Hematopoietic Gene Therapy Research Articles

Lentiviral MGMTP140K-mediated in vivo selection employing a ubiquitous chromatin opening element (A2UCOE) linked to a cellular promoter

Notwithstanding recent successes, insertional mutagenesis as well as silencing and variegation of transgene expression still represent considerable obstacles to hematopoietic gene therapy. This also applies to O6-methylguanine DNA methyltransferase (MGMT)-mediated myeloprotection, a concept recently proven clinically effective in the context of glioblastoma therapy. To improve on this situation we here evaluate a SIN-lentiviral vector expressing the MGMTP140K-cDNA from a combined A2UCOE/PGK-promoter. In a murine in vivo chemoselection model the A2UCOE.PGK.MGMT construct allowed for significant myeloprotection as well as robust and stable selection of transgenic hematopoietic cells. In contrast, only transient enrichment and severe myelotoxicity was observed for a PGK.MGMT control vector. Selection of A2UCOE.PGK.MGMT-transduced myeloid and lymphoid mature and progenitor cells was demonstrated in the peripheral blood, bone marrow, spleen, and thymus. Unlike the PGK and SFFV promoters used as controls, the A2UCOE.PGK promoter allowed for sustained vector copy number-related transgene expression throughout the experiment indicating an increased resistance to silencing, which was further confirmed by CpG methylation studies of the PGK promoter. Thus, our data support a potential role of the A2UCOE.PGK.MGMT-vector in future MGMT-based myeloprotection and chemoselection strategies, and underlines the suitability of the A2UCOE element to stabilize lentiviral transgene expression in hematopoietic gene therapy.

VISPA: a Computational pipeline for the identification and analysis of genomic vector integration sites

The analysis of the genomic distribution of viral vector genomic integration sites is a key step in hematopoietic stem cell-based gene therapy applications, allowing to assess both the safety and the efficacy of the treatment and to study the basic aspects of hematopoiesis and stem cell biology. Identifying vector integration sites requires ad-hoc bioinformatics tools with stringent requirements in terms of computational efficiency, flexibility, and usability. We developed VISPA (Vector Integration Site Parallel Analysis), a pipeline for automated integration site identification and annotation based on a distributed environment with a simple Galaxy web interface. VISPA was successfully used for the bioinformatics analysis of the follow-up of two lentiviral vector-based hematopoietic stem-cell gene therapy clinical trials. Our pipeline provides a reliable and efficient tool to assess the safety and efficacy of integrating vectors in clinical settings.

A glance on recent progresses in diagnosis and treatment of primary immunodeficiencies/ Progrese recente în diagnosticul şi tratamentul imunodeficienţelor primare

Primary immunodeficiencies (PIDs)* belong to the group of rare diseases which need more awareness by the relevant medical disciplines. Below a review on recent progresses in diagnosis and treatment of PIDs is given. Reducing the regrettable delay in diagnosis of PIDs (worldwide) is possible only when awareness is increased by doctors who may encounter patients with PID. This review shall serve this purpose. Progresses in understanding what the link might be between one genetic defect presenting in various phenotypes or how various gene defects may manifest by very similar PID phenotypes helps building awareness. Knowledge of PID favours early diagnosis, a cornerstone of optimal, sometimes life-long care at justifiable costs. The complexity of PIDs calls for clinical laboratory and clinical diagnostic performed by experts only. Exciting laboratory diagnostic progresses in early diagnosis of the most severe forms of PID are reviewed below. Progresses in curative therapies for PIDs, such as hematopoietic stem cell transplantation and gene therapies, are mentioned in short. About 80% of PID patients suffer from an antibody deficiency syndrome and can profit from non-curative replacement therapies with human immunoglobulin G concentrates. Modes of application, safety and hints for dosing of replacement therapies to reduce frequencies of severe infections are mentioned below. Thanks to the increasing quality of care, patients survive adolescence. A glance is given on the problems of transition to the adult medicine setting.

Concurrent Measures of Fusion and Transduction Efficiency of Primary CD34+ Cells with Human Immunodeficiency Virus 1-Based Lentiviral Vectors Reveal Different Effects of Transduction Enhancers

Lentiviral vectors (LVs) are used for various gene transfer applications, notably for hematopoietic gene therapy, but methods are lacking for precisely evaluating parameters that control the efficiency of transduction in relation to the entry of vectors into target cells. We adapted a fluorescence resonance energy transfer-based human immunodeficiency virus-1 fusion assay to measure the entry of nonreplicative recombinant LVs in various cell types, including primary human hematopoietic stem progenitor cells (HSPCs), and to quantify the level of transduction of the same initially infected cells. The assay utilizes recombinant LVs containing β-lactamase (BLAM)-Vpr chimeric proteins (BLAM-LVs) and encoding a truncated form of the low-affinity nerve growth factor receptor (ΔNGFR). After infection of target cells with BLAM-LVs, the vector entry rapidly leads to BLAM-Vpr release into the cytoplasm, which is measured by cleavage of a fluorescent substrate using flow cytometry. Parallel cultures of the same infected cells show transduction efficiency resulting from ΔNGFR expression. This LV-based fusion/transduction assay is a dynamic and versatile tool, revealing, for instance, the postentry restrictions of LVs known to occur in cells of hematopoietic origin, especially human HSPCs. Furthermore, this BLAM-LV assay allowed us to evaluate the effect of cytokine prestimulation of HSPCs on the entry step of LVs. The assay also shows that transduction enhancers such as Vectofusin-1 or Retronectin can partially relieve the postentry block, but their effects differ in how they promote LV entry. In conclusion, one such assay should be useful to study hematopoietic postentry restrictions directed against LVs and therefore should allow improvements in various LV-based gene therapy protocols.

Hepatic lentiviral gene transfer is associated with clonal selection, but not with tumor formation in serially transplanted rodents

Lentiviral (LV) vectors are promising tools for long-term genetic correction of hereditary diseases. In hematopoietic stem cell gene therapies adverse events in patients due to vector integration-associated genotoxicity have been observed. Only a few studies have explored the potential risks of LV gene therapy targeting the liver. To analyze hepatic genotoxicity in vivo, we transferred the fumarylacetoacetate hydrolase (FAH) gene by LV vectors into FAH((-/-)) mice (n = 97) and performed serial hepatocyte transplantations (four generations). The integration profile (4,349 mapped insertions) of the LV vectors was assessed by ligation-mediated polymerase chain reaction and deep sequencing. We tested whether the polyclonality of vector insertions was maintained in serially transplanted mice, linked the integration sites to global hepatocyte gene expression, and investigated the effects of LV liver gene therapy on the survival of the animals. The lifespan of in vivo gene-corrected mice was increased compared to 2-(2-nitro-4-trifluoromethylbenzoyl)-1,3-cyclohexanedione (NTBC) control animals and unchanged in serially transplanted animals. The integration profile (4,349 mapped insertions) remained polyclonal through all mouse generations with only mild clonal expansion. Genes close to the integration sites of expanding clones may be associated with enhanced hepatocyte proliferation capacity. We did not find evidence for vector-induced tumors. LV hepatic gene therapy showed a favorable risk profile for stable and long-term therapeutic gene expression. Polyclonality of hepatocyte regeneration was maintained even in an environment of enforced proliferation.

31. Unimolecular Microchimerism Achieved through Hematopoietic Stem Cell Transplantation Gene Therapy Facilitates Adjunctive Therapy with Genetically-Modified Mesenchymal Stem Cells in Hemophilia A

31. Unimolecular Microchimerism Achieved through Hematopoietic Stem Cell Transplantation Gene Therapy Facilitates Adjunctive Therapy with Genetically-Modified Mesenchymal Stem Cells in Hemophilia A

Infectivity enhancement of different HIV-1-based lentiviral pseudotypes in presence of the cationic amphipathic peptide LAH4-L1

Lentiviral vectors (LVs) are promising delivery systems for gene therapy. To enhance the efficiency of target cell transduction by LVs, protocols often include the addition of culture additives. In this study, the cationic amphipathic peptide LAH4-L1 (KKALLAHALHLLALLALHLAHALKKA), a DNA transfection agent, was evaluated for its capacity to improve LV infectivity in cell lines and primary cells. Results show that LAH4-L1 enhances infectivity of all LV pseudotypes tested, particularly GALVTR-LVs. More importantly, LAH4-L1 promotes the transduction of CD34+ hematopoietic stem cells with GALVTR-LVs as efficiently as Retronectin, a culture additive used in ex vivo clinical protocols involving LVs. The action of LAH4-L1 relies both on the GALVTR-LV adhesion and post-adhesion steps. LAH4-L1 represents a new and attractive transduction enhancer for hematopoietic gene therapy protocols.

Lentiviral Transduction of CD34+ Cells Induces Genome-Wide Epigenetic Modifications

Epigenetic modifications may occur during in vitro manipulations of stem cells but these effects have remained unexplored in the context of cell and gene therapy protocols. In an experimental model of ex vivo gene modification for hematopoietic gene therapy, human CD34+ cells were cultured shortly in the presence of cytokines then with a gene transfer lentiviral vector (LV) expected to transduce cells but to have otherwise limited biological effects on the cells. At the end of the culture, the population of cells remained largely similar at the phenotypic level but some epigenetic changes were evident. Exposure of CD34+ cells to cytokines increased nuclear expression of epigenetic regulators SIRT1 or DNMT1 and caused genome-wide DNA methylation changes. Surprisingly, the LV caused additional and distinct effects. Large-scale genomic DNA methylation analysis showed that balanced methylation changes occurred in about 200 genes following culture of CD34+ cells in the presence of cytokines but 900 genes were modified following addition of the LV, predominantly increasing CpG methylation. Epigenetic effects resulting from ex vivo culture and from the use of LV may constitute previously unsuspected sources of biological effects in stem cells and may provide new biomarkers to rationally optimize gene and cell therapy protocols.

Current translational and clinical practices in hematopoietic cell and gene therapy

Clinical trials over the last 15 years have demonstrated that cell and gene therapies for cancer, monogenic and infectious disease are feasible and can lead to long-term benefit for patients. However, these trials have been limited to proof-of-principle and were conducted on modest numbers of patients or over long periods of time. In order for these studies to move towards standard practice and commercialization, scalable technologies for the isolation, ex vivo manipulation and delivery of these cells to patients must be developed. Additionally, regulatory strategies and clinical protocols for the collection, creation and delivery of cell products must be generated. In this article we review recent progress in hematopoietic cell and gene therapy, describe some of the current issues facing the field and discuss clinical, technical and regulatory approaches used to navigate the road to product development.

Hematopoietic Stem Cell and Gene Therapy Corrects Primary Neuropathology and Behavior in Mucopolysaccharidosis IIIA Mice

Mucopolysaccharidosis IIIA (MPS IIIA or Sanfilippo disease) is a neurodegenerative disorder caused by a deficiency in the lysosomal enzyme sulfamidase (SGSH), catabolizing heparan sulfate (HS). Affected children present with severe behavioral abnormalities, sleep disturbances, and progressive neurodegeneration, leading to death in their second decade. MPS I, a similar neurodegenerative disease accumulating HS, is treated successfully with hematopoietic stem cell transplantation (HSCT) but this treatment is ineffectual for MPS IIIA. We compared HSCT in MPS IIIA mice using wild-type donor cells transduced ex vivo with lentiviral vector-expressing SGSH (LV-WT-HSCT) versus wild-type donor cell transplant (WT-HSCT) or lentiviral-SGSH transduced MPS IIIA cells (LV-IIIA-HSCT). LV-WT-HSCT results in 10% of normal brain enzyme activity, near normalization of brain HS and GM2 gangliosides, significant improvements in neuroinflammation and behavioral correction. Both WT-HSCT and LV-IIIA-HSCT mediated improvements in GM2 gangliosides and neuroinflammation but were less effective at reducing HS or in ameliorating abnormal HS sulfation and had no significant effect on behavior. This suggests that HS may have a more significant role in neuropathology than neuroinflammation or GM2 gangliosides. These data provide compelling evidence for the efficacy of gene therapy in conjunction with WT-HSCT for neurological correction of MPS IIIA where conventional transplant is ineffectual.

Stem Cell Factor-Displaying Simian Immunodeficiency Viral Vectors Together with a Low Conditioning Regimen Allow for Long-Term Engraftment of Gene-Marked Autologous Hematopoietic Stem Cells in Macaques

Although clinical benefits have been reported in several human hematopoietic gene therapy trials, a remaining important goal is the transition to nonmyeloablative pretransplantation conditioning to decrease toxicity. Previous attempts at reduced intensity conditioning in nonhuman primates have resulted in only temporary vector marking of autologous blood cells or their persistence at low levels, well below the thresholds for clinical efficacy. In addition, we reasoned that lentiviral vector particles displaying cytokines at their surface have the potential to preserve stem cell fitness better than current ex vivo transduction protocols, which involve exposure to cytokine overstimulation. Here we show that the classically nonmyeloablative agent fludarabine (30 mg/m(2)/day for 3 days) together with low-level total body irradiation (2 Gy) and the use of a stem cell factor-displaying simian immunodeficiency virus-based vector, resulted in sustained, single-copy vector marking of autologous blood cells in two macaques over 3 years posttransplantation at levels averaging 1% of all lineages. This percentage is within the range of anticipated efficacy levels for hemophilia and related diseases and forms a basis for further improvement.

The case for newborn screening for severe combined immunodeficiency and related disorders

Early detection of primary immunodeficiency is recognized as important for avoiding infectious complications that compromise outcomes. In particular, severe combined immunodeficiency (SCID) is fatal in infancy unless affected infants can be diagnosed before the onset of devastating infections and provided with an immune system through allogenic hematopoietic cell transplantation, enzyme replacement, or gene therapy. A biomarker of normal T cell development, T cell receptor excision circles (TRECs), can be measured in DNA isolated from the dried blood spots routinely obtained for newborn screening; infants identified as lacking TRECs can thus receive confirmatory testing and prompt intervention. Early results of TREC testing of newborns in five states indicate that this addition to the newborn screening panel can be successfully integrated into state public health programs. A variety of cases with typical SCID genotypes and other T lymphocytopenic conditions have been detected in a timely manner and referred for appropriate early treatment.

A Chemical Approach for Ex Vivo Expansion of Human Hematopoietic Stem Cells

Abstract 481Hematopoietic stem cells (HSCs) have been applied to treatment of a wide variety of malignant and non-malignant blood disorders. Because of low risk of graft versus-host disease, use of human cord blood (hCB) as a source of HSCs continues to increase. However, wide application of hCB is limited by the relatively low number of HSCs obtained from a single CB unit. Numerous attempts have been made to expand hCB HSCs in cultures to acquire a sufficient number of transplantable HSCs. Chemical approaches have especially showed promise for ex vivo expansion of HSCs. As an example, Boitano et al. reported that SR1, a chemically synthesized purine derivative, induced hCB HSC expansion by antagonizing the aryl hydrocarbon receptor.In this study, we initially screened several tens of thousands of small-molecule compounds for the capability to induce STAT5 activation but obtained active compounds showed no positive effect on proliferation of hCB CD34+ hematopoietic stem and progenitor cells (HSPCs). However, a derivative of the STAT5 activating compounds proved to increase the population of CD34+CD38– cells when added to hCB CD34+ cell culture. Thus, we optimized the chemical structure of the compound and identified MISK303 as the most potent compound. We next evaluated the effects of MISK303 on the ex vivo expansion of hCB CD34+ cells in detail; hCB CD34+ cells were cultured in serum-free medium supplemented with MISK303 in addition to rhTPO and rhSCF for 7 days and analyzed the cellular phenotype of the cultured cells by flow cytometry and colony assays. Although the total and CD34+ cell number when cultured with MISK303 was comparable to that cultured with DMSO (vehicle), the cultures with MISK303 exhibited a >2-fold increase in the number of CD34+CD38– cells and contained 1.7-fold more high proliferative potential colony-forming cells (HPP-CFCs; >1mm in diameter) compared to those with DMSO. Correspondingly, in the NOD/SCID repopulation assay, hCB CD34+ cells cultured with MISK303 for 7 days displayed up to 2-fold higher levels of engraftment compared to control cultures and uncultured HSPCs. These data suggest that MISK303 promotes the net expansion of hematopoietic stem and progenitor cells. Furthermore, combination of SR1 and MISK303 had additive effects; cell cultures with SR1 and MISK303 showed a >4-fold increase in the number of CD34+CD38– cells. In accordance with the data, MISK303 had no antagonizing activity against the aryl hydrocarbon receptor. We also observed that a chemically-synthesized TPO receptor (c-mpl) agonist could be substituted with rhTPO and provided better expansion of CD34+CD38– cells in combination with SR1 and MISK303.To investigate the molecular mechanism by which MISK303 increases HSPCs, we first conducted gene expression profiling of CD34+ cells cultured with MK303 by DNA microarray analysis. Treatment of CD34+ cells with MISK303 for 24 hours led to up-regulation of 343-genes (>2-fold) and down-regulation of 112 genes (<0.5-fold). We also profiled effect of MISK303 over 311 protein kinases and found no inhibitory activity. We are now validating the expression of differentially expressed genes identified by DNA microarray analysis.In conclusion, we have conducted high-throughput screening and identified MK303, a novel chemically synthesized small-molecule compound, which induces the expansion of human HSCs ex vivo. The approach using MISK303 will facilitate the development of novel and efficient technologies for hematopoietic stem cell and gene therapies. Disclosures:No relevant conflicts of interest to declare.

MicroRNA-150-regulated vectors allow lymphocyte-sparing transgene expression in hematopoietic gene therapy

Endogenous microRNA (miRNA) expression can be exploited for cell type-specific transgene expression as the addition of miRNA target sequences to transgenic cDNA allows for transgene downregulation specifically in cells expressing the respective miRNAs. Here, we have investigated the potential of miRNA-150 target sequences to specifically suppress gene expression in lymphocytes and thereby prevent transgene-induced lymphotoxicity. Abundance of miRNA-150 expression specifically in differentiated B and T cells was confirmed by quantitative reverse transcriptase PCR. Mono- and bicistronic lentiviral vectors were used to investigate the effect of miRNA-150 target sequences on transgene expression in the lymphohematopoietic system. After in vitro studies demonstrated effective downregulation of transgene expression in murine B220(+) B and CD3(+) T cells, the concept was further verified in a murine transplant model. Again, marked suppression of transgene activity was observed in B220(+) B and CD4(+) or CD8(+) T cells whereas expression in CD11b(+) myeloid cells, lin(-) and lin(-)/Sca1(+) progenitors, or lin(-)/Sca1(+)/c-kit(+) stem cells remained almost unaffected. No toxicity of miRNA-150 targeting in transduced lymphohematopoietic cells was noted. Thus, our results demonstrate the suitability of miRNA-150 targeting to specifically suppress transgene expression in lymphocytes and further support the concept of miRNA targeting for cell type-specific transgene expression in gene therapy approaches.

Concise Review: Managing Genotoxicity in the Therapeutic Modification of Stem Cells

The therapeutic use of procedures for genetic stem cell modification is limited by potential adverse events related to uncontrolled mutagenesis. Prominent findings have been made in hematopoietic gene therapy, demonstrating the risk of clonal, potentially malignant outgrowth on the basis of mutations acquired during or after therapeutic genome modification. The incidence and the growth rate of insertional mutants have been linked to the "stemness" of the target cells and vector-related features such as the integration pattern, the architecture, and the exact content of transgene cassettes. Milieu factors supporting the survival and expansion of mutants may eventually allow oncogenic progression. Similar concerns apply for medicinal products based on pluripotent stem cells. Focusing on the genetic stress induced by insertional mutagenesis and culture adaptation, we propose four conclusions. (a) Mutations occurring in the production of stem cell-based medicines may be unavoidable and need to be classified according to their risk to trigger the formation of clones that are sufficiently long-lived and mitotically active to acquire secondary transforming mutations. (b) The development of rational prevention strategies depends upon the identification of the specific mutations forming such "dominant clones" (which can also be addressed as cancer stem cell precursors) and a better knowledge of the mechanisms underlying their creation, expansion, and homeostatic control. (c) Quantitative assay systems are required to assess the practical value of preventive actions. (d) Improved approaches for the genetic modification of stem cells can address all critical steps in the origin and growth control of mutants.

589. Evaluation of Biopotency of rHIV Lentiviral Vectors Produced for Ex Vivo Hematopoietic Gene Therapy

589. Evaluation of Biopotency of rHIV Lentiviral Vectors Produced for Ex Vivo Hematopoietic Gene Therapy

Hematopoietic gene therapy restores thymidine phosphorylase activity in a cell culture and a murine model of MNGIE.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an autosomal recessive disorder caused by mutations in the TYMP gene, which encodes thymidine phosphorylase (TP). TP dysfunction results in systemic thymidine (dThd) and deoxyuridine (dUrd) overload, which selectively impair mitochondrial DNA replication. Allogeneic hematopoietic transplantation has been used to treat MNGIE patients; however, this approach has serious adverse effects, including the toxicity of myeloablative conditioning, graft rejection and graft-versus-host disease. With the aim of testing the feasibility of gene therapy for MNGIE, we transduced TP-deficient B-lymphoblastoid cells from two MNGIE patients, with lentiviral vectors carrying a functional copy of the human TYMP DNA coding sequence. This restored TP activity in the cells, which reduced the excretion of dThd and dUrd and their concentrations when added in excess. Additionally, lentiviral-mediated hematopoietic gene therapy was used in partially myeloablated double Tymp/Upp1 knockout mice. In spite of the relatively low levels of molecular chimerism achieved, high levels of TP activity were observed in the peripheral blood of the transplanted mice, with a concomitant reduction of nucleoside concentrations. Our results suggest that hematopoietic gene therapy could be an alternative treatment for this devastating disorder in the future.

Quantification of lentiviral vector copy numbers in individual hematopoietic colony-forming cells shows vector dose-dependent effects on the frequency and level of transduction

Lentiviral vectors are effective tools for gene transfer and integrate variable numbers of proviral DNA copies in variable proportions of cells. The levels of transduction of a cellular population may therefore depend upon experimental parameters affecting the frequency and/or the distribution of vector integration events in this population. Such analysis would require measuring vector copy numbers (VCN) in individual cells. To evaluate the transduction of hematopoietic progenitor cells at the single-cell level, we measured VCN in individual colony-forming cell (CFC) units, using an adapted quantitative PCR (Q-PCR) method. The feasibility, reproducibility and sensitivity of this approach were tested with characterized cell lines carrying known numbers of vector integration. The method was validated by correlating data in CFC with gene expression or with calculated values, and was found to slightly underestimate VCN. In spite of this, such Q-PCR on CFC was useful to compare transduction levels with different infection protocols and different vectors. Increasing the vector concentration and re-iterating the infection were two different strategies that improved transduction by increasing the frequency of transduced progenitor cells. Repeated infection also augmented the number of integrated copies and the magnitude of this effect seemed to depend on the vector preparation. Thus, the distribution of VCN in hematopoietic colonies may depend upon experimental conditions including features of vectors. This should be carefully evaluated in the context of ex vivo hematopoietic gene therapy studies.

Ex Vivo Expansion of Hematopoietic Stem and Progenitor Cells Using a Natural Product, Garcinol

AbstractAbstract 1174Ex vivo expansion of hematopoietic stem and progenitor cells (HSPCs) have recently been explored to optimize autologous and allogeneic HSPC transplantation and shown to be effective in the field of stem cell biology. However, to our knowledge, identification of culture conditions that allow HSPCs expansion and long-term hematopoietic reconstitution have remained incomplete, and clinical methods to expand human HSPCs have yet to be realized.In this study, we assumed that some small molecule compounds may preferentially activate signals that are required for optimal HSPC expansion and facilitate self-renewal of hematopoietic stem cells (HSCs). Thus, we evaluated the effects of several biologically active compounds on the ex vivo expansion of CD34+ hematopoietic stem and progenitor cells from human cord blood (hCB) and identified Garcinol, a plant-derived natural product as a novel modulator of HSPC proliferation.We cultured hCB CD34+ cells in serum-free medium supplemented with human thrombopoietin, human stem cell factor and Garcinol for 7 days and analyzed the cellular phenotype of the cultured cells by flow cytometry and colony assay. Although the total number of cells cultured with Garcinol was similar to those cultured without Garcinol, the cultures with Garcinol showed >2-fold increase in the number of CD34+CD38- hematopoietic stem and progenitor cells and contained 2-fold more high-proliferative-potential colony-forming cells (HPP-CFCs; >1mm in diameter) compared to control cultures. Correspondingly, SCID-repopulating cells (SRCs) were increased 2-fold during a 7-day culture with Garcinol compared to cultures without Garcinol. These findings suggest that Garcinol efficiently promotes the net expansion of HPSCs.To investigate the structure-activity relationship of Garcinol, we synthesized the chemical derivatives of Garcinol and evaluated the effect of Garcinol and its derivatives, Isogarcinol and O, O'-dimethylisogarcinol, on the proliferation of CD34+CD38- cells. Although Isogarcinol exhibited almost the same activity as Garcinol, O, O'-dimethyl isogarcinol was scarcely effective in the CD34+CD38- cell proliferation. Correspondingly, O, O'-dimethylisogarcinol had no effect on numbers of HPP-CFCs. These results indicate that dihydroxybenzoyl moiety is crucial for the positive effect of Gacinol on HSPCs.Garcinol has been reported to be a potent inhibitor of histone acetyltransferases (HAT). Thus, we estimated the HAT activity in cells treated with Garcinol and its derivatives. Garcinol and Isogarcinol inhibited HAT activity while O, O'-dimethylisogarcinol showed much less HAT inhibitory activity as compared to Garcinol and Isogarcinol, which suggested that HAT inhibitory activity of Garcinol is correlate with the expansion of HPSCs. We are now investigating gene expression profiling in cells cultured with Garcinol using DNA microarray analysis and Q-PCR.In conclusion, we have identified Garcinol, a plant-derived small-molecule compound, which exhibits inhibitory effect on HAT activity, as a novel stimulator of HSPC expansion. The results reported here indicate that Garcinol would be applied as a useful tool for the development of novel and efficient technologies for hematopoietic stem cell and gene therapies. Disclosures:No relevant conflicts of interest to declare.

Methotrexate supports in vivo selection of human embryonic stem cell derived-hematopoietic cells expressing dihydrofolate reductase

Human embryonic stem cells (hESCs) 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 hESCs 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.