"Hepatic Diseases — Hitting the Target with Inhibitory RNAs."

Beverly L. Davidson, Ph.D.

Department of Internal Medicine, University of Iowa, Iowa City, IA 52242

What is all the noise about RNA silencing? And how does silencing, or impairing translation of target RNAs, help us develop effective therapies for chronic and acute liver diseases? As with many important advances in molecular medicine, the answer, once found, is elegantly simple. Deliver to cells a small duplex or hairpin RNA with sequences that match only those of your target, and let natural cellular mechanisms do the rest (Figure 1). Voila! Target RNA degradation and inhibit downstream expression. Unlike antisense-DNA–based approaches, inhibition by RNA silencing is highly efficient, because it takes advantage of endogenous cellular machinery to degrade the unwanted messages.

Figure 1. New Tools for Silencing.

Gene expression can be silenced with small inhibitory RNA (siRNA) and short hairpin RNA (shRNA) that are targeted to the RNA of interest. Song and colleagues [1] generated siRNAs against Fas RNA sequences and transfected them into cells in tissue culture and into hepatocytes in mice. After entering the cell, siRNA is incorporated into the cytoplasmic-RNA–induced silencing complex (RISC) along with Fas messenger RNA (mRNA), causing cleavage of Fas mRNA and inhibiting the expression of Fas protein. McCaffrey and colleagues [2] transfected cells with plasmids that contained an shRNA expression cassette. Plasmids reaching the nucleus are transcribed by cellular RNA polymerases, resulting in shRNAs specific to hepatitis B virus (HBV) mRNAs. Once the shRNAs reach the cytoplasm, they are incorporated into the RISC along with HBV mRNA targets, with subsequent inhibition of viral gene expression and viral replication.

Two recent articles [1, 2] demonstrate for the first time the power of small inhibitory RNAs (siRNAs) in animal models of liver disease. In one study [1], Song and colleagues evaluated the effect of inhibiting the cell-surface Fas receptor in mouse models of autoimmune hepatitis. (On binding its ligand, Fas initiates a cascade of events leading to cell death and is a key mediator of hepatocellular damage.) They found that siRNAs directed against Fas caused the specific loss of Fas messenger RNA (mRNA) in tissue-culture cells, leaving other RNAs in the Fas-directed apoptosis pathway intact. Also, Fas-specific siRNA delivered to mouse hepatocytes in vivo, before the injection of a hepatotoxic plant lectin, protected against hepatocellular damage and fibrosis. Serum alanine and aspartate aminotransferase levels were dramatically lower in the lectin-treated animals that received Fas-specific siRNA than in lectin-treated mice given saline or control siRNAs. In addition, in a model of chronic hepatitis, indicators of fibrosis were reduced by the administration of Fas-specific siRNA. Mice were also
protected when the Fas-specific siRNA was given after the initiation of lectin-induced damage, indicating its therapeutic potential in a relevant clinical setting. Currently, patients with autoimmune disease have a poor prognosis. The ability of Fas-specific siRNA to temper the destructive role of Fas could find widespread clinical application in acute and chronic liver diseases.

In the second study, McCaffrey and colleagues [2] used inhibitory RNA in the form of hairpins (short hairpin RNAs, or shRNAs) specific to hepatitis B virus (HBV) RNAs. HBV infection remains an important public health burden despite the existence of a successful vaccine, with hundreds of millions of people chronically infected. As is the case with some other viruses, the parental genome of HBV leads to the production of overlapping RNAs. A strategically chosen inhibitory RNA could therefore degrade more than one HBV transcript (Figure 2). The experimental data show that HBV replication can be inhibited in tissue-culture cells. In in vivo studies using a recently developed mouse model of HBV infection, shRNAs targeting HBV sequences also inhibited viral replication and the production of core antigen in hepatocytes. The treatment also reduced serum levels of HBV surface antigen. The reduction in viral burden would be expected to improve virus-induced abnormalities.

Figure 2. Getting the Most from Short Hairpin RNAs (shRNAs).

The top line depicts the RNA that serves as a template for replication of the hepatitis B virus genome and overlapping transcripts (the bottom three lines). The shRNAs (arrows) could target one (A), two (B), three (C), or multiple (D) RNAs. The shRNAs targeted to region C were the most efficient at silencing the expression of the hepatitis B virus gene. Adapted from McCaffrey et al. [2]

These proof-of-principle experiments, together with future developments in the hepatic delivery of small inhibitory nucleic acids or their analogues to larger models and humans, will provide an important foundation for liver-based molecular medicine.

Source Information:

From the Department of Internal Medicine, University of Iowa, Iowa City, 52242

References:

1. Song E, Li S-K, Wang J, et al. "RNA interference targeting". Nature Medicine 2003; 9: 347-351.

2. McCaffrey AP, Nakai H, Pandey K, et al. "Inhibition of hepatitis B virus in mice by RNA interference". Nature Biotechnology 2003; 21: 639-644.

References on RNA as a Therapeutic Agent:

1. DeCarvalho S, and Rand HJ, "Comparative Effects of Liver and Tumour Ribonucleic Acids on the Normal Liver and the Novikoff Hepatoma Cells of the Rat", Nature, vol. 189, no. 4767, pp. 815-817 (March 11, 1961).

2. Niu MC, Cordova CC, and Niu LC, "Ribonucleic Acid-Induced Changes in Mammalian Cells", Proc. Natl. Acad. Sci. USA, vol. 47, pp. 1689-1700 (October, 1961).

3. DeCarvalho S, "Effect of RNA from Normal Human Marrow on Leukaemic Marrow In-Vivo", Nature, vol. 197, no. 4872, pp. 1077-1080 (March 16, 1963).

4. Frenster JH, "Nuclear Polyanions as De-Repressors of Synthesis of Ribonucleic Acid", Nature, vol. 206, no. 4985, pp. 680-683 (May 15, 1965).

5. Czihak G, "Evidence for inductive properties of the micromere RNA in sea urchin embryos",
Naturwissenschaften vol. 52, no. 6, pp. 141-142 (1965).

6. Kolodny GM, "Evidence for transfer of macromolecular RNA between mammalian cells in culture", Exp. Cell Res. vol. 65, no. 2, pp. 313-324 (April, 1971).

7. Kolodny GM, "Cell to cell transfer of RNA into transformed cells", J. Cell Physiol. vol. 79, no. 1, pp. 147-150 (February, 1972).

8. Kolodny GM, Culp LA, and Rosenthal LJ, "Secretion of RNA by normal and transformed cells", Exp. Cell Res. vol. 73, no. 1, pp. 65-72 (July, 1972).

9. Frenster JH, and Herstein PR, "RNA in Gene De-Repression", in: Proc. AAAS Symposium, December 28-30, 1972, "The Role of RNA in Reproduction and Development", edit. by Niu MC, and Segal SJ, pp. 330-338, North-Holland Publishing Co. Amsterdam, 1973.

10. Kern DH, Chow N, and Pilch YH, "Lymphocyte Populations Participating in Cellular Antitumor Immune Responses Mediated by Immune RNA", J. Natl. Cancer Inst. 60, 335-344 (1978).

11. Frenster JH, "Oncogenes as Molecular Targets within Active Chromatin", in: AACR-NCI-EORTC International Conference: "Molecular Targets and Cancer Therapeutics: Discovery, Development, and Clinical Validation", Washington, DC, November 16-19, 1999, and Published in: Clinical Cancer Research, vol. 5, suppl. l, p. 3855s, (624), (November, 1999).

12. Frenster JH, "Activation of DNA Transcription within Repressed Chromatin", 14th John Innes Symposium, September 5-8, 2001.

13. Maruo S, Nanbo A, and Takada K, "Replacement of the Epstein-Barr Virus Plasmid with the EBER Plasmid in Burkitt's Lymphoma cells", J. Virology, vol. 75, no. 20, pp. 9977-9982 (October, 2001).

14. Frenster JH, "Yeast  RNA  Re-Programming  of  Already-Active  Mammalian Chromatin", RNA2002, p. 592, (June, 2002).

15. Lanz RB, Chua SS, Barron N, Söder BM, DeMayo F, and O'Malley BW, "Steroid Receptor RNA Activator Stimulates Proliferation as Well as Apoptosis In Vivo", Mol. Cell. Biol., vol. 23, no. 20, pp. 7163-7176 (October, 2003).

16. Park W-S, Hayafune M, Miyano-Kurosaki N, and Takaku H, "Specific HIV-1 env gene silencing by small interfering RNAs in human peripheral blood mononuclear cells", Gene Therapy,  vol. 10, no. 24, pp. 2046-2050 (November, 2003).

17. Santulli-Marotto S, Nair SK, Rusconi C, Sullenger B,  and Gilboa E, "Multivalent RNA Aptamers That Inhibit CTLA-4 and Enhance Tumor Immunity", Cancer Research 63, 7483-7489, November 1, 2003.

18. Coughlin CM, Vance BA, Grupp SA, and Vonderheide RH, "RNA-transfected CD40-activated B cells induce functional T cell responses against viral and tumor antigen targets: implications for pediatric immunotherapy".

19. Frenster JH, "Ultrastructural Probes of Active DNA Sites, and the RNA Activators of DNA".

20. Frenster JH,  and Herstein PR, "Gene De-Repression", New Eng. J. Med. 288: 1224-1229 (June 7, 1973).