Editing Combinations of Transcription Factor Binding Sites to Maximise Fetal Hemoglobin Production

Editing Combinations Of Transcription Factor Binding Sites To Maximise Fetal Hemoglobin Production

Natural mutations in the HBG1/2 promoters increase HbF production, by either creating new binding sites for activators or impairing the binding of transcriptional repressors. For example, a variant at -175 facilitates TAL1 activator binding, and mutations around -115 and -200 impair the binding of the repressors BCL11A and LRF respectively. We have also shown that editing at -123 and -124 increases HbF levels via creating a new site for the activator KLF1. While most studies target just one transcription factor binding site to enhance HbF production, we reasoned that introducing multiple changes might lead to higher HbF.

Towards this end, we cloned specific sgRNAs targeting all 4 clusters (-115, -123, -175, and -200) in Lentiviral vectors. Initially, HUDEP2 cells expressing ABE8e were transduced with each sgRNA (with GFP reporter) and expanded for 5 days. Subsequently, these individually transduced cells were transduced again with the same set of sgRNAs (RPF reporter) in all the combinations and expanded for another 5 days. Editing efficiency in each region ranged from 40-90%, in both single round and sequentially transduced cells. We observed unintended insertions and deletions (InDels), presumably due to the simultaneous binding of two sgRNA guides within the same promoter, at low levels. Most InDels occurred in the -200 region. Targeting the HBG1/2 locus can also cause concurrent nicking at both the HBG2 and HBG1 promoters, which can result in a deletion of ~4.9Kb that includes HBG2. Such deletions were observed most often in cells edited in the -115 and -123 and -124 region, regardless of the transduction sequence of the two sgRNAs. Interestingly, the -175 and -123and -124 combination did not induce as many unintended edits. To reduce the level of unintended gene modifications, we used a nickase-deficient ABE8e (dABE8e). dABE8e exhibited a slightly lower base editing efficiency across various targets but generated fewer large deletions and InDels in most editing combinations.

Analysis of HbF elevation revealed that among the individually targeted regions, the -175 TAL1 edit was most effective, followed by the -123 and -124 KLF1 binding site creation, and disruption of either of the repressor binding sites, in both ABE8e and dABE8e edited cells. The combined disruption of both repressor binding sites (-200 LRF and -115 BCL11A) resulted in higher HbF levels than targeting them individually. We also considered the impact of introducing an activator binding site and disrupting a repressor binding site. Targeting either the -200 LRF or the -115 BCL11A site, combined with the introduction of the -175 TAL1 binding site, resulted in higher HbF levels compared to the introduction of the KLF1 binding site.

Finally, creating two activator sites by targeting the -175 TAL1 and -123 and 124 KLF1 regions generated the highest levels of HbF, in both ABE8e and dABE8e expressing cells. This higher level of HbF production may result not only from activation via TAL1 and KLF1 but also possibly the binding of KLF1 to the -123 and -124 region reducing BCL11A binding to the -115 site via steric hindrance.

In summary, these findings show that combinatorial targeting of the key regulatory elements at the gamma-globin promoter is most effective at increasing HbF levels. Confirmation of these results in primary hematopoietic stem and progenitor cells, and assessment of the efficiency of editing in this context will inform the best strategies for using base editing in the treatment of severe β-thalassemia major and sickle cell disease.