Sickle Mutation Research Articles

Serum Fructosamine is a Valid Measure of Glycemic Status in the Presence of the Sickle Cell Mutation

Background: The glycosylated hemoglobin (HbA1c) test can be unreliable in the presence of hemoglobinopathies and remains cost-prohibited and impractical in many developing countries. The increasing burden of type 2 diabetes (T2D) in sub-Saharan Africa (SSA) where sickle cell anemia, the most common inherited hemoglobinopathy, is prevalent calls for alternative tests for the management of T2D. In the present study, we evaluated the utility of serum fructosamine (FRA) as a biomarker of glycemic control that is not affected by abnormal hemoglobin or red blood cell (RBC) turnover. Methods: The accuracy of FRA in monitoring and diagnosing T2D was evaluated using the Area under the Receiver Operating Characteristics (AUROC) and other measures in a training dataset of 618 Nigerians [155 (25·1%) carrying sickle cell trait (HbAS) and 463 (74·9%) without sickle cell trait (HbAA)]. The FRA’s diagnostic cut-off was validated in an independent cohort of 634 West Africans. Findings: FRA was similar between HbAS and HbAA participants (Median = 287 vs 275 umol/L, p=0·11) despite statistically different HbA1c median among the two groups. FRA was highly correlated with both HbA1c (r HbAA = 0·80, r HbAS =0·82, p<0·001) and fasting glucose (FG) (r HbAA =0·72, r HbAS = 0·83, p<0·001) independent of sickle cell trait (SCT). With optimal thresholds of 352·5 umol/L and 309·5 umol/L, FRA had 81·5% [CI 95%:0·76-0·87] and 82·4%, [CI 95% 0·77-0·88] sensitivities and 97·4% [CI 95%:0·95-1·00] and 92·4% [95% CI 0·90-0·95] specificities for identifying individuals with controlled glycemic index and individuals with T2D respectively and independently of SCT. The diagnosis cut-off was validated in a multi-ethnic population with a good positive predictive value (94·6%). Interpretation: We showed that FRA is an effective alternative to HbA1c for monitoring and diagnosing T2D in populations where SCT is prevalent. Funding: National Institutes of Health. Conflict of Interest: The authors have no conflict of interest to disclose. Ethical Approval: The study protocol was approved by the institutional ethicsreview board (IRB) of the National Institutes of Health/National Human Genome Research Institute (protocol HG-09-N070) and the IRBs of each participating institution, including University of Lagos, Lagos, Nigeria; College of Medicine, University of Ibadan, Ibadan, Nigeria; University of Nigeria Teaching Hospital, Enugu, Nigeria; University of Science and Technology, Kumasi, Ghana; University of Ghana Medical School, Accra, Ghana. Written informed consent was obtained from each participant prior to enrollment.

Efficacy and Safety of Voxelotor in Sickle Cell Disease: A Systematic Review

Introduction: Sickle cell disease (SCD) is caused by mutation of beta-globin chain alleles, with the involvement of at least one sickle mutation. Sickling of red cells leads to hemolytic anemia, vaso-occlusions, and inflammation. Voxelotor (GBT440) is a hemoglobin modulator that prevents polymerization by increasing the affinity of hemoglobin with oxygen. We performed a systematic review to evaluate the efficacy and safety of voxelotor in SCD patients. Methods: PRISMA guidelines were followed to perform the literature search and selection of articles for this systematic review. A search was performed using databases including PubMed, Cochrane, Web of Science, Embase, and clinicaltrials.gov. We used the following keywords, "Voxelotor" OR "Benzaldehydes" OR "GBT440" AND "Sickle Cell Anemia" from the inception of literature till 04/25/2020. Out of 475 articles, we screened and included three clinical trials and a case series measuring the efficacy (i-e, change in Hemoglobin (Hb), Hb modification, etc.) and safety (adverse events) in clinical terms (N=359). We excluded case reports, pre-clinical studies, review articles, and meta-analysis. RESULTS: We included data on 359 patients, with 12-67 years of age. In Blyden et al. 2018, authors presented a case series of 7 patients with advanced SCD treated with 700 mg-1500 mg voxelotor. With treatment, vaso-occlusive episodes related hospitalizations decreased by 67%, hemoglobin levels, and markers of hemolysis improved in all patients. Authors in Hutchaleelaha et al. 2019 randomly assigned 24 participants to a once-daily dose of 900 mg, 600 mg, 300 mg voxelotor, and placebo for 15 days. With treatment, hemoglobin modification was maximum in the 900 mg voxelotor group. Headache and diarrhea were the only adverse events related to voxelotor treatment. No grade 3 adverse events were reported. In phase I/II trial by Howard et al. 2019, (n=54) 38 patients were followed for 28 days, and 16 patients were followed for &amp;gt;90 days. The compliance for study drug was 91%. In the 28-day cohort, treatment with 1000 mg of voxelotor showed maximum improvement in hemoglobin level, reticulocyte count, and unconjugated bilirubin. In &amp;gt;90-day cohort, the improvement in hemoglobin, unconjugated bilirubin, and reticulocyte count were statistically significant in favor of 900 mg voxelotor treatment as compared to placebo (p&amp;lt;0.05). LDH showed variability with treatment. Vaso-occlusive episodes seen in voxelotor groups were reported when the treatment was on hold or after the last dose. No grade ≥3 adverse events were reported. In a randomized placebo-controlled phase III clinical trial by Vichinsky et al. 2019, two doses of voxelotor 1500 mg (N=90) and 900 mg (N=92) were compared with placebo (N=92). 12-65 years old SCD patients were followed for 24 weeks. After treatment, improvement in hemoglobin was statistically significant in favor of 1500 mg voxelotor vs. placebo. Moreover, markers of hemolysis, reticulocyte count, and indirect bilirubin levels were also significantly improved in favor of 1500mg voxelotor treatment vs. placebo. The incidence of vaso-occlusive crisis episodes was similar in 1500 mg, 700 mg, and placebo groups (p&amp;gt;0.05). Treatment-related adverse events were seen in 94%, 93%, and 89% of participants in 1500mg, 700mg, and placebo groups, respectively. (Table 1) There are 6 ongoing clinical trials registered on clinicaltrials.gov (n=665) to determine the efficacy and safety of high doses of voxelotor and its use in children below 12 years. (Table 2) Conclusion: Voxelotor has an acceptable safety profile in sickle cell disease patients of 12 years or older. Voxelotor has shown a dose-dependent improvement in hemoglobin levels and markers of hemolysis, which is associated with a reduction in end-organ damage. Moreover, the increase in hemoglobin was not associated with an increase in vaso-occlusive crisis episodes, in contrast to the other hemoglobin modulator (senicapoc). Additional large prospective multicenter randomized clinical trials are needed to confirm these results. Disclosures Anwer: Incyte, Seattle Genetics, Acetylon Pharmaceuticals, AbbVie Pharma, Astellas Pharma, Celegene, Millennium Pharmaceuticals.:Honoraria, Research Funding, Speakers Bureau.

Base Editing Eliminates the Sickle Cell Mutation and Pathology in Hematopoietic Stem Cells Derived Erythroid Cells

Sickle cell disease (SCD) is a chronic, life-altering multisystem disorder that affects millions of individuals worldwide. Strategies for genetic therapy of autologous SCD hematopoietic stem cells (HSCs) include lentiviral vector (LV) delivery of an anti-sickling β-like globin gene and genome editing, either to revert the SCD mutation by homology-directed repair (HDR) or to induce the expression of fetal hemoglobin (HbF, α2γ2) in red blood cell (RBC) by non-homologous end-joining repair (NHEJ). Base editing is a newer technology that offers the potential for facile generation of more precise genetic alterations with improved safety features. Adenosine base editors (ABEs) convert A-T base pairs to G-C pairs at loci targeted by a single guide (sg) RNA and Cas9 nickase. In contrast to LV vectors and standard Cas9 genome editing, ABEs function through mechanisms that are independent of double-stranded DNA breaks (DSBs), which can cause large deletions, structural DNA rearrangements and TP53-mediated DNA damage responses leading to cell death or malignant transformation. Base editors cannot generate the T-to-A transversion required to revert the mutant SCD codon (Val, GTG) to wild-type (Glu, GAG). However, ABEs can convert the Val codon to Ala (GCG) to generate the naturally occurring, non-sickling variant hemoglobin "Makassar" (HbG). Hemoglobin Makassar heterozygotes and 1 reported homozygote exhibit normal RBC indices, indicating that the variant is benign. We used protein directed evolution to generate a new ABE (ABE8e-NRCH) that accesses a nearby CACC PAM to convert HbS alleles to HbG efficiently in heterologous cells. To demonstrate therapeutic proof of concept, we electroporated ABE8e-NRCH mRNA and targeting sgRNA or ABE8e-NRCH/sgRNA ribonucleoprotein (RNP) complex into 3 different SCD donor CD34+ hematopoietic stem and progenitor cells (HSPCs). After 48 hours, conversion of the HbS allele to HbG was 58±5% with ABE8e-NRCH mRNA/sgRNA and 34±5% with RNP (n=3). On target editing was maximal at 144 hours: 80±2% with ABE8e-NRCH mRNA/sgRNA and 44±7% with ABE8e-NRCH protein (n=3). The indel rate resulting from inadvertent DSBs was &amp;lt;1%, and missense bystander mutations were not detected. In vitro differentiation of base-edited CD34+ cells generated late stage erythroid cells with 76% HbG/14% HbS protein after editing with ABE8e-NRCH mRNA/sgRNA, and 52% HbG/37% HbS after editing with RNP. The sickling rate of in vitro-generated reticulocytes exposed to hypoxia (2% O2) was 52% in controls derived from unedited CD34+ cells, 16% after editing with ABE8e-NRCH mRNA/sgRNA and 27% after editing with RNP. To assess base editing in repopulating HSCs, we transplanted ABE8e-NRCH-edited SCD CD34+ cells into NBSGW mice. The editing frequency before transplantation was ~64% for ABE8e-NRCH mRNA/sgRNA and 40% for RNP at 48 hours after electroporation. Mice were euthanized after 16 weeks and base editing was analyzed in human donor-derived cells. Overall editing rates were preserved in repopulating cells: 68%±3% for ABE8e-NRCH mRNA/sgRNA and 36%±3% for RNP (n=4). The editing frequencies were similar in donor cell-derived myeloid, erythroid, and B cell-lineages, indicating that base editing did not alter hematopoietic development. Bone marrow erythroblasts derived from base-edited and control CD34+ HSPCs exhibited similar maturation profiles and enucleation. Erythroblasts generated in vivo from HSPCs from SCD patient exhibited potentially therapeutic levels of HbG protein: 58±3% with ABE8e-NRCH mRNA/sgRNA and 27%±3% with RNP (n=4). Adenine base editor conversion of the HbS allele to the Makassar variant in autologous HSCs represents a new therapeutic approach for SCD. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Henderson:Trilink Biotech: Current Employment. Mccaffrey:Trilink Biotech: Current Employment. Liu:Pairwise Plants: Consultancy, Patents &amp; Royalties; Prime Medicine: Consultancy, Patents &amp; Royalties; Editas Medicine: Consultancy, Patents &amp; Royalties; Beam Therapeutics: Consultancy, Patents &amp; Royalties. Weiss:Rubius Inc.: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Beam Therapeuticcs: Consultancy, Current equity holder in private company.

Adenine Base Editing of the Sickle Allele in CD34+ Hematopoietic Stem and Progenitor Cells Eliminates Hemoglobin S

While there are several small molecule, gene therapy, and gene editing approaches for treating sickle cell disease (SCD), these strategies do not result in the direct elimination of the causative sickle β-globin (HbS) variant itself. The reduction or complete removal of this pathologic globin variant and expression of normal β-hemoglobin (HbB) or other non-polymerizing β-globin variant may increase the likelihood of beneficial outcomes for SCD patients. Adenine base editors (ABEs) can precisely convert the mutant A-T base pair responsible for SCD to a G-C base pair, thus generating the hemoglobin variant, Hb G-Makassar, a naturally occurring β-globin variant that is not associated with human disease. Our studies have identified ABEs that can achieve highly efficient Makassar editing (&amp;gt;70%) of the sickle mutation in both sickle trait (HbAS) and homozygous sickle (HbSS) patient CD34+ cells with high cell viability and recovery and without perturbation of immunophenotypic hematopoietic stem and progenitor cell (HSPC) frequencies after ex vivo delivery of guide RNA and mRNA encoding the ABE. Furthermore, Makassar editing was retained throughout erythropoiesis in bulk in vitro erythroid differentiated cells (IVED) derived from edited CD34+ cells. To gain an understanding of allelic editing at a single clone resolution, we assessed editing frequencies of clones from both single cell expansion in erythroid differentiation media, as well as from single BFU-E colonies. We found that we could achieve &amp;gt;70% of colonies with bi-allelic Makassar editing and approximately 20% of colonies with mono-allelic Makassar editing, while &amp;lt;3% of colonies remained completely unedited. Previously, conventional hemoglobin capillary electrophoresis and high-performance liquid chromatography (HPLC) were unable to distinguish between HbS and HbG-Makassar. Here, we developed an ultra-high-performance liquid chromatography (UPLC) method that resolves sickle globin (HbS) from Hb G-Makassar globin in IVED cells. The Makassar globin variant was further confirmed by liquid chromatography mass spectrometry (LC-MS). By developing this new method to resolve these two β-globin variants in edited HbSS cells, we were able to detect, in bulk IVED cultures, &amp;gt;80% abundance Hb G-Makassar of total β-globins, which corresponded to a concomitant reduction of HbS levels to &amp;lt;20%. Furthermore, we were also able to determine globin abundance as well as allelic editing at the level of single clones and found that HbS was completely eliminated in &amp;gt;70% of cells that had bi-allelic Makassar editing. Moreover, in the approximately 20% of colonies that were found to be mono-allelically edited for the Makassar variant, there was a 60:40 ratio of Hb G-Makassar:HbS globin abundance in individual clones, at levels remarkably similar to the HbA(wildtype HbB):HbS levels found in HbAS individuals, with minimal observable in vitro sickling when exposed to hypoxia. Thus, with our ABEs, we were able to reduce HbS to &amp;lt;40% on a per cell basis in &amp;gt;90% of IVED cells and found that in vitro sickling under hypoxia inversely correlated with the level of Hb G-Makassar globin variant installation and corresponding reduction in HbS levels. By converting HbS to Hb G-Makassar, our direct and precise editing strategy replaces a pathogenic β-globin with one that has been shown to have normal hematologic parameters. Coupled with autologous stem cell transplant, this next generation gene editing strategy presents a promising new modality for treating patients with SCD. Disclosures Chu: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lam:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Packer:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Olins:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liquori:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Marshall:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yan:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Decker:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gantzer:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Haskett:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bohnuud:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Born:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barrera:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Slaymaker:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gaudelli:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hartigan:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ciaramella:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company.

Supramolecular nanosubstrate-mediated delivery system enables CRISPR-Cas9 knockin of hemoglobin beta gene for hemoglobinopathies.

Leveraging the endogenous homology-directed repair (HDR) pathway, the CRISPR-Cas9 gene-editing system can be applied to knock in a therapeutic gene at a designated site in the genome, offering a general therapeutic solution for treating genetic diseases such as hemoglobinopathies. Here, a combined supramolecular nanoparticle (SMNP)/supramolecular nanosubstrate-mediated delivery (SNSMD) strategy is used to facilitate CRISPR-Cas9 knockin of the hemoglobin beta (HBB) gene into the adeno-associated virus integration site 1 (AAVS1) safe-harbor site of an engineered K562 3.21 cell line harboring the sickle cell disease mutation. Through stepwise treatments of the two SMNP vectors encapsulating a Cas9•single-guide RNA (sgRNA) complex and an HBB/green fluorescent protein (GFP)-encoding plasmid, CRISPR-Cas9 knockin was successfully achieved via HDR. Last, the HBB/GFP-knockin K562 3.21 cells were introduced into mice via intraperitoneal injection to show their in vivo proliferative potential. This proof-of-concept demonstration paves the way for general gene therapeutic solutions for treating hemoglobinopathies.

Sickle cell disease in Sri Lanka: clinical and molecular basis and the unanswered questions about disease severity

BackgroundThough case reports and limited case series of Sickle cell disease in Sri Lanka have been reported previously, no attempt has been made hitherto to undertake a comprehensive genotypic-phenotypic analysis of this “rare” group of patients.ResultsAll accessible Sickle cell disease patients, totaling 60, including, 51 Sickle β-thalassaemia and 9 homozygous sickle patients were enrolled from seven thalassaemia treatment centres between December 2016–March 2019. The majority of patients were of Sinhalese ethnicity (n = 52, 86.67%). Geographically, two prominent clusters were identified and the distribution of Sickle haemoglobin in the island contrasted markedly with the other haemoglobinopathies. 3/ 9 homozygous sickle patients and 3/ 51 Sickle β-thalassaemia patients were receiving regular transfusion. Joint pain was the commonest clinical symptom among all sickle cell disease patients (n = 39, 65.0%). Dactylitis was significantly more common in homozygous sickle patients compared with the Sickle β-thalassaemia groups (p 0.027). Two genetic backgrounds sickle mutation were identified namely, Arab Indian and Benin. Among the regulators of Foetal hemoglobin in Sickle patients of the present study rs1427407 G > T seemed to be the most prominent modifier, with a significant association with Foetal haemoglobin levels (p 0.04).ConclusionsOverall, the clinical course of the Asian version of Sickle cell disease in Sri Lanka appears to be milder than that described in India.

Enabling routine \u03b2-thalassemia Prevention and Patient Management by scalable, combined Thalassemia and Hemochromatosis Mutation Analysis

BackgroundBeta (β)-thalassemia is one of the most common inherited disorders worldwide, with high prevalence in the Mediterranean, the Middle East and South Asia. Over the past 40 years, awareness and prevention campaigns in many countries have greatly reduced the incidence of affected child births. In contrast, much remains to be done in South-Asia. Thus, for Pakistan, an estimated ~ 7000 children annually are born with thalassemia, with no sign of improvement. Although there is good agreement that intermarriage of carriers significantly contributes to the high prevalence of the disorder, effective tools for molecular screening and diagnosis on which to base prevention programs are not readily available.MethodsHere, we present results for a novel LeanSequencing™ process to identify a combination of 18 β-thalassemia mutations (including the sickle cell anemia mutation, HbS, and structural variants HbC and HbE) and 2 hemochromatosis mutations in a multi-ethnic population of 274 pediatric and adolescent patients treated at Afzaal Memorial Thalassemia Foundation in Karachi, Pakistan.ResultsWe found substantial differences in the predominance of disease-causing mutations among the principal ethnic groups in our cohort. We also found the hemochromatosis mutation H63D C > G in 61 (or 22.1%) of our patients including 6 (or 2.2%) homozygotes.ConclusionsTo our knowledge, this is the first screen combining a large set of β-thalassemia and hemochromatosis mutations, so as to facilitate the early identification of patients who may be at increased potential risk for complications from iron overload and thereby to improve the prospective management of thalassemia patients.

XmnI Polymorphism in Sickle Cell Disease in North Morocco

Sickle cell disease is one of the most common severe monogenic disorders in the world. The –158 XmnI polymorphism (C>T) of the Gγ-globin gene promoter is known to be associated with increased expression of the Gγ-globin gene, thus, higher production of Hb F and lesser clinical severity. This study aims to determine the frequency of the XmnI polymorphism and its association with Hb F levels as a modulating factor of sickle cell disease severity in north Moroccan patients. Three hundred and eight subjects carrying the sickle cell mutation and 160 healthy individuals were recruited at the regional hospital of Larache, Morocco. The complete blood count and the Hb F levels were analyzed. The XmnI polymorphism was determined by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique and statistical analysis were done using the Statistical Package for Social Sciences software version 20. Our results estimated the allelic frequency of the XmnI polymorphism in our population at 15.8%. Out of 468 samples, 7.6% were homozygous [+/+] and 16.4% were heterozygous [+/−] for the XmnI polymorphism. This polymorphism was revealed at 20.6% in SS patients, 24.2% in AS carriers, 28.6% in Hb S (HBB: c.20A>T)/β-thalassemia (β-thal) patients and 22.5% in AA subjects. The north Moroccan sickle cell disease patients have shown a low frequency of the XmnI polymorphism. This was later found to be associated with high Hb F levels and mild clinical severity.

Generation of hematopoietic stem/progenitor cells with sickle cell mutation from induced pluripotent stem cell in serum-free system.

IntroductionSickle cell disease (SCD) is a monogenic disease and it is estimated that 300,000 infants are born annually with it. Most treatments available are only palliative, whereas the allogeneic hematopoietic stem cell transplantation offers the only potential cure for SCD.ObjectiveGeneration of human autologous cells, when coupled with induced pluripotent stem cell (iPSC) technology, is a promising approach for developing study models. In this study, we provide a simple and efficient model for generating hematopoietic cells using iPSCs derived from a sickle cell anemia patient and an inexpensive in-house-prepared medium.MethodThis study used iPSCs previously generated from peripheral blood mononuclear cells (PBMCs) from a patient with sickle cell anemia (iPSC_scd). Hematopoietic and erythroid differentiation was performed in two steps. Firstly, with the induction of hematopoietic differentiation through embryoid body formation, we evaluated the efficiency of two serum-free media; and secondly, the induction of hematopoietic stem/progenitor cells to erythroid progenitor cells was performed.ResultsThe patient-specific cell line generated CD34+/CD45+ and CD45+/CD43+ hematopoietic stem/progenitor cells and erythroid progenitors, comprising CD36+, CD71+ and CD235a+ populations, as well as the formation of hematopoietic colonies, including erythroid colonies, in culture in a semi-solid medium.ConclusionIn conjunction, our results described a simple serum-free platform to differentiate human the iPSCs into hematopoietic progenitor cells. This platform is an emerging application of iPSCs in vitro disease modeling, which can significantly improve the search for new pharmacological drugs for sickle cell disease.

Continuous evolution of SpCas9 variants compatible with non-G PAMs.

The targeting scope of Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants is largely restricted to protospacer-adjacent motif (PAM) sequences containing Gs. Here, we report the evolution of three new SpCas9 variants that collectively recognize NRNH PAMs (where R = A or G and H = A, C, or T) using phage-assisted non-continuous evolution (PANCE), three new phage-assisted continuous evolution (PACE) strategies for DNA binding, and a secondary selection for DNA cleavage. The targeting capabilities of these evolved variants and SpCas9-NG were characterized in HEK293T cells using a library of 11,776 genomically integrated protospacer-sgRNA pairs containing all possible NNNN PAMs. The evolved variants mediate indel formation and base editing in human cells and enable the A•T-to-G•C base editing of a sickle-cell anemia mutation using a previously inaccessible CACC PAM. These new evolved SpCas9s, together with previously reported variants, in principle enable targeting the majority of NR PAM sequences and substantially reduce the fraction of genomic sites that are inaccessible by Cas9-based methods.

Biallelic correction of sickle cell disease-derived induced pluripotent stem cells (iPSCs) confirmed at the protein level through serum-free iPS-sac/erythroid differentiation.

New technologies of induced pluripotent stem cells (iPSCs) and genome editing have emerged, allowing for the development of autologous transfusion therapies. We previously demonstrated definitive β‐globin production from human embryonic stem cell (hESC)‐derived erythroid cell generation via hemangioblast‐like ES‐sacs. In this study, we demonstrated normal β‐globin protein production from biallelic corrected sickle cell disease (SCD) iPSCs. We optimized our ES/iPS‐sac method for feeder cell‐free hESC maintenance followed by serum‐free ES‐sac generation, which is preferred for electroporation‐based genome editing. Surprisingly, the optimized protocol improved yields of ES‐sacs (25.9‐fold), hematopoietic‐like spherical cells (14.8‐fold), and erythroid cells (5.8‐fold), compared with our standard ES‐sac generation. We performed viral vector‐free gene correction in SCD iPSCs, resulting in one clone with monoallelic and one clone with biallelic correction, and using this serum‐free iPS‐sac culture, corrected iPSC‐generated erythroid cells with normal β‐globin, confirmed at DNA and protein levels. Our serum‐free ES/iPS‐sac protocol with gene correction will be useful to develop regenerative transfusion therapies for SCD.

Blood rheology biomarkers in sickle cell disease.

Sickle cell disease (SCD) is the most common inherited blood disorder, affecting approximately 100,000 patients in the U.S. and millions more worldwide. Patients with SCD experience a wide range of clinical complications, including frequent pain crises, stroke, and early mortality, all originating from a single-point mutation in the β-globin subunit. The RBC changes resulting from the sickle mutation lead to a host of rheological abnormalities that diminish microvascular blood flow, and produce severe anemia due to RBC hemolysis, and ischemia from vaso-occlusion initiated by sticky, rigid sickle RBCs. While the pathophysiology and mechanisms of SCD have been investigated for many years, therapies to treat the disease are limited. In addition to RBC transfusion, there are only two US Food and Drug Administration (FDA)-approved drugs to ameliorate SCD complications: hydroxyurea (HU) and L-glutamine (Endari™). The only curative therapy currently available is allogeneic hematopoietic stem cell transplantation (HSCT), which is generally reserved for individuals with a matched related donor, comprising only 10–15% of the total SCD population. Potentially curative advanced gene therapy approaches for SCD are under investigation in ongoing clinical trials. The ultimate goal of any curative treatment should be to repair the hemorheological abnormalities caused by SCD, and thus normalize blood flow and prevent clinical complications. Our mini-review highlights a set of key hemorheological biomarkers (and the current and emerging technologies used to measure them) that may be used to guide the development of novel curative and palliative therapies for SCD, and functionally assess outcomes.Impact statementSevere impairment of blood rheology is the hallmark of SCD pathophysiology, and one of the key factors predisposing SCD patients to pain crises, organ damage, and early mortality. As novel therapies emerge to treat or cure SCD, it is crucial that these treatments are functionally evaluated for their effect on blood rheology. This review describes a comprehensive panel of rheological biomarkers, their clinical uses, and the technologies used to obtain them. The described technologies can produce highly sensitive measurements of the ability of current treatments to improve blood rheology of SCD patients. The goal of curative therapies should be to achieve blood rheology biomarkers measurements in the range of sickle cell trait individuals (HbAS). The use of the panel of rheological biomarkers proposed in this review could significantly accelerate the development, optimization, and clinical translation of novel therapies for SCD.

Pharmacological interventions for painful sickle cell vaso-occlusive crises in adults.

Pharmacological interventions for painful sickle cell vaso-occlusive crises in adults.

Characterization of a Genetically Engineered HUDEP2 Cell Line Harboring a Sickle Cell Disease Mutation As a Potential Research Tool for Preclinical Sickle Cell Disease Drug Discovery

Sickle cell disease (SCD) is a genetic disorder caused by a missense mutation in the beta-globin gene [Glu6Val] resulting in the formation of HbS (sickle hemoglobin). HbS polymerizes under low oxygen tension causing an array of severe pathophysiological complications, including vaso-occlusion, pain and hemolytic anemia. Traditionally, research efforts toward studying anti-sickling effects of various pathways and compounds in vitro have been made using sickle cell patient derived samples. However, such studies are limited based on sample availability and a large degree of phenotypic variability. Thus, a cultured cell system with a sickle mutation mimicking human sickle cell erythroid cells is a potentially useful tool that can be used to interrogate anti-sickling compound action and to study various facets of sickle cell phenotype. In this study, we generated a genetically engineered sickle mutation (GAG to GTG) in HUDEP2 cell line using CRISPR Cas9 gene editing and homology directed repair mechanism. HUDEP2 cells are immortalized human erythroid progenitor cells, obtained from the Riken Institute and were cultured as described (Kurita et al 2013). CRISPR Cas9 RNP complex with modified guide RNA along with ssODN were electroporated in HUDEP2 cells. Cells were also treated with SCR7, a NHEJ inhibitor to facilitate homology- directed repair events. Cells were sorted for clonal selection, cultured and further screened for homozygous knock-in mutations using digital droplet PCR. The mutation was further confirmed by sanger sequencing. 12 clones consisting of homozygous mutation were identified, with 6 of these clones selected for further characterization. Cells were differentiated for 7 days in culture and hemolysates were analyzed using HPLC. Heterozygote clone showed 32.3% HbS which was comparable to sickle cell trait patient derived blood samples whereas homozygous clones (ssHUDEP2) showed between 71-85% HbS. These results were further confirmed for the presence of HbS using mass spectrometry. Mutant HUDEP2 cell growth and differentiation were similar to the wild-type HUDEP2 cells. To evaluate sickling in these cells we optimized the differentiation protocol to improve the maturation of these cells by withdrawing SCF and doxycycline gradually. Hemoglobin polymerization and resulting shape change in sickle HUDEP cells was determined by subjecting mature ssHUDEP2 cells to hypoxia (1% oxygen) for 3 hours. Upon subjecting to hypoxia, ssHUDEP2 cells showed morphological changes indicative of HbS polymerization. These morphological changes were absent in wild type cells under similar conditions of hypoxia. In addition, ssHUDEP2 cells pre-treated with 25µM GBT440, a known anti-sickling compound, for 1 hour prior to hypoxia incubation elicited a reduction in the shape change associated with hypoxia, suggesting inhibition of hemoglobin polymerization and sickling. These results suggest that genetically-engineered sickle mutant HUDEP cells represent a preclinical model for development of anti-sickling compounds for the potential treatment of Sickle Cell Disease and to explore novel mechanistic pathways. Disclosures Gupta: Sanofi: Employment. Sturtevant:Sanofi: Employment. Vieira:Sanofi: Employment. Krishnamoorthy:Sanofi: Employment. Demers:Sanofi: Employment.

Burden of Illness of Sickle Cell Disease in Countries of the Middle East: A Systematic Literature Review

Background: Sickle cell disease (SCD) is a group of hereditary hemolytic disorders characterized by abnormal hemoglobin molecules that distort red blood cells (RBCs) into a rigid, sickle-like shape. The global burden of SCD is substantial. In North America and Europe, the incidence and prevalence of SCD range from approximately 0.00008% to 0.274% and 0.01% to 0.05%, respectively. In these regions, the median age at death for patients with the disease ranges from 40 to 60 years, and childhood mortality has steadily dropped in recent years. Sickle cell disease is more common in certain ethnic groups and is most prominent in malaria-endemic regions, as individuals who are heterozygous for the sickle cell mutation are relatively protected against malaria infection. One such region is the Middle East, where the incidence and prevalence rates of SCD in some countries are amongst the highest in the world, and childhood mortality rates for patients with SCD are relatively high. However, the overall burden of illness-encompassing various aspects of disease and their impact on costs and health outcomes-is largely uncharacterized for SCD in Middle Eastern countries. Understanding the burden of illness of SCD in this region could help to inform and prioritize health care policies and assist in the allocation of health care resources. Objective: To understand the burden of illness of SCD in Middle Eastern countries by identifying available information using a systematic literature review (SLR). Methods: A systematic literature search of English-language studies published within five years of the search date (July 2018) was conducted using PubMed and EMBASE databases. Pre-defined population, intervention, comparator, and outcomes (PICO) criteria were used to identify relevant studies conducted in Middle Eastern countries. All patient populations with SCD receiving any approved or guideline-recommended medical interventions were of interest. Studies that reported any outcomes related to burden of illness such as epidemiology, treatment patterns, prognosis, health-related quality of life (HRQoL), and economic information were included. All study types were included except for case reports and conference meeting minutes. Results: Twenty-three studies from Middle Eastern countries met the inclusion criteria (Figure 1). Of these studies, eight were from Saudi Arabia, one was from the United Arab Emirates, six were from Bahrain, three were from Lebanon, one was from Oman, one was from Kuwait, and three were from Egypt. Epidemiological information was captured for six countries, treatment pattern information was captured for three countries, disease prognosis information was captured for six countries, HRQoL information was captured for two countries, and cost information was captured for one country. Overall, the incidence and prevalence of SCD in Middle Eastern countries both ranged from approximately 0.04% to 2.1%. Vaso-occlusive crises (VOC), acute chest syndrome (ACS), acute splenic sequestration events, viral infections, and anemic/hemolytic events were the most common causes of hospitalization; however, the rates generally differed between countries. Limited information was available for disease prognosis; factors related to high mortality included age, frequency of hospitalizations, and length of stay in the ICU. Information on HRQoL was even further limited; only two studies captured by the search reported this type of data. In these two studies, SCD patients experienced a reduced QoL compared with non-diseased controls. The reported reductions in QoL for SCD patients were associated with poor education, age of onset of ACS and blood transfusion in the three-month period preceding the study, and specific physical symptoms including fever and pain. Finally, the only published economic information identified by the search was the cost of screening for SCD with high-performance liquid chromatography (HPLC) in Lebanon ($3.50 per newborn). Conclusions: Substantial data gaps across all aspects of burden of illness were identified. Moreover, the data captured-especially for HRQoL and costs-were limited and reported in very few countries. These findings highlight the importance of further characterizing the burden of illness of SCD in the Middle East, where the incidence and prevalence rates of the disease are relatively high. Disclosures Bailey: Novartis: Employment. Gibbs:Novartis: Employment. Dani:Novartis: Employment. Mendell:Novartis: Consultancy. Thompson:Novartis: Consultancy.

Preclinical Evaluation for Engraftment of Gene-Edited CD34+ Cells with a Sickle Cell Disease Mutation in a Rhesus Transplantation Model

Sickle cell disease (SCD) is caused by a 20A&amp;gt;T mutation in the β-globin gene. State-of-the-art genome editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin (Hb) while simultaneously eliminating sickle Hb. We have demonstrated efficient gene correction in SCD CD34+ cells with SCD mutation-specific guide RNA, Cas9 mRNA/protein, and single strand donor DNA, resulting in ~30% gene correction and ~50% indels at the DNA level, and ~60% normal β-globin production at the protein level in in vitro erythroid differentiation (ASH 2018). Gene correction by homology directed repair is thought to be enhanced by cell proliferation; however, cell proliferation might reduce stemness of HSCs. To investigate this hypothesis, we sought to evaluate engraftment of gene-edited CD34+ HSCs in a non-human primate model. To model SCD gene correction, a β-to-βs globin conversion was designed in rhesus macaques. Mobilized rhesus CD34+ cells (n=2) were electroporated using the GMP-compliant, FDA Master File-supported, and scalable MaxCyte GT System to deliver rhesus β-globin-targeting guide RNA (the same target site as the SCD mutation-specific guide RNA), SpCas9 protein, and single strand donor DNA including a SCD mutation (20A&amp;gt;T). We also added an adjuvant to improve gene conversion efficiencies. Following erythroid differentiation, gene correction efficiency was evaluated at DNA levels by deep sequencing and at protein levels by reverse-phase HPLC. We observed high-efficiency genome editing without the adjuvant (20-30% gene conversion and 61-64% indels), and further enhanced genome editing with the adjuvant (51-59% gene conversion and 36-39% indels). After erythroid differentiation, we observed production of βs-globin protein (~100%) but not normal β-globin in gene-edited cells. We then evaluated engraftment of gene-edited rhesus CD34+ cells with β-to-βs globin conversion (n=2, 13U005 and 12U011). Mobilized rhesus CD34+ cells (3.4-3.8e7) were pre-stimulated for 2 days, and edited cells were cryopreserved after electroporation with editing tools. Small aliquots of edited cells (before and after cryopreservation) were differentiated into erythroid cells in vitro, resulting in 17-26% of gene conversion and 57-71% of indels at the DNA level and 50-100% of β-globin production at the protein level, with no difference observed between aliquots taken before and after cryopreservation. Following 9.5 Gy total body irradiation, the frozen edited CD34+ cells (1.6-2.2e7) were injected into autologous macaques. We observed robust recovery of blood counts in 13U005, while peripheral blood recovery was delayed in 12U011, who was supported by serial whole blood transfusion. We observed 7-11% of gene conversion and 44-54% of indels in both granulates and lymphocytes in 13U005 1 month post-transplant. Around 15% sickle Hb production in red blood cells was detected by Hb electrophoresis in 13U005 three months post-transplant and ~7% in 12U011 two months post-transplant. Interestingly, ~10% of fetal Hb production was observed in 12U001, likely due to stress hematopoiesis. In summary, we developed a rhesus β-to-βs globin conversion model with HSC-targeted genome editing strategies. The gene-edited rhesus CD34+ cells are engraftable for at least 3 months post-transplant. Although further follow-up is necessary for transplanted animals, these findings are helpful in designing HSC-targeted gene correction trials. Figure Disclosures Li: MaxCyte, Inc: Employment. Allen:MaxCyte, Inc: Employment. Peshwa:MaxCyte, Inc: Employment.

Paediatric haematologists' attitudes regarding haematopoietic cell transplantation as treatment for sickle cell disease.

Beginning early in childhood, patients with sickle cell disease [SCD; a group of genetic haemoglobin disorders characterized by the sickle or HbS mutation (HBB E7V)] are at risk of life-threatening and debilitating health events. Despite the high morbidity and mortality of this disease, haematopoietic cell transplantation (HCT), a curative therapy for SCD, remains underutilized. A variety of factors, including the limited availability of suitable donors, play a role in this trend, but do not fully explain the low frequency with which this therapy is employed. The objective of this study was to identify paediatric haematologists' attitudes about HCT as a treatment option for SCD, and to describe the impact of these attitudes on their practices of discussing HCT with families of children affected by this disease. A nationwide survey of paediatric haematologists in the United States was conducted between February and May 2016. Two hundred and eighty-seven surveys were included in the final analysis (response rate 20%). On average, respondents reported informing 42% of families about HCT as a treatment option (N=248, 95% confidence interval: 38-46). Clinician attitudes about the cost and safety of HCT were associated with practices of discussing this therapy with families. These findings suggest that clinician attitudes and referral practices may play a role in the underutilization of this therapy in the SCD population.

Francisella novicida Cas9 interrogates genomic DNA with very high specificity and can be used for mammalian genome editing

Genome editing using the CRISPR/Cas9 system has been used to make precise heritable changes in the DNA of organisms. Although the widely used Streptococcus pyogenes Cas9 (SpCas9) and its engineered variants have been efficiently harnessed for numerous gene-editing applications across different platforms, concerns remain regarding their putative off-targeting at multiple loci across the genome. Here we report that Francisella novicida Cas9 (FnCas9) shows a very high specificity of binding to its intended targets and negligible binding to off-target loci. The specificity is determined by its minimal binding affinity with DNA when mismatches to the target single-guide RNA (sgRNA) are present in the sgRNA:DNA heteroduplex. FnCas9 produces staggered cleavage, higher homology-directed repair rates, and very low nonspecific genome editing compared to SpCas9. We demonstrate FnCas9-mediated correction of the sickle cell mutation in patient-derived induced pluripotent stem cells and propose that it can be used for precise therapeutic genome editing for a wide variety of genetic disorders.

Association of sickle cell trait with measures of cognitive function and dementia in African Americans.

ObjectiveThe incidence and prevalence of cognitive decline and dementia are significantly higher among African Americans compared with non-Hispanic Whites. The aim of this study was to determine whether inheritance of the sickle cell trait (SCT) i.e. heterozygosity for the sickle cell mutation increases the risk of cognitive decline or dementia Among African Americans.MethodsWe studied African American participants enrolled in the Atherosclerosis Risk in Communities study. SCT genotype at baseline and outcome data from cognitive assessments at visits 2, 4 and 5, and an MRI performed at visit 5 were analyzed for the association between SCT and risk of cognitive impairment and/or dementia.ResultsThere was no significant difference in risk factors profile between participants with SCT (N = 176) and those without SCT (N = 2532). SCT was not independently associated with a higher prevalence of global or domain-specific cognitive impairment at baseline or with more rapid cognitive decline. Participants with SCT had slightly lower incidence of dementia (HR = 0.63 [0.38, 1.05]). On the other hand, SCT seems to interact with the apolipoprotein E ε4 risk allele resulting in poor performance on digit symbol substitution test at baseline (z-score = −0.08, Pinteraction = 0.05) and over time (z-score = −0.12, Pinteraction = 0.04); and with diabetes mellitus leading to a moderately increased risk of dementia (HR = 2.06 [0.89, 4.78], Pinteraction = 0.01).ConclusionsSCT was not an independent risk factor for prevalence or incidence of cognitive decline or dementia, although it may interact with and modify other putative risk factors for cognitive decline and dementia.

In Vivo Outcome of Homology-Directed Repair at the HBB Gene in HSC Using Alternative Donor Template Delivery Methods

Gene editing following designer nuclease cleavage in the presence of a DNA donor template can revert mutations in disease-causing genes. For optimal benefit, reversion of the point mutation in HBB leading to sickle cell disease (SCD) would permit precise homology-directed repair (HDR) while concurrently limiting on-target non-homologous end joining (NHEJ)-based HBB disruption. In this study, we directly compared the relative efficiency of co-delivery of a novel CRISPR/Cas9 ribonucleoprotein targeting HBB in association with recombinant adeno-associated virus 6 (rAAV6) versus single-stranded oligodeoxynucleotides (ssODNs) to introduce the sickle mutation (GTC or GTG; encoding E6V) or a silent change (GAA; encoding E6optE) in human CD34+ mobilized peripheral blood stem cells (mPBSCs) derived from healthy donors. In vitro, rAAV6 outperformed ssODN donor template delivery and mediated greater HDR correction, leading to both higher HDR rates and a higher HDR:NHEJ ratio. In contrast, at 12–14 weeks post-transplant into recipient, immunodeficient, NOD, B6, SCID Il2rγ−/− Kit(W41/W41) (NBSGW) mice, a ∼6-fold higher proportion of ssODN-modified cells persisted in vivo compared to recipients of rAAV6-modified mPBSCs. Together, our findings highlight that methodology for donor template delivery markedly impacts long-term persistence of HBB gene-modified mPBSCs, and they suggest that the ssODN platform is likely to be most amenable to direct clinical translation.