Liver-Targeted Anti-HBV Single-Stranded Oligonucleotides with Locked Nucleic Acid Potently Reduce HBV Gene Expression In Vivo

Abstract

Chronic hepatitis B infection (CHB) is an area of high unmet medical need. Current standard-of-care therapies only rarely lead to a functional cure, defined as durable hepatitis B surface antigen (HBsAg) loss following treatment. The goal for next generation CHB therapies is to achieve a higher rate of functional cure with finite treatment duration. To address this urgent need, we are developing liver-targeted single-stranded oligonucleotide (SSO) therapeutics for CHB based on the locked nucleic acid (LNA) platform. These LNA-SSOs target hepatitis B virus (HBV) transcripts for RNase-H-mediated degradation. Here, we describe a HBV-specific LNA-SSO that effectively reduces intracellular viral mRNAs and viral antigens (HBsAg and HBeAg) over an extended time period in cultured human hepatoma cell lines that were infected with HBV with mean 50% effective concentration (EC50) values ranging from 1.19 to 1.66 μM. To achieve liver-specific targeting and minimize kidney exposure, this LNA-SSO was conjugated to a cluster of three N-acetylgalactosamine (GalNAc) moieties that direct specific binding to the asialoglycoprotein receptor (ASGPR) expressed specifically on the surface of hepatocytes. The GalNAc-conjugated LNA-SSO showed a strikingly higher level of potency when tested in the AAV-HBV mouse model as compared with its non-conjugated counterpart. Remarkably, higher doses of GalNAc-conjugated LNA-SSO resulted in a rapid and long-lasting reduction of HBsAg to below the detection limit for quantification, i.e., by 3 log10 (p < 0.0003). This antiviral effect depended on a close match between the sequences of the LNA-SSO and its HBV target, indicating that the antiviral effect is not due to non-specific oligonucleotide-driven immune activation. These data support the development of LNA-SSO therapeutics for the treatment of CHB infection.

PAPD5/7 Are Host Factors That Are Required for Hepatitis B Virus RNA Stabilization

“…Next, we reasoned that if PAPD5 and PAPD7 are cellular targets of HBV‐active DHQ molecules, oligonucleotide‐mediated knockdown of either one or both of these proteins should phenocopy the posttranscriptional effect of the DHQ molecules on the stability of HBV transcripts . For optimal knockdown of PAPD5 and PAPD7, a recently described hepatocyte‐targeted SSO with LNA platform was used that allows efficient knockdown of target genes in hepatocytes or the dHepaRG ASGPR1/2 cell line (engineered to overexpress the ASGPR1/2 receptors to improve SSO LNA uptake) . The dHepaRG ASGPR1/2 cell line was infected with HBV and treated with increasing amounts of a control LNA or with LNAs targeting PAPD5 and PAPD7, either alone or in combination.…”

“…Differentiated HepaRG hepatoma (dHepaRG) and dHepaRG asialoglycoprotein receptor 1/2 (ASGPR1/2) cell lines were infected by HBV genotype D as described . Primary human hepatocytes (PHHs) isolated by the collagenase perfusion method from chimeric urokinase‐type plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice with humanized livers were obtained from PhoenixBio (Hiroshima, Japan).…”

“…Differentiated HepaRG hepatoma (dHepaRG) and dHepaRG asialoglycoprotein receptor 1/2 (ASGPR1/2) cell lines were infected by HBV genotype D as described. (20,28) Primary human hepatocytes (PHHs) isolated by the collagenase perfusion method from chimeric urokinase-type plasminogen activator/severe combined immunodeficiency (uPA/SCID) mice with humanized livers were obtained from PhoenixBio (Hiroshima, Japan). The cells were either seeded on type I collagen-coated 96-well plates or in 24-well plates at a respective concentration of 7 × 10 4 and 4 × 10 5 cells per well in culture media provided by Phoenix Bio.…”

“…(20) For optimal knockdown of PAPD5 and PAPD7, a recently described hepatocyte-targeted SSO with LNA platform was used that allows efficient knockdown of target genes in hepatocytes or the dHepaRG ASGPR1/2 cell line (engineered to overexpress the ASGPR1/2 receptors to improve SSO LNA uptake). (28) The dHep-aRG ASGPR1/2 cell line was infected with HBV and treated with increasing amounts of a control LNA or with LNAs targeting PAPD5 and PAPD7, either alone or in combination. Separate knockdown of PAPD5 or PAPD7 led to only a partial reduction in HBV gene expression at the mRNA level ( Fig.…”

Can we cure hepatitis B virus with novel direct‐acting antivirals?

“…Non‐conjugated SSOs naturally accumulate in different tissues leading to cell toxicity. Thus, locked nucleic acid SSOs (LNA‐SSOs) have been engineered to be liver‐specific and more stable, reducing viral replication and HBsAg both in vitro and in vivo . Nucleic acid polymers (NAPs) are single SSOs that work independently of their sequence and thus, with an antisense effect.…”