Biallelic correction of sickle cell disease-derived iPSCs confirmed at the protein level through serum-free iPS-sac/erythroid differentiation.

Abstract New technologies of induced pluripotent stem cells (iPSCs) and genome editing have emerged, allowing for the development of autologous transfusion therapies. We previously demonstrated definitive β‐globin production from human embryonic stem cell (hESC)‐derived erythroid cell generation via hemangioblast‐like ES‐sacs. In this study, we demonstrated normal β‐globin protein production from biallelic corrected sickle cell disease (SCD) iPSCs. We optimized our ES/iPS‐sac method for feeder cell‐free hESC maintenance followed by serum‐free ES‐sac generation, which is preferred for electroporation‐based genome editing. Surprisingly, the optimized protocol improved yields of ES‐sacs (25.9‐fold), hematopoietic‐like spherical cells (14.8‐fold), and erythroid cells (5.8‐fold), compared with our standard ES‐sac generation. We performed viral vector‐free gene correction in SCD iPSCs, resulting in one clone with monoallelic and one clone with biallelic correction, and using this serum‐free iPS‐sac culture, corrected iPSC‐generated erythroid cells with normal β‐globin, confirmed at DNA and protein levels. Our serum‐free ES/iPS‐sac protocol with gene correction will be useful to develop regenerative transfusion therapies for SCD.


| INTRODUCTION
The development of human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs) 1 has spurred rapid progress in the fields of stem cell biology and regenerative medicine. ES/iPSC technology is a powerful tool for disease modeling and drug discovery, 2 improving speed, reliability, and specificity in the development of new therapies. 3 ES/iPSCs could potentially serve as an alternative source of (a) red blood cells (RBCs) and platelets for transfusion, and (b) hematopoietic stem/progenitor cells (HSPCs) for transplantation. RBC transfusion from healthy donors is well established to treat acute and chronic anemia, but it carries the risks of immune-based hemolysis, infectious disease transmission, and rarely graft-vs-host disease, in addition to issues concerning donor screening and blood collection. 4 An in vitro culture system for RBC generation from CD34+ HSPCs and ES/iPSCs is being developed for an alternative RBC source. [5][6][7][8] Primary human CD34+ cells can be efficiently differentiated into erythroid cells in vitro, 5 but CD34+ HSPCs must be collected prior to each differentiation culture since they lose self-renewal abilities after differentiation. In contrast, ES/iPSCs can undergo large-scale expansion followed by erythroid cell differentiation, making them a better candidate for an alternative RBC source. [6][7][8] Autologous RBC transfusion could minimize the risk of alloimmunization by using erythroid cells differentiated from patient-generated iPSCs that have been genetically modified. Therefore, transfusion of RBCs generated from autologous, gene-corrected iPSCs would be an ideal therapeutic option for hereditary anemia, including sickle cell disease (SCD).
SCD is caused by a point mutation in the β-globin gene (HBB: c.20A>T), which induces hemoglobin polymerization in hypoxic conditions, causing RBCs to become deformed and lead to symptoms such as hemolytic anemia, vaso-occlusion, pain crisis, multiorgan damage, and early mortality. 9 Since SCD is the most common single gene disorder, it is an attractive target for gene therapy approaches, such as gene correction. In the last decade, genome editing tools such as zinc finger nucleases (ZFNs) have been developed that allow for sitespecific DNA breakage. 10 Correction of the SCD mutation has been reported in SCD iPSCs using ZFNs, 11,12 demonstrating normal β-globin production at RNA levels when differentiated in vitro into erythroid cells 11 and teratoma formation following mouse injection. 12 The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR associated protein 9 (Cas9) system was recently developed for site-specific DNA cleavage, improving the efficiency, ease of design, and feasibility of genome editing. 13,14 In the CRISPR/ Cas9 system, a guide RNA is utilized for the delivery of the Cas9 endonuclease to a specific DNA region, such as a disease-related mutation site. Following delivery, the guide RNA and Cas9 protein complex cleave the DNA at the target site, leaving the opportunity for either non-homologous end-joining or homology directed repair to occur. Efficient, site-specific DNA breakage at the mutated target site increases the frequency of homology directed repair when a normal donor DNA sequence is provided along with guide RNA and Cas9 protein, allowing for gene correction.
The combination of iPSC technology and CRISPR/Cas9-based gene correction could be used as a possible regenerative medicine strategy for treating SCD. This approach has been recently described by using a guide RNA/Cas9 ribonucleoprotein (RNP) and an adeno-associated virus type 6 (AAV6) donor vector demonstrating a biallelic correction of the SCD mutation with normal β-globin production at the RNA expression level 15 ; however, β-globin protein production was not confirmed.
The iPSC-derived RBC generation is currently limited by insufficient expansion of erythroid cells per iPSC, primitive globin production (ζ-and ε-globins), and reliance on xeno-materials such as mouse feeder cells and fetal bovine serum (FBS). Takayama et al developed a 15-day protocol using culture media containing FBS and vascular endothelial growth factor (VEGF) to produce functional megakaryocytes from hESCs through the formation of hESC-derived-sacs (ES-sacs), 16 which are hemangioblast-like structures composed of hematopoietic-like spherical cells, containing a CD34+CD45+ HSPC population, and endothelial-like extracellular cells. We previously demonstrated that ESsac-derived spherical cells can be differentiated into definitive erythroid cells that produce mainly γ-globin and β-globin (with low levels of ε-globin) at both the RNA and protein levels. 8,17 The current understanding is that definitive RBCs (producing γ-globin and β-globin) are primarily generated by HSPCs, whereas primitive RBCs (producing ε-globin) are derived from hemangioblast precursor cells in yolk sacs. 18 We hypothesized that this ES-sac-based erythroid differentiation method would allow us to generate definitive erythroid cells from gene-corrected SCD iPSCs. In addition, we sought to generate hESC-derived definitive HSPCs/RBCs via ES-sacs using serum-free media, since it would reduce variability due to different FBS lots and would represent an important step toward xeno-free clinical application.
Here, we optimized the ES-sac generation protocol using feeder cell-free hES/iPSC maintenance, a preferable method for electroporation-based gene correction, followed by a serum-free ES-sac culture. The optimized protocol improved the yields of hematopoieticlike spherical cells as well as β-globin-producing erythroid cells from hES/iPSCs. We demonstrated viral vector-free biallelic β-globin gene correction in SCD iPSCs followed by erythroid differentiation, which allowed for normal β-globin production at the protein level.

Significance statement
The sickle mutation in induced pluripotent stem cells (iPSCs) derived from a patient with sickle cell disease has successfully been corrected using an improved serum-free method for the generation of ES/iPS-sacs, a hemangioblast-like structure containing hematopoietic stem and progenitor cells that can be differentiated into erythroid cells containing mostly definitive globins, including γand β-globins.
was maintained in Basal Medium Eagle (BME, Life Technologies) supplemented with 10% FBS (Thermo Fisher Scientific) and 2 mM L-glutamine (Thermo Fisher Scientific). The OP9 mouse bone marrow stromal cell line (ATCC) was maintained in Minimum Essential Medium alpha no nucleosides (MEM alpha, no nucleosides; Life technologies) supplemented with 20% FBS. Both C3H10T1/2 and OP9 cells were irradiated (50 Gy) before being used as feeder cells.

| Reverse transcription quantitative polymerase chain reaction
After erythroid differentiation, erythroid cells were collected and evaluated to determine RNA expression levels of ε-globin, γ-globin, β-globin, and α-globin, as previously described. 17 Total RNA was extracted from erythroid cells produced after differentiation using TRIZol LS (Life Technologies). Reverse transcription was performed using the SuperScript III kit (Life Technologies). Quantitative PCR assay was performed using gene-specific primers and probes in the QuantStudio 6 Flex Real-Time PCR System (ThermoFisher Scientific). The primer and probe sequences  Table 1. We used a control plasmid containing one copy of ε-, γ-, β-, and α-globin cDNA and calculated relative amounts of ε-, γ-, and β-globin RNA, which were standardized by α-globin signals.

| Reversed-phase high-performance liquid chromatography
We performed reversed-phase high-performance liquid chromatography (RP-HPLC) for globin protein analysis, as previously described. 8,20,21 Briefly, we collected ES/iPS-sac-derived erythroid cells, and after three washes with phosphate-buffered saline (Corning, New York), the cells were lysed using HPLC grade water, vortexed, and mixed with 10% of 100 mM Tris (2-carboxyethyl) phosphine (Thermo Fisher Scientific).  The difference between the two groups was evaluated by a two-tailed t-test. The difference between more than two groups was evaluated by one-way analysis of variance using Dunnett's test when compared with a control group, or Tukey's honest significant difference test when compared among all the groups. A P value of <.05 or <.01 was deemed significant.

| RESULTS
3.1 | hESCs maintained on Matrigel and differentiated using a KSR-based media improves ESsac and spherical cell generation with similar levels of β-globin production after erythroid differentiation Since feeder cell-free iPSC maintenance is optimal for electroporationbased delivery of gene correction tools, we evaluated feeder cell-free culture for hESC maintenance followed by serum-free ES-sac generation. In hESC maintenance, mouse embryonic fibroblast (MEF) feeder cells were switched to Matrigel (MT) protein coating, and in ES-sac generation, FBS was replaced by KSR. 22  greater amounts of hematopoietic-like spherical cells (P < .01), which was probably due to more efficient ES-sac generation (P < .01) ( Figure S1). In both conditions, ES-sacs included slightly lower percentages of a CD34+CD45+ population (containing HSPC) (P < .05) and slightly lower percentages of a CD34−GPA+ population (producing a more primitive erythropoiesis producing ε-globin, γ-globin, and no β-globin 17 ) (P < .05), compared with our standard MEF-FBS condition.
We 3.3 | Gene correction of the SCD mutation in the β-globin gene in SCD iPSCs confirmed at DNA and protein levels To investigate a regenerative medicine strategy for SCD, we performed gene correction of the SCD mutation in the β-globin gene on an iPSC line generated from SCD patient bone marrow stromal cells 8 followed by iPS-sac generation and erythroid differentiation ( Figure 2A). To perform gene correction in SCD iPSCs, Matrigel condition was used for iPSC maintenance, since a feeder cell-free condition is optimal for the delivery of CRISPR/Cas9 tools with electroporation.
Two million SCD iPSCs were used for electroporation to deliver a single-stranded donor DNA encoding the normal β-globin sequence, a plasmid encoding both high-fidelity Cas9 and a guide RNA targeting the SCD mutation site, and a GFP marker gene. After electroporation, we sorted GFP-positive cells by flow cytometry, expanded single clones, and performed restriction enzyme-based genetic screening.
Using the optimized MT-KSR condition, we generated iPS-sacs from non-corrected SCD iPSCs (without gene correction), mCOR-iPSCs, and bCOR-iPSCs. The iPS-sac-derived spherical cells were differentiated into erythroid cells using KSR-based serum-free erythroid differentiation media. 21 Greater amounts of spherical cells (after iPSsac generation) and erythroid cells (after erythroid differentiation from spherical cells) were produced from the mCOR-iPSCs (P < .01), which could potentially be due to the genetic variance among clones ( Figure 2D). We also observed variance in the type of cell populations produced by the clones: higher percentages of CD34+CD45+ HSPCs in the bCOR-iPSC-derived spherical cells (P < .01), and higher percentages of both CD34−GPA+ primitive and CD34+GPA− definitive cell populations in iPS-sacs (P < .01) ( Figure 2F). However, after erythroid differentiation from iPS-sac-derived spherical cells, we obtained red colored pellets from all three iPSC clones, demonstrating robust hemoglobinization ( Figure 2E). We evaluated β-globin production at the protein level by RP-HPLC, which demonstrated only β S -globin in SCD-iPSC-derived erythroid cells, both β S -globin and normal β-globin in mCOR-iPSC-derived erythroid cells, and only normal β-globin in bCOR-iPSC-derived erythroid cells ( Figure 2G and Figure S3B). These Using a method previously developed by our group, we performed viral vector-free CRISPR/Cas9-based gene correction of the SCD mutation in a SCD iPSC line (derived from SCD patient bone marrow stromal cells) that was maintained using our optimized MT-ES culture condition. 8 Our restriction enzyme-based screening system for iPSC genome editing identified 2 clones of gene-corrected iPSC lines from a screening of 96 clones. The gene correction efficiency in human iPSCs was strongly improved by these methods compared with our previous publication 11 ; however, this efficiency is much lower than gene correction in human CD34+ HSPCs. 39 Unlike HSPCs, corrected iPSCs can be isolated by cloning steps even at lower efficiencies of gene correction. Using our optimized serum-free ES-sac generation followed by serum-free erythroid differentiation, genecorrected iPSCs can efficiently generate ES-sacs, ES-sac-derived spherical cells, and spherical cell-derived erythroid cells, which demonstrate similar amounts of normal β-globin production at the protein level compared with the β S -globin production from non-corrected SCD iPSCs. Interestingly, both normal β-globin and β S -globin peaks were detected in RP-HPLC from monoallelic corrected iPSCs.
Recently, another group also reported a biallelic gene correction at the RNA level in SCD iPSCs using guide RNA/Cas9 RNP electroporation and AAV6 donor vector transduction. 15 However, massive DNA synthesis from AAV vectors could result in various random integrations of rearranged vector sequences in target cells. 40 Here, we report viral vector-free biallelic correction and quantitative data of corrected β-globin protein production in erythroid cells differentiated from gene-corrected human SCD iPSCs. 11,15,41,42

| SUMMARY
In summary, we demonstrate that compared with the FBS-based media, our serum-free culture media improves the yield of ES/iPS-sacs containing CD34+CD45+ HSPCs, allowing for a greater production of erythroid cells expressing β-globin protein. Furthermore, we performed viral vector-free gene correction of the SCD mutation in an SCD iPSC line using this optimized serum-free ES/iPS-sac culture, and the biallelic corrected iPSCs generated iPS-sac-derived erythroid cells with normal β-globin production, which was confirmed at DNA and protein levels.
Our serum-free ES/iPS-sac protocol has the potential to contribute to the development of a xeno-free strategy for iPSC-based regenerative medicine, which is needed for the safe and reliable clinical application.