bluuebunny/crossref_metadata_2025_split huggingface dataset:
TALEN®-Based Gene Editing in Hematopoietic Stem Cells
Authors: Busser Brian W , Temburni Sonal , Boyne Alex , Juillerat Alexandre , Poirot Laurent , Duchateau Philippe 
Published: October 2019

Abstract
Autologous gene therapy in human hematopoietic stem cells (HSCs) has shown the potential to transform the treatment of genetic diseases of the blood and immune system. However, viral vector delivery has shown issues with insertional mutagenesis, unregulated transgene expression and durability. Genome editing via homology directed repair (HDR) in human HSCs can mitigate these issues by correcting and repairing the endogenous gene permitting precise spatio-temporal gene expression. Transcription activator-like effector nuclease (TALEN®) are a designer nuclease that enables the site-specific introduction of double-stranded breaks (DSBs) in the genome. Repair of these DSBs occurs largely through one of two pathways, non-homologous end joining (NHEJ) and HDR. NHEJ is an error-prone pathway that often results in insertions or deletions (indels) whereas HDR is a precise mechanism that uses a homologous piece of DNA (usually the homologous chromosome or sister chromatid) to accurately repair the lesion by recombination. However, HDR can be manipulated by supplying an exogenous DNA template with homology arms that start at the DSB as single-stranded oligonucleotides or as double-stranded DNA to introduce any genetic modifications encoded in the template DNA. Recent studies have shown efficient HDR in numerous cell types using adeno-associated virus (AAV) to deliver template DNA. Here we combined TALEN® with AAV containing a repair template to identify the conditions required to efficiently modify disease-relevant alleles in HSCs. We first describe a strategy to remove the nuclease target site in the template DNA even when the target site was located in regulatory sequences. Optimization of the transfection, transduction and culturing conditions of HSCs shows highly efficient allele modification using low doses of AAV and nuclease. Importantly, alleles more often show evidence of HDR as opposed to NHEJ. Further, colony forming unit assays and a xenograft model confirm that modified HSCs are multipotent. Lastly, we show that differentiated progeny of modified HSCs produce similar amounts of mRNA from the modified allele as wild-type unmodified alleles. In total, our results show that combining TALEN® with repair template delivery via AAV is a robust gene editing system for HSCs.

Disclosures
Busser: Cellectis: Employment, Patents & Royalties: Cellectis. Temburni:Cellectis: Employment, Patents & Royalties: Cellectis. Boyne:Cellectis, Inc: Employment, Patents & Royalties: Cellectis. Juillerat:Cellectis: Employment, Patents & Royalties: Cellectis. Poirot:Cellectis: Employment, Patents & Royalties: Cellectis. Duchateau:Cellectis: Employment, Patents & Royalties: Cellectis.
CRISPR/Cas9 Unsettle PIP4K2A and α, β, and γ Globin Genes Expression
Authors: Malimpensa Danaê , Faber Eliel , Corat Marcus A.F. , Lana Marlous V.G. , Strauss Bryan E. , Costa Fernando F. , Jorge Susan Elisabeth , Sonati Maria de Fátima 
Published: October 2019

Abstract
Phosphatidylinositol-phosphate kinases (PIPKins) belong to a family of lipid kinase enzymes that generate lipid messengers derivate from inositol, including the second messenger phosphatidylinositol-4,5-biphosphate [PI(4,5)P2], which participates in several cell regulation processes including gene expression. Previous studies developed in our laboratory suggested that the enzyme phosphatidylinositol-4-phosphate kinase-II-alpha (PIP4KIIα) may be related with the expression of the globin genes. Thus, the aim of this work was to evaluate the expression levels of the α, β and γ globin genes after disruption and overexpression of the PIP4KIIα gene (PIP4KIIA) in KU812 erythroleukemic cell line. The PIP4KIIα gene disruption was performed by using CRISPR/Cas9 and CRISPR/dCas9 techniques: KU812 cells were electro-transfected (Lonza 4D Nucleofector) with CRISPR/Cas9 lentiviral plasmid construct containing 3 gRNA targets to exon 8 of PIP4KIIA to knockout the gene; RNA samples from the pool of cells were collected and quantified by qPCR after 9 (t1) and 14 days (t2) of transfection. To overexpress PIP4K2A, CRISPR/dCas9 system (five lentiviral plasmids - 3 gRNAs targeting 214 nt upstream the transcriptional start site of PIP4K2A and 2 SAM accessorial factors) was transfected (Lipofectamine 3000) and RNA quantified by qPCR 24 (h1), 48 (h2) and 72 (h3) hours after the procedure. β-ACTIN and GAPDH genes were used as endogenous controls for qPCR. Our results with transfected cells without selection revealed, as expected, a reduction of PIP4KIIA compared to the control (no transfected cells - t1 and t2). The α globin genes presented a variation of 42% (increase in t1) and a 60% reduction in t2 (RQ = 1.42; RQ = 0.40, respectively), while β globin genes presented a 123% increase in t1 and remained similar to the control at t2 (RQ = 2.23; RQ = 1.01). The γ globin genes presented variation of 18% and 59% reduction in t1 and t2, respectively (RQ = 0.82; RQ = 0.41). The overexpression results suggested that PIP4KIIA leads to a higher γ globin expression in h2 and h3 (RQ = 3.26; RQ = 2.01) as well as a lower β globin expression in h1, h2 and h3 (RQ = 0.54; RQ = 0.002; RQ = 0.18), while a globin remained similar to the control at all analysed times (RQ variation of 1.26; 0.82 and 1.25, respectively). These results, although preliminary, suggest a possible relationship between PIP4KIIα and the regulation of expression of the globin genes. Financial Support: Fapesp, CNPq, CAPES, Faepex-Unicamp.

Disclosures
No relevant conflicts of interest to declare.
Chemically Controlled, Immunosuppression-Resistant, Anti-Bcma CAR-T Cells for Treatment of Antibody-Mediated Autoimmunity
Authors: Rajavel Sowndharya , Ito Cade E. , Abe Keith , Guerrero Valerie , Uenishi Gene I. , Scharenberg Andrew M. , Cost Gregory J. 
Published: October 2019

Abstract
Auto-reactive antibody production by plasma cells is the direct cause of many auto-immune diseases. In such cases elimination of plasma cells would ameliorate the disease. Chimeric antigen receptor T (CAR-T) cells with cytotoxicity toward cells expressing B-cell maturation antigen (BCMA) have shown remarkable promise for the treatment of multiple myeloma, a plasma cell neoplasm. Elimination of non-malignant plasma cells is a side-effect of anti-BCMA CAR-T treatment of multiple myeloma, suggesting the use of these anti-BCMA CAR T cells for auto-immune indications. Unfortunately, CAR-T administration requires use of lymphodepletion to achieve efficient cell engraftment, and is often accompanied by cytokine release syndrome (CRS), a potentially life-threatening side-effect.
As lymphodepletion and CRS pose morbidity/mortality risks that are unacceptable for therapy of many auto-immune diseases, we have utilized CRISPR-Cas9 gene editing to develop a controllable CAR-T cell platform that provides for (1) engraftment with non-cytotoxic transient immunosuppression; and (2) small-molecule dependent CAR T-cell expansion. We have implemented this platform using a unique dual targeting approach in which a BCMA CAR transgene is integrated into the TRAC locus, and additional payloads are integrated into a second locus, thus also enabling an allogeneic manufacturing process. Transgene integration occurred in >50% of cells individually with several percent of cells targeted at two loci. TRAC-targeted, anti-BCMA CAR T cells demonstrated CAR-dependent, target-cell-BCMA-dependent cytotoxicity towards both high-BCMA- and low-BCMA-expressing cell lines and in multiple myeloma cells xenografted into NSG mice. Drug-regulation properties and immunosuppression resistance are the subject of ongoing experiments. Anti-BCMA CAR T cells that are chemically controlled, incapable of graft-versus-host disease, and insensitive to immunosuppression may be an attractive treatment option a variety of antibody-mediated auto-immune conditions.

Disclosures
Rajavel: Casebia Therapeutics: Employment. Ito:Casebia Therapeutics: Employment. Abe:Casebia Therapeutics: Employment. Guerrero:Casebia Therapeutics: Employment. Uenishi:Casebia Therapeutics: Employment. Scharenberg:Casebia Therapeutics: Employment; Generation Bio: Equity Ownership; Alpine Immune Sciences: Equity Ownership. Cost:Casebia Therapeutics: Employment.
Secdrug: A Computational Method for Discovering Novel Secondary Drug Combinations Using Large-Scale Pharmacogenomics Databases
Authors: Chen Li , Mukherjee Ujjal Kumar , Rankine Emily , Van Ness Brian G. , Mitra Amit Kumar 
Published: October 2019

Abstract
Drug resistance is a major obstacle in achieving complete and sustained therapeutic effect in cancer chemotherapy. Chemo-resistance may also lead to over-dosing and unwanted exposure to ineffective anti-tumor agents thereby increasing the risk of negative side-effects and the cost of drug development. Therefore, our goal is to utilize a large-scale pharmacogenomics database (Genomics of Drug Sensitivity in Cancer or GDSC, the largest public resource of drug-sensitivity data on over 250 drugs in more than 1000 human cancer cell lines of common and rare types of adult and childhood cancers of diverse origin) and develop a prediction method to identify novel secondary drug combination regimens that may effectively reverse drug resistance.
We utilized a greedy algorithm-based set-covering computational optimization method followed by a regularization technique to seek all secondary drugs that could kill maximum number of cell lines of the test disease (B-Cell cancers) resistant to the test drug (the Proteasome inhibitor/PI drug Bortezomib/Bz/Velcade) in a sequential manner ordered by the number of cell lines killed. The predicted top secondary drug combinations in PI-resistant B-cell cancers are shown in Figure 1.
To validate our prediction results, we treated human multiple myeloma cell lines (HMCLs) highly resistant to the proteasome inhibitors Bortezomib, Carfilzomob, Oprozomib and Ixazomib with the predicted best secondary drugs. Figure 2 depicts In vitro chemo-sensitivity profiles of PI-resistant HMCLs and other B-cell cancer cell lines showing percent survival compared to untreated control at increasing concentrations of secondary drugs, as single agents or in combination.
Furthermore, for each drug in the predicted drug combination, we identified differentially expressed (DE) genes by comparing the expression profiles between extraordinary- sensitive and resistant cell lines. These significantly regulated DE genes were used to identify pathways associated with the successful drug combinations.
Finally, we developed an R software package secDrug based on this computational pipeline for predicting novel secondary therapies in chemotherapy-resistant cancers. secDrug takes a query of any cancer type and any test drug, and outputs a list of the top secondary drug combinations with confidence score and biological pathway visualization. Thus, secDrug has potential application in clinical decision-making for discovering resistance-reversing cancer chemotherapy regimens.

Disclosures
No relevant conflicts of interest to declare.
Potential Long-Term Treatment of Hemophilia a By Early Postnatal Co-Transplantation of Cord Blood Derived Endothelial Colony-Forming Cells and Placental Mesenchymal Stem Cells
Authors: Gao Kewa , Kumar Priyadarsini , Hao Dake , Reynaga Lizette , Farmer Diana Lee , Rose Melanie , Nolta Jan A. , Zhou Jianda , Zhou Ping , Wang Aijun 
Published: October 2019

Abstract
Hemophilia A (HA) is an X-linked recessive disorder caused by mutations in the Factor VIII (FVIII) gene leading to deficient blood coagulation. As a monogenic disorder, HA is an ideal target for gene or cell-based therapy. Both mesenchymal stem cells (MSCs) and endothelial progenitor cells (EPCs) have been used independently to correct the HA phenotype, but poor survival and insufficient retention of transplanted cells in the long term limit their application. In this study, we sought to determine whether co-transplantation of endothelial colony-forming cells (ECFCs) with placental mesenchymal stem cells (PMSCs) can achieve stable, long-term engraftment. To investigate stem cell engraftment and therapeutic potential, ECFCs and PMSCs were transduced with a B domain deleted factor VIII (BDD-FVIII) expressing lentiviral vector. To distinguish between PMSCs and ECFCs in the co-transplantation group, a Td-Tomato containing lentiviral vector was used to label PMSCs while a green fluorescent protein (GFP) labeling vector was used to label ECFCs. They were then transplanted intramuscularly either as ECFC only group, PMSC only group or co-transplantation of ECFCs and PMSCs group into neonatal (3 days) or adult (12 weeks) immune deficient NOD scid gamma (NSG) mice. Using an in vivo bioluminescence imaging system, we investigated cell engraftment for up to 26 weeks after transplantation. ECFC only and the co-transplantation groups achieved better engraftment than PMSCs only group in both neonatal and adult mice during the entire study period. The co-transplantation group showed higher engraftment rate than the ECFC only group at 16 weeks and 20 weeks post-transplantation. In addition, transplantation of ECFCs only or in combination with PMSCs in neonatal period achieved higher engraftment than transplantation in adult period. Immunohistochemical analyses were further employed to determine the fate and functionality of the engrafted cells. We found that engrafted ECFCs maintained endothelial phenotype and expressed high level of FVIII at 26 weeks post-transplantation. The presence of ECFCs in the co-transplantation group also promoted formation of functional vasculature after transplantation. To study the functional reversal of HA phenotype we co-transplanted ECFCs and PMSCs into F8 knock-out HA mice. The transplanted cells reduced the blood loss volume from 562.13±19.84µl of blood to 155.78±44.93µl in a tail clip assay in these HA mice. This work demonstrates that co-transplantation of ECFCs with PMSCs at the neonatal period is a promising strategy to achieve stable, long-term engraftment of transplanted cells, and thus holds great promise for the treatment of HA and other genetic diseases and tissue regeneration applications.

Disclosures
No relevant conflicts of interest to declare.
PIM Inhibitor SMI-4a Induces Cell Apoptosis and Cell Cycle Arrest in B-Cell Acute Lymphocytic Leukemia Cells Via the JAK2/STAT3 Pathway
Authors: Kuang Xingyi , Xiong Jie , Wang Weili , Li Xinyao , Lu Tingting , Wang Jishi 
Published: October 2019

Abstract
The serine/threonine PIM protein kinases are critical regulators of turmorigenesis in mutiple hematologic malignancies and solid cancers. We used real-time PCR to detect the expression of PIM in B-cell acute lymphocytic leukemia (B-ALL) patients, and found the expression of PIM in B-ALL patients was significantly higher than that in normal controls. SMI-4a is a pan-PIM small molecule inhibitor, and this agent exhibits demonstrable preclinical antitumour activity in a wide range of hematologic malignant cell lines. To further explore the effect of SMI-4a on B-ALL cells, B-ALL cell lines CCRF-SB and Sup-B15 were treated with this small molecule inhibitor, and the results showed that SMI-4a inhibited B-ALL cell proliferation in a dose- and time-dependent manner. Moreover, SMI-4a significantly promoted B-ALL cell apoptosis and caused cell cycle arrest in the G0/G1 phase. The results of Western blot showed that SMI-4a increased the expression of Caspase-3, Caspase-9, Bax and P21, and decreased the expression of Bcl-2 and CDK4. Furthermore, we found that SMI-4a significantly inhibits the activation of the JAK2/STAT3 pathway and HO-1 interferes with the JAK2/STAT3 pathway to inhibit SMI-4a-induced ALL cell apoptosis. Finally, xenograft experiments in NOD/SCID mice were operated to investigate the potential role of SMI-4a in B-ALL tumorigenesis in vivo. To observe the effect of SMI-4a on tumor growth in vivo, NOD/SCID mice were transplanted with B-ALL devied cells, and the tumor-bearing mice were intraperitoneally injected with saline and SMI-4a, respectively. As a result, tretment with SMI-4a resulted in a significant inhibition on tumor growth. In addition, PIM inhibtor obviously reduced the volume and weight of B-ALL cell-derived tumors. TUNEL assay revealed the proportion of apoptotic cells was higher in the SMI-4a-treated group than in the control group. Taken together, our data showed PIM inhibitor (SMI-4a) significantly inhibits the growth of B-ALL cells in vitro and in vivo and promotes apoptosis and cell cycle arrest. This suppressive effect is mediated partly through inhibiting the JAK2/STAT3 pathway activation.

Disclosures
No relevant conflicts of interest to declare.
Downstream Pathway of AML1-ETO Targeted ARHGEF12 Might be Targetable for Rock Inhibitor to Improve Therapy Outcome
Authors: Xie Yangyang , Wu Ruichi , Chen Xiaoxiao , Sun Xiao-Jian , Shen Shuhong 
Published: October 2019

Abstract
As a transcription factor AML1-ETO modulate gene expression of its targets. Scanning our ChIP-seq data we found AML1-ETO bind to an AML1 binding motif in the promotor region of ARHGEF12. Exploring acute myeloid leukemia (AML) microarray databases we confirmed that the expression of ARHGEF12 was up-regulated consistently in AML1-ETO-positive AML patient samples. To confirmed this finding, we quantitated expression of ARHGEF12 in pediatric AML samples from Shanghai children's medical center. Not out of expectation, all AML1-ETO-positive cases have higher ARHGEF12. Moreover, the ARHGEF12 expression was an independent poor prognostic factor for overall survival comparing between patients with higher and lower expressed ARHGEF12 (P=0.0258) (Figure A).
To understand the role of ARHGEF12 in leukemogenic function of AML1-ETO fusion gene, we knocked down ARHGEF12 expression in kasumi-1 cells by shRNA. Growth of kasumi-1 cells in vitro was significantly inhibited with cell cycle exit and more apoptosis. Further, we transformed fetal liver cells of Arhgef12 conditional knockout mice to leukemia model by retrovirus conducted AML1-ETO9a(AE9a) expression. Induced Arhgef12 deletion delayed full blown of leukemia in vivo. The overall survival prolonged 15.5 days (median 83.5 vs. 68 days, P=0.0072). (Figure B) ARHGEF12 is a well-known guanine nucleotide exchange factor for RhoA GTPase. Arhgef12 deletion decreased activated GTP-RhoA and MYPT1 phosphorylation, which is one of ROCK kinase substrates. Spleen cells from leukemic mice were cultured in vitro, the ROCK inhibitor Y-27632 significantly decreased the cell proliferation, which mimic the tamoxifen-induced Arhgef12 knockout phenotype. Y-27632 combined with tamoxifen treated cells almost eradicate alive leukemia cells on the ninth day in vitro (Figure C) and delay leukemogenesis in vivo (Figure D).
These findings suggest that ARHGEF12 as a transcriptional target of the AML1-ETO fusion protein and plays an essential role in leukemogenesis. The ARHGEF12-RHOA-ROCK pathway may serve as a new therapeutic target for AML1-ETO+ AML
Figure. Figure.

Disclosures
No relevant conflicts of interest to declare.
Good Care and Long-Term Follow-up in Young Hemophiliacs
Authors: Iscan Zeynep , Koc Basak , Kilicoglu Onder , Atalar Ata , Taser Omer , Cetik Gulten , Zulfikar Bulent 
Published: October 2019

Abstract
Introduction: Hemophilia is a hereditary bleeding disorder that presents with primarily hemarthrosis and hematoma. The most common and frequent complication, especially in severe hemophilia, where factor VIII or IX is not administered prophylactically, is the hemophilic arthropathy that develops as a result of recurrent bleeding. In this study, the relationship between arthropathy development and timing of the prohylaxis and orthopedic interventions in hemophiliacs was presented.
Materials and Methods: The data of 99 haemophilia (HA) and hemophilia B (HB) patients under the age of 25 years at our center were reviewed retrospectively. Age, type of disease, factor level, follow-up duration, prophylaxis dose and frequency, annual bleeding rate (ABR), target joints, arthropathic joints, applied radioactive synovectomy and orthopedic interventions were recorded.
Results: Seventy-six HA and 23 HB were included. The median age was 14 (range 1-25) years old. Ten of them (10.1%) were mild, 15 (15.2%) were moderate and 74 (74.7%) were severe. Severe hemophiliacs were most of the patients in both types. Eighty-nine (89.9%) patients including 17 of mild-to-moderate type with target joint and / or surgical intervention were under prophylaxis and 10 (10.1%) were receiving on-demand therapy. The annual bleeding rate was 1.38. Twelve (12%) patients (8 HA, 4 HB) with degenerative hemophilic arthropathy had a mean age of 22 years old and a mean age of application was 12.3 years, a mean annual bleeding rate was 1.54 and a mean follow up and prophylaxis time was 9.5 years. Six patients (4 HA, 2 HB) with median age 21.8 years (range: 18-25) had 9 major orthopedic surgeries (arthrodesis (2), radial head resection (2), TEP, contracture releasing, acyloplasty, deformity correction, arthroscopic sinovectomy) and 22 patients with median age of 17.8 years had 29 minor orthopedic interventions (radiosynovectomy (28), closed reduction to femur fracture). Orthopedic interventions were performed at an advanced age, often who met with prophylaxis too late.
Discussion: In our study, it has been shown that who had prophylaxis at an early age and prophylaxis for a longer period had less arthropathies which is the most important complication and additionally less arthroplasties. The fact that, the prevalence of permanent musclosceletal deformity was 59% before year 2000 (historical data) which reduced to 12% nowadays shows the sensitivity of hemophilia care in our center. Early and regular prophylaxis remains the most current treatment modality for the prevention of hemophilia complications.

Disclosures
No relevant conflicts of interest to declare.
MiR-221 Sensitizes Chronic Myeloid Leukemia Cells to Imatinib By Targeting STAT5
Authors: Xu Xiucai , Jiang Xiaoxiao , Xia Lijun 
Published: October 2019

Abstract
Imatinib, a tyrosine-kinase inhibitors (TKIs), is the first-line treatment in the early chronic phase of chronic myeloid leukemia (CML). In the last decade, the survival of patients with CML has significantly increased with the use of TKIs. However, the drug resistance is one major barrier in CML treatment. At present, although the research on CML TKIs resistance mechanism has made great progress, the underlying mechanism is complex and still not fully elucidated, especially the lack of understanding of the upstream common mechanism. MicroRNAs (miRNAs) are involved in various processes from the development to drug resistance of tumors, including chronic myeloid leukemia (CML). In this work, we examined the STAT5-related miRNA-expression profile in CML cell lines (K562 and imatinib-resistant K562/G) by quantitative real-time reverse-transcriptase polymerase chain reactions. MiR-221 expression was markedly decreased in K562/G cells and peripheral blood mononuclear cells from patients with treatment failure, when compared to K562 cells and patients with optimal responses respectively. We also observed the expression of STAT5 expression inversely correlated with miR-221 expression in CML cells. Additionally, STAT5 was validated in this study as a direct target of miR-221 in dual-luciferase reporter vector assays. MiR-221 restoration and STAT5 knockdown in K562/G cells increased the sensitivity of CML cells to imatinib by reducing the Bcl2: Bax ratio. Collectively, our data suggest that miR-221-STAT5 axis played crucial roles in controlling the sensitivity of CML cells to imatinib.

Disclosures
No relevant conflicts of interest to declare.