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 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 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 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.

Effects of Sickle Cell Disease on Hematopoietic Stem Cell Function and the Bone Marrow Microenvironment.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 1227-1227
Author(s):  
Elisabeth H. Javazon ◽  
Leslie S. Kean ◽  
Jennifer Perry ◽  
Jessica Butler ◽  
David R. Archer
Keyword(s):  
Stem Cells ◽  
Bone Marrow ◽  
Stem Cell ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Cell Function ◽  
Donor Cell ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Cell Engraftment

Abstract Gene therapy and stem cell transplantation are attractive potential therapies for sickle cell disease (SCD). Previous studies have shown that the sickle environment is highly enriched for reactive oxygen species (ROS), but have not addressed whether or not the increased ROS may alter the bone marrow (BM) microenvironment or affect stem cell function. Using the Berkeley sickle mouse model, we examined the effects of sickle cell disease on hematopoietic stem cell function and the bone marrow microenvironment. We transplanted C57BL/6 (control) BM into C57BL/6 and homozygous sickle mice. Recipients received 2 × 106 BM cells and a conditioning regimen consisting of busulfan, anti-asialo GM1, and co-stimulation blockade (anti-CD40L and CTLA4-Ig). Following transplantation, sickle mice demonstrated increased donor cell engraftment in the peripheral blood compared to normal mice (58.3% vs. 33.1%, respectively). Similarly, BMT in a fully allogeneic system also resulted in enhanced engraftment in sickle recipients. Next we analyzed whether or not engraftment defects exist within the BM stem cell population of sickle mice. In vitro colony forming assays showed a significant decrease in progenitor colony formation in sickle compared to control BM. By flow cytometry, we determined that there was a significant decrease in the KSL (c-Kit+, Sca-1+, Lineage−) progenitor population within the BM of sickle mice. Cell cycle analysis of the KSL population demonstrated that significantly fewer sickle KSL cells were in G0 phase compared to control, suggesting that there are fewer quiescent stem cells in the BM of sickle mice. To assess the potential role of ROS and glutathione depletion in sickle mice, we tested the engraftment efficiency of KSL cells from untreated and n-acetyl-cysteine (NAC) treated control, hemizygous sickle (hemi), and sickle mice in a competitive repopulation experiment. Peripheral chimerism showed an engraftment defect from both hemizygous and homozygous sickle mice such that control KSL cells engrafted > hemi > sickle at a ratio of 1 : 0.4 : 0.25. Treatment with NAC for four months prior to transplantation partially restored KSL engraftment (control : hemi : sickle; 1 : 0.97 : 0.56 ). We have demonstrated that congenic and allogeneic BMT into sickle mice result in increased donor cell engraftment in the sickle recipients. Both the decreased number of KSL cells and the decreased percentage of quiescent KSL cells in the sickle mice indicate that more stem cells in the transgenic sickle mouse model are mobilized from the BM environment. The engraftment defect of sickle KSL cells that was partially ameliorated by NAC treatment suggests that an altered redox environment in sickle mice may contribute to the engraftment deficiencies that we observed.

Relationship of Targeted Busulfan Exposure to Engraftment in Children Undergoing Myeloablative Matched Sibling Donor Transplantation for Sickle Cell Disease.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2152-2152
Author(s):  
Marianne E. McPherson ◽  
Don Hutcherson ◽  
Ellen Olson ◽  
Ann Haight ◽  
John Horan ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Significant Risk ◽  
Systemic Exposure ◽  
Therapeutic Drug ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Donor Chimerism ◽  
Increased Risk

Abstract BACKGROUND: Allogeneic hematopoietic stem cell transplantation (HSCT) for sickle cell disease (SCD) provides curative therapy but carries significant risk of transplant-related morbidity and mortality, graft failure, and disease recurrence. Past trials of myeloablative HSCT with busulfan in SCD patients showed a 10–13% rate of rejection and 8–9% rate of partial chimerism with busulfan steady-state concentration (Css) targeted to 400–600 ng/ml (area under curve (AUC) 575–877 mmol•min/L). In thalassemia and other diseases, total systemic exposure to busulfan is a critical determinant of engraftment yet is limited by risk of toxicity. Therapeutic drug monitoring (TDM) is used to measure variability in individual patient pharmacokinetics and allows dose adjustments to achieve desired level of busulfan exposure. A retrospective analysis of busulfan AUC in SCD patients during HSCT was performed to determine if higher systemic busulfan levels resulted in more effective engraftment or increased toxicities. METHODS: A retrospective review of busulfan pharmacokinetics, engraftment status, and clinical toxicities in a cohort of SCD patients who received MSD bone marrow grafts at a single pediatric institution was performed. All patients received a myeloablative preparative regimen with busulfan (initially dosed for total 14 mg/kg), cyclophosphamide (200 mg/kg), antithymocyte globulin (90 mg/kg), and graft versus host disease prophylaxis with methotrexate and cyclosporine. Anticonvulsant prophylaxis with phenytoin or with levitiracetam plus lorazepam was given during busulfan administration. Busulfan was administered every 6 hours for 16 doses. Busulfan AUC was determined for the first dose and adjustments were made on subsequent doses if needed. Total AUC was calculated as a weighted average of total doses. Target AUC varied widely over the study period; however, for the last 3 years, the intended AUC has been targeted to 900–1100 mmol•min/L (Css 618–753 ng/ ml). Busulfan-associated toxicities recorded were: hepatic veno-occlusive disease (VOD), interstitial pneumonitis (IP), and seizure during or 1 day following busulfan administration. RESULTS: Twenty-seven consecutive hemoglobin SS SCD patients received HSCT with MSD between December 1993 and August 2007. All patients transplanted since May 1996 (n=25) had busulfan TDM performed. Median age was 8.8 years (range 3.3–17.4 years). Event-free survival was 96% with a median follow-up time of 3.9 years. Engraftment occurred in all cases with no subsequent graft rejection. Full donor chimerism (>95% donor leukocytes) was seen in 21 (84%) patients, and high partial donor chimerism (50–95% donor) in 4 (16%) at >12 months post-HSCT. Busulfan was administered orally in 18 (72%) and intravenously in 7 (28%). Dose adjustments were made in 17 patients (8 increased, 9 decreased). Median dose increase was 16.3% (range 9.7–76.5%), and median decrease was 14.3% (range 4.2–57.1%). Five (20%) patients had dose alterations >20% of initial dose (2 increased, 3 decreased). Median total dose received was 13.9 mg/kg (range 10.8–24.0). Median AUC for first dose was 968 mmol•min/L (range 595–1379) and median total AUC was 992 mmol•min/L (range 780–1305). Busulfan-associated toxicities were observed in 9 (36%) patients: 8 with mild-moderate VOD, 1 with IP. No seizures occurred during busulfan administration. Patients with busulfan-associated toxicity had mean total AUC of 1044.7 mmol•min/L (range 821–1305) versus 962.4 mmol•min/L (range 780–1184) in those without toxicity (p=0.185). Patients with partial donor chimerism had lower total AUC (mean 861.5 mmol•min/L, range 780–932) compared to those with full donor chimerism (mean 1017 mmol•min/L, range 885–1305; p=0.0379) (Figure). Since the implementation of a target busulfan AUC range of 900–1100 mmol•min/L, all patients (n=10) achieved full donor chimerism. CONCLUSIONS: We conclude that higher busulfan levels are associated with decreased incidence of partial donor chimerism in SCD patients during myeloablative HSCT with MSD bone marrow grafts. A target AUC range of 900–1100 mmol•min/L, attainable with TDM and dose adjustment, provides effective engraftment without increased risk of busulfan-related toxicity. Figure: Total busulfan AUC in patients with full donor chimerism (mean 1017 mmol•min/L) vs. partial chimerism (mean 861.5 mmol•min/L, p=0.0379). Figure:. Total busulfan AUC in patients with full donor chimerism (mean 1017 mmol•min/L) vs. partial chimerism (mean 861.5 mmol•min/L, p=0.0379).

Hematopoietic Stem Cell Transplantation for Sickle Cell Disease in Childhood: A Single Center Experience with 45 Patients

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 3103-3103 ◽  
Author(s):  
Laurence Dedeken ◽  
Phu-Quoc Le ◽  
Nadira Azzi ◽  
Cecile Brachet ◽  
Catherine Heijmans ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Stem Cell ◽  
Sickle Cell Disease ◽  
Hematopoietic Stem Cell Transplantation ◽  
Stem Cell Transplantation ◽  
Cord Blood ◽  
Sickle Cell ◽  
Cell Disease ◽  
Hematopoietic Stem

Abstract Abstract 3103 Despite improvement in medical management, sickle cell disease (SCD) is still associated with high risk of morbidity, chronic disability and early death. Allogeneic hematopoietic stem cell transplantation (HSCT) remains the only curative approach. Since November 1988, 45 patients (median age: 8.3 years; range: 1.7–15.3 years) with severe SCD underwent related HSCT in our unit. Thirty-five received bone marrow transplant, 3 cord blood, 6 bone marrow and cord blood and 1 peripheral blood stem cells. Two donors result from preimplantation genetic diagnosis with HLA selection. All were HLA-identical sibling except one who had one class II mismatch. All had one or more severe manifestations: 24 patients presented more than 2 vaso-occlusive crises per year, 11 recurrent acute chest syndrome, 19 cerebral vasculopathy and 4 erythroid alloimmunisation. Conditioning regimen consisted of the standard combination of busulfan, cyclophosphamide and from November 1991 antithymocyte globulins (ATG) were added: ATG Fresenius first and from July 2000 ATG Merieux. Since 1995 all patients were treated with hydroxyurea (HU) prior to transplantation for a median duration of 2.7 years (range: 0.8–10.7 years). Acute graft versus host disease (GVHD) was observed in 11 patients (3 grade III and 2 grade IV). Ten patients were treated for CMV reactivation and 4 for EBV reactivation. Only one patient had presented a probable invasive fungal disease. After median follow-up of 6.5 years, 10 patients had presented chronic GVHD, none was extensive. Only one required therapy beyond 2 years from transplant. Engraftment was successful in 42/45. One rejection occurred 15 months after transplantation. Since HU introduction before transplant (1995), no graft failure occurred. Important mixed chimerism is present in 2 patients (AA donor) who remain free of any sickle cell disease symptoms. Two deaths occurred: 1 unexplained death 6 years after HSCT in a child free of any treatment and 1 cerebral hemorrhage 18 days after transplant in a child with severe cerebral vasculopathy. Growth was normal after transplant. As expected, gonadal function was impaired in the majority of girls. However 3 girls had spontaneous normal puberty and one had two spontaneous pregnancies with normal outcome. Our results are very encouraging showing excellent outcomes. Both the overall survival (OS: 95.6%) and the event-free survival (EFS: 86.7%) are comparable to the other published studies, ranging from 93 to 97%, and 82 to 86 % respectively. Since 1995, all the 33 patients engrafted successfully. Previous treatment with HU may have contributed to successful engraftment. After 5.3 years of follow-up, their OS and EFS are both at 96.9%. The difference in outcome before and after 1995 is strongly significant for EFS (58.3% vs 96.9%, p=0.003). Severe cerebral vasculopathy with its risk of CNS hemorrhage remains a true challenge. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Bone Marrow as a Hematopoietic Stem Cell Source for Gene Therapy in Sickle Cell Disease: Evidence from Rhesus and SCD Patients

2017 ◽  
Vol 28 (3) ◽  
pp. 136-144 ◽  
Author(s):  
Naoya Uchida ◽  
Atsushi Fujita ◽  
Matthew M. Hsieh ◽  
Aylin C. Bonifacino ◽  
Allen E. Krouse ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Bone Marrow ◽  
Stem Cell ◽  
Sickle Cell Disease ◽  
Hematopoietic Stem Cell ◽  
Sickle Cell ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Stem Cell Source ◽  
Cell Source

scholarly journals Novel Lentiviral Vectors for Gene Therapy of Sickle Cell Disease Combining Gene Addition and Gene Silencing Strategies

Blood ◽  
2021 ◽  
Vol 138 (Supplement 1) ◽  
pp. 3973-3973
Author(s):  
Megane Brusson ◽  
Anne Chalumeau ◽  
Pierre Martinucci ◽  
Valentina Poletti ◽  
Fulvio Mavilio ◽  
...  
Keyword(s):  
Sickle Cell Disease ◽  
Gene Silencing ◽  
Sickle Cell ◽  
Globin Gene ◽  
Insertion Site ◽  
Lentiviral Vectors ◽  
Erythroid Cell ◽  
Erythroid Cells ◽  
Cell Disease ◽  
Hematopoietic Stem

Abstract Sickle cell disease (SCD) is due to a mutation in the β-globin (HBB) gene causing the production of the sickle β S-globin chain. The sickle Hb (HbS, a 2β S2) polymerizes, leading to the formation of sickle-shaped red blood cells that cause vaso-occlusions and organ damage. Transplantation of autologous hematopoietic stem/progenitor cells (HSPCs) transduced with lentiviral vectors (LV) expressing an anti-sickling β-globin transgene (βAS LV) is a promising curative treatment; however, it is partially effective in SCD patients, who still present elevated HbS levels. Here, we aim to improve LVs to boost therapeutic β-like globin levels without increasing the mutagenic vector load in HSPCs. We developed 2 novel LVs expressing βAS together with an artificial microRNA (amiR) targeting either the fetal Hb (HbF) repressor BCL11A (βAS/amiRBCL11A) or the β S-globin (βAS/amiRHBB). By downregulating BCL11A, amiRBCL11A re-activates the expression of the endogenous anti-sickling fetal γ-globin, which, together with βAS, should improve the clinical course of SCD; β S-globin downregulation should favor βAS incorporation in Hb tetramers, increase therapeutic Hb levels and ameliorate the SCD phenotype. First, we developed βAS/amiRBCL11A LV by inserting the amiR in multiple position of the βAS intron 2 under the control of HBB promoter/enhancers to limit BCL11A downregulation to the erythroid lineage and reduce potential amiR-related cellular toxicity and off-target effects. We showed that amiR insertion site did not affect LV titer nor βAS expression in a human erythroid cell line (HUDEP2). BCL11A downregulation in HUDEP2 led to γ-globin gene de-repression and a high proportion of HbF + cells (RTqPCR, HPLC, flow cytometry). Importantly, the total amount of therapeutic β-like globins was substantially higher in βAS/amiRBCL11A LV- than in βAS LV-transduced cells, with no impairment in cell viability or erythroid differentiation. In parallel, we designed 17 amiRs targeting HBB and generated the corresponding βAS/amiRHBB LVs. We tested these LVs in HUDEP2 and selected 2 amiRs efficiently downregulating β-globin at mRNA and protein levels (RT-qPCR and Western Blot). Of note, we modified the βAS transgene by inserting silent mutations that prevent its recognition by the amiR (βASm). Finally, we tested βAS/amiRBCL11A and βAS/amiRHBB LVs in HSPCs from SCD patients. HSPC-derived erythroid cells transduced with βAS/amiRBCL11A LV showed increased HbF levels, although HbS levels remained high. To further reduce β S-globin levels, we targeted the β S-globin mRNA using the βAS/amiRHBB LV. Efficient HSPC transduction by βASm/amiRHBB LV led to a substantial decrease of β S-globin transcripts in HSPC-derived erythroid cells compared to the βAS LV-transduced cells (RTqPCR) at a VCN/cell of 2. Notably, the amiR specifically down-regulated β S-globin, without affecting βAS expression. In βASm/amiRHBB- vs βAS LV-transduced cells, HPLC analysis showed that β S-globin downregulation led to a significant decrease of HbS, which represented 58% and 71% of the total Hb, respectively). This was associated with a significant increase of the therapeutic Hb in βASm/amiRHBB LV- vs βAS LV-transduced erythroid cells (38% and 27% of the total Hb, respectively). Importantly, we observed a substantial reduction of the proportion of HbS-positive cells in βASm/amiRHBB- vs βAS LV-transduced samples (from 96% to 70%; Figure 1A). The increased incorporation of βAS in Hb tetramers and the decrease in β S-globin led to a better correction of the sickling phenotype in mature RBCs derived from HSPCs transduced with βASm/amiRHBB LV- compared to βAS LV (55% and 84% of sickling cells, respectively; Figure 1B). A clonal assay of hematopoietic progenitors showed no impairment in HSPC viability and differentiation towards the erythroid and myeloid lineages upon transduction with bifunctional LVs. βASm/amiRHBB LV showed a standard lentiviral integration profile. Finally, we performed RNAseq to further evaluate the safety of our therapeutic strategy. In conclusion, we created a LV able to concomitantly silence the β S-globin and express βAS, achieving clinically relevant levels of therapeutic Hb and efficient correction of the sickling phenotype. Therefore, the combination of gene addition and gene silencing strategies can improve the efficacy of current therapeutic approaches, representing a novel treatment for SCD. Figure 1 Figure 1. Disclosures Cavazzana: Smart Immune: Other: co-founder.

Murine bone marrow mesenchymal stromal cells have reduced hematopoietic maintenance ability in sickle cell disease

Blood ◽  
2021 ◽  
Author(s):  
Alice Tang ◽  
Ana Nicolle Strat ◽  
Mahmudur Rahman ◽  
Helen Zhang ◽  
Weili Bao ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Bone Marrow ◽  
Stem Cell ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Mesenchymal Stromal Cells ◽  
Stromal Cells ◽  
Tissue Injury ◽  
Cell Disease ◽  
Hematopoietic Stem

Sickle Cell Disease (SCD) is characterized by hemolytic anemia, which can trigger oxidative stress, inflammation, and tissue injury that contributes to disease complications. Bone marrow mesenchymal stromal cells (MSCs) tightly regulate hematopoietic stem cell (HSC) homeostasis in health and disease but their functionality in SCD remains unclear. We identified for the first time murine SCD MSCs to have altered gene signatures, reduced stem cell properties, and increased oxidative stress, due in part to hemolysis. Murine SCD MSCs had lower HSC maintenance ability in vitro and in vivo as manifested by increased HSC mobilization and decreased HSC engraftment following transplant. Activation of TLR4 through p65 in MSCs further contributed to MSC dysfunction. Transfusions led to improved MSC and HSC oxidative state in SCD mice. Improving the regulation between MSCs and HSCs has vital implications for enhancing clinical HSC transplantation and gene therapy outcomes and for identification of new molecular targets for alleviating SCD complications.

scholarly journals Common Myeloid Progenitors As Biomarkers of Hbf Response to Hydroxyurea in Sickle Cell Disease

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4827-4827
Author(s):  
Caterina P. Minniti ◽  
Seda S. Tolu ◽  
Kai Wang ◽  
Zi Yan ◽  
Andrew Crouch ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Sickle Cell Disease ◽  
Sickle Cell ◽  
Research Funding ◽  
Inverse Correlation ◽  
Healthy Controls ◽  
Healthy Individuals ◽  
Cell Disease ◽  
Hematopoietic Stem ◽  
Cd34 Cells

Background Hydroxyurea (HU) is used to treat sickle cell disease (SCD) in part because of its ability to increase hemoglobin F (HbF) concentration, but the mechanism by which HU induces HbF, and the low or lack of HbF response in a fraction of the patient remains unclear. HU causes myelo-suppression and induces stress hematopoiesis, which is associated with increase production of HbF. Earlier research has shown that HbF levels in SCD patients are inversely correlated with reticulocytes, which can be secondary to: 1) HbF-induced decreased hemolysis with less needs for red blood cell (RBC) production, and 2) Myelo-suppression. It has also been shown that the number of CD34+ cells is generally lower in HU treated patients, but the overall response of the hematopoietic system in relationship to HbF has not been characterized. Here, we prospectively isolated hematopoietic stem and progenitor cells (HSPCs) from HU-treated SCD patients and characterize their hematopoietic and HbF responses. Methods Peripheral blood (PB) was collected from 19 HbSS who had been HU for >3 years and from 12 healthy controls. Frozen mono-nuclear cells were analyzed by flow cytometry using CD49f, 90 45Ra, 123, 235a, 38, 34, 33 and lineage antibodies. The number of 49f+ long-term Hematopoietic Stem Cells (LT-HSC), Multipotent Progenitors (MPPs), Common Myeloid Progenitors (CMPs), Megakaryocyte-Erythroid Progenitors (MEPs), and Granulocyte-Monocyte Progenitors (GMPs) per uL of blood or per CD34+ cells was then quantified. Results The percentages of reticulocytes per uL of blood were found to correlate positively with the concentration per uL of blood of all stem and progenitor cell populations tested (CD34 (R2 = 0.6583), LT-HSC (R2 = 0.3532), MPP (R2 = 0.2603), CMP (R2 = 0.5889), MEP (R2 = 0.2411), and GMP (R2 = 0.6911)). Statistically significant (p<0.05) inverse correlations were also observed between HbF levels and the number of CD34+/uL ( R2 = 0.2931), and the number of CMP/uL (R2 = 0.3732). Normalization of the data to the number of circulating CD34+ cells revealed that there was a strong inverse correlation between HbF levels and the percentage of circulating CMPs (R2 = 0.5424), and importantly, that this correlation was specific to the CMP population since Hb F did not correlates with any of the other HSPC populations analyzed . Analysis of the PB of 12 healthy individuals revealed that, as in SCD patients, the percentage of CMPs varied between < 10% and >60% of the total circulating CD34+ cells. Further analysis revealed that the CMP percentages in both SCD and healthy controls appeared characteristics of each individual tested since the measurements were remarkably correlated (R2 >0.7) when they were repeated on blood samples collected at intervals of two-weeks or one-year. Discussion The positive correlation between the reticulocyte and HSPC populations that we observed was previously unreported and suggests that, in first approximation, the reticulocytes could serve as a proxy for the levels of circulating HSPCs which could help assess the degree of bone marrow suppression in compliant non-responding HU-treated patients. We identified an inverse correlation between percentages of HbF and circulating CMPs in HU-treated SCD patients that is specific to these progenitors. The specificity of the correlation suggests that the major mechanism for the correlation is unlikely to be differential mobilization of CMPs to the PB since inducing mobilization generally affect all HSPCs. A depletion of the CMPs in the bone marrow of high Hb F responders is therefore a more likely mechanism. A possible mechanism for the correlation is that HU acts directly on CMPs by accelerating their differentiation leading to the relative depletion of these progenitors in high F individuals, and initiating the reprogramming of gene expression that ultimately results in high level of gamma-globin expression. Alternatively, the similar range of variability in the percentage of CMPs in HU-treated SCD patients and in healthy individuals never exposed to HU, and the observation that the percentage of circulating CMP seem to be an intrinsic characteristic of each individual suggest that the percentage of circulating CMPs might be a genetically determined marker associated with the ability to produce HbF in response to HU therapy, rather than being a consequence of the treatment. Figure Disclosures Minniti: Doris Duke Foundation: Research Funding. Manwani:Novartis: Consultancy; Pfizer: Consultancy; GBT: Consultancy, Research Funding.

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