD patients with and without albuminuria.

The prevention and treatment of acute chest syndrome (ACS) is a major clinical concern in sickle cell disease (SCD). However, the mechanism underlying the pathogenesis of ACS remains elusive. We tested the hypothesis that the hemolysis byproduct hemin elicits events that induce ACS. Infusion of a low dose of hemin caused acute intravascular hemolysis and autoamplification of extracellular hemin in transgenic sickle mice, but not in sickle-trait littermates. The sickle mice developed multiple symptoms typical of ACS and succumbed rapidly. Pharmacologic inhibition of TLR4 and hemopexin replacement therapy prior to hemin infusion protected sickle mice from developing ACS. Replication of the ACS-like phenotype in nonsickle mice revealed that the mechanism of lung injury due to extracellular hemin is independent of SCD. Using genetic and bone marrow chimeric tools, we confirmed that TLR4 expressed in nonhematopoietic vascular tissues mediated this lethal type of acute lung injury. Respiratory failure was averted after the onset of ACS-like symptoms in sickle mice by treating them with recombinant hemopexin. Our results reveal a mechanism that helps to explain the pathogenesis of ACS, and we provide proof of principle for therapeutic strategies to prevent and treat this condition in mice.

Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS). We examined the oxidative effects of sickle cell disease on hematopoietic stem cell function in a sickle mouse model. In vitro colony-forming assays showed a significant decrease in progenitor colony formation derived from sickle compared to control bone marrow (BM). Sickle BM possessed a significant decrease in the KSL (c-kit+, Sca-1+, Lineage−) progenitor population, and cell cycle analysis showed that there were fewer KSL cells in the G0 phase of the cell cycle compared to controls. We found a significant increase in both lipid peroxidation and ROS in sickle-derived KSL cells. In vivo analysis demonstrated that normal bone marrow cells engraft with increased frequency into sickle mice compared to control mice. Hematopoietic progenitor cells derived from sickle mice, however, demonstrated significant impairment in engraftment potential. We observed partial restoration of engraftment by n-acetyl cysteine (NAC) treatment of KSL cells prior to transplantation. Increased intracellular ROS and lipid peroxidation combined with improvement in engraftment following NAC treatment suggests that an altered redox environment in sickle mice affects hematopoietic progenitor and stem cell function.