title: Correcting the PiZ defect in the alpha 1-antitrypsin gene of human cells by targeted homologous recombination.
abstract: BACKGROUND
  Approximately 1% of people in North America carry the PiZ defect in the gene encoding the serum protein alpha 1-antitrypsin (AAT). Homozygotes are subject to early onset emphysema and liver disease. Here we demonstrate a means of correcting this gene defect, a point mutation in chromosome 14, by targeted homologous recombination.
  
  
  METHODS
  To target recombination to the AAT locus in human cells we use a normal complementary DNA cleaved once approximately 400 base pairs upstream of the PiZ mutation site in exon V. After transfection, targeting is monitored using a polymerase chain reaction assay devised to uniquely amplify successful homologous recombinants.
  
  
  RESULTS
  When excercised on human PiZZ GM2522 fibroblasts, targeting replaces exon V of the endogenous gene, containing the PiZ mutation, with the exon V counterpart of a normal complementary DNA, simultaneously displacing the mutated exon V away from its promoter. By reversing the targeting strategy and using human PiM hepatoma cells, we have been able to supplant a normal copy of the AAT gene with a mutated PiZ version. This has allowed us to culture human HepG2 cells expressing the pathologic phenotype of the PiZ defect. Immunocytologic staining demonstrates the presence of inclusions in the liver cells due to aggregation of the PiZ isoform encoded by the recombined AAT gene under control of the endogenous gene promoter.
  
  
  CONCLUSIONS
  (a) Targeting to the AAT locus is observed, using the methods we have developed, at rates that render feasible the selection of targeted cells and isolation of clones bearing a corrected AAT gene. (b) Correction of the exon V PiZ mutation alone, regardless of an accompanying exon III mutation, is sufficient to prevent aggregation of the PiZ protein in human liver cells. (c) Targeted homologous recombination offers advantages in potential gene therapy to correct AAT defects.
authors.affiliation: Department of Pathology, Mount Sinai School of Medicine, City University of New York, New York.
authors.forename: E
authors.initials: E
authors.lastname: Savransky
authors.forename: P
authors.initials: P
authors.lastname: Hytiroglou
authors.forename: N
authors.initials: N
authors.lastname: Harpaz
authors.forename: S N
authors.initials: SN
authors.lastname: Thung
authors.forename: E M
authors.initials: EM
authors.lastname: Johnson
mesh_terms: D000818:Animals; D001483:Base Sequence; D006528:Carcinoma, Hepatocellular; D002460:Cell Line; D002522:Chlorocebus aethiops; D017931:DNA Primers; D005091:Exons; D005347:Fibroblasts; D006720:Homozygote; D006801:Humans; D008099:Liver / Q000378:metabolism; D008113:Liver Neoplasms; D008969:Molecular Sequence Data; D009154:Mutation; D016133:Polymerase Chain Reaction; D011995:Recombination, Genetic; D014162:Transfection; D014407:Tumor Cells, Cultured; D000515:alpha 1-Antitrypsin / Q000096:biosynthesis / Q000235:genetics*; D019896:alpha 1-Antitrypsin Deficiency
chemical_list: D017931:DNA Primers; C043392:SERPINA1 protein, human; D000515:alpha 1-Antitrypsin