bluuebunny/crossref_metadata_2025_split huggingface dataset:
Long-Term Phenotypic Correction of Hemophilia A Mice Following Intravenous Injection of miRNA-Regulated Lentiviral Vectors.
Authors: Matsui Hideto , Ozelo Margareth , Hegadorn Carol , Labelle Andrea , Burnett Erin , Tuttle Angie , Brown Brian , Naldini Luigi , Hough Christine , Lillicrap David 
Published: April 2020

Abstract
Hemophilia A is an excellent candidate disorder for the use of gene therapy as a treatment modality. To date, although lentiviral delivery of the factor VIII (FVIII) transgene has the potential to provide sustained therapeutic correction of the hemophilia A phenotype, this has not been achieved in adult animals because of the anti-FVIII immune response. We have used lentiviral vectors to deliver the canine FVIII transgene to hemophilia A neonates and although no anti-FVIII immune response occurred, and indeed the treated mice displayed long-term tolerance to the canine FVIII antigen, this strategy did not provide sustained therapeutic levels of plasma FVIII. To overcome these limitations, we modified our lentiviral vector and the protocol for viral delivery to enhance transduction of hepatocytes and direct transgene expression away from antigen presenting cells. We engineered lentiviral vectors that encode the B-domain deleted canine FVIII cDNA under the transcriptional control of either a non-viral ubiquitous promoter or two different liver-restricted promoters. However, no plasma FVIII was detected in any of the adult hemophilia A mice after intravenous injection of the various lentiviral vectors because of an anti-canine FVIII immune response. An alternate pseudotype (GP64) was used to enhance transduction of hepatocytes and a target sequence for a hematopoietic-specific microRNA was incorporated into the transgene to prevent FVIII expression in antigen presenting cells that may arise from promoter trapping. When hemophilia A mice received intravenous infusions of these modified vectors, where the cFVIII trangene is under the control of either of the liver-restricted promoters, all treated mice (n=4) showed sustained FVIII expression (mean FVIII levels 28.2±2.4 mU/mL) for more than 150 days (last time analyzed) without developing anti-FVIII antibodies. Moreover, temporary depletion of Kuppfer cells prior to viral administration resulted in a 3-fold elevation of levels of plasma FVIII (mean FVIII levels 83.3±2.1mU/mL; n=4). Analysis of the biodistribution of the integrated FVIII transgene and expression of canine FVIII mRNA indicate an enhanced restriction of FVIII expression in hepatocytes with the use of the modified lentiviral vectors. These results demonstrate, for the first time, the long-term therapeutic potential of modified lentiviral vectors for treating adult pre-clinical animal models of hemophilia A.
In Vitro and In Vivo Selection of Genetically Modified Cells Using shRNA Against HPRT and Treatment with 6-thioguanine.
Authors: Porter Christopher C. , DeGregori James 
Published: April 2020

Abstract
Inefficient transduction, poor long term expression, and engraftment failure of ex vivo manipulated cells have slowed the practical advancement of gene therapy trials. Thus, the ability to select for or amplify a population of cells that has been modified to express a gene of interest might enhance the effectiveness of gene therapy. Strategies for in vivo expansion of genetically modified cells that have been studied to date have relatively high toxicity or low efficacy in selection of hematopoietic stem cells. We hypothesized that resistance to the purine analog 6-thioguanine (6TG) could be programmed via lentiviruses, and that treatment with 6TG would allow for selection of genetically modified cells in vitro and in vivo. Using short hairpin RNAs, we achieved efficient knockdown of hypoxanthine phosphoribosyl transferase (HPRTkd), the enzyme required for 6TG cytotoxicity, in the murine hematopoietic progenitor cell line FL5.12. In so doing we were able to provide Fl5.12 cells with resistance to 6TG. In the presence of 6TG, HPRTkd cells continued to proliferate for at least 30 days, whereas control transduced cells ceased proliferating after 7-10 days. 6TG treatment of mixed cultures of GFP+-HPRTkd cells and untransduced cells resulted in selective outgrowth of HPRTkd cells. Knockdown of HPRT in FL5.12 cells was found to attenuate the checkpoint activation, cell cycle arrest and apoptosis seen in control transduced cells when treated with 6TG. Knockdown of HPRT in murine primary hematopoietic cells also allowed for selection of transduced cells with 6TG ex vivo. Furthermore, and most importantly, after transduction of whole bone marrow and transplantation into sub-lethally irradiated recipient mice, a single, short course of treatment with 6TG resulted in up to 12 fold greater percentages of circulating transduced granulocytes as compared to untreated controls. These results suggest that genetically modified hematopoietic stem cells can be selected in vivo using 6TG. This strategy may be useful for therapy of a variety of hematopoietic diseases, particularly those that affect hematopoietic progenitors. The benefits of this strategy include the following: 1) the use of a lentivirus with a self inactivating long terminal repeat, 2) a very short cassette encoding drug resistance, making the vector easier to manipulate, and 3) a very well tolerated and relatively non-toxic medication for selection.
Efficient Transfer of CD40L mRNA into Mantle Cell Lymphoma for the Production of a Whole Tumor Cell Vaccine.
Authors: Brody Joshua D. , Li Linhong , Feller Stephanie , Fratantoni Joseph , Levy Ronald 
Published: April 2020

Abstract
Mantle cell lymphoma (MCL) is an aggressive non-Hodgkin’s lymphoma with the worst long-term prognosis of any NHL subtype. Current therapeutic options are unsatisfactory. MCL patients’ malignant B cells are ineffective antigen-presenting cells (APCs), perhaps resulting from low level expression of the immune co-stimulatory molecules that are essential to activate T cells upon interaction with the T-cell receptor. The MCL cells can be engineered to be effective APCs and thereby function as a therapeutic cellular vaccine in combination with chemotherapy and/or stem cell transplantation to eradicate residual disease. However, primary MCL cells are difficult targets for gene transfer by both viral and non-viral methodologies. Ligation of CD40 resulting from co-culturing with hCD40L expressing murine fibroblasts was shown to be superior to a panel of other immune stimulants and cytokines in upregulating co-stimulatory markers and inducing anti-tumor T cell responses (Hoogendoorn et al. 2005). We now report on a technology platform, based on electroporation of mRNA for CD40L, for the introduction of CD40L protein expression and subsequent induction of immune co-stimulatory molecules by MCL tumor cells. Primary MCL malignant B cells were obtained from patients’ lymph node biopsies by mechanical dissociation, placed in single cell suspension and cryopreserved prior to modification. Full-length 5′-end capped hCD40L mRNA transcript was generated by in vitro transcription with a commercially available T7 polymerase kit. The transfected MCL cells were immunostained with fluorophore-conjugated monoclonal antibodies against hCD40L, hCD80 and 86 then analyzed by FACS. Data showed hCD40L could be detected in ≥ 80% of the transfected MCL cells as early as 2 hrs post transfection. At 3 days post manipulation, hDC40L expression could be detected on approximately 30% of the transfected MCL cells. Cell viability remained at approximately 80% during the 3 day in vitro culturing. FACS analysis of the immune co-stimulatory molecules revealed that forced expression of hCD40L caused an up-regulation of CD80/86, which was increased approximately 10 fold compared to the expression levels in naïve, non modified cells. The increased expression level of CD80/86 was maintained for 3 days. Furthermore, when the hCD40L modified MCL cells were mixed with allogeneic PBMC, they stimulated IFN-γ production at a level 4 fold higher than was observed with naïve, non modified MCL cells mixed with allogeneic PBMC. This provides proof-of-concept that MCL cells modified by mRNA-hCD40L transfection have the potential to be used as a cellular vaccine. Such transduced cells function to protect animals from tumor challenge. The process can be scaled up to produce &gt;2×1010 modified tumor cells. This simple, non-viral cell manipulation system is practical and will be a useful tool for immunotherapy of human hematopoietic malignancies such as MCL.
Targeted Killing of Glioblastoma Multiforme In Vivo by IL-13 Zetakine Redirected CTLs Made Glucocorticoid Resistant with Zinc Finger Nucleases.
Authors: Reik Andreas , Holmes Michael C. , Zhou Yuanyue , Mendel Matthew , Liu Pei-Qi , Lee Gary , Paschon David , Rebar Edward , Ando Dale , DiGiusto David , Gregory Philip D. , Jensen Michael C. 
Published: April 2020

Abstract
Genetic modification of cytolytic T-lymphocytes (CTL) for enhancing their functional immunobiology is a promising immunotherapeutic approach for the treatment of cancer and infectious disease. CTLs modified to express a chimeric antigen receptor comprising an extracellular IL13 domain and cytoplasmic CD3 domain (IL13-zetakine) can be re-directed both in vitro and in animal models to target glioblastoma multiforme (GBM), which is characterized by high expression of IL13Ralpha2. Patient-derived IL13-zetakine/HyTK expressing CD8+ CTL clones have entered early stage clinical trials. However, their clinical application is frequently limited in this patient population by the pervasive use of dexamethasone, a potent glucocorticoid analogue employed in the management of cerebral edema. Thus iatrogenic dexamethasone-mediated T-cell functional anergy and apoptosis in these patients is a barrier to realizing the full clinical utility of this adoptive therapy strategy. We hypothesized that knocking out the expression of the glucocorticoid receptor would render therapeutic CTLs resistant to the effects of synthetic glucocorticoids, including dexamethasone. We therefore developed engineered zinc finger nucleases (ZFNs) to specifically disrupt the glucocorticoid receptor (GR) locus in the human genome. ZFNs include the cleavage domain of the restriction enzyme FokI linked to an engineered zinc finger DNA-binding domain and can be designed to cleave a predetermined site in the genome. Natural repair of such DNA breaks via the error-prone non-homologous end joining pathway results in the inactivation of the target gene at frequencies which permit the isolation of knock out clones. Employing adenovirally delivered and transiently expressed ZFNs targeting exon 3 of the human GR gene, we isolated IL13-zetakine+ CD8+T-cells containing a biallelically mutated GR locus. These cells were characterized by the absence of full length GR protein, lack of glucocorticoid hormone-induced gene regulation and resistance to glucocorticoid hormone-mediated immunosupression and apoptosis. Importantly, the ZFN-modified, glucocorticoid-resistant CTLs demonstrated zetakine re-directed cytolytic activity and tumor cell specificity in chromium release assays in vitro and in an orthotopic mouse model of GBM in vivo. These results indicate that glucocorticoid-resistant IL13-zetakine targeted CTLs should retain function in cancer patients receiving glucocorticoids. A clinical trial to test this hypothesis is currently under development.
A Prospective Study of Imatinib 400 mg vs 800 mg Frontline in High Risk Ph+ Chronic Myeloid Leukemia (CML) Patients.
Authors: Baccarani Michele , Castagnetti Fausto , Porkka Kimmo , Nielsen Johan L. , Hjorth-Hansen Henrik , Haznedaroglu Ibrahim , Nagler Arnon , Rege-Cambrin Giovanna , Specchia Giorgina , Amabile Marilina , Testoni Nicoletta , Palandri Francesca , Pane Fabrizio , Alimena Giuliana , Martinelli Giovanni , Rosti Gianantonio , Simonsson Bengt 
Published: April 2020

Abstract
Background: imatinib mesylate (IM) is the drug of choice for the front-line treatment of Ph+ CML, at a dose of 400 mg daily (M. Baccarani et al Blood2006;108:1809–1820). Several biological and clinical observations suggest that increasing the dose may improve the results. The cytogenetic response to IM 400 mg daily is significantly related with Sokal relative risk. High risk patients could benefit of a dose increase front-line.
Aim: to compare the effects of 400 mg and 800 mg daily in previously untreated, early chronic phase patients, high Sokal risk. The primary efficacy variable of the study is the complete cytogenetic response (CCgR) rate after 12 months, on an intention-to-treat analysis.
Patients and Methods: this is a multicentric international study running in Italy, Sweden, Denmark, Finland, Norway, Turkey and Israel, approved by the local Ethic Committees, conducted according to Helsinki Declaration and Good Clinical Practice. 215 patients with confirmed Ph+ CML previously untreated, high risk according to Sokal formulation (J. Sokal et al Blood1984;63:789–799) were enrolled over a 3-year period and were randomized (1:1) to receive IM 400 or 800 mg daily. Cytogenetic response was assessed by conventional cytogenetics and FISH analysis after 3, 6 and 12 months. Molecular response was evaluated every three months.
Results: as of August 2007, 137 patients are evaluable for CCgR rate at 12 months (primary efficacy variable). Patients in CCgR at that time were 78/137 (57%). Treatment failures during the study (no complete hematologic response or 100% Ph+ at 6 months, or loss of response) were 24/137 (17%), patients off-treatment for protocol violations or refusal were 10/137 (7%), patients off-treatment for toxicity were 7/137 (5%).
Conclusions: the results of this preliminary analysis show that the CCgR rate at 12 months is overall 57%, in line with the results of the IRIS trial (imatinib 400 mg daily - T. Hughes et al, NEJM 2003, 349;15: 1423–1432) in the same risk category (69% all risks, 49% high Sokal risk). At the time of writing is too early to analyze the results by arm: a second analysis will be performed in November (datalock, October 31) and the results will be presented on site.
Angiogenesis Assessed in Tissue Microarrays of Diagnostic Follicular Lymphoma Correlates with Outcome.
Authors: Clear Andrew J. , Lee Abigail M. , Morris Kelly J. , Calaminici Maria , Macdougall Finlay , Proto-Siqueira Rodrigo , Goff Lindsey K. , Lister Thomas A. , Gribben John G. 
Published: April 2020

Abstract
Micro-vessel density (MVD) is a powerful prognostic factor in cancers but its value in haematological malignancies is more controversial. To examine its prognostic value in follicular lymphoma (FL) we assessed number and distribution of blood vessels by high-throughput tissue microarray and automated image analysis measurements in tissue microarrays (TMA) of paraffin-embedded, diagnostic lymph node biopsies taken from fifty-nine FL patients. These patients were selected from those who died from lymphoma progression less than 5 years after diagnosis (short survivor group) (n=34) and those who survived more than 15 years from diagnosis (long survivor group) (n=25). Immunohistochemistry was used in TMAs to study the number and location of vessels staining positive for the endothelial cell markers CD31 and CD34 and pericyte coverage using PDGFR. Interactive quantification using image analysis software was used to provide details of absolute numbers of vessels from each patient, as well as vessel size and their location. Image stacks of immunofluorescence stained sections were obtained using laser-scanning confocal microscopy to trace the tumour vasculature. Results demonstrated that both total vessel count and mean vessel area were significantly different between the two groups. Samples from the long survivor group were significantly more likely to have fewer (p=0.025), but larger vessels (p=0.002) than those from the short surviving group. The differences in vessel size and number were more prominent in inter-follicular vessels compared with those inside the neoplastic follicles. The smaller and more numerous blood vessels seen in the poorer prognostic sub-group likely reflects active, sprouting angiogenesis as confirmed by confocal microscopy. This study validates the use of TMAs to examine angiogenesis and demonstrates the powerful prognostic value of assessing MVD in FL. These results suggest that sprouting angiogenesis represents a therapeutic target in this disease and ongoing studies are investigating the mechanisms contributing to alteration in angiogenesis in different prognostic subgroups in FL and in transformation of this disease. The prognostic significance of MVD assessment in TMA is currently being evaluated in a validation set of FL samples.
Majority of Transformed Lymphomas Have High SUVs on PET Scanning Similar to Diffuse Large B Cell Lymphoma (DLBCL).
Authors: Noy Ariela , Weissler Matthew , Schoder Heiko , Gonen Mithat , Yeung Henry 
Published: April 2020

Abstract
Background: In our previous work, we correlated non-Hodgkins (NHL) histology with intensity of FDG uptake (SUVmax) on positron emission tomography in untreated patients: an SUVmax greater than 10 predicted an aggressive lymphoma with &gt;80% certainty and an SUVmax greater than 13 predicted an aggressive lymphoma with &gt;90% certainty. We sought to evaluate SUVmax in a series of patients with transformed lymphoma.
Methods: All PET scans for lymphoma indications at our institution 1999-5/2007 were evaluated. Patients were included if they had biopsy proven transformation. FDG-PET scans were included if they were no more than sixty days prior to or 90 days after biopsy proven transformation. Patients had no therapy for sixty days prior to PET scan unless there was documented progression of disease after the last treatment.
Results: Among the indolent diagnoses (n= 44), the following specific histologies were identified: 9 follicular lymphoma grade 1 (FL I), 9 FL grade 2 (FL 2), 7 small lymphocytic lymphoma / chronic lymphocytic leukemia (SLL/CLL), 11 marginal zone lymphoma (MZL) including one of mucosa associated lymphoid tumor (MALT) type, one mycoses fungoides, and one indolent lymphoma otherwise not specified. 5 FL 3 (FL 3), including one specified as grade 3a (FL 3a) were considered indolent if they preceded a diagnosis of DLBCL. Among the aggressive diagnoses (n= 28), the following specific histologies were identified: 37 DLBCL, one follicular lymphoma grade IIIa included as it was preceded by FLI, one large peripheral T-cell lymphoma, and 5 Large cell lymphoma(LCL), not otherwise characterized on account of biopsy size. Standard uptake values (SUV) were measured at the biopsy site when possible (n=38). In 6 patients the biopsy site was completely excised before the PET scan. The SUV of biopsy site ranged from 3-38, with a median of 10 and mean of 15. Among evaluable patients, 17/36 (47%) biopsies had an SUV above 10; and 12/36 (33%), above 13. The SUVmax for a transformed aggressive lymphoma ranged from 3.2–40, with a median of 12 and mean of 15. 27/44 (61%) patients had an SUVmax above 10; and 21/44 (48%), above 13.
Conclusions: Similar to patients with de-novo aggressive lymphoma, the majority of patients with transformed lymphomas have high SUVmax for a given pre-treatment staging study. Therefore, transformation to aggressive lymphoma should be suspected in patients with indolent lymphoma found to have high SUVs on FDG PET, and biopsies should be directed to the site of greatest PET avidity whenever feasible.
Modulation of CD20 Expression in Rituximab-Sensitive (RSCL) and Rituximab Resistant Cell Lines (RRCL) Using IL-4 and Bryostatin-1.
Authors: Tsai Ping-Chiao , Hernandez-Ilizaliturri Francisco J. , Olejniczak Scott H. , Naveen Bangia , Czuczman Myron S. 
Published: April 2020

Abstract
While the use of rituximab in combination with chemotherapy resulted in an improved survival among various subtypes of B-cell lymphomas, a significant number of patients fail to respond or relapse as a consequence of intrinsic or acquired resistance. A change in CD20 antigen density expression is a potential mechanism to explain rituximab resistance. Moreover, several groups of investigators are focused in understanding the mechanisms that regulate CD20 expression and to develop therapeutic strategies to up-regulate CD20 expression (i.e. IL-4, GM-CSF or Bryostatin-1). Up-regulation of CD20 in DB and Ramos cells by Bryostatin-1 was found to be PKC and Erk dependent. In an attempt to characterize the mechanisms responsible for rituximab resistance we developed several RRCL derived from rituximab-sensitive RL and Raji cells. We have demonstrated a significant down-regulation of CD20mRNA and CD20 surface antigen in RRCL when compared to RSCL. In our present work we evaluated the mechanisms involved in the mRNA down-regulation of CD20 and the potential of IL-4 and Bryostatin-1 in modulating CD20 antigen expression among a panel of RRCL. To this end, RSCL and RRCL were treated with either 5ng/ml of IL-4 or 3 different doses of Bryostatin-1(1, 3 or 5ng/ml). Nuclear and cytosolic extract were also obtained from RSCL RL and RRCL RL-4RH after 24, 48 and 74 hrs exposure to IL-4 (5ng/ml) or control. Differences in the expression of key regulatory transcription factors for B-cell lymphocyte development (PU.1, Oct-2, Pax5, E2A and EBF) were studied by Western Blotting. Previously, we found a significant down-regulation of CD20 antigen in the RRCL. An up-regulation of cytosolic and surface CD20 was detected in RRCL exposed to either IL-4 or Bryostatin by western blotting and flow cytometric analysis. Besides, a higher expression of Pax5, PU.1, EBF was found in nuclear fractions of RRCL when compared to RSCL. In vitro exposure of RRCL to IL-4 decreases the expression of PU.1, Oct-2, Pax5 and EBF in nuclear extracts from RSCL or RRCL when compared with controls treated cells. Our data suggest that rituximab resistance is associated with the up-regulation of transcription factors PU.1, Oct-2, Pax5 and EBF and concomitant suppression of CD20 antigen. Future study of how these agents induce CD20 expression in RRCL will provide a potential means to reversing resistance towards rituximab treatment.