Browsing by Author "Bissig, Karl-Dimiter"

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    Differentiation of spontaneously contracting cardiomyocytes from non-virally reprogrammed human amniotic fluid stem cells
    (Public Library of Science, 2017) Velasquez-Mao, Aaron J.; Tsao, Christopher J.M.; Monroe, Madeline N.; Legras, Xavier; Bissig-Choisat, Beatrice; Bissig, Karl-Dimiter; Ruano, Rodrigo; Jacot, Jeffrey G.; Bioengineering
    Congenital heart defects are the most common birth defect. The limiting factor in tissue engineering repair strategies is an autologous source of functional cardiomyocytes. Amniotic fluid contains an ideal cell source for prenatal harvest and use in correction of congenital heart defects. This study aims to investigate the potential of amniotic fluid-derived stem cells (AFSC) to undergo non-viral reprogramming into induced pluripotent stem cells (iPSC) followed by growth-factor-free differentiation into functional cardiomyocytes. AFSC from human second trimester amniotic fluid were transfected by non-viral vesicle fusion with modified mRNA of OCT4, KLF4, SOX2, LIN28, cMYC and nuclear GFP over 18 days, then differentiated using inhibitors of GSK3 followed 48 hours later by inhibition of WNT. AFSC-derived iPSC had high expression of OCT4, NANOG, TRA-1-60, and TRA-1-81 after 18 days of mRNA transfection and formed teratomas containing mesodermal, ectodermal, and endodermal germ layers in immunodeficient mice. By Day 30 of cardiomyocyte differentiation, cells contracted spontaneously, expressed connexin 43 and β-myosin heavy chain organized in sarcomeric banding patterns, expressed cardiac troponin T and β-myosin heavy chain, showed upregulation of NKX2.5, ISL-1 and cardiac troponin T with downregulation of POU5F1, and displayed calcium and voltage transients similar to those in developing cardiomyocytes. These results demonstrate that cells from human amniotic fluid can be differentiated through a pluripotent state into functional cardiomyocytes.
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    Somatic genome editing with CRISPR/Cas9 generates and corrects a metabolic disease
    (Springer Nature, 2017) Jarrett, Kelsey E.; Lee, Ciaran M.; Yeh, Yi-Hsien; Hsu, Rachel H.; Gupta, Rajat; Zhang, Min; Rodriguez, Perla J.; Lee, Chang Seok; Gillard, Baiba K.; Bissig, Karl-Dimiter; Pownall, Henry J.; Martin, James F.; Bao, Gang; Lagor, William R.; Bioengineering
    Germline manipulation using CRISPR/Cas9 genome editing has dramatically accelerated the generation of new mouse models. Nonetheless, many metabolic disease models still depend upon laborious germline targeting, and are further complicated by the need to avoid developmental phenotypes. We sought to address these experimental limitations by generating somatic mutations in the adult liver using CRISPR/Cas9, as a new strategy to model metabolic disorders. As proof-of-principle, we targeted the low-density lipoprotein receptor (Ldlr), which when deleted, leads to severe hypercholesterolemia and atherosclerosis. Here we show that hepatic disruption of Ldlr with AAV-CRISPR results in severe hypercholesterolemia and atherosclerosis. We further demonstrate that co-disruption of Apob, whose germline loss is embryonically lethal, completely prevented disease through compensatory inhibition of hepatic LDL production. This new concept of metabolic disease modeling by somatic genome editing could be applied to many other systemic as well as liver-restricted disorders which are difficult to study by germline manipulation.
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    Targeting the Apoa1 locus for liver-directed gene therapy
    (Cell Press, 2021) De Giorgi, Marco; Li, Ang; Hurley, Ayrea; Barzi, Mercedes; Doerfler, Alexandria M.; Cherayil, Nikitha A.; Smith, Harrison E.; Brown, Jonathan D.; Lin, Charles Y.; Bissig, Karl-Dimiter; Bao, Gang; Lagor, William R.; Bioengineering
    Clinical application of somatic genome editing requires therapeutics that are generalizable to a broad range of patients. Targeted insertion of promoterless transgenes can ensure that edits are permanent and broadly applicable while minimizing risks of off-target integration. In the liver, the Albumin (Alb) locus is currently the only well-characterized site for promoterless transgene insertion. Here, we target the Apoa1 locus with adeno-associated viral (AAV) delivery of CRISPR-Cas9 and achieve rates of 6% to 16% of targeted hepatocytes, with no evidence of toxicity. We further show that the endogenous Apoa1 promoter can drive robust and sustained expression of therapeutic proteins, such as apolipoprotein E (APOE), dramatically reducing plasma lipids in a model of hypercholesterolemia. Finally, we demonstrate that Apoa1-targeted fumarylacetoacetate hydrolase (FAH) can correct and rescue the severe metabolic liver disease hereditary tyrosinemia type I. In summary, we identify and validate Apoa1 as a novel integration site that supports durable transgene expression in the liver for gene therapy applications.