Title of Abstract
Hereditary tyrosinemia type 1 (HT1) is an autosomal recessive disorder caused by deficiency of fumarylacetoacetate hydrolase (FAH). Our work has previously shown that ex vivo hepatocyte-directed gene therapy using an integrating lentiviral vector to replace the defective Fah gene can cure liver disease in small and large animal models of HT1. In this study, we hypothesized that ex vivo hepatocyte-directed gene editing using CRISPR/Cas9 could be used to correct a mouse model of HT1 in which a single point mutation results in loss of FAH function. To achieve high transduction efficiencies of the target hepatocytes, we utilized a lentiviral vector (LV) to deliver both the Streptococcus pyogenes Cas9 nuclease and target guide RNA, and an adeno-associated viral vector to deliver a 1.2kb homology template (AAV-HT). Cells were isolated from Fah-/- mice and cultured in the presence of LV and AAV vectors. Transduction of cells with LV-Cas9 induced significant indels at the target locus, and correction of the point mutation in Fah-/- cells using AAV-HT was completely depended on LV-Cas9. Next, hepatocytes transduced ex vivo by LV-Cas9 and AAV-HR were then transplanted into syngeneic Fah-/- mice that had undergone two-thirds partial hepatectomy or sham hepatectomy. Recipient animals were cycled on/off the protective drug NTBC to provide a selective advantage for corrected FAH+ cells to proliferate. Interestingly, a significant improvement was observed in weight stability off NTBC between animals that received partial hepatectomy or not. After six months, mice were euthanized and thorough biochemical and histological examinations performed. All transplanted mice became weight stable off NTBC. Biochemical markers of liver injury were significantly improved over non-transplanted control animals. Histological examination of mice revealed normal tissue architecture and immunohistochemistry showed robust repopulation of recipient animals with FAH+ cells, with increased corrected cells in mice that had undergone partial hepatectomy. In summary, this is the first report of ex vivo hepatocyte-directed gene repair using CRISPR/Cas9 to demonstrate curative therapy in an animal model of liver disease.
Since coming to Mayo Clinic in 2012, my research has focused exclusively on liver-directed regenerative medicine. Specifically, my research interest has been on developing novel gene and cell therapies for metabolic liver diseases. During this time, I have been able to expand and develop a unique skill set that includes in vivo and ex vivo gene manipulation with viral vectors, stem cell and differentiated cell transplantation, and noninvasive cell imaging in small and large animals using nuclear imaging.
CaitlinVanLith; Rebekah Guthman; Clara Nicolas; Kari Allen; DongJin Joo; Scott Nyberg; Joseph Lillegard; Raymond Hickey.
All Author Affiliations
Mayo Clinic, Rochester, Minnesota.