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

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

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 1000. Down-Regulating the Epithelial Glycoprotein-2 Promoter Utilizing Engineered Zinc Finger Protein Transcription Factors

2006 ◽  
Vol 13 ◽  
pp. S385
Author(s):  
Willemijn M. Gommans ◽  
Pamela M.J. McLaughlin ◽  
Robbert Cool ◽  
Beatrice I. Lindhout ◽  
Bert van der Zaal ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Finger Protein ◽  
Protein Transcription

scholarly journals Regulation of endogenous gene expression using small molecule-controlled engineered zinc-finger protein transcription factors

Gene Therapy ◽  
2007 ◽  
Vol 14 (18) ◽  
pp. 1362-1369 ◽  
Author(s):  
C L Dent ◽  
G Lau ◽  
E A Drake ◽  
A Yoon ◽  
C C Case ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factors ◽  
Zinc Finger ◽  
Small Molecule ◽  
Zinc Finger Protein ◽  
Endogenous Gene ◽  
Finger Protein ◽  
Protein Transcription

Engineering zinc finger protein transcription factors to downregulate the epithelial glycoprotein-2 promoter as a novel anti-cancer treatment

2007 ◽  
Vol 46 (5) ◽  
pp. 391-401 ◽  
Author(s):  
Willemijn M. Gommans ◽  
Pamela M.J. McLaughlin ◽  
Beatrice I. Lindhout ◽  
David J. Segal ◽  
D.J. Wiegman ◽  
...  
Keyword(s):  
Transcription Factors ◽  
Cancer Treatment ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
Finger Protein ◽  
Anti Cancer ◽  
Protein Transcription

Gene regulation in planta by plant-derived engineered zinc finger protein transcription factors

2005 ◽  
Vol 57 (3) ◽  
pp. 411-423 ◽  
Author(s):  
Rachel Holmes-Davis ◽  
Guofu Li ◽  
Andrew C. Jamieson ◽  
Edward J. Rebar ◽  
Qiang Liu ◽  
...  
Keyword(s):  
Gene Regulation ◽  
Transcription Factors ◽  
Zinc Finger ◽  
Zinc Finger Protein ◽  
In Planta ◽  
Finger Protein ◽  
Protein Transcription
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