bluuebunny/crossref_metadata_2025_split huggingface dataset:
Combining HPFH Mutations in Human Adult HSCs to Enhance Reactivation of Fetal Hemoglobin
Authors: Psatha Nikoletta , Georgakopoulou Afrodite , Li Chang , Sova Pavel , Wang Hongjie , Lieber Andre , Papayannopoulou Thalia 
Published: November 2019

Despite the significant advances in developing novel therapies for hemoglobinopathies, developing a universal and robust therapeutic approach, able to achieve an event-free result in all β0/β0 thalassemic and sickle cell patients is a work in progress. It is known that increase of Fetal hemoglobin (HbF) can lead to an ameliorated clinical picture up to transfusion independency. Inactivation via genome editing of γ-globin suppressors or introduction of Hereditary Persistence of Fetal Hemoglobin (HPFH) mutations in the promoter of hemoglobin gamma (HBG) have been shown to significantly increase the endogenous HbF expression. Here we studied the effect in HbF reactivation by targeting the binding site of the two main HbF silencers, BCL11a and LRF and how it compares to the well-established editing of the BCL11a erythroid enhancer. Furthermore, in order to achieve higher levels of fetal hemoglobin, we assayed the effect of all combinations of these cis and trans acting mutations. We first observed that all single knock outs had similar effects in HbF reactivation. Interestingly, by targeting both sites in the gamma globin promoter we observed a decrease in HbF induction compared to each single edit, suggesting a possible binding of a gamma globin activator in the in-between region. However, editing of the BCL11a erythroid enhancer and either of the two silencer binding sites yielded maximum levels of fetal hemoglobin, with pancellular HbF expression, both in vitro and in vivo following xeno-transplantation in NSGW mice. Collectively, our data suggest that the combination of two fetal globin reactivation mutations, one cis and one trans, have the potential to significantly increase HbF both totally and on per cell basis. This strategy has the potential to induce higher levels of HbF reactivation with a clinical benefit in patients with beta globin disorders.

Disclosures
No relevant conflicts of interest to declare.
Enumerating Functional HSC Clones in the Zebrafish Using Gestalt
Authors: Teets Emily M , Gregory Charles Thomas , Shaffer Jami L , Blachly James S. , Blaser Bradley W 
Published: November 2019

Hematopoietic stem cells (HSCs) are functionally and genetically diverse. This diversity decreases as a function of age and with the development of myeloid neoplasia. Numerous systems have been developed to genetically "barcode" HSCs and quantify HSC clone numbers in the setting of native hematopoiesis, but no experimental and computational framework has been established to perform this analysis on a large number of biological replicates. Here we have employed the transgenic zebrafish system, Genome Editing of Synthetic Target Arrays for Lineage Tracing (GESTALT), to develop a framework scaled to permit the investigation of putative oncogenes or niche factors with moderate to small effect sizes. The GESTALT line carries a single copy of an array of 10 tandem CRISPR target sites that is highly variable after being repaired by non-homologous end joining. GESTALT embryos were injected with sgRNAs and Cas9 protein to generate genetic barcodes at the GESTALT locus. Fifty-six zebrafish were raised to adulthood and GESTALT variants were read from a sample of peripheral blood by amplicon sequencing. In a training set of 35 fish we developed a computational model to discriminate informative barcodes from uninformative GESTALT variants in each sample. This model identifies variants shared among samples and excludes them as uninformative. Experimentally-defined thresholds were used to minimize inter-sample correlation (mean Pearson Correlation Coefficient = 0.0021) while maximizing the number of retained informative barcodes in each sample. Bootstrapping and k-means clustering were used to classify samples with a high or low fraction of informative (FI) barcodes (FI cutoff = 0.65). 24 samples within the training set were classified in the High-FI group (mean FI = 0.93 ± 0.02, compared to 0.40 ± 0.04 in the Low-FI group). We then evaluated 3 methods of quantifying GESTALT HSC clones from all sequenced barcodes in the High-FI samples and found that counting barcodes with a VAF>0.02 produced the most normal distribution across the training set (Shapiro-Wilk test for normality, W = 0.95, p = 0.21). 4.3 ± 0.38 GESTALT HSC clones were detected in the training set. In an independent validation set of 21 samples, the number of GESTALT HSC clones was 3.4 ± 0.55, (p = 0.2). This study provides the largest reference dataset to date for HSC genetic barcoding experiments in the zebrafish and addresses the validity of barcode sequences, sample selection and clone enumeration in a systematic, objective way. The GESTALT barcoding system and our analysis framework compare favorably with other methods of studying HSC clonal diversity under conditions of native hematopoiesis in terms of scale, cost, labor and barcode validity. Future studies aiming to understand the mechanisms of HSC clonal evolution in the context of health, ageing and disease will benefit from this framework for enumerating functional HSC clones.

Disclosures
No relevant conflicts of interest to declare.
Inflammatory Cytokine Responsive Enzymatic Mutagenesis Fuels Myeloproliferative Neoplasm Pre-Leukemia Stem Cell Evolution
Authors: Jamieson Catriona , Jiang Qingfei , Holm Frida , Isquith Jane , Mark Adam , Mason Cayla , Ma Wenxue , Diep Raymond H , Pham Jessica , Reynoso Moreno Eduardo , Nasamran Chanond A , Guorong Xu , Birmingham Amanda , Crews Leslie A , Alexandrov Ludmil , Pineda Gabriel , Whisenant Thomas , Fisch Kathleen M 
Published: November 2019

Innate immune anti-viral adenosine to inosine (A-to-I) base editing enzymes (editases) promote hematopoietic stem cell (HSC) self-renewal and protect the human genome from retroviral integration in response to inflammatory cytokine signaling. However, hyper-editing has been linked to therapeutic resistance and cancer progression. Because myeloproliferative neoplasm (MPN) progression is typified by increased JAK2/STAT-mediated cytokine signaling, we investigated the cell type and context specific role of adenosine deaminase acting on RNA1 (ADAR1) editaseactivity in MPN pre-leukemia stem cell (pre-LSC) evolution into acute myeloid leukemia stem cells (LSCs). Here we show by whole transcriptome sequencing (RNA-seq) of 113 FACS-purified hematopoietic stem cells and progenitors from 78 individuals, including 54 MPN and AML patients and 24healthy young and aged individuals, that anti-viral signaling pathway activation and splice isoform switching from ADAR1p110 to JAK2/STAT-inducible ADAR1p150 RNA editase activation contributes to MPN progression. Pre-LSC evolution to LSC was characterized by ADAR1p150 upregulation, distinctive RNA editome patterns, STAT3 hyper-editing, increased replating as a measure of self-renewal. Moreover, LSC generation was typified by beta-catenin self-renewal pathway upregulation, which was recapitulated by lentiviral ADAR1p150 overexpression and reversed by lentiviral ADAR1p150 shRNA knockdown. Our studyunderscores the importance of inflammatory-cytokine fueled enzymatic mutagenesis in human MPN pre-LSC evolution to LSC. Thus, this study sets the stage for developing predictive RNA editome biomarkers of LSC generation to guidetherapeutic strategies aimed at preventing progression of hematopoietic malignancies.

Disclosures
Crews: Ionis Pharmaceuticals: Research Funding.
Low-Dose Methotrexate Could Reduce Severe Early Immune Reactions but Probably Increase Garft Failure in Umbilical Cord Blood Transplantation
Authors: Huilan Liu , Zhu Xiaoyu , Song Kaidi , Wan Xiang , Sun Guangyu , Tang Baolin , Tong Juan , Yao Wen , Zhang Xuhan , Geng Liangquan , Sun Zimin 
Published: November 2019

Early immune disorders, including preengraftment syndrome (PES)and acute graft-versus-host disease(aGvHD)are problematic in cord blood transplantation (CBT), and optimal prophylaxis has not been established. Here, we prospectively investigated whether intensive GvHD prophylaxis by adding low-dose methotrexate (MTX)at day 3 after CBThas a prognostic impact on CBT. 16 consecutive single-unit CBT recipients treated for high-risk hematologic malignancies (10 cases) and non-malignancies (6 cases) between February 2019 and March 2019. The patients, 6 female and 10 males, had a median age of 9 years (range 4-40) and a median weight of 26 kg (range 14-68). Myeloablative preparative regimen comprised fludarabine, busulfan and cyclophosphamide for malignancies and reduced-intensity-conditioning comprised fludarabine, cyclophosphamide and total-body irradiation (4 Gy) for non-malignancies. Graft-versus-host disease (GvHD) prophylaxis was cyclosporine and mycophenolate mofetil plus MTX 3 mg/m2 on day+3. 14 patients achieved engraftment at a median of day 19 and had more than 95% (complete) donor chimerism on day+14.Two patients without hematopoietic reconstitution were engrafted after a second CBT. PES was characterized by high-grade fever, rash, pulmonary edema, weight gain, liver and renal dysfunction developed on a median of day 8 in 11 of the 16 evaluable patients, including 2 who did not achieve engraftment. The PES patients received intravenous corticosteroid at a median dose of 1 mg/kg/day, and of these, 2 patients were requiredBasiliximab to improve clinical symptoms. Grade I to IV and aGvHD developed in 4 and only 1 patient developed severe aGvHD with Grade Ⅲ. Follow-up so far, one death occurred at day 103 because of pneumonia. 15 patients are alive well at a median follow-up of 135 days (131-163). Adding low-dose MTX at day +3 may be offer one optimal regimen to reduce severe early immune reactions and improve outcomes in CBT.

Disclosures
No relevant conflicts of interest to declare.
Cost Effective Cardio Protective Intervention in Haploidentical Hematopoietic Stem Cell Transplantation in Children - a Study from a Tertiary Care Centre in India
Authors: Raj Revathi , Uppuluri Ramya , Vellaichamy Venkateswaran , Ravichandran Nikila , Melarkode Ramanan Kesavan 
Published: November 2019

Cost effective cardio protective intervention in haploidentical hematopoietic stem cell transplantation in children - A study from a tertiary care center in India
Haploidentical hematopoietic stem cell transplantation with post transplant cyclophosphamide (PTCy) is increasingly being offered to children with life threatening blood disorders with no matched family or unrelated donors. High dose cyclophophamide is an alkylating agent known to cause severe cardiac toxicity. Hepatic metabolism of cyclophosphamide results in the production of phosphoramide which is toxic to the myocardium and acrolein which is a bladder irritant. The metabolites deplete antioxidants and augment the inflammatory damage to the cardiac myocytes. N-acetylcysteine (NAC) helps replenish the oxidant pool in the body and provides cardio protective and overall superior outcomes.
We present retrospective data on 103 children between the ages of nine months to eighteen years who underwent haploidentical transplantation with PTCy between May 2015 and May 2019. All children received 50 mg/kg of cyclophosphamide on day 3 and day 4 after infusion of stem cells with Mesna starting with the first dose of cyclophosphamide as a continuous infusion till 24 hours after the second cyclophosphamide dose. All children had a cardiac ECHO performed by a pediatric cardiologist before conditioning and on day 5 after completion of Mesna. The first 30 children (Group 1) did not receive continuous infusion of NAC and the subsequent 83 children (Group 2) were treated with NAC at a dose of 10 mg/kg/ hour from the start of cyclophosphamide. Disease free survival, graft rejection and cardio toxicity were recorded. The follow up period ranged from 3 months to 4 years. In Group 1 the disease free survival was 46%, graft rejection rate was 13.5% and adverse cardiac events occurred in 10%. In Group 2 the disease fee survival was 67.5%, graft rejection rate was 3.6% and there were no adverse cardiac events.
In conclusion, the introduction of N-acetylcysteine provides a simple and yet cost effective way to eliminate cardiac toxicity even in infants and small children. This is the first interventional study to help safe delivery of a chemotherapeutic agent at a cost of 25 USD for a 10 kg child. We would recommend the use of NAC in all patients undergoing PTCy based transplantation.

Disclosures
No relevant conflicts of interest to declare.
Short-Term Noncryopreserved Hematopoietic Stem Cells: Single Center Experience of Autologous Transplantation
Authors: Voloshin Sergei , Garifullin Andrey , Kuzyaeva Anastasiya , Shuvaev Vasily , Schmidt Alexander , Kuvshinov Alexey , Balashova Valentina , Chubukina Zhanna , Belyakova Elena , Rysev Georgiy , Chechetkin Alexander , Rugal' Viktor 
Published: November 2019

Background: High-dose chemotherapy (HDCT) followed by autologous transplantation of hematopoietic stem cells (auto-HSCT) is an important component of treatment in multiple myeloma (MM). There is a standard method of controlled cryopreservation of HSC suspension. We found that the storage of native HSC suspension with temperature fluctuations from +3 °C to +5 °C during 72 - 120 hours does not significantly affect the content of CD34+ cells in the product, the index 7AAD- (7-AAD (7-aminoactinomycin - D) is a fluorescent marker that penetrates damaged cell membranes and binds to double-stranded DNA. Through 7AAD does not penetrate intact membranes, so living cells are not stained 7AAD with flow cytometry), and colony-forming ability (CFA) of HSC, as well as the recovery time of hematopoiesis in MM patients after auto-HSCT.
Aim: To evaluate the effectiveness and safety of the method of storage of non-cryopreserved peripheral blood stem cells.
Methods: 39 patients with MM were included in this study(male/female ratio 1.36:1). All the patients get standard immunochemotherapy programs and were in remission at the time of auto-HSCT. Patients were divided into two groups depending on the method of stem cell storage: group 1 - non-cryopreserved (n=20), group 2 - cryopreserved (standard) (n=19). An effectivity and safety were evaluated in such parameters as the number of CD34+ and 7AAD- cells, CFA after apheresis and before reinfusion of HSC. Also, we evaluated the number of platelets concentrate transfusions, the timing of engraftment of granulocytic and megakaryocytic blood sprouts, the length of hospital stays after auto-HSCT.
Results: The results are presented in the comparison table of the evaluated parameters. Our data showed significantly reduce of episodes febrile neutropenia and cases of enteropathy.
Conclusion: Thus, the proposed method of storage of HSC is not inferior to the traditional method with cryopreservation on such parameters as CD34+, 7AAD-, CFA, the number of platelets concentrate transfusions, terms of hematopoiesis restoration, length of hospital stay after HSCT, the number of complications.
Table.

Disclosures
Shuvaev: Fusion Pharma: Consultancy; BMS: Consultancy; Novartis: Consultancy; Pfize: Honoraria.
Brain Anomalies in Children with Severe Factor VIII Deficiency- a Pilot Study
Authors: Al-Huniti Ahmad , Harshman Lyndsay A , Novak Marci , Nopoulos Peggy , Staber Janice M 
Published: November 2019

Introduction: Bleeding resulting from factor VIII deficiency (hemophilia A) can occur in any tissue including the brain. While significant progress has been made in prevention and treatment of hemophilia-related joint and muscle disease with prophylaxis, there is a paucity of research to understand, treat, or prevent brain disease in hemophilia patients. People with hemophilia are recognized to be at risk for neuropsychiatric and neurocognitive disorders even when treated with factor VIII (FVIII) primary prophylaxis. This is especially problematic given that brain injury is difficult to detect early, and consequences are often delayed. Therefore, our study aims to evaluate brain structure and cognitive function in pediatric patients with severe FVIII deficiency in order to improve outcomes.
Methods: After IRB approval, a single-center pilot study enrolled seven pediatric subjects with severe FVIII deficiency and 23 healthy controls to evaluate brain structure and function. Inclusion criteria included males aged 6 - 16 years. All subjects with severe hemophilia A had a FVIII level of less than 1% completed at the Iowa Hemophilia and Thrombosis Center. Any subject was excluded due to a known diagnosis of traumatic brain injury, brain tumor, major chromosomal anomalies, or intellectual disability. Subjects with a known diagnosis of intracranial hemorrhage or FVIII inhibitor were excluded. Neurocognitive assessments included the Behavioral Regulation Index of Executive Function (BRIEF) which measures executive function. Magnetic resonance imaging sequences included T1, T2, and diffusion tensor imaging. Freesurfer and Brain Research: Analysis of Images, Networks, and Systems (BRAINS2) software were used to preprocess imaging and generate volumetric data.
Results: Mean age for PWH was 10.4 years and 11.8 years for controls (p = 0.24). Both groups had similar height, weight, and socioeconomic status. Despite normal IQ, PWH demonstrate impaired behavior regulation and globally impaired executive composition compared to controls (p = 0.001 and p = 0.038, respectively). Volumetric brain quantification revealed subjects with hemophilia had decreased whole brain volumes compared to controls (p = 0.019). Our analysis suggests that these differences are in discrete regions. Future analysis will investigate if volumetric differences correlate with neurocognitive assessments.
Conclusions: PWH demonstrated abnormal brain structure and function compared to healthy controls. Further evaluation is warranted to understand why and how FVIII deficiency and its treatment alter neurological outcomes.

Disclosures
Staber: UniQure: Honoraria; Genentech: Honoraria; Bayer: Honoraria; Spark: Honoraria; Novo Nordisk: Honoraria.
Identifying Novel Immunodominant Epitopes within Human Parainfluenza Virus-3 Matrix Protein
Authors: Harris Katherine Marie , Keller Michael , Bollard Catherine M. , Horn Sarah , Lazarski Christopher , Sani Gelina 
Published: November 2019

Human Parainfluenza Virus-3 (HPIV3) is a common cause of severe respiratory illness in immunocompromised patients and has no approved antiviral therapies. Recent preclinical studies demonstrated T-cell recognition of HPIV3 viral proteins, and a phase I study adoptively transferring virus specific T cells (VSTs) targeting HPIV3 to patients following bone marrow transplantation is underway (NCT03180216). Hence, we sought to identify immunodominant epitopes within the Matrix protein based on the extent of the T-cell responses.
VSTs were generated from the peripheral blood of healthy donors by ex vivo expansion after stimulation with a 15-mer peptide library encompassing HPIV3 Matrix protein, which has been identified as the HPIV3 antigen eliciting the most robust response (Mclaughlin et al., J Cytotherapy 2016). Epitope mapping was performed using IFN-g ELIspot with combinatorial 15-mer peptide pools (Straathof et al., J Immunology 2005). Flow cytometry was used to characterize the products with intracellular cytokine staining.
Initial mapping with the peptide pools showed activity in 16 of the 19 pools. Pools which elicited activity in 5 or more products had mean spots per well (ASC)/1x10^5 cells range 89 to 217, versus 0.6 ASC/1x10^5 in the negative controls. We identified 13 novel immunodominant epitopes (Table) within Matrix protein identified in 9 tested VST products, with 4 clustered epitopes (peptides 82-85) at the C-terminus, which contains a leucine residue critical for regulating virion release (Ding et al, 2017). Five (55%) donors recognized epitopes in this domain. Individual peptides which elicited activity had mean (ASC)/1x10^5 cells range 13 to 224, versus 0.4 ASC/1x10^5 in the negative controls. VST products were polyfunctional releasing both IFN-γ and TNF-α in response to the newly identified epitopes, and preliminary flow cytometry data showed that the majority of these epitope specific responses were HLA Class II restricted.
In conclusion, we have identified several novel epitopes within the HPIV3 Matrix protein. Comprehensive classification of the breadth and stability of HPIV3 epitopes is critical for the development of third-party "off the shelf" virus specific T cell therapeutics. Therefore, these results will facilitate the development of clinical studies using partially-HLA VSTs as an "off the shelf' treatment for the treatment of immune suppressed patients with severe respiratory infection related to HPIV3.
Figure

Disclosures
Bollard: Cellectis: Membership on an entity's Board of Directors or advisory committees; NexImmune: Equity Ownership; Torque: Equity Ownership; Caballetta: Equity Ownership; Mana Therapeutics: Equity Ownership; self: Patents & Royalties: patent on HIV T cells. Lazarski:self: Patents & Royalties: IP related to virus T cells.
Catheter Related Thrombosis (CRT) in Hematologic Malignancy: A Retrospective Study of Venous Thromboembolism Management
Authors: Desai Ruchi , Tandon Ankita , Jaglal Michael 
Published: November 2019

Introduction: Central Venous Catheters (CVC) improve quality of life for patients with hematologic malignancy but long-term presence may result in CRT. Optimal treatment of CRT is uncertain. Here, we focus on a retrospective study of CRT in patients with hematologic malignancy to identify clinical features associated with increased risk for recurrent thrombosis.
Methods: Demographic and management information of patients with CRT and hematologic malignancy was extracted from the medical record of a single academic medical center. Data was described using summary statistics. Nonparametric Cox proportional hazard models were utilized for thrombosis recurrence and bleeding occurrence. Unadjusted models were utilized for CRT treatment, type of anticoagulation, platelet count, and other factors.
Results: 85 patients were included for analysis. Summary statistics are noted in Table 1. Median platelet count differed across initial treatment approach [Anticoagulation (AC): 177 (106.5- 662), Anticoagulation and Catheter Removal (AC+): 112 (61.75-304), Catheter Removal only 24 (20.25-35.5), No treatment 30.5 (16.75-10.8), p<0.0001]. For CRT treatment, patients with AC+ have more than a 660 % chance of thrombosis recurrence compared to AC as reference group. This significant difference is also seen in the Kaplan Meir curves for CRT treat (log-rank p=0.0484), Figure 1. Of note, 15 patients treated with AC+ developed recurrent thrombosis, the majority of which (53%, 8/15) occurred within 100 days. Of these patients, 62% (5/8 patients) had at least 2 days delay in starting anticoagulation while no delays in starting anticoagulation were noted in patients who were primarily treated with AC. Of the 8 patients who received no therapy for CRT, 7/8 patients (87%) had noted brachial (peripheral deep).
Discussion: Significant variability in management of CRT in patients with hematologic malignancy is noted. Platelet count at time of thrombosis may influence treatment decision. Our data indicate that treatment with anticoagulation is superior to treatment without anticoagulation. The difference in thrombosis recurrence seen in patients who received AC v. AC+ could potentially be explained by delays in starting AC in the latter group. Interestingly, patients who did not receive any therapy for CRT had no recurrence, which may be explained by the fact that the majority had peripheral deep thrombosis, which are less likely to have deep recurrences. In addition, type of anticoagulation used for treatment was notHowever, our data is limited by its population of single institutional patients and retrospective nature. Prospective, studies are needed to better delineate differences in outcomes.

Disclosures
Jaglal: NOVARTIS: Consultancy.