Materials
All siRNA used in this study were synthesized by Suzhou Ribo Life Science Co. Ltd. (Kunshan, China). In order to enhance their stability and specificity, chemical modifications with methoxy group or fluorine at the 2’ site hydroxyl groups, or phosphorothioate at the phosphonate backbone were placed at certain sites of the sense and antisense strands of siRNA. For Cy5-labeled siRNA, the Cy5 fluorophore was placed at the 5’ end of the sense strand. TRIzol® was purchased from Thermo Fisher Scientific (Eugene, OR, USA). Reverse transcription kit and real-time PCR kit (UltraSYBR Mixture) were purchased from Promega Corporation (Fitchburg, Wisconsin, USA) and Beijing ComWin Biotech Co., Ltd. (Beijing, China), respectively. RNAlater® were purchased from Sigma–Aldrich (St Louis, MO, USA). DAPI (4’, 6-diamidino-2-phenylindole, for staining nuclei) was from Zhongshan Golden Bridge Biotechnology Co., Ltd., Beijing, China, and fluorescein isothiocyanate-labeled phalloidin (for staining F-actin) was from Sigma-Aldrich (MO, USA). Pentobarbital sodium was provided by Peking University Laboratory Animal Center.
Formulation of lipid nanoparticles (LNPs)
The Lipidoid component of RBP131 was synthesized by Suzhou Ribo Life Science Co. Ltd. RBP131 formulation was prepared according to a modified protocol adopted from reference,57 or an example described in patent literature.34 Briefly, LC8, cholesterol (Sigma–Aldrich, St Louis, MO), and “16:0 PEG2000 PE” (MW 2749.391, Sigma–Aldrich) were prepared in ethanol and mixed to yield a molar ratio or 59:29:12. Mixed lipids were added to 200 mM sodium acetate buffer (pH 5.2) to yield a solution containing 25% ethanol, resulting in spontaneous formation of empty lipidoid nanoparticles. Then empty lipidoid nanoparticles and siRNA solution in 25% ethanol were rapidly mixed together using a peristaltic pump at ~1:15 (wt/wt) siRNA/total lipids, followed by incubating at 50 °C for approximate 20 min. Finally, ethanol removal and buffer exchange of siRNA-containing lipidoid nanoparticles was achieved by dialyzing against 1 × PBS for 2 h in 100 KD MWCO cassettes (Float-A-Lyzer® G2, Spectrum Laboratories Inc. New Brunswick, New Jersey, USA). If needed, 100 KD MWCO ultrafiltration tube (Amicon® Ultra-15 or Ultra-4, EMD Millipore, Billerica, Massachusetts, USA), or 1 × PBS (or normal saline) can be used to concentrate or dilute the solution to the desired concentration. Alternatively, a KrosFlo® Research IIi Tangential Flow Filtration System can be also used to prepare RBP131/siRNA formulation on large scale. In addition, a lyophilization technology was established, by which the liquid RBP131/siRNA formulation can be lyophilized. Sterile water was used to reconstitute the lyophilized powder before administration.
Detection of encapsulation efficiency
siRNA encapsulation efficiency was determined by modified RiboGreen RNA assay (Invitrogen, Carlsbad, CA).58 Briefly, siRNA entrapment was determined by comparing the signal of the RNA binding dye RiboGreen in formulation samples in the absence and presence of the detergent Triton-X100. In the absence of detergent, the signal comes from accessible (unentrapped) siRNA only. In the presence of detergent, the signal comes from total siRNA. In all preparations used for this study, the encapsulation efficiency was above 90%.
Characterization of particle sizes and zeta potential
The particle sizes and zeta potentials of the RBP131/siRNA complexes were measured using a Zetasizer Nano ZS (Malvern Instruments, Inc., Worcestershire, UK) at a wavelength of 677 nm with a constant angle of 90° at room temperature. The complex was prepared according to the above-mentioned protocol. Typically, ~0.8 ml of solution was used to detect size and zeta potential.
In situ determination of pKa using TNS
TNS (6-(p-Toluidino)-2-naphthalenesulfonic acid sodium salt) assay was used to detect the pKa value of RBP131 formulation. Empty RBP131 vesicles were prepared in 1 × PBS with a concentration of ~6 mM total lipids. TNS was prepared as a 100 μM stock solution in distilled water. RBP131 vesicles were diluted to 30 μM lipid in 2 mL of buffered solutions containing 10 mM HEPES, 10 mM MES, 10 mM ammonium acetate, 130 mM NaCl, where the pH ranged from 3.0 to 11.0. An aliquot of the TNS solution was added to give a final concentration of 1 μM and following vortex mixing fluorescence intensity was measured at room temperature in a fluorometer (Synergy™ HT, BioTek, Winooski, VT, USA) using excitation and emission wavelengths of 321 and 445 nm. A sigmoidal best fit analysis was applied to the fluorescence data and the pKa was measured as the pH gave rise to half-maximal fluorescence intensity.
Cryogenic electron microscopy (Cryo-EM)
Cryo-EM samples were made by Vitrobot (FEI Company) at a controlled temperature (25 °C) and at saturation. Cryo-EM grids were frozen using a Vitrobot Mark IV (FEI) as follows: 6 μL of the sample was applied to a glow discharged Quantifoil R1.2/1.3 holey carbon 300 mesh gold grid. To remove excess solution and produce a thin liquid film the drop is blotted manually in the Vitrobot. The blotted sample is then plunged into liquid ethane (−183 °C) to form a vitrified specimen and transferred to liquid nitrogen (−196 °C) for storage. Cryo-EM data of vitrified specimens were recorded on a Titan Krios (FEI) operated at 300 kV, equipped with a Gatan K2 Summit camera. SerialEM was used for automated data collection.
Cell culture
HepG2.2.15 is a widely-used cell line for studying HBV in vitro. It contains 2 head to tails stably integrated HBV of the genotype D. It was kept by Suzhou Ribo Life Science Ltd. Co. and is cultured with DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% fetal bovine serum, 100 units/ml penicillin, and 100 µg/ml streptomycin at 37 °C in a humidified atmosphere of 5% CO2. Hek293A was used in psiCHECK™ assay, which was cultured with the same protocol of HepG2.2.15.
Off-target effect evaluation in vitro
The off-target effect is a critical issue for RNAi therapeutic development, since off-target may trigger serious toxicity in vivo.59,60 siRNA may induce off-target effect via multiple mechanisms, such as (1) passenger (sense) strand of siRNA bind with undesired mRNA target in a complete-match manner, and inhibits its translation (siRNA working pathway); (2) either passenger (sense) or guide (antisense) strand of siRNA recognizes undesired target mRNA via seed-region matching (miRNA working mechanism); (3) gene disturbing resulting from immune-response stimulated by siRNA molecule. These weaknesses can be surrounded by positioning proper chemical modifications at specific sites of both the passenger and the guide strands of siRNA. Lead siRNA sequence targeting HBV genes (SR16-X2) was screened in this study, and rational chemical modifications (generating a molecule called SR16-X2M2) were used to enhance its performance. psiCHECK™ dual-luciferase reporter system (Promega, Madison, WI, USA)21 was employed to evaluate the on-target and off-target activity of unmodified siRNA and chemically-modified siRNA. Briefly, both firefly and renilla luciferase expression sequences were incorporated into the plasmid, and the target sequence of SR16-X2 (without modification) and SR16-X2M2 (siHBV, with modification) was inserted into the plasmid at the 3’-UTR of renilla luciferase. Then the unmodified or chemically-modified siRNAs were transfected into Hek293A cells at concentration gradients of 100, 25, 6.25, 1.5625, 0.3906, 0.0977, 0.0244, 0.0061, 0.0015, 0.0004, 0.0001 nM, together with the psiCHECK™ plasmid, by lipofectamine 2000. Twenty-four hours later, the cells were lysed with Passive Lysis Buffer (Promega), 10 μL of lysate of each treatment was transformed to 96-well plate, and the activities of both firefly and renilla luciferases were evaluated with the Dual-Luciferase Reporter Assay System (Promega). Finally, renilla luciferase activity was normalized to the firefly luciferase activity, and siRNA silencing activity was calculated by comparison with the negative control sample. The IC50 was calculated with GraphPad Prism 8.0 software.
Serum stability assay
Serum is rich of RNase, therefore fetal bovine serum (FBS, Sigma) was used to evaluate the stability of RNase-resistance capability of unmodified and modified siRNA. Unmodified SR16-X2 or modified SR16-X2M2 were incubated in 10% FBS (v/v, in 1 × PBS) at 37 °C. Samples were collected and immediately frozen at −20 °C at 0, 2, 4, 6, 8, 12, 24, and 48 h post-incubation. Then all samples were diluted 2 times with DEPC water and mixed with loading buffer, followed by separating in 20% non-denaturing PAGE (polyacrylamide gel electrophoresis) for 120 min at a constant voltage of 150 V. Finally, gels were stained with Sybr Gold for 20 min, exposed by Vtlber Lourmat imaging system (Frence).
Anti-HBV effects of SR16-X2M2 in vitro
In vitro activity of SR16-X2M2, the lead compound selected to perform preclinical evaluation was assessed with HepG2.2.15. siRNA was transfected with lipofectamine 2000 at the concentrations of 50, 25, and 12.5 nM, respectively. Twenty-four hours later, the expression of X gene and HBsAg were determined by RT-qPCR and ELISA, respectively.
Animals
C57BL/6 mice (6–8 weeks old, 18–22 grams) were purchased from Vital River Laboratory Animal Technology Co., Ltd. (Beijing, China). ApoE−/− mice (C57BL/6 background, 6–8 weeks old, 18–22 grams) and LDLR−/− mice (C57BL/6 background, 6–8 weeks old, 18–22 grams) were purchased from Changzhou Cavens Laboratory animal Co., Ltd. (Changzhou, Jiangsu, China). Animals were maintained in the Peking University Laboratory Animal Center (an AAALAC-accredited and specific pathogen-free (SPF) experimental animal facility). ApoE−/− and LDLR−/− were ApoE (apolipoprotein E) and LDLR (low-density lipoprotein receptor) knockout mice, respectively. They originally came from Jackson Laboratories. HBV transgenic mice (C57BL/6j-TgN(AlblHBV)44Bri) used in this study were also kept in Peking University Laboratory Animal Center. In addition, efficacy of RB-HBV008 was also assessed with other two disease murine models. The first one was a transgenic mouse model, C57BL/6J-M-Tg (HBV C1.0), which was established by Fudan University.47 The second one was pAAV-HBV mouse model that was established by hydrodynamic injection of pAAV-HBV1.3mer plasmid DNA (10 μg/mouse) that contains a 1.3-fold-overlength genome of HBV.48 It was kept by WuXi AppTec, a leading global contract research outsourcing provider. Toxicity study with SD rats was performed by WestChina-Frontier PharmaTech Co., Ltd., and the animals were kept in their facility. All procedures involving experimental animals were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee (IACUC) of Peking University, or Fudan University, or WuXi AppTec, or WestChina-Frontier PharmaTech.
ApoB knockdown in normal C57BL/6 mice
Apolipoprotein B (apo B) is encoded by the APOB gene and dominantly expressed in the liver. It is the primary apolipoprotein of chylomicrons, VLDL, IDL, and LDL particles, which is responsible for carrying fat molecules, including cholesterol, around the body to all cells within all tissues. Here, siRNA-against apoB (siApoB) was used to evaluate the delivery efficiency of RBP131. Male C57BL/6 mice were randomly divided to nine groups with eight mice per group. Then they were given 1 × PBS, naked siRNA, MC3/siRNA complex (positive control, PosCtl), and RBP131/siApoB complex, respectively. The dose of naked siRNA and MC3/siRNA were 1 and 0.1 mg/kg, respectively. The doses of RBP131/siRNA were 1.0, 0.5, 0.25, 0.1, 0.05, 0.01 mg/kg, respectively. After 72 h, animals were sacrificed by cervical isolation. Blood samples were collected by retroorbital eye bleed. Tissue samples were subjected to RNAlater® (Sigma–Aldrich), kept at 4 °C for 24 h, and then transferred to −80 °C for storage. Subsequently, tissues were homogenized, followed by adding RNAVzol (Vigorous Biotechnology Beijing Co., Ltd., Beijing, China) and extracting total RNA according to the manufacturer’s protocol. After finishing reverse transcription with total RNA (1 μg), cDNA (50 ng) was quantified by RT-PCR system using SYBR Green PCR Master Mix. The expression level of apoB or HBV gene was analyzed using the Ct (cycle threshold) values with the standard protocol. β-actin was selected as the reference gene. Then a fitting sigmoid curve for apoB mRNA knockdown was generated with GraphPad Prism software according to a function of Y = Bottom + (Top-Bottom)/(1 + 10^((LogIC50-X) × HillSlope)), by which the ED50 of RBP131/siApoB was calculated.
To evaluate lipid accumulation in the liver resulting from the knockdown of the apoB gene, lipids in the liver were extracted and detected. When mice were sacrificed, a piece of the liver was collected and stored at −80 °C. Then 50 mg of the liver tissue was measured with analytical balance (Mettler–Toledo International, Inc., Columbus, OH, USA), followed by adding 1 ml solution of chloroform and methanol (2:1, v/v), homogenizing with homogenizer (Superfine Homogenizers, Fluko®, Essen, Germany), and keeping at 4 °C overnight. Then 0.3 ml of deionized water was added into the sample, followed by mixing with vortex and centrifuging for 10 min at 8,000 rpm at 4 °C (Eppendorf® Centrifuge 5417 R, Eppendorf, Hamburger, Germany). Then solution at the organic phase (~200 μl) was collected and volatilized to dryness by centrifuging with concentrator 5301 (Eppendorf®, Hamburger, Germany). Subsequently, the dried sample was re-dissolved with 600 μl of 1 × PBS containing 5% (v/v) triton, incubated at 60 °C for 30 min, and ultrasonically treated for 30 min. The procedures of incubation at 60 °C and ultrasonic treatment were repeated once until the lipids were totally dissolved. Finally, the TG and CHO levels in the liver were detected with commercial detecting Kits (Biosino Bio-Technology and Science Incorporation, Beijing, China) according to the manufacturer’s protocols.
In parallel, pieces of the livers were embedded with OCT. Eight-micrometer cryosections were prepared and stained with Oil Red O. Then they were observed using an inverted fluorescence microscope (Olympus X71, Olympus, Tokyo, Japan), from which the lipid accumulation was also analyzed.
In addition, serum samples were analyzed by Beijing DIAN Clinical Laboratory Co. Ltd., which is a company providing preclinical and clinical analysis services. Concentrations of TG and CHO in serum were detected with a biochemistry analyzer.
To evaluate the longevity of gene silencing triggered by RBP131/siRNA, a duration assay was performed. Three groups of mice were given RBP131/siApoB complex at the doses of 1.0, 0.5, and 0.25 mg/kg, respectively. Normal saline was included as a control. The concentration of total cholesterol (CHO) was examined on days 2, 4, 7, 14, 21, and 28 post-administrations according to the above-mentioned methods.
Gene knockdown with ApoE−/− and LDLR−/− knockout mice
In order to explore the mechanism of RBP131 in mediating siRNA delivery into hepatocytes, wild type C57BL/6, ApoE−/− (ApoE knockout), and LDLR−/− (LDLR knockout) mice were employed. Three kinds of mice were divided into four groups (six mice per group), respectively. For each kind of mice, RBP131/siApoB was dosed at 0.5, 0.1, and 0.05 mg/kg, respectively. 1 × PBS was included as a negative control. Expression level of apoB mRNA was analyzed at 72 h post-administration by quantitative RT-PCR.
Inhibition of HBV mRNA expression in transgenic mice
HBV transgenic mice C57BL/6J-TgN(AlblHBV) 44Bri/J containing HBV genome S, pre-S and X domains,46 were from Peking University Health Science Center (Beijing, China). Seven to ten-week-old mice were employed to evaluate the gene silencing efficiency of anti-HBV siRNA (siHBV) encapsulated by RBP131. Here, six groups of mice (eight animals per group, with half males and half females) were dosed with saline, naked siHBV, MC3-loaded siHBV (MC3/siHBV), empty RBP131, RBP131-loaded scramble siRNA (or NC siRNA), and RBP131-loaded siHBV (RB-HBV008), respectively. The dose of siRNA was 1 mg/kg. The dose of empty RBP131 (lipids) was the same as the dose of lipids contained in RB131/siHBV complex. Seventy-two hours later, mice were sacrificed by cervical dislocation. Liver samples were collected and the expression level of HBV gene was analyzed according to the above-mentioned protocol.
In vivo efficacy of RB-HBV008 in disease models
To evaluate in vivo anti-HBV efficacy of RB-HBV008, three HBV mouse models were applied. The first one is C57BL/6J-TgN(AlblHBV) 44Bri/J, which is a transgenic mouse model containing partial HBV genome, as described before. The second one is C57BL/6J-M-Tg (HBV C1.0), which is another transgenic mouse model containing 1.0 copies of HBV genome. It can generate most virus elements, e.g., HBsAg and HBcAg. However, it cannot product the HBV virion. The third one is pAAV-HBV mouse model. This model is established by hydrodynamic injection of pAAV-HBV1.3mer plasmid DNA that contains a 1.3-fold-overlength genome of HBV. It can generate all virus elements, such as HBsAg, HBeAg, HBV DNA, as well as virions. For efficacy evaluation in C57BL/6J-M-Tg (HBV C1.0) and pAAV-HBV mouse models, single or multiple doses of RBP131/siRNA formulation were intravenously injected into the mice at indicated dosages, which was shown in the figures or figure legends. For multiple dosing experiments, siRNA was weekly or biweekly injected into the mice, which was also shown in the figure legends. At indicated time points after treatment, serum and/or tissue samples were collected. Multiple parameters, e.g., HBsAg, HBeAg, virus DNA, and virus mRNA, were analyzed by ELISA or real-time PCR.
Toxicity study in rodent
Male CD-1 mice were randomly divided into seven groups with eight mice per group. Then they were given following formulations at the dose volume of 10 mL/kg respectively: (1) without treatment; (2) 1 × PBS, (3) lipopolysaccharides (LPS) (5 mg/kg); (4) poly I:C (10 mg/kg); (5) RBP131/siRNA complex (1 mg/kg); (6) RBP131/siRNA complex (3 mg/kg); (7) RBP131/siRNA complex (5 mg/kg). LPS and poly I: C were administered via intraperitoneal (i.p.) injection. Other samples were administered via intravenous (i.v.) injection. Blood samples were collected at 3, 24, and 48 h after injection from the animals. Weights of the body, the liver, and the spleen were recorded to calculate organ coefficients. Immunoassays were used to measure the levels of cytokines (TNF-α, IFN-γ, IL-1β, IL-2, IL-5, IL-6, IL-12(p70), MCP-1, KC, GM-CSF) in a 96-well plate using Luminex® assays (high-throughput multiplex bead-based assays) with Luminex 100 system (Luminex Corporation, Austin, Texas, USA). Clinical chemistry of ALT (alanine transaminase), AST (aspartate transaminase), CRE (creatinine), and BUN (blood urea nitrogen) was measured by biochemical analyzer. Meanwhile, livers of the mice sacrificed at 48 h were fixed in 10% formaldehyde, embedded in paraffin, sectioned with Microtome (Leica, Germany), stained with H&E, and at last observed histological change with an optical microscope (Olympus X71, Olympus, Tokyo, Japan).
Toxicity evaluation was also performed with SD rats. Animals were divided into two groups with 10 animals (5 males and 5 females) per group. One group received 1 × PBS treatment, another received three doses of RBP131-formulated siRNA at the dose of 5 mg/kg. Three doses were administrated on day 1, day 6, and day 12, and the experiment was terminated on day 14. Dosing volume was kept at 20 mL/kg. When the animals were sacrificed on day 14, blood biochemistry was analyzed. In addition, the main organs include of the liver were fixed with 10% formaldehyde, then embedded in paraffin, at last, observed histological change with an optical microscope.
Statistical analysis
The data were expressed as the mean ± SD or mean ± SEM, as indicated in figure legends. One-way or two-way analysis of variance (ANOVA) was used when there were multiple comparisons. A two-tailed Student’s t test was used for single comparisons. The specific statistical methods are indicated in the figure legends. Significance was defined as *P < 0.05, **P < 0.01, and ***P < 0.001.
