Project

Functional correction of the f8 mutation in a hemophilia a patient's ipscs with crispr-cas9

Project (M.A., Biological Sciences (Stem Cell))--California State University, Sacramento, 2019.

Hemophilia A (HA) is an X-linked disorder that occurs in 1 in 5,000 male births. A lack of the blood clotting protein factor VIII (FVIII) in the blood plasma in HA patients due to mutations in the F8 gene results in excessive and prolonged hemorrhage. FVIII is primarily produced by the liver sinusoidal endothelial cells in a non-diseased person and is essential to the extrinsic pathway of the blood coagulation cascade. The severity of HA is delineated by the amount of circulating FVIII. Normal FVIII level is established as 100-200 ng/mL. Mild, moderate and severe hemophilia A is classified as 6-49%, 1-5% and <1% of normal FVIII plasma levels, respectively. Severe cases may represent up to 70% of all HA cases and are characterized by spontaneous bleeding in joints and soft tissue, arthropathy, and elevated risk of intracranial hemorrhage.
 Currently, the standard treatment for HA is FVIII replacement therapy. The short half-life of FVIII after intravenous administration entails multiple weekly infusions, creating challenges in maintaining sufficient hemostasis and impairing quality of life. Lifelong infusions also make treating HA cost prohibitive. These issues with the current treatment for HA necessitate better treatment options.
 The goal of this project was to generate vascularized liver organoids derived from HA patient induced pluripotent stem cells (iPSCs) as a vehicle for FVIII delivery upon transplantation. To determine if gene editing in our iPSCs is feasible, we inserted the reporter GFP-luciferase into the genomic safe harbor site AAVS1 in wild-type human iPSCs. These gene edited cells were subsequently differentiated into hepatic and endothelial progenitor cells, following previously established protocols. The differentiated cells maintained expression of the transgene GFP as seen under a fluorescent microscope. To test liver organoids for delivery of FVIII, we first generated liver organoids from the iPSC-derived hepatic and endothelial progenitor cells using a 3D culture system. Immunostaining showed that these liver organoids were positive for the hepatoblast marker α-fetoprotein and endothelial cell marker CD31. Our therapeutic approach involves transplantation of the liver organoids. We began optimization of liver organoid engraftment in an animal model. We generated liver organoids from early and later stage hepatic cells with the endothelial cells and transplanted these organoids onto the mesentery of the immune deficient FRG (Fah−/−/Rag2−/−/Il2rg −/−) mice. Our study showed that we can successfully target the AAVS1 site in iPSC’s genome for gene editing and that early stage hepatic cells are more suitable for engraftment than their late stage counterparts. Further studies will need to be done as proof-of-concept that liver organoids can be used as a cell therapy for HA.

Hemophilia A (HA) is an X-linked disorder that occurs in 1 in 5,000 male births. A lack of the blood clotting protein factor VIII (FVIII) in the blood plasma in HA patients due to mutations in the F8 gene results in excessive and prolonged hemorrhage. FVIII is primarily produced by the liver sinusoidal endothelial cells in a non-diseased person and is essential to the extrinsic pathway of the blood coagulation cascade. The severity of HA is delineated by the amount of circulating FVIII. Normal FVIII level is established as 100-200 ng/mL. Mild, moderate and severe hemophilia A is classified as 6-49%, 1-5% and <1% of normal FVIII plasma levels, respectively. Severe cases may represent up to 70% of all HA cases and are characterized by spontaneous bleeding in joints and soft tissue, arthropathy, and elevated risk of intracranial hemorrhage. Currently, the standard treatment for HA is FVIII replacement therapy. The short half-life of FVIII after intravenous administration entails multiple weekly infusions, creating challenges in maintaining sufficient hemostasis and impairing quality of life. Lifelong infusions also make treating HA cost prohibitive. These issues with the current treatment for HA necessitate better treatment options. The goal of this project was to generate vascularized liver organoids derived from HA patient induced pluripotent stem cells (iPSCs) as a vehicle for FVIII delivery upon transplantation. To determine if gene editing in our iPSCs is feasible, we inserted the reporter GFP-luciferase into the genomic safe harbor site AAVS1 in wild-type human iPSCs. These gene edited cells were subsequently differentiated into hepatic and endothelial progenitor cells, following previously established protocols. The differentiated cells maintained expression of the transgene GFP as seen under a fluorescent microscope. To test liver organoids for delivery of FVIII, we first generated liver organoids from the iPSC-derived hepatic and endothelial progenitor cells using a 3D culture system. Immunostaining showed that these liver organoids were positive for the hepatoblast marker α-fetoprotein and endothelial cell marker CD31. Our therapeutic approach involves transplantation of the liver organoids. We began optimization of liver organoid engraftment in an animal model. We generated liver organoids from early and later stage hepatic cells with the endothelial cells and transplanted these organoids onto the mesentery of the immune deficient FRG (Fah−/−/Rag2−/−/Il2rg −/−) mice. Our study showed that we can successfully target the AAVS1 site in iPSC’s genome for gene editing and that early stage hepatic cells are more suitable for engraftment than their late stage counterparts. Further studies will need to be done as proof-of-concept that liver organoids can be used as a cell therapy for HA.

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