scholarly journals Heterologous expression of adenovirus E3-gp19K in an E1a-deleted adenovirus vector inhibits MHC I expression in vitro, but does not prolong transgene expression in vivo

Gene Therapy ◽  
1997 ◽  
Vol 4 (4) ◽  
pp. 351-360 ◽  
Author(s):  
DB Schowalter ◽  
JC Tubb ◽  
M Liu ◽  
CB Wilson ◽  
MA Kay
Keyword(s):  
Heterologous Expression ◽  
Transgene Expression ◽  
Adenovirus Vector ◽  
Mhc I

scholarly journals Polyinosinic acid enhances delivery of adenovirus vectors in vivo by preventing sequestration in liver macrophages

2008 ◽  
Vol 89 (5) ◽  
pp. 1097-1105 ◽  
Author(s):  
Hidde J. Haisma ◽  
Jan A. A. M. Kamps ◽  
Gera K. Kamps ◽  
Josee A. Plantinga ◽  
Marianne G. Rots ◽  
...  
Keyword(s):  
Gene Therapy ◽  
Kupffer Cells ◽  
Transgene Expression ◽  
Liver Toxicity ◽  
Viral Vector ◽  
Adenovirus Vector ◽  
Liver Macrophages ◽  
Polyinosinic Acid

Adenovirus is among the preferred vectors for gene therapy because of its superior in vivo gene-transfer efficiency. However, upon systemic administration, adenovirus is preferentially sequestered by the liver, resulting in reduced adenovirus-mediated transgene expression in targeted tissues. In the liver, Kupffer cells are responsible for adenovirus degradation and contribute to the inflammatory response. As scavenger receptors present on Kupffer cells are responsible for the elimination of blood-borne pathogens, we investigated the possible implication of these receptors in the clearance of the adenovirus vector. Polyinosinic acid [poly(I)], a scavenger receptor A ligand, was analysed for its capability to inhibit adenovirus uptake specifically in macrophages. In in vitro studies, the addition of poly(I) before virus infection resulted in a specific inhibition of adenovirus-induced gene expression in a J774 macrophage cell line and in primary Kupffer cells. In in vivo experiments, pre-administration of poly(I) caused a 10-fold transient increase in the number of adenovirus particles circulating in the blood. As a consequence, transgene expression levels measured in different tissues were enhanced (by 5- to 15-fold) compared with those in animals that did not receive poly(I). Finally, necrosis of Kupffer cells, which normally occurs as a consequence of systemic adenovirus administration, was prevented by the use of poly(I). No toxicity, as measured by liver-enzyme levels, was observed after poly(I) treatment. From our data, we conclude that poly(I) can prevent adenovirus sequestration by liver macrophages. These results imply that, by inhibiting adenovirus uptake by Kupffer cells, it is possible to reduce the dose of the viral vector to diminish the liver-toxicity effect and to improve the level of transgene expression in target tissues. In systemic gene-therapy applications, this will have great impact on the development of targeted adenoviral vectors.

scholarly journals Construction of adenovirus vectors simultaneously expressing four multiplex, double-nicking guide RNAs of CRISPR/Cas9 and in vivo genome editing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Tomoko Nakanishi ◽  
Aya Maekawa ◽  
Mariko Suzuki ◽  
Hirotaka Tabata ◽  
Kumiko Sato ◽  
...  
Keyword(s):  
Target Gene ◽  
Animal Experiments ◽  
Adenovirus Vector ◽  
Mouse Embryonic Fibroblast ◽  
Newborn Mouse ◽  
Guide Rnas ◽  
One Step ◽  
Target Effect

AbstractSimultaneous expression of multiplex guide RNAs (gRNAs) is valuable for knockout of multiple genes and also for effective disruption of a gene by introducing multiple deletions. We developed a method of Tetraplex-guide Tandem for construction of cosmids containing four and eight multiplex gRNA-expressing units in one step utilizing lambda in vitro packaging. Using this method, we produced an adenovirus vector (AdV) containing four multiplex-gRNA units for two double-nicking sets. Unexpectedly, the AdV could stably be amplified to the scale sufficient for animal experiments with no detectable lack of the multiplex units. When the AdV containing gRNAs targeting the H2-Aa gene and an AdV expressing Cas9 nickase were mixed and doubly infected to mouse embryonic fibroblast cells, deletions were observed in more than 80% of the target gene even using double-nicking strategy. Indels were also detected in about 20% of the target gene at two sites in newborn mouse liver cells by intravenous injection. Interestingly, when one double-nicking site was disrupted, the other was simultaneously disrupted, implying that two genes in the same cell may simultaneously be disrupted in the AdV system. The AdVs expressing four multiplex gRNAs could offer simultaneous knockout of four genes or two genes by double-nicking cleavages with low off-target effect.

scholarly journals Deciphering the tumor-specific immunopeptidome in vivo with genetically engineered mouse models

2021 ◽  
Author(s):  
Tyler Jacks ◽  
Alex Jaeger ◽  
Lauren Stopfer ◽  
Emma Sanders ◽  
Demi Sandel ◽  
...  
Keyword(s):  
Transcript Abundance ◽  
Genetically Engineered ◽  
Ductal Adenocarcinoma ◽  
Affinity Tag ◽  
Class I Mhc ◽  
Mhc I ◽  
Novel Approach ◽  
In Vitro Investigation

Abstract Effective immunosurveillance of cancer requires the presentation of peptide antigens on major histocompatibility complex Class I (MHC-I). Recent developments in proteomics have improved the identification of peptides that are naturally presented by MHC-I, collectively known as the “immunopeptidome”. Current approaches to profile tumor immunopeptidomes have been limited to in vitro investigation, which fails to capture the in vivo repertoire of MHC-I peptides, or bulk tumor lysates, which are obscured by the lack of tumor-specific MHC-I isolation. To overcome these limitations, we report here the engineering of a Cre recombinase-inducible affinity tag into the endogenous mouse MHC-I gene and targeting of this allele to the KrasLSL-G12D/+; p53fl/fl (KP) mouse model (KP; KbStrep). This novel approach has allowed us to isolate tumor-specific MHC-I peptides from autochthonous pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LUAD) in vivo. With this powerful analytical tool, we were able to profile the evolution of the LUAD immunopeptidome through tumor progression and show that in vivo MHC-I presentation is shaped by post-translational mechanisms. We also uncovered novel, putative LUAD tumor associated antigens (TAAs). Many peptides that were recurrently presented in vivo exhibited very low expression of the cognate mRNA, provoking reconsideration of antigen prediction pipelines that triage peptides according to transcript abundance. Beyond cancer, the KbStrep allele is compatible with a broad range of Cre-driver lines to explore antigen presentation in vivo in the pursuit of understanding basic immunology, infectious disease, and autoimmunity.

Abstract 124: Parthenogenetic Stem Cell-derived Cardiomyocytes Express Major Histocompatibility Complex-I only after Inflammatory Stimulation

2014 ◽  
Vol 115 (suppl_1) ◽  
Author(s):  
Satish Galla ◽  
Michael Didie ◽  
Vijayakumar Muppala ◽  
Ralf Dressel ◽  
Wolfram Hubertus Zimmermann
Keyword(s):  
Major Histocompatibility Complex ◽  
Heart Muscle ◽  
Immunological Response ◽  
Type I ◽  
Major Histocompatibility ◽  
Adrenergic Stimulation ◽  
Mhc I ◽  
Histocompatibility Complex

Background: Pluripotent parthenogenetic stem cells (PSCs) can be directed towards a cardiac fate and utilized in tissue engineered heart repair. In vivo applications of tissue engineered allografts are compromised by expression of mismatching major histocompatibility complex proteins (MHC; encoded in the murine H2 locus). Here we investigated whether PSC-derived cardiomyocytes (CM) express MHC-I. Methods: Mouse PSCs (A3-line from B6D2F1 strain with haploidentical H2K d -locus) expressing a CM-specific neomycin-resistance and GFP were differentiated and purified for CM by addition of G418 (85% purity by FACS for actinin). To simulate heart muscle biology in vitro, we made use of engineered heart muscle (EHM) constructed from PSC-derived CM (75%), growth-inhibited murine embryonic fibroblasts (MEF (25%); NMRI mice), and collagen type I. MHC class-I H2K d (MHC-I) expression was assessed on CM and Non myocytes before EHM assembly and from enzymatically digested EHMs (cultured for 10 days) by FACS. Interferon gamma (IFNγ) was added for 48 h to stimulate MHC-I expression. As a reference, we investigated MHC-I expression in CM from neonatal mice and adult mouse hearts by FACS and by immunofluorescence staining. Results: EHM showed a positive ionotropic response to beta-adrenergic stimulation which could be reduced by muscarinergic stimulation. A3-CM, in contrast to Non myocytes, showed negligible expression of MHC-I (1±0.5% vs. 60±10% positive cells; n=3). EHM culture did not change MHC-I expression in CM. IFNγ treatment resulted in a marked increase of MHC-I-expression in CM monolayer culture (40±6%; n=3) and in EHM (30±8%; n=3). For comparison, 30% (n=2) neonatal CM expressed MHC-I while MHC-I was not detectable in adult CM. Conclusion: PSC-derived CM show a similarly low expression of MHC-I as adult CM and respond with MHC-I upregulation to IFNγ stimulation. This suggests a mature immunological response in PSC-CM with important implications for in vivo applications, i.e., MHC-I matching will likely be a prerequisite for successful allografting of PSC-EHM.

scholarly journals Persistent transgene expression in mouse liver following in vivo gene transfer with a ΔE1/ΔE4 adenovirus vector

Gene Therapy ◽  
1997 ◽  
Vol 4 (5) ◽  
pp. 393-400 ◽  
Author(s):  
Q Wang ◽  
G Greenburg ◽  
D Bunch ◽  
D Farson ◽  
MH Finer
Keyword(s):  
Gene Transfer ◽  
Mouse Liver ◽  
Transgene Expression ◽  
Adenovirus Vector ◽  
In Vivo Gene Transfer

scholarly journals Design of Epitope-Based Peptide Vaccine against Pseudomonas aeruginosa Fructose Bisphosphate Aldolase Protein Using Immunoinformatics

2020 ◽  
Vol 2020 ◽  
pp. 1-11
Author(s):  
Mustafa Elhag ◽  
Ruaa Mohamed Alaagib ◽  
Nagla Mohamed Ahmed ◽  
Mustafa Abubaker ◽  
Esraa Musa Haroun ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Peptide Vaccine ◽  
Multidrug Resistant ◽  
Fructose Bisphosphate ◽  
Mhc I ◽  
Mhc Ii ◽  
Fructose Bisphosphate Aldolase ◽  
Hospital Acquired

Pseudomonas aeruginosa is a common pathogen that is responsible for serious hospital-acquired infections, ventilator-associated pneumonia, and various sepsis syndromes. Also, it is a multidrug-resistant pathogen recognized for its ubiquity and its intrinsically advanced antibiotic-resistant mechanisms. It usually affects immunocompromised individuals but can also infect immunocompetent individuals. There is no vaccine against it available till now. This study predicts an effective epitope-based vaccine against fructose bisphosphate aldolase (FBA) of Pseudomonas aeruginosa using immunoinformatics tools. The protein sequences were obtained from NCBI, and prediction tests were undertaken to analyze possible epitopes for B and T cells. Three B cell epitopes passed the antigenicity, accessibility, and hydrophilicity tests. Six MHC I epitopes were found to be promising, while four MHC II epitopes were found promising from the result set. Nineteen epitopes were shared between MHC I and II results. For the population coverage, the epitopes covered 95.62% worldwide excluding certain MHC II alleles. We recommend in vivo and in vitro studies to prove its effectiveness.

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