Baboon Envelope Pseudotyped “Nanoblades” Carrying Cas9/gRNA Complexes Allow Efficient Genome Editing in Human T, B, and CD34+ Cells and Knock-in of AAV6-Encoded Donor DNA in CD34+ Cells

Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into human blood cells can be challenging. Here, we have utilized “nanoblades,” a new technology that delivers a genomic cleaving agent into cells. These are modified murine leukemia virus (MLV) or HIV-derived virus-like particle (VLP), in which the viral structural protein Gag has been fused to Cas9. These VLPs are thus loaded with Cas9 protein complexed with the guide RNAs. Highly efficient gene editing was obtained in cell lines, IPS and primary mouse and human cells. Here, we showed that nanoblades were remarkably efficient for entry into human T, B, and hematopoietic stem and progenitor cells (HSPCs) thanks to their surface co-pseudotyping with baboon retroviral and VSV-G envelope glycoproteins. A brief incubation of human T and B cells with nanoblades incorporating two gRNAs resulted in 40 and 15% edited deletion in the Wiskott-Aldrich syndrome (WAS) gene locus, respectively. CD34+ cells (HSPCs) treated with the same nanoblades allowed 30–40% exon 1 drop-out in the WAS gene locus. Importantly, no toxicity was detected upon nanoblade-mediated gene editing of these blood cells. Finally, we also treated HSPCs with nanoblades in combination with a donor-encoding rAAV6 vector resulting in up to 40% of stable expression cassette knock-in into the WAS gene locus. Summarizing, this new technology is simple to implement, shows high flexibility for different targets including primary immune cells of human and murine origin, is relatively inexpensive and therefore gives important prospects for basic and clinical translation in the area of gene therapy.

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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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Differential Requirements for Survivin in Hematopoietic Cell Development.

Blood ◽

10.1182/blood.v104.11.1257.1257 ◽

2004 ◽

Vol 104 (11) ◽

pp. 1257-1257

Author(s):

Yanfei Xu ◽

Sandeep Gurbuxani ◽

Ganesan Keerthivasan ◽

Amittha Wickrema ◽

John D. Crispino

Keyword(s):

Cell Cycle ◽

Red Blood Cells ◽

Blood Cells ◽

Caspase 3 ◽

Terminal Differentiation ◽

Dependent Manner ◽

Erythroid Cells ◽

Hematopoietic Stem ◽

Stage Of Development ◽

Primary Mouse

Abstract The development of the complete repertoire of blood cells from a common progenitor, the hematopoietic stem cell, is a tightly controlled process that is regulated, in part, by the activity of lineage specific transcription factors. Despite our knowledge of these factors, the mechanisms that regulate the formation and growth of distinct, but closely related lineages, such as erythroid cells and megakaryocytes, remain largely uncharacterized. Here we show that Survivin, a member of the inhibitor of apoptosis (IAP) family that also plays an essential role in cytokinesis, is differentially expressed during erythroid versus megakaryocyte development. Erythroid cells express Survivin throughout their maturation, up to the terminal stage of differentiation (orthochromatic), even after the cells exit the cell cycle. This is surprising because Survivin is generally expressed in a cell cycle dependent manner and not thought to be expressed in terminally differentiated cells. In contrast, purified murine megakaryocytes express nearly 5-fold lower levels of Survivin mRNA compared to erythroid cells. To investigate whether Survivin is involved in the differentiation and/or survival of hematopoietic progenitors, we infected primary mouse bone marrow cells with retroviruses harboring either the human Survivin cDNA or a mouse Survivin shRNA, and then induced erythroid and megakaryocyte differentiation in both liquid culture and colony-forming assays. These studies revealed that overexpression of Survivin promoted the terminal differentiation of red blood cells, while its reduction, by RNA interference, inhibited their differentiation. In contrast, downregulation of Survivin facilitated the expansion of megakaryocytes, and its overexpression antagonized megakaryocyte formation. In addition, consistent with a role for survivin in erythropoiesis, downregulation of Survivin expression in MEL cells led to a block in terminal differentiation. Finally, since caspase activity is known to be required for erythroid maturation, we investigated whether survivin associated with cleaved caspase-3 in erythroid cells. Immunofluorescence revealed that Survivin and cleaved caspase-3 co-localized to discrete foci within the cytoplasm of erythroid cells at the orthochromatic stage of development. Based on these findings, we hypothesize that Survivin cooperates with cleaved caspase-3 in terminal maturation of red blood cells. Together, our findings demonstrate that Survivin plays multiple, distinct roles in hematopoiesis.

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A Modified HIV1-Based Lentiviral Vector Can Transduce Rhesus Hematopoietic Repopulating Cells as Efficiently as An SIV Vector in An Autologous Transplantation Model.

Blood ◽

10.1182/blood.v114.22.692.692 ◽

2009 ◽

Vol 114 (22) ◽

pp. 692-692

Author(s):

Naoya Uchida ◽

Phillip W Hargrove ◽

Kareem Washington ◽

Coen J. Lap ◽

Matthew M. Hsieh ◽

...

Keyword(s):

T Cells ◽

Blood Cells ◽

Conflicts Of Interest ◽

Lentiviral Vector ◽

Autologous Transplantation ◽

Vector System ◽

Transduction Efficiency ◽

Large Animal ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 692 HIV1-based vectors transduce rhesus hematopoietic stem cells poorly due to a species specific block by restriction factors, such as TRIM5αa which target HIV1 capsid proteins. The use of simian immunodeficiency virus (SIV)-based vectors can circumvent this restriction, yet use of this system precludes the ability to directly evaluate HIV1-based lentiviral vectors prior to their use in human clinical trials. To address this issue, we previously developed a chimeric HIV1 vector (χHIV vector) system wherein the HIV1-based lentiviral vector genome is packaged in the context of SIV capsid sequences. We found that this allowed χHIV vector particles to escape the intracellular defense mechanisms operative in rhesus hematopoietic cells as judged by the efficient transduction of both rhesus and human CD34+ cells. Following transplantation of rhesus animals with autologous cell transduced with the χHIV vector, high levels of marking were observed in peripheral blood cells (J Virol. 2009 Jul. in press). To evaluate whether χHIV vectors could transduce rhesus blood cells as efficiently as SIV vectors, we performed a competitive repopulation assay in two rhesus macaques for which half of the CD34+ cells were transduced with the standard SIV vector and the other half with the χHIV vector both at a MOI=50 and under identical transduction conditions. The transduction efficiency for rhesus CD34+ cells before transplantation with the χHIV vector showed lower transduction rates in vitro compared to those of the SIV vector (first rhesus: 41.9±0.83% vs. 71.2±0.46%, p<0.01, second rhesus: 65.0±0.51% vs. 77.0±0.18%, p<0.01, respectively). Following transplantation and reconstitution, however, the χHIV vector showed modestly higher gene marking levels in granulocytes (first rhesus: 12.4% vs. 6.1%, second rhesus: 36.1% vs. 27.2%) and equivalent marking levels in lymphocytes, red blood cells (RBC), and platelets, compared to the SIV vector at one month (Figure). Three to four months after transplantation in the first animal, in vivo marking levels plateaued, and the χHIV achieved 2-3 fold higher marking levels when compared to the SIV vector, in granulocytes (6.9% vs. 2.8%) and RBCs (3.3% vs. 0.9%), and equivalent marking levels in lymphocytes (7.1% vs. 5.1%) and platelets (2.8% vs. 2.5)(Figure). Using cell type specific surface marker analysis, the χHIV vector showed 2-7 fold higher marking levels in CD33+ cells (granulocytes: 5.4% vs. 2.7%), CD56+ cells (NK cells: 6.5% vs. 3.2%), CD71+ cells (reticulocyte: 4.5% vs. 0.6%), and RBC+ cells (3.6% vs. 0.9%), and equivalent marking levels in CD3+ cells (T cells: 4.4% vs. 3.3%), CD4+ cells (T cells: 3.9% vs. 4.6%), CD8+ cells (T cells: 4.2% vs. 3.9%), CD20+ cells (B cells: 7.6% vs. 4.8%), and CD41a+ cells (platelets: 3.5% vs. 2.2%) 4 months after transplantation. The second animal showed a similar pattern with higher overall levels (granulocytes: 32.8% vs. 19.1%, lymphocytes: 24.4% vs. 17.6%, RBCs 13.1% vs. 6.8%, and platelets: 14.8% vs. 16.9%) 2 months after transplantation. These data demonstrate that our χHIV vector can efficiently transduce rhesus long-term progenitors at levels comparable to SIV-based vectors. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in the large animal model, especially for granulocytic or RBC diseases. Disclosures: No relevant conflicts of interest to declare.

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Long-Term Reconstitution of Transduced Rhesus CD34+ Cells Mobilized by G-CSF and Plerixafor.

Blood ◽

10.1182/blood.v116.21.1449.1449 ◽

2010 ◽

Vol 116 (21) ◽

pp. 1449-1449

Author(s):

Naoya Uchida ◽

Aylin Bonifacino ◽

Allen E Krouse ◽

Sandra D Price ◽

Ross M Fasano ◽

...

Keyword(s):

Stem Cells ◽

Stem Cell ◽

Progenitor Cells ◽

Blood Cells ◽

Transgene Expression ◽

Hematopoietic Stem ◽

Expression Levels ◽

Cd34 Cells ◽

One Year

Abstract Abstract 1449 Granulocyte colony-stimulating factor (G-CSF) in combination with plerixafor (AMD3100) produces significant mobilization of peripheral blood stem cells in the rhesus macaque model. The CD34+ cell population mobilized possesses a unique gene expression profile, suggesting a different proportion of progenitor/stem cells. To evaluate whether these CD34+ cells can stably reconstitute blood cells, we performed hematopoietic stem cell transplantation using G-CSF and plerixafor-mobilized rhesus CD34+ cells that were transduced with chimeric HIV1-based lentiviral vector including the SIV-capsid (χHIV vector). In our experiments, G-CSF and plerixafor mobilization (N=3) yielded a 2-fold higher CD34+ cell number, compared to that observed for G-CSF and stem cell factor (SCF) combination (N=5) (8.6 ± 1.8 × 107 vs. 3.6 ± 0.5 × 107, p<0.01). Transduction rates with χHIV vector, however, were 4-fold lower in G-CSF and plerixafor-mobilized CD34+ cells, compared to G-CSF and SCF (13 ± 4% vs. 57 ± 5%, p<0.01). CD123+ (IL3 receptor) rates were higher in CD34+ cells mobilized by G-CSF and plerixafor (16.4%) or plerixafor alone (21.3%), when compared to G-CSF alone (2.6%). To determine their repopulating ability, G-CSF and plerixafor-mobilized CD34+ cells were transduced with EGFP-expressing χHIV vector at MOI 50 and transplanted into lethally-irradiated rhesus macaques (N=3). Blood counts and transgene expression levels were followed for more than one year. Animals transplanted with G-CSF and plerixafor-mobilized cells showed engraftment of all lineages and earlier recovery of lymphocytes, compared to animals who received G-CSF and SCF-mobilized grafts (1200 ± 300/μl vs. 3300 ± 900/μl on day 30, p<0.05). One month after transplantation, there was a transient development of a skin rash, cold agglutinin reaction, and IgG and IgM type plasma paraproteins in one of the three animals transplanted with G-CSF and plerixafor cells. This animal had the most rapid lymphocyte recovery. These data suggested that G-CSF and plerixafor-mobilized CD34+ cells contained an increased amount of early lymphoid progenitor cells, compared to those arising from the G-CSF and SCF mobilization. One year after transplantation, transgene expression levels were 2–5% in the first animal, 2–5% in the second animal, and 5–10% in the third animal in all lineage cells. These data indicated G-CSF and plerixafor-mobilized CD34+ cells could stably reconstitute peripheral blood in the rhesus macaque. Next, we evaluated the correlation of transgene expression levels between in vitro bulk CD34+ cells and lymphocytes at one month, three months, and six months post-transplantation. At one and three months after transplantation, data from G-CSF and plerixafor mobilization showed higher ratio of %EGFP in lymphocytes to that of in vitro CD34+ cells when compared to that of G-CSF and SCF mobilization. At six months after transplantation the ratios were similar. These results again suggest that G-CSF and plerixafor-mobilized CD34+ cells might include a larger proportion of early lymphoid progenitor cells when compared to G-CSF and SCF mobilization. In summary, G-CSF and plerixafor mobilization increased CD34+ cell numbers. G-CSF and plerixafor-mobilized CD34+ cells contained an increased number of lymphoid progenitor cells and a hematopoietic stem cell population that was capable of reconstituting blood cells as demonstrated by earlier lymphoid recovery and stable multilineage transgene expression in vivo, respectively. Our findings should impact the development of new clinical mobilization protocols. Disclosures: No relevant conflicts of interest to declare.

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High Transgene Expression Rates After Extended Follow up Among Rhesus Macaque Recipients of Autologous Hematopoietic Stem Cells Transduced with a Modified HIV1-Based Lentiviral Vector

Blood ◽

10.1182/blood.v118.21.3118.3118 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3118-3118

Author(s):

Naoya Uchida ◽

Phillip W. Hargrove ◽

Coen J. Lap ◽

Oswald Phang ◽

Aylin C. Bonifacino ◽

...

Keyword(s):

Peripheral Blood ◽

Blood Cells ◽

Transgene Expression ◽

Vector System ◽

Large Animal ◽

Peripheral Blood Cells ◽

Hematopoietic Stem ◽

Integration Pattern ◽

Cd34 Cells ◽

Integration Sites

Abstract Abstract 3118 Hematopoietic stem cell (HSC)-targeted gene therapy is potentially curative for the hemoglobin disorders; however, highly efficient, lineage specific globin expression remains elusive, and large animal models thus remain important for further development toward clinical application. We previously constructed a chimeric HIV1 vector (χHIV vector) system to circumvent a species specific restriction to HIV1-based vectors wherein the HIV1 vector genome is packaged in the context of the simian immunodeficiency virus (SIV) capsid for efficient transduction of rhesus CD34+ cells in vitro (J Virol. 2009) and in vivo (ASH 2009). In this study, we sought to evaluate transduction efficiency and vector integration pattern among long-term repopulating cells in the rhesus HSC transplantation model. We followed up transgene expression rates among peripheral blood cells of three animals for 1.5–2 years. For two animals (RQ7307 and RQ7280), half of the CD34+ cells were transduced with a standard SIV vector and the other half with the χHIV vector using the same protocol. Transduced cells were transplanted into lethally irradiated rhesus macaques, as previously described (J Virol. 2009). The transgene expression rates in peripheral blood cells plateaued 3–4 months after transplantation and similar transgene expression rates continued in all cell lineages for at least 1.5 years (Figure). The χHIV vector demonstrated that 2–3 fold higher transgene expression rates were seen in granulocytes (RQ7307: 8.6±0.2% vs. 3.1±0.1%, RQ7280: 27.9±0.7% vs. 18.4±0.2%) and RBCs (RQ7307: 3.3±0.1% vs. 0.9±0.0%, RQ7280: 10.0±0.1% vs. 4.0±0.1%), and equivalent transgene expression rates in lymphocytes (RQ7307: 7.8±0.2% vs. 4.5±0.1%, RQ7280: 22.4±0.5% vs. 17.6±0.3%) and platelets (RQ7307: 3.1±0.1% vs. 2.7±0.1%, RQ7280: 12.3±0.2% vs. 16.8±0.2%), compared to the SIV vector. The average vector copy numbers in transduced cells were 4.6–5.7 for the χHIV vector and 1.5–2.0 for the SIV vector in both transplanted animals, evaluated by Southern blot analysis. We then performed transplantation of rhesus CD34+ cells which were transduced with the χHIV vector alone to evaluate transgene expression and vector integration pattern. Transgene expression rates among peripheral blood cells in this animal (RQ7387) plateaued 1–3 months after transplantation, with stable high transgene expression rates of 51.7±1.2% in granulocytes, 54.7±0.1% in lymphocytes, 22.1±0.2% in RBCs, and 19.1±0.1% platelets for 2 years after transplantation. Multi-lineage marking was observed by flow cytometric analysis. We then evaluated integration sites for the χHIV vector in the recipient of χHIV vector alone transduced cells by linear amplification mediated-PCR, using peripheral blood cells of RQ7387 in 0.5–1.5 years after transplantation. We found a total of 344 integration sites for the χHIV vector, and our data demonstrated that the χHIV vector integrated into gene regions, especially introns, when compared to the integration pattern of computer-generated random controls (p<0.001). On the other hand, our data revealed fewer integrations of the χHIV vector into ≤30kb upstream of genes (p<0.001) and into the upstream regions of transcription start sites. Most of the integration sites had low gene density (0–10 genes within 1 Mb upstream or downstream of integration sites, p<0.01), compared to that of random controls. No specific trend was noted for the number of integration sites around CpG islands and the number of CISs around integration sites. These data suggest that the χHIV vector has integration patterns comparable to HIV1 and SIV vectors. In summary, our χHIV vector shows efficient transduction for rhesus long-term repopulating cells, achieving sufficient levels for therapeutic effects in gene therapy trials for globin disorders. This χHIV vector system should allow preclinical testing of HIV1-based therapeutic vectors in large animal models. Disclosures: No relevant conflicts of interest to declare.

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Direct growth suppression of myeloid bone marrow progenitor cells but not cord blood progenitors by human cytomegalovirus in vitro

Blood ◽

10.1182/blood.v88.7.2510.bloodjournal8872510 ◽

1996 ◽

Vol 88 (7) ◽

pp. 2510-2516 ◽

Cited By ~ 11

Author(s):

M Holberg-Petersen ◽

H Rollag ◽

S Beck ◽

I Overli ◽

G Tjonnfjord ◽

...

Keyword(s):

Bone Marrow ◽

Cord Blood ◽

Clinical Isolate ◽

Human Cytomegalovirus ◽

Mononuclear Cells ◽

Colony Growth ◽

Structural Protein ◽

Hematopoietic Stem ◽

Cd34 Cells

Recently, considerable interest has arisen as to use cord blood (CB) as a source of hematopoietic stem cells for allogenic transplantation when bone marrow (BM) from a familial HLA-matched donor is not available. Because human cytomegalovirus (HCMV) has been shown to inhibit the proliferation of BM progenitors in vitro, it was important to examine whether similar effect could be observed in HCMV-infected CB cells. Therefore, the effect of HCMV challenge on the proliferation of myeloid progenitors from BM and CB was compared using both mononuclear cells (MNC) and purified CD34+ cells. A clinical isolate of HCMV inhibited the colony formation of myeloid BM progenitors responsive to granulocyte-macrophage colony-stimulating factor (CSF), granulocyte-CSF, macrophage-CSF, interleukin-3 (IL-3) and the combination of IL-3 and stem cell factor (SCF). In contrast, colony growth of CB progenitors was not affected. In addition, HCMV inhibited directly the growth of purified BM CD34+ cells responsive to IL-3 and SCF in single cell assay by 40%, wheras the growth of CD34+ progenitors obtained from CB was not suppressed. The HCMV lower matrix structural protein pp65 and HCMV DNA were detected in both CB and BM CD34+ cells after in vitro challenge. However, neither immediate early (IE)-mRNA nor IE proteins were observed in infected cells. Cell cyclus examination of BM and CB CD34+ cells revealed that 25.7% of BM progenitors were in S + G2/ M phase wheras only 10.7% of the CB progenitors. Thus, a clinical isolate of HCMV directly inhibited the proliferation of myeloid BM progenitors in vitro wheras CB progenitors were not affected. This difference in the susceptibility of CB and BM cells to HCMV may partly be caused by the slow cycling rate of naive CB progenitors compared to BM progenitors at the time of infection.

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Preclinical Studies for Sickle Cell Disease Gene Therapy Using Bone Marrow CD34+ Cells Modified with a βAS3-Globin Lentiviral Vector

Blood ◽

10.1182/blood.v118.21.3119.3119 ◽

2011 ◽

Vol 118 (21) ◽

pp. 3119-3119

Author(s):

Fabrizia Urbinati ◽

Zulema Romero Garcia ◽

Sabine Geiger ◽

Rafael Ruiz de Assin ◽

Gabriela Kuftinec ◽

...

Keyword(s):

Stem Cells ◽

Gene Therapy ◽

Sickle Cell Disease ◽

Sickle Cell ◽

Blood Cells ◽

Globin Gene ◽

Cell Disease ◽

Hematopoietic Stem ◽

Cd34 Cells

Abstract Abstract 3119 BACKGROUND: Sickle cell disease (SCD) affects approximately 80, 000 Americans, and causes significant neurologic, pulmonary, and renal injury, as well as severe acute and chronic pain that adversely impacts quality of life. Because SCD results from abnormalities in red blood cells, which in turn are produced from adult hematopoietic stem cells, hematopoietic stem cell transplant (HSCT) from a healthy (allogeneic) donor can benefit patients with SCD, by providing a source for life-long production of normal red blood cells. However, allogeneic HSCT is limited by the availability of well-matched donors and by immunological complications of graft rejection and graft-versus-host disease. Thus, despite major improvements in clinical care, SCD continues to cause significant morbidity and early mortality. HYPOTHESIS: We hypothesize that autologous stem cell gene therapy for SCD has the potential to treat this illness without the need for immune suppression of current allogeneic HSCT approaches. Previous studies have demonstrated that addition of a β-globin gene, modified to have the anti-sickling properties of fetal (γ-) globin (βAS3), to bone marrow (BM) stem cells in murine models of SCD normalizes RBC physiology and prevents the manifestations of sickle cell disease (Levassuer Blood 102 :4312–9, 2003). The present work seeks to provide pre-clinical evidence of efficacy for SCD gene therapy using human BM CD34+ cells modified with the bAS3 lentiviral (LV) vector. RESULTS: The βAS3 globin expression cassette was inserted into the pCCL LV vector backbone to confer tat-independence for packaging. The FB (FII/BEAD-A) composite enhancer-blocking insulator was inserted into the 3' LTR (Ramezani, Stem Cells 26 :32–766, 2008). Assessments were performed transducing human BM CD34+ cells from healthy or SCD donors with βAS3 LV vectors. Efficient (1–3 vector copies/cell) and stable gene transmission were determined by qPCR and Southern Blot. CFU assays demonstrated that βAS3 gene modified SCD CD34+ cells are fully capable of maintaining their hematopoietic potential. To demonstrate the effectiveness of the erythroid-specific bAS3 gene in the context of human HSPC (Hematopoietic Stem and Progenitor Cells), we optimized an in vitro model of erythroid differentiation of huBM CD34+ cells. We successfully obtained an expansion up to 700 fold with >80% fully mature enucleated RBC derived from CD34+ cells obtained from healthy or SCD BM donors. We then assessed the expression of the βAS3 globin gene by isoelectric focusing: an average of 18% HbAS3 over the total globin present (HbS, HbA2) per Vector Copy Number (VCN) was detected in RBC derived from SCD BM CD34+. A qRT-PCR assay able to discriminate HbAS3 vs. HbA RNA, was also established, confirming the quantitative expression results obtained by isoelectric focusing. Finally, we show morphologic correction of in vitro differentiated RBC obtained from SCD BM CD34+ cells after βAS3 LV transduction; upon induction of deoxygenation, cells derived from SCD patients showed the typical sickle shape whereas significantly reduced numbers were detected in βAS3 gene modified cells. Studies to investigate risks of insertional oncogenesis from gene modification of CD34+ cells by βAS3 LV vectors are ongoing as are in vivo studies to demonstrate the efficacy of βAS3 LV vector in the NSG mouse model. CONCLUSIONS: This work provides initial evidence for the efficacy of the modification of human SCD BM CD34+ cells with βAS3 LV vector for gene therapy of sickle cell disease. This work was supported by the California Institute for Regenerative Medicine Disease Team Award (DR1-01452). Disclosures: No relevant conflicts of interest to declare.

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Enhanced Homology-directed Repair for Highly Efficient Gene Editing in Hematopoietic Stem/Progenitor Cells

Blood ◽

10.1182/blood.2020008503 ◽

2021 ◽

Author(s):

Suk See De Ravin ◽

Julie Brault ◽

Ronald J Meis ◽

Siyuan Liu ◽

Linhong Li ◽

...

Keyword(s):

Gene Editing ◽

Hematopoietic Stem ◽

Vector Integration ◽

Homology Directed Repair ◽

Highly Efficient ◽

Donor Control ◽

Efficient Gene ◽

Cd34 Cells ◽

Viable Approach

Lentivector gene therapy for X-linked chronic granulomatous disease (X-CGD) has proven to be a viable approach, but random vector integration and subnormal protein production from exogenous promoters in transduced cells remain concerning for long-term safety and efficacy. A previous genome editing-based approach using SpCas9 and an oligodeoxynucleotide donor to repair genetic mutations demonstrated the capability to restore physiological protein expression, but lacked sufficient efficiency in quiescent CD34+ hematopoietic cells for clinical translation. Here, we show transient inhibition of p53-binding protein 1 (53BP1) significantly increased (2.3-fold) long-term homology directed repair (HDR) to achieve highly efficient (80% gp91phox+ cells compared to healthy donor control) long-term correction of X-CGD CD34+ cells.

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Highly Efficient Editing of the Beta-Globin Gene in Patient Derived Hematopoietic Stem and Progenitor Cells to Treat Sickle Cell Disease

Blood ◽

10.1182/blood-2018-99-117371 ◽

2018 ◽

Vol 132 (Supplement 1) ◽

pp. 2192-2192

Author(s):

So Hyun Park ◽

Ciaran M Lee ◽

Daniel P. Dever ◽

Timothy H Davis ◽

Joab Camarena ◽

...

Keyword(s):

Sickle Cell Disease ◽

Progenitor Cells ◽

Gene Editing ◽

Globin Gene ◽

Cell Disease ◽

Gene Correction ◽

Hematopoietic Stem ◽

Genome Wide ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract Sickle cell disease (SCD) is an inherited blood disorder associated with a debilitating chronic illness. SCD is caused by a point mutation in the β-globin gene (HBB). A single nucleotide substitution converts glutamic acid to a valine that leads to the production of sickle hemoglobin (HbS), which impairs the function of red blood cells. Here we show that delivery of Streptococcus pyogenes (Sp) Cas9 protein and CRISPR guide RNA as a ribonucleoprotein complex (RNP) together with a short single-stranded DNA donor (ssODN) template into CD34+ hematopoietic stem and progenitor cells (HSPCs) from SCD patients' bone marrow (BM) was able to correct the sickling HBB mutation, with up to 33% homology directed repair (HDR) without selection. Further, CRISPR/Cas9 cutting of HBB in SCD HSPCs induced gene conversion between the HBB sequences in the vicinity of the target locus and the homologous region in δ-globin gene (HBD), with up to 4.4% additional gene correction mediated by the HBD conversion in cells with Cas9 cutting only. The erythrocytes derived from gene-edited cells showed a marked reduction of the HbS level, increased expression of normal adult hemoglobin (HbA), and a complete loss of cell sickling, demonstrating the potential in curing SCD. We performed extensive off-target analysis of gene-edited SCD HSPCs using the in-silico prediction tool COSMID and unbiased, genome-wide assay Guide-Seq, revealing a gross intrachromosomal rearrangement event between the on- and off-target Cas9 cutting sites. We used a droplet digital PCR assay to quantify deletion and inversion events from Day 2 to Day 12 after RNP delivery, and found that large chromosomal deletion decreased from 1.8% to 0.2%, while chromosomal inversion maintained at 3.3%. We demonstrated that the use of high-fidelity SpCas9 (HiFi Cas9 by IDT) significantly reduced off-target effects and completely eliminated the intrachromosome rearrangement events, while maintaining the same level of on-target gene editing, leading to high-efficiency gene correction with increased specificity. In order to determine if gene-corrected SCD HSPCs retain the ability to engraft, CD34+ cells from the BM of SCD patients were treated with Cas9/gRNA RNP and ssODN donor for HBB gene correction, cryopreserved at Day 2 post genome editing, then intravenously transplanted into NSG mice shortly after thawing. These mice were euthanized at Week 16 after transplantation, and the BM was harvested to determine the engraftment potential. An average of 7.5 ±5.4% of cells were double positive for HLA and hCD45 in mice injected with gene-edited CD34+ cells, compared to 16.8 ±9.3% with control CD34+ cells, indicating a good level of engraftment of gene-corrected SCD HSPCs. A higher fraction of human cells were positive for CD19 (66 ±28%), demonstrating lymphoid lineage bias. DNA was extracted from unsorted cells, CD19 or CD33 sorted cells for gene-editing analysis; the HBB editing rates were respectively 29.8% HDR, 2.4% HBD conversion, and 42.8% non-homologous end joining (NHEJ) pretransplantation, and editing rates at Week 16 posttransplantation were respectively 8.8 ±12% HDR, 1.8 ±1.7% HBD conversion, and 24.5 ±12% NHEJ. The highly variable editing rate and indel diversity in gene-edited cells at Week 16 in all four transplanted mice suggest clonal dominance of a limited number of HSPCs after transplantation. Taken together, our results demonstrate highly efficient gene and phenotype correction of the sickling mutation in BM HSPCs from SCD patients mediated by HDR and HBD conversion, and the ability of gene-edited SCD HSPCs to engraft in vivo. We also demonstrate the importance of genome-wide analysis for off-target analysis and the use of HiFi Cas9. Our results provide further evidence for the potential of moving genome editing-based SCD treatment into clinical practice. Acknowledgments: This work was supported by the Cancer Prevention and Research Institute of Texas grants RR140081 and RP170721 (to G. B.), and the National Heart, Lung and Blood Institute of NIH (1K08DK110448 to V.S.) Disclosures Porteus: CRISPR Therapeutics: Consultancy, Membership on an entity's Board of Directors or advisory committees.

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Administration of Granulocyte Colony-Stimulating Factor Post-Transplant Impedes Engraftment of CRISPR-Cas9 Edited Long-Term Repopulating Hematopoietic Stem and Progenitor Cells

Blood ◽

10.1182/blood-2021-147352 ◽

2021 ◽

Vol 138 (Supplement 1) ◽

pp. 2936-2936

Author(s):

Daisuke Araki ◽

Christi Salisbury-Ruf ◽

Waleed Hakami ◽

Keyvan Keyvanfar ◽

Richard H. Smith ◽

...

Keyword(s):

Gene Editing ◽

Treated Group ◽

Untreated Group ◽

Self Renewal ◽

Hematopoietic Stem ◽

Post Transplant ◽

Cell Engraftment ◽

Cd34 Cells ◽

Stem And Progenitor Cells

Abstract Transplantation of genetically modified autologous hematopoietic stem and progenitor cells (HSPCs) holds a curative potential for subjects with inherited blood disorders. In recent years, transfer of a therapeutic gene to HSPCs has been successfully achieved using replication-incompetent integrating lentiviral vectors. More recently, advances have emerged to more precisely edit cellular genomes by specific correction of mutations or targeted gene addition at endogenous genomic loci. However, cellular processes triggered in HSPCs by the programmable nucleases utilized in these gene editing approaches may negatively impact their ability to reconstitute and maintain hematopoiesis long-term in recipient hosts. Granulocyte colony-stimulating factor (G-CSF) use after autologous HSPC transplantation is generally recommended to shorten the duration of severe neutropenia. However, little is known about the safety and efficacy of G-CSF use after transplantation of genetically modified autologous HSPCs. G-CSF is the principal cytokine regulating granulopoiesis, but also plays an important role in regulating hematopoietic stem cell (HSC) function (Schuttpelz, Leukemia 2014). Studies have suggested that G-CSF can exacerbate HSC damage caused by chemotherapeutic agents and irradiation by promoting differentiation at the expense of self-renewal and by inducing cellular senescence (van Os, Stem Cells 2000; Li, Cell Biosci 2015). Here, we asked whether G-CSF use after transplantation of gene edited HSPCs may negatively affect their long-term repopulating (LTR) and self-renewal capacities. To assess the effect of G-CSF use post-transplant on HSPC repopulating function after gene editing, mobilized human CD34+ cells were stimulated for 2 days, electroporated with AAVS1-specific sgRNA/Cas9 ribonucleoprotein complexes, and subsequently transplanted into NSG mice following busulfan conditioning. We subcutaneously injected G-CSF (125 mcg/kg/day) or PBS from post-transplant day 1 to 14 and compared hematopoietic reconstitution between both groups. The use of G-CSF initially increased human CD45+ cells in peripheral blood (PB) at 2 weeks post-transplant by enhancing CD13+ myeloid cell proliferation from committed progenitors (Fig. A). However, starting at 10 weeks post-transplant when hematopoiesis begins to emerge from the most primitive HSPCs, administration of G-CSF resulted in a 3 to 4-fold reduction in PB human cell engraftment compared to untreated mice (Fig. A). Similarly, G-CSF treated mice had significantly lower bone marrow (BM) and splenic engraftment at the endpoint (22 weeks) analysis, with comparable editing efficiency and lineage composition detected within human CD45+ cells (Fig. B, C). Importantly, percentages of immunophenotypic HSCs were 2-fold lower within the BM of G-CSF treated mice relative to the untreated group (Fig. D). To determine whether the negative effect of G-CSF post-transplant is specific to CRISPR-Cas9 gene editing, similar experiments were conducted using unmanipulated CD34+ cells or CD34+ cells transduced with a lentivirus vector expressing GFP. Interestingly, we found no differences in engraftment levels or immunophenotypic HSC frequencies between G-CSF treated and untreated mice. To assess the self-renewal capacity and quantify the frequency of gene edited LTR-HSCs, human CD45+ cells obtained from the BM of primary mice were serially transplanted into secondary recipient (NBSGW) mice at limiting dilution and BM engraftment was analyzed at 20 weeks post-transplant (total period of engraftment was 42 weeks). Notably, the secondary mice in the untreated group showed significantly superior human CD45+ cell engraftment compared with those in G-CSF treated group at the highest dose tested (Fig. E). The extreme limiting dilution analysis indicated that the frequency of LTR-HSCs was 5.1-fold higher (p = 0.011) in the untreated group compared with G-CSF treated group (Fig. F, G). Considering total engraftment in primary mice and the frequency of edited LTR-HSCs in secondary mice, we estimated the frequency of edited LTR-HSCs was reduced by 10-fold with G-CSF administration post-transplant. Collectively, our data suggest that G-CSF use post-transplant significantly reduces LTR and self-renewal capacities of CRISPR-Cas9 gene edited HSPCs. This understanding could have important clinical implications in HSPC gene therapy protocol. Figure 1 Figure 1. Disclosures No relevant conflicts of interest to declare.

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