当前位置： SCI文献检索 > Molecular Therapy-Nucleic Acids期刊下所有文献 > Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting.

Priming Human Repopulating Hematopoietic Stem and Progenitor Cells for Cas9/sgRNA Gene Targeting.

Abstract：

:Engineered nuclease-mediated gene targeting through homologous recombination (HR) in hematopoietic stem and progenitor cells (HSPCs) has the potential to treat a variety of genetic hematologic and immunologic disorders. Here, we identify critical parameters to reproducibly achieve high frequencies of RNA-guided (single-guide RNA [sgRNA]; CRISPR)-Cas9 nuclease (Cas9/sgRNA) and rAAV6-mediated HR at the β-globin (HBB) locus in HSPCs. We identified that by transducing HSPCs with rAAV6 post-electroporation, there was a greater than 2-fold electroporation-aided transduction (EAT) of rAAV6 endocytosis with roughly 70% of the cell population having undergone transduction within 2 hr. When HSPCs are cultured at low densities (1 × 105 cells/mL) prior to HBB targeting, HSPC expansion rates are significantly positively correlated with HR frequencies in vitro as well as in repopulating cells in immunodeficient NSG mice in vivo. We also show that culturing fluorescence-activated cell sorting (FACS)-enriched HBB-targeted HSPCs at low cell densities in the presence of the small molecules, UM171 and SR1, stimulates the expansion of gene-edited HSPCs as measured by higher engraftment levels in immunodeficient mice. This work serves not only as an optimized protocol for genome editing HSPCs at the HBB locus for the treatment of β-hemoglobinopathies but also as a foundation for editing HSPCs at other loci for both basic and translational research.

journal_name

Mol Ther Nucleic Acids

journal_title

Molecular therapy. Nucleic acids

authors

Charlesworth CT,Camarena J,Cromer MK,Vaidyanathan S,Bak RO,Carte JM,Potter J,Dever DP,Porteus MH

doi

10.1016/j.omtn.2018.04.017

subject

Has Abstract

pub_date

2018-09-07 00:00:00

pages

89-104

issn

2162-2531

pii

S2162-2531(18)30095-7

journal_volume

12

pub_type

杂志文章

相关文献

Molecular Therapy-Nucleic Acids文献大全
  • The Underlying Mechanisms of Noncoding RNAs in the Chemoresistance of Hepatocellular Carcinoma.

    abstract：:Hepatocellular carcinoma (HCC) is one of the most lethal human malignancies. Chemotherapeutic agents, such as sorafenib and lenvatinib, can improve the outcomes of HCC patients. Nevertheless, chemoresistance has become a major hurdle in the effective treatment of HCC. Noncoding RNAs (ncRNAs), including mircoRNAs (miRN...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章,评审

    doi：10.1016/j.omtn.2020.05.011

    authors： Wang M,Yu F,Chen X,Li P,Wang K

    更新日期：2020-09-04 00:00:00

  • Transcriptional Factor Yin Yang 1 Promotes the Stemness of Breast Cancer Cells by Suppressing miR-873-5p Transcriptional Activity.

    abstract：:Transcription factor Yin Yang 1 (YY1) is upregulated in multiple tumors and plays essential roles in tumor proliferation and metastasis. However, the function of YY1 in breast cancer stemness remains unclear. Herein, we found that YY1 expression was negatively correlated with the overall survival and relapse-free surv...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2020.06.018

    authors： Guo Q,Wang T,Yang Y,Gao L,Zhao Q,Zhang W,Xi T,Zheng L

    更新日期：2020-09-04 00:00:00

  • Schizandrin A Protects Human Retinal Pigment Epithelial Cell Line ARPE-19 against HG-Induced Cell Injury by Regulation of miR-145.

    abstract：:Diabetic retinopathy (DR) is a serious complication of diabetes, which is the main cause of blindness among adults. Traditional Chinese medicines (TCMs) have been proven to delay the development of DR. Nonetheless, the effect of Schizandrin A (SchA) on DR remains uninvestigated. The present study aimed to probe the pr...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2019.10.026

    authors： Dong Y,Qian C,Wan G,Yan P,Liang S,Wang J

    更新日期：2020-03-06 00:00:00

  • Long Noncoding RNA OIP5-AS1 Promotes the Progression of Liver Hepatocellular Carcinoma via Regulating the hsa-miR-26a-3p/EPHA2 Axis.

    abstract：:Numerous studies have suggested that dysregulated long noncoding RNAs (lncRNAs) contributed to the development and progression of many cancers. lncRNA OIP5 antisense RNA 1 (OIP5-AS1) has been reported to be increased in several cancers. However, the roles of OIP5-AS1 in liver hepatocellular carcinoma (LIHC) remain to ...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2020.05.032

    authors： Ma YS,Chu KJ,Ling CC,Wu TM,Zhu XC,Liu JB,Yu F,Li ZZ,Wang JH,Gao QX,Yi B,Wang HM,Gu LP,Li L,Tian LL,Shi Y,Jiang XQ,Fu D,Zhang XW

    更新日期：2020-09-04 00:00:00

  • Erratum: Fructose Promotes Uptake and Activity of Oligonucleotides with Different Chemistries in a Context-Dependent Manner in mdx Mice.

    abstract：:[This corrects the article DOI: 10.1038/mtna.2016.46.]. ...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 已发布勘误

    doi：10.1016/j.omtn.2020.08.028

    authors： Cao L,Han G,Lin C,Gu B,Gao X,Moulton HM,Seow Y,Yin H

    更新日期：2020-11-19 00:00:00

  • circINSR Promotes Proliferation and Reduces Apoptosis of Embryonic Myoblasts by Sponging miR-34a.

    abstract：:As a diverse and abundant class of endogenous RNAs, circular RNAs (circRNAs) participate in processes including cell proliferation and apoptosis. Nevertheless, few researchers have investigated the function of circRNAs in bovine muscle development. Based on existing sequencing data, we identified circINSR. The localiz...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2019.12.032

    authors： Shen X,Zhang X,Ru W,Huang Y,Lan X,Lei C,Chen H

    更新日期：2020-03-06 00:00:00

  • lncRNA lnc-TSI Inhibits Metastasis of Clear Cell Renal Cell Carcinoma by Suppressing TGF-β-Induced Epithelial-Mesenchymal Transition.

    abstract：:The transforming growth factor-β (TGF-β)/Smads signal plays an important role in cancer metastasis by mediating the epithelial-mesenchymal transition (EMT) in cancer cells. lnc-TSI is a recently identified long noncoding RNA that negatively regulates the TGF-β/Smads signal. The present study was conducted to test the ...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2020.08.003

    authors： Wang P,Chen W,Ma T,Lin Z,Liu C,Liu Y,Hou FF

    更新日期：2020-08-05 00:00:00

  • PAK1 Promotes the Proliferation and Inhibits Apoptosis of Human Spermatogonial Stem Cells via PDK1/KDR/ZNF367 and ERK1/2 and AKT Pathways.

    abstract：:Spermatogonial stem cells (SSCs) have significant applications in reproductive and regenerative medicine. However, nothing is known about genes in mediating human SSCs. Here we have explored for the first time the function and mechanism of P21-activated kinase 1 (PAK1) in regulating the proliferation and apoptosis of ...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2018.06.006

    authors： Fu H,Zhang W,Yuan Q,Niu M,Zhou F,Qiu Q,Mao G,Wang H,Wen L,Sun M,Li Z,He Z

    更新日期：2018-09-07 00:00:00

  • Endogenous MicroRNA Competition as a Mechanism of shRNA-Induced Cardiotoxicity.

    abstract：:Gene knockdown using short hairpin RNAs (shRNAs) is a promising strategy for targeting dominant mutations; however, delivering too much shRNA can disrupt the processing of endogenous microRNAs (miRNAs) and lead to toxicity. Here, we sought to understand the effect that excessive shRNAs have on muscle miRNAs by treatin...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2019.12.007

    authors： Course MM,Gudsnuk K,Desai N,Chamberlain JR,Valdmanis PN

    更新日期：2020-03-06 00:00:00

  • Characterization of Transcriptome Transition Associates Long Noncoding RNAs with Glioma Progression.

    abstract：:Long noncoding RNAs (lncRNAs) have been implicated in cancer biogenesis and prognosis. However, we still lack knowledge on their function during glioma progression. In this study, we analyzed the lncRNA expression profile across 907 glioma patients in grades II, III, and IV. Widespread dynamic expression of lncRNAs du...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2018.10.009

    authors： Lin X,Jiang T,Bai J,Li J,Wang T,Xiao J,Tian Y,Jin X,Shao T,Xu J,Chen L,Wang L,Li Y

    更新日期：2018-12-07 00:00:00

  • A Dual-Functioning 5'-PPP-NS1shRNA that Activates a RIG-I Antiviral Pathway and Suppresses Influenza NS1.

    abstract：:Retinoic acid-inducible gene-I (RIG-I) is a cytosolic pathogen sensor that is crucial against a number of viral infections. Many viruses have evolved to inhibit pathogen sensors to suppress host innate immune responses. In the case of influenza, nonstructural protein 1 (NS1) suppresses RIG-I function, leading to viral...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2020.01.025

    authors： Singh N,Ranjan P,Cao W,Patel J,Gangappa S,Davidson BA,Sullivan JM,Prasad PN,Knight PR,Sambhara S

    更新日期：2020-03-06 00:00:00

  • A Hyperactive Transposase Promotes Persistent Gene Transfer of a piggyBac DNA Transposon.

    abstract：:Nonviral vector systems are used increasingly in gene targeting and gene transfer applications. The piggyBac transposon represents an alternative integrating vector for in vivo gene transfer. We hypothesized that this system could achieve persistent gene transfer to the liver when administered systemically. We report ...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1038/mtna.2012.12

    authors： Burnight ER,Staber JM,Korsakov P,Li X,Brett BT,Scheetz TE,Craig NL,McCray PB Jr

    更新日期：2012-10-16 00:00:00

  • A Kinetic Model Explains Why Shorter and Less Affine Enzyme-recruiting Oligonucleotides Can Be More Potent.

    abstract：:Antisense oligonucleotides complementary to RNA targets promise generality and ease of drug design. The first systemically administered antisense drug was recently approved for treatment and others are in clinical development. Chemical modifications that increase the hybridization affinity of oligonucleotides are reas...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1038/mtna.2013.72

    authors： Pedersen L,Hagedorn PH,Lindholm MW,Lindow M

    更新日期：2014-02-18 00:00:00

  • Generation of Genetically Stable Human Direct-Conversion-Derived Neural Stem Cells Using Quantity Control of Proto-oncogene Expression.

    abstract：:As the human lifespan has increased due to developments in medical technology, the number of patients with neurological diseases has rapidly increased. Therefore, studies on effective treatments for neurological diseases are becoming increasingly important. To perform these studies, it is essential to obtain a large n...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2018.12.009

    authors： Daekee K,Mi-Jung H,Minjun J,Hee-Jin A,Kwang-Won S,Kyung-Sun K

    更新日期：2019-03-01 00:00:00

  • Mbd2 Mediates Retinal Cell Apoptosis by Targeting the lncRNA Mbd2-AL1/miR-188-3p/Traf3 Axis in Ischemia/Reperfusion Injury.

    abstract：:Recent studies reported that DNA methylation was involved in retinal cell death. Methyl-CpG binding domain protein 2 (Mbd2) is one of the DNA methylation readers. Its role and mechanism of regulation remain unclear. The ischemia/reperfusion (I/R) model in mice primary culture retinal ganglion cells (RGCs) and Mbd2 kno...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2020.01.011

    authors： Ge Y,Zhang R,Feng Y,Li H

    更新日期：2020-03-06 00:00:00

  • Pip6-PMO, A New Generation of Peptide-oligonucleotide Conjugates With Improved Cardiac Exon Skipping Activity for DMD Treatment.

    abstract：:Antisense oligonucleotides (AOs) are currently the most promising therapeutic intervention for Duchenne muscular dystrophy (DMD). AOs modulate dystrophin pre-mRNA splicing, thereby specifically restoring the dystrophin reading frame and generating a truncated but semifunctional dystrophin protein. Challenges in the de...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1038/mtna.2012.30

    authors： Betts C,Saleh AF,Arzumanov AA,Hammond SM,Godfrey C,Coursindel T,Gait MJ,Wood MJ

    更新日期：2012-08-14 00:00:00

  • Circular RNA circTNPO3 Regulates Paclitaxel Resistance of Ovarian Cancer Cells by miR-1299/NEK2 Signaling Pathway.

    abstract：:Circular RNAs (circRNAs) were recently reported to be involved in the pathogenesis of ovarian cancer (OC); however, the molecular mechanisms of circRNAs in tumor progression and paclitaxel (PTX) resistance of OC remain largely undetermined. Here, we focused on circTNPO3 (hsa_circ_0001741), which is located on chromoso...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2020.06.002

    authors： Xia B,Zhao Z,Wu Y,Wang Y,Zhao Y,Wang J

    更新日期：2020-09-04 00:00:00

  • Dual Myostatin and Dystrophin Exon Skipping by Morpholino Nucleic Acid Oligomers Conjugated to a Cell-penetrating Peptide Is a Promising Therapeutic Strategy for the Treatment of Duchenne Muscular Dystrophy.

    abstract：:The knockdown of myostatin, a negative regulator of skeletal muscle mass may have important implications in disease conditions accompanied by muscle mass loss like cancer, HIV/AIDS, sarcopenia, muscle atrophy, and Duchenne muscular dystrophy (DMD). In DMD patients, where major muscle loss has occurred due to a lack of...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1038/mtna.2012.54

    authors： Malerba A,Kang JK,McClorey G,Saleh AF,Popplewell L,Gait MJ,Wood MJ,Dickson G

    更新日期：2012-12-18 00:00:00

  • Multi-pathway Protective Effects of MicroRNAs on Human Chondrocytes in an In Vitro Model of Osteoarthritis.

    abstract：:Osteoarthritis (OA) is the most common degenerative joint disease. One of the main pathogenic factors of OA is thought to be inflammation. Other factors associated with OA are dysregulation of microRNAs, reduced autophagic activity, oxidative stress, and altered metabolism. microRNAs are small non-coding RNAs that are...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2019.07.011

    authors： Al-Modawi RN,Brinchmann JE,Karlsen TA

    更新日期：2019-09-06 00:00:00

  • Lipidoid Nanoparticles Containing PD-L1 siRNA Delivered In Vivo Enter Kupffer Cells and Enhance NK and CD8(+) T Cell-mediated Hepatic Antiviral Immunity.

    abstract：:Effective clinical application of antiviral immunotherapies necessitates enhancing the functional state of natural killer (NK) and CD8(+) T cells. An important mechanism for the establishment of viral persistence in the liver is the activation of the PD-1/PD-L1 inhibitory pathway. To examine the role of hepatic myeloi...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1038/mtna.2012.63

    authors： Dolina JS,Sung SS,Novobrantseva TI,Nguyen TM,Hahn YS

    更新日期：2013-02-19 00:00:00

  • miR-223 Regulates Cell Proliferation and Invasion via Targeting PDS5B in Pancreatic Cancer Cells.

    abstract：:Emerging evidence has demonstrated that miR-223 is critically involved in the progression of pancreatic cancer (PC); however, the underlying mechanisms are not fully elucidated. In the present study, we explored the molecular basis of miR-223-mediated tumor progression in PC. We performed numerous approaches including...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2019.01.009

    authors： Ma J,Cao T,Cui Y,Zhang F,Shi Y,Xia J,Wang ZP

    更新日期：2019-03-01 00:00:00

  • Development of Lentiviral Vectors Simultaneously Expressing Multiple siRNAs Against CCR5, vif and tat/rev Genes for an HIV-1 Gene Therapy Approach.

    abstract：:Gene therapy holds considerable promise for the functional cure of HIV-1 infection and, in this context, RNA interference (RNAi)-based approaches represent powerful strategies. Stable expression of small interfering RNAs (siRNAs) targeting HIV genes or cellular cofactors has the potential to render HIV-1 susceptible c...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1038/mtna.2016.24

    authors： Spanevello F,Calistri A,Del Vecchio C,Mantelli B,Frasson C,Basso G,Palù G,Cavazzana M,Parolin C

    更新日期：2016-04-19 00:00:00

  • The Bioactivity of D-/L-Isonucleoside- and 2'-Deoxyinosine-Incorporated Aptamer AS1411s Including DNA Replication/MicroRNA Expression.

    abstract：:In this study, chemical modification of 2'-deoxyinosine (2'-dI) and D-/L-isothymidine (D-/L-isoT) was performed on AS1411. They could promote the nucleotide-protein interaction by changing the local conformation. Twenty modified sequences were obtained, FCL-I and FCL-II showed the most noticeable activity improvement....

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2017.09.010

    authors： Fan X,Sun L,Li K,Yang X,Cai B,Zhang Y,Zhu Y,Ma Y,Guan Z,Wu Y,Zhang L,Yang Z

    更新日期：2017-12-15 00:00:00

  • Histone-targeted Polyplexes Avoid Endosomal Escape and Enter the Nucleus During Postmitotic Redistribution of ER Membranes.

    abstract：:Nonviral gene delivery is a promising therapeutic approach because of its safety and controllability, yet limited gene transfer efficacy is a common issue. Most nonviral strategies rely upon endosomal escape designs; however, endosomal escape is often uncorrelated with improved gene transfer and membranolytic structur...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1038/mtna.2015.2

    authors： Ross NL,Munsell EV,Sabanayagam C,Sullivan MO

    更新日期：2015-02-10 00:00:00

  • Targeting Nicotinamide N-Methyltransferase and miR-449a in EGFR-TKI-Resistant Non-Small-Cell Lung Cancer Cells.

    abstract：:Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) are used clinically as target therapies for lung cancer patients, but the occurrence of acquired drug resistance limits their efficacy. Nicotinamide N-methyltransferase (NNMT), a cancer-associated metabolic enzyme, is commonly overexpressed in var...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2018.03.011

    authors： Bach DH,Kim D,Bae SY,Kim WK,Hong JY,Lee HJ,Rajasekaran N,Kwon S,Fan Y,Luu TT,Shin YK,Lee J,Lee SK

    更新日期：2018-06-01 00:00:00

  • Cross-Species Suppression of Hepatoma Cell Growth and Migration by a Schistosoma japonicum MicroRNA.

    abstract：:Schistosoma japonicum eggs trapped in host liver secretes microRNA (miRNA)-containing extracellular vesicles (EVs) that can be transferred to host cells. Recent studies demonstrated that miRNAs derived from plants can modulate gene expression and phenotype of mammalian cells in a cross-kingdom manner. In this study, w...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2019.09.006

    authors： Lin Y,Zhu S,Hu C,Wang J,Jiang P,Zhu L,Li Z,Wang S,Zhang Y,Xu X,Pan W

    更新日期：2019-12-06 00:00:00

  • Overexpression of microRNA-203 Suppresses Proliferation, Invasion, and Migration while Accelerating Apoptosis of CSCC Cell Line SCL-1.

    abstract：:Cutaneous squamous cell carcinoma (CSCC) is a malignant proliferation of cutaneous epithelium that has been observed to have an alarming rise in incidence. Numerous studies have demonstrated microRNAs (miRNAs or miRs) as important biomarkers in the diagnosis, prognosis, and treatment of CSCC. This study aims to invest...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2020.04.014

    authors： Ting W,Feng C,Zhang M,Long F,Bai M

    更新日期：2020-09-04 00:00:00

  • Molecular Signatures and Networks of Cardiomyocyte Differentiation in Humans and Mice.

    abstract：:Cardiomyocyte differentiation derived from embryonic stem cells (ESCs) is a complex process involving molecular regulation of multiple levels. In this study, we first identify and compare differentially expressed gene (DEG) signatures of ESC-derived cardiomyocyte differentiation (ESCDCD) in humans and mice. Then, the ...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2020.07.011

    authors： Wang Y,Yi N,Hu Y,Zhou X,Jiang H,Lin Q,Chen R,Liu H,Gu Y,Tong C,Lu M,Zhang J,Zhang B,Peng L,Li L

    更新日期：2020-09-04 00:00:00

  • Enhanced Transduction of Human Hematopoietic Stem Cells by AAV6 Vectors: Implications in Gene Therapy and Genome Editing.

    abstract：:We have reported that of the 10 most commonly used adeno-associated virus (AAV) serotype vectors, AAV6 is the most efficient in transducing primary human hematopoietic stem cells (HSCs) in vitro, as well as in vivo. More recently, polyvinyl alcohol (PVA), was reported to be a superior replacement for human serum album...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2020.03.009

    authors： Yang H,Qing K,Keeler GD,Yin L,Mietzsch M,Ling C,Hoffman BE,Agbandje-McKenna M,Tan M,Wang W,Srivastava A

    更新日期：2020-06-05 00:00:00

  • MicroRNA-129 Inhibits Glioma Cell Growth by Targeting CDK4, CDK6, and MDM2.

    abstract：:Glioblastoma is the most common malignant primary brain tumor among adults and one of the most lethal cancers. It is characterized by the deregulation of signaling pathways involving proliferation, growth, survival, and other factors. MicroRNAs (miRNAs) play a role in the regulation of genes by affecting the 3' untran...

    journal_title：Molecular therapy. Nucleic acids

    pub_type： 杂志文章

    doi：10.1016/j.omtn.2019.11.033

    authors： Moradimotlagh A,Arefian E,Rezazadeh Valojerdi R,Ghaemi S,Jamshidi Adegani F,Soleimani M

    更新日期：2020-03-06 00:00:00