Kerstin Jahn-Hofmann

Hepatocyte-targeted RNAi Therapeutics for the Treatment of Chronic Hepatitis B Virus Infection

RNA interference (RNAi)-based therapeutics have the potential to treat chronic hepatitis B virus (HBV) infection in a fundamentally different manner than current therapies. Using RNAi, it is possible to knock down expression of viral RNAs including the pregenomic RNA from which the replicative intermediates are derived, thus reducing viral load, and the viral proteins that result in disease and impact the immune systems ability to eliminate the virus. We previously described the use of polymer-based Dynamic PolyConjugate (DPC) for the targeted delivery of siRNAs to hepatocytes. Here, we first show in proof-of-concept studies that simple coinjection of a hepatocyte-targeted, N-acetylgalactosamine-conjugated melittin-like peptide (NAG-MLP) with a liver-tropic cholesterol-conjugated siRNA (chol-siRNA) targeting coagulation factor VII (F7) results in efficient F7 knockdown in mice and nonhuman primates without changes in clinical chemistry or induction of cytokines. Using transient and transgenic mouse models of HBV infection, we show that a single coinjection of NAG-MLP with potent chol-siRNAs targeting conserved HBV sequences resulted in multilog repression of viral RNA, proteins, and viral DNA with long duration of effect. These results suggest that coinjection of NAG-MLP and chol-siHBVs holds great promise as a new therapeutic for patients chronically infected with HBV.

Solid‐Phase Synthesis of Sequence‐Defined T‐, i‐, and U‐Shape Polymers for pDNA and siRNA Delivery

Viral proteins are far more effective in mediating the transport of viral nucleic acids into cells than the currently available synthetic polymers for gene transfer. To mimic viral delivery processes, functional domains such as endosomolytic agents and targeting ligands have been conjugated to polymers. The chemistry of such conjugates, however, lacks the molecular precision of sequence-defined viral proteins, regarding both the polydispersity of the polymer and the conjugation sites. The common practice to apply such polydisperse mixtures in transfections may obscure accurate structure–activity relationships. It is questionable whether polydisperse macromolecules will ever compete successfully with their viral counterparts. Herein we communicate on the solid-phase-supported synthesis of a small library of sequence-defined polymers and their use for pDNA and siRNA delivery. Solid-phase-supported macromolecule assembly has already been applied for nucleic acid carriers. Hartmann, Bçrner, and colleagues published solid-phase-based syntheses of polyamidoamines employing alternating condensation steps using succinic anhydride and diamino-N-methyldipropylamine or protected spermine. To combine the advantages of peptide synthesis with the broader chemical diversity of synthetic polymers, we designed artificial Fmoc/Boc-protected amino acids with defined diaminoethane units. The protonatable diaminoethane motif has unique properties as a “proton sponge” for the endosome buffering and destabilization responsible for the high transfection activity of polyethylenimines (PEI) 7] and other polymers. The biological activity of diaminoethane units is far superior to that of diaminopropane units, which are completely protonated at physiological pH. 8a] The three artificial amino acids (Stp, Gtp, and Gtt; Figure 1a) were applied together with lysines (as branching units), cysteines (bioreversible disulfide-forming units), and

Host Scavenger Receptor SR-BI Plays a Dual Role in the Establishment of Malaria Parasite Liver Infection

An obligatory step of malaria parasite infection is Plasmodium sporozoite invasion of host hepatocytes, and host lipoprotein clearance pathways have been linked to Plasmodium liver infection. By using RNA interference to screen lipoprotein-related host factors, we show here that the class B, type I scavenger receptor (SR-BI) is the strongest regulator of Plasmodium infection among these factors. Inhibition of SR-BI function reduced P. berghei infection in Huh7 cells, and overexpression of SR-BI led to increased infection. In vivo silencing of liver SR-BI expression in mice and inhibition of SR-BI activity in human primary hepatocytes reduced infection by P. berghei and by P. falciparum, respectively. Heterozygous SR-BI(+/-) mice displayed reduced P. berghei infection rates correlating with liver SR-BI expression levels. Additional analyses revealed that SR-BI plays a dual role in Plasmodium infection, affecting both sporozoite invasion and intracellular parasite development, and may therefore constitute a good target for malaria prophylaxis.

Targeted siRNA Delivery and mRNA Knockdown Mediated by Bispecific Digoxigenin-binding Antibodies

Bispecific antibodies (bsAbs) that bind to cell surface antigens and to digoxigenin (Dig) were used for targeted small interfering RNA (siRNA) delivery. They are derivatives of immunoglobulins G (IgGs) that bind tumor antigens, such as Her2, IGF1-R, CD22, and LeY, with stabilized Dig-binding variable domains fused to the C-terminal ends of the heavy chains. siRNA that was digoxigeninylated at its 3′end was bound in a 2:1 ratio to the bsAbs. These bsAb–siRNA complexes delivered siRNAs specifically to cells that express the corresponding antigen as demonstrated by flow cytometry and confocal microscopy. The complexes internalized into endosomes and Dig-siRNAs separated from bsAbs, but Dig-siRNA was not released into the cytoplasm; bsAb-targeting alone was thus not sufficient for effective mRNA knockdown. This limitation was overcome by formulating the Dig-siRNA into nanoparticles consisting of dynamic polyconjugates (DPCs) or into lipid-based nanoparticles (LNPs). The resulting complexes enabled bsAb-targeted siRNA-specific messenger RNA (mRNA) knockdown with IC50 siRNA values in the low nanomolar range for a variety of bsAbs, siRNAs, and target cells. Furthermore, pilot studies in mice bearing tumor xenografts indicated mRNA knockdown in endothelial cells following systemic co-administration of bsAbs and siRNA formulated in LNPs that were targeted to the tumor vasculature.

Hepatitis B virus genome replication triggers toll-like receptor 3-dependent interferon responses in the absence of hepatitis B surface antigen

The hepatitis B virus (HBV) has been described as stealth virus subverting immune responses initially upon infection. Impaired toll-like receptor signaling by the HBV surface antigen (HBsAg) attenuates immune responses to facilitate chronic infection. This implies that HBV replication may trigger host innate immune responses in the absence of HBsAg. Here we tested this hypothesis, using highly replicative transgenic mouse models. An HBV replication-dependent expression of antiviral genes was exclusively induced in HBsAg-deficient mice. These interferon responses attributed to toll-like receptor 3 (TLR3)-activated Kupffer and liver sinusoidal endothelial cells and further controlled the HBV genome replication. However, activation of TLR3 with exogenous ligands indicated additional HBs-independent immune evasion events. Our data demonstrate that in the absence of HBsAg, hepatic HBV replication leads to Tlr3-dependent interferon responses in non-parenchymal liver cells. We hypothesize that HBsAg is a major HBV-mediated evasion mechanism controlling endogenous antiviral responses in the liver. Eradication of HBsAg as a therapeutic goal might facilitate the induction of endogenous antiviral immune responses in patients chronically infected with HBV.

398 VIRAL REPLICATION IN HBV-TRANSGENIC MICE LACKING THE HBsAg SIGNIFICANTLY INDUCES INTERFERON RESPONSES

Methods: Explanted and transplanted liver from 21 patients (5 with HBV-related, 6 with HBV/HDV-related and 10 with HCVrelated chronic liver disease) were analysed. All but one donor were HBsAg negative, 6 were anti-HBc positive, 14 were negative for all serum markers. From transplanted livers, biopsy specimens obtained before LT, in post-perfusion period and at end of LT were available. HBVDNA was tested in all liver tissue specimens by nested-PCR amplifications specific for 4 different HBV genomic regions. Total HBVDNA, HBV cccDNA and HDV RNA were quantified by specific real time-PCR approaches. Results: At the end of surgery, two of the 5 HBsAg-positive patients (both donors were anti-HBc positive), 1 of the 6 HBV/HDV coinfected patients (the donor was HBsAg-positive) and 2 of the 10 HCV-infected patients (one donor was anti-HBc positive and the other anti-HBc negative) showed the presence of quantifiable amounts of HBVDNA in the liver (range: 6x10-1x10 copies/cells). Sequencing analysis of the isolated HBV genomes showed that they were donor viral strains. HBV cccDNA could be detected and quantified (range: 2x10-2x10 copies/cell) in all the HBV-positive patients. None of the HBV/HDV-infected patients showed HDV RNA in the transplanted liver. Conclusions: 1. HBV genomes infecting the transplanted liver cause the reinfection of the recipients in the course of LT; 2. HDV does not re-infect the transplanted liver at the time of LT; 3. occult HBV infection (namely, presence of intrahepatic HBVDNA in HBsAg negative individuals) may be present in trasplanted liver and may persist during LT independently of the anti-HBc status of the donor.