scholarly journals Development of a Clinically Applicable Method for High-Efficiency Gene Correction of Plerixafor-Mobilized CD34+ Cells from Patients with Sickle Cell Disease

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 2191-2191 ◽  
Author(s):  
Naoya Uchida ◽  
Linhong Li ◽  
Juan J Haro-Mora ◽  
Selami Demirci ◽  
Tina Nassehi ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Single Strand ◽  
Gene Correction ◽  
Guide Rna ◽  
Protein Levels ◽  
Cd34 Cells ◽  
Cas9 Mrna

Abstract Sickle cell disease (SCD) is caused by a 20A>T mutation in the β-globin gene, and can be cured by therapeutic β-globin gene addition into hematopoietic stem cells (HSCs) with lentiviral transduction. However, this method relies upon random integration, leaving the SCD mutation intact and potentially inducing insertional mutagenesis. Genome editing technologies have the potential to correct the SCD mutation without integration, producing adult hemoglobin (Hb) while simultaneously eliminating sickle Hb. In this study, we investigated CRISPR/Cas9-based gene correction for SCD CD34+ cells. Plerixafor-mobilized SCD CD34+ cells were transfected by electroporation using the GMP-compliant, FDA Master File-supported, and scalable MaxCyte GT System to deliver SCD mutation-specific guide RNA at 200mg/ml, SpCas9 mRNA at 200mg/ml or protein at 120mg/ml, and single strand donor DNA with a normal β-globin sequence at 80, 120, or 200mg/ml. We chose Cas9 mRNA and single strand donor DNA due to the ease of clinical grade large-scale production and to avoid the need for viral vector manufacturing. Following erythroid differentiation, gene correction efficiency was evaluated at DNA levels by deep sequencing and at protein levels by reverse-phase HPLC. Cell viability was reduced to 76-87% after electroporation, compared to 90% in the control. We observed high-efficiency genome editing (29-34% gene correction and 49-58% indels) with Cas9 mRNA, showing donor DNA concentration dependence, and editing levels were comparable to Cas9 protein (39% correction and 43% indels). 15-23% Biallelic and 17-26% monoallelic gene correction were detected at the clonal level by colony assay. After erythroid differentiation, up to 54% normal β-globin production was observed with Cas9 mRNA (Figure), comparable to Cas9 protein (67%), while βs-globin amounts were markedly reduced under both conditions (6-10%). Similar correction efficiencies were obtained from two additional SCD patients' CD34+ cells at DNA levels (28-35%) and protein levels (33-56%). These data demonstrate that Cas9 mRNA and single strand donor DNA allow for efficient gene correction in SCD CD34+ cells, exceeding the therapeutic threshold of 20% in SCD. We then evaluated off-target effects on the δ-globin gene, which was reported as a major off-target site in β-globin gene editing due to high homology; however, almost no off-target effects (0.6-1.3% indels) were detected. Interestingly, gene conversion in the 9T>C polymorphism (11bp upstream of SCD mutation) on the β-globin gene was observed, and this conversion always occurred with SCD gene correction (26-33% of SCD gene correction), suggesting that gene conversion is strongly affected by distance from the target site. In addition, we evaluated genome editing among subpopulations of CD34+ cells from 3 healthy donors under the same conditions (normal β-globin to SCD mutation). We observed similar editing efficiencies (conversion and indels) among more immature (CD34+CD133+CD90+) and relatively differentiated populations (CD34+CD133+CD90-, CD34+CD133-, and CD34-) as well as among cells at different phases of the cell cycle (G0/G1, S, and G2/M), suggesting that similar gene correction efficiencies are obtained in all CD34+ cell populations, including the HSC population. We have begun efforts to evaluate gene-corrected SCD CD34+ cell engraftment in the mouse xenograft model, as similarly corrected X-CGD CD34+ cells were engrafted in immunodeficient mice. To examine the effects of indels in the β-globin gene, we next evaluated Hb production from genome-edited SCD CD34+ cells (2 patients) without donor DNA. Editing without donor DNA resulted in 63-70% indels (compared to 26-29% correction and 46-53% indels with donor DNA) and increased non-adult Hb production (small amounts of fetal Hb and significant amounts of a Hb variant), which will require further investigation to characterize. In summary, we observed efficient gene correction in SCD CD34+ cells with a simple Cas9 mRNA, single strand donor DNA, and guide RNA method, resulting in ~30% gene correction and ~50% indels. After erythroid differentiation, the majority of Hb detected was adult Hb; we detected up to 54% normal β-globin production with a marked reduction of βs-globin to ~10%. Evaluation of engraftment potential is required for gene-corrected CD34+ cells, but these methods would be clinically applicable for gene correction in SCD. Figure. Figure. Disclosures Li: MaxCyte, Inc.: Employment. Allen:MaxCyte, Inc.: Employment. Peshwa:MaxCyte, Inc.: Employment.

Preclinical Evaluation for Engraftment of Gene-Edited CD34+ Cells with a Sickle Cell Disease Mutation in a Rhesus Transplantation Model

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 609-609
Author(s):  
Naoya Uchida ◽  
Linhong Li ◽  
Tina Nassehi ◽  
Morgan Yapundich ◽  
Jackson Gamer ◽  
...  
Keyword(s):  
Gene Conversion ◽  
Genome Editing ◽  
Erythroid Differentiation ◽  
Cell Disease ◽  
Gene Correction ◽  
Guide Rna ◽  
Post Transplant ◽  
Cd34 Cells ◽  
Before And After

Sickle cell disease (SCD) is caused by a 20A>T mutation in the β-globin gene. State-of-the-art genome editing technologies have the potential to correct the SCD mutation in hematopoietic stem cells (HSCs), producing adult hemoglobin (Hb) while simultaneously eliminating sickle Hb. We have demonstrated efficient gene correction in SCD CD34+ cells with SCD mutation-specific guide RNA, Cas9 mRNA/protein, and single strand donor DNA, resulting in ~30% gene correction and ~50% indels at the DNA level, and ~60% normal β-globin production at the protein level in in vitro erythroid differentiation (ASH 2018). Gene correction by homology directed repair is thought to be enhanced by cell proliferation; however, cell proliferation might reduce stemness of HSCs. To investigate this hypothesis, we sought to evaluate engraftment of gene-edited CD34+ HSCs in a non-human primate model. To model SCD gene correction, a β-to-βs globin conversion was designed in rhesus macaques. Mobilized rhesus CD34+ cells (n=2) were electroporated using the GMP-compliant, FDA Master File-supported, and scalable MaxCyte GT System to deliver rhesus β-globin-targeting guide RNA (the same target site as the SCD mutation-specific guide RNA), SpCas9 protein, and single strand donor DNA including a SCD mutation (20A>T). We also added an adjuvant to improve gene conversion efficiencies. Following erythroid differentiation, gene correction efficiency was evaluated at DNA levels by deep sequencing and at protein levels by reverse-phase HPLC. We observed high-efficiency genome editing without the adjuvant (20-30% gene conversion and 61-64% indels), and further enhanced genome editing with the adjuvant (51-59% gene conversion and 36-39% indels). After erythroid differentiation, we observed production of βs-globin protein (~100%) but not normal β-globin in gene-edited cells. We then evaluated engraftment of gene-edited rhesus CD34+ cells with β-to-βs globin conversion (n=2, 13U005 and 12U011). Mobilized rhesus CD34+ cells (3.4-3.8e7) were pre-stimulated for 2 days, and edited cells were cryopreserved after electroporation with editing tools. Small aliquots of edited cells (before and after cryopreservation) were differentiated into erythroid cells in vitro, resulting in 17-26% of gene conversion and 57-71% of indels at the DNA level and 50-100% of β-globin production at the protein level, with no difference observed between aliquots taken before and after cryopreservation. Following 9.5 Gy total body irradiation, the frozen edited CD34+ cells (1.6-2.2e7) were injected into autologous macaques. We observed robust recovery of blood counts in 13U005, while peripheral blood recovery was delayed in 12U011, who was supported by serial whole blood transfusion. We observed 7-11% of gene conversion and 44-54% of indels in both granulates and lymphocytes in 13U005 1 month post-transplant. Around 15% sickle Hb production in red blood cells was detected by Hb electrophoresis in 13U005 three months post-transplant and ~7% in 12U011 two months post-transplant. Interestingly, ~10% of fetal Hb production was observed in 12U001, likely due to stress hematopoiesis. In summary, we developed a rhesus β-to-βs globin conversion model with HSC-targeted genome editing strategies. The gene-edited rhesus CD34+ cells are engraftable for at least 3 months post-transplant. Although further follow-up is necessary for transplanted animals, these findings are helpful in designing HSC-targeted gene correction trials. Figure Disclosures Li: MaxCyte, Inc: Employment. Allen:MaxCyte, Inc: Employment. Peshwa:MaxCyte, Inc: Employment.

Co-encapsulation of Cas9 mRNA and guide RNA in polyplex micelles enables genome editing in mouse brain

2021 ◽  
Vol 332 ◽  
pp. 260-268
Author(s):  
Saed Abbasi ◽  
Satoshi Uchida ◽  
Kazuko Toh ◽  
Theofilus A. Tockary ◽  
Anjaneyulu Dirisala ◽  
...  
Keyword(s):  
Genome Editing ◽  
Mouse Brain ◽  
Guide Rna ◽  
Cas9 Mrna

scholarly journals An improved method for precise genome editing in zebrafish using CRISPR-Cas9 technique

2021 ◽  
Author(s):  
Eugene V. Gasanov ◽  
Justyna Jędrychowska ◽  
Michal Pastor ◽  
Malgorzata Wiweger ◽  
Axel Methner ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Target Site ◽  
Proof Of Concept ◽  
Intervention Site ◽  
End Joining ◽  
Guide Rna ◽  
Guide Rnas ◽  
Cas9 Nuclease ◽  
Non Homologous End Joining

AbstractCurrent methods of CRISPR-Cas9-mediated site-specific mutagenesis create deletions and small insertions at the target site which are repaired by imprecise non-homologous end-joining. Targeting of the Cas9 nuclease relies on a short guide RNA (gRNA) corresponding to the genome sequence approximately at the intended site of intervention. We here propose an improved version of CRISPR-Cas9 genome editing that relies on two complementary guide RNAs instead of one. Two guide RNAs delimit the intervention site and allow the precise deletion of several nucleotides at the target site. As proof of concept, we generated heterozygous deletion mutants of the kcng4b, gdap1, and ghitm genes in the zebrafish Danio rerio using this method. A further analysis by high-resolution DNA melting demonstrated a high efficiency and a low background of unpredicted mutations. The use of two complementary gRNAs improves CRISPR-Cas9 specificity and allows the creation of predictable and precise mutations in the genome of D. rerio.

Pomalidomide Transcriptionally Reprograms Adult Erythroid Progenitors Independently of Ikaros Proteasomal Degradation

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 160-160
Author(s):  
Brian M. Dulmovits ◽  
Abena O. Appiah-Kubi ◽  
Julien Papoin ◽  
John Hale ◽  
Mingzhu He ◽  
...  
Keyword(s):  
Cord Blood ◽  
Board Of Directors ◽  
Peripheral Blood ◽  
Globin Gene ◽  
Erythroid Differentiation ◽  
Erythroid Progenitors ◽  
Advisory Committees ◽  
Cd34 Cells ◽  
Terminal Erythroid Differentiation ◽  
Adult Peripheral Blood

Abstract Pomalidomide, a second-generation immunomodulatory drug, is a fetal hemoglobin (HbF) inducing agent with potential implications for the treatment of β-hemoglobinopathies such as sickle cell disease (SCD). However, its mechanism of action remains unknown. Through an in-depth characterization of human erythropoiesis and globin gene regulatory networks, we now provide evidence that pomalidomide alters transcription networks involved in erythropoiesis and globin switching, thereby leading to a partial reprogramming of adult hematopoietic progenitors toward fetal-like erythropoiesis. Adult peripheral blood CD34+ cells from normal individuals were differentiated toward the red cell lineage using an adapted 3-phase culture system. At day 14 of culture, we observed a reciprocal globin gene switch at the mRNA and protein levels. These results were confirmed by high performance liquid chromatography of hemolysates (HbF/(HbF+HbA): 31.7 ± 1.4% vs. 6.5 ± 0.7% pomalidomide and vehicle, respectively). Next, we studied erythroid differentiation using flow cytometric analyses of the cell surface markers interleukin-3R (IL-3R), glycophorin A (GPA), CD34 and CD36 for early erythroid precursors (BFU-E and CFU-E) as well as GPA, α4-integrin and band3 for terminal erythroid differentiation. While there were no changes in terminal erythroblast maturation, an accumulation of BFU-E in pomalidomide-treated cultures at days 2 and 4 of differentiation was seen, indicating a delay at the BFU-E to CFU-E transition, and also, that pomalidomide exerts its effect in the early-stages of erythropoiesis. Indeed, treatment with pomalidomide during the phase of the culture system that generates erythroid progenitors led to significantly more γ-globin expression than treatment during the phase which proerythroblasts undergo terminal erythroid differentiation. At the molecular level, pomalidomide was found to rapidly and robustly decrease Ikaros (IKZF1) expression exclusively by post-translational targeting to the proteasome. Moreover, pomalidomide selectively reduced the expression of components of key globin regulatory pathways including BCL11A, SOX6, KLF1, GATA1 and LSD1 while not affecting others (e.g. CoREST, GATA2, GFI1B, and HDAC1). Pomalidomide had a transient effect on GATA1 and KLF1 expression. While shRNA knockdown of Ikaros using two different lentiviral constructs delayed erythroid differentiation, it failed to appreciably stimulate HbF production or alter BCL11A expression. These results suggest that the loss of Ikaros alone is insufficient to recapitulate the phenotype observed in pomalidomide-treated conditions. We next compared the expression levels of proteins involved in globin gene regulation among untreated peripheral blood, pomalidomide-treated peripheral blood and untreated cord blood-derived erythroid cells. We found striking similarities between cord blood and pomalidomide-treated adult cells at day 4 of differentiation. Indeed, BCL11A, KLF1, SOX6, LSD1 and GATA1 showed decreased expression levels both in cord blood and pomalidomide-treated adult peripheral blood, while the levels of CoREST, HDAC1 and GATA2 remained unchanged indicating that pomalidomide partially reprograms adult erythroid cells to a fetal-like state. Taken together, our results show that the mechanism underlying reactivation of HbF by pomalidomide involves Ikaros-independent reprogramming of adult erythroid progenitors. Finally, we found that this mechanism is conserved in SCD-derived CD34+ cells. Our work has broad implications for globin switching, as we provide direct evidence that Ikaros does not play a major role in the repression of γ-globin during adult erythropoiesis, and further supports the previously held notion that globin chain production is determined prior to or at the level of CFU-E. Disclosures Allen: Celgene: Research Funding; Bristol Myers Squibb: Equity Ownership; Onconova: Membership on an entity's Board of Directors or advisory committees; Alexion: Membership on an entity's Board of Directors or advisory committees; Takeda: Membership on an entity's Board of Directors or advisory committees.

scholarly journals High efficiency gene correction in hematopoietic cells by donor-template-free CRISPR/Cas9 genome editing

2017 ◽  
Vol 53 ◽  
pp. S64 ◽  
Author(s):  
Duran Sürün ◽  
Nina Kurrle ◽  
Hubert Serve ◽  
Harald von Melchner ◽  
Frank Schnütgen
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Hematopoietic Cells ◽  
Gene Correction ◽  
Template Free ◽  
Donor Template

scholarly journals Therapeutic Crispr/Cas9 Genome Editing for Treating Sickle Cell Disease

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 4703-4703 ◽  
Author(s):  
So Hyun Park ◽  
Ciaran M Lee ◽  
Harshavardhan Deshmukh ◽  
Gang Bao
Keyword(s):  
Sickle Cell Disease ◽  
Genome Editing ◽  
Sickle Cell ◽  
Proof Of Concept ◽  
Cell Disease ◽  
Gene Correction ◽  
Mutation Site ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Target Activity

Abstract Introduction Sickle cell disease (SCD) is one of the most common monogenic disorders, affecting millions worldwide. SCD is caused by a point mutation in the β-globin gene (HBB). A single nucleotide substitution from A to T in the codon for the sixth amino acid in the β-globin protein converts a glutamic acid to a valine that leads to the production of sickle hemoglobin (HbS), which impairs the function of the red blood cells (RBCs). Allogeneic hematopoietic stem cell transplantation (HSCT) is the only available cure, but it is feasible for only a small subpopulation (<15%) of patients and may be associated with a high risk. Here, we show that targeted genome editing can potentially provide a permanent cure for SCD by correcting the sickle mutation in clinically relevant hematopoietic stem and progenitor cells (HSPCs) for autologous transplantation. Methods For proof-of-concept, we designed CRISPR/Cas9 systems and donor templates to introduce the sickle mutation into wild-type (WT) HBB of mobilized peripheral blood CD34+ cells. To assess genome-editing outcomes mediated by CRISPR/Cas9 systems, we developed a novel digital droplet PCR (ddPCR) assay that can quantify the rates of non-homologous end joining (NHEJ) and homology directed repair (HDR) events simultaneously following the generation of DNA double strand breaks. The assay enables rapid and accurate quantification of gene modifications in HSPCs by CRISPR/Cas9 genome-editing. Specifically, Streptococcus pyogenes (Spy) Cas9 proteins, guide RNAs (gRNA), and single-stranded DNA (ssDNA) donor templates were delivered into CD34+ cells by nucleofection with optimized conditions. Different gRNAs targeting HBB near the SCD mutation site were tested, and the optimal gRNA was chosen based on high on-target activity and proximity to the mutation site. The optimal DNA donor design and concentration were determined based on the frequency of HDR events and viability/growth rate of edited cells. Treated samples and untreated controls were assayed as both single cell clones and in bulk culture. In 2-phase liquid culture, genome editing frequencies at both DNA and mRNA levels were quantified by ddPCR to confirm persistence of edited cells in the heterozygous population over time. The expression of globins and other erythroid markers were monitored using flow cytometry and real time PCR to determine if genome editing had any effect on the kinetics of erythropoiesis. Colony formation assays were used to determine the number and type of colonies following induction of differentiation. Colony ddPCR was performed to determine the genotype of edited cells. Wright/Giemsa stain was used to confirm terminal maturation of erythrocytes into enucleated RBC. Native polyacrylamide gel electrophoresis (PAGE) and high performance liquid chromatography (HPLC) were used to confirm translation of edited β-globin protein and formation of HbS. Results and Discussion We found that the efficiency of site-specific gene correction could be substantially improved by optimizing the CRISPR/Cas9 systems for genome editing. For example, with optimization, we achieved ~30% HDR rates in CD34+ cells with >80% cell viability. The HDR-modified alleles persisted in the population over the course of differentiation, and the edited CD34+ cells retained differentiation potential. Genotyping of individual erythroid colonies confirmed that up to 35% of colonies are either homozygous or heterozygous for HDR alleles. Following differentiation, treated cells express modified HBB mRNA and HbS. In addition, the off-target activity of the HBB-specific gRNAs was determined using both bioinformatics tools and unbiased genome-wide mapping techniques. Ongoing work includes the validation of gene correction in SCD patient derived HSPCs, characterization of modified cells in vitro and in vivo to assess the therapeutic potential, and analysis of long-term genotoxicity. Conclusions Based on the proof-of-concept study, we demonstrate that using the optimized CRISPR/Cas9 system and donor template, an HDR rate of ~30% can be achieved in CD34+ cells. The gene corrected cells have the potential to differentiate into erythroid cells that permanently produce WT β-globin. Our findings provide promising evidence for clinical translation of the HSPCs genome correction strategy in treating SCD patients, as well as correcting gene defects underlying other inherited single-gene disorders. Disclosures No relevant conflicts of interest to declare.

scholarly journals Strategies to Increase On-Target and Reduce Off-Target Effects of the CRISPR/Cas9 System in Plants

2019 ◽  
Vol 20 (15) ◽  
pp. 3719 ◽  
Author(s):  
Zahra Hajiahmadi ◽  
Ali Movahedi ◽  
Hui Wei ◽  
Dawei Li ◽  
Yasin Orooji ◽  
...  
Keyword(s):  
Genome Editing ◽  
High Efficiency ◽  
Zinc Finger Nucleases ◽  
Transcription Activator ◽  
Guide Rna ◽  
Short Palindromic Repeat ◽  
External Forces ◽  
Time And Energy ◽  
Reduced Time ◽  
Target Effects

The CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeat-associated protein 9) is a powerful genome-editing tool in animals, plants, and humans. This system has some advantages, such as a high on-target mutation rate (targeting efficiency), less cost, simplicity, and high-efficiency multiplex loci editing, over conventional genome editing tools, including meganucleases, transcription activator-like effector nucleases (TALENs), and zinc finger nucleases (ZFNs). One of the crucial shortcomings of this system is unwanted mutations at off-target sites. We summarize and discuss different approaches, such as dCas9 and Cas9 paired nickase, to decrease the off-target effects in plants. According to studies, the most effective method to reduce unintended mutations is the use of ligand-dependent ribozymes called aptazymes. The single guide RNA (sgRNA)/ligand-dependent aptazyme strategy has helped researchers avoid unwanted mutations in human cells and can be used in plants as an alternative method to dramatically decrease the frequency of off-target mutations. We hope our concept provides a new, simple, and fast gene transformation and genome-editing approach, with advantages including reduced time and energy consumption, the avoidance of unwanted mutations, increased frequency of on-target changes, and no need for external forces or expensive equipment.

Genome Editing Recreates Hereditary Persistence of Fetal Hemoglobin in Primary Human Erythroblasts

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 640-640 ◽  
Author(s):  
Elizabeth Traxler ◽  
Yu Yao ◽  
Chunliang Li ◽  
Jeremy Grevet ◽  
Peng Huang ◽  
...  
Keyword(s):  
Real Time ◽  
Genome Editing ◽  
Sickle Cell ◽  
Real Time Pcr ◽  
Fetal Hemoglobin ◽  
Erythroid Differentiation ◽  
Globin Mrna ◽  
Benign Condition ◽  
Cd34 Cells ◽  
Hereditary Persistence

Abstract Manipulating the developmental switch from γ- to β-globin expression that occurs after birth has been intensively investigated as therapeutic strategy for sickle cell anemia and β-thalassemia. Rare individuals with a benign condition termed hereditary persistence of fetal hemoglobin (HPFH) exhibit an attenuated or absent γ-to-β switch, resulting in high levels of fetal hemoglobin (α2γ2) in all red blood cells (RBCs) throughout life. Moreover, individuals with HPFH and homozygosity for sickle cell disease (SCD) mutations exhibit few or no clinical manifestations of the latter. We used genome editing to induce a naturally occurring 13-nucleotide (-102 to -114) deletional HPFH mutation in the γ-globin (HBG1) gene promoter. Heterozygosity for this mutation is associated with HbF levels &gt; 30% in adults. We used the clustered, regularly interspaced, short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9) system to create small deletions around -102 to -114 in the γ-globin genes in peripheral blood CD34+ cells from healthy donors. We delivered guide RNA (gRNA) and Cas9 using lentiviruses, sorted transduced hematopoietic progenitors by FACS, and cultured them using a 3-phase erythroid differentiation protocol. Real time PCR showed that γ-globin mRNA increased more than 10-fold in Cas9/gRNA transduced cells compared to controls. HbF flow cytometry and high-performance liquid chromatography (HPLC) demonstrated that induced γ-globin chains were effectively incorporated into hemoglobin tetramers. HPLC revealed 1-3% HbF in negative controls and an increase to 15% in cells transduced with gRNA and Cas9. Expression of erythroid differentiation markers CD235 and CD71 were unaffected, suggesting that the γ-globin increase is not due to impaired erythroid maturation. Next generation sequencing demonstrated that a single gRNA created one predominant mutation that co-segregated with high HbF expression and represented over 50% of the sequencing coverage. Interestingly, this mutation is identical to the 13-nucleotide HPFH deletion. We also tested the gRNA mutation efficiency after transient expression of gRNA and Cas9 in human CD34+ cells by electroporation followed by analysis of single burst-forming unit-erythroid (BFU-E) colonies formed in methylcellulose. Genomic DNA analysis revealed that one gRNA targeted 50% of HBG1 alleles, and cells that received two overlapping gRNAs demonstrated 80% mutation frequency. Real-time PCR of mRNA from edited BFU-Es showed that mutations stimulated γ-globin mRNA expression to 19-55% total globin synthesis, whereas control colonies contained 1-5% γ-globin. Together, our data demonstrate that the CRISPR-Cas9 system can generate precisely the -102 to -114 HPFH mutation at high efficiency in primary human progenitor cells and thereby induce the expression of HbF to potentially therapeutic levels. This work provides proof of concept for targeted genome editing for γ-globin activation as a therapy for patients with β hemoglobinopathies. Disclosures Weiss: Biogen: Research Funding; GlaxoSmithKline: Consultancy; Rubius: Membership on an entity's Board of Directors or advisory committees.

scholarly journals Ex Vivo Expansion with E478 Increases the Rate of CRISPR-Mediated Homology Directed Repair of the Beta-Globin Gene in Human Hematopoietic Stem Cells By >10-Fold and Improves In Vivo Engraftment By >120-Fold

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 4634-4634
Author(s):  
Kevin A. Goncalves ◽  
Megan D. Hoban ◽  
Sharon L. Hyzy ◽  
Katia S. George ◽  
Anthony E. Boitano ◽  
...  
Keyword(s):  
Ex Vivo ◽  
Globin Gene ◽  
Fold Increase ◽  
Ex Vivo Expansion ◽  
Gene Correction ◽  
Employment Equity ◽  
Hematopoietic Stem ◽  
Cd34 Cells ◽  
Equity Ownership

Background . Site-specific gene correction of hematopoietic stem cells (HSCs) via homology directed repair (HDR) has the potential to precisely repair defective genes and provide life-long cures for a variety of blood-based diseases. It is possible to obtain high levels of HDR during in vitro HSC culture, but these cells fail to robustly engraft in vivo, suggesting that the procedure of HDR compromises HSC function or that true HSCs are not undergoing HDR. Cells need to be actively cycling in order to undergo HDR, but conditions that allow HSC replication in vitro without compromising HSC number and function remain elusive. Thus, most HDR protocols minimize time in culture, potentially limiting HDR rates and cell yield. We recently reported that ex vivo expansion of HSCs with an aryl hydrocarbon receptor (AHR) antagonist is a clinically validated method to expand HSCs. The AHR antagonist-expanded CD34+ cell therapy, MGTA-456, results in rapid and durable recovery in patients with hematologic malignancies and inherited metabolic diseases (Wagner et al Cell Stem Cell 2016; Orchard et al AAN 2019). To apply this technology to gene-modified HSCs, we developed a novel AHR antagonist, E478, which expands NSG-engrafting cells 10-fold compared to uncultured primary human mobilized peripheral blood (mPB) CD34+cells in limit dilution studies. We previously showed that expansion with E478 results in up to 10-fold higher engraftment of lentiviral vector (LVV)-transduced cells and CRISPR/Cas9 knockout cells (Hoban et al ASGCT 2019). Here, we demonstrate that ex vivo expansion of mPB CD34+ cells with E478 results in >10-fold increase in rate of HDR and >120-fold increase in NSG engraftment of HDR+ cells compared to conventional approaches. Results . To determine whether more active cycling would lead to higher rates of HDR, we cultured cells for 1, 2, 3, and 4 days prior to electroporation with CRISPR gRNA targeting the beta-globin gene and transduction with a GFP-containing adeno-associated virus (AAV) donor template. Cell cycle analysis revealed that 33±1.8% of cells enriched for HSCs (CD34+CD90+ cells) remain quiescent after 2 days in culture, whereas 0.92±0.06% of CD34+CD90+ cells were quiescent after 3 and 4 days in culture (n=2 mPB donors). We then assessed HDR rates and HSC number after 1, 2, 3, and 4 days of additional culture. Compared to a conventional HDR protocol utilizing a 2-day pre-stimulation period followed by 1 day of culture after electroporation (herein called a 2+1 culture), we observed up to 8-fold increase in HDR with longer pre-stimulation periods, but this was accompanied with differentiation of CD34+CD90+ cells and loss of engraftment in NSG mice (79% decrease, p<0.001). We next evaluated whether E478 could increase the dose of HSCs and maintain high HDR rates. We cultured mPB CD34+ cells with E478 for a 4 day pre-stimulation, performed HDR, and continued the expansion for 4 days with E478 (herein called 4+4 culture). With the 4+4 protocol, we observed a 6-fold increase in the rate of HDR in vitro and a 134-fold increase in the number of CD34+CD90+ cells with E478 relative to 2+1 conditions with DMSO vehicle (n=2, p<0.01). Transplant of these cells into sublethally-irradiated NSG mice resulted in a 4-fold higher rate of engraftment (Figure A, p<0.01, n=8 mice), 12-fold higher rates of HDR (Figure B, p<0.001) and >120-fold increase in the number of HDR+ NSG-engrafting cells relative to 2+1 cultures (Figure C, p<0.001). Further, a 2+1 culture with E478 led to an 8-fold increase in number of HDR+ NSG-engrafting cells (p<0.001) relative to standard 2+1 approaches without a small molecule. Multi-lineage engraftment was observed in all groups. Studies using E478 with bone marrow from patients with sickle cell disease are in progress and will be presented. Conclusions. We demonstrate that ex vivo HSC expansion with E478 enables higher rates of HDR and a high dose of HDR+ HSCs, leading to >120-fold increase in the engraftment of HDR+ HSCs compared to conventional 2+1 approaches. Culture with E478 is a promising approach to realize the full potential of targeted gene correction in HSCs for a variety of genetic diseases. Disclosures Goncalves: Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Hoban:Magenta Therapeutics: Employment, Equity Ownership. Hyzy:Magenta Therapeutics: Employment, Equity Ownership. George:Magenta Therapeutics: Employment, Equity Ownership. Boitano:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties. Cooke:Magenta Therapeutics: Employment, Equity Ownership, Patents & Royalties.

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