Use of Base Editing Technology to Cure Sickle Cell Anemia (Article Sample)

A Glimmer for Sickle-Celled Patients? Stem cell editing is a prominent consideration in genetic disease correction, with Sickle cell disease (SCD) being the latest beneficiary. Newby et al (2021) discover the potential one-time treatment for SCD patients by an autologous ex vivo base editing approach and transplantation of the human HSPCs which result in haematological rescue and phenotypic correction. C onverting the HBBS allele to a benign variant, without introducing double-stranded DNA breaks (DSBs) to eliminate the root cause of SCD, could be the much-needed breakthrough to alleviate the lethal effects of the SCD mutation. Various approaches for autologous therapies have been utilized in the effort to treat Sickle cell disease (SCD) in clinical trials. SCD is a mutational disorder that, under low oxygen concentration, causes haemoglobin polymerisation within the red blood cells (RBCs), which results in characteristic sickle-shaped RBCs and haemolysis, inflammation, and microvascular constrictions. The symptoms include anaemia, severe pain, weakened immunity, multi-organ failure, and premature death [1]. What are the therapeutic approaches used in clinical trials? Haematopoietic stem cell (HSC) transplants are potential remedies for SCD yet the availability of optimally matched donors and the efforts involved can lead to graft rejection or graft-versus-host disease [2]. Numerous experiments have been focused on ex vivo modification of autologous HSCs towards SCD treatment, with those showing early clinical promise including ectopic expression of an anti-sickling β-like globin gene by lentiviral vectors [3] and induction of foetal haemoglobin (HbF) by suppression [4] or Cas9-mediated disruption [5] of BCL 11A. However, lentiviral vectors possess insertional mutagenesis risks and may not effectively suppress the expression of pathological βS. Also, genetic manipulation to induce the expression of HbF does not eliminate βS, and with the mediation of DSBs, carries the risks associated with uncontrolled mixtures of indels (insertions and deletions), translocations, loss of large chromosomal segments, chromothripsis, and p53 activation. Further, Cas9 nuclease-mediated homology-directed repair can correct HBBS but is difficult to efficiently repopulate HSCs, also requiring DSBs. -14643101563429Figure SEQ Figure \* ARABIC 1| The edited region of HBB with the target A at protospacer position 7 shown in blue along with potential bystander edits in green (silent), brown (silent), and red (non-silent).00Figure SEQ Figure \* ARABIC 1| The edited region of HBB with the target A at protospacer position 7 shown in blue along with potential bystander edits in green (silent), brown (silent), and red (non-silent).How does HBBS base editing work? The HBBS is targeted by base editing using a phage-assisted continuous evolution (PACE)- generated Cas9-NRCH3 which recognizes a CACC protospacer-adjacent motif (PAM; Fig. 1a). The study combined TadA-8e with CaS9-NRCH nickase to generate ABE8e-NRCH by plasmid lipofection achieving 58% conversion of HBBS to HBBG. The ex vivo procedure used is similar to that currently used for HSC editing in clinical trials, with the ABEs electroporated as mRNA or RNP to reduce the duration of exposure to the editing agent, hence minimizing off-target editing compared to DNA delivery. In the research, HSCs were also edited using single electroporation and transplanted into adult mice after 24 hours to reduce the duration of in-vitro culture and any associated loss of multipotency. For the assessment of the effects of editing on sickling, purified reticulocytes were incubated from ex vivo differentiation of unedited SCD CD34 cells in 2% oxygen. Editing reduced sickling frequency from 47.7% to 16.3%, proving that HBBS-to-HBBG conversion minimized sickling. The reticulocytes that were differentiated from cells treated with ABE8e-NRCH RNP showed similar results, but with lesser efficiencies. The experiment also used flow cytometry to track the expression of the cell-surface maturation markers CD49d, CD235a, and BAND3, finding no differences in the expression of the markers between edited and unedited cells. This led to the conclusion that based editing does not alter erythropoiesis. What happens when human HSPCs is transplanted into mice? The experiment further investigated whether the delivery of ABE8e-NRCH into SCD CD34 cells can convert HBBS to HBBG in HSCs used to repopulate the animal bone marrow by electroporation of ABE8e-NRCH and sgRNA in the RNA or RNP forms. It was discovered that the disruption of targeted genes through DSBs or deletion can alter the engraftment and maintenance of certain lineages. An assessment of the human haematopoietic lineages in recipient mouse bone marrow after transplantation revealed that the engraftment and differentiation potential of CD34 cells was unaltered by base editing. Human lineage-specific antibodies were also used to purify donor-derived mononuclear cells from mouse bone marrow. The results indicated that ABE8e-NRCH-mediated conversion of HBBS to HBBG in HSCs repopulation did not impede their engraftment or multipotency. How did transplanting mouse HSPCs into other mice fair? This process is somewhat difficult owing to the short lifespan of human RBCs circulation in mice [6]. For physiological phenotypic evaluation, the lineage-negative (Lin-) HSpCs from the Townes SCD mouse model was edited, with endogenous adult α- and β- like globin genes replaced by human globin genes hence SCD phenotypes [7]. The ABE8e-NRCH RNP was electroporated into HBBS/SHSpCs from Townes mice and then transplanted the cells into irradiated adult recipient mice 24hours later. 10 weeks after transplantation, the CD45.2 showed donor engraftment was above 90% in all mice. After measuring the effects of base editing on haemoglobin composition in circulating RBCs, there was no difference in oxygen binding in blood from mice that received unedited HBBS/S after transplantation, suggesting that βG- containing haemoglobin binds oxygen normally. What was done to rescue the SCD transplanted mice? After performing blood counts on all the patient groups of mice, there were disruptions in total haemoglobin concentration and cell counts of reticulocytes, RBCs, and white blood cells (WBCs) in mice that received unedited mouse HBBS/SHSPCs, abnormalities consistent with haemolytic anaemia and inflammations in patients with SCD [7]. The good news is that transplantation of base-edited HBBS/SHSPCs rescued the haematological defects in the patients, restoring all blood tested parameters to healthy level controls. Further secondary transplantations were done to confirm editing of long-term repopulating HSCs and determine the level of HBBS-to-HBBG base editing required to rescue SCD-associated haematological abnormalities. The mice that received at least 60% from recipients of edited HSCs maintained HBBG allele frequencies, demonstrating durable base editing of long-term repopulating mouse HSCs, sufficient enough to rescue haematological phenotypes. Therefore, it is imminent to claim the efficiency of base editing strategy, with 80% in editing HSPCs and 68% in bone marrow-repopulating HSCs, minimal bystander edits or indels, and yielding non-sickling RBCs without disruption of globin gene regulation or haematopoiesis. Why should the base editing approach be fancied? This approach favours the elimination of disease-causing mutation through precise HBBS-to-HBBG editing, reducing the concentration of sickle haemoglobin in RBCs more efficiently than the lentiviral expression of β-like globin or HbF induction (both of which leave HBBS alleles intact. Besides, base editing greatly avoids DSBs formed by nucleases, leading to uncontrolled mixtures of indels at the target site as well as large deletions, translocations, chromosomal loss, chromothripsis, and p53 DNA damage response activation. Treatment of HSPCs with ABE8e-NRCH did not cause a detected p53 response or large deletions, unlike the Ca...