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Successful expansion of bone marrow (BM) hematopoietic stem and progenitor cells (HSPCs) would benefit many HSPC transplantation and gene therapy/editing applications. However, current expansion technologies have been limited by a loss of multipotency and self-renewal properties ex vivo. We hypothesized that an ex vivo vascular niche would provide prohematopoietic signals to expand HSPCs while maintaining multipotency and self-renewal. To test this hypothesis, BM autologous CD34+ cells were expanded in endothelial cell (EC) coculture and transplanted in nonhuman primates. CD34+C38− HSPCs cocultured with ECs expanded up to 17-fold, with a significant increase in hematopoietic colony-forming activity compared with cells cultured with cytokines alone (colony-forming unit-granulocyte-erythroid-macrophage-monocyte; p < .005). BM CD34+ cells that were transduced with green fluorescent protein lentivirus vector and expanded on ECs engrafted long term with multilineage polyclonal reconstitution. Gene marking was observed in granulocytes, lymphocytes, platelets, and erythrocytes. Whole transcriptome analysis indicated that EC coculture altered the expression profile of 75 genes in the BM CD34+ cells without impeding the long-term engraftment potential. These findings show that an ex vivo vascular niche is an effective platform for expansion of adult BM HSPCs.

Engineered T cell therapies have been remarkably successful in the treatment of B cell malignancies, yet lack of control over these “living drugs” can lead to significant toxicities or limited efficacy. One particular challenge is the achievement of durable anti-tumor responses because the reduction of tumor burden results in reduced antigen stimulation and therefore reduced antigen-dependent T cell expansion. Interleukin-15 (IL15) drives T and NK cell expansion and persistence in an antigen-independent manner, however unregulated expression of this cytokine may compromise the safety and efficacy of cellular immunotherapy. To address these issues, we engineered T cells with a pharmacologically controllable, membrane-bound IL15 that supports antigen-independent T cell expansion and has the potential to reduce the safety risks associated with continuous exposure to soluble IL15. Our approach utilizes Drug Responsive Domains (DRDs) which are fully human protein domains that are inherently unstable in the cell but are reversibly stabilized when bound to specific FDA-approved drugs. Fusion of a DRD to a protein of interest confers drug-dependent, reversible regulation of protein expression and function. We developed a DRD based on the carbonic anhydrase 2 (CA2) protein, which is stabilized in the presence of the FDA-approved drug acetazolamide (ACZ). Upon gene transfer of membrane-bound IL15 fused to a CA2 DRD, regulated IL15 expression on T cells was detected only in the presence of ACZ. In the absence of ACZ, the level of IL15 detected on the surface of gene-modified T cells is not substantially different from the level detected on untransduced T cells. ACZ treatment of gene-modified T cells increases surface IL15 expression in a dose-dependent manner. We observed prolonged survival and up to 15-fold expansion of IL15-CA2 DRD-transduced T cells in the absence of supplemental cytokines or antigen stimulation. In contrast, vehicle-treated IL15-CA2 DRD modified T cells and untransduced T cells did not survive or expand in vitro. Importantly, both IL15-CA2-transduced T cells and unengineered, co-infused NK cells survived and persisted significantly more in vivo in ACZ-treated but not vehicle-treated non-tumor-bearing NSG mice. Our results demonstrate that a novel membrane-bound IL15-CA2 fusion protein coupled with ACZ treatment induces antigen-independent T cell expansion and augments bystander NK cell persistence. Regulatable IL15 expression has significant implications for both T and NK cell therapies by providing more durable cell expansion and cytolytic activity in vivo, and thus the potential to significantly reduce cell dosing while maintaining clinical efficacy in patients. Citation Format: Steven Shamah, Kutlu Elpek, Tucker Ezell, Michelle Fleury, Michael Gallo, Jennifer Gori, Scott Heller, Mara Inniss, Meghan Langley, Grace Olinger, Celeste Richardson, Karen Tran, Dhruv Sethi, Dexue Sun, Vipin Suri. Drug responsive domain regulation of IL15-engineered T cells provides pharmacological control over antigen-independent cell expansion [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 6604.

Genome editing using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated 9 (Cas9) technology is revolutionizing the study of gene function and likely will give rise to an entire new class of therapeutics for a wide range of diseases. Achieving this goal requires not only characterization of the technology for efficacy and specificity but also optimization of its delivery to the target cells for each disease indication. In this review we survey the various methods by which the CRISPR-Cas9 components have been delivered to cells and highlight some of the more clinically relevant approaches. Additionally, we discuss the methods available for assessing the specificity of Cas9 editing; an important safety consideration for development of the technology.

Pluripotent stem cells (PSCs) represent an alternative hematopoietic stem cell (HSC) source for treating hematopoietic disease. The limited engraftment of human PSC–derived (hPSC-derived) multipotent progenitor cells (MPP) has hampered the clinical application of these cells and suggests that MPP require additional cues for definitive hematopoiesis. We hypothesized that the presence of a vascular niche that produces Notch ligands jagged-1 (JAG1) and delta-like ligand-4 (DLL4) drives definitive hematopoiesis. We differentiated hes2 human embryonic stem cells (hESC) and Macaca nemestrina–induced PSC (iPSC) line-7 with cytokines in the presence or absence of endothelial cells (ECs) that express JAG1 and DLL4. Cells cocultured with ECs generated substantially more CD34 + CD45 + hematopoietic progenitors compared with cells cocultured without ECs or with ECs lacking JAG1 or DLL4. EC-induced cells exhibited Notch activation and expressed HSC-specific Notch targets RUNX1 and GATA2. EC-induced PSC-MPP engrafted at a markedly higher level in NOD/SCID/IL-2 receptor γ chain–null (NSG) mice compared with cytokine-induced cells, and low-dose chemotherapy-based selection further increased engraftment. Long-term engraftment and the myeloid-to-lymphoid ratio achieved with vascular niche induction were similar to levels achieved for cord blood–derived MPP and up to 20-fold higher than those achieved with hPSC-derived MPP engraftment. Our findings indicate that endothelial Notch ligands promote PSC-definitive hematopoiesis and production of long-term engrafting CD34+ cells, suggesting these ligands are critical for HSC emergence.

Temozolomide (TMZ) is one of the most potent chemotherapy agents for the treatment of glioblastoma. Unfortunately, almost half of glioblastoma tumors are TMZ resistant due to overexpression of methylguanine methyltransferase (MGMT(hi)). Coadministration of O6-benzylguanine (O6BG) can restore TMZ sensitivity, but causes off-target myelosuppression. Here, we conducted a prospective clinical trial to test whether gene therapy to confer O6BG resistance in hematopoietic stem cells (HSCs) improves chemotherapy tolerance and outcome.We enrolled 7 newly diagnosed glioblastoma patients with MGMT(hi) tumors. Patients received autologous gene-modified HSCs following single-agent carmustine administration. After hematopoietic recovery, patients underwent O6BG/TMZ chemotherapy in 28-day cycles. Serial blood samples and tumor images were collected throughout the study. Chemotherapy tolerance was determined by the observed myelosuppression and recovery following each cycle. Patient-specific biomathematical modeling of tumor growth was performed. Progression-free survival (PFS) and overall survival (OS) were also evaluated.Gene therapy permitted a significant increase in the mean number of tolerated O6BG/TMZ cycles (4.4 cycles per patient, P0.05) compared with historical controls without gene therapy (n = 7 patients, 1.7 cycles per patient). One patient tolerated an unprecedented 9 cycles and demonstrated long-term PFS without additional therapy. Overall, we observed a median PFS of 9 (range 3.5-57+) months and OS of 20 (range 13-57+) months. Furthermore, biomathematical modeling revealed markedly delayed tumor growth at lower cumulative TMZ doses in study patients compared with patients that received standard TMZ regimens without O6BG.These data support further development of chemoprotective gene therapy in combination with O6BG and TMZ for the treatment of glioblastoma and potentially other tumors with overexpression of MGMT.Clinicaltrials.gov NCT00669669.R01CA114218, R01AI080326, R01HL098489, P30DK056465, K01DK076973, R01HL074162, R01CA164371, R01NS060752, U54CA143970.

Background Nonmyeloablative allogeneic hematopoietic stem cell (HSC) transplantation can cure malignant and nonmalignant diseases affecting the hematopoietic system, such as severe combined immunodeficiencies, aplastic anemia and hemoglobinopathies. Although nonmyeloablative is favored over myeloablative transplantation for many patients, graft rejection remains problematic. One strategy for decreasing rejection is to protect donor activated T cells in the graft from methotrexate (MTX) by genetically modifying the cells to express MTX-resistant dihydrofolate reductase (Tyr22-DHFR), leaving the immunosuppressive effects of MTX to act solely on activated host T lymphocytes, shifting the balance to favor allogeneic engraftment. Methods To evaluate MTX resistance of Tyr22-DHFR+ T lymphocytes in vivo, we transplanted dogs with autologous CD34+ cells modified with yellow fluorescent protein (YFP) and DHFR-green fluorescent protein (GFP) lentivirus vectors. Dogs were then treated with a standard MTX regimen days 1, 3, 6 and 11) following immune activation with a foreign antigen as a surrogate assay to mimic early transplantation. Results DHFR-GFP+ gene marking was maintained in CD3+CD25+ and CD4+ T lymphocytes after MTX treatment, whereas the level of T lymphocytes that expressed only a fluorescent reporter (YFP+) decreased. These data show that Tyr22-DHFR expression protects T lymphocytes from MTX toxicity in dogs, highlighting a clinically relevant application for preserving donor T lymphocytes during post-transplantation immunosuppression. Conclusions The findings of the present study have implications for the clinical translation of MTX-resistant T cells to facilitate engraftment of allogeneic cells following nonmyeloablative conditioning and to minimize the risk of rejection. In summary, Tyr22-DHFR expression in T lymphocytes provides chemoprotection from MTX-mediated elimination in the context of immune activation in vivo. Copyright © 2013 John Wiley & Sons, Ltd.

Induced pluripotent stem cell (iPSC) therapeutics are a promising treatment for genetic and infectious diseases. To assess engraftment, risk of neoplastic formation, and therapeutic benefit in an autologous setting, testing iPSC therapeutics in an appropriate model, such as the pigtail macaque (Macaca nemestrina; Mn), is crucial. Here, we developed a chemically defined, scalable, and reproducible specification protocol with bone morphogenetic protein 4, prostaglandin-E2 (PGE2), and StemRegenin 1 (SR1) for hematopoietic differentiation of Mn iPSCs. Sequential coculture with bone morphogenetic protein 4, PGE2, and SR1 led to robust Mn iPSC hematopoietic progenitor cell formation. The combination of PGE2 and SR1 increased CD34+CD38−Thy1+CD45RA−CD49f+ cell yield by 6-fold. CD34+CD38−Thy1+CD45RA−CD49f+ cells isolated on the basis of CD34 expression and cultured in SR1 expanded 3-fold and maintained this long-term repopulating HSC phenotype. Purified CD34high cells exhibited 4-fold greater hematopoietic colony-forming potential compared with unsorted hematopoietic progenitors and had bilineage differentiation potential. On the basis of these studies, we calculated the cell yields that must be achieved at each stage to meet a threshold CD34+ cell dose that is required for engraftment in the pigtail macaque. Our protocol will support scale-up and testing of iPSC-derived CD34high cell therapies in a clinically relevant nonhuman primate model.

Chemotherapy with 1,3-bis (2-chloroethyl)-1-nitrosourea (BCNU) and temozolomide (TMZ) is commonly used for the treatment of glioblastoma multiforme (GBM) and other cancers. In preparation for a clinical gene therapy study in patients with glioblastoma, we wished to study whether these reagents could be used as a reduced-intensity conditioning regimen for autologous transplantation of gene-modified cells. We used an MGMT(P140K)-expressing lentivirus vector to modify dog CD34(+) cells and tested in four dogs whether these autologous cells engraft and provide chemoprotection after transplantation. Treatment with O(6)-benzylguanine (O6BG)/TMZ after transplantation resulted in gene marking levels up to 75%, without significant hematopoietic cytopenia, which is consistent with hematopoietic chemoprotection. Retrovirus integration analysis showed that multiple clones contribute to hematopoiesis. These studies demonstrate the ability to achieve stable engraftment of MGMT(P140K)-modified autologous hematopoietic stem cells (HSCs) after a novel reduced-intensity conditioning protocol using a combination of BCNU and TMZ. Furthermore, we show that MGMT(P140K)-HSC engraftment provides chemoprotection during TMZ dose escalation. Clinically, chemoconditioning with BCNU and TMZ should facilitate engraftment of MGMT(P140K)-modified cells while providing antitumor activity for patients with poor prognosis glioblastoma or alkylating agent-sensitive tumors, thereby supporting dose-intensified chemotherapy regimens.

Tumor antigen directed T-cells, using chimeric antigen receptors (CAR), have shown remarkable clinical responses in B-cell malignancies, particularly acute lymphoblastic leukemia, diffuse large B-cell lymphoma and multiple myeloma. A number of challenges remain, however, in safe and effective cell therapy for durable responses broadly in hematologic malignancies and solid tumors. The durability of response is often reduced by antigen positive or antigen negative escape while the immunosuppressive tumor microenvironment can reduce the potency of the engineered T-cells. Cytokines can be effectively used to improve T-cell expansion and persistence to prevent antigen positive escape, enhance epitope spreading to prevent antigen negative escape, and to relieve the immunosuppressive tumor microenvironment. Indeed, anti-tumor responses with cytokine immunotherapy as well as cytokine-aided cell therapy have been observed. However, the utility of cytokines is often limited by systemic toxicity associated with their potent pharmacological effects. Locally restricted, on demand production of cytokines coupled with antigen directed T-cells can enable safe and effective cell therapy for the treatment of hematologic malignancies as well as solid tumors. Among the cytokines, Interleukin 12 (IL12) and Interleukin 15 (IL15) are of particular interest due to their role in remodeling the tumor microenvironment and improving T-cell persistence. To determine the effect of cytokine expression on CAR-T control of tumor growth, we generated bicistronic constructs expressing CD19-CAR and IL12 or CD19-CAR and membrane bound IL15 (mbIL15). The constructs were configured to provide a high level of CAR expression with high or low levels of cytokine expression for dose exploration. Even at low cytokine levels and low cell dose, expression of IL12 along with CD19-CAR in human T-cells durably regressed disseminated CD19+ Nalm6-luc tumors in Nod-scid IL2Rgnull (NSG mice), compared to CD19-CAR-expressing cells alone and improved survival at both low and high doses (n=8/group, p To enable regulation of the cytokines, we used destabilizing domain (DD) technology which is based on fully human protein domains that are inherently unstable in the cell but are reversibly stabilized by binding of small molecule ligands. Moreover, fusion with DDs can confer ligand dependent, reversible regulation to any protein of interest. We fused IL12 and mbIL15 to destabilizing domains derived from Estrogen Receptor (ER) and Phosphodiesterase 5 (PDE5) which can be regulated by safe, FDA approved drugs. We identified ER-based DDs that, when fused with IL12, yielded low cytokine levels in the basal state in T-cells. The addition of an ER stabilizing ligand, however, led to a rapid, dose dependent, 10-fold induction of secreted IL12. We also identified PDE5-regulated mbIL15 constructs that displayed rapid and dose dependent upregulation of cell surface mbIL15 levels in T-cells when treated with PDE5-stabilizing ligands. We are currently evaluating ER-regulated IL12 and PDE5-regulated mbIL15 in immunocompetent models to evaluate whether these regulated cytokines can enable safe and effective cytokine usage to improved breadth, depth and durability of anti-tumor responses to cellular therapies. Disclosures Suri: Obsidian Therapeutics: Employment, Equity Ownership. Gori:Obsidian Therapeutics: Employment.

Human embryonic stem cells (hESCs) provide a novel source of hematopoietic and other cell populations suitable for gene therapy applications. Preclinical studies to evaluate engraftment of hESC-derived hematopoietic cells transplanted into immunodeficient mice demonstrate only limited repopulation. Expression of a drug-resistance gene, such as Tyr22-dihydrofolate reductase (Tyr22-DHFR), coupled to methotrexate (MTX) chemotherapy has the potential to selectively increase the engraftment of gene-modified, hESC-derived cells in mouse xenografts. Here, we describe the generation of Tyr22-DHFR-GFP-expressing hESCs that maintain pluripotency, produce teratomas and can differentiate into MTXr-hemato-endothelial cells. We demonstrate that MTX administered to nonobese diabetic/severe combined immunodeficient/IL-2Rgammac(null) (NSG) mice after injection of Tyr22-DHFR-hESC-derived cells significantly increases human CD34(+) and CD45(+) cell engraftment in the bone marrow (BM) and peripheral blood of transplanted MTX-treated mice. These results demonstrate that MTX treatment supports selective, long-term engraftment of Tyr22-DHFR cells in vivo, and provides a novel approach for combined human cell and gene therapy.

Methotrexate (MTX) dose-escalation studies were conducted in C57BL/6 mice to determine the chemoprotective effect of transplantation using bone marrow transduced with lentivirus vectors expressing a drug-resistant variant of murine dihydrofolate reductase (DHFR). Methotrexate-resistant dihydrofolate reductase [tyrosine-22 (Tyr22)DHFR] and enhanced green fluorescent protein (GFP) coding sequences were inserted into self-inactivating lentiviral vectors as part of a genetic fusion or within the context of a bicistronic expression cassette. MTX-treated animals that received Tyr22DHFR-transduced marrow recovered to normal hematocrit levels by 3 weeks post-transplant and exhibited significant GFP marking in myeloid and lymphoid lineage-derived peripheral blood mononuclear cells (PBMCs). In contrast, MTX-treated animals transplanted with control GFP-transduced marrow exhibited extremely reduced hematocrits with severe marrow hypoplasia and did not survive MTX dose escalation. To minimize cell manipulation, we treated unfractionated marrow in an overnight exposure. Transduction at a multiplicity of infection of 10 resulted in up to 11% vector-modified PBMCs in primary recipients and successful repopulation of secondary recipients with vector-marked cells. Experimental cohorts exhibited sustained proviral expression with stable GFP fluorescence intensity. These results demonstrate the effectiveness of lentivirus vectors for chemoprotection in a well developed animal model, with the potential for further preclinical development toward human application.

Genome editing via CRISPR/Cas9 promises to provide a novel class of therapies for a variety of human diseases. To unlock the potential of the CRISPR/Cas9 technology, a deeper understanding of its efficacy in different primary cell types is required. A cell type of particular interest for gene editing is the human T cell due to its central role in the evolving cancer immunotherapy field. To better understand the utility of Cas9-mediated gene editing for engineering human T cells, we surveyed a variety of delivery modalities including electroporation of RNA and administration of ribonucleic acid-protein complexes. Additionally, we assessed the functionality of different Cas9 variants in human T cells. Here we report our findings including genome editing in human T cells using CRISPR/Cas technology.

Transplantation of genetically corrected autologous hematopoietic stem/progenitor cells (HSPCs) is an effective treatment for patients with inherited hematologic disease. Genome editing with CRISPR/Cas9 ribonucleoprotein (RNP) precisely modifies gene targets in human cells, including HSPCs. In order to translate this technology to a clinical setting, gene editing must be reproducible and efficient across multiple patient donors without compromising HSPC viability, multipotency, and long-term engraftment capability. To determine the reproducibility of Cas9 RNP mediated gene editing in HSPCs, human CD34+ cells obtained from 15 different patient donors (cord blood n=12, mobilized peripheral blood [mPB] n=3) were electroporated with S. pyogenes Cas9 RNP targeting the β-hemoglobin (HBB) or AAVS1 genetic locus. DNA sequence analysis of 15 separate experiments demonstrated that Cas9 supported up to 72% gene editing in cord blood CD34+ cells and up to 61% gene editing in adult mPB CD34+ cells (mean % editing by DNA sequencing: 57% ± 8%). Cas9 induced multiple modifications that comprise insertions and deletions at the HBB locus, and some of the lesions were repaired through an HDR repair mechanism that used the homologous sequences from the endogenous HBD gene as a template (Gene Conversion). Gene edited CD34+ cells retained viability and hematopoietic colony forming cell (CFC) activity ex vivo, with no significant differences between treatment groups or across donors. Indels were detected in one (BFU-E/GEMM: 50%-75%, CFU-GM/M: 50-66%) or both (BFU-E/GEMM: 12-38%, CFU-GM/M: 12-50%) alleles, at high frequencies in clonal erythroid and myeloid colonies (range of 63-100% of individual colonies assayed across experiments). To evaluate engraftment potential, both Cas9 modified (unsorted) and untreated control human CD34+ cells were transplanted into immunodeficient mice. Twelve weeks after transplantation, up to 34% human CD45+ cell engraftment was detected in the peripheral blood of recipients of Cas9 RNP treated CD34+ cells. There was no significant difference in the engraftment of Cas9 RNP treated and untreated control CD34+ cells which were detected in blood, spleen, and bone marrow. Human lymphoid, myeloid, and erythroid cells were detected in blood and hematopoietic organs with no difference in lineage distribution between cohorts. Analysis of the bone marrow from both groups revealed an average of 20% human CD45+ and 13% CD34+CD45+ cell engraftment long-term. Analysis of human gDNA revealed that up to 20% gene editing in the marrow of treated mice and edited cells were also detected in the blood and spleen. In summary, these data show that Cas9 RNP gene edited primary human CD34+ HSPCs retained the ability to engraft long-term and reconstitute hematopoiesis in vivo at levels higher than reported previously. These findings also show that electroporation of HSPCs with Cas9 RNP mediated highly reproducible gene editing levels across multiple donors and did not alter hematopoietic reconstitution or differentiation properties in comparison to untreated control CD34+ cells.

Human embryonic stem cells (hES Cs) are an attractive alternative cell source for hematopoietic gene therapy applications as the cells are easily modified with lentiviral or other vectors and can be subsequently induced to differentiate into hematopoietic progenitor cells. However, demonstration of the full hematopoietic potential of hESC-derived progeny is challenging due to low marrow engraftment and the difficulty of detecting cells in the peripheral blood of human/mouse xenografts. Methotrexate (MTX) chemotherapy coupled with expression of a drug resistant dihydrofolate reductase such as Tyr22 (Tyr22DHFR) has the potential to selectively increase engraftment of gene-modified human hematopoietic cells in mice, which would allow for better phenotypic characterization of hESC-derived cells in vivo. We showed that hES Cs transduced with Tyr22DHFR-GFP encoding lentivirus vectors differentiate into MTX resistant (MTXr) hemato-endothelial cells. MTX treatment of immunodeficient mice infused with Tyr22DHFR hESC-derived hemato-endothelial cells increased the long-term engraftment of human cells in the bone marrow of MTX-treated mice. In contrast to previous studies, these results indicate that MTX administration has the potential to support in vivo selection that is maintained after cessation of treatment. The MTX/Tyr22DHFR system may therefore be useful for enrichment of gene-modified cell populations in human stem cell and gene therapy applications.

The development of technology to generate induced pluripotent stem (iPS) cells constitutes one of the most exciting scientific breakthroughs because of the enormous potential for regenerative medicine. However, the safety of iPS cell-related products is a major concern for clinical translation. Insertional mutagenesis, possible oncogenic transformation of iPS cells or their derivatives, or the contamination of differentiated iPS cells with undifferentiated cells, resulting in the formation of teratomas, have remained considerable obstacles. Here, we demonstrate the utility of suicide genes to safeguard iPS cells and their derivatives. We found suicide genes can control the cell fate of iPS cells in vitro and in vivo without interfering with their pluripotency and self-renewal capacity. This study will be useful to evaluate the safety of iPS cell technology in a clinically highly relevant, large animal model and further benefit the clinical use of human iPS cells.

Organ-specific endothelial cells (ECs) are both conduits for delivery of nutrients and also establish an instructive vascular niche. The vascular niche produces paracrine factors, (i.e., angiocrine factors), that balance self-renewal and differentiation of hematopoietic stem/progenitor cells (HSPCs) (1,2). Activation of Akt-mTOR pathway in sinusoidal ECs (SECs) stimulates physiological expression of angiocrine factors, including Kit-ligand, Notch-ligands, Wnts, FGFs, BMPs and TGFb, that expand long-term repopulating HSPCs. Activation of MAPkinase in ECs upregulates expression of GM-CSF, M-CSF, IL6, IL7, SDF-1 and G-CSF (..others) to accelerate HSPC multi-lineage differentiation. We developed an ex vivo vascular niche in which HSPC/EC co-cultures are maintained and expanded in serum-free conditions. This vascular niche platform produces physiologic levels of angiocrine factors that balance expansion/differentiation of human cord blood, mobilized peripheral blood, and steady state bone marrow HSPCs that maintain their ability to reconstitute hematopoiesis in vivo. In contrast to our vascular platform, co-culture with bone marrow-derived mesenchymal does not support long-term expansion of HSPCs. In collaboration with Drs. Kiem and Gori at Hutchinson Cancer Center, we have shown that ECs expand repopulating nonhuman primate marrow-derived HSPCs. Transplantation of the vascular-niche expanded gene-modified HSPCs reconstituted long-term multi-lineage hematopoiesis in autologous transplantation setting in nonhuman primates. Importantly, intravenous co-infusion of the vascular niche with HSPCs did not cause infusional toxicity. Vascular niche-expanded HSPCs supported robust hematopoietic recovery underscoring the essential function of vascular niche-signals in hematopoietic reconstitution without provoking fibrosis (3). The ECs also supplies key signals that induce emergence of HSPCs from hemogenic ECs. To prove this point, we transduced adult human or mouse ECs with Runx1/Spi1/Gfi1/FosB transcription factors along with vascular niche-induction allowing for conversion of these ECs into stable and long-term engraftable HSPCs, including functional immune cells (4). Importantly, transition through a pluripotent state results in poorly engraftable hematopoietic cells that are unstable and upon exposure to pathophysiological stressors differentiate aberrantly into other cell-types. Remarkably, signals from vascular niche support specification of repopulating multipotent-HSPCs from both human and nonhuman primate pluripotent stem cells (5). In summary, we developed and characterized a vascular niche platform that provides physiologically relevant levels of key angiocrine factors that stimulate safe clinical-scale expansion of authentic adult, cord blood, and primitive HSPCs under GMP-grade culture conditions. We are currently translating the vascular niche platform to the clinical setting, to evaluate the potential of co-transplantation of HSPCs with vascular niche cells to reconstruct injured EC niches thereby accelerating short- and long-term hematopoietic recovery. This first-in-man clinical application will set the stage for repopulation with true hematopoietic stem cells, thereby enabling use of a vascular niche for treatment of a wide range of acquired, inherited, and malignant hematopoietic diseases. 1. Butler JM …… Rafii S. Endothelial cells are essential for the self-renewal and repopulation of Notch-dependent hematopoietic stem cells. Cell Stem Cell, 3:251-64, 2010. 2. Nolan D........Rafii S. Molecular and cellular signatures of tissue-specific vascular heterogeneity in organ maintenance and regeneration. Developmental Cell, 26(2):204-19, 2013. 3. Ding BS …..Rafii S. Divergent angiocrine signals from vascular niche balance liver regeneration and fibrosis.Nature 505(7481):97-102, 2014. 4. Sandler VM, Lis R ...... Butler JM, Scandura JM, Rafii S. Reprogramming of Human Endothelium Into Engraftable Hematopoietic Progenitors by Vascular Niche Induction.Nature, 511(7509):312-8, 2014. 5. Gori J., Butler JM, .....Rafii S, Kiem HP. Vascular niche promotes hematopoietic multipotent progenitor formation from pluripotent stem cells. Journal of Clinical Investigation, 125(3): 1243-54, 2015. Disclosures Rafii: Angiocrine Bioscience: Consultancy, Equity Ownership.

Transplantation of genetically modified autologous hematopoietic stem/progenitor cells (HSPCs) has proven to be an effective clinical treatment for patients with hematologic disease. Genome editing with the CRISPR/Cas9 platform has been shown to precisely alter endogenous gene targets in multiple human cell lines and animal models. Here, we compared Cas9-induced gene modification in primary human HSPCs, including mobilized peripheral blood and steady state bone marrow CD34+ cells. Co-delivery of Streptococcus pyogenes or Staphylococcus aureus Cas9 paired with locus-specific guide RNAs induced targeted gene editing in CD34+ cells and hematopoietic progeny, as determined by T7E1 assay and DNA sequence analysis. Multiple gene targets and delivery strategies were evaluated in these primary human CD34+ cells. Importantly, hematopoietic progeny differentiated from Cas9 gene-edited HSPCs maintained ex vivo hematopoietic colony forming potential. This study provides further evidence of Cas9-mediated genome editing in clinically relevant primary cell populations.

Sleeping Beauty (SB) is a gene-insertion system reconstructed from transposon sequences found in teleost fish and is capable of mediating the transposition of DNA sequences from transfected plasmids into the chromosomes of vertebrate cell populations. The SB system consists of a transposon, made up of a gene of interest flanked by transposon inverted repeats, and a source of transposase. Here we carried out a series of studies to further characterize SB-mediated transposition as a tool for gene transfer to chromosomes and ultimately for human gene therapy. Transfection of mouse 3T3 cells, HeLa cells, and human A549 lung carcinoma cells with a transposon containing the neomycin phosphotransferase (NEO) gene resulted in a several-fold increase in drug-resistant colony formation when co-transfected with a plasmid expressing the SB transposase. A transposon containing a methotrexate-resistant dihydrofolate reductase gene was also found to confer an increased frequency of methotrexate-resistant colony formation when co-transfected with SB transposase-encoding plasmid. A plasmid containing a herpes simplex virus thymidine kinase gene as well as a transposon containing a NEO gene was used for counterselection against random recombinants (NEO+TK+) in medium containing G418 plus ganciclovir. Effective counterselection required a recovery period of 5 days after transfection before shifting into medium containing ganciclovir to allow time for transiently expressed thymidine kinase activity to subside in cells not stably transfected. Southern analysis of clonal isolates indicated a shift from random recombination events toward transposition events when clones were isolated in medium containing ganciclovir as well as G418. We found that including both transposon and transposase functions on the same plasmid substantially increased the stable gene transfer frequency in Huh7 human hepatoma cells. The results from these experiments contribute technical and conceptual insight into the process of transposition in mammalian cells, and into the optimal provision of transposon and transposase functions that may be applicable to gene therapy studies.

Chemical factors that simultaneously maintain and expand definitive hematopoietic stem cells (HSCs) have the potential to greatly improve the clinical outcome in patients transplanted with allogeneic umbilical cord blood or gene corrected autologous CD34+ cells, in which the number of cells available for transplantation is limited. The recently discovered small molecules UM729 and UM171 effectively expand HSCs and synergize with StemRegenin1 (SR1) to enhance human HSC expansion, leading to successful long-term engraftment of SCID repopulating cells (SRCs) in immunodeficient mice. In order to facilitate translation of these small molecules for clinical use, we sought to determine the effect of UM729 and UM171 on expansion and transduction of nonhuman primate CD34+ cells from various sources. We hypothesized that UM171 would expand definitive CD34+ HSCs from macaque umbilical cord blood (Mn UCB), thereby increasing the definitive HSC cell dose available for engraftment. Here, we compared engraftment of the progeny of 250,000 Mn UCB CD34+ cells after a 10-day expansion with cytokines with or without SR1, UM729, or UM171 in immunodeficient mice. Although all expansion conditions led to >44-fold increase in the number of CD34+ cells across all cohorts, mice transplanted with UM171 treated Mn UCB CD34+ cells had a significantly higher level of engraftment compared to all other groups (41% nonhuman primate CD45+ cells in peripheral blood at 10 weeks post transplantation, P Disclosures: No relevant conflicts of interest to declare.

Clinical use of human pluripotent stem cell (PSC)-hematopoietic stem cells (HSCs) is impeded by low engraftment potential. This block suggests that additional vascular derived angiocrine signals and hematopoietic cues must be provided to produce authentic HSCs. In addition, gene modification of induced (i)PSCs with a chemotherapy resistance transgene would provide a selective mechanism to stabilize or increase engraftment of HSCs. We therefore hypothesized that modifying iPSCs to express the O6-benzylguanine (O6BG)-resistant P140K variant of methylguanine methyltransferase (MGMT), would support in vivo selection of early-engrafted iPSC-HSCs. We further postulated that Akt-activated human endothelial cells afforded by transduction of the E4ORF1 gene (E4ORF1+ECs) through angiocrine upregulation of Notch and IGF ligands would provide the necessary signals under xenobiotic-free conditions to promote definitive hematopoiesis. This vascular induction platform could drive the emergence of true HSCs. We focused on pigtail macaque (Mn)iPSCs, as a scalable, clinically relevant nonhuman primate model. MniPSCs modified to express P140K had 15-fold higher MGMT levels compared to levels in human peripheral blood mononuclear cells. P140K-MniPSCs differentiated into chemoresistant CD34+ hematopoietic progenitors (50% CD34+) with a predominant long-term (LT)-HSC-like phenotype (CD34+CD38-Thy1+CD45RA-CD49f+). Hematopoietic progenitors maintained colony forming potential after O6BG and bis-chloroethylnitrosourea (BCNU) treatment. HSCs expanded on E4ORF1+ECs maintained colony forming potential, in contrast to cells cultured with cytokines alone, with a 22-fold increase in CD34+ cell content and 10-fold increase in LT-HSC-like cells. Importantly, MniPSC-HSCs expanded with the E4ORF1+ECs had long-term engraftment in NSG mice at levels comparable to Mn bone marrow HSC engrafted mice. O6BG/BCNU treatment increased engraftment to 35% CD45+ cells the blood of mice transplanted with E4ORF1+EC expanded P140K-MniPSC-HSCs, which was maintained 16 weeks post transplantation. Primate CD45+ cell levels in the blood after selection were significantly higher for this cohort compared to mice transplanted with P140K-MniPSC-HSCs expanded in the “cytokines alone” condition (18% vs. 3% CD45+, P Table 1 CD34+ and CD45+ engraftment and gene marking in the bone marrow of mice transplanted with nonhuman primate HPSCs from MniPSCs and bone marrow. HSCs E4ORF1+ECs O6BG/BCNU Mean %CD34+ Mean %CD45+ % gene marking in CFCs (lentivirus+) total lentivirus+ CFCs per 105 cells GFP-MniPSC + - 3 16 9 ± 2 13 ± 2 P140K-MniPSC + - 4 19 12 ± 5 17 ± 7 P140K-MniPSC - + 0.4 24 3 ± 2 2 ± 1 P140K-MniPSC + + 15 37 27 ± 24 111 ± 96 Mn BM CD34+ - - 2 21 0 0 Disclosures: Nolan: Angiocrine Bioscience: Employment. Ginsberg:Angiocrine Bioscience: Employment. Rafii:Angiocrine Bioscience: Founder Other.

The narrow species tropism of hepatitis C virus (HCV) limits animal studies. We found that pigtail macaque (Macaca nemestrina) hepatic cells derived from induced pluripotent stem cells support the entire HCV life cycle, although infection efficiency was limited by defects in the HCV cell entry process. This block was overcome by either increasing occludin expression, complementing the cells with human CD81, or infecting them with a strain of HCV with less-restricted requirements for CD81. Using this system, we can modify viral and host cell genetics to make pigtail macaques a suitable, clinically relevant model for the study of HCV infection.

Top of pageAbstract Methotrexate (MTX) impedes tumor development by inhibiting dihydrofolate reductase (DHFR), an enzyme that catalyzes the conversion of 7,8-dihydrofolate to 5,6,7,8-tetrahydrofolate (THF). MTX depletes cells of THF, a required precursor for de novo purine and thymidine nucleotide synthesis, and has toxic effects on normal cells of the hematopoietic and gastrointestinal systems. To decrease MTX sensitivity, we introduced a MTX-resistant murine DHFR variant into mouse bone marrow by lentiviral transduction, so as to efficiently transduce non-dividing hematopoietic stem cells. We constructed HIV-1-based lentiviral vectors containing an enhanced green fluorescent protein (eGFP) gene with or without a murine tyr22 DHFR cDNA under transcriptional control of the human EF1-|[alpha]| promoter. These VSV-G-protein pseudotyped lentiviral vectors contained a self-inactivating 3|[prime]| LTR, rev response element (RRE) and woodchuck hepatitis virus posttranscriptional regulatory element (WPRE) to enhance transduction efficiency. Viral vector was produced by co-transfection in 293T cells and concentrated 100-fold by centrifugation to a titer of 1 x 108 IU/ml, as determined by MTX-resistant colony formation and by flow cytometry for GFP expression. We transduced unfractionated bone marrow from normal Bl/6 Ly5.1 mice with either DHFR-GFP or GFP virus for 12 hours at a multiplicity of infection of 10 in the presence of cytokines (mSCF, IL-6, IL-3). Congenic Ly5.2 recipients received sub-lethal irradiation (700 cGy) prior to transplantation of 3 x 106 transduced marrow cells. MTX or PBS were administered daily starting twenty- four hours after transplantation in three recipient cohorts. Animals that received DHFR-virus transduced marrow recovered to normal hematocrit levels by week 3 in spite of MTX administration (44.7% +/- 2.5). In contrast, animals that received GFP-transduced marrow with subsequent MTX administration exhibited extremely reduced hematocrits (16.5% +/- 0.5) at week 3 and did not survive MTX dose-escalation to 2 mg/kg/day. Animals on MTX therapy that received DHFR-GFP transduced marrow exhibited increased donor cell (Ly5.1) engraftment and significant GFP marking in lymphoid (CD3, B220) and myeloid (GR-1) populations, while recipients that received GFP-transduced marrow exhibited lower levels of peripheral blood mononuclear cells and GFP+ leukocytes. Studies are currently underway to determine transgene copy number in repopulated marrow cells and assess MTX toxicity for the gastrointestinal tract. We conclude that transplantation of lentivirally transduced MTXr-DHFR transgenic marrow supports bone marrow repopulation during MTX chemotherapy in irradiated recipients, providing significant chemoprotection that permits MTX dose-escalation. These pre-clinical results support the development of a clinical gene therapy protocol to provide chemoprotection by lentiviral gene transfer.