Percentage Of GFP Research Articles

Construction and Optimization of CD19 Chimeric Antigen Receptor T Cells Derived from C57BL/6J Mice

To explore the stimulation conditions, optimal culture time and infection time of C57BL/6J mice CD3+ T cells in vitro, so as to improve the infection efficiency of CD19 chimeric antigen receptor T cells (mCD19 CAR-T). Purified C57BL/6J mice CD3+ T cells were cultured in anti-CD3/CD28 coated, anti-CD3 coated+soluble anti-CD28 and anti-CD3 coated, respectively. The cells were stimulated in above three conditions for 12 h and 24 h, following with 24 h, 48 h and 72 h incubation and then the number of cell clones was recorded. C57BL/6J mice CD3+ T cells were stimulated for 12 h, 24 h, and 36 h under the above three conditions, then interleukin (IL)-2 (100 U/ml) was added. The number of cell clones was recorded under microscope at 24 h, 48 h, and 72 h of culture. After 24 h of stimulation, CD3+ T cells derived from C57BL/6J mice were infected with retrovirus for 48 h to establish mCD19 CAR-T cells, and the percentage of GFP+ CAR-T cells was detected by flow cytometry. The infection efficiency of mCD19 CAR-T cells derived from C57BL/6J mice was only 5.23% under the optimized conditions of mCD19 CAR-T cells derived from BALB/c mice. The number of clones of C57BL/6J mice CD3+ T cells was the highest in anti-CD3 coated+soluble anti-CD28 group after stimulated for 24 h and followed cultured for 48 h. After 24 hours of stimulation under the above conditions and 48 hours of culture with IL-2, the number of T cell proliferating clones in the anti-CD3 coated+soluble anti-CD28 group was significantly increased compared with the same group without IL-2, and the infection efficiency of CAR-T cells in this group reached 17.63%±4.17%. The optimal conditions for constructing CAR-T cells from C57BL/6J mice CD3+ T cells are different from those of BABL/c mice. T cells stimulated by anti-CD3 coated+soluble anti-CD28+IL-2 can obtain mCD19 CAR-T cells with the highest efficiency after retrovirus infection.

Efferocytosis is restricted by axon guidance molecule EphA4 via ERK/Stat6/MERTK signaling following brain injury

BackgroundEfferocytosis is a process that removes apoptotic cells and cellular debris. Clearance of these cells alleviates neuroinflammation, prevents the release of inflammatory molecules, and promotes the production of anti-inflammatory cytokines to help maintain tissue homeostasis. The underlying mechanisms by which this occurs in the brain after injury remain ill-defined.MethodsWe used GFP bone marrow chimeric knockout (KO) mice to demonstrate that the axon guidance molecule EphA4 receptor tyrosine kinase is involved in suppressing MERTK in the brain to restrict efferocytosis of resident microglia and peripheral-derived monocyte/macrophages.ResultsSingle-cell RNAseq identified MERTK expression, the primary receptor involved in efferocytosis, on monocytes, microglia, and a subset of astrocytes in the damaged cortex following brain injury. Loss of EphA4 on infiltrating GFP-expressing immune cells improved functional outcome concomitant with enhanced efferocytosis and overall protein expression of p-MERTK, p-ERK, and p-Stat6. The percentage of GFP+ monocyte/macrophages and resident microglia engulfing NeuN+ or TUNEL+ cells was significantly higher in KO chimeric mice. Importantly, mRNA expression of Mertk and its cognate ligand Gas6 was significantly elevated in these mice compared to the wild-type. Analysis of cell-specific expression showed that p-ERK and p-Stat6 co-localized with MERTK-expressing GFP + cells in the peri-lesional area of the cortex following brain injury. Using an in vitro efferocytosis assay, co-culturing pHrodo-labeled apoptotic Jurkat cells and bone marrow (BM)-derived macrophages, we demonstrate that efferocytosis efficiency and mRNA expression of Mertk and Gas6 was enhanced in the absence of EphA4. Selective inhibitors of ERK and Stat6 attenuated this effect, confirming that EphA4 suppresses monocyte/macrophage efferocytosis via inhibition of the ERK/Stat6 pathway.ConclusionsOur findings implicate the ERK/Stat6/MERTK axis as a novel regulator of apoptotic debris clearance in brain injury that is restricted by peripheral myeloid-derived EphA4 to prevent the resolution of inflammation.

A novel role of EphA4/Stat6/Mertk signaling in cleaning up the brain after trauma

Abstract Traumatic brain injury (TBI) is a major cause of disabilities and deaths from acute injuries in the US and worldwide. Neuroinflammation and neuronal apoptosis are hallmarks of TBI; however, the mechanism of clearance of apoptotic debris by efferocytosis remains ill-defined. Our previous studies demonstrated that EphA4 receptor in peripheral-derived macrophage (PDM) mediates the pro-inflammatory response after injury. Here, we aimed to evaluate whether PDM-specific EphA4 restricts efferocytosis following TBI. We generated wild-type (WT) and EphA4-knockout (KO), GFP +bone marrow (BM) chimeric mice and performed Controlled cortical impact injury. We observed that KO mice displayed improved motor function, cerebral blood flow, reduced cortical cell death as measured by TUNEL, and enhanced efferocytosis compared to WT mice. The percentage of GFP +/Iba1 −expressing PDMs that engulf NeuN +or TUNEL +cells was significantly higher in KO mice (p<0.05, n=5). We further performed an in vitro efferocytosis assay using pHrodo-labeled apoptotic Jurkat cells co-cultured with GFP +WT and KO BM-derived macrophages. We found a significant increase in efferocytosis efficiency in KO compared to WT cells (p<0.05, n=4). In addition, mRNA isolated from KO cells show increased expression of the efferocytosis receptor Mertk and its ligands Gas6 and Pros1 and phospho-array analysis shows increased p-Stat6, a key regulator of Mertk/Gas6. Interestingly, the use of Stat6 and Mertk inhibitors attenuated efferocytosis effects in KO macrophages in-vitro. Our findings implicate the EphA4/Stat6/Mertk axis as a novel regulator of apoptotic debris clearance that may be targeted to augment resolution of inflammation following TBI. National Institute of Neurological Disorders and Stroke of the National Institutes of Health, NS096281, NS119540, and NS121103 (MHT)

Magic-TT (Magnetism-induced cell target transplantation) Improves the Effect of Intra-Bone Marrow Transplantation Resulting in Rapid Hematopoietic Reconstitution in Allogenic Murine Transplantation Model

Background: Intra-bone marrow transplantation has been proven to be efficient in improving the homing of transplanted cells, resulting in faster hematopoietic reconstitution and a reduction in severity of GVHD. However, because of staggered blood vessels in bone, many transplanted cells leak out to peripheral circulation. In our previous study (Blood 2015 126:5404), it has been initially proven that MagIC-TT could enhance target transplantation to femurs by flow cytometry and pathological examination. After that, by bioluminescent imagine system, we further proved that most intra-bone marrow transplanted cells moved to lung immediately without MagIC-TT (Figure 1 A); while the cells were retained in the bone marrow with MagIC-TT (Figure 1 B), but if the magnetic field withdrawal 1 hour after, the cells also moved to the lung quickly(Figure 1 C). In this study, the therapeutic effect of intra-bone marrow transplantation combined with MagIC-TT was explored in allogenic murine transplantation model.MethodsMice and cell preparation: Sixty female BALB/c mice with specific pathogen free (SPF) were randomly separated in 4 groups (16 and 14 mice in 5×10e6M and 1×10e7 M group respectively; 16 and 14 mice in 5×10e6W and 1×10e7 W group respectively, M/W referring to MagIC-TT/non-MagIC-TT). The recipients received 7.5Gy irradiation before transplantation. Twelve SPF C57BL/6 CAG-eGFP transgenic mice were used as donors. The bone marrow were magnetized by nanoparticles and then adjusted to the concentration of 5×10e6/20μL and 1×10e7/20μl for use. Every mouse was injected with 20μL, 5×10e6 or 1×10e7 cells.Intra-bone marrow injection and fixed magnetic field After anesthetizing the mice and disinfecting the knee joint, the femoral puncture was operated with the medulla-puncture needle invented by our team (Chinese patent No.201620090904.2). In M groups, special magnetic fields were applied to the femurs of mice during and after injection. The effects of intra-bone marrow injection had been proven by X-ray and bioluminescent imaging system, etc .The therapeutic effects The therapeutic effects were determined by the survival of mice, the percentage of GFP+ cells in peripheral blood, blood routine test etc. to evaluate the hematopoietic reconstitution.ResultsSurvival: The survival rate was 66.7% and 33.3% in M groups and W groups on the 40th day after transplantation (P=0.096) (Figure 2 A), which was 62.5% and 71.4% in 5×10e6M and 1×10e7M, 37.5% and 28.6% in 5×10e6W and 1×10e7W, respectively(Figure 2 B).Donor cells percentage in peripheral blood (Figure 3 A): Flow cytometry analysis was performed on the 5th, 13rd, 17th, and 25thday after transplantation. The percentage of donor GFP+ cells grew much faster on d5~d13 in M groups than that in W groups (P<0.05). Pairwise comparisons showed that 10e7M group grew faster than 10e7W (P<0.05), donor GFP+ cells in 5×10e6M grew even faster than those in both 5×10e6W and 1×10e7W (P<0.05). No significant difference was found between 5×10e6M and 1×10e7M.Blood Routine Test: Generally, WBC in M groups reached growth peak on d9-13 vs d13-17 in W groups, and the WBC number of M groups are higher than those in W group (P<0.05) (Figure 3B). This trend also existed for HGB and PLT, but not as significant as WBC (Figure 3 C, 3 D) .ConclusionMagIC-TT can improve the effect of intra-bone marrow transplantation by avoiding transplanted cells leakage to peripheral circulation, resulting in faster hematopoietic reconstitution in allo-murine transplantation model. The mechanism of MagIC-TT and its relation with reduction of complication are under further study. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

BNP increases the number of cardiomyocytes in physiological and pathological conditions

Brain natriuretic peptide (BNP) treatment protects the heart after myocardial infarction (MI). As cardiomyocytes (CMs) express BNP receptors, the aim of this study was to determine BNP effect on CM's fate in neonatal and in adult infarcted hearts. New born (NB) unmanipulated pups and adult infarcted mice were injected with BNP every 2 days. NB mice were sacrificed eleven days after birth. MI was induced in C57BL/6 and Myh6 MerCreMer mice by permanent ligation of the left anterior descending coronary artery and sacrificed 24 hours and 10 days after surgery. An increased number of CMs was detected in BNP-treated NB mice (+ 18% P = 0.002) and in the infarcted zone (ZI) of adult hearts, 10 days after MI (+ 43%; P = 0.07). In BNP-treated pups, the number of CMs expressing BrdU (+ 22% P = 0.04), phospho histone H3 (pH3) (+ 31%; P = 0.04) and Aurora B (+ 47% P = 0.001) were increased. Expressions of mRNAs coding for cyclins E1 (+ 110% P = 0.01), A2 (+ 126% P = 0.002) and B2 (+70% P = 0.08) were increased in CMs isolated from BNP treated hearts. Accordingly, in vitro BNP treatment of neonatal CMs led to increased number of CMs (+ 21% P = 0.003), and CMs expressing Aurora B (+ 58% P = 0.01), pH3 (+ 75% P = 0.04) or Ki67 (+ 20% P = 0.03). Interestingly, BNP treatment on these cells was associated with p38 MAPK inhibition (-42% P = 0.01). In BNP-treated mice 24 hours after MI, a decreased concentration of cardiac Troponin T in the plasma (− 70% P = 0.04) was measured, whereas the percentage of BrdU+ (+ 92% P = 0.03), pH3+ (+ 51%; n = 2) and Aurora B+ CMs (+ 419% P = 0.03) increased in ZI of BNP-treated hearts, 10 days after MI. In Myh6 MerCreMer mice, the percentage of GFP+ CMs expressing BrdU also increased in ZI (+ 15% P < 0.05). In conclusion, BNP treatment protects CMs from cell death after MI. In addition, it stimulates the proliferation of neonatal and adult pre-existing CMs after MI.

Abstract B31: Detection of viable tumor cells from cryopreserved buffy coat using the VTX-1 Liquid Biopsy Platform

Abstract Introduction: Several circulating tumor cell (CTC) technologies have been developed amidst growing interests in the “liquid biopsy” field. The fully automated VTX-1 platform is developed to gently isolate intact CTCs from whole blood with high recovery and viability, compatible with many applications (1-3). However, many CTC technologies require processing of fresh patient samples at off-site facilities. Here we present encouraging preliminary results showing viable cell isolation by VTX-1 from cryopreserved buffy coat, using a telomerase-selective replicating adenovirus (TelomeScan). This widens potential applications of viable CTC detection for retrospective clinical studies and drug development. Methods: Buffy coat preparation: Buffy coat was prepared by centrifugation of healthy donor blood collected in EDTA, heparin, or heparin CPT vacutainers. Cell spiking and processing: 1) Cancer cells were spiked into freshly prepared buffy coat and processed with the Vortex chip at different dilutions. 2) Cancer cells were labeled and spiked into healthy donor blood, and buffy coat was prepared and processed by VTX-1 either immediately or following cryopreservation up to 1 week. Imaging was evaluated for cell recovery. Viable, telomerase-activated tumor cell detection: VTX-1 enriched cells were directly collected in chamber slides and infected with the TelomeScan adenovirus (OBP-401) for 24 h (4). The percentage of GFP (+), viable, telomerase-expressing cells was evaluated alongside cell recovery. Results: MCF7 cells were spiked into freshly prepared buffy coat, resuspended in 8mL, and processed at 5X, 10X, and 20X dilutions. The best compromise between recovery, WBC count, and processing time was obtained for the 10X dilution with 46% recovery. The majority of spiked cells (MCF7 and H1299) collected from buffy coat were shown to be viable, as demonstrated by infection with the telomerase-selective replicating adenovirus (TelomeScan, OBP-401). Cryopreservation of the buffy coat samples showed a slight reduction following cryopreservation. Discussion: The optimal performance of the Vortex chip on buffy coat samples has previously been shown at 10X dilution. This capability of the VTX-1 was further demonstrated through infection with a telomerase-selective replicating adenovirus. This combined workflow has shown seamless integration from whole blood. Noteworthy recovery and sensitivity were also observed with cryopreserved buffy coat. Patient biobanked samples are currently being pursued. The overall advantages are: a) viable cell detection, b) direct collection of cells into culture media ready for in vitro testing, c) no requirements for fixation, labeling, backflushing, d) a very low WBC background eliminating false positives, and e) the potential to retrospectively analyze clinical biobanked samples at a centralized laboratory.

UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling.

SummaryNeural stem cell (NSC) proliferation and differentiation in the developing brain is a complex process precisely regulated by intrinsic and extrinsic signals. Although epigenetic modification has been reportedly involved in the regulation of the cerebral cortex, whether UTX, an H3K27me3 demethylase, regulates the development of cerebral cortex during the embryonic period is unclear. In this study, we demonstrate that Utx deficiency by knockdown and conditional knockout increases NSC proliferation and decreases terminal mitosis and neuronal differentiation. Furthermore, we find that impairment of cortical development caused by lack of Utx is less significant in males than in females. In addition, UTX demethylates H3K27me3 at the Pten promoter and promotes Pten expression. P-AKT and P-mTOR levels are increased in the Utx conditional knockout cortices. Finally, Utx or Pten overexpression can rescue the impairment of brain development caused by Utx loss. These findings may provide important clues toward deciphering brain diseases.

Folate receptor mediated genetic modification of human mesenchymal stem cells via folic acid-polyethylenimine-grafted poly(N-3-hydroxypropyl)aspartamide.

Mesenchymal stem cells (MSCs) are targeted as vehicles for cell mediated gene therapy. Here we report on a macromolecular carrier, which was designed aiming at successful targeted gene delivery into MSCs through the mediation of folate receptor and reduced cytotoxicity compared to established cationic polymer vector - polyethylenimine with a weight average molecular weight (Mw) of 25,000 Dalton (PEI25K). The carrier PHPA-PEI1800-FA was synthesized in a two-step procedure. PHPA-PEI1800 was prepared by grafting polyethylenimine with a Mw of 1800 Dalton (PEI1800) onto the α,β-poly(N-3-hydroxypropyl)-D,L-aspartamide (PHPA) backbone. PHPA-PEI1800-FA was obtained by chemically conjugating folic acid onto PHPA-PEI1800. The grafting degree of PEI1800 was 3.9±0.2% in relation to the CH groups of PHPA and the molar ratio of folic acid conjugated to PEI1800 (χFA) was 1.8±0.1 as calculated by NMR spectroscopy. The copolymers were biodegradable and exhibited lower cytotoxicity than PEI25K. Compared to PHPA-PEI1800, PHPA-PEI1800-FA led to a significantly higher transfection efficiency in human MSCs, which could be attributed to the mediation of folate receptor during the transfection process as confirmed by folic acid competition assay. Both marker gene (GFP) and therapeutic gene (VEGF) were delivered into human MSCs from multi-donors using PHPA-PEI1800-FA. The percentage of GFP+ MSCs showed an average value of 2.85±1.60% but a large variation for different samples. The VEGF expression level of the PHPA-PEI1800-FA transfected cells was significantly higher than that of either untransfected or naked DNA transfected cells. Conclusively, PHPA-PEI1800-FA is a suitable vector to deliver genes into human MSCs through the interaction with folate receptor.

Bone marrow endothelial progenitor cells activate hepatic stellate cells and aggravate carbon tetrachloride induced liver fibrosis in mice via paracrine factors.

Bone marrow derived endothelial progenitor cells (BM-EPCs) are increased in chronic liver disease (CLD). Their role in hepatic fibrosis and regeneration remains an area of intense studies. We investigated the migration and secretory functions of BM-EPCs in fibrotic mice liver. Bone marrow cells from C57BL6-GFP mice were transplanted into the femur of irradiated C57BL6 mice, followed by CCl4 doses for 8 weeks, to develop hepatic fibrosis (n=36). Transplanted C57BL6 mice without CCl4 treatment were used as controls. EPCs were analyzed in BM, blood and liver by flow cytometry and immunofluorescence. VEGF and TGF-β were analysed in the hepatic stellate cells (HSCs) and BM-EPCs co-cultures using ELISAs. There was a significant migration of EPCs from BM to blood and to the liver (P≤0.01). Percentage of GFP+ CD31+ EPCs and collagen proportionate area was substantially increased in the liver at 4th week of CCl4 dosage compared to the controls (19.8% vs 1.9%, P≤0.05). Levels of VEGF (533.6pg/ml) and TGF-β (327.44pg/ml) also increased significantly, when HSCs were treated with the EPC conditioned medium, as compared to controls (25.66pg/ml and 5.87pg/ml, respectively; P≤0.001). Present findings suggest that BM-EPCs migrate to the liver during CCl4-induced liver injury and contribute to fibrosis.

HDAC Inhibitors Lead to Significant Survival Benefit in a Novel Fusion Protein TBLR1-Rarα Induced Murine Promyelocytic Leukemia Model

Introduction: TBLR1-RARα is the tenth fusion gene of acute promyelocytic leukemia (APL) first identified in a rare case of APL with t(3;17)(q26;q21) chromosomal translocation in our previous study. The characteristics of its basic structure and functions had been clarified in our previous study. In this study, we successfully established a novel TBLR1-RARα leukemia mouse model (TR mouse) which fully recapitulated the most relevant features of human APLs. The therapeutic effects of retinoic acid (ATRA), arsenic trioxide (As2O3), cytarabine (Ara-C) and histone deacetylase inhibitors (HDACi) on TR mice were examined. The differentially expressed genes (DEGs) between TR mice and normal mice were compared to explore the possible mechanisms and better therapeutic targets for this kind of APL.Methods: pMSCV-TBLR1-RARα-Flag-IRES-GFP (MSCV-TR) and pMSCV-IRES-GFP (vehicle) retroviral plasmids were constructed and transfected 293T packaging cells to produce retroviruses. Lin- cells from C57BL/6 mice bone marrow were purified and infected with MSCV-TR and vehicle retroviral supernatant. For in vitro assay, the GFP+ lin- cells sorted and incubated with or without different concentrations of ATRA were analyzed for the differentiation and proliferation capacity by cell morphology, myeloid markers (CD11b and GR-1) and colony formation assay. For the in vivo experiment, GFP+ lin- cells transfected with indicated retroviral vectors were injected intravenously to lethally irradiated C57BL/6 mice to establish an APL mouse model. Cell surface markers were analyzed by flow cytometry. In treatment assays, GFP+ spleen cells from TR leukemia mice were injected intravenously into recipient mice. The mice were randomly separated into groups and received different treatment with ATRA, As2O3, As2O3 in combination with ATRA, Ara-C, Ara-C in combination with ATRA, chidamide and NL101, respectively. The percentage of GFP+ cells in peripheral blood and body weight were measured dynamically. The survival time of every group was recorded and compared. RNA-seq assay was used to identify DEGs between TR mice and normal mice.Results: In vitro assays indicated that TBLR1-RARα could either block the differentiation of HSCs at a relatively early stage or enhanced the clonogenic potential of cells. The TBLR1-RARα leukemia mouse model was successfully established. During the ten-month observational period, 3 out of 15 mice transplanted with TBLR1-RARα expressing cells developed an APL-like disease. Development of leukemia was not observed in any of the mice in control group. All the leukemia mice had a body weight loss as well as splenomegaly and hepatomegaly. The phenotype analysis revealed that the progenitor markers Sca-1, CD34 and C-kit were positive, the myeloid lineage markers Gr-1 and CD11b were also positive, erythroid lineage marker Ter119 was weekly positive, but the lymphatic lineage marker B220, CD3,CD4 and CD8 were all negative. TR mice treated with 1.5-2.5 mg/kg ATRA alone or together with 2.0 mg/kg As2O3 didn’t survive longer than that of control group, although in vitro differentiation experiment showed that the leukemia cells were sensitive to ATRA. Leukemic mice receiving Ara-C treatment had a much longer survive time. Surprisingly, HDAC inhibitors (12.5 and 25 mg/kg chidamide and 30 mg/kg NL-101) could significantly prolong the survival time of TR mice. Thousands of DEGs had been identified between TR mice and wild type mice, which were widely involved in multiple pathways and participated in various biological functions.Conclusion: The TBLR1-RARα leukemia mouse model was successfully established for the first time, and its main characteristics were clarified. Although the leukemia cells were sensitive to ATRA in vitro, TR mice didn’t benefit from ATRA or As2O3 treatment in vivo. Besides Ara-C, HDAC inhibitors, such as chidamide and NL-101 exhibited potency therapeutic values for TR mice, which provided a new strategy for this kind of refractory APL. What’ more, lots of genes that might be related with the process of leukemogenesis and new therapeutic targets for TR leukemia were identified. This model would serve as a versatile tool to study the mechanisms of leukemogenesis and help to design better strategies for APLs in further studies. DisclosuresNo relevant conflicts of interest to declare.

The MEK/ERK Inhibitor Trametinib Reduces Fibrosis in a Transduction-Transplantation Model of Mutated Calreticulin

▪Insertion or deletion mutations in calreticulin (CALR) are present in the majority of JAK2V617F-negative MPN patients. We utilized a murine retroviral transduction-transplantation model to express the 52bp CALR deletion mutation (CALRDEL) in both BALB/c and C57B/6 backgrounds. As described previously (Marty et al., Blood 2016;127:1317), recipients of CALRDEL-transduced marrow developed persistent thrombocytosis without leukocytosis or erythrocytosis by two months post-transplant. Mice were euthanized at six and nine months post-transplant to evaluate the tempo of disease progression. At six months CALRDEL mice had impressive expansion of megakaryocytes expressing the CALRDEL mutant protein in the bone marrow (BM) without fibrosis or significant splenomegaly. By nine months BM fibrosis and splenomegaly were present. Both whole BM and spleen cells were able to serially transplant the MPN phenotype into secondary recipients.When cultured in collagen-based media supplemented with thrombopoietin, CALRDEL BM cells produced an increased number of megakaryocyte colonies as compared to empty vector. The increased colony formation potential of CALRDEL bone marrow cells was limited to megakaryocytes, we found no increase in colony formation from CALRDEL hematopoietic stem and progenitor cells in methylcellulose with cytokines supporting erythroid and GM colony formation. However, CALRDEL enhanced the serial replating ability of LKS (lineageneg, c-kit+ Sca-1+) cells.Both pSTAT5 and pERK were increased in whole spleen lysates from CALRDEL mice as compared to wild-type BALB/c mice. Therefore, we tested the impact of ruxolitinib, a JAK1/2 inhibitor, and trametinib, a MAPK/ERK inhibitor, on the MPN phenotype of CALRDEL mice. At six months post-transplant mice were treated with either ruxolitinib (90mg/kg PO BID), trametinib (3mg/kg PO daily), or vehicle for 40 days. Ruxolitinib reduced pSTAT5 but caused a paradoxical increase in pERK in whole spleen lysates, while trametinib reduced pERK but not pSTAT5. Trametinib caused a transient increase in platelets and white cells. In spite of pharmacodynamic evidence of effective dosing, ruxolitinib had no significant effect on platelet or leukocyte count but did reduce hemoglobin slightly. Both ruxolitinib and trametinib reduced spleen weight. Ruxolitinib reduced the fraction of the mutant CALRDEL allele (inferred from percentage of GFP+ cells) in the spleen but not the bone marrow, while trametinib had no impact on disease allele burden in any organ. Neither ruxolitinib nor trametinib reduced the expansion of megakaryocytes in the bone marrow but trametinib significantly reduced marrow fibrosis (average score MF-2.5 for vehicle, MF-1.75 for ruxolitinib, MF-1 for trametinib). To assess the role of STAT5 in the pathogenesis of the ET-like MPN induced by the CALRDEL mutant, we transduced BM from syngeneic Balb/c donors carrying a floxed Stat5ab allele in combination with a Stat5ab null allele (Mx-Cre;Stat5abfl/-; Walz et al., Blood 2012;119:3550). Haploinsufficiency for Stat5ab significantly delayed the development of ET-like MPN and attenuated thrombocytosis, implicating JAK2-STAT5 signaling directly in the pathogenesis of this disease.In summary, this CALRDELmouse model results in an MPN phenotype resembling essential thrombocythemia followed by myelofibrosis. CALRDELresults in expansion of megakaryocytes and platelets without expansion of other myeloid cell types. Both pSTAT5 and pERK are increased in our CALRDEL model and pharmacologic inhibition of pERK results in reduction of fibrosis without reducing megakaryocytes. These studies implicate pERK as a potential anti-fibrosis therapeutic target in MPN. DisclosuresNo relevant conflicts of interest to declare.

Histones Are Rapidly Loaded onto Unintegrated Retroviral DNAs Soon after Nuclear Entry.

Chromosomal structure of nuclear DNA is usually maintained by insertion of nucleosomes into preexisting chromatin, both on newly synthesized DNA atreplication forks and at sites of DNA damage. But during retrovirus infection, a histone-free DNA copy of the viral genome is synthesized that must be loaded with nucleosomes de novo. Here, we show that core histones are rapidly loaded onto unintegrated Moloney murine leukemia virus DNAs. Loading of nucleosomes requires nuclear entry, but does not require viral DNA integration. The histones associated with unintegrated DNAs become marked by covalent modifications, with a delay relative to thetime of core histone loading. Expression from unintegrated DNA can be enhanced by modulation of the histone-modifying machinery. The data show that histone loading onto unintegrated DNAs occurs very rapidly after nuclear entry and does not require prior establishment of an integrated provirus.

564. The Cytotoxic Effect of RNA-Guided Endonuclease Cas9 on Human Hematopoietic Stem and Progenitor Cells (HSPCs)

The CRISPR (clustered regulatory interspaced short palindromic repeats) - CRISpR-associated protein 9 (Cas9) system (CRISPR/Cas9) has recently emerged as an efficient and powerful approach widely used for targeted genetic engineering. RNA-guided endonuclease Cas9 in combination with a single guide RNA (sgRNA) targets the sequence-specific DNA binding domains and generates double strand breaks (DSBs), which are repaired through non-homologous end joining (NHEJ) or homologous recombination (HR). Although CRISPR/CAS9 system very efficiently disrupts target locations in the genome of multiple model organisms, editing in some primary cell types, especially hematopoietic stem and progenitor cells (HSPCs) has been more challenging, with lower efficiencies particularly in functional engrafting HSPC. However, the exact mechanisms have not been elucidated. To optimize editing via CRISPR/Cas9 in human HSPCs, we generated several different lentiviral vectors (Figure 1Figure 1); a negative control GFP only non-editing vector (LeGO-GFP), U6 promoter-AAVS1 targeting sgRNA-EF1a promoter-GFP (AAVS1-GFP), U6-EFS promoter-Cas9-EF1a-GFP (Cas9-GFP), U6-AAVS1 targeting sgRNA-EFS-Cas9-EF1a-GFP (AAVS1-Cas9-GFP), U6-EFS-human codon-optimized Cas9-EF1a-GFP (hCas9-GFP), and U6-AAVS1 targeting sgRNA-EFS-human codon-optimized Cas9-EF1a-GFP (AAVS1-hCas9-GFP). These lentiviral vectors simultaneously deliver sgRNA targeting a “safe harbor” genome location (AAVS1 in these experiments), Cas9 and a fluorescent marker, however, we replaced P2A with EF1a promoter between Cas9 and a fluorescent marker, because we found dim or none fluorescent marker expression using P2A co-expression systems. Lentivirus-transduced HSPCs were analyzed by flow cytometry based on their GFP expression in a time-dependent manner (3, 6, 9, 12 days). All vectors resulted in a gradual decrease in the percentage of GFP+ cells over time, however, standard Cas9 containing vectors (Cas9-GFP and AAVS1-Cas9-GFP) showed most significant decreases (Figure 2Figure 2). Vectors containing the human codon-optimized version of Cas9 (hCas9-GFP, AAVS1-hCas9-GFP) showed intermediate decreases, and vectors without Cas9, whether or not they expressed sgRNA (LeGO, AAVS1-GFP), showed the least decrease in GFP+ cells, with 30% of the cells still expressing GFP at 12 days following transduction. These results led us to investigate the cytotoxicity of Cas9 on HSPCs, using Vivid and Annexin V staining. Both standard Cas9 and human codon-optimized Cas9 expressing HSPCs showed strong Annexin V positivity and a gradual increase in Vivid+ cells, explaining the loss of GFP+ cells with Cas9-expressing vectors. On the other hand, HSPCs transduced with the non-Cas9-expressing vectors remained Vivid and Annexin V low until through 12 days. Our findings suggest that sustained expression of Cas9 in human HSPCs, for instance via an integrating vector, has significant toxicity, and that an alternative Cas9 delivery method for HSPCs is required. We have tried integrase-defective lentivirus (IDLV) and mRNA transfection to deliver Cas9 into HSPCs, and data will be presented. This approach will facilitate the use of CRISPR/Cas9 system to engineer genes of clinical significance in HSPCs with a therapeutically meaningful efficacy.View Large Image | Download PowerPoint SlideView Large Image | Download PowerPoint Slide

126. Identification of Human Papillomavirus Entry Receptors with CRISPR-Cas9 Library

Human papillomaviruses (HPVs) are a family of small nonenveloped viruses that induce mostly benign papillomas. However, infection of several high-risk HPV types, most prominently HPV 16 and 18, can cause cervical cancer and other epithelial tumors. The cell biology of HPV entry is still a subject of scientific debate, and in particular, the cellular receptors for HPV infection have not been firmly determined. The present study was designed to use CRISPR-Cas9 gene knock-out library to identify cellular proteins that may be used by HPV for entry. HEK293 cells were initially transduced with CRISPR-Cas9 knockout screening libraries, and then selected with puromycin for two weeks. The cells were then transduced with HPV-16 pseudoviruses containing the GFP marker gene, and GFP-negative cells were subsequently selected by flow cytometry. This process was repeated multiple rounds to enrich the GFP− cell population, and the percentage of GFP+ cells went down from over 90% to less than 10% during this enrichment process. A combination of PCR and DNA sequencing was then used to determine the genes that had been knocked out from this cell population. Our data show that solute carrier family 35 (adenosine 3’-phospho 5’-phosphosulfate transporter) member B2 (SLC35B2), malectin (MLEC), xylosylprotein beta 1,4-galactosyltransferase and transmembrane protein 8C (TMEM8C) were the most frequently knocked out genes in these cells. We then validated the involvement of one of these genes, SLC35B2 in HPV entry, by either deleting genes from fresh HEK293 cells or by reintroducing the gene back to the cells that had it knocked out. Interestingly, SLC35B2 knockout was found to affect entry of some other viruses such as herpes simplex viruses. In conclusions, CRISPR-Cas9 knockout screening system is a useful tool for identifying cellular receptors for viruses such as HPV, in which the entry mechanism has not been fully elucidated.

MiR-181b in Chronic Lymphocytic Leukemia B Cells Is Regulated By Cellular Interaction with CD4+ T Cells and Increases the CTL Toxicity Versus the Leukemic Clone

Abstract Clinical progression of chronic lymphocytic leukemia (CLL) is characterized by gradual reduction of the ratio T/B cells, along with immune cell dysfunction due, at least in part, to T cell defects, such as decreased expression of CD40L and reduced signaling via the TCR CD3. This compromise the ability of T cells to respond and to eliminate leukemic cell from CLL patients. Enhanced activation of either allogenic or autologous T cells can drive the death of CLL cells in vitro and in human subjects. Changes in microRNAs expression also characterize clinical progression of CLL with a strong decrease of miR-181b/a and miR-130a associated with the more aggressive phase of the disease. The miR-181b targets anti-apoptotic proteins, such as BCL-2 and MCL1 and its expression correlates with those protein levels in CLL. In this study we demonstrate that the expression of those microRNAs in CLL-B cells, are regulated by T cells. We co-cultured allogenic pure CLL-B cells with either activated (CD2, CD3 and CD28 antibodies, used to mimic antigen-presenting cells) or not activated CD4+ T cells from healthy donors. We observed a significant increase of mir-181b/a and miR-130a expression in CLL B-cells after co-culture with activated CD4+ T cells in 8 out of 11 cases. A significant increase of these miRs was also determined in purified CLL B-cells after 4 days activation of peripheral blood mononuclear cells (PBMCs) from CLL patients, even if in minor rate. By the use of specific antibodies, co-culture with Hela CD40 expressing cells and transwell experiments, we established that this effect is a T/B contact-dependent signaling mediated through CD40L-CD40 interaction. We determine that increased expression of the 3 miRs occurs at the transcriptional level. Since the expression of miR-181b showed the most significant variation in previous experiments it was selected for further analyses. We next investigated the in vivo role of the miR-181b in highly immunodeficient mice. The CLL cell line, MEC-01, infected with either the LV-miR-181b_coGFP or the LV-CTRL_coGFP was intravenously inoculated in NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ (NSG) mice. Mice were sacrificed after 4 weeks and assayed for percentage of GFP+ cells in bone marrow and spleen compartments. The miR-181b did not show any specific effect into the leukemic clone. However when the same cells were inoculated in an environment hosting mature T cells, miR-181b consistently influences the death of leukemic cells (Fig 1B), suggesting that T cells are required to potentiate the apoptotic role of this miRNA. To explain what we observed in vivo, we mixed in vitro MEC-01 infected with either the LV-miR-181b or the LV-CTRL and CD8+ T cells from healthy donors. After few hours of contact T cells showed stronger cytotoxic effect on MEC-01 carrying miR-181b as compared to the control. Mixed lymphocyte reaction CD40L-activated CLL and T cells is used to generate effector CTLs. Therefore we grew T cell with CD40L-activated MEC-01 in which the expression of miR-181b was either shut down by lentiviral vector or unchanged as control. After one week, we monitored by cytofluorimetry the CD38 surface marker on T cells since its expression has been associated with more active CTLs and, by ELISA, the release of IL-10, the inhibitor of the potent inducer of CTLs INF-g. We demonstrate that activated MEC-01 with higher expression of miR-181b leads to an increase of the cell number expressing CD38 and this was accompanied by a reduced release of IL-10 from B cells through down-regulation of c-FOS, which we show to be target of the miR-181b and to promote the transcription of the IL-10. In conclusion, our data suggest a role of the miR-181b in the immune response against CLL-B cells. We show that an efficient activation of CD4+ T cells through CD3-complex pathway and a right CD40L-CD40 interaction lead to a significant increase of the some miRNAs deregulated over the progression of chronic lymphocytic leukemia, namely miR-181b. This miRNA potentiates the cytotoxicity of T cells favoring the killing of the leukemic clone. Disclosures No relevant conflicts of interest to declare.

Effect of ADAR1 on the development of MLL-AF9 induced murine AML

目的建立敲除RNA腺苷脱氨酶1(ADAR1)的小鼠MLL-AF9融合基因急性髓系白血病（AML）模型，初步探讨ADAR1对AML发病的影响。方法采用免疫磁珠法富集介导雌激素受体-重组酶Cre (ER-Cre)的ADAR1lox/lox及其对照ADAR1lox/lox小鼠骨髓Lineage−（Lin−）细胞，用携带MSCVMLL/AF9-IRES-GFP的逆转录病毒感染上述Lin−细胞，流式细胞术检测感染效率，移植相同数量细胞至致死剂量和半致死剂量照射受体小鼠中，建立MLL-AF9诱导的AML模型。移植48 h后诱导ADAR1敲除，体内实验分为实验组（ER-Cre;ADAR1lox/lox＋他莫昔芬）和对照组(①ER-Cre;ADAR1lox/lox +空载体、②ADAR1lox/lox+他莫昔芬、③ADAR1lox/lox＋空载体)，第10、15、20天分别检测小鼠外周血GFP+细胞比例，观察各组小鼠的存活情况。体外实验分组同上，将他莫昔芬改为4-羟基他莫昔芬，观察各组AML细胞并检测其凋亡情况。结果成功建立敲除ADAR1的MLL-AF9融合基因AML小鼠模型。与对照组比较，体内实验中实验组AML小鼠在各时间点外周血GFP+细胞比例均降低，存活时间明显延长，差异均有统计学意义(P值均<0.05)；体外实验中实验组细胞总数、GFP+细胞比例均降低，Annexin Ⅴ+7-AAD+和Annexin Ⅴ+细胞比例均升高，差异均有统计学意义(P值均<0.05)。结论敲除ADAR1可减缓AML的发病，增加AML细胞凋亡。ADAR1在MLL-AF9诱导的AML发生和维持过程中起关键作用。

The Vascular Niche Is Involved in Regulating Leukemic Stem Cells in Murine Chronic Myelogenous Leukemia

Chronic myelogenous leukemia (CML) is effectively controlled by tyrosine kinase inhibitors (TKIs) such as imatinib mesylate, leading to a hematologic remission in >90% of patients. However, the majority of patients relapse once TKI therapy is discontinued, suggesting that CML leukemia stem cells (LSC) are not eradicated. We recently showed that modulation of the osteoblastic niche can lead to reduction of LSC in CML (Krause et al., Nat. Med. 2013;19:1513), but the role of the vascular hematopoietic stem cell niche in CML has not been well defined.E-selectin is expressed on bone marrow (BM) endothelium within the vascular niche, whereas loss of E-selectin expression or treatment with GMI-1271, an E-selectin small molecule antagonist, enhances HSC quiescence and self-renewal (Winkler et al., Nat. Med. 2012;18:1651). E-selectin also plays a critical role in the homing and engraftment of CML LSCs (Krause et al., Blood 2014;123:1361) through E-selectin ligands expressed on the LSCs, including CD44 (Krause et al., Nat Med. 2006;12:1175). We, therefore, hypothesized that E-selectin blockade with GMI-1271 may overcome niche-mediated resistance to TKIs and eradicate CML LSC.Using the well-described murine retroviral transduction/transplantation model of CML we showed that the white blood cell count (WBC) of mice with BCR-ABL1-induced CML-like leukemia was significantly reduced by treatment with imatinib plus GMI-1271 (or imatinib alone) and there was a trend towards WBC reduction by treatment with GMI-1271 alone (P=0.07). The percentage of GFP+ Mac-1+ cells in peripheral blood on day 16 post-transplant was decreased by imatinib or GMI-1271 alone or by combined treatment with imatinib and GMI-1271. Spleen weights were significantly reduced by combined treatment with imatinib plus GMI 1271. Furthermore, the BM GFP+ (BCR-ABL1+) Lin– c-Kit+ Sca-1+ population, which contains the LSCs in this model, was significantly reduced in animals treated with GMI-1271 compared to vehicle controls. As expected, treatment with imatinib alone had no effect on BM LSC frequency, and there was no added benefit in the reduction of LSC when imatinib and GMI-1271 were combined. In addition, the survival of mice treated with imatinib plus GMI-1271 was significantly prolonged compared to vehicle-treated animals, with ~20% of mice treated with GMI-1271 alone or the combination of imatinib and GMI-1271 exhibiting long-term low-burden disease despite discontinuation of treatment on day 28 post-transplant. In these primary recipients neither BCR-ABL1+ myeloid cells nor BCR-ABL1+ LSC were mobilized to peripheral organs. However, fewer BCR-ABL1+ LSC were found in the spleen of mice treated with GMI-1271 compared to imatinib-treated mice. There was a significant reduction in the frequency of cycling BCR-ABL1+ LSC in mice treated with GMI-1271 and imatinib.To assess directly the effect of E-selectin inhibition on LSC frequency and function, we transplanted BM from primary leukemic mice treated with vehicle, imatinib, GMI-1271 or the combination of imatinib and GMI-1271 into irradiated secondary recipient mice. There was a significant reduction of WBC and a trend towards reduction of BCR-ABL1+ myeloid cells in secondary recipients of BM from donors treated with GMI-1271 alone or in combination with imatinib, but not by imatinib alone.These data suggest that modulation of the vascular niche and, specifically, inhibition of E-selectin may be a possible strategy to target LSC in CML, possibly via a reduction in S-G2/M as cells arrest prior to apoptosis, even when imatinib is discontinued. Further studies on the effects of GMI-1271 on homing of LSC and the more exact mechanism of LSC reduction by GMI-1271 are being performed. DisclosuresMagnani:GlycoMimetics Inc.: Employment, Equity Ownership, Membership on an entity’s Board of Directors or advisory committees. Krause:Glycomimetics. Inc.: Research Funding.

Inhibition Of LSD1 As a Therapeutic Strategy For The Treatment Of Acute Myeloid Leukemia

Lysine specific demethylase 1 (LSD1) is a histone H3K4me1/2 demethylase found in various transcriptional co-repressor complexes. These complexes include Histone Deacetylases (HDAC1/2) and Co-Repressor for Element-1-Silencing Transcription factor (CoREST). LSD1 mediated H3K4 demethylation can result in a repressive chromatin environment that silences gene expression. LSD1 has been shown to play a role in development in various contexts. LSD1 can interact with pluripotency factors in human embryonic stem cells and is important for decommissioning enhancers in stem cell differentiation. Beyond embryonic settings, LSD1 is also critical for hematopoietic differentiation. LSD1 is overexpressed in multiple cancer types and recent studies suggest inhibition of LSD1 reactivates the all-trans retinoic acid receptor pathway in acute myeloid leukemia (AML). These studies implicate LSD1 as a key regulator of the epigenome that modulates gene expression through post-translational modification of histones and through its presence in transcriptional complexes.The current study describes the anti-tumor effects of a novel LSD1 inhibitor (GSK2879552) in AML. GSK2879552 is a potent, selective, mechanism-based, irreversible inhibitor of LSD1. Screening of over 150 cancer cell lines revealed that AML cells have a unique requirement for LSD1. While LSD1 inhibition did not affect the global levels of H3K4me1 or H3K4me2, local changes in these histone marks were observed near transcriptional start sites of putative LSD1 target genes. This increase in the transcriptionally activating histone modification correlated with a dose dependent increase in gene expression. Treatment with GSK2879552 promoted the expression of cell surface markers, including CD11b and CD86, associated with a differentiated immunophenotype in 12 of 13 AML cell lines. For example, in SKM-1 cells, increases in cell surface expression of CD86 and CD11b occurred after as early as one day of treatment with EC50 values of 13 and 7 nM respectively. In a separate study using an MV-4-11 engraftment model, increases in CD86 and CD11b were observed as early as 8 hours post dosing.GSK2879552 treatment resulted in a potent anti-proliferative growth effect in 19 of 25 AML cell lines (average EC50 = 38 nM), representing a range of AML subtypes. Potent growth inhibition was also observed on AML blast colony forming ability in 4 out of 5 bone marrow samples derived from primary AML patient samples (average EC50 = 205 nM). The effects of LSD1 inhibition were further characterized in an in vivo mouse model of AML induced by transduction of mouse hematopoietic progenitor cells with a retrovirus encoding MLL-AF9 and GFP. Primary AML cells were transplanted into a cohort of secondary recipient mice and upon engraftment, the mice were treated for 17 days. After 17 days of treatment, control treated mice had 80% GFP+ cells in the bone marrow whereas treated mice possessed 2.8% GFP positive cells (p<0.012). The percentage of GFP+ cells continued to decrease to 1.8% by 1-week post therapy. Remarkably, in a preliminary assessment for survival, control-treated mice succumbed to AML by 28 days post transplant, while treated mice showed prolonged survival.Together, these data demonstrate that pharmacological inhibition of LSD1 may provide a promising treatment for AML by promoting differentiation and subsequent growth inhibition of AML blasts. GSK2879552 is currently in late preclinical development and clinical trials are anticipated to start in 2014.All studies were conducted in accordance with the GSK Policy on the Care, Welfare and Treatment of Laboratory Animals and were reviewed the Institutional Animal Care and Use Committee either at GSK or by the ethical review process at the institution where the work was performed. Disclosures:Kruger:GlaxoSmithKline Pharmaceuticals: Employment. Mohammad:GlaxoSmithKline Pharmaceuticals: Employment. Smitheman:GlaxoSmithKline Pharmaceuticals: Employment. Liu:GlaxoSmithKline Pharmaceuticals: Employment. Pappalardi:GlaxoSmithKline Pharmaceuticals: Employment. Federowicz:GlaxoSmithKline Pharmaceuticals: Employment. Van Aller:GlaxoSmithKline Pharmaceuticals: Employment. Kasparec:GlaxoSmithKline Pharmaceuticals: Employment. Tian:GlaxoSmithKline Pharmaceuticals: Employment. Suarez:GlaxoSmithKline Pharmaceuticals: Employment. Rouse:GlaxoSmithKline Pharmaceuticals: Employment. Schneck:GlaxoSmithKline Pharmaceuticals: Employment. Carson:GlaxoSmithKline Pharmaceuticals: Employment. McDevitt:GlaxoSmithKline Pharmaceuticals: Employment. Ho:GlaxoSmithKline Pharmaceuticals: Employment. McHugh:GlaxoSmithKline Pharmaceuticals: Employment. Miller:GlaxoSmithKline Pharmaceuticals: Employment. Johnson:GlaxoSmithKline Pharmaceuticals: Employment. Armstrong:Epizyme Inc.: Has consulted for Epizyme Inc. Other. Tummino:GlaxoSmithKline Pharmaceuticals: Employment.

Targeted Gene Addition in Human Epithelial Stem Cells by Zinc-finger Nuclease-mediated Homologous Recombination

Preclinical and clinical studies showed that autologous transplantation of epidermis derived from genetically modified epithelial stem cells (EpSCs) leads to long-term correction of inherited skin adhesion defects. These studies were based on potentially genotoxic retroviral vectors. We developed an alternative gene transfer strategy aimed at targeting a "safe harbor" locus, the adeno-associated virus integration site 1 (AAVS1), by zinc-finger nuclease (ZFN)-induced homologous recombination (HR). Delivery of AAVS1-specific ZFNs and a GFP-expressing HR cassette by integration-defective lentiviral (LV) vectors (IDLVs) or adenoviral (Ad) vectors resulted in targeted gene addition with an efficiency of > 20% in a human keratinocyte cell line, > 10% in immortalized keratinocytes, and < 1% in primary keratinocytes. Deep sequencing of the AAVS1 locus showed that ZFN-induced double-strand breaks are mostly repaired by nonhomologous end joining (NHEJ) in primary cells, indicating that poor induction of the HR-dependent DNA repair pathway may be a significant limitation for targeted gene integration. Skin equivalents derived from unselected keratinocyte cultures coinfected with a GFP-IDLV and a ZFN-Ad vector were grafted onto immunodeficient mice. GFP-positive clones were observed in all grafts up to 18 weeks post-transplantation. By histological and molecular analysis, we were able to demonstrate highly efficient targeting of the AAVS1 locus in human repopulating EpSCs.

Platelets: An Efficient Depot for Generation and Distribution of Lysosomal Idua in Enzyme-Deficient MPS I Mice.

Abstract 3157Proviral integration into hematopoietic stem cells (HSC) by lentivirus vector (LV)-mediated gene transfer can provide the benefit of life-long therapeutic effect, yet it can also bring the risk of insertional oncogenesis. Platelets are terminally differentiated, enucleated cells full of secretory granules. Restricting transgene expression to platelets may reduce the risk for activating oncogenes in HSCs and most of their progeny, and take advantage of their rapid turnover and professional secretion function. In this study, we evaluated the feasibility of employing megakaryocyte-platelet as a depot for the over-expression and release of alpha-L-iduronidase (IDUA), the lysosomal enzyme deficient in patients with Mucopolysaccharidosis type I (MPS I, or Hurler Syndrome). Utilizing a human megakaryocytic DAMI cell line, we found that a hybrid human ankyrin-1 promoter (K) containing ALAS2 intron 8 enhancer and HS40 core element from human alpha LCR could introduce robust intracellular IDUA expression (over 500-fold of background) and significant enzyme release (120-fold). Upon introduction to megakaryocytic differentiation as verified by FACS analysis with PI staining and morphologic evaluation, the elevated intracellular IDUA levels remained unchanged, while released IDUA increased steadily by 30-fold. We then evaluated in vivo IDUA production/release from platelets in an enzyme-deficient MPS I mouse model. Lineage-negative bone marrow (BM) cells were isolated from MPS I mice using immunomagnetic cell sorting, and transduced twice with LV-K-IDUA-ires-GFP (KIiG) for a total multiplicity of infection at 30. Five-months after 1° transplantation, GFP+ platelets were detected by FACS analysis with CD41-PE staining and observed by Image X analysis. To generate animals with various transgene-dosages, 2° transplantation was performed into MPS I mice using a serial dilution of BM from 1° MPS/KIiG (51–65% transduction efficiency) with BM of MPS I. FACS analysis revealed that the percentage of GFP+ platelets was correlated directly to transduction efficiencies in 2° recipients as determined by real-time qPCR of BM 5 months post transplantation. Moreover, the intra-platelet IDUA enzyme activities were associated linearly with the changes of GFP+% platelets (r2=0.95) among MPS I mice with different transgene-dosages, suggesting that the lysosomal IDUA from transgene overexpression could be sorted and packaged into platelets with proper catalytic function. Noticeably 1% gene transfer efficiency as shown by 1% GFP+ platelets was sufficient to introduce the IDUA levels comparable to those found in wild-type platelets. To determine if platelet-derived IDUA could be released, we conducted Ca-induced platelet activation as verified by FACS analysis with Annexin V-APC and CD41-PE staining, resulting in linear correlation (r2=0.98) of released IDUA with intra-platelet IDUA. Interestingly, the plasma IDUA levels were also linearly correlated with GFP+% platelets (r2=0.96) among MPS I with different transgene-dosages. Competitive uptake assay using lymphoblastoid cells derived from a MPS I patient (LCLmps) demonstrated steady increase of intracellular IDUA with increasing amounts of extracellular enzyme levels, which was blocked by the presence of mannose-6-phosphate (M6P). This suggested that platelet-released IDUA retained its intercellular trafficking capability via M6P receptor. Finally normalization of aberrant lysosomal morphology was observed in LCLmps exposed to platelet-released IDUA as determined by in situ immunostaining with LysoTracker, indicating functional proficiency of platelet-derived IDUA to cross-correct deficit in patient’s cells. These results demonstrate for the first time that megakaryocytes/platelets are capable of over-producing, packaging and storing a lysosomal enzyme which retains proper catalytic activity, lysosomal enzyme trafficking and endogenous M6PR-mediated uptake, as well as the ability of cross-correction in patient’s cells. This data warrants further evaluation for the potential application of megakaryocytes/platelets in treating lysosomal storage diseases, especially for those, such as Gaucher disease, when the desirable enzyme is sensitive to neutral pH in serum and protection of enzyme in platelets may provide continuous, real-time enzyme release by low physiological levels of platelet activation. Disclosures:No relevant conflicts of interest to declare.