Ingo Roehl

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.

RNA Interference-Mediated Silencing of the Respiratory Syncytial Virus Nucleocapsid Defines a Potent Antiviral Strategy

ABSTRACT We describe the design and characterization of a potent human respiratory syncytial virus (RSV) nucleocapsid gene-specific small interfering RNA (siRNA), ALN-RSV01. In in vitro RSV plaque assays, ALN-RSV01 showed a 50% inhibitory concentration of 0.7 nM. Sequence analysis of primary isolates of RSV showed that the siRNA target site was absolutely conserved in 89/95 isolates, and ALN-RSV01 demonstrated activity against all isolates, including those with single-mismatch mutations. In vivo, intranasal dosing of ALN-RSV01 in a BALB/c mouse model resulted in potent antiviral efficacy, with 2.5- to 3.0-log-unit reductions in RSV lung concentrations being achieved when ALN-RSV01 was administered prophylactically or therapeutically in both single-dose and multidose regimens. The specificity of ALN-RSV01 was demonstrated in vivo by using mismatch controls; and the absence of an immune stimulatory mechanism was demonstrated by showing that nonspecific siRNAs that induce alpha interferon and tumor necrosis factor alpha lack antiviral efficacy, while a chemically modified form of ALN-RSV01 lacking measurable immunostimulatory capacity retained full activity in vivo. Furthermore, an RNA interference mechanism of action was demonstrated by the capture of the site-specific cleavage product of the RSV mRNA via rapid amplification of cDNA ends both in vitro and in vivo. These studies lay a solid foundation for the further investigation of ALN-RSV01 as a novel therapeutic antiviral agent for clinical use by humans.

In vitro and in vivo efficacy of SYL040012, a novel siRNA compound for treatment of glaucoma.

Glaucoma is a progressive ocular syndrome characterized by degeneration of the optic nerve and irreversible visual field loss. Elevated intraocular pressure (IOP) is the main risk factor for glaucoma. Increased IOP is the result of an imbalance between synthesis and outflow of aqueous humor (AH). Blocking β2 adrenergic receptor (ADRB2) has shown to reduce IOP by decreasing production of AH at the ciliary body (CB). SYL040012 is a siRNA designed to specifically silence ADRB2 currently under development for glaucoma treatment. Here, we show that SYL040012 specifically reduces ADRB2 expression in cell cultures and eye tissues. The compound enters the eye shortly after administration in eye drops and is rapidly distributed among structures of the anterior segment of the eye. In addition, SYL040012 is actively taken up by cells of the CB but not by cells of systemic organs such as the lungs, where inhibition of ADRB2 could cause undesirable side effects. Moreover, SYL040012 reduces IOP in normotensive and hypertensive animal models and the effect appears to be long lasting and extremely well tolerated both locally and systemically.

Purification of Small Interfering RNA Using Nondenaturing Anion-Exchange Chromatography

A manufacturing and purification process for duplex oligonucleotides was established, which shortens and simplifies currently used procedures, yielding a product of higher purity. The reported procedure is based on nondenaturing anion-exchange (AEX) chromatography, which is performed on the annealed duplex rather than the individual single strands. The duplex is formed early in the process by annealing of the crude single strands directly after solid-phase synthesis. Two 30 μmol manufacturing runs using duplex purification were performed on 2 different AEX resins and compared with a manufacturing run of the same scale using conventional single-strand chromatography. The same pooling strategy was employed for all purifications. Content of optimal duplex (duplex exclusively comprising full-length single strands) was 90.5% and 90.2% for the batches obtained by duplex purification and 86.1% for the batch obtained by single-strand purification. Maximum chromatographic recoveries were 67% for the duplex purification and 68% for the single-strand purification. Hence, the manufacture of small interfering RNA (siRNA) using duplex purification was simpler and faster than conventional single-strand purification and provided better purity and similar yield of final siRNA.

Nucleic Acid Polymers with Accelerated Plasma and Tissue Clearance for Chronic Hepatitis B Therapy

REP 2139 is a nucleic acid polymer (NAP) currently under clinical development for chronic hepatitis B (HBV) therapy. This preclinical study investigated different REP 2139 analogs that would display reduced accumulation in the serum and tissues, while retaining an antiviral effect against HBV infection. REP 2139 analogs were evaluated in human plasma, CD-1 mice, cynomolgus monkeys, and Pekin ducks. Discrete ribose transformation to 2′OH in selected riboadenosines resulted in a slow degradation in acidified human plasma that plateaued after 48 hr. REP 2165, a REP 2139 analog containing three unmodified riboadenosines equally spaced throughout the polymer, showed similar plasma clearance and tissue distribution as REP 2139 in mice and cynomolgus monkeys after a single dose. Interestingly, after repeated administration, accumulation of REP 2165 in plasma and organs was reduced, indicating a dramatically faster rate of clearance from organs after therapy was ended in both species. Both REP 2139 and REP 2165 were well tolerated at clinically relevant doses, with no alterations in liver, kidney, or hematological function. In chronic duck HBV (DHBV) infection, REP 2165 displayed significantly reduced liver accumulation after repeated dosing but retained antiviral activity similar to REP 2139. These results indicate the therapeutic potential of REP 2165 against chronic HBV infection in patients is similar to REP 2139, but with significantly reduced drug accumulation and improved tissue clearance.

Activity of nucleic acid polymers in rodent models of HBV infection

ABSTRACT Nucleic acid polymers (NAPs) block the release of HBsAg from infected hepatocytes. These compounds have been previously shown to have the unique ability to eliminate serum surface antigen in DHBV‐infected Pekin ducks and achieve multilog reduction of HBsAg or HBsAg loss in patients with chronic HBV infection and HBV/HDV coinfection. In ducks and humans, the blockage of HBsAg release by NAPs occurs by the selective targeting of the assembly and/or secretion of subviral particles (SVPs). The clinically active NAP species REP 2055 and REP 2139 were investigated in other relevant animal models of HBV infection including woodchucks chronically infected with WHV, HBV transgenic mice and HBV infected SCID‐Hu mice. The liver accumulation of REP 2139 in woodchucks following subcutaneous administration was examined and was found to be similar to that observed in mice and ducks. However, in woodchucks, NAP treatment was associated with only mild (36–79% relative to baseline) reductions in WHsAg (4/10 animals) after 3–5 weeks of treatment without changes in serum WHV DNA. In HBV infected SCID‐Hu mice, REP 2055 treatment was not associated with any reduction of HBsAg, HBeAg or HBV DNA in the serum after 28 days of treatment. In HBV transgenic mice, no reductions in serum HBsAg were observed with REP 2139 with up to 12 weeks of treatment. In conclusion, the antiviral effects of NAPs in DHBV infected ducks and patients with chronic HBV infection were weak or absent in woodchuck and mouse models despite similar liver accumulation of NAPs in all these species, suggesting that the mechanisms of SVP assembly and or secretion present in rodent models differs from that in DHBV and chronic HBV infections. HighlightsNAPs display similar liver accumulation in ducks, mice, non‐human primates and woodchucks.In woodchuck and mouse models of HBV infection, WHsAg or HBsAg response to NAP treatment is weak or absent.SVP assembly/secretion occurring chronic HBV infection may differ from that in rodent models of HBV infection.