Biosafety in Handling Gene Transfer Vectors

Swindle, Scott

doi:10.1002/cphg.54

Cited by 3 publications

(7 citation statements)

References 68 publications

(52 reference statements)

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“…Wildtype (WT) , GATA6 +/- , GATA6 -/- , and GATA6 R456G/R456G hiPSCs were processed for differentiation into cardiomyocytes (hiPSC-CMs) by modulation of the Wnt signaling pathway and subsequent metabolic selection via glucose deprivation ( Sharma et al, 2018b ). This protocol yields hiPSC-CMs that express first and second heart field genes ( Zhang et al, 2019 ).…”

Section: Resultsmentioning

confidence: 99%

“…The TNNT2-GFP iPSC line was generated by homology-directed repair, using a plasmid with the endogenous TNNT2 sequence connected to a GSSSS linker region, attached to the GFP gene, and the entire construct was flanked by 500 bp homology arms. PGP1 iPSCs were transfected as described ( Sharma et al, 2018b ) to obtain homozygous integration of the GFP tag in both TNNT2 alleles. Edited clones were selected using puromycin and expanded and genotyped as previously described ( Sharma et al, 2018b ) and differentiated into cardiomyocytes as described ( Sharma et al, 2018a ).…”

Section: Methodsmentioning

confidence: 99%

“…PGP1 iPSCs were transfected as described ( Sharma et al, 2018b ) to obtain homozygous integration of the GFP tag in both TNNT2 alleles. Edited clones were selected using puromycin and expanded and genotyped as previously described ( Sharma et al, 2018b ) and differentiated into cardiomyocytes as described ( Sharma et al, 2018a ).…”

Section: Methodsmentioning

confidence: 99%

“…GATA6 R456G/R456G hiPSCs were generated by homology-directed repair using a 2 µg plasmid expressing Cas9 (PX459v2 from Addgene) that was co-transfected with 2 µg plasmid expressing guide RNA (provided in Key Resource Table) and 2 µg single-stranded oligonucleotide (HDR template) using a stem cell nucleofector kit (Amaxa). Edited clones were selected using puromycin and expanded and genotyped as previously described ( Sharma et al, 2018b ) and differentiated into cardiomyocytes as described ( Sharma et al, 2018a ).…”

Section: Methodsmentioning

confidence: 99%

“…The hiPSC-CMs were generated from GATA6 mutant hiPSCs using a small-molecule mediated differentiation approach that modulates Wnt signaling ( Sharma et al, 2018a ). Cells began beating at approximately day 7 post-differentiation.…”

Section: Methodsmentioning

confidence: 99%

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GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm

Sharma

Wasson

Willcox

et al. 2020

eLife

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Damaging GATA6 variants cause cardiac outflow tract defects, sometimes with pancreatic and diaphragmic malformations. To define molecular mechanisms for these diverse developmental defects, we studied transcriptional and epigenetic responses to GATA6 loss of function and missense variants during cardiomyocyte differentiation of isogenic human induced pluripotent stem cells. We show that GATA6 is a pioneer factor in cardiac development, regulating SMYD1 that activates HAND2, and KDR that with HAND2 orchestrates outflow tract formation. Loss of function variants perturbed cardiac genes and also endoderm lineage genes that direct PDX1 expression and pancreatic development. Remarkably, an exon 4 GATA6 missense variant, highly associated with extra-cardiac malformations, caused ectopic pioneer activities, profoundly diminishing GATA4, FOXA1/2 and PDX1 expression and increasing normal retinoic acid signaling that promotes diaphragm development. These aberrant epigenetic and transcriptional signatures illuminate the molecular mechanisms for cardiovascular malformations, pancreas and diaphragm dysgenesis that arise in patients with distinct GATA6 variants.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 3 more Smart Citations

GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm

Sharma

Wasson

Willcox

et al. 2020

eLife

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The Horizon of Gene Therapy in Modern Medicine: Advances and Challenges

Arjmand

Larijani

Hosseini

et al. 2019

Advances in Experimental Medicine and Biology

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Viral Shedding in Mice following Intravenous Adenovirus Injection: Impact on Biosafety Classification

LaRocca

Jacobsen

Inoko

et al. 2023

Viruses

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There have been numerous advances in gene therapy and oncolytic virotherapy in recent years, especially with respect to cutting-edge animal models to test these novel therapeutics. With all of these advances, it is important to understand the biosafety risks of testing these vectors in animals. We performed adenovirus-based viral shedding studies in murine models to ascertain when it is appropriate to downgrade the animals from Biosafety Level (BSL) 2 to BSL 1 for experimental handling and transport. We utilized intravenous injections of a replication-competent adenovirus and analyzed viral shedding via the collection of buccal and dermal swabs from each animal, in addition to obtaining urine and stool samples. The adenovirus hexon copy number was determined by qPCR, and plaque formation was analyzed to assess the biologic activity of viral particles. Our results demonstrate that after 72 h following viral inoculation, there is no significant quantity of biologically active virus shedding from the animals. This observation suggests that on day 4 following adenovirus injection, mice can be safely downgraded to BSL 1 for the remainder of the experiment with no concern for hazardous exposure to laboratory personnel.

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Biosafety in Handling Gene Transfer Vectors

Abstract: This unit is devoted to safety issues that must be considered when generating and working with the most common vectors under development for human gene therapy today. © 2018 by John Wiley & Sons, Inc.

Cited by 3 publications

References 68 publications

GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm

GATA6 mutations in hiPSCs inform mechanisms for maldevelopment of the heart, pancreas, and diaphragm

The Horizon of Gene Therapy in Modern Medicine: Advances and Challenges

Viral Shedding in Mice following Intravenous Adenovirus Injection: Impact on Biosafety Classification

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