249. High Levels of Protein Expression in the Liver Could Be Detrimental for Long-Term Transgene Expression

Previous studies suggest that parental obesity may adversely impact long-term metabolic health of the offspring. We tested the hypothesis that parental (paternal + maternal) obesity impairs cardiac function in the offspring early in life. Within 1-3 days after weaning, offspring from obese rats fed a high fat diet (HFD-Offs) and age-matched offspring from lean rats (ND-Offs) were submitted to echocardiography and cardiac catheterization for assessment of pressure-volume relationships. Then, hearts were digested and isolated cardiomyocytes were used to determine contractile function, calcium transients, proteins related to calcium signaling, and mitochondrial bioenergetics. Female and male HFD-Offs were heavier (72±2 and 61±4 vs 57±2 and 49 ±1 g), hyperglycemic (112±8 and 115±12 vs 92±10 and 96±8 mg/dL), with higher plasma insulin and leptin concentrations compared to female and male ND-Offs. Compared to male controls, male HFD-Offs exhibited similar systolic function but impaired diastolic function as indicated by increased IVRT (22±1 vs. 17±1), E/E' ratio (29±2 vs. 23±1) and Tau (5.7±0.2 vs. 4.8±0.2). The impaired diastolic function was associated with reduced resting free Ca2+ levels and phospholamban protein expression, increased activated matrix metalloproteinase 2 and reduced SIRT3 protein expression, mitochondrial ATP reserve and ATP-linked respiration. These results indicate that male and female Offs from obese parents have multiple metabolic abnormalities early in life (1-3 days after weaning) and that male, but not female, Offs have impaired diastolic dysfunction as well as reductions in cardiac SIRT3, resting free Ca+2 levels and mitochondrial biogenesis.

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Construction and optimization of herpes simplex virus vectors for central nervous system gene delivery based on CRISPR/Cas9-mediated genome editing

Current Gene Therapy ◽

10.2174/1566523219666210618154326 ◽

2021 ◽

Vol 21 ◽

Author(s):

Xinwei Huang ◽

Xiuqing Li ◽

Lijuan Yang ◽

Pengfei Wang ◽

Jingyuan Yan ◽

...

Keyword(s):

Gene Expression ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Transgene Expression ◽

Mouse Hippocampus ◽

Simplex Virus ◽

Hsv 1

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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Discovery of increased epidermal DNAH10 expression after regeneration of dermis in a randomized with-in person trial — reflections on psoriatic inflammation

Scientific Reports ◽

10.1038/s41598-019-53874-z ◽

2019 ◽

Vol 9 (1) ◽

Author(s):

Heli Lagus ◽

Mariliis Klaas ◽

Susanna Juteau ◽

Outi Elomaa ◽

Juha Kere ◽

...

Keyword(s):

Protein Expression ◽

Psoriatic Skin ◽

Dermal Matrix ◽

Thickness Skin ◽

Dermal Regeneration ◽

Unaffected Skin ◽

One Year ◽

To Receive

AbstractBecause molecular memories of past inflammatory events can persist in epidermal cells, we evaluated the long-term epidermal protein expression landscapes after dermal regeneration and in psoriatic inflammation. We first characterized the effects of two dermal regeneration strategies on transplants of indicator split-thickness skin grafts (STSGs) in ten adult patients with deep burns covering more than 20% of their body surface area. After fascial excision, three adjacent areas within the wound were randomized to receive a permanent dermal matrix, a temporary granulation-tissue-inducing dressing or no dermal component as control. Control areas were covered with STSG immediately, and treated areas after two-weeks of dermis formation. Epidermis-dermis-targeted proteomics of one-year-follow-up samples were performed for protein expression profiling. Epidermal expression of axonemal dynein heavy chain 10 (DNAH10) was increased 20-fold in samples having had regenerating dermis vs control. Given the dermal inflammatory component found in our dermal regeneration samples as well as in early psoriatic lesions, we hypothesized that DNAH10 protein expression also would be affected in psoriatic skin samples. We discovered increased DNAH10 expression in inflammatory lesions when compared to unaffected skin. Our results associate DNAH10 expression with cell proliferation and inflammation as well as with the epidermal memory resulting from the previous regenerative signals of dermis. This study (ISRCTN14499986) was funded by the Finnish Ministry of Defense and by government subsidies for medical research.

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Long-term multilineage expression in peripheral blood from a Moloney murine leukemia virus vector after serial transplantation of transduced bone marrow cells

Blood ◽

10.1182/blood.v95.3.829.003k18_829_836 ◽

2000 ◽

Vol 95 (3) ◽

pp. 829-836 ◽

Cited By ~ 8

Author(s):

Timothy W. Austin ◽

Suzan Salimi ◽

Gabor Veres ◽

Franck Morel ◽

Heini Ilves ◽

...

Keyword(s):

Bone Marrow ◽

Peripheral Blood ◽

Blood Cells ◽

Transgene Expression ◽

Bone Marrow Cells ◽

Leukemia Virus ◽

Moloney Murine Leukemia Virus ◽

Marrow Cells

Using a mouse bone marrow transplantation model, the authors evaluated a Moloney murine leukemia virus (MMLV)-based vector encoding 2 anti-human immunodeficiency virus genes for long-term expression in blood cells. The vector also encoded the human nerve growth factor receptor (NGFR) to serve as a cell-surface marker for in vivo tracking of transduced cells. NGFR+ cells were detected in blood leukocytes of all mice (n=16; range 16%-45%) 4 to 5 weeks after transplantation and were repeatedly detected in blood erythrocytes, platelets, monocytes, granulocytes, T cells, and B cells of all mice for up to 8 months. Transgene expression in individual mice was not blocked in the various cell lineages of the peripheral blood and spleen, in several stages of T-cell maturation in the thymus, or in the Lin−/loSca-1+ and c-kit+Sca-1+ subsets of bone marrow cells highly enriched for long-term multilineage-reconstituting activity. Serial transplantation of purified NGFR+c-kit+Sca-1+bone marrow cells resulted in the reconstitution of multilineage hematopoiesis by donor type NGFR+ cells in all engrafted mice. The authors concluded that MMLV-based vectors were capable of efficient and sustained transgene expression in multiple lineages of peripheral blood cells and hematopoietic organs and in hematopoietic stem cell (HSC) populations. Differentiation of engrafting HSC to peripheral blood cells is not necessarily associated with dramatic suppression of retroviral gene expression. In light of earlier studies showing that vector elements other than the long-terminal repeat enhancer, promoter, and primer binding site can have an impact on long-term transgene expression, these findings accentuate the importance of empirically testing retroviral vectors to determine lasting in vivo expression.

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