Oaklands Hospital

Background:Sickle Cell Disease (SCD) is a devastating disorder caused by a single base change in the ß‐globin gene (HBB) that is one component of the adult hemoglobin tetramer (HbA, which contains 2 a‐globin and 2 ß‐globin proteins). SCD is a recessive disorder – if one of the 2 alleles of HBB is normal, the disease is not present; thus gene correction needs to correct only one of the two HBB alleles in any given stem cell.Aims:To correct the sickle allele in human hematopoietic stem cellsMethods:To correct the sickle mutation, we developed a process that directly introduces a preassembled Cas9 ribonucleoprotein (RNP) complex (composed of recombinant Cas9 protein and single guide RNA) along with a single‐stranded DNA oligonucleotide donor template (ssODN) for homology‐directed repair (HDR). We studied gene correction after long‐term xenografting of human sickle HSCs. After electroporation with RNP and ssODN, sickle human HSCs were injected into immunodeficient NBSGW mice, which permit engraftment of human HSCs and erythroid differentiation in the bone marrow.Results:Sixteen to 20 weeks after injection, we analyzed engrafted human cells in the bone marrow of 43 mice in 4 cohorts. We quantified editing in xenografted cells, and found an average of 23.4% of HBB alleles with the corrected genotype, and 65.2% with insertion/deletion mutations (indels). To assess the distribution of HBB alleles within populations of xenografted cells, we inferred HBB genotypes using RNA‐Seq data from 359 individual clonal BFU‐E colonies derived from marrow CD34+ cells (figure 1A). This reveals that a substantial proportion of corrected (HDR) alleles are heterozygous with indel alleles equivalent to ß‐thalassemia mutations, confirming that corrected (wild type) HBB alleles in edited HSCs are distributed such that many erythrocytes have only a single wild type HBB allele. Data from the colony analysis, extrapolated to the entire experimental group, indicates that the 23.4% of corrected HBB alleles are distributed among >30% of HSCs, producing a comparable proportion of functional erythrocytes. SCD is a recessive disorder; based upon observations of mixed chimerism after allogeneic transplantation, this proportion of normal cells is consistent with cure.From engrafted NBSGW mice, we immunoselected human erythroid cells with anti‐CD235a (GlycophorinA), carried out RNA‐Seq, and inferred HBB genotypes in the erythroblast population. For each individual mouse, we compared the proportion of corrected HBB alleles in the xenografted bone marrow, in CD34+ cells immunoselected from marrow, and in CD235a+ erythroblasts. To control for enrichment of alleles corrected by HDR, we distinguished two types of HDR: “PAM‐only” HDR events mutate the Cas9 PAM without the conversion tract reaching the sickle mutation, whereas “sickle‐corrected” HDR events mutate the Cas9 PAM and also correct the sickle mutation. We observe a marked enrichment of “sickle‐corrected” HDR alleles in erythroblasts when compared to marrow or CD34+ cells from the same mouse, but no enrichment of “PAM‐only” HDR alleles (figure 1B).Summary/Conclusion:In summary, we have developed a Cas9‐mediated gene editing protocol that reproducibly yields a cell product in which the sickle mutation is corrected to wild type at a level that is sufficient to cure SCD. Evidence we have presented indicates that cells carrying the corrected ß‐globin gene are enriched during in vivo erythroid differentiation. These results support moving this therapeutic approach to the clinic.image

Background:SUCCESSOR (SUSTAIN Chart‐review of Crizanlizumab to Evaluate Sickle‐cell Study One‐year Retrospective) reviewed medical records of patients who completed the SUSTAIN study at US sites to assess subsequent cases of significant pain crisis events and generate real‐world data on treatment patterns and health care resource utilization upon completion of crizanlizumab treatment. SUSTAIN was a phase 2, randomized, double‐blind, placebo‐controlled, 52‐week study that compared the effect of crizanlizumab, a P‐selectin inhibitor, versus placebo on the frequency of sickle cell pain crises (SCPCs, or vaso‐occlusive crises [VOCs] leading to a health care visit) in patients with any genotype of sickle cell disease (SCD).Aims:To determine the 52‐week post‐SUSTAIN occurrence of VOCs, time to first VOC, frequency of blood transfusions, and use of opioids and hydroxyurea in patients after withdrawal of treatment with crizanlizumab or placebo.Methods:SUCCESSOR is a multicenter, retrospective cohort study of patients (≥18 years old) who participated in SUSTAIN to evaluate SCD‐related outcomes up to 52 weeks following trial completion. SUCCESSOR included the SUSTAIN per‐protocol population who received at least 12 of the 14 study drug doses, completed a visit at least 14 days after the final dose, and had no major protocol deviations that impacted efficacy assessments. The study period was from September 2015 to March 2017 and patients’ data were obtained from medical records. Crizanlizumab was not administered in the 52 weeks post‐SUSTAIN. An exploratory analysis was conducted to evaluate outcomes in the retrospective SUCCESSOR study using a subset of SUSTAIN patients. Patient consent was obtained prior to data collection if required by local and/or central research ethics review.Results:In SUCCESSOR, data for 48 patients were analyzed (15, 18, and 15 patients who were previously randomized in SUSTAIN to receive crizanlizumab 5 mg/kg, crizanlizumab 2.5 mg/kg, and placebo, respectively). The median age was 31.5 years (range 19–65 years) and 69% were female. One patient had HbSβ+, 4 had HbSβ0, 10 had HbSC, 1 had HbS‐Lepore disease; all remaining patients had HbSS. In the 52 weeks post‐SUSTAIN, VOCs were reported by 73% of patients who previously received crizanlizumab 5 mg/kg, 83% of patients who previously received crizanlizumab 2.5 mg/kg, and 80% of patients previously treated with placebo. The mean annual rate of VOCs was 2.7, 3.9, and 5.4 in patients previously on crizanlizumab 5 mg/kg, 2.5 mg/kg, and placebo, respectively. Median time to first VOC post‐SUSTAIN was 6.1, 2.7, and 2.6 months in patients previously on crizanlizumab 5 mg/kg, 2.5 mg/kg, and placebo, respectively, similar to differences observed for the same patients during SUSTAIN (6.9, 2.2, and 1.4 months for crizanlizumab 5 mg/kg, 2.5 mg/kg, and placebo, respectively). In SUCCESSOR, all but 1 patient utilized opioids, and 79% did not require blood transfusions. Hydroxyurea was used by 60%, 50%, and 80% of patients previously on crizanlizumab 5 mg/kg, 2.5 mg/kg, and placebo, respectively. There were no deaths.Summary/Conclusion:In SUCCESSOR across all patient groups, 80% of patients experienced a VOC in the 52 weeks post‐SUSTAIN. This retrospective cohort study indicates that after withdrawal of crizanlizumab, patients previously treated with 5 mg/kg maintained a similar time to first VOC compared with time to first VOC while on treatment in SUSTAIN. Further research is needed to establish whether these findings are due to a prolonged effect of crizanlizumab or to other factors.image

To review recent evidence for the role of dietary carbohydrate in de novo lipogenesis (DNL) and nonalcoholic fatty liver disease (NAFLD). A large body of evidence suggests that increased hepatic DNL is a significant pathway contributing to the development of NAFLD. Dietary carbohydrates, in particular, fructose, have been shown to stimulate DNL and increase liver fat, although it is debated whether this is due to excess energy or fructose per se. Recent dietary intervention studies conducted in energy balance show that high-fructose diets increase DNL and liver fat, whereas fructose restriction decreases DNL and liver fat. The association of high-carbohydrate and high-sugar diets with NAFLD may in part be explained by the effect of sugar on increasing hepatic DNL.

Sickle cell anaemia (SCA) is a progressive vascular disease characterized by episodic vaso-occlusive pain. Despite the broad impact of inflammation on acute and chronic clinical manifestations of SCA, no directed anti-inflammatory therapies currently exist. Statins are cholesterol-lowering agents shown to confer protection from vascular injury by suppressing inflammation. We previously documented a reduction in soluble biomarkers of inflammation in patients with sickle cell disease treated with simvastatin. To determine the potential clinical efficacy of simvastatin, we treated 19 SCA patients with single daily dose simvastatin for 3months and assessed changes from baseline in the frequency and intensity of diary-reported pain and levels of circulating nitric oxide metabolites (NOx), high sensitivity C-reactive protein (hs-CRP), vascular cell adhesion molecule 1 (VCAM-1), intercellular adhesion molecule 1 (ICAM-1), ICAM-3, E-selectin, and vascular endothelial growth factor (VEGF). Treatment with simvastatin resulted in a significant reduction in the frequency of pain (P=0·0003), oral analgesic use (P=0·003) and circulating hs-CRP (P=0·003), soluble (s)E-selectin (P=0·01), sICAM-1 (P=0·02), sICAM-3 (P=0·02) and sVEGF (P=0·01). Simvastatin had no effect on pain intensity or levels of NOx, sP-selectin and sVCAM-1. The observed reductions in pain rate and markers of inflammation were greatest in subjects receiving hydroxycarbamide (HC), suggesting a synergistic effect of simvastatin. These results provide preliminary clinical datato support a larger trial of simvastatin in SCA.

Transfusions are the primary therapy for thalassemia but have significant cumulative risks. In 2004, the Centers for Disease Control and Prevention (CDC) established a national blood safety monitoring program for thalassemia. This report summarizes the population and their previous nonimmune and immune transfusion complications. The CDC Thalassemia Blood Safety Network is a consortium of centers longitudinally following patients. Enrollment occurred from 2004 through 2012. Demographics, transfusion history, infectious exposures, and transfusion and nontransfusion complications were summarized. Logistic regression analyses of factors associated with allo- and autoimmunization were employed. The race/ethnicity of these 407 thalassemia patients was predominantly Asian or Caucasian. The mean ± SD age was 22.3 ± 13.2 years and patients had received a mean ± SD total number of 149 ± 103.4 units of red blood cells (RBCs). Multiorgan dysfunction was common despite chelation. Twenty-four percent of transfused patients had previous exposure to possible transfusion-associated pathogens including one case of babesia. As 27% were immigrants, the infection source cannot be unequivocally linked to transfusion. Transfusion reactions occurred in 48%, including allergic, febrile, and hemolytic; 19% were alloimmunized. Common antigens were E, Kell, and C. Years of transfusion was the strongest predictor of alloimmunization. Autoantibodies occurred in 6.5% and were associated with alloimmunization (p < 0.0001). Local institutional policies, not patient characteristics, were major determinants of blood preparation and transfusion practices. Hemosiderosis, transfusion reactions, and infections continue to be major problems in thalassemia. New pathogens were noted. National guidelines for RBC phenotyping and preparation are needed to decrease transfusion-related morbidity.

The KIR complex appears to be evolving rapidly in humans, and more than 50 different haplotypes have been described, ranging from four to 14 KIR loci. Previously it has been suggested that most KIR haplotypes consist of framework genes, present in all individuals, which bracket a variable number of other genes. We used a new technique to type 793 families from the United Kingdom and United States for both the presence/absence of all individual KIR genes as well as copy number and found that KIR haplotypes are even more complex. It is striking that all KIR loci are subject to copy number variation (CNV), including the so-called framework genes, but CNV is much more frequent in KIR B haplotypes than KIR A haplotypes. These two basic KIR haplotype groups, A and B, appear to be following different evolutionary trajectories. Despite the great diversity, there are 11 common haplotypes, derived by reciprocal recombination near KIR2DL4, which collectively account for 94% of KIR haplotypes determined in Caucasian samples. These haplotypes could be derived from combinations of just three centromeic and two telomeric motifs, simplifying disease analysis for these haplotypes. The remaining 6% of haplotypes displayed novel examples of expansion and contraction of numbers of loci. Conventional KIR typing misses much of this additional complexity, with important implications for studying the genetics of disease association with KIR that can now be explored by CNV analysis.

Using cell culture models, we have investigated the relative importance of cystic fibrosis transmembrane conductance regulator (CFTR) and calcium-activated chloride channels (CaCC) in Cl secretion by mucous and serous cells of human airway glands. In transepithelial recordings in Ussing chambers, the CFTR inhibitor CFTR(inh)-172 abolished 60% of baseline Cl secretion in serous cells and 70% in mucous. Flufenamic acid (FFA), an inhibitor of CaCC, reduced baseline Cl secretion by ∼20% in both cell types. Methacholine and ATP stimulated Cl secretion in both cell types, which was largely blocked by treatment with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and partially by mucosal FFA or CFTR(inh)-172 with the exception of methacholine responses in mucous cells, which were not blocked by FFA and partially (∼60%) by CFTR(inh)-172. The effects of ionomycin on short-circuit current (I(sc)) were less than those of ATP or methacholine. Forskolin stimulated Cl secretion only if Cl in the mucosal medium was replaced by gluconate. In whole cell patch-clamp studies of single isolated cells, cAMP-induced Cl currents were ∼3-fold greater in serous than mucous cells. Ionomycin-induced Cl currents were 13 times (serous) or 26 times (mucous) greater than those generated by cAMP and were blocked by FFA. In serous cells, mRNA for transmembrane protein 16A (TMEM16A) was ∼10 times more abundant than mRNA for CFTR. In mucous cells it was ∼100 times more abundant. We conclude: 1) serous and mucous cells both make significant contributions to gland fluid secretion; 2) baseline Cl secretion in both cell types is mediated predominantly by CFTR, but CaCC becomes increasingly important after mediator-induced elevations of intracellular Ca; and 3) the high CaCC currents seen in patch-clamp studies and the high TMEM16A expression in intact polarized cells sheets are not reflected in transepithelial current recordings.

Background/Aims: While the Cl^- efflux assays are relatively straightforward, their ability to assess the efficacy of phenotypic correction in cystic fibrosis (CF) tissue or cells may be limited. Accurate assessment of therapeutic efficacy, i.e., correlating wild type CF transmembrane conductance regulator (CFTR) levels with phenotypic correction in tissue or individual cells, requires a sensitive assay. Methods: Radioactive chloride (³⁶Cl) efflux was compared to Ussing chamber analysis for measuring cAMP-dependent Cl^- transport in mixtures of human normal (16HBE14o-) and cystic fibrosis (CF) (CFTE29o- or CFBE41o-, respectively) airway epithelial cells. Cell mixtures with decreasing amounts of 16HBE14o- cells were evaluated. Results: Efflux and Ussing chamber studies on mixed populations of normal and CF airway epithelial cells showed that, as the number of CF cells within the population was progressively increased, the cAMP-dependent Cl^- decreased. The ³⁶Cl efflux assay was effective for measuring Cl^- transport when ≥ 25% of the cells were normal. If < 25% of the cells were phenotypically wild-type (wt), the ³⁶Cl efflux assay was no longer reliable. Polarized CFBE41o- cells, also homozygous for the ΔF508 mutation, were used in the Ussing chamber studies. Ussing analysis detected cAMP-dependent Cl^- currents in mixtures with ≥1% wild-type cells indicating that Ussing analysis is more sensitive than ³⁶Cl efflux analysis for detection of functional CFTR. Conclusions: Assessment of CFTR function by Ussing analysis is more sensitive than ³⁶Cl efflux analysis. Ussing analysis indicates that cell mixtures containing 10% 16HBE14o- cells showed 40-50% of normal cAMP-dependent Cl^- transport that drops off exponentially between 10-1% wild-type cells.

[Bob Palmer’s (2006)][1] article considering the advances (or lack of them) in the management of anorexia nervosa over the past 10 years comes at a time of renewed interest in the subject of eating disorders. This has been contributed to by NICE guidelines ([National Collaborating Centre for