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pBABEpuro_p53 Retroviral Vector

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[#RTV-401] pBABEpuro_p53 Retroviral Vector

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(1) Construction of replication-competent oncolytic retroviral vectors expressing R peptide-truncated 10A1 envelope glycoprotein.[TOP]

Pubmed ID :30898575
Publication Date : //
Replication-deficient retroviral (RDR) vectors have been generally used for gene therapy, but clinically beneficial transduction efficiency is difficult to achieve with these vectors. In recent times, attention has been focused on the use of murine leukemia virus (MLV)-based replication-competent retroviral (RCR) vectors. RCR vectors have been shown to achieve efficient tumor reduction in a wide variety of cancer models. Most RCR vectors have been developed from amphotropic 4070 A MLV env, which is broadly applied in basic research. In this study, we generated RCR vectors based on Moloney MLV by replacing the native env gene in a full-length viral genome with the 10A1 env gene. 10A1 MLV can infect a wide variety of cells. Unlike amphotropic MLV, the 10A1 MLV can use amphotropic MLV receptor Pit2 or gibbon ape leukemia virus (GaLV) receptor Pit1. The resulting construct MoMLV-10A1-EGFP was able to replicate in 293 T, NIH3T3, and Mus dunni cells. To evaluate the potential of MoMLV-10A1 vector as a therapeutic agent, we incorporated the yeast cytosine deaminase (CD) suicide gene into vectors. The resulting vector MoMLV-10A1-CD could inhibit the growth of human 293 T cells upon 5-fluorocytosine (5-FC) administration. In addition, to lyse tumor cells by syncytium, MoMLV-10A1-R(-)-EGFP was generated by replacing wild-type 10A1 env with the 16-amino acid R peptide-truncated 10A1 env gene. Syncytium formation was observed in the TE671 human tumor cells, 293 T and PG13 cells upon transfection of the MoMLV-10A1-R(-)-EGFP vector. This result suggests that replication of this vector could be oncolytic in itself. We also found that syncytium could contribute to enhance cell-to-cell transmission of the retroviral vectors. Our results thus show that the MoMLV-10A1 vectors can be potentially useful for cancer gene therapy.

Authors : Lee Eun Sik, Jin Sae Young, Kang Byeng Kwon, Jung Yong-Tae,



(2) Ebola Virus Entry: From Molecular Characterization to Drug Discovery.[TOP]

Pubmed ID :30893774
Publication Date : //
Ebola Virus Disease (EVD) is one of the most lethal transmissible infections, characterized by a high fatality rate, and caused by a member of the family. The recent large outbreak of EVD in Western Africa (2013⁻2016) highlighted the worldwide threat represented by the disease and its impact on global public health and the economy. The development of highly needed anti-Ebola virus antivirals has been so far hampered by the shortage of tools to study their life cycle , allowing to screen for potential active compounds outside a biosafety level-4 (BSL-4) containment. Importantly, the development of surrogate models to study Ebola virus entry in a BSL-2 setting, such as viral pseudotypes and Ebola virus-like particles, tremendously boosted both our knowledge of the viral life cycle and the identification of promising antiviral compounds interfering with viral entry. In this context, the combination of such surrogate systems with large-scale small molecule compounds and haploid genetic screenings, as well as rational drug design and drug repurposing approaches will prove priceless in our quest for the development of a treatment for EVD.

Authors : Salata Cristiano, Calistri Arianna, Alvisi Gualtiero, Celestino Michele, Parolin Cristina, Palù Giorgio,



(3) Lentiviral Vectors for the Treatment and Prevention of Cystic Fibrosis Lung Disease.[TOP]

Pubmed ID :30875857
Publication Date : //
Despite the continued development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs for the treatment of cystic fibrosis (CF), the need for mutation agnostic treatments remains. In a sub-group of CF individuals with mutations that may not respond to modulators, such as those with nonsense mutations, CFTR gene transfer to airway epithelia offers the potential for an effective treatment. Lentiviral vectors are well-suited for this purpose because they transduce nondividing cells, and provide long-term transgene expression. Studies in primary cultures of human CF airway epithelia and CF animal models demonstrate the long-term correction of CF phenotypes and low immunogenicity using lentiviral vectors. Further development of CF gene therapy requires the investigation of optimal CFTR expression in the airways. Lentiviral vectors with improved safety features have minimized insertional mutagenesis safety concerns raised in early clinical trials for severe combined immunodeficiency using γ-retroviral vectors. Recent clinical trials using improved lentiviral vectors support the feasibility and safety of lentiviral gene therapy for monogenetic diseases. While work remains to be done before CF gene therapy reaches the bedside, recent advances in lentiviral vector development reviewed here are encouraging and suggest it could be tested in clinical studies in the near future.

Authors : Marquez Loza Laura I, Yuen Eric C, McCray Paul B,



(4) A retrospective molecular study of Cryptosporidium species and genotypes in HIV-infected patients from Thailand.[TOP]

Pubmed ID :30867022
Publication Date : //
Opportunistic infections represent a serious health problem for HIV-infected people. Among enteric infections, cryptosporidiosis, a severe and life-threatening diarrheal disease, is of particular importance in low economic settings where access to anti-retroviral therapy is limited. Understanding transmission routes is crucial in establishing preventive measures, and requires the use of informative genotyping methods. In this study, we performed a retrospective analysis of Cryptosporidium species in 166 stool samples collected from 155 HIV-infected patients during 1999-2004 at the Siriraj Hospital in Bangkok, Thailand.

Authors : Sannella Anna Rosa, Suputtamongkol Yupin, Wongsawat Ekkarat, Cacciò Simone M,



(5) Restorative Mechanism of Neural Progenitor Cells Overexpressing Arginine Decarboxylase Genes Following Ischemic Injury.[TOP]

Pubmed ID :30853827
Publication Date : //
Cell replacement therapy using neural progenitor cells (NPCs) following ischemic stroke is a promising potential therapeutic strategy, but lacks efficacy for human central nervous system (CNS) therapeutics. In a previous study, we reported that the overexpression of human arginine decarboxylase (ADC) genes by a retroviral plasmid vector promoted the neuronal differentiation of mouse NPCs. In the present study, we focused on the cellular mechanism underlying cell proliferation and differentiation following ischemic injury, and the therapeutic feasibility of NPCs overexpressing ADC genes (ADC-NPCs) following ischemic stroke. To mimic cerebral ischemia , we subjected the NPCs to oxygen-glucose deprivation (OGD). The overexpressing ADC-NPCs were differentiated by neural lineage, which was related to excessive intracellular calcium-mediated cell cycle arrest and phosphorylation in the ERK1/2, CREB, and STAT1 signaling cascade following ischemic injury. Moreover, the ADC-NPCs were able to resist mitochondrial membrane potential collapse in the increasingly excessive intracellular calcium environment. Subsequently, transplanted ADC-NPCs suppressed infarct volume, and promoted neural differentiation, synapse formation, and motor behavior performance in an tMCAO rat model. The results suggest that ADC-NPCs are potentially useful for cell replacement therapy following ischemic stroke.

Authors : Kim Jae Young, Kim Jong Youl, Kim Jae Hwan, Jung Hosung, Lee Won Taek, Lee Jong Eun,



(6) Rapid establishment of stable retroviral packaging cells and recombinant susceptible target cell lines employing novel transposon vectors derived from Sleeping Beauty.[TOP]

Pubmed ID :30852270
Publication Date : //
Viral vector particles derived from murine leukemia virus (MLV) mediate highly efficient stable gene transfer used in gene therapeutic approaches and in the generation of transgenic cell lines. However, the establishment of stable viral packaging cells (VPCs) is a time-consuming challenge. To overcome this limitation, we successfully generated novel Sleeping Beauty-derived transposon vectors entailing envelope and packaging expression cassettes as well as a transfer vector. Upon multiplexed transposition in human cells, VPC bulk populations yielding titers of over 1 × 10 transduction-competent vectors were established within three weeks. In contrast, conventional plasmid-based establishment of VPCs, conducted in parallel, took much longer and yielded significantly lower vector productivity and vector fitness. The generated MLV vectors decorated with the envelope proteins of ecotropic MLV PVC-211mc mediated efficient transduction of Chinese hamster ovary (CHO) cells. Cell susceptibility was further elevated upon recombinant expression of the murine ecotropic receptor mCAT employing a transposon vector.

Authors : Berg Karen, Schäfer Vanessa Nicole, Bartnicki Natalie, Eggenschwiler Reto, Cantz Tobias, Stitz Jörn,



(7) Overexpression of TEL-MN1 Fusion Enhances Resistance of HL-60 Cells to Idarubicin.[TOP]

Pubmed ID :30840968
Publication Date : //
The translocation t(12; 22) (p13;q12) is a recurrent but infrequent chromosome abnormality in human myeloid malignancies. To date, the role of TEL-MN1 fusion in leukemogenic process and drug resistance is still largely unknown.

Authors : Gong Shenglan, Guo Mengqiao, Tang Gusheng, Yang Jianmin, Qiu Huiying,



(8) Insights Into an Unexplored Component of the Mosquito Repeatome: Distribution and Variability of Viral Sequences Integrated Into the Genome of the Arboviral Vector .[TOP]

Pubmed ID :30809249
Publication Date : //
The Asian tiger mosquito is an invasive mosquito and a competent vector for public-health relevant arboviruses such as Chikungunya (), Dengue and Zika () viruses. Unexpectedly, the sequencing of the genome of this mosquito revealed an unusually high number of integrated sequences with similarities to non-retroviral RNA viruses of the and genera. These Non-retroviral Integrated RNA Virus Sequences (NIRVS) are enriched in piRNA clusters and coding sequences and have been proposed to constitute novel mosquito immune factors. However, given the abundance of NIRVS and their variable viral origin, their relative biological roles remain unexplored. Here we used an analytical approach that intersects computational, evolutionary and molecular methods to study the genomic landscape of mosquito NIRVS. We demonstrate that NIRVS are differentially distributed across mosquito genomes, with a core set of seemingly the oldest integrations with similarity to . Additionally, we compare the polymorphisms of NIRVS with respect to that of fast and slow-evolving genes within the genome. Overall, NIRVS appear to be less polymorphic than slow-evolving genes, with differences depending on whether they occur in intergenic regions or in piRNA clusters. Finally, two NIRVS that map within the coding sequences of genes annotated as RNA-dependent RNA polymerase and the nucleocapsid-encoding gene, respectively, are highly polymorphic and are expressed, suggesting exaptation possibly to enhance the mosquito's antiviral responses. These results greatly advance our understanding of the complexity of the mosquito repeatome and the biology of viral integrations in mosquito genomes.

Authors : Pischedda Elisa, Scolari Francesca, Valerio Federica, Carballar-Lejarazú Rebeca, Catapano Paolo Luigi, Waterhouse Robert M, Bonizzoni Mariangela,



(9) A Phase I-II Study Using Rexin-G Tumor-Targeted Retrovector Encoding a Dominant-Negative Cyclin G1 Inhibitor for Advanced Pancreatic Cancer.[TOP]

Pubmed ID :30705966
Publication Date : //
Rexin-G is a replication-incompetent retroviral vector displaying a cryptic -binding peptide for targeting abnormal Signature () proteins in tumors and encoding a dominant-negative human cyclin G1 construct. Herein we report on the safety and antitumor activity of escalating doses of Rexin-G in gemcitabine-refractory pancreatic adenocarcinoma, with one 10-year survivor. For the safety analysis (n = 20), treatment-related grade 1 adverse events included fatigue (n = 6), chills (n = 2), and headache (n = 1), with no organ damage and no DLT. No patient tested positive for vector-neutralizing antibodies, antibodies to gp70, replication-competent retrovirus (RCR), or vector integration into genomic DNA of peripheral blood lymphocytes (PBLs). For the efficacy analysis (n = 15), one patient achieved a complete response (CR), two patients had a partial response (PR), and 12 had stable disease (SD). Median progression-free survival (PFS) was 2.7, 4.0, and 5.6 months at doses 0-I, II, and III, respectively. Median overall survival (OS) and 1-year OS rate at dose 0-I were 4.3 months and 0%, and at dose II-III they were 9.2 months and 33.3%. To date, one patient is still alive with no evidence of cancer 10 years after the start of Rexin-G treatment. Taken together, these data suggest that Rexin-G, the first targeted gene delivery system, is uniquely safe and exhibits significant antitumor activity, for which the FDA granted fast-track designation.

Authors : Chawla Sant P, Bruckner Howard, Morse Michael A, Assudani Nupur, Hall Frederick L, Gordon Erlinda M,



(10) In vitro study of HAX1 gene therapy by retro viral transduction as a therapeutic target in severe congenital neutropenia.[TOP]

Pubmed ID :30698159
Publication Date : //
Severe congenital neutropenia (SCN) is a primary immunodeficiency disease in which a number of underlying gene defects are responsible for abnormalities in neutrophil development. The HCLS1-associated protein X1 (HAX1) mutation is associated with an autosomal-recessive form of SCN. Considering the potential of gene therapy approaches for the treatment of monogenic disorders, in this study we aimed to develop retroviral vectors expressing coding sequences (CDS) to be used for the removal of the genetic blockade in deficient hematopoietic cells. Following amplification of CDS with primers containing appropriate restriction sites, HAX1 CDS was cloned into an intermediate vector using TA-cloning. The sequence was transferred into a retroviral vector, followed by retroviral packaging in Plat-A cells. To show HAX1 protein expression, HEK293T cells were exposed to 10 multiplicity of infection (MOI) of retroviral particles and HAX1 expression was confirmed in these cells, using indirect intracellular flow cytometry. This vector was applied for in vitro transduction of hematopoietic stem cell with HAX1 mutation; after 11 days, cultured cells were analyzed for CD66acde and CD177 (neutrophil surface markers) expression. Increased neutrophil production in HAX1 viral vector-expressing hematopoietic cells was observed as compared to control vector transduced cells. Hence, according to the results, this type of therapy could be considered a potential treatment protocol for the disease.

Authors : Farajifard Hamid, Zavvar Mahdi, Rajaei Taraneh, Noorbakhsh Farshid, Nikougoftar-Zarif Mahin, Azadmanesh Kayhan, Kompani Farzad, Rezaei Nima,