Therapeutics

Hepatitis C

SomaGenics’ lead therapeutic indication is hepatitis C. The hepatitis C virus (HCV) currently afflicts an estimated 3.9 million people in the US and 175 million worldwide. Infection with HCV becomes chronic in 75-85% of individuals, with 70% of those eventually presenting chronic liver disease. HCV is recognized as a major cause of end-stage liver disease such as liver cancer (1-5% of HCV infections) and cirrhosis (10-20%). HCV is the leading indication for liver transplantation in the Western world.

Microscopic view of Hepatitis C Virus particles

Hepatitis C Virus particles

Current HCV therapy, a combination of pegylated interferon-alpha and ribavirin, is only partially effective; of those patients infected with the most common strain, genotype 1, only about half achieve a sustained viral response. Unfortunately, HCV genotype 1 comprises 77% of cases worldwide, and even more in the West. Because the standard of care is associated with severe adverse effects including flu-like symptoms, hematologic abnormalities and depression, patient compliance is poor.

Several new drugs currently in clinical trials inhibit specific viral proteins (protease or polymerase), mechanisms of action that are distinct from those of the standard of care. While some of these drugs show promising interim results, all are vulnerable to the development of viral resistance through mutation. SomaGenics’ shRNA-based HCV drug candidate, now in pre-clinical development, has potent antiviral activity against all common HCV genotypes and is expected to be much less vulnerable to viral escape.HCV SEM image

HCV has many features that make it an ideal target for shRNA-based therapeutics:

  • Chronic, slow viral infection
  • Effective treatment is known to clear the infection
    • No reservoir of silently infected cells is known, unlike with HIV
    • No DNA component as with HIV
  • Target organ (liver) is accessible to i.v. delivery of RNA drugs
    • Provides a platform for subsequent targeting of additional liver or metabolic diseases
  • Cocktail of shRNAs targeting multiple conserved sites minimizes chance of viral escape
  • Independent mechanism of action makes shRNA complementary to other drugs

Targeting HCV by sshRNAs

Enhanced Serum Stability

Improvement of stability and safety (drug-like properties) of sshRNA by chemical modification: Introduction of smaller loops and natural chemical modifications at specific sites of the sshRNA greatly improves stability in 10% human serum, as shown in the denaturing polyacrylamide gel above. FL, full-length.

Good in vitro Safety Profiles

The same chemical modification pattern essentially eliminates immune stimulatory tendencies, especially in blunt-ended sshRNAs, as shown above in comparison to poly IC as a positive control.

In vivo activity in a reporter mouse model

The ability of the sshRNAs to inhibit HCV IRES activity in vivo when formulated with lipid nanoparticles (LNP, from Tekmira Pharmaceuticals) was assessed using an HCV IRES-dependent bioluminescence (luciferase) mouse model. A single administration of sshRNA resulted in a dose-dependent reduction of luciferase activity in mouse liver, reaching more than 90% knock-down with 2.5 mg/kg sshRNA. This knock-down was sequence-specific and durable, with inhibition lasting for several weeks.

Efficient liver uptake of formulated HCV sshRNAs in mice that can support HCV infection

uPA-SCID mice with chimeric human-mouse livers have previously been used to support stable, high-level infections with HCV or HBV. A good correlation has been established between the antiviral potency of various agents in HCV-infected, chimeric uPA-SCID mice and the clinical efficacy of these agents in HCV-infected patients. To evaluate the efficacy of sshRNAs in this model, chimeric uPA-SCID mice were injected with single, 5.0 mg/kg doses of formulated sshRNA via ordinary intravenous injection in a collaboration with Roche and Tekmira. Analysis of liver samples showed substantial amounts (5–6%) of the injected sshRNA remaining in the liver 48 hours after treatment.

Efficient inhibition of HCV replication by sshRNAs in vivo

To further determine the activity of the HCV sshRNAs against HCV virus replication in a chronic in vivo HCV infection model, SomaGenics researchers and collaborators tested lead sshRNAs in chimeric uPA-SCID mice that were infected with HCV genotype. The livers of these mice contained mostly human hepatocytes, allowing the establishment of a stable HCV infection with high levels of viral replication in all mice. Injection of single, 2.5 mg/kg doses of individual LNP-formulated sshRNA led to viral load reductions of up to 2 orders of magnitude, and when given in combination, the two sshRNAs decreased viral load by 2.5 orders of magnitude after two doses. The inhibition was specific, as a scrambled control sshRNA had no effect. The HCV load reduction was also durable, remaining at least an order of magnitude lower than the pretreatment level even 3 weeks after treatment (p<0.01-0.05). The LNP-formulated sshRNAs were well tolerated, as evidenced by lack of treatment-related elevation of serum ALT or AST levels, no reduction in serum human-albumin concentration, and no treatment-associated body weight loss, morbidity or mortality in the study animals.

The above findings were published in the Journal of Virology (J. Virol. JVI.00105-14; 2014) and presented at the annual meetings of the European Association for the Study of Liver (Barcelona, Spain, 18-22 April, 2012) and the Oligonucleotide Therapeutics Society (Boston, USA, 28-31 October, 2012).

Pipeline

SomaGenics has discovery-phase programs in hepatitis B, hepatitis delta and wound healing.

References:

  1. Wang, C. H. Contag, H. Ilves, B. H. Johnston, and R. L. Kaspar,  Small hairpin RNAs efficiently inhibit hepatitis C IRES-mediated gene expression in human tissue culture cells and a mouse model. Mol. Therapy, 12:562-8 (2005). PMID 15953767
  2. H. Ilves, R. L. Kaspar, Q. Wang, A. A. Seyhan, A. V. Vlassov, C. H. Contag, D. Leake, and B. H. Johnston (2006) Inhibition of hepatitis C IRES-mediated gene expression by small hairpin RNAs in human hepatocytes and mice. Ann. N.Y. Acad. Sci., 1082: 52-55. PMID 17145925
  3. A.V. Vlassov, B. Korba, K. Farrar, S. Mukerjee, A. A. Seyhan, H. Ilves, R. Kaspar, S. A. Kazakov, and B. H. Johnston (2007) shRNAs targeting hepatitis C: effects of sequence and structural features, and comparison with siRNA. Oligonucleotides 17:223-236. PMID 17638526
  4. A. A. Seyhan, B. N. Alizadeh and B. H. Johnston (2007) RNA interference-mediated inhibition of Semliki Forest virus replication in mammalian cells.  Oligonucleotides 17(4): 473-84. PMID 18095875
  5. Ge Q., Dallas A., Ilves H., Shorenstein J., Behlke M.A., Johnston B.H. (2009) Effects of chemical modification on the potency, serum stability, and immunostimulatory properties of short shRNAs. RNA 16(1):118-30 (Epub Nov 30, 2009). PMID 19948766
  6. Ge Q., Ilves H., Dallas A., Kumar P., Shorenstein J., Kazakov S.A., and Johnston B.H. (2009) Minimal-length short hairpin RNAs: The relationship of structure and RNAi activity. RNA 16(1):106-17 (Epub Dec. 1, 2009). PMID 19952116
  7. Dallas, A., Ilves, H., Ge, Q., Kumar, P., Shorenstein, J., Kazakov, S. A.; Cuellar, Trinna L.; McManus, Michael T.; Behlke, Mark A.;, Johnston, B. H. (2012) Right- and Left-loop short shRNAs have distinct and unusual mechanisms of gene silencing. Nucleic Acids Res. 40: 9255–9271. (Epub July 18, 2012) PMID: 22810205
  8. Dallas A., Ilves H., Shorenstein J., Judge A, Spitler R, Contag C, Ping Wong S,  Harbottle RP, MacLachlan I, and Johnston B.H. (2013) Minimal-length synthetic shRNAs (sshRNAs) formulated with lipid nanoparticles are potent inhibitors of hepatitis C virus IRES-linked gene expression in mice.  Mol. Therapy–Nucleic Acids 17;2:e123. PMID: 24045712
  9. Ma H. , Dallas A., Ilves H., Shorenstein J., Maclachlan I, Klumpp K, and Johnston B.H. (2014) Formulated minimal-length synthetic shRNAs are potent inhibitors of Hepatitis C virus in chimeric human-liver mice.  Gastroenterology, 146(1):63-66.e5. PMID: 24076507
  10. A.Dallas, H. Ilves, H. Ma, D. J. Chin, I. MacLachlan, K. Klumpp, and B. H. Johnston (2014) Inhibition of hepatitis C virus in chimeric mice by sshRNAs:  sequence analysis of surviving virus shows added selective pressure of combination therapy. J. Virology (in press)