Zinc finger nucleases for targeted mutagenesis and repair of the sickle-cell disease mutation: An in-silico study

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.

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In silico modeling of malaria and sickle-cell disease

Drug Discovery Today Disease Models ◽

10.1016/j.ddmod.2015.05.001 ◽

2015 ◽

Vol 16 ◽

pp. 17-22 ◽

Cited By ~ 2

Author(s):

Dmitry A. Fedosov

Keyword(s):

Sickle Cell Disease ◽

Sickle Cell ◽

In Silico ◽

Cell Disease ◽

In Silico Modeling

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901. Transactivation of Fetal Hemoglobin Genes by Engineered Zinc-Finger Protein Transcription Factors for the Treatment of Sickle Cell Disease

Molecular Therapy ◽

10.1016/j.ymthe.2004.06.807 ◽

2004 ◽

Vol 9 ◽

pp. S344

Keyword(s):

Transcription Factors ◽

Sickle Cell Disease ◽

Zinc Finger ◽

Sickle Cell ◽

Zinc Finger Protein ◽

Fetal Hemoglobin ◽

Cell Disease ◽

Finger Protein ◽

Protein Transcription

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In-Silico structural analysis of wild-type human hemoglobin and its mutation resulting in sickle cell anemia

Asian Journal of Medical Sciences ◽

10.3126/ajms.v11i4.28486 ◽

2020 ◽

Vol 11 (4) ◽

pp. 1-8

Author(s):

Soumitra Nath ◽

Nirmal Paul ◽

Manabesh Nath ◽

Bibhas Deb

Keyword(s):

Sickle Cell Disease ◽

Point Mutation ◽

Sickle Cell ◽

Sickle Cell Anemia ◽

In Silico ◽

Gene Networks ◽

Wild Type ◽

Cell Disease ◽

Globin Chains ◽

Sequence Of Events

Background: Hemoglobin (HbA) is a metalloprotein having a heme prosthetic group, two α-globin chains, and two β-globin chains. A point mutation in the 6th position of wild-type HBB gene (GAG>GTG) substitutes Glutamic acid (E) to Valine (V) in the β-chains of hemoglobin molecule (HbS) resulting in Sickle Cell Anemia. Structural deformities in the β-globin gene leads to disruptive conformation of red blood cells, disturbs the oxygen transportation and causes functional abnormalities. Aims and Objective: The purpose of the study was to understand the structural, genetic and metabolic effect of the point mutation on HBB gene that causes sickle cell disease. Materials and Methods: The 3D structures of HbA and HbS proteins were retrieved from PDB and the changes in their physical properties were analyzed using Swiss PDB viewer and Molegro molecule viewer. Gene networks were constructed using GeneMANIA server to study genetic and metabolic interactions of the HBB gene. Results: In-silico studies showed that the normal bond length between Glu6-Glu7 in HbA molecule is 1.32Å and that of HbS is 1.33Å. After comparing the two proteins, it was observed that sickle cell hemoglobin suffers a change in bond angle from 122.4º in HbA to 119.35º in HbS. Comparative energy minimization of Glu6 and Val6 in wild-type and mutant hemoglobin respectively yielded 8.78 and -9.083 KJ/mol net energy values, suggesting a more reactive HbA and less reactive HbS. Gene networks were determined on the basis of physical, genetic and co-expressive interactions of HBB gene which revealed a strong connection between HBB and HBA1 genes within the association constituted of various metabolic functions. Conclusion: Sickle cell disease results from a sequence of events which start with a single nucleotide substitution that ultimately leads to severe anemia and other cardiovascular problems. Incorporation of computational exercises correlates to a better probability of discovering precision medicine through target site-specific drug designing.

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Seamless correction of the sickle cell disease mutation of theHBBgene in human induced pluripotent stem cells using TALENs

Biotechnology and Bioengineering ◽

10.1002/bit.25018 ◽

2013 ◽

Vol 111 (5) ◽

pp. 1048-1053 ◽

Cited By ~ 84

Author(s):

Ning Sun ◽

Huimin Zhao

Keyword(s):

Stem Cells ◽

Sickle Cell Disease ◽

Induced Pluripotent Stem Cells ◽

Sickle Cell ◽

Pluripotent Stem Cells ◽

Cell Disease ◽

Disease Mutation ◽

Induced Pluripotent

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Preliminary Safety and Efficacy Results from Precizn-1: An Ongoing Phase 1/2 Study on Zinc Finger Nuclease-Modified Autologous CD34+ HSPCs for Sickle Cell Disease (SCD)

Blood ◽

10.1182/blood-2021-151650 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2930-2930

Author(s):

Asif Alavi ◽

Lakshmanan Krishnamurti ◽

Mehrdad Abedi ◽

Isobelle Galeon ◽

David Reiner ◽

...

Keyword(s):

Sickle Cell Disease ◽

Zinc Finger ◽

Sickle Cell ◽

Ex Vivo ◽

Globin Gene ◽

Cell Disease ◽

Platelet Recovery ◽

Clin Neurol ◽

F Cells

Abstract Introduction Sickle cell disease (SCD) is caused by pathologic variants in both alleles of the β-globin gene, affecting ~100,000 patients in the US (Strouse. Handb Clin Neurol 2016;138:311-24). Elevated fetal hemoglobin (HbF) levels ameliorate symptoms and improve survival in patients with SCD (Hebert. Am J Hematol 2020;95:1235-45). SAR445136 (BIVV003) is a novel therapeutic product comprising autologous CD34+ HSPCs modified ex vivo by zinc finger nucleases (ZFN) and targeting the BCL11A gene erythroid-specific enhancer (ESE) to increase endogenous HbF production. Methods PRECIZN-1 (NCT03653247) is an ongoing first-in-human, open label, single arm, multi-site study evaluating safety and tolerability of SAR445136 (n=8; aged 18-40 years), with severe SCD across 6 US sites. Eligible subjects underwent mobilization and apheresis with plerixafor 240 ug/kg/day for up to 3 days to collect autologous CD34+ HSPCs to achieve a minimum of 10 × 10 6 CD34+ HSPC/kg for manufacturing of the SAR445136 dose. Additional apheresis cycles were allowed to achieve the minimum cell dose and rescue aliquots. Autologous HSPCs were transfected ex vivo with ZFN mRNAs targeting the ESE region of the BCL11A locus to manufacture SAR445136. A single IV infusion of 3-20 × 10 6 CD34+ HSPC/kg was administered at least 72 hours after the final busulfan myeloablation dose. Subjects were monitored for stem cell engraftment and hematopoietic recovery, adverse events (AEs), clinical and laboratory hemolysis markers, total Hb and HbF, percentage of F cells and sickle-cell related events post-SAR445136 infusion. Health-related quality of life (HRQoL) was assessed via the PROMIS-57 survey at screening, Weeks 26 and 52, and early termination visit. Results Of the 7 subjects that underwent mobilization and apheresis to date (25 June 2021), 5 achieved successful target yields ranging from 3.4-13.8 x 10 6 CD34+ HSPC/kg per apheresis day (mean: 6.49 x10 6 CD34+ HSPC/kg per apheresis day) in one apheresis cycle (4.45-10.9 x 10 6 CD34+ HSPC/kg per 2-day cycle). One subject failed to mobilize; one discontinued due to intercurrent cholangitis. Baseline patient characteristics of the 4 patients infused are in Table 1. Pre-apheresis peripheral blood WBC ranged from 23.2-36.9 x 10 3/μL (mean: 28.7 x 10 3/μL) and % CD34+ was 0.09-0.36% (0.22%) with absolute CD34+ counts of 20-80/μL (mean: 60/μL). Four of the mobilized subjects were successfully infused with SAR445136 at a single dose ranging from 3.2-9.7 x 10 6 CD34+ HSPC/kg (mean: 5.17 x 10 6 CD34+ HSPC/kg). All 4 subjects engrafted with a median time to neutrophil and platelet recovery of 21.5 and 24.5 days, respectively. No rescue doses were required. All 4 patients improved clinically since SAR445136 infusion, with no recurrence of previous vaso-occlusive crises (VOCs). Total Hb stabilized at 9-10 g/dL by week 26 post SAR445136 infusion along with improvements in the clinical markers of hemolysis in all 4 subjects. Percent HbF levels were 1-11% at screening, increasing to 15-29% by Week 13 in all 4 subjects, to 14-39% by Week 26 in the 3 subjects with at least 26 weeks follow up; and persisting at 35% in 1 subject with 65 weeks follow up (Figure 1). Percent F cells increased to 49-94% in 3 subjects with at least 26 weeks follow up, persisting at 90% in 1 subject with 65 weeks follow up. The fourth subject had 87.5% F cells at 13 weeks follow up. Although preliminary, a trend of improvement exceeding the proposed minimally clinically important difference in all PROMIS-57 HRQoL domains except sleep disturbance was observed in the 3 subjects with 26 weeks follow up, whose scores were "worse" than the norm at baseline (≤2 points per domain). SAR445136 was generally well tolerated with no infusion related reactions. The AEs reported were consistent with plerixafor mobilization and busulfan myeloablation therapy. No AEs or SAEs were reported as related to SAR445136. Conclusions As of 25 June 2021, these preliminary proof-of-concept efficacy and safety results confirm the potential therapeutic value of ZFN-mediated modification of the BCL11A ESE region and SAR445136 infusion to address current unmet needs of patients with SCD. All 4 infused patients had no SCD related events including VOCs following SAR445136 infusion, as well as increases in total Hb, HbF, and %F cells, and clinical improvements in PROMIS-57 domains. SAR445136 is generally well tolerated in the 4 subjects infused to date, with no related AEs or SAEs reported. Figure 1 Figure 1. Disclosures Abedi: BMS/Celgene: Membership on an entity's Board of Directors or advisory committees, Speakers Bureau; Seattle Genetics: Speakers Bureau; Abbvie: Speakers Bureau. Galeon: Sanofi: Current Employment. Reiner: Sanofi: Current Employment. Smith: Sanofi: Current Employment. Wang: Sanofi: Current Employment. Ramezi: Sanofi: Current Employment. Rendo: Sanofi: Current Employment, Other: May hold shares and/or stock options . Walters: AllCells, Inc: Consultancy; Vertex pharmaceuticals: Consultancy; Ensoma, Inc.: Consultancy; BioLabs, Inc: Consultancy.

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