Cholesterol Conjugation Strategies to Enhance In Vivo Antisense Oligonucleotide-Mediated Exon Skipping in the Mouse Retina

Abstract

Purpose:

Antisense oligonucleotide (AON)-mediated exon skipping is a potential therapeutic approach to certain inherited retinal diseases, including Usher’s Syndrome Type 2 A. Heteroduplex AONs (HAONs) have been reported to enhance RNA-modulating activity in vivo; this study evaluated whether the HAON strategy improves Ush2a exon skipping activity in the mouse retina.

Methods:

AONs of different chemical makeup targeting Ush2a exon 12 were formulated as HAONs with and without lipid conjugation. Activity was evaluated in 2D and 3D cell models and in the mouse eye following intravitreal administration.

Results:

Lipid conjugation was required to increase activity in the mouse retina. However, exon-skipping effects observed for cholesterol (Chol)-conjugated HAONs in vivo were not reflected in cell models. A single dose of HAONs bearing a Chol on either strand or the Chol–AON conjugate produced up to a 4.6-fold increase in Ush2a exon 12 skipping in mouse retina over AON alone, with effects lasting at least 28 days without major tolerability findings.

Conclusion:

Chol conjugation—either to a HAON construct or directly to the AON—can substantially enhance the potency of retina-targeted exon-skipping AON therapies in vivo.

Keywords

antisense conjugate delivery therapeutic splicing retina

Introduction

Antisense oligonucleotide (AON) therapeutics that modulate RNA—through exon skipping or inclusion, A→I editing, upregulation, and knockdown—are emerging treatments for genetic disease.^1–8 With substantial structure/activity principles identified for single-stranded AONs, especially for the class of RNase H-competent gapmers,^8–10 emphasis is shifting to ligand-assisted targeting, delivery, and endosomal escape.¹¹

Heteroduplex oligonucleotides (HAONs/HDOs), in which an AON is paired with a complementary sense strand, can improve pharmacodynamics in vivo and even support uptake over the blood–brain barrier; sense strand conjugation to tocopherol (Toc) or cholesterol (Chol) is essential.^12–16 Duplexes of gapmers with RNA (or DNA¹⁷)-based sense strands enable intracellular release of the active AON, in which altered trafficking has been proposed as a key mechanism.¹⁸ The HAON concept has also been extended to splice-modulating AONs.^15,19,20

Mutations in the USH2A gene may lead to Usher’s Syndrome Type 2A, a rare inherited retinal disease.^21,22 Skipping of mouse Ush2a exon 12 would be equivalent to skipping of human USH2A exon 13, which is a potentially therapeutic approach for a subgroup of Usher’s Syndrome Type 2A patients.²³

Here we report our evaluation of HAONs with DNA-based sense strands for Ush2a exon 12 skipping in the mouse retina and compared Chol positioning on the sense strand, on the AON, or on both strands and assessed activity and tolerability after intravitreal (IVT) injection in mice.

Materials and Methods

Oligonucleotides

Oligonucleotides as indicated in Figure 1 and Table 1 obtained from EuroGentec (*) and Gene Tools (**) were used as received; the other compounds were synthesized in-house by ProQR according to standard procedures using commercially available phosphoramidite monomers. After completion of the solid-phase synthesis, the solid support was incubated in a sealed container with aq. NH₄OH (28%–30%) at 55°C for 18 h to allow cleavage and deprotection. The support was removed by filtration, and the solution was washed with water. The crude oligonucleotide was analyzed by LCMS to confirm the identity and determine the crude purity. Oligonucleotides were purified by anion exchange (IEX) chromatography using Source 15Q resin (buffer A: 25 mM NaOH, buffer B: 25 mM NaOH + 2.5 M NaCl, with a 10→90% gradient in 15 column volume (CV). Fractions were analyzed by ultra-performance liquid chromatography (UPLC) analysis using an ACQUITY UPLC BEH C18 Column (Waters, 186002350) at 80°C (buffer A: 385 mM hexafluoroisopropanol (HFIP), 14.5 mM triethylamine (TEA), and 5% MeOH in water; buffer B was identical but with 50% MeOH; with a gradient of 30%–50% over 12 min with a flow rate of 1 mL/min). Fractions with >85% purity were pooled, desalted, and concentrated using vivaspin ultrafiltration with a 3 kDa molecular weight cut-off. The identities and purities of all oligonucleotides were confirmed using LCMS. The final desalted oligonucleotides were lyophilized.

Table 1.

Oligonucleotides in This Study

Name	Sequence (5′→3′)	MW_calc	MW_found	Purity (%)
	Oligonucleotides for mouse Ush2a exon 12 skipping experiments
MOE	eA _S eA _S eC _S eU _S eC _S eU _S eG _S eG _S eA _S eG _S eG _S eA _S eA _S eU _S eU _S eU _S eA _S eA _S eA _S eU _S eC	8,372.2	8,373.5	96
Chol-MOE	Chol _O eA _S eA _S eC _S eU _S eC _S eU _S eG _S eG _S eA _S eG _S eG _S eA _S eA _S eU _S eU _S eU _S eA _S eA _S eA _S eU _S eC	8,964.4	*	*
PMO	oA _N oA _N oC _N oU _N oC _N oU _N oG _N oG _N oA _N oG _N oG _N oA _N oA _N oU _N oU _N oU _N oA _N oA _N oA _N oU _N oC	**	**	**
Sense	mG _S mA _S mU _S dU _O dU _O dA _O dA _O dA _O dU _O dU _O dC _O dU _O dU _O dU _O dU _O dA _O dG _O dA _S mG _S mU _S mU	6,685.7	6,684.6	93
Toc-sense	Toc _O mG _S mA _S mU _S dU _O dU _O dA _O dA _O dA _O dU _O dU _O dC _O dU _O dU _O dU _O dU _O dA _O dG _O dA _S mG _S mU _S mU	7,178.6	7,177.4	97
Chol-sense	Chol _O mG _S mA _S mU _S dU _O dU _O dA _O dA _O dA _O dU _O dU _O dC _O dU _O dU _O dU _O dU _O dA _O dG _O dA _S mG _S mU _S mU	7,277.8	7,276.3	98
SCR	eA _S eG _S eA _S eG _S eA _S eA _S eU _S eG _S eA _S eA _S eU _S eC _S eU _S eC _S eU _S eG _S eA _S eA _S eU _S eU _S eC	8,372.2	8,371.8	99

For chemical structures, see Figure 1.

*Obtained from EuroGentec and used as received.

**Obtained from Gene Tools and used as received.

C, 5-methyl-C; Chol, cholesterol; d, DNA; e, 2′-MOE; m, 2′-O-Me; MW, molecular weight; _N, N,N-dimethylaminophosphorodiamidate; _O, phosphodiester; o, PMO; _S, phosphorothioate; Toc, tocopherol; U, 5-methyl-U (T).

Preparation of (H)AONs

Equimolar amounts of sense and AONs were diluted in nuclease-free water (for in vitro applications) or phosphate-buffered saline (PBS) diluted with 7% nuclease-free water (for in vivo applications). In a T100 Thermocycler (Bio-Rad), samples were incubated at 98°C for 5 min, cooled to 24°C, decreasing the temperature by 8°C/s and incubated at 24°C for 90 min. Finally, samples were cooled down and stored at 4°C until application to cells or animals. For in vivo applications, the osmolality of all test items was confirmed to be between 270 and 400 mOsm/kg using a Micro-Osmometer model 3320 (Advanced Instruments).

Animals

Wild-type female C57Bl6/J mice (Janvier, France) of different ages (phosphorodiamidate morpholino oligomer [PMO] 7-day study: 8–12 weeks, 2′-MOE 7-day study: 10 weeks, 28-day study: 33 weeks) at the start of treatment were randomly assigned to different groups, with balanced age distribution. Mice were group-housed (5 per cage) under pathogen-free conditions with a reversed light–dark cycle and cage enrichment in standard open polysulfone type II cages at constant temperature and humidity according to recommendations of the Federation of European Laboratory Animal Science Associations. Water and standard chow were provided ad libitum. Mouse experiments were approved by the local Ethics Committee of Innoser Laboratories BV, Leiden, The Netherlands) and conformed to the European Community regulations (EEC 86/609). The authors confirm adherence to the tenets of the Declaration of Helsinki in view of the use of human(-derived) cell lines and the Association for Research in Vision and Ophthalmology (ARVO) statement for the Use of Animals in Ophthalmical and Vision Research.

Mouse IVT injection, necropsy, and collection of retinae

Test items were administered bilaterally to the vitreous by means of single bolus IVT injection according to procedures described earlier.²³ In short, mice were anesthetized via intraperitoneal injection (10 μL/g body weight) of ketamine (10 mg/mL) and xylazine (0.5 mg/mL). Pupillary dilation was achieved using a topical mydriatic regimen consisting of one drop each of tropicamide (1%) and phenylephrine hydrochloride (2.5%) to facilitate visualization of the intraocular injection needle during IVT administration. The mouse head was secured in a custom-built nose bar and rotated horizontally to optimally present the eye. Eyelids were retracted, and the globe was stabilized using forceps. IVT injections were performed under direct visualization using a Leica M80 stereomicroscope. Injections were administered at an angle of approximately 45° relative to the lens to avoid damage to the lens and retinal tissue. A 2.5-μL Hamilton syringe (VWR; HAMI7632-01) fitted with a 32G needle (length 15 mm, point style 4/tap; VWR; HAMI7803-04) was used. The injection apparatus was rinsed with ethanol followed by PBS between animals. Upon visualization of the needle bevel within the vitreous chamber through the dilated pupil, 1 μL of test article was injected. Immediately thereafter, an anterior corneal counter-puncture was performed prior to needle withdrawal to reduce intraocular pressure and minimize reflux of the injected material. Following needle retraction, vascular integrity was assessed by inspecting for intraocular hemorrhage. Both eyes were treated with ophthalmic ointment (Duodrops; Medpets) to protect the cornea during recovery. After injection, mice were closely monitored for 1 h after injection. No signs of discomfort or adverse occurrences in the injected eyes were observed. Bodyweights were monitored after injection, over the course of the study and at necropsy, and did not decrease significantly. Mice were sacrificed by CO₂ asphyxiation at the indicated timepoints. Eyes used for histology were collected and fixed in Hartman’s fixative for a maximum of 24 h. Retinal tissue used for RNA isolation was removed, snap-frozen in liquid nitrogen, and stored at −80°C until RNA extraction.

RNA isolation from mouse retinae

Mouse retinae were homogenized in RLT Plus buffer (Qiagen; #74136) supplemented with 1% beta-mercaptoethanol with the MagNA Lyser (Roche) using beads (Roche; #0335894100). Two homogenization cycles at 5000 rpm (approx. 1400 g) for 30 s each were performed with 1 min cooling on ice after each cycle. The homogenate was used for RNA extraction with the RNeasy Plus Micro Kit (Qiagen; #74136) following the manufacturer’s protocol. RNA concentrations were measured using a NanoDrop 2000/2000c Spectrophotometer (Thermo Scientific), and RNA was stored at −80°C until further use.

Quantification of murine Ush2a exon 12 skipping by digital PCR

Equal amounts of total RNA obtained from retinal tissue were used as templates for random hexamer-primed cDNA synthesis with the Verso cDNA Synthesis Kit (Thermo Fisher; #AB1453B) according to the manufacturer’s instructions. cDNA was used as template for digital PCR (dPCR) with the QIAcuity Four dPCR system (Qiagen) or digital droplet PCR (ddPCR) system (Bio-Rad) according to the manufacturers’ instructions. Detailed information on primers and Taqman probes for dPCR and ddPCR assays is summarized in Table 2. For dPCR, the following PCR program was used: 1 cycle of enzyme activation for 2 min at 95°C, 40 cycles of denaturation for 15 s at 95°C and annealing/extension for 30 s at 60°C. After PCR, the plate was imaged using a 500 ms of exposure duration and a gain of 6 for all channels. Droplets for ddPCR were generated using the QX200 droplet generator (Bio-Rad) according to the manufacturer’s instructions. PCR was performed in a T100 Thermocycler (Bio-Rad) using the following PCR program: 1 cycle of enzyme activation for 10 min at 95°C, 40 cycles of denaturation for 30 s at 95°C and annealing/extension for 1 min at 60°C following enzyme deactivation for 10 min at 98°C and cooling down to 4°C. Fluorescence was quantified in the QX200 droplet reader (Bio-Rad). Copy numbers were analyzed with the QIAcuity Software Suite (Qiagen; version 2.0.20) for dPCR and QuantaSoft software (Bio-Rad; version 1.7.4.0917) for ddPCR. Thresholds were set manually. Ush2a exon 12 skipping was calculated by dividing Ush2a copies missing exon 12 by the total number of Ush2a (exons 8–9) copies. Data obtained using both methods, dPCR and ddPCR, for the same samples showed that the amounts of exon skipping detected were not different between those two methods (data not shown).

Table 2.

List of Taqman Assays

Commercial assays
Target	Species	Label	Reference	Supplier
Ush2a exon 8–9	mouse	5′-VIC	Mm01316803_m1	Applied Biosystems

Custom assays
Target	Species	Sequence	Supplier
Ush2a Δexon 12	mouse	Forward primer: 5′-GACCGTGGATGGAGACATTAC-3′Reverse primer: 5′-GCATTGCTGGGAGAACTGTA-3′Taqman probe:5′-/56-FAM/CAGTGCTCG/ZEN/TGCAAAGCGAATGTT/3IABkFQ/-3′	Integrated DNA Technologies

Histology

Lenses were removed from eyes and fixed in Hartman’s fixative. Tissue was embedded in paraffin directly or stored in 4% formaldehyde until processing. Tissue blocks were sectioned by cutting on the sagittal axis at three different depths and attached to glass slides according to standard procedures.

Visualization of AON localization in the mouse retina

Slides cut from fixed ocular tissue were hybridized with a fully complementary DNA/LNA Cy5-labeled probe (5′-Cy5-GATTTAAATTCCTCCAGAGTT-3′, with LNA underlined). Hoechst 33342 solution was used for nuclear staining. Microscopic images were acquired using an LSM800 confocal laser scanning microscope (Zeiss), with the excitation and emission of the fluorochromes Hoechst 33342 (excitation 405 nm, detection wavelength 410–617 nm) and Cy5 (excitation 640 nm, detection wavelength 645–700 nm). Airyscan images and Z-stacks (around 11 stacks of 0.3–0.5 µm thick) of retinal regions of the eye [ganglion cell layer (GCL), inner nuclear layer, outer nuclear layer (ONL)] were obtained using the 20× Plan-Apochromat oil objective.

Histopathology evaluation

Morphology of the retina was assessed after hematoxylin/phloxine/saffron (HPS) staining of slides cut from fixed retinal tissue following standard procedures. Light microscopy images of the HPS-stained sections were made with a 20× objective using an Axio Observer.A1 light microscope and ZEN blue software (Zeiss). Based on these images, the integrity of the retinal layers, including the GCL, inner nuclear layer, and ONL, was assessed.

Statistics

GraphPad Prism 9 was used for data visualization and statistical analysis. An ordinary one-way ANOVA was used, followed by Dunnett’s multiple comparisons test in case all groups were compared with one baseline condition and Tukey’s multiple comparisons in case all groups were compared with each other.

Statistical significance: ns, not significant; *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.

Results

In vitro HAON evaluation

In unassisted uptake assays, HAONs containing a DNA-based sense strand did not improve over AON-only controls using CEP290-targeting splice-switching sequences of different chemistries (2′-O-[2-methoxyethyl] phosphorothioate (2′-MOE PS), morpholino (PMO)) in ARPE-19 cells and retinal organoids (Supplementary Data). These data suggest that standard culture conditions do not recapitulate the features required for (lipid-conjugated) HAON benefit.

Short-term in vivo HAON evaluation (7 days)

Wild-type mouse eyes received a single IVT injection of 0.3 nmol (equimolar to 2.5 µg) of the Ush2a 2′-MOE PS AON (“MOE”) formulated as HAONs with DNA sense strands (Fig. 1). At day 7, conjugation of the sense strand was a differentiating factor: MOE/Toc-sense and MOE/Chol-sense produced 3.0- and 4.4-fold increases in Ush2a exon 12 skipping, respectively, over MOE alone (Fig. 2A). By contrast, an unconjugated duplex reduced activity, indicating that ligand conjugation is essential for HAON benefit in vivo.

PMO-based HAONs underperform (28 days)

An isosequential PMO formulated as HAON (0.3 nmol IVT) showed very low exon skipping at day 28 across all PMO groups compared with MOE alone (Fig. 2B). Given the minimal protein binding of naked PMOs,²⁴ due to its neutral phosphorodiamidate backbone, poor construct uptake may have limited activity. In contrast to prior work that employs cationic peptide or dendrimer conjugates to enhance ocular PMO delivery,^25–28 we discontinued PMO HAONs in favor of 2′-MOE PS chemistry.

Chol placement governs MOE HAON performance (28 days)

To assess durability and the impact of ligand position, we evaluated at day 28 (0.3 nmol IVT) the following: MOE/sense unconjugated duplex, MOE/Chol-sense, Chol-MOE/sense, Chol-MOE/Chol-sense, and the single-stranded Chol-MOE conjugate. On day 7, skipping levels persisted or improved. MOE alone increased from 11% Ush2a exon 12 skipping (day 7, Fig. 2A) to 18% (day 28, Fig. 3A). Notably, placing Chol on the MOE strand in an HAON (Chol-MOE/sense) yielded 85% skipping, a 4.6-fold increase over MOE alone and better than MOE/Chol-sense (68%). The single-stranded Chol-MOE also performed well (60%). In contrast, the double-Chol HAON (Chol-MOE/Chol-sense) showed only a modest, nonsignificant increase (28%).

Ocular distribution and histology

Fluorescent in situ hybridization (FISH) qualitatively correlated tissue distribution with activity. Constructs with the highest exon skipping (MOE/Chol-sense, Chol-MOE/sense, Chol-MOE) showed a stronger AON signal across retinal layers, including the ONL containing photoreceptors where Ush2a is expressed. The double-Chol HAON displayed a weaker ONL signal, mirroring its modest activity (Fig. 3B). Because the ONL is among the layers farthest from the vitreous, detectable signal in the GCL and inner nuclear layer (INL) was expected as material distributed into the retina.

Histopathological assessment at day 28 indicated preserved retinal architecture for most groups. Minor local retinal thinning occurred in one of four eyes in the MOE/sense duplex group. Local thinning and occasional macrophages in the vitreous were observed in several eyes treated with Chol-MOE and Chol-MOE/sense, warranting further tolerability evaluation (Fig. 3C, Supplementary Table S3).

Mechanistic considerations

The performance of Chol-bearing constructs supports a trafficking-driven mechanism in vivo. Conjugating the ligand to the sense strand is commonly preferred to avoid potential interference with antisense hybridization or intracellular routing. Nevertheless, Chol-MOE/sense exceeded MOE/Chol-sense, indicating that conjugation to the antisense strand did not interfere with the AON’s activity; either as the intact conjugate the ligand did not interfere, or the ligand may have been cleaved due to the conjugate’s design containing a metabolically susceptible phosphodiester linkage between AON and ligand to release the unconjugated AON after cleavage, preventing any potential hindrance.^29–31 Presence of such cleavable linkage does not explain the weaker activity of the double Chol HAON (Chol-MOE/Chol-sense), which arguably may reflect altered ligand metabolism or release kinetics, and/or disrupted interactions with proteins required for productive uptake. Overall, eye-specific pharmacokinetics likely contribute, as the vitreous and retinal layers present delivery environments distinct from systemic tissues towards which Chol conjugates typically have been employed.

Discussion and Conclusion

Whereas the in vitro observed effect of chemical changes to oligonucleotides themselves often translates to in vivo models, such an observation has, to the best of our knowledge, not been reported for heteroduplex delivery strategies without the use of a transfection reagent. In addition, in a recent study on lipid–oligonucleotide conjugates for systemic muscle treatment, underperformance in vivo compared with in vitro was observed,³² underscoring the importance of evaluation in models that best recapitulate circumstances relevant to the intended enhanced delivery effect. In the mouse retina, HAONs improved exon skipping efficacy only when a lipid ligand is present. After a single IVT dose, Chol-enabled constructs markedly enhanced and sustained Ush2a exon 12 skipping at day 28 after a single dose of 0.3 nmol with the following rank order: Chol-MOE/sense (85%) > MOE/Chol-sense (68%) > Chol-MOE (60%) ≫ Chol-MOE/Chol-sense (28%) > MOE (18%). FISH analysis supported improved ONL exposure for the most active test items. PMO-based HAONs were found less effective than isosequential 2′-MOE PS-based ones. Overall, Chol conjugation—on the sense strand or directly on the antisense strand—drives potent, durable exon skipping in the mouse retina with acceptable short-term tolerability, whereas placing Chol on both strands is counterproductive.

Despite these promising results, the short duration of our experiments with limited numbers of animals, the unknown fate of conjugated Chol, and its effect on longer term tolerability present limitations of our research into the feasibility of the (H)AON approach. While sex differences may exist in terms of susceptibility to retinal degradation,³³ male mice have the disadvantage of often requiring solitary housing due to aggressive behavior.^34,35 Thus, for practical and ethical reasons, test groups consisted of female mice only. Related to the test items, exogenous Chol may affect the retina, which has a tightly regulated lipid environment; repeated or high-dose exposure to exogenous lipids could disrupt retinal homeostasis. Excess Chol has been linked to photoreceptor degeneration, inflammation, cellular senescence, and age-related macular degeneration (AMD)-like pathology.^36–38 In addition, Chol can be metabolized into oxysterols, some of which are neurotoxic and implicated in inflammation.^38–40 Chol alternatives may display improved characteristics,^40,41 and conjugation with such alternatives could be considered.

Ultimately, the described approach’s feasibility to benefit patients will be determined by additional factors; apart from its translation into disease-relevant functional outcomes, extensive and larger (long-term) relevant preclinical safety (e.g., electroretinogram (ERG), optical coherence tomography (OCT)) and pharmacokinetic studies in both sexes are required, as an exon-skipping AON therapy would constitute a lifelong treatment requiring repeated administration.

Authors’ Contributions

P.C.d.V.: Conceptualization (lead), data curation (equal), supervision, writing—original draft, writing—review and editing (equal), project administration (lead); F.N.B.: Data curation (equal), formal analysis, investigation (equal), visualization (equal); D.B.: Investigation (equal); H.J.A.A.: Investigation (equal); M.T.T.: Investigation (equal), data curation, visualization (equal); W.B.: Conceptualization (supporting), writing—review and editing (equal), data curation (equal), project administration (supporting); G.J.P.: Writing—review and editing (equal), conceptualization (supporting).

Supplemental Material

sj-docx-1-opt-10.1177_10807683261469047 — Supplemental material for Cholesterol Conjugation Strategies to Enhance In Vivo Antisense Oligonucleotide-Mediated Exon Skipping in the Mouse Retina

Supplemental material, sj-docx-1-opt-10.1177_10807683261469047 for Cholesterol Conjugation Strategies to Enhance In Vivo Antisense Oligonucleotide-Mediated Exon Skipping in the Mouse Retina by Peter C. de Visser, Fabian N. Bangel, Dionne Blangé, Herma J.A. Anthonijsz, Monica T. Tartjiono, Wouter Beumer, and Gerard J. Platenburg

Footnotes

Disclosure Statement

All authors were employees of ProQR Therapeutics at the time of this research.

Funding Information

No funding was received for this article.

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