Abstract
Familial hypercholesterolemia is an inherited disease in humans that is caused by a defect in the receptor for low density lipoproteins (LDLR). The existence of an animal model for this disease, the Watanabe heritable hyperlipidemic (WHHL) rabbit, makes it an attractive candidate for developing new therapies that involve gene transfer into liver. As a first step toward the development of these therapies, we report the use of retrovirus-mediated gene transfer to correct the genetic defect in hepatocytes isolated from WHHL rabbits. A series of retroviral vectors that express the gene for human LDLR were constructed, each differing in the transcriptional elements used to drive LDLR expression. Helper-free amphotropic virus stocks representing each construct were then used to infect primary cultures of hepatocytes that were isolated from newborn WHHL rabbits. The efficiency of transduction, as measured by Southern analysis of integrated proviral sequences, ranged from 20% to 100%. Expression of human LDLR was analyzed by blot hybridization analysis of total cellular RNA and by biochemical and in situ analyses of transduced cultures for receptor function. The vector in which the expression of LDLR was driven by the viral long terminal repeat sequence produced the greatest quantity of LDLR RNA and protein in WHHL hepatocytes; LDLR activity approached normal levels in these cultures.
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Selected References
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- Bilheimer D. W., Goldstein J. L., Grundy S. M., Starzl T. E., Brown M. S. Liver transplantation to provide low-density-lipoprotein receptors and lower plasma cholesterol in a child with homozygous familial hypercholesterolemia. N Engl J Med. 1984 Dec 27;311(26):1658–1664. doi: 10.1056/NEJM198412273112603. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Chirgwin J. M., Przybyla A. E., MacDonald R. J., Rutter W. J. Isolation of biologically active ribonucleic acid from sources enriched in ribonuclease. Biochemistry. 1979 Nov 27;18(24):5294–5299. doi: 10.1021/bi00591a005. [DOI] [PubMed] [Google Scholar]
- Clayton D. F., Darnell J. E., Jr Changes in liver-specific compared to common gene transcription during primary culture of mouse hepatocytes. Mol Cell Biol. 1983 Sep;3(9):1552–1561. doi: 10.1128/mcb.3.9.1552. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Cone R. D., Weber-Benarous A., Baorto D., Mulligan R. C. Regulated expression of a complete human beta-globin gene encoded by a transmissible retrovirus vector. Mol Cell Biol. 1987 Feb;7(2):887–897. doi: 10.1128/mcb.7.2.887. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Demetriou A. A., Whiting J. F., Feldman D., Levenson S. M., Chowdhury N. R., Moscioni A. D., Kram M., Chowdhury J. R. Replacement of liver function in rats by transplantation of microcarrier-attached hepatocytes. Science. 1986 Sep 12;233(4769):1190–1192. doi: 10.1126/science.2426782. [DOI] [PubMed] [Google Scholar]
- Dzierzak E. A., Papayannopoulou T., Mulligan R. C. Lineage-specific expression of a human beta-globin gene in murine bone marrow transplant recipients reconstituted with retrovirus-transduced stem cells. Nature. 1988 Jan 7;331(6151):35–41. doi: 10.1038/331035a0. [DOI] [PubMed] [Google Scholar]
- Enat R., Jefferson D. M., Ruiz-Opazo N., Gatmaitan Z., Leinwand L. A., Reid L. M. Hepatocyte proliferation in vitro: its dependence on the use of serum-free hormonally defined medium and substrata of extracellular matrix. Proc Natl Acad Sci U S A. 1984 Mar;81(5):1411–1415. doi: 10.1073/pnas.81.5.1411. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Feinberg A. P., Vogelstein B. A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal Biochem. 1983 Jul 1;132(1):6–13. doi: 10.1016/0003-2697(83)90418-9. [DOI] [PubMed] [Google Scholar]
- Goldstein J. L., Basu S. K., Brown M. S. Receptor-mediated endocytosis of low-density lipoprotein in cultured cells. Methods Enzymol. 1983;98:241–260. doi: 10.1016/0076-6879(83)98152-1. [DOI] [PubMed] [Google Scholar]
- Gunning P., Ponte P., Okayama H., Engel J., Blau H., Kedes L. Isolation and characterization of full-length cDNA clones for human alpha-, beta-, and gamma-actin mRNAs: skeletal but not cytoplasmic actins have an amino-terminal cysteine that is subsequently removed. Mol Cell Biol. 1983 May;3(5):787–795. doi: 10.1128/mcb.3.5.787. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hanly S. M., Bleecker G. C., Heintz N. Identification of promoter elements necessary for transcriptional regulation of a human histone H4 gene in vitro. Mol Cell Biol. 1985 Feb;5(2):380–389. doi: 10.1128/mcb.5.2.380. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Korman A. J., Frantz J. D., Strominger J. L., Mulligan R. C. Expression of human class II major histocompatibility complex antigens using retrovirus vectors. Proc Natl Acad Sci U S A. 1987 Apr;84(8):2150–2154. doi: 10.1073/pnas.84.8.2150. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Kost T. A., Theodorakis N., Hughes S. H. The nucleotide sequence of the chick cytoplasmic beta-actin gene. Nucleic Acids Res. 1983 Dec 10;11(23):8287–8301. doi: 10.1093/nar/11.23.8287. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ledley F. D., Darlington G. J., Hahn T., Woo S. L. Retroviral gene transfer into primary hepatocytes: implications for genetic therapy of liver-specific functions. Proc Natl Acad Sci U S A. 1987 Aug;84(15):5335–5339. doi: 10.1073/pnas.84.15.5335. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McKnight S. L., Gavis E. R., Kingsbury R., Axel R. Analysis of transcriptional regulatory signals of the HSV thymidine kinase gene: identification of an upstream control region. Cell. 1981 Aug;25(2):385–398. doi: 10.1016/0092-8674(81)90057-x. [DOI] [PubMed] [Google Scholar]
- Mito M., Ebata H., Kusano M., Onishi T., Saito T., Sakamoto S. Morphology and function of isolated hepatocytes transplanted into rat spleen. Transplantation. 1979 Dec;28(6):499–505. doi: 10.1097/00007890-197912000-00013. [DOI] [PubMed] [Google Scholar]
- Orkin S. H. Molecular genetics and potential gene therapy. Clin Immunol Immunopathol. 1986 Jul;40(1):151–156. doi: 10.1016/0090-1229(86)90080-2. [DOI] [PubMed] [Google Scholar]
- Pitas R. E., Innerarity T. L., Weinstein J. N., Mahley R. W. Acetoacetylated lipoproteins used to distinguish fibroblasts from macrophages in vitro by fluorescence microscopy. Arteriosclerosis. 1981 May-Jun;1(3):177–185. doi: 10.1161/01.atv.1.3.177. [DOI] [PubMed] [Google Scholar]
- Price J., Turner D., Cepko C. Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc Natl Acad Sci U S A. 1987 Jan;84(1):156–160. doi: 10.1073/pnas.84.1.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Price J., Turner D., Cepko C. Lineage analysis in the vertebrate nervous system by retrovirus-mediated gene transfer. Proc Natl Acad Sci U S A. 1987 Jan;84(1):156–160. doi: 10.1073/pnas.84.1.156. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Southern E. M. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J Mol Biol. 1975 Nov 5;98(3):503–517. doi: 10.1016/s0022-2836(75)80083-0. [DOI] [PubMed] [Google Scholar]
- Stacey A., Arbuthnott C., Kollek R., Coggins L., Ostertag W. Comparison of myeloproliferative sarcoma virus with Moloney murine sarcoma virus variants by nucleotide sequencing and heteroduplex analysis. J Virol. 1984 Jun;50(3):725–732. doi: 10.1128/jvi.50.3.725-732.1984. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Watanabe Y. Serial inbreeding of rabbits with hereditary hyperlipidemia (WHHL-rabbit). Atherosclerosis. 1980 Jun;36(2):261–268. doi: 10.1016/0021-9150(80)90234-8. [DOI] [PubMed] [Google Scholar]
- Wilson J. M., Jefferson D. M., Chowdhury J. R., Novikoff P. M., Johnston D. E., Mulligan R. C. Retrovirus-mediated transduction of adult hepatocytes. Proc Natl Acad Sci U S A. 1988 May;85(9):3014–3018. doi: 10.1073/pnas.85.9.3014. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamamoto T., Bishop R. W., Brown M. S., Goldstein J. L., Russell D. W. Deletion in cysteine-rich region of LDL receptor impedes transport to cell surface in WHHL rabbit. Science. 1986 Jun 6;232(4755):1230–1237. doi: 10.1126/science.3010466. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Yamamoto T., Davis C. G., Brown M. S., Schneider W. J., Casey M. L., Goldstein J. L., Russell D. W. The human LDL receptor: a cysteine-rich protein with multiple Alu sequences in its mRNA. Cell. 1984 Nov;39(1):27–38. doi: 10.1016/0092-8674(84)90188-0. [DOI] [PubMed] [Google Scholar]
- van Berkel T. J., Nagelkerke J. F., Kruijt J. K. The effect of Ca2+ and the trifluoperazine on the processing of human acetylated low density lipoprotein by non-parenchymal liver cells. FEBS Lett. 1981 Sep 14;132(1):61–66. doi: 10.1016/0014-5793(81)80427-9. [DOI] [PubMed] [Google Scholar]