scholarly journals Zinc Finger Nuclease-Mediated Disruption of the BCL11A Erythroid Enhancer Results in Enriched Biallelic Editing, Increased Fetal Hemoglobin, and Reduced Sickling in Erythroid Cells Derived from Sickle Cell Disease Patients

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 974-974 ◽  
Author(s):  
Samuel Lessard ◽  
Pauline Rimmele ◽  
Hui Ling ◽  
Kevin Moran ◽  
Benjamin Vieira ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Cell Therapy ◽  
Single Cell ◽  
Zinc Finger ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Potential Efficacy

High fetal hemoglobin (HbF) levels are associated with decreased severity and mortality in sickle cell disease (SCD) and beta thalassemia (BT). We have developed a novel gene-edited cell therapy using autologous hematopoietic stem and progenitor cells (HSPCs) that have been genetically modified with zinc finger nucleases (ZFNs) to reactivate HbF expression. The ZFNs target the binding motif of GATA1 (GATAA) within an intronic erythroid-specific enhancer (ESE) of BCL11A, which encodes a major transcriptional repressor of HbF. Previously, we reported successful ZFN-mediated editing of the BCL11A ESE and reactivation of HbF in both dual (granulocyte colony-stimulating factor (G-CSF) and plerixafor) and single plerixafor mobilized HSPCs(Holmes 2017, Moran 2018). Both related drug candidates, ST-400 and BIVV003, are currently in phase 1/2a clinical trials for transfusion-dependent BT (NCT03432364) and SCD (NCT03653247), respectively. Here, we performed extensive genetic and phenotypic characterization of ZFN-edited HSPCs from healthy and SCD donors. We performed single-cell characterization of BCL11A ESE-edited HSPCs from 4 healthy donors. Briefly, individual HSPCs were sorted and cultured in erythroid differentiation medium. Genomic DNA and protein lysate were collected at day 14 and 20, respectively. In total, we successfully genotyped 961 single-cell derived colonies by next-generation sequencing. The distribution was highly skewed towards biallelic-edited cells (P<3x10-149) representing 94% of edited clones, suggesting that ZFN-expressing cells are likely to become edited at both alleles. We found that each edited allele contributed additively to an increase in HbF% of 15% (P=1x10-80) as measured by UPLC. Clones harboring GATAA-disrupting indels on both alleles displayed on average 34% more HbF% than WT clones (P=1x10-112). In contrast, clones with biallelic indels that left the motif intact displayed a more modest increase (13%, P=1x10-6). Overall, our data revealed that >90% of edited cells were biallelic, displaying on average 27-38% more HbF% despite variation in donor baseline levels. We observed a strong enrichment of biallelic-edited homozygotes (same indel pattern at both alleles) compared to an expected random distribution (161 vs 24; P<1x10-5). These clones may harbor larger deletions not captured by sequencing, as reported previously using CRISPR/Cas9 (Kosicki 2018). To address this question, we used a combination of a small amplicon sequencing assay design covering an informative SNP and a 12kb amplicon Nextera assay. We found that 27% of initially assigned homozygote clones were bona fide homozygotes (44/161) with the remaining harboring indels not originally captured. Nevertheless, most indels remained small, with 91% of indels <50bp, and deletions and insertions >1kb together consisting of less than 1% of alleles. The largest deletion was 4kb, but no indel extended outside the enhancer region of BCL11A or altered the coding region (>26 kb away). Moreover indels >50bp were not associated with enucleation levels (P=0.77), suggesting that they did not alter erythroid function. Overall, these results are consistent with previous data showing that ZFN-mediated gene editing does not impair HSPC function in vitro based on colony forming unit (CFU) production, and that injection of BIVV003 into immune-deficient NBSGW mice results in robust long-term engraftment with no impact on the number of HSPCs or their progeny, including erythrocytes. Finally, BCL11A ESE editing in HSPCs mobilized from one SCD donor resulted in a 3-fold HbF increase consistent across technical duplicates, without impacting CFU production or erythroid enucleation. Importantly, clonal analysis revealed a similar enrichment of biallelic editing (P=6x10-4) and additive HbF up-regulation, with biallelic edited cells reaching 28% more HbF% than unedited cells (50% vs 22%, P=7x10-5). Furthermore, enucleated cells differentiated from edited HSPCs showed attenuation of sickling under hypoxic conditions supporting the potential efficacy of BIVV003. Experiments in HSPCs from additional SCD donors are ongoing. Overall, our data have shown that ZFN-mediated disruption of BCL11A ESE results in enriched biallelic editing with on-target small indels, reactivates HbF and reduces sickling, supporting the potential efficacy and specificity of BIVV003 as a novel cell therapy for SCD. Disclosures Lessard: Sanofi: Employment. Rimmele:Sanofi: Employment. Ling:Sanofi: Employment. Moran:Sanofi: Employment. Vieira:Sanofi: Employment. Lin:Sanofi: Employment. Hong:Sanofi: Employment. Reik:Sangamo Therapeutics: Employment. Dang:Sangamo Therapeutics: Employment. Rendo:Sanofi: Employment. Daak:Sanofi: Employment. Hicks:Sanofi: Employment.

scholarly journals Genome editing using CRISPR-Cas9 to create the HPFH genotype in HSPCs: An approach for treating sickle cell disease and β-thalassemia

2016 ◽  
Vol 113 (38) ◽  
pp. 10661-10665 ◽  
Author(s):  
Lin Ye ◽  
Jiaming Wang ◽  
Yuting Tan ◽  
Ashley I. Beyer ◽  
Fei Xie ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Genome Editing ◽  
Sickle Cell ◽  
Globin Gene ◽  
Autologous Transplantation ◽  
Clinical Manifestations ◽  
Fetal Hemoglobin ◽  
Compound Heterozygous ◽  
Cell Disease ◽  
Hematopoietic Stem

Hereditary persistence of fetal hemoglobin (HPFH) is a condition in some individuals who have a high level of fetal hemoglobin throughout life. Individuals with compound heterozygous β-thalassemia or sickle cell disease (SCD) and HPFH have milder clinical manifestations. Using RNA-guided clustered regularly interspaced short palindromic repeats-associated Cas9 (CRISPR-Cas9) genome-editing technology, we deleted, in normal hematopoietic stem and progenitor cells (HSPCs), 13 kb of the β-globin locus to mimic the naturally occurring Sicilian HPFH mutation. The efficiency of targeting deletion reached 31% in cells with the delivery of both upstream and downstream breakpoint guide RNA (gRNA)-guided Staphylococcus aureus Cas9 nuclease (SaCas9). The erythroid colonies differentiated from HSPCs with HPFH deletion showed significantly higher γ-globin gene expression compared with the colonies without deletion. By T7 endonuclease 1 assay, we did not detect any off-target effects in the colonies with deletion. We propose that this strategy of using nonhomologous end joining (NHEJ) to modify the genome may provide an efficient approach toward the development of a safe autologous transplantation for patients with homozygous β-thalassemia and SCD.

scholarly journals Complementary Base Editing Approaches for the Treatment of Sickle Cell Disease and Beta Thalassemia

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 3352-3352 ◽  
Author(s):  
Ling Lin ◽  
Adrian P. Rybak ◽  
Conrad Rinaldi ◽  
Jonathan Yen ◽  
Yanfang Fu ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Beta Thalassemia ◽  
Beta Globin ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Base Editing ◽  
Gamma Globin

Sickle cell disease (SCD) and Beta thalassemia are disorders of beta globin production and function that lead to severe anemia and significant disease complications across a multitude of organ systems. Autologous transplantation of hematopoietic stem cells engineered through the upregulation of fetal hemoglobin (HbF) or correction of the beta globin gene have the potential to reduce disease burden in patients with beta hemoglobinopathies. Base editing is a recently developed technology that enables precise modification of the genome without the introduction of double strand DNA breaks. Gamma globin gene promoters were comprehensively screened with cytosine and adenine base editors (ABE) for the identification of alterations that would derepress HbF. Three regions were identified that significantly upregulated HbF, and the most effective nucleotide residue conversions are supported by natural variation seen in patients with hereditary persistence of fetal hemoglobin (HPFH). ABEs have been developed that significantly increase the level of HbF following nucleotide conversion at key regulatory motifs within the HBG1 and HBG2 promoters. CD34+ hematopoietic stem and progenitor cells (HSPC) were purified at clinical scale and edited using a process designed to preserve self-renewal capacity. Editing at two independent sites with different ABEs reached 94 percent and resulted in up to 63 percent gamma globin by UPLC. The levels of HbF observed should afford protection to the majority of SCD and Beta thalassemia patients based on clinical observations of HPFH and non-interventional therapy that links higher HbF dosage with milder disease (Ngo et al, 2011 Brit J Hem; Musallam et al, 2012 Blood). Directly correcting the Glu6Val mutation of SCD has been a recent goal of genetic therapies designed for the SCD population. Current base editing technology cannot yet convert mutations like those that result from the A-T transversion in sickle beta globin; however, ABE variants have been designed to recognize and edit the opposite stranded adenine residue of valine. This results in the conversion of valine to alanine and the production of a naturally occurring variant known as Hb G-Makassar. Beta globin with alanine at this position does not contribute to polymer formation, and patients with Hb G-Makassar present with normal hematological parameters and red blood cell morphology. SCD patient fibroblasts edited with these ABE variants achieve up to 70 percent conversion of the target adenine. CD34 cells from healthy donors were then edited with a lead ABE variant, targeting a synonymous mutation in an adjacent proline that resides within the editing window and serves as a proxy for editing the SCD mutation. The average editing frequency was 40 percent. Donor myeloid chimerism documented at these levels in the allogeneic transplant setting exceeds the 20 percent that is required for reversing the sickle phenotype (Fitzhugh et al, 2017 Blood). These next generation editing approaches provide a promising new modality for treating patients with Beta thalassemia and SCD. Disclosures No relevant conflicts of interest to declare.

scholarly journals Adenosine Base Editing of γ-Globin Promoters Induces Fetal Hemoglobin and Inhibit Erythroid Sickling

Blood ◽  
2020 ◽  
Vol 136 (Supplement 1) ◽  
pp. 21-22
Author(s):  
Thiyagaraj Mayuranathan ◽  
Jonathan S. Yen ◽  
Gregory A. Newby ◽  
Yu Yao ◽  
Shaina N. Porter ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Erythroid Cell ◽  
Cell Disease ◽  
Private Company ◽  
Hematopoietic Stem ◽  
Benign Condition ◽  
Base Editing

Rare variants in the γ-globin (HBG2 and HBG1) promoters cause sustained postnatal expression of fetal hemoglobin (HbF, α2γ2) in red blood cells (RBCs). This benign condition is termed hereditary persistence of fetal hemoglobin (HPFH). Individuals with HPFH variants are protected from β-hemoglobinopathies including sickle cell disease and β-thalassemia. Our group and others have used CRIPSR/Cas9-mediated non-homologous end joining to generate HPFH-like insertion-deletion (indel) mutations in the γ-globin promoter. However, simultaneous double-stranded breaks (DSBs) in the tandem duplicated γ-globin genes can result in loss or inversion of the intervening genetic material and/or chromosomal rearrangements. More generally, Cas9-associated DSBs can elicit a cytotoxic DNA repair response leading to cell death or evoke p53 loss with malignant transformation. Base editor (BE) proteins represent a promising approach to install precise nucleotide substitutions without DSBs. Adenosine base editors (ABEs), consisting of catalytically impaired Cas9 fused to a modified adenosine deaminase, create targeted A:T-to-G:C mutations. Here we describe the use of ABEs to recapitulate naturally occurring HPFH variants in hematopoietic stem cells (HSCs). We electroporated ABE7.10-single guide (sg) RNA ribonucleoprotein (RNP) complex into mobilized peripheral blood CD34+ hematopoietic stem and progenitor cells (HSPCs) to recreate 3 different HPFH variants in the HBG1/2 promoters (-198 T>C, -175 T>C and -113 A>G). Measured editing frequency was maximal on day 10 after electroporation and transferred to erythroid differentiation media. 20% editing efficiency was observed for the -198 site, 58% for -175 and 50% for -113. Indel frequencies were <2% at each of the three sites, reflecting a low rate of DSBs. Fetal hemoglobin levels in erythroid cells generated in vitro from A base-edited CD34+ HSPCs were 26±4% (-198 T>C), 60±10% (-175 T>C), and 42±7% (-113 A>G) versus14±2% in unedited control cells. Base editing at the -175 site in sickle cell disease (SCD) donor CD34+ HSPCs resulted in the induction of HbF to 55% in erythroid progeny compared to 6% in controls. After exposure to hypoxia (2% oxygen), reticulocytes generated from -175 T>C-edited CD34+ HSPCs exhibited sickling rates of 24%, compared to 52% in controls. Thus, creation of this variant, which generates a de novo binding site for the transcriptional activator TAL1, reactivates erythroid cell HbF to levels that inhibit sickle hemoglobin polymerization and cell sickling. To assess base editing in HSCs, we used ABE RNP to modify the -175 site in SCD donor CD34+ HSPCs, followed by transplantation into NBSGW mice. The editing frequency in CD34+ HSPCs before transplantation was ~30% and declined to approximately 20% in bone marrow-repopulating donor cells at 16 weeks post-transplantation. Editing frequencies were similar in CD34+ donor cell-derived myeloid, erythroid, and B cells, indicating that hematopoietic differentiation was not altered. Bone marrow erythroblasts derived from base-edited and control CD34+ HSPCs exhibited similar maturation profiles and enucleation. Erythroblasts generated in vivo from SCD patient HSPCs exhibited 32±2% HbF compared to unedited controls (4±1%) (n=4, P>0.0001). Our studies provide proof of concept that adenosine base editors can be used therapeutically for β-hemoglobinopathies. Specifically, generation of the -175 T>C HPFH mutation in patient HSCs followed by autologous transplantation represents a new therapeutic approach for SCD and β-thalassemia. Disclosures Yen: Beam Therapeutics: Current equity holder in publicly-traded company, Ended employment in the past 24 months. Sharma:Spotlight Therapeutics: Consultancy; Magenta Therapeutics: Other: Research Collaboration; CRISPR Therapeutics, Vertex Pharmaceuticals, Novartis: Other: Clinical Trial PI. Liu:Pairwise Plants: Consultancy, Patents & Royalties; Editas Medicine: Consultancy, Patents & Royalties; Beam Therapeutics: Consultancy, Patents & Royalties; Prime Medicine: Consultancy, Patents & Royalties. Weiss:Beam Therapeuticcs: Consultancy, Current equity holder in private company; Esperion Therapeutics: Consultancy, Current equity holder in private company; Novartis: Consultancy, Current equity holder in private company; Cellarity Inc.: Consultancy, Current equity holder in private company; Rubius Inc.: Consultancy, Current equity holder in private company.

scholarly journals HbF Levels in Sickle Cell Disease Are Associated with Proportion of Circulating Hematopoietic Stem and Progenitor Cells and CC-Chemokines

Cells ◽  
2020 ◽  
Vol 9 (10) ◽  
pp. 2199
Author(s):  
Caterina P. Minniti ◽  
Seda S. Tolu ◽  
Kai Wang ◽  
Zi Yan ◽  
Karl Robert ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Progenitor Cells ◽  
Sickle Cell ◽  
Fetal Hemoglobin ◽  
Treatment Modalities ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Multipotent Cells ◽  
Stem And Progenitor Cells ◽  
Highly Correlated

The concentration of circulating hematopoietic stem and progenitor cells has not been studied longitudinally. Here, we report that the proportions of Lin-CD34+38- hematopoietic multipotent cells (HMCs) and of Lin-CD34+CD38+ hematopoietic progenitors cells (HPCs) are highly variable between individuals but stable over long periods of time, in both healthy individuals and sickle cell disease (SCD) patients. This suggests that these proportions are regulated by genetic polymorphisms or by epigenetic mechanisms. We also report that in SCD patients treated with hydroxyurea, the proportions of circulating HMCs and HPCs show a strong positive and negative correlation with fetal hemoglobin (HbF) levels, respectively. Titration of 65 cytokines revealed that the plasma concentration of chemokines CCL2, CCL11, CCL17, CCL24, CCL27, and PDGF-BB were highly correlated with the proportion of HMCs and HPCs and that a subset of these cytokines were also correlated with HbF levels. A linear model based on four of these chemokines could explain 80% of the variability in the proportion of circulating HMCs between individuals. The proportion of circulating HMCs and HPCs and the concentration of these chemokines might therefore become useful biomarkers for HbF response to HU in SCD patients. Such markers might become increasingly clinically relevant, as alternative treatment modalities for SCD are becoming available.

scholarly journals Pathogenic BCL11A variants provide insights into the mechanisms of human fetal hemoglobin silencing

PLoS Genetics ◽  
2021 ◽  
Vol 17 (10) ◽  
pp. e1009835
Author(s):  
Yong Shen ◽  
Rick Li ◽  
Kristian Teichert ◽  
Kara E. Montbleau ◽  
Jeffrey M. Verboon ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Zinc Finger ◽  
Sickle Cell ◽  
Neurodevelopmental Disorder ◽  
Fetal Hemoglobin ◽  
Missense Variant ◽  
Loss Of Function ◽  
Cell Disease ◽  
Missense Variants

Increased production of fetal hemoglobin (HbF) can ameliorate the severity of sickle cell disease and β-thalassemia. BCL11A has been identified as a key regulator of HbF silencing, although its precise mechanisms of action remain incompletely understood. Recent studies have identified pathogenic mutations that cause heterozygous loss-of-function of BCL11A and result in a distinct neurodevelopmental disorder that is characterized by persistent HbF expression. While the majority of cases have deletions or null mutations causing haploinsufficiency of BCL11A, several missense variants have also been identified. Here, we perform functional studies on these variants to uncover specific liabilities for BCL11A’s function in HbF silencing. We find several mutations in an N-terminal C2HC zinc finger that increase proteasomal degradation of BCL11A. We also identify a distinct C-terminal missense variant in the fifth zinc finger domain that we demonstrate causes loss-of-function through disruption of DNA binding. Our analysis of missense variants causing loss-of-function in vivo illuminates mechanisms by which BCL11A silences HbF and also suggests potential therapeutic avenues for HbF induction to treat sickle cell disease and β-thalassemia.

scholarly journals Investigational curative gene therapy approaches to sickle cell disease

2021 ◽  
Vol 5 (23) ◽  
pp. 5452-5452
Author(s):  
David A. Williams ◽  
Erica Esrick
Keyword(s):  
Stem Cells ◽  
Gene Therapy ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Clinical Manifestations ◽  
Fetal Hemoglobin ◽  
Unrelated Donor ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Sickle Hemoglobin

Abstract Sickle cell disease (SCD) is an inherited blood condition resulting from abnormal hemoglobin production. It is one of the most common genetic diseases in the world. The clinical manifestations are variable and range from recurrent acute and debilitating painful crises to life-threatening pulmonary, cardiovascular, renal, and neurologic complications. The only curative treatment of SCD at this time is bone marrow transplantation (also called hematopoietic stem cell transplantation) using healthy blood stem cells from an unaffected brother or sister or from an unrelated donor if one can be identified who is a match in tissue typing. Unfortunately, only a minority of patients with sickle cell has such a donor available. The use of autologous hematopoietic stem cells and alternative types of genetic modifications is currently under study in clinical research trials for this disease. The approaches include the use of viral vectors to express globin genes that are modified to prevent sickle hemoglobin polymerization or to express interfering RNAs to “flip the switch” in adult red cells from adult β-sickle hemoglobin to fetal hemoglobin using a physiologic switch, and several gene editing approaches with the goal of inducing fetal hemoglobin or correcting/modifying the actual sickle mutation. In this audio review, we will discuss these different approaches and review the current progress of curative therapy for SCD using gene therapy.

Hemoglobinopathies

Hematology ◽  
2003 ◽  
Vol 2003 (1) ◽  
pp. 14-39 ◽  
Author(s):  
George F. Atweh ◽  
Joseph DeSimone ◽  
Yogen Saunthararajah ◽  
Hassana Fathallah ◽  
Rona S. Weinberg ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Globin Gene ◽  
Fetal Hemoglobin ◽  
Experimental Models ◽  
Adult Patients ◽  
Main Theme ◽  
Cell Disease ◽  
Hematopoietic Stem

Abstract The outlook for patients with sickle cell disease has improved steadily during the last two decades. In spite of these improvements, curative therapies are currently available only to a small minority of patients. The main theme of this chapter is to describe new therapeutic options that are at different stages of development that might result in further improvements in the outlook for patients with these disorders. Dr. Joseph DeSimone and his colleagues had previously made the important observation that the hypomethylating agent 5-azacytidine can reverse the switch from adult to fetal hemoglobin in adult baboons. Although similar activity was demonstrated in patients with sickle cell disease and β-thalassemia, concern about the toxicity of 5-azacytidine prevented its widespread use in these disorders. In Section I, Dr. DeSimone discusses the role of DNA methylation in globin gene regulation and describe recent clinical experience with decitabine (an analogue of 5-azacytidine) in patients with sickle cell disease. These encouraging studies demonstrate significant fetal hemoglobin inducing activity of decitabine in patients who fail to respond to hydroxyurea. In Section II, Dr. George Atweh continues the same theme by describing recent progress in the study of butyrate, another inducer of fetal hemoglobin, in patients with sickle cell disease and β-thalassemia. The main focus of his section is on the use of a combination of butyrate and hydroxyurea to achieve higher levels of fetal hemoglobin that might be necessary for complete amelioration of the clinical manifestations of these disorders. Dr. Atweh also describes novel laboratory studies that shed new light on the mechanisms of fetal hemoglobin induction by butyrate. In Section III, Dr. Ronald Nagel discusses the different available transgenic sickle mice as experimental models for human sickle cell disease. These experimental models have already had a significant impact on our understanding of the pathophysiology of sickle cell disease. Dr. Nagel describes more recent studies in which transgenic sickle mice provide the first proof of principle that globin gene transfer into hematopoietic stem cells inhibits in vivo sickling and ameliorates the severity of the disease. Although stroke in adult patients with sickle cell disease is not as common as in children, adult hematologists, like their pediatric colleagues, need to make management decisions in adult patients with a stroke or a history of stroke. Dr. Robert Adams has led several large clinical studies that investigated the role of transfusions in the prevention of stroke in children with sickle cell disease. Much less is known, however, about the prevention of first or subsequent strokes in adult patients with sickle cell disease. In Section IV, Dr. Adams provides some general guidelines for the management of adult patients with stroke while carefully distinguishing between recommendations that are evidence-based and those that are anecdotal in nature.

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