1. Fig. 1. Identification of an shRNA that rescues Fah deficiency.
Identification of an shRNA that rescues Fah deficiency. (A) The tyrosine catabolic pathway. Genetic deficiency of Fah causes hereditary tyrosinemia type 1 (HT1) due to accumulation of FAA in hepatocytes. The disease can be treated pharmacologically (NTBC) or by shRNA knockdown of the genes required for making FAA. CEHPOBA (4-[(2-carboxyethyl)-hydroxyphosphinyl]-3-oxobutyrate) inhibits FAH and causes accumulation of FAA. TAT, tyrosine aminotransferase; MAI, maleylacetoacetate isomerase. (B) Lentiviral construct. shRNAs targeting Hpd, Hgd, or Tat were expressed from a U6 promoter. Vector expresses a green fluorescent protein (GFP) reporter. LTR, long terminal repeat; UbC, ubiquitin promoter. (C) Experimental timeline. Neonatal Fah−/− mice were injected with shTat, shHpd, and shHgd lentiviruses or a vector devoid of any shRNA (control) and kept on NTBC until weaning. NTBC was then withdrawn to permit liver injury and selection of resistant hepatocytes. (D) Mouse weights during selection starting at 5 weeks of age. Gray bars represent periods of intermittent NTBC therapy. Only the control and shHgd and shTat cohorts were given NTBC after week 6. Data are means − SD (downward tick) (n = 4 to 6). (E) Mice were injected with a nonselectable vector (control) or shHpd. Liver tissues were stained for the reporter GFP and for α-fetoprotein (AFP), which is highly expressed in mutant Fah−/− hepatocytes. Yellow arrow denotes the absence of AFP within a selected nodule compared with AFP-positive surrounding tissue (black arrows). Scale bars, 100 μm. (F) Polymerase chain reaction (PCR) amplification of genomic liver DNA with primers flanking the lentiviral-shRNA sequence (two primer sets).
Sean Nygaard et al., Sci Transl Med 2016;8:342ra79
Published by AAAS