scholarly journals 15. A Novel Fast Production Pipeline for High Capacity Adenoviral Vectors to Deliver All Components of CRISPR/Cas9 System for Somatic Gene Editing Using One Single Viral Vector with Multiple Guide RNAs

2016 ◽  
Vol 24 ◽  
pp. S8
Author(s):  
Eric Ehrke-Schulz ◽  
Theo Leitner ◽  
Stephan Dávid ◽  
Jing Liu ◽  
Maren Schiwon ◽  
...  
Keyword(s):  
Gene Editing ◽  
Viral Vector ◽  
High Capacity ◽  
Adenoviral Vectors ◽  
Guide Rnas ◽  
Production Pipeline ◽  
Somatic Gene

scholarly journals Native ribonucleases process sgRNA transcripts to create catalytic Cas9/sgRNA complexes in planta

2020 ◽  
Author(s):  
Will B Cody ◽  
Herman B. Scholthof
Keyword(s):  
Catalytic Activity ◽  
Gene Editing ◽  
Viral Vector ◽  
Fusion Transcript ◽  
In Vitro Assays ◽  
In Planta ◽  
Guide Rnas ◽  
Catalytically Active

The current CRISPR/Cas9 gene editing dogma for single guide RNAs (sgRNA) delivery is based on the premise that 5′ and 3′ nucleotide overhangs negate Cas9/sgRNA catalytic activity in vivo. This has led to engineering strategies designed to either avoid or remove extraneous nucleotides on the 5′ and 3′ termini. Previously, we used a Tobacco mosaic virus viral vector to express both GFP and a sgRNA from a single virus-derived mRNA in Nicotiana benthamiana. This vector yielded high levels of GFP and catalytically active sgRNAs. Here, in an effort to understand the biochemical interactions of this result, we used in vitro assays to demonstrate that nucleotide overhangs 5′, but not 3′, proximal to the sgRNA do in fact inactivate Cas9 catalytic activity at the specified target site. Next we showed that in planta sgRNAs bound to Cas9 are devoid of the expected 5′ overhangs transcribed by the virus. Furthermore, when a plant nuclear promoter was used for expression of the GFP-sgRNA fusion transcript it also produced indels when delivered with Cas9. These results reveal that 5′ "auto-processing" of progenitor sgRNAs occurs natively in plants. Towards a possible mechanism for the perceived "auto-processing", we found, using in vitro generated RNAs and those isolated from plants, that the 5′ to 3′ exoribonuclease XRN1 can degrade elongated progenitor sgRNAs whereas the mature sgRNA end-products are resistant. Comparisons with other studies suggest that sgRNA "auto-processing" may be a phenomenon not unique to plants, but other eukaryotes as well.

scholarly journals Danio Rerio as Model Organism for Adenoviral Vector Evaluation

Genes ◽  
2019 ◽  
Vol 10 (12) ◽  
pp. 1053
Author(s):  
Paola Gulías ◽  
Jorge Guerra-Varela ◽  
Manuela Gonzalez-Aparicio ◽  
Ana Ricobaraza ◽  
Africa Vales ◽  
...  
Keyword(s):  
Danio Rerio ◽  
Viral Vector ◽  
Early Stage ◽  
High Capacity ◽  
Model Organism ◽  
Adenoviral Vectors ◽  
Regulatory Agencies ◽  
In Vivo Model ◽  
Efficient Alternative

Viral vector use is wide-spread in the field of gene therapy, with new clinical trials starting every year for different human pathologies and a growing number of agents being approved by regulatory agencies. However, preclinical testing is long and expensive, especially during the early stages of development. Nowadays, the model organism par excellence is the mouse (Mus musculus), and there are few investigations in which alternative models are used. Here, we assess the possibility of using zebrafish (Danio rerio) as an in vivo model for adenoviral vectors. We describe how E1/E3-deleted adenoviral vectors achieve efficient transduction when they are administered to zebrafish embryos via intracranial injection. In addition, helper-dependent (high-capacity) adenoviral vectors allow sustained transgene expression in this organism. Taking into account the wide repertoire of genetically modified zebrafish lines, the ethical aspects, and the affordability of this model, we conclude that zebrafish could be an efficient alternative for the early-stage preclinical evaluation of adenoviral vectors.

scholarly journals CRISPR/Cas9 Flexes Its Muscles: In Vivo Somatic Gene Editing for Muscular Dystrophy

2016 ◽  
Vol 24 (3) ◽  
pp. 414-416 ◽  
Author(s):  
Thierry VandenDriessche ◽  
Marinee K Chuah
Keyword(s):  
Muscular Dystrophy ◽  
Gene Editing ◽  
Somatic Gene

Production and Quality Control of High-Capacity Adenoviral Vectors

Cell Biology ◽  
2006 ◽  
pp. 445-456 ◽  
Author(s):  
G SCHIEDNER ◽  
F KREPPEL ◽  
S KOCHANEK
Keyword(s):  
Quality Control ◽  
High Capacity ◽  
Adenoviral Vectors

70 XBP1 DYSREGULATION BY CRISPR/Cas9-MEDIATED GENE EDITING DURING PORCINE EMBRYO EARLY DEVELOPMENT

2017 ◽  
Vol 29 (1) ◽  
pp. 142
Author(s):  
K. Gutierrez ◽  
W. G. Glanzner ◽  
N. Dicks ◽  
R. C. Bohrer ◽  
L. G. Currin ◽  
...  
Keyword(s):  
Er Stress ◽  
Gene Editing ◽  
Early Embryo ◽  
Early Embryo Development ◽  
Unfolded Protein ◽  
Guide Rnas ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Er Homeostasis

Early developing embryos are very sensitive to their developmental milieu. For instance, variations in temperature, pH, or culture media composition can trigger endoplasmic reticulum (ER) stress. Endoplasmic reticulum stress has been shown to reduce early embryo development and embryo quality. In response to ER stress, embryos activate coping mechanisms, such as the unfolded protein response, to re-establish ER homeostasis. The X box binding protein (XBP1) is one of the main transducers of the unfolded protein response. Under ER stress, XBP1 mRNA is unconventionally spliced by IRE1α to yield its activated isoform (XBP1s), which allows expression of genes involved in protein folding, transport, and degradation. XBP1s has been detected in oocytes and early stage embryos of different species, including Drosophila, Xenopus, zebrafish, mice, and pigs, suggesting an important role during early embryo development. In this study, we used the CRISPR/Cas9 gene editing technology to investigate the effect of XBP1 dysregulation during development of porcine embryos in vitro. Pig zygotes were produced by intracytoplasmic sperm injection using in vitro-matured oocytes. Treatments consisted of (a) Cas9 mRNA (Cas9) + 1 single guide RNAs targeting XBP1 gene region 1 (sgRNA-1); (b) Cas9 + 1 single guide RNAs targeting XBP1 gene region 2 (sgRNA-2); (c) Cas9 + sgRNA-1 + sgRNA-2; (d) Cas9 alone; and (e) sgRNA-1 + sgRNA-2. After injection, embryos were cultured in vitro for 5 to 7 days to assess development and cell numbers. Experiments were repeated 5 or more times, and data were analysed by ANOVA and means compared using Student’s t-test or Tukey–Kramer Honestly Significant Difference test. Embryo cleavage was similar between the groups (a = 59.8 ± 4.9%, b = 58.8 ± 5.3%, c = 68.86 ± 2.2%, d = 66.4 ± 5.9%, and e = 70.10 ± 1.9%), but development to the blastocyst stage was substantially reduced (P < 0.05) in the groups injected with Cas9 + sgRNAs (a = 18 ± 4.5%, b = 16 ± 1.5%, and c = 5.3 ± 2.8%) compared with controls (d = 33.7 ± 6.2% and e = 31.4 ± 1.2%). Moreover, we observed that only 22.7% of the embryos treated with Cas9 + sgRNA-1 + sgRNA-2 were able to develop beyond 8-cell stage compared with 62.5% in the control group injected with Cas9 alone. These findings suggest that XBP1 activity is required for maintenance of ER homeostasis and development of porcine embryos beyond the main period of embryo genome activation.

scholarly journals Differential localization and functional role of calsequestrin in growing and differentiated myoblasts.

1995 ◽  
Vol 128 (3) ◽  
pp. 341-354 ◽  
Author(s):  
M Raichman ◽  
M C Panzeri ◽  
E Clementi ◽  
P Papazafiri ◽  
M Eckley ◽  
...  
Keyword(s):  
Functional Role ◽  
Heterologous Protein ◽  
Viral Vector ◽  
Binding Protein ◽  
High Capacity ◽  
Peptide Hormone ◽  
L6 Cells ◽  
Er Retention ◽  
Before And After

Calsequestrin (CSQ) is the low affinity, high capacity Ca(2+)-binding protein concentrated within specialized areas of the muscle fiber sarcoplasmic reticulum (a part of the ER) where it is believed to buffer large amounts of Ca2+. Upon activation of intracellular channels this Ca2+ pool is released, giving rise to the [Ca2+]i increases that sustain contraction. In order to investigate the ER retention and the functional role of the protein, L6 rat myoblasts were infected with a viral vector with or without the cDNA of chicken CSQ, and stable clones were investigated before and after differentiation to myotubes. In the undifferentiated L6 cells, expression of considerable amounts of heterologous CSQ occurred with no major changes of other ER components. Ca2+ release from the ER, induced by the peptide hormone vasopressin, remained however unchanged, and the same occurred when other treatments were given in sequence to deplete the ER and other intracellular stores: with the Ca2+ pump blocker, thapsigargin; and with the Ca2+ ionophore, ionomycin, followed by the Na+/H+ ionophore, monensin. The lack of effect of CSQ expression on the vasopressin-induced [Ca2+]i responses was explained by immunocytochemistry showing the heterologous protein to be localized not in the ER but in large vacuoles of acidic content, positive also for the lysosomal enzyme, cathepsin D, corresponding to a lysosomal subpopulation. After differentiation, all L6 cells expressed small amounts of homologous CSQ. In the infected cells the heterologous protein progressively decreased, yet the [Ca2+]i responses to vasopressin were now larger with respect to both control and undifferentiated cells. This change correlated with the drop of the vacuoles and with the accumulation of CSQ within the ER lumen, where a clustered distribution was observed as recently shown in developing muscle fibers. These results provide direct evidence for the contribution of CSQ, when appropriately retained, to the Ca2+ capacity of the rapidly exchanging, ER-located Ca2+ stores; and for the existence of specific mechanism(s) (that in L6 cells develop in the course of differentiation) for the ER retention of the protein. In the growing L6 myoblasts the Ca(2+)-binding protein appears in contrast to travel along the exocytic pathway, down to post-Golgi, lysosome-related vacuoles which, based on the lack of [Ca2+]i response to ionomycin-monensin, appear to be incompetent for Ca2+ accumulation.

scholarly journals Herpes Simplex Virus Type 1 Thymidine Kinase Sequence Fused to the lacZ Gene Increases Levels of β-Galactosidase Activity per Genome of High-Capacity but Not First-Generation Adenoviral Vectors In Vitro and In Vivo

2008 ◽  
Vol 83 (4) ◽  
pp. 2004-2010 ◽  
Author(s):  
M. Puntel ◽  
R. J. Barrett ◽  
S. Mondkar ◽  
V. Saxena ◽  
K. M. Kroeger ◽  
...  
Keyword(s):  
Herpes Simplex Virus ◽  
Herpes Simplex ◽  
Transgene Expression ◽  
First Generation ◽  
High Capacity ◽  
Adenoviral Vectors ◽  
Simplex Virus

ABSTRACT Increased transgene expression per vector genome is an important goal in the optimization of viral vectors for gene therapy. Herein we demonstrate that herpes simplex virus type 1 (HSV1) thymidine kinase (TK) gene sequences (1,131 bp) fused to the 3′ end of lacZ increase transgene expression from high-capacity adenoviral vectors (HCAd), but not from first-generation (Ad) vectors. The woodchuck hepatitis virus posttranscriptional regulatory element (WPRE), in contrast, increased transgene expression levels from Ad but not HCAd vectors. The differential activity of the HSV1 TK gene and WPRE sequences was detected both in vitro and in vivo and suggests potentially different mechanisms of action or the interaction of these elements with vector genomic sequences.

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