Precise and error-prone CRISPR-directed gene editing activity in human CD34+ cells varies widely among patient samples

Differences in engraftment potential of hematopoietic stem cells (HSCs) in distinct phases of cell cycle may result from the inability of cycling cells to home to the bone marrow (BM) and may be influenced by the rate of entry of BM-homed HSCs into cell cycle. Alternatively, preferential apoptosis of cycling cells may contribute to their low engraftment potential. This study examined homing, cell cycle progression, and survival of human hematopoietic cells transplanted into nonobese diabetic severe combined immunodeficient (NOD/SCID) recipients. At 40 hours after transplantation (AT), only 1% of CD34+ cells, or their G0(G0CD34+) or G1(G1CD34+) subfractions, was detected in the BM of recipient mice, suggesting that homing of engrafting cells to the BM was not specific. BM of NOD/SCID mice receiving grafts containing approximately 50% CD34+ cells harbored similar numbers of CD34+ and CD34− cells, indicating that CD34+ cells did not preferentially traffic to the BM. Although more than 64% of human hematopoietic cells cycled in culture at 40 hours, more than 92% of cells recovered from NOD/SCID marrow were quiescent. Interestingly, more apoptotic human cells were detected at 40 hours AT in the BM of mice that received xenografts of expanded cells in S/G2+M than in recipients of G0/G1 cells (34.6% ± 5.9% and 17.1% ± 6.3%, respectively; P < .01). These results suggest that active proliferation inhibition in the BM of irradiated recipients maintains mitotic quiescence of transplanted HSCs early AT and may trigger apoptosis of cycling cells. These data also illustrate that trafficking of transplanted cells to the BM is not selective, but lodgment of BM-homed cells may be specific.

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Intrathymic and extrathymic development of human plasmacytoid dendritic cell precursors in vivo

Blood ◽

10.1182/blood.v99.8.2752 ◽

2002 ◽

Vol 99 (8) ◽

pp. 2752-2759 ◽

Cited By ~ 64

Author(s):

Kees Weijer ◽

Christel H. Uittenbogaart ◽

Arie Voordouw ◽

Franka Couwenberg ◽

Jurgen Seppen ◽

...

Keyword(s):

Dendritic Cells ◽

Fetal Liver ◽

Plasmacytoid Dendritic Cell ◽

T And B Cells ◽

Human Thymus ◽

Human Cd34 ◽

Cd34 Cells ◽

Fetal Liver Cells ◽

Thymus Graft

Abstract The development of plasmacytoid dendritic cells (pDC2) from human CD34+ stem cells in vivo was studied in RAG-2−/− interleukin (IL)-2Rγ−/− mice that lack functional T and B cells and natural killer cells. CD34+ cells isolated from fetal liver or thymus were labeled with 5- and 6-carboxyfluorescein diacetate succinimidyl ester (CFSE) and were injected into a human thymus grafted subcutaneously in the RAG-2−/− IL-2Rγ−/− mice. One to 4 weeks later the CFSE label was found not only in T cells but also in CD123+/high CD4+CD45RA+ pDC2, indicating that the CD34+ cells can develop into pDC2 within a thymus. In addition to pDC2, CFSE-labeled dendritic cells with a mature phenotype, determined by the cell surface markers CD11c, CD83, and CD80, were found in the injected human thymus graft. pDC2 was not found in the periphery of mice carrying a human thymic graft, indicating that the intrathymic pDC2 failed to emigrate from the thymus. We also demonstrate that pDC2 can develop outside the thymus because relatively high percentages of pDC2 were found in the periphery after the intravenous injection of CD34+CD38−fetal liver cells in RAG-2−/− IL-2Rγ−/−mice without a human thymus graft. These data indicate that the thymus and the peripheral pDC2 develop independently of each other.

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Kinetics and Effect of Integrin Expression on Human CD34+Cells During Murine Leukemia Virus-Derived Retroviral Transduction with Recombinant Fibronectin for Stem Cell Gene Therapy

Human Gene Therapy ◽

10.1089/hum.2008.159 ◽

2009 ◽

Vol 20 (7) ◽

pp. 777-783 ◽

Cited By ~ 2

Author(s):

Yasuomi Horiuchi ◽

Masafumi Onodera ◽

Yoshitaka Miyagawa ◽

Ban Sato ◽

Keiko Onda ◽

...

Keyword(s):

Murine Leukemia Virus ◽

Leukemia Virus ◽

Integrin Expression ◽

Retroviral Transduction ◽

Human Cd34 ◽

Cd34 Cells ◽

Cell Gene ◽

Stem Cell Gene ◽

Stem Cell Gene Therapy ◽

Murine Leukemia

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Critical Parameters Influencing the Expansion and Differentiation of Cultured Human CD34 Cells

Hematopoietic Stem Cell Transplantation ◽

10.1201/b14000-22 ◽

2000 ◽

pp. 330-340

Keyword(s):

Critical Parameters ◽

Human Cd34 ◽

Cd34 Cells

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Truncated Erythropoietin Receptors Confer an In Vivo Selective Advantage in Gene-Modified Erythroid Cells Expressing Fetal Hemoglobin Due to BCL11A Interference

Blood ◽

10.1182/blood-2019-122770 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 2063-2063

Author(s):

Naoya Uchida ◽

Claire Drysdale ◽

Morgan Yapundich ◽

Jackson Gamer ◽

Tina Nassehi ◽

...

Keyword(s):

Rhesus Macaques ◽

Fetal Hemoglobin ◽

Selective Advantage ◽

Erythroid Cells ◽

Post Transplant ◽

Human Cd34 ◽

Cd34 Cells ◽

Hbf Induction

Hematopoietic stem cell gene therapy for hemoglobin disorders, such as sickle cell disease, requires high-level gene marking and robust therapeutic globin expression in erythroid cells (>20% of γ- or β-globin production) for widespread successful clinical application. We previously demonstrated that lentiviral transduction of a truncated human erythropoietin receptor (thEpoR) gene allows for erythropoietin-dependent selective proliferation of gene-modified human erythroid cells during in vitro differentiation (ASH 2017). In this study, we sought to evaluate whether thEpoR can enhance the phenotypic effect of a therapeutic vector in erythroid cells in xenograft mouse and autologous non-human primate transplantation models. To investigate this hypothesis, we designed lentiviral vectors encoding both thEpoR and BCL11A-targeting micro RNA-adapted short hairpin RNA (shmiBCL11A), driven off an erythroid specific ankyrin 1 (ANK1) promoter. Both selective proliferation and high-level fetal hemoglobin (HbF) induction were observed in in vitro erythroid differentiation cultures using transduced human CD34+ cells. Healthy donor CD34+ cells were transduced with shmiBCL11A vector, thEpoR-shmiBCL11A vector, and GFP vector (control). Transduced cells were transplanted into immunodeficient NBSGW mice. Five months post-transplant, xenograft bone marrow cells were evaluated for human cell engraftment (human CD45+) and vector copy number (VCN) in both human CD34+ progenitor cells and glycophorin A+ (GPA+) erythroid cells. HbF production was also measured in GPA+ erythroid cells by reverse phase HPLC. We observed efficient transduction in transduced CD34+ cells in vitro (VCN 2.1-5.1) and similar human cell engraftment among all groups (84-89%). The VCN with thEpoR-shmiBCL11A transduction was 3-fold higher in human erythroid cells when compared to CD34+ cells (p<0.01), but not with shmiBCL11A or GFP vectors. HbF levels were significantly elevated in thEpoR-shmiBCL11A vector (43±6%, p<0.01) when compared to no transduction control (1±0%), but not for either shmiBCL11A vector (3±1%) or GFP vector (1±0%). These data demonstrate selective proliferation of gene-modified erythroid cells, as well as enhanced HbF induction with thEpoR-shmiBCL11A transduction. We then performed autologous rhesus CD34+ cell transplantation using either shmiBCL11A vector (142562 and RA0706, n=2, compared to a GPA promoter-derived shmiBCL11A vector) or thEpoR-shmiBCL11A vector (ZL50 and ZM24, n=2, compared to a Venus-encoding vector). Transduced CD34+ cells were transplanted into autologous rhesus macaques following 2x5Gy total body irradiation. Efficient transduction was observed in CD34+ cells in vitro among all 4 macaques (VCN 3.8-8.7) using a high-density culture protocol (Uchida N, Mol Ther Methods Clin Dev. 2019). In shmiBCL11A transduction animals, engraftment of gene-modified cells (VCN 0.2-1.0) and robust HbF induction (14-16%) were observed 1 month post-transplant. However, VCN and HbF levels were reduced down to VCN ~0.1 and HbF ~0.4% in both animals 6 months post-transplant. In contrast, a thEpoR-shmiBCL11A transduction animal (ZL50) resulted in engraftment of gene-modified cells (VCN 0.8-1.0) and robust HbF induction (~18%) 1 month post-transplant, with both gene marking and HbF levels remaining high at VCN 0.6-0.7 and HbF ~15% 4 months post-transplant. These data suggest that shmiBCL11A transduction results in transient HbF induction in gene-modified erythroid cells, while thEpoR-based selective advantage allows for sustained HbF induction with shmiBCL11A. In summary, we developed erythroid-specific thEpoR-shmiBCL11A expressing vectors, enhancing HbF induction in gene-modified erythroid cells in xenograft mice and rhesus macaques. While further in vivo studies are desirable, the use of thEpoR appears to provide a selective advantage for gene-modified erythroid cells in gene therapy strategies for hemoglobin disorders. Disclosures No relevant conflicts of interest to declare.

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Dose‐response relationship of human CD34+ cells in an athymic rat model of myocardial infarction

The FASEB Journal ◽

10.1096/fasebj.24.1_supplement.824.8 ◽

2010 ◽

Vol 24 (S1) ◽

Author(s):

Ben Brooks ◽

Jeff McKee ◽

Deborah Rice ◽

Bernhard Baumgartner ◽

Delara Motlagh ◽

...

Keyword(s):

Myocardial Infarction ◽

Dose Response ◽

Rat Model ◽

Response Relationship ◽

Dose Response Relationship ◽

Human Cd34 ◽

Cd34 Cells ◽

Relationship Of

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Adenine Base Editing of the Sickle Allele in CD34+ Hematopoietic Stem and Progenitor Cells Eliminates Hemoglobin S

Blood ◽

10.1182/blood-2020-141805 ◽

2020 ◽

Vol 136 (Supplement 1) ◽

pp. 47-47

Author(s):

S. Haihua Chu ◽

Daisy Lam ◽

Michael S. Packer ◽

Jennifer Olins ◽

Alexander Liquori ◽

...

Keyword(s):

High Performance Liquid Chromatography ◽

Liquid Chromatography ◽

Base Pair ◽

High Performance ◽

Gene Editing ◽

Publicly Traded ◽

Hematopoietic Stem ◽

Cd34 Cells ◽

Adenine Base

While there are several small molecule, gene therapy, and gene editing approaches for treating sickle cell disease (SCD), these strategies do not result in the direct elimination of the causative sickle β-globin (HbS) variant itself. The reduction or complete removal of this pathologic globin variant and expression of normal β-hemoglobin (HbB) or other non-polymerizing β-globin variant may increase the likelihood of beneficial outcomes for SCD patients. Adenine base editors (ABEs) can precisely convert the mutant A-T base pair responsible for SCD to a G-C base pair, thus generating the hemoglobin variant, Hb G-Makassar, a naturally occurring β-globin variant that is not associated with human disease. Our studies have identified ABEs that can achieve highly efficient Makassar editing (>70%) of the sickle mutation in both sickle trait (HbAS) and homozygous sickle (HbSS) patient CD34+ cells with high cell viability and recovery and without perturbation of immunophenotypic hematopoietic stem and progenitor cell (HSPC) frequencies after ex vivo delivery of guide RNA and mRNA encoding the ABE. Furthermore, Makassar editing was retained throughout erythropoiesis in bulk in vitro erythroid differentiated cells (IVED) derived from edited CD34+ cells. To gain an understanding of allelic editing at a single clone resolution, we assessed editing frequencies of clones from both single cell expansion in erythroid differentiation media, as well as from single BFU-E colonies. We found that we could achieve >70% of colonies with bi-allelic Makassar editing and approximately 20% of colonies with mono-allelic Makassar editing, while <3% of colonies remained completely unedited. Previously, conventional hemoglobin capillary electrophoresis and high-performance liquid chromatography (HPLC) were unable to distinguish between HbS and HbG-Makassar. Here, we developed an ultra-high-performance liquid chromatography (UPLC) method that resolves sickle globin (HbS) from Hb G-Makassar globin in IVED cells. The Makassar globin variant was further confirmed by liquid chromatography mass spectrometry (LC-MS). By developing this new method to resolve these two β-globin variants in edited HbSS cells, we were able to detect, in bulk IVED cultures, >80% abundance Hb G-Makassar of total β-globins, which corresponded to a concomitant reduction of HbS levels to <20%. Furthermore, we were also able to determine globin abundance as well as allelic editing at the level of single clones and found that HbS was completely eliminated in >70% of cells that had bi-allelic Makassar editing. Moreover, in the approximately 20% of colonies that were found to be mono-allelically edited for the Makassar variant, there was a 60:40 ratio of Hb G-Makassar:HbS globin abundance in individual clones, at levels remarkably similar to the HbA(wildtype HbB):HbS levels found in HbAS individuals, with minimal observable in vitro sickling when exposed to hypoxia. Thus, with our ABEs, we were able to reduce HbS to <40% on a per cell basis in >90% of IVED cells and found that in vitro sickling under hypoxia inversely correlated with the level of Hb G-Makassar globin variant installation and corresponding reduction in HbS levels. By converting HbS to Hb G-Makassar, our direct and precise editing strategy replaces a pathogenic β-globin with one that has been shown to have normal hematologic parameters. Coupled with autologous stem cell transplant, this next generation gene editing strategy presents a promising new modality for treating patients with SCD. Disclosures Chu: Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lam:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Packer:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Olins:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Liquori:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Marshall:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Lee:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Yan:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Decker:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gantzer:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Haskett:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Bohnuud:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Born:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Barrera:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Slaymaker:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Gaudelli:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Hartigan:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company. Ciaramella:Beam Therapeutics: Current Employment, Current equity holder in publicly-traded company.

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