We present plans for the construction of a retroviral vector for delivery of an appropriately regulated human alpha-1-antitrypsin (A1Pi) cDNA specifically to liver cells. Targeting will be accomplished by use of the liver-specific asialoglycoprotein (ASGP) receptor which mediates efficient uptake of a number of ASGP by recognition of properly spaced terminal galactose residues. Three approaches will be tested to modify the Moloney murine leukemia virus (MoMLV) envelope protein to contain appropriate ligands for interaction with the ASGP receptor: 1) treatment of MoMLV with neuraminidase to expose terminal galactose residues, 2) reductive lactosamination of MoMLV to increase the number of terminal galactose residues, and 3) engineering hybrid envelope proteins that contain signals known to be recognized by this receptor. A human hepatoma cell line, Hep G2, which expresses the ASGP receptor will be used to measure internalization and establish infectivity of the modified viruses. To achieve appropriately regulated expression, we shall ligate cloned cDNA coding for human A1Pi with previously characterized (or putative) liver-specific regulatory elements (promoters and enhancers) from albumin, A1Pi, or alpha- fetoprotein genes. After characterizing expression following DNA transfection into various cell types, the most promising constructs will be incorporated into various cell types, the most promising constructs will be incorporated into a deleted MLV genome (retaining LTRs and packaging signal) so that the regulatory elements should function internally to direct liver- specific A1Pi expression. Replacement of the LTR enhancer by a liver-specific enhancer will also be investigated. Recombinant virus will initially be produced in standard packaging cell lines and A1Pi expression will be characterized after infection of hepatic and other cell types in vitro. Once adequate control of expression has been achieved, we shall generate A1Pi-transducing retrovirus containing the above ASGP receptor-tropic envelope protein, to package the recombinant retroviral genome. This virus will then be used to infect nice undergoing liver regeneration in an effort to demonstrate stable hepatic integration and expression of A1Pi cDNA.
| Maxwell, I H; Brown, J L; Maxwell, F (1991) Inefficiency of expression of luciferase reporter from transfected murine leukaemia proviral DNA may be partially overcome by providing a strong polyadenylation signal. J Gen Virol 72 ( Pt 7):1721-4 |
