Browsing by Subject "genome editing"

Now showing 1 - 4 of 4

Open Access
Bezafibrate Enhances AAV Vector-Mediated Genome Editing in Glycogen Storage Disease Type Ia.
(Molecular therapy. Methods & clinical development, 2019-06) Kang, Hye-Ri; Waskowicz, Lauren; Seifts, Andrea M; Landau, Dustin J; Young, Sarah P; Koeberl, Dwight D
Glycogen storage disease type Ia (GSD Ia) is a rare inherited disease caused by mutations in the glucose-6-phosphatase (G6Pase) catalytic subunit gene (G6PC). Absence of G6Pase causes life-threatening hypoglycemia and long-term complications because of the accumulations of metabolic intermediates. Bezafibrate, a pan-peroxisome proliferator-activated receptor (PPAR) agonist, was administered in the context of genome editing with a zinc-finger nuclease-containing vector (AAV-ZFN) and a G6Pase donor vector (AAV-RoG6P). Bezafibrate treatment increased survival and decreased liver size (liver/body mass, p < 0.05) in combination with genome editing. Blood glucose has higher (p < 0.05) after 4 h of fasting, and liver glycogen accumulation (p < 0.05) was lower in association with higher G6Pase activity (p < 0.05). Furthermore, bezafibrate-treated mice had increased numbers of G6PC transgenes (p < 0.05) and higher ZFN activity (p < 0.01) in the liver compared with controls. PPAR-α expression was increased and PPAR-γ expression was decreased in bezafibrate-treated mice. Therefore, bezafibrate improved hepatocellular abnormalities and increased the transduction efficiency of AAV vector-mediated genome editing in liver, whereas higher expression of G6Pase corrected molecular signaling in GSD Ia. Taken together, bezafibrate shows promise as a drug for increasing AAV vector-mediated genome editing.
Open Access
Editing the Neuronal Genome: a CRISPR View of Chromatin Regulation in Neuronal Development, Function, and Plasticity.
(Yale J Biol Med, 2016-12) Yang, Marty G; West, Anne E
The dynamic orchestration of gene expression is crucial for the proper differentiation, function, and adaptation of cells. In the brain, transcriptional regulation underlies the incredible diversity of neuronal cell types and contributes to the ability of neurons to adapt their function to the environment. Recently, novel methods for genome and epigenome editing have begun to revolutionize our understanding of gene regulatory mechanisms. In particular, the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has proven to be a particularly accessible and adaptable technique for genome engineering. Here, we review the use of CRISPR/Cas9 in neurobiology and discuss how these studies have advanced understanding of nervous system development and plasticity. We cover four especially salient applications of CRISPR/Cas9: testing the consequences of enhancer mutations, tagging genes and gene products for visualization in live cells, directly activating or repressing enhancers in vivo, and manipulating the epigenome. In each case, we summarize findings from recent studies and discuss evolving adaptations of the method.
Open Access
Examination of Endogenous Rotund Expression and Function in Developing Drosophila Olfactory System Using CRISPR-Cas9-Mediated Protein Tagging.
(G3 (Bethesda), 2015-10-23) Li, Qingyun; Barish, Scott; Okuwa, Sumie; Volkan, Pelin C
The zinc-finger protein Rotund (Rn) plays a critical role in controlling the development of the fly olfactory system. However, little is known about its molecular function in vivo. Here, we added protein tags to the rn locus using CRISPR-Cas9 technology in Drosophila to investigate its subcellular localization and the genes that it regulates . We previously used a reporter construct to show that rn is expressed in a subset of olfactory receptor neuron (ORN) precursors and it is required for the diversification of ORN fates. Here, we show that tagged endogenous Rn protein is functional based on the analysis of ORN phenotypes. Using this method, we also mapped the expression pattern of the endogenous isoform-specific tags in vivo with increased precision. Comparison of the Rn expression pattern from this study with previously published results using GAL4 reporters showed that Rn is mainly present in early steps in antennal disc patterning, but not in pupal stages when ORNs are born. Finally, using chromatin immunoprecipitation, we showed a direct binding of Rotund to a previously identified regulatory element upstream of the bric-a-brac gene locus in the developing antennal disc.
Open Access
Pathogenesis of Hepatic Tumors following Gene Therapy in Murine and Canine Models of Glycogen Storage Disease.
(Molecular therapy. Methods & clinical development, 2019-12) Kang, Hye-Ri; Gjorgjieva, Monika; Smith, Stephanie N; Brooks, Elizabeth D; Chen, Zelin; Burgess, Shawn M; Chandler, Randy J; Waskowicz, Lauren R; Grady, Kylie M; Li, Songtao; Mithieux, Gilles; Venditti, Charles P; Rajas, Fabienne; Koeberl, Dwight D
Glycogen storage disease type Ia (GSD Ia) is caused by mutations in the glucose-6-phosphatase (G6Pase) catalytic subunit gene (G6PC). GSD Ia complications include hepatocellular adenomas (HCA) with a risk for hepatocellular carcinoma (HCC) formation. Genome editing with adeno-associated virus (AAV) vectors containing a zinc-finger nuclease (ZFN) and a G6PC donor transgene was evaluated in adult mice with GSD Ia. Although mouse livers expressed G6Pase, HCA and HCC occurred following AAV vector administration. Interestingly, vector genomes were almost undetectable in the tumors but remained relatively high in adjacent liver (p < 0.01). G6Pase activity was decreased in tumors, in comparison with adjacent liver (p < 0.01). Furthermore, AAV-G6Pase vector-treated dogs with GSD Ia developed HCC with lower G6Pase activity (p < 0.01) in comparison with adjacent liver. AAV integration and tumor marker analysis in mice revealed that tumors arose from the underlying disorder, not from vector administration. Similarly to human GSD Ia-related HCA and HCC, mouse and dog tumors did not express elevated α-fetoprotein. Taken together, these results suggest that AAV-mediated gene therapy not only corrects hepatic G6Pase deficiency, but also has potential to suppress HCA and HCC in the GSD Ia liver.