875. Long-Term Transgene Expression and Survival by Genetically Modified Fibroblasts In Vitro

Aims: We aim to define parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors and optimize the expression cassettes to achieve robust and sustained expression in CNS. Background: Engineered, attenuated Herpes simplex virus (HSV) vectors are promising vehicles for gene delivery to the peripheral and central nervous systems. The virus latent promoter (LAP) is commonly used to drive exogenous gene expression; however, parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors have not been fully understood. Objective: This study aimed to construct attenuated HSV-1 vectors using the CRISPR-Cas9 system and examine the influence of transgene cassette construction and insertion site on transgene expression and vector safety. Method: In this study, we used a CRISPR-Cas9 system to accurately and efficiently edit attenuated HSV-1 strain 1716, and constructed two series of recombinant virus LMR and LMRx with different sets of gene cassettes insertion in Exon1(LAP2) and 2.0 kb intron downstream of LAP, respectively. The transgene expression and viral gene transcriptional kinetics were compared in in-vitro cell lines. The reporter gene expression and safety profiles of each vector were further evaluated in the mouse hippocampus gene transduction model. Result: The in-vitro cell line analysis indicated that the insertion of a gene expression cassette would disrupt virus gene transcription. Mouse hippocampus transducing analysis suggested that complete expression cassette insertion at 2.0 kb intron could achieve robust and longtime gene expression than the other constructs. Recombinants with gene expression cassettes lacked Poly (A), which induced significant neuronal inflammation due to persistent viral antigen expression and microglia activation. Conclusion: Our results indicated that the integrity of LAT transcripts was not necessary for the establishment of long-term latent expression. Exogenous strong promoters (like cBh promoter) could remain active during latency when placed in Exon1 or 2.0 Kb Intron of LAT locus, although their transcriptional activity declined with time. Consistent with previous research, the foreign gene expression would last much longer when the gene cassette was located downstream of Exon1, which suggested a role of LAP2 in maintaining promoter activity during latency. Besides, over-transcription of the downstream part of LAT may induce continuous activation of the attenuated vectors, suggesting an important role of LAT in maintaining viral reactivation potential.

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Long-Term Transgene Expression in Proliferating Cells Mediated by Episomally Maintained High-Capacity Adenovirus Vectors

Journal of Virology ◽

10.1128/jvi.78.1.9-22.2004 ◽

2004 ◽

Vol 78 (1) ◽

pp. 9-22 ◽

Cited By ~ 49

Author(s):

Florian Kreppel ◽

Stefan Kochanek

Keyword(s):

Transgene Expression ◽

High Capacity ◽

Target Cells ◽

Resting Cells ◽

Proliferating Cells ◽

Daughter Cells ◽

Adenovirus Vectors

ABSTRACT High-capacity “gutless” adenovirus vectors (HC-AdV) mediate long-term transgene expression in resting cells in vitro and in vivo because of low toxicity and immunogenicity. However, in proliferating cells, expression is transient since HC-AdV genomes do not possess elements that allow for replication and segregation of the replicated genomes to daughter cells. We developed a binary HC-AdV system that, under certain conditions, allows for significantly prolonged episomal maintenance of HC-AdV genomes in proliferating tissue culture cells, resulting in sustained transgene expression. After transduction of target cells the linear HC-AdV genomes were circularized by the DNA recombinase FLPe, which was expressed from the second HC-AdV. The oriP/EBNA-1 replication system derived from Epstein-Barr virus, as well as the human replication origin from the lamin B2 locus, were used as cis elements to test for replication of the 28-kb circular vector genomes with or without selective pressure. Depending on the system, up to 98% of the circularized genomes were replicated and segregated to daughter cells, as demonstrated by Southern assays and as confirmed by monitoring EGFP transgene expression. Surprisingly, in the absence of FLPe recombinase, a small but significant number of HC-AdV genomes spontaneously circularized after transduction of target cells. These circles, found to contain end-to-end joined adenovirus termini, replicated with increased efficiency compared to vectors circularized by FLPe. After further improvements, this HC-AdV system might be suitable for gene therapy applications requiring long-term transgene expression.

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Lentivirus-mediated gene transfer induces long-term transgene expression of BMP-2 in vitro and new bone formation in vivo

Molecular Therapy ◽

10.1016/j.ymthe.2004.10.019 ◽

2005 ◽

Vol 11 (3) ◽

pp. 390-398 ◽

Cited By ~ 102

Author(s):

Osamu Sugiyama ◽

Dong Sung An ◽

Sam P.K. Kung ◽

Brian T. Feeley ◽

Seth Gamradt ◽

...

Keyword(s):

Gene Transfer ◽

Bone Formation ◽

Transgene Expression ◽

New Bone Formation

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Searching for Promoters to Drive Stable and Long-Term Transgene Expression in Fibroblasts for Syngeneic Mouse Tumor Models

International Journal of Molecular Sciences ◽

10.3390/ijms21176098 ◽

2020 ◽

Vol 21 (17) ◽

pp. 6098

Author(s):

Dina V. Antonova ◽

Irina V. Alekseenko ◽

Anastasiia K. Siniushina ◽

Alexey I. Kuzmich ◽

Victor V. Pleshkan

Keyword(s):

Tumor Microenvironment ◽

Cancer Cells ◽

Cancer Progression ◽

Transgene Expression ◽

Close Contact ◽

Mouse Tumor ◽

Cancer Associated Fibroblasts

Tumor is a complex system of interactions between cancer cells and other cells of the tumor microenvironment. The cancer-associated fibroblasts (CAFs) of the tumor microenvironment remain in close contact with the cancer cells and play an important role in cancer progression. Genetically, CAFs are more stable than cancer cells, making them an attractive target for genetic modification in gene therapy. However, the efficiency of various promoters for transgene expression in fibroblasts is scarcely studied. We performed a comparative analysis of transgene long-term expression under the control of strong cytomegalovirus promoter (pCMV), constitutive cell promoter of the PCNA gene (pPCNA), and the potentially fibroblast-specific promoter of the IGFBP2 gene (pIGFBP2). In vitro expression of the transgene under the control of pCMV in fibroblasts was decreased soon after transduction, whereas the expression was more stable under the control of pIGFBP2 and pPCNA. The efficiency of transgene expression was higher under pPCNA than that under pIGFBP2. Additionally, in a mouse model, pPCNA provided more stable and increased transgene expression in fibroblasts as compared to that under pCMV. We conclude that PCNA promoter is the most efficient for long-term expression of transgenes in fibroblasts both in vitro and in vivo.

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Genetic Manipulation of Non-Falciparum Human Malaria Parasites

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.680460 ◽

2021 ◽

Vol 11 ◽

Author(s):

Taís Baruel Vieira ◽

Thafne Plastina Astro ◽

Roberto Rudge de Moraes Barros

Keyword(s):

Genetic Manipulation ◽

Rhesus Macaques ◽

Genetically Modified ◽

Resistance Mechanisms ◽

The Other ◽

Malaria Parasites ◽

Human Malaria

The development of genetic manipulation of Plasmodium falciparum in the 1980s was key to study malaria biology. Genetically modified parasites have been used to study several aspects of the disease, such as red blood cell invasion, drug resistance mechanisms, gametocyte development and mosquito transmission. However, biological and genetic differences between P. falciparum and the other human malaria parasites make P. falciparum a poor model to study different species. The lack of robust systems of long-term in vitro culture of P. vivax and the other human malaria parasites lagged the genetic manipulation of these species. Here we review the efforts to generate genetically modified non-falciparum human malaria parasites, in vivo and in vitro. Using in vivo models – infection of non-human primates such as rhesus macaques and saimiri monkeys – researchers were able to generate transgenic lines of P. knowlesi, P. cynomolgi, and P. vivax. The development of long-term in vitro culture of P. knowlesi in the 2000’s, using rhesus and human red blood cells, created a platform to genetically manipulate non-falciparum malaria parasites. Recently, the use of CRISPR/Cas9 technology to genome edit P. knowlesi provides another tool to non-falciparum malaria research, extending the possibilities and allowing researchers to study different aspects of the biology of these parasites and understand the differences between these species and P. falciparum.

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A Cre-lox approach for transient transgene expression in neural precursor cells and long-term tracking of their progeny in vitro and in vivo

BMC Developmental Biology ◽

10.1186/1471-213x-7-45 ◽

2007 ◽

Vol 7 (1) ◽

pp. 45 ◽

Cited By ~ 7

Author(s):

Cédric G Geoffroy ◽

Olivier Raineteau

Keyword(s):

Transgene Expression ◽

Precursor Cells ◽

Neural Precursor Cells ◽

Neural Precursor

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Evidence for keratinocyte stem cells in vitro: Long term engraftment and persistence of transgene expression from retrovirus-transduced keratinocytes

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.95.8.4356 ◽

1998 ◽

Vol 95 (8) ◽

pp. 4356-4361 ◽

Cited By ~ 157

Author(s):

T. M. Kolodka ◽

J. A. Garlick ◽

L. B. Taichman

Keyword(s):

Stem Cells ◽

Transgene Expression ◽

Keratinocyte Stem Cells

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BacMam Virus Transduced Cardiomyoblasts Can Be Used for Myocardial Transplantation Using AP-PEG-A Microcapsules: Molecular Cloning, Preparation, andIn VitroAnalysis

Journal of Biomedicine and Biotechnology ◽

10.1155/2010/858094 ◽

2010 ◽

Vol 2010 ◽

pp. 1-12 ◽

Cited By ~ 3

Author(s):

Arghya Paul ◽

Afshan Afsar Khan ◽

Dominique Shum-Tim ◽

Satya Prakash

Keyword(s):

Transgene Expression ◽

Genetically Modified ◽

Limiting Factors ◽

Efficient Delivery ◽

Osmotic Stability ◽

Massive Cell Death ◽

Poly Ethylene ◽

Optimized Conditions ◽

Massive Cell

The potential of genetically modified cardiomyoblasts in treating damaged myocardium is well known. However, efficient delivery of these cells is of major concern during treatment. The limiting factors are the massive cell death that occurs soon after their intramyocardial transplantation into the beating heart. To address these problems, we generated recombinant baculoviruses (BacMam viruses) which efficiently transduced cardiomyoblast cells under optimized conditions. These genetically modified cells were then protected in a new polymeric microcapsule using poly-ethylene-glycol (PEG), alginate, and poly-L-lysine (PLL) polymers for efficient delivery. Results showed that microcapsules maintain cell viability and support cell proliferation for at least 30 days. The capsules exhibit strong immunoprotective potential and have high mechanical and osmotic stability with more than 70% intact capsules. The encased transduced cells showed a rapid transgene expression inside the capsule for at least 15 days. However, preclinical studies are needed to further explore its long-term functional benefits.

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Ultrastructural investigations of MDL 19,660-induced effects on the canine platelet and megakaryocyte

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100159412 ◽

1990 ◽

Vol 48 (3) ◽

pp. 374-375

Author(s):

D.E. Loudy ◽

J. Sprinkle-Cavallo ◽

J.T. Yarrington ◽

F.Y. Thompson ◽

J.P. Gibson

Keyword(s):

Antidepressant Drug ◽

Beagle Dogs ◽

Long Term Effects ◽

Short Term ◽

Platelet Counts ◽

Toxicological Studies ◽

Induced Aggregation ◽

Internal Architecture

Previous short term toxicological studies of one to two weeks duration have demonstrated that MDL 19,660 (5-(4-chlorophenyl)-2,4-dihydro-2,4-dimethyl-3Hl, 2,4-triazole-3-thione), an antidepressant drug, causes a dose-related thrombocytopenia in dogs. Platelet counts started to decline after two days of dosing with 30 mg/kg/day and continued to decrease to their lowest levels by 5-7 days. The loss in platelets was primarily of the small discoid subpopulation. In vitro studies have also indicated that MDL 19,660: does not spontaneously aggregate canine platelets and has moderate antiaggregating properties by inhibiting ADP-induced aggregation. The objectives of the present investigation of MDL 19,660 were to evaluate ultrastructurally long term effects on platelet internal architecture and changes in subpopulations of platelets and megakaryocytes.Nine male and nine female beagle dogs were divided equally into three groups and were administered orally 0, 15, or 30 mg/kg/day of MDL 19,660 for three months. Compared to a control platelet range of 353,000- 452,000/μl, a doserelated thrombocytopenia reached a maximum severity of an average of 135,000/μl for the 15 mg/kg/day dogs after two weeks and 81,000/μl for the 30 mg/kg/day dogs after one week.

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