Reduction in ocular complement factor B protein in mice and monkeys by systemic administration of factor B antisense oligonucleotide

ASO synthesis and chemistry

The ASOs used were 20 nucleotides long and chemically modified with phosphorothioate in the backbone, five residues at each terminus, and a central deoxynucleotide region of ten residues (5–10–5 gapmer). 2′-O-methoxyethyl modified antisense phosphorothioate oligonucleotides (2′-MOE ASOs) were synthesized at Ionis Pharmaceuticals, Inc. (Carlsbad, CA) as described previously [38]. Oligonucleotides were synthesized using an Applied Biosystems 380B automated DNA synthesizer (PerkinElmer Life and Analytical Sciences–Applied Biosystems, Foster City, CA) and purified as previously described [39]. The ASO sequences were as follows: murine factor B ASO, ION-516323, 5′-TCC ACC CAT GTT GTG CAA GC-3′; control ASO ION-141923, 5′-CCT TCC CTG AAG GTT CCT CC-3′; monkey ASO, ION-588548, 5′-TTA ATT CAA TCC CAC GCC CC-3′. The ASOs were dissolved in PBS (137 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 2 mM KH₂PO₄, pH7.4; Ca-Mg-; Invitrogen, Thermo Fisher Scientific, Carlsbad, CA) for the in vivo experiments.

Culture of hepatocytes and treatment with ASOs

Mouse or monkey primary hepatocytes were prepared using the standard collagenase procedure described previously [40]. Electroporation of ASOs was performed using the HT-200 BTX Electroporator with the ElectroSquare Porator (ECM 830) voltage source in 96-well electroporation plates (BTX, 2 mm; Harvard Apparatus, Holliston, MA). Cells were harvested 16 h after electroporation. Cells were electroporated in the presence of factor B ASOs at the indicated concentrations and plated. Sixteen hours after transfection, total cellular RNA was isolated, and the amount of factor B mRNA was quantified using a quantitative real-time PCR (qRT-PCR) assay (TaqMan, Applied Biosystems, Thermo Fisher Scientific).

Quantitative real-time PCR

The cultured cells were lysed, and total RNA was extracted with a QIAGEN (Valencia, CA) RNeasy column. Animal tissues were homogenized in a guanidine isothiocyanate solution (Invitrogen) supplemented with 8% 2-mercaptoethanol (Sigma-Aldrich, St Louis, MO). Total RNA was prepared using the RNeasy mini kit instructions (QIAGEN). The qRT-PCR analyses were performed using an ABI Prism 7700 sequence detector (Applied Biosystems). PCR results were normalized to total RNA measure with Quant-iT RiboGreen RNA Reagent (Molecular Probes, Eugene, OR). The sequences of primers and probes used were as follows: for mouse factor B: forward: 5′-GGG CAA ACA GCA ATT TGT GA-3′, reverse: 5′-TGG CTA CCC ACC TTC CTT GT-3′, probe: 5′-Fam-CTG GAT ACT GTC CCA ATC CCG GTA TTC C-Tamra-3′; monkey factor B: forward: 5′-CGA AGA AGC TCA GTG AAA TCA A-3′, reverse: 5′-TGC CTG GAG GGC CCT CTT-3′, probe: 5′-Fam- AGA CCA CAA GTT GAA GTC-Tamra-3′; mouse cyclophilin A: forward: 5′-TCG CCG CTT GCT GCA-3′, reverse: 5′-ATC GGC CGT GAT GTC GA-3′, probe: 5′-CCA TGG TCA ACC CCA CCG TG TTC-Tamra-3′; monkey cyclophilin A: forward: 5′-CGA CGG CGA GCC TTT G-3′, reverse: 5′-TCT GCT GTC TTT GGA ACC TTG TC-3′, probe: 5′-CGC GTC TCC TTC GAG CTG TTT GC-Tamra-3′. PCR conditions, holding stage: 15 min at 50 °C, 5 min at 95 °C. Cycling stage: 15 msec at 95 °C, 1 min at 60 °C for 40 cycles. PCR results were normalized to total RNA measure with Quant-iT RiboGreen RNA Reagent (Molecular Probes).

Animal studies

Mouse studies: Male wild-type C57BL/6J mice (Jackson Laboratories, Bar Harbor, ME) were housed four animals per cage at 22–25 °C with a 12 h: 12 h light-dark cycle and free access to food and water. The ASO drugs were prepared in PBS and were administered by subcutaneous injection. Dosages are reported as the weekly dose for all the animal studies, and the mice were dosed subcutaneously once per week. On the day of necropsy, the mice were euthanized with isoflurane inhalation. Plasma was collected by cardiac puncture and frozen at −70 °C or less. Liver and whole eyes were collected when the mice were euthanized approximately 72 h after dosing was completed.

Monkey study: Male cynomolgus monkeys received loading doses of 0 (vehicle control, n = 6) or 40 mg/kg of ION-588548 (n = 4), every 2 days during the first week of the study (on days 1, 3, 5, and 7) followed by once a week administration thereafter, commencing on day 14 (40 mg/kg/wk). Doses were administered by subcutaneous injection for 12 consecutive weeks and calculated based on the most recently recorded bodyweight. Plasma for factor B analysis was collected at days −6, 1, 30, 58, 72, and 86. At the terminal necropsy, liver samples and whole eyes were collected for factor B mRNA and protein analysis. On the day of the necropsy, the monkeys were sedated, weighed, and anesthetized followed by exsanguination and necropsy. The study was conducted at Korea Institute of Toxicology (KIT).

Study approval

All the animal studies were conducted under protocols approved by the Institutional Animal Care and Use Committee (IACUC) of KIT and Ionis Pharmaceuticals. The monkey study was conducted at an Association for Assessment and Accreditation of Laboratory Animal Care (AAALAC) accredited facility that has an Animal Welfare Assurance issued by the Office of Laboratory Animal Welfare (OLAW), is registered with the United States Department of Agriculture (USDA), and has an Institutional Animal Care and Use Committee (IACUC) that is responsible for compliance with applicable laws and regulations regarding the use of laboratory animals. The study protocol was approved by the IACUC at the testing facility before dose administration.

Factor B western blot

Blood was collected under anesthesia via cardiac puncture into sample tubes coated with the anticoagulant EDTA. Intracardiac blood collection was performed once at the end of the study, before the necropsy. Blood was centrifuged at 4,000 ×g for 15 min, and platelet-poor plasma was collected and stored at –80 °C before western blotting. One microliter of plasma samples from all groups was analyzed with sodium dodecyl sulfate–polyacrylamide gel electrophoresis (PAGE) followed by immunoblotting with antibodies to factor B. The antifactor B antibody was obtained from Sigma (Cat #HPA001817), was produced in rabbits, and was specific to human, mouse, and rat full-length factor B protein.

In the perfusion experiments, 9-week-old, wild-type C57BL/6J mice were subjected to trans-cardial perfusion with Dulbecco’s PBS (137.9 mM NaCl, 2.7 mM KCl, 8 mM Na₂HPO₄, 1.5 mM KH₂PO₄, pH7.3; DPBS, no calcium, no magnesium; Invitrogen, Thermo Fisher Scientific, Cat.: 14190144) to remove the blood from the tissues. Briefly, a catheter infusion set was connected to a 20 ml syringe, and the needle was inserted in the left ventricle. The right atria was cut, and 15–20 ml of DPBS+10 U/ml heparin (Sigma, Cat: H3393) was slowly pumped through the heart. A steady flow of about 10 ml/min was applied. Following perfusion with DPBS+heparin, an additional 5 ml of DPBS was pumped through the heart to wash out any residual heparin. The eye and liver samples were collected and snap frozen in liquid nitrogen.

One eye from each animal was used for protein extraction and western blot analysis. The eye was homogenized in buffer containing protease inhibitors and incubated for 30 min on ice. The eye homogenate was then centrifuged for 10 min at 17,949 ×g. The resulting supernatant was used for western blot analysis. The plasma and the protein extracts from the eye and liver were analyzed with sodium dodecyl sulfate PAGE followed by immunoblotting with an anti-human polyclonal antibody against factor B (Sigma HPA001817, 1:1,000). To ensure equal loading on the gel, an antibody anti-apolipoprotein E (ApoE; Abcam, Cambridge, MA, 1:5,000) was used for the plasma samples. Anti-alpha tubulin antibody (Abcam, ab7291, 1:10,000) was used for the eye and liver samples. The quantification of the western blot images was performed using Image J software. Recombinant factor B (Abcam, ab151392) was included as a control.

Plasma factor B analysis

Plasma factor B levels were analyzed at the Complement Laboratory at National Jewish Health. Factor B levels were measured with radial immunodiffusion (RID; CL.0012), using an anti-human Factor B antibody preparation incorporated into an agarose gel to form immunoprecipitation in a ring around the application well. The area of the precipitin ring (minus the area of the well) is proportional to the amount of antigen (factor B) that is present in the test specimen. The reference range for cynomolgus monkeys is 127.6–278.5 µg/ml.

Statistical analysis

Values presented represent the mean ± standard error of the mean (SEM). Statistical differences between groups were determined using one-way ANOVA with Tukey honest significant difference (HSD) multiple comparisons. A p value of less than 0.05 was considered statistically significant.

Identification of mouse factor B ASO (ION-516323) and monkey factor B ASO (ION-588548)

ASOs are short synthetic strings of nucleotides designed to prevent the expression of a targeted protein by selectively binding to the RNA that encodes the targeted protein with high affinity and thus preventing translation [41]. The factor B ASO hybridizes to its complementary mRNA target through well-characterized Watson–Crick base pairing and triggers mRNA cleavage by RNase H1, leading to the inhibition of target RNA translation and subsequent reduction in target protein levels [39].

To identify active ASOs that specifically target mouse or monkey factor B mRNA for in vivo studies, we designed and screened in vitro more than 150 ASOs for each species. The efficacy of the ASOs was determined in mouse or monkey primary hepatocytes as previously described [42]. All the ASOs tested were 20 base chimeric 2’-MOE phosphorothioate oligonucleotides incorporating a 5–10–5 design, that is, five 2′-O-methoxyethyl nucleotides at the 5′ end, ten deoxynucleotides in the center, five 2′-O-methoxyethyl nucleotides at the 3′ end, and phosphorothioate backbone substitution throughout [39]. An example of the range of activities of several of the ASOs tested is presented in Appendix 1. Active ASOs were further evaluated in dose–response studies. The IC₅₀ achieved for the ASOs selected for in vivo studies were 0.5 μM for ION-516323 (mouse) and 0.7 μM for ION-588548 (monkey).

Administration of factor B ASO in wild-type mice effectively reduces liver factor B mRNA and plasma factor B protein levels

To establish the relationship between liver factor B mRNA reduction by the factor B ASO and plasma factor B levels, the factor B ASO were administered to wild-type C57BL/6J mice, four mice for each treatment group, by subcutaneous injections at doses of 12, 25, 50, 75, or 100 mg/kg/wk, for 6 weeks (Figure 1A). Liver factor B mRNA levels were reduced in a dose-dependent manner, reaching a maximum reduction of 95±0.1% of saline (Figure 1B). The calculated ED₅₀ of the murine factor B ASO (ION-516323) was 18 mg/kg/wk (Figure 1C). Liver factor B mRNA levels in the control ASO-treated (50 mg/kg/week) mice were not different from those in the saline-treated control group. ASO drugs are distributed broadly to many organs with the exception of the central nervous system (CNS) and the eyes, as ASO drugs are unable to pass the blood–brain or blood–retina barrier [43]. Therefore, as expected, the ocular mRNA levels of factor B were not changed with systemic administration of the factor B ASO (Figure 1D). Although the factor B mRNA levels were measurable in the whole eye preparations, the ocular level of factor B mRNA was less than 1% (0.86+0.03%) of the liver levels.

We next analyzed the effect of the administration of the factor B ASO on plasma factor B levels with western blot. The analysis showed that administration of the factor B ASO resulted in a dose-dependent reduction in the plasma factor B levels (Figures 1E,F). The reduction in the factor B mRNA level correlated with the reduction in factor B plasma protein (Figure 1G).

Administration of factor B ASO effectively reduces ocular factor B protein levels in mice and in cynomolgus monkeys

To evaluate the effect of systemically administered factor B ASOs on ocular factor B levels, species-specific factor B ASOs were administered to mice or cynomolgus monkeys by subcutaneous injection for 6 or 13 weeks, respectively. In both species, following the subcutaneous administration of the ASO, the ASO binds to plasma proteins and transfer rapidly from blood to tissues with a distribution half-life of 1–2 h. Once the ASO is in tissues, clearance is dramatically slower than that observed in plasma, with tissue half-life reported to range from about 1 to 4 weeks [44]. The long half-lives of ASOs allow for infrequent (once weekly or less) dosing, coupled with a robust safety profile, including chronic toxicology [45].

Wild-type C57BL/6J mice (n = 4 per treatment group) received 25, 50, 75, and 100 mg/kg/wk the factor B ASO by subcutaneous injections for 6 weeks. A control mismatch ASO at 100 mg/kg/wk was used as negative control. As observed in the previous experiment, the administration of the factor B ASO resulted in a significant and dose-dependent reduction in the factor B mRNA levels in the liver. The maximum reduction achieved was 79±10% with factor B ASO ION-516323 (Appendix 2). Plasma factor B protein levels were analyzed with a western blot using 1 µl of plasma. As shown in Figure 2A, the levels of factor B protein were reduced in a dose-dependent manner. Moreover, systemic administration of the factor B ASO resulted in marked reductions in ocular factor B protein levels in mice and in the 50 and 100 mg/kg/week treatment groups. Total whole eye protein extract was purified from all mice from the various treatment groups. Ocular factor B protein levels were determined with western blot (Figure 2B). The robust reduction in ocular protein corresponded with the reductions of factor B in the plasma.

To demonstrate that the major source of ocular factor B protein is the circulating factor B, we removed all blood from wild-type mice by trans-cardial perfusion with PBS. Subsequently, we evaluated the level of ocular factor B protein level with western blot. The results show that the ocular factor B levels are significantly reduced following perfusion (Figure 3A,B). However, the factor B levels in the perfused livers were comparable to the levels in the unperfused tissues (Figure 3C,D). In addition, we compared factor B expression in the murine liver and eye. Using qRT-PCR and western blot analysis, we determined at the mRNA and protein levels that ocular factor B expression is about ten- to 100-fold less than the hepatic factor B expression (data not shown).

The in vivo efficacy of the monkey-specific factor B ASO, ION-588548 was also evaluated with subcutaneous administration to cynomolgus monkeys. Two- to four-year-old male monkeys were dosed with 40 mg/kg/wk of ION-588548 (n = 4) or saline (n = 6) for 12 weeks. Plasma, eye, and liver samples were collected after the animals were euthanized and analyzed for factor B mRNA and protein levels. As expected, a significant reduction in the hepatic complement factor B mRNA levels was observed following the administration of the factor B ASO (−66±7% compared to saline control). Similar to the observations in mice, the ocular factor B mRNA levels in animals treated with the factor B ASO were not significantly different from those in animals treated with saline (−19±8% of saline control). Moreover, the monkeys treated with the factor B ASO demonstrated a time-dependent reduction in plasma factor B levels, achieving an 80±6% reduction in plasma factor B at day 72 (Figure 4). Furthermore, the level of factor B protein in the monkey eye, as determined with western blot, was dramatically reduced after the systemic factor B ASO treatment, reaching undetectable levels (Figure 5).