Antisense Oligonucleotides Targeting a NOTCH3 Mutation in Male Mice Ameliorate the Cortical Osteopenia of Lateral Meningocele Syndrome

Abstract

Lateral Meningocele Syndrome (LMS) is a monogenic disorder associated with NOTCH3 pathogenic variants that result in the stabilization of NOTCH3 and a gain-of-function. A mouse model (Notch3^em1Ecan) harboring a 6691TAATGA mutation in the Notch3 locus that results in a functional outcome analogous to LMS exhibits cancellous and cortical bone osteopenia. We tested Notch3 antisense oligonucleotides (ASOs) specific to the Notch3^{6691- TAATGA} mutation for their effects on Notch3 downregulation and on the osteopenia of Notch3^em1Ecan mice. Twenty-four mouse Notch3 mutant ASOs were designed and tested for toxic effects in vivo, and 12 safe ASOs were tested for their impact on the downregulation of Notch3^6691-TTATGA and Notch3 mRNA in osteoblast cultures from Notch3^em1Ecan mice. Three ASOs downregulated Notch3 mutant transcripts specifically and were tested in vivo for their effects on the bone microarchitecture of Notch3^em1Ecan mice. All three ASOs were well tolerated. One of these ASOs had more consistent effects in vivo and was studied in detail. The Notch3 mutant ASO downregulated Notch3 mutant transcripts in osteoblasts and bone marrow stromal cells and had no effect on other Notch receptors. The subcutaneous administration of Notch3 mutant ASO at 50 mg/Kg decreased Notch^6691-TTATGA mRNA in bone without apparent toxicity; microcomputed tomography demonstrated that the ASO ameliorated the cortical osteopenia of Notch3^em1Ecan mice but not the cancellous bone osteopenia. In conclusion, a Notch3 ASO that downregulates Notch3 mutant expression specifically ameliorates the cortical osteopenia in Notch3^em1Ecan mice. ASOs may become useful strategies in the management of monogenic disorders affecting the skeleton.

1. INTRODUCTION

Notch receptors (Notch1 through 4) are determinants of cell fate and function in multiple cellular organizations including the skeleton [1, 2]. Notch1, 2 and 3 and low levels of Notch4 transcripts are detected in bone cells, where each receptor plays a distinct role influencing the fate of cells of the osteoblast and osteoclast lineages [3]. Interactions of Notch receptors with ligands of the Jagged and Delta-like families result in the cleavage of NOTCH and activation of signaling following the release of the NOTCH intracellular domain (NICD), and its translocation into the nucleus to induce the transcription of target genes [4–6]. These include genes encoding Hairy Enhancer of Split (HES)1, 5 and 7 and HES-related with YRPW motif (HEY)1, 2 and L [7].

Each Notch receptor has distinct patterns of cellular expression and activity and pathogenic variants associated with each receptor result in distinct clinical syndromes. NOTCH3 is expressed in mural vascular cells where it plays a critical function in vascular homeostasis [8]. Pathogenic variants of NOTCH3 associated with mutations in its extracellular domain cause cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) [8–11]. In bone, Notch3 is expressed in osteocytes and osteoblasts and its activation in these cells increases intracortical bone remodeling [12].

Lateral meningocele syndrome (LMS), also known as Lehman Syndrome, (Online Mendelian Inheritance in Man 130720) is a devastating disorder associated with pathogenic variants of NOTCH3 and presenting with meningoceles, distinct facial features, developmental delay, decreased muscle mass, cardiac valve defects, short stature, scoliosis and craniofacial abnormalities [13–16]. LMS is associated with mutations or deletions in exon 33 of NOTCH3, that result in the creation of a STOP codon upstream of sequences coding for the proline (P), glutamic acid (E), serine (S) and threonine (T) (PEST) domain. As a result, the PEST domain is not translated and NOTCH3 is presumably stable, and a gain-of-function occurs [14]. Autosomal dominant inheritance and de novo heterozygous mutations are reported. Treatment of LMS is not available. To understand mechanisms operating in the pathogenesis of LMS and develop potential therapeutic agents to influence the outcome of LMS, we created a mouse model, Notch3^em1Ecan (syn Notch3^tm1.1Ecan), that recapitulates the genetic variant from LMS. Notch3^em1Ecan mice harbor a 6691-TAATGA mutation in exon 33 that results in a STOP codon upstream of the PEST domain [17]. The mice exhibit a NOTCH3 gain-of-function, cortical and cancellous bone osteopenia [17].

A number of approaches have been devised to downregulate Notch signaling and could serve to temper conditions where a gain-of-function exits. Often Notch inhibitors are not specific for Notch signaling or specific to a given Notch receptor [18–23]. Antibodies to the negative regulatory region (NRR) of Notch have been effective at preventing the activation of individual Notch receptors, including NOTCH3, but they do not target the Notch pathogenic variants and as a consequence they downregulate wild type as well as the mutant Notch receptor [24–26].

The use of antisense oligonucleotides (ASO) is a novel therapeutic approach of RNA modulation, and ASOs have been effective in a variety of tissues including the central and peripheral nervous system, retina, liver, muscle and bone [27–36]. ASOs are single-stranded synthetic nucleic acids that bind target mRNA by Watson-Crick pairing resulting in mRNA degradation by RNase H [37, 38]. In previous studies, we employed the systemic administration of ASOs targeting either wild type Notch2 or Notch3 to downregulate these genes in mice harboring Notch2 or Notch3 mutations resulting in a gain-of-function [36, 39]. Although the approach was successful in ameliorating the osteopenia of both mouse models, the ASOs employed were not allele selective and they did not target the mutant gene specifically.

The purpose of the present work was to evaluate if by targeting the mutant allele in heterozygous Notch3^em1Ecan mice, we could preserve the wild type allele creating a more normal genetic outcome. Because Notch3^em1Ecan mice present with osteopenia, we asked whether a more favorable genetic outcome would ameliorate this aspect of the Notch3^em1Ecan phenotype. Ionis pharmaceuticals designed and synthesized ASOs that would target specifically the Notch3^6691-TAATGA mutation harbored by the Notch3^em1Ecan mouse. Notch3 mutant specific ASOs were tested in vitro and in vivo for their tolerability and for effects on the downregulation of wild type and mutant Notch3 transcripts and on the skeletal microarchitecture of heterozygous Notch3^em1Ecan mice.

2. MATERIALS AND METHODS

2.1. Notch3^em1Ecan Mutant Mice

Heterozygous Notch3^em1Ecan (syn Notch3^tm1.1Ecan) mice in a C57BL/6 genetic background were used to test the efficacy of ASOs in vitro and in vivo. Notch3^em1Ecan mice harbor a tandem termination at bases 6691–6696 (ACCAAG→TAATGA) in exon 33 of Notch3 that results in the same functional outcome as the one found in LMS [17]. Genotypes were determined by PCR analysis of tail DNA using forward primer 5’-GTGCTCAGCTTTGGTCTGCTC-3’ and either reverse primer 5’-CGCAGGAAGCGCGCTCATTA-3’ for Notch3^em1Ecan mutant or 5’-CGCAGGAAGCGGGCCT TGG-3’ for the wild type allele (Integrated DNA Technologies, Coralville, IA). To characterize and study the effect of Notch3 ASOs in Notch3^em1Ecan mice, heterozygous Notch3^em1Ecan mutants were crossed with wild type mice to generate ~50% heterozygous Notch3^em1Ecan mice and 50% control littermates. Studies were approved by the Institutional Animal Care and Use Committee of UConn Health.

2.2. Notch3 Antisense Oligonucleotides

ASOs used in the current study consist of a central stretch of 10 DNA nucleotides flanked on either side by 3 nucleotides containing constrained ethyl (cEt) modifications. Additionally, the phosphodiester internucleotide linkages were replaced with phosphorothioate (PS). Twenty-four ASOs targeting the Notch3^6691-TAATGA mutant pre-mRNA were designed in silico by Ionis Pharmaceuticals (Carlsbad, CA). ASOs covering at least one of the nucleotides that are mutated in the Notch3^em1Ecan mouse were designed (Figure 1). A control ASO that does not hybridize to any specific mRNA sequence was included in all experiments. Oligonucleotides were synthesized at Ionis Pharmaceuticals using an AKTA Oligopilot, as described previously [40]. Initially, 24 Notch3 mutant ASOs were tested for toxicity at Ionis Pharmaceuticals (Carlsbad, CA) in 8-week-old BALB/c mice. To this end, male mice were administered ASOs at a dose of 50 mg/Kg subcutaneously once a week for a total of 4.5 weeks (5 doses) and euthanized 72 h after the last dose of ASO. Body weights were determined weekly, and liver, kidney, and spleen were weighed after euthanasia and normalized to body weight and compared with organs from control mice. Blood was obtained by cardiac puncture, and plasma was collected for the measurement of alanine aminotransferase, aspartate aminotransferase, total bilirubin, albumin, and blood urea nitrogen. These procedures were performed at, and approved by, the Animal Care and Use Committee of Ionis Pharmaceuticals. Twelve well tolerated mouse ASOs targeting the mutant Notch3 sequence, and a control ASO that does not hybridize to any specific mRNA sequence, were selected for further study.

Figure 1.

Twenty-four ASOs were designed to target the mouse mutant Notch3 pre-mRNA specifically and induce degradation of the LMS-causing transcript. Grey bars represent the site targeted by each ASO. The colored letters inside the grey bars represent the residues that are mutated in the LMS compared to the wild-type mouse Notch3 transcript. The wild-type mouse Notch3 pre-mRNA sequence is reported at top of the figure. Arrows indicate the three ASOs studied in detail (ASO 14, 17 and 18).

2.3. Osteoblast-enriched Cell Cultures

Osteoblasts were isolated following the digestion of parietal bones from 3 to 5 day old control and Notch3^em1Ecan mice with liberase TL 1.2 units/ml (Sigma-Aldrich St. Louis, MO) for 20 min at 37°C for 5 consecutive reactions [41]. The cells isolated from the last 3 digestions were pooled and seeded at a density of 10 × 10⁴ cells/cm² [42]. Osteoblast-enriched cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with non-essential amino acids (both from Thermo Fisher Scientific, Waltham, MA) and 10% heat-inactivated fetal bovine serum (FBS; Atlanta Biologicals, Norcross, GA) in a humidified 5% CO₂ atmosphere at 37°C. Confluent cells were exposed to DMEM supplemented with 10% heat-inactivated FBS, 100 μg/ml ascorbic acid and 5 mM β-glycerophosphate (both from Sigma-Aldrich) in the absence or presence of Notch3 mutant or control ASOs at various doses and periods of time as indicated in text and legends.

2.4. Bone Marrow Stromal Cell Cultures

Femurs and tibiae from 4 to 8 week old Notch3^em1Ecan mice and littermate controls were dissected, the epiphysis removed and bone marrow stromal cells recovered by centrifugation [43]. Cells were pooled and seeded at a density of 1.25 × 10⁶ cells/cm² in α-minimum essential medium (α-MEM; Thermo Fisher Scientific) containing heat-inactivated 15% FBS and cultured at 37°C in a humidified 5% CO₂ incubator. At confluence, cells were exposed to α-MEM supplemented with 10% FBS, 100 μg/ml ascorbic acid and 5 mM β-glycerophosphate and cultured in the absence or presence of Notch3 mutant or control ASOs at distinct doses and periods of time as indicated in text and legends.

2.5. In vivo Administration of Notch3 ASOs

One month old male Notch3^em1Ecan heterozygous mutant and sex-matched wild type littermates were administered Notch3 mutant specific ASOs or control ASO suspended in PBS subcutaneously a dose of 50 mg/Kg once a week for 4 consecutive weeks to 1 month old Notch3^em1Ecan and control mice, and mice were euthanized at 2 months of age.

2.6. Microcomputed Tomography (μCT)

Bone microarchitecture of femurs from experimental and control mice was determined using a μCT 40 microcomputed tomography instrument (Scanco Medical AG, Bassersdorf, Switzerland), which was calibrated periodically using a phantom provided by the manufacturer [44, 45]. Femurs were placed in 70% ethanol and scanned at high resolution, energy level of 55 kVp, intensity of 145 μA and integration time of 200 ms. One hundred and sixty slices at the distal femoral metaphysis were acquired at an isotropic voxel size of 216 μm³ and a slice thickness of 6 μm and chosen for analysis of cancellous bone microarchitecture. Trabecular bone volume fraction and microarchitecture were evaluated starting ~1.0 mm proximal from the femoral condyles. Contours were manually drawn a few voxels away from the endocortical boundary every 10 slices to define the region of analysis. The remaining slice contours were iterated automatically. Trabecular regions were assessed for total volume, bone volume, bone volume fraction (bone volume/total volume), trabecular thickness, trabecular number, trabecular separation, connectivity density and structure model index, using a Gaussian filter (σ = 0.8), and a threshold of 240 permil equivalent to 355.5 mg/cm³ hydroxyapatite [44, 45]. Femoral cortical bone architecture was performed along the cortex of the femoral midshaft, excluding the marrow cavity. Contours were iterated across 100 slices and analysis of bone volume/total volume, porosity, cortical thickness, total cross-sectional and cortical bone area, and polar moment inertia were performed using a Gaussian filter (σ = 0.8, support = 1), and a threshold of 400 permil equivalent to 704.7 mg/cm³ hydroxyapatite.

2.7. Quantitative Reverse Transcription-Polymerase Chain Reaction (qRT-PCR)

Total RNA was extracted from cells, homogenized femurs or tibiae, following the removal of the bone marrow by centrifugation with the RNeasy kit or microRNeasy Kit (Qiagen, Valencia, CA), in accordance with manufacturer’s instructions [46–49]. The integrity of the RNA was assessed in random samples by microfluidic electrophoresis on an Experion instrument (BioRad, Hercules, CA), and only RNA with a quality indicator number equal to or higher than 7.0 was used for subsequent analysis. Equal amounts of RNA were reverse-transcribed using the iScript RT-PCR kit (BioRad) and amplified in the presence of specific primers (IDT) (Table 1A) with the iQ SYBR Green Supermix or SsoAdvanced Universal SYBR Green Supermix (BioRad) at 60°C for 35 cycles. Transcript copy number was estimated by comparison with a serial dilution of cDNA for Notch1 (from J.S. Nye, Cambridge, MA), Notch2 (from Thermo Fisher Scientific) and Notch4 (from Y. Shirayoshi, Tottori, Japan) [50, 51]. Notch3 wild type copy number was estimated by comparison to a serial dilution of a 100 to 200 base pair synthetic DNA template (IDT) cloned into pcDNA3.1 (Thermo Fischer Scientific) by isothermal single reaction assembly using commercially available reagents (New England BioLabs, Ipswich, MA) [52].

Table 1.

Primers used for qRT-PCR determinations. GenBank accession numbers identify transcript recognized by primer pairs.

A. Conventional qRT-PCR
Gene	Strand	Sequence	GenBank Accession Number
Notch1	Forward Reverse	5'-GTCCCACCCATGACCACTACCCAGTTC-3' 5'-GGGTGTTGTCCACAGGGGA-3'	NM_008714
Notch2	Forward Reverse	5'-TGACGTTGATGAGTGTATCTCCAAGCC-3' 5'-GTAGCTGCCCTGAGTGTTGTGG-3'	NM_010928
Notch3*	Forward Reverse	5'-CCGATTCTCCTGTCGTTGTCTCC-3' 5'-TGAACACAGGGCCTGCTGAC-3'	NM_008716
Notch4	Forward Reverse	5'-CCAGCAGACAGACTACGGTGGAC-3' 5'-GCAGCCAGCATCAAAGGTGT-3'	NM_010929
Rpl38	Forward Reverse	5'-AGAACAAGGATAATGTGAAGTTCAAGGTTC-3' 5'-CTGCTTCAGCTTCTCTGCCTTT-3'	NM_001048057; NM_001048058; NM_023372
B. qRT-PCR using fluorescent tagged PCR products
Gene	Strand/Flurophore	Sequence	GenBank Accession Number
Notch3	Forward Reverse HEX Fluorophore	5'-AGGACATGGAGAGGAATA-3' 5'-GGTCAAATAAGGATGCTC-3' 5'-ACCAAGGCCCGCTTCC-3'	Not Applicable
Notch3 6691-TAATGA	Forward Reverse FAM Fluorophore	5'-AGGACATGGAGAGGAATA-3' 5'-GGTCAAATAAGGATGCTC-3' 5'-AGTAGCCCCTAATGAGCGCG-3'	Not Applicable

^*Used for Notch3 wild type mRNA determination in Figure 4, Panel B.

In experiments where cells or tissues were obtained from Notch3^em1Ecan mice with the intent to determine an ASO effect on Notch3^6691-TAATGA mutant transcripts, fluorescent tagged products were used to perform RT-PCR. Total RNA was reverse transcribed with Moloney murine leukemia virus reverse transcriptase in the presence of reverse primers of Notch3 (5’-ATAAGGATGCTCGC TGGGAACC-3’) and Rpl38 (Table 1A). Notch3 cDNA was amplified by qPCR in the presence of SsoAdvanced Universal Probes Supermix (BioRad) gene expression assay mix, Notch3 and Notch3 mutant primers and HEX labeled Notch3 and FAM labeled Notch3^6691-TAATGA probes (Table 1B) (BioRad) at 95°C for 10 secs then 60°C for 30 secs and repeated for 45 cycles [53]. Notch3 or Notch3^6691-TAATGA mutant transcript copy number was estimated by comparison to a serial dilution of a 100 to 200 base pair (bp) synthetic DNA fragment (IDT) with or without the 6691TAATGA mutation in the Notch3 locus cloned into pcDNA3.1(−) and copy number was corrected for Rpl38 expression [52].

2.8. Statistics

Data are expressed as individual sample values, and as means ± SD. Data represent biological replicates except for osteoblast-enriched and stromal cell cultures, which represent technical replicates. qRT-PCR values were derived from two technical replicates of technical or biological replicates as indicated in the text and legends. Statistical differences were determined by unpaired t test for pairwise comparisons or two-way analysis of variance for multiple comparisons with Holm Šidák post-hoc analysis using GraphPad Prism version 9.3.1 for Mac OS, GraphPad Software (San Diego, CA).

3. RESULTS

3.1. Effect of Notch3 ASOs on Notch3 Expression in Cells of the Osteoblast Lineage

Twenty-four ASOs to target the Notch3^6691-TTATGA mutation were synthesized and tested initially for toxicity in vivo following their administration to BALB/c mice at 50 mg/Kg/week for 4.5 weeks. Most of the ASOs tested were well tolerated in mice, with only a few showing mild elevation in liver transaminase levels (Figure 2). We selected 12 safe ASOs and examined them further for activity and specificity in vitro (Table 2). The effect of Notch3 mutant ASOs was tested in cells of the osteoblast lineage from Notch3^em1Ecan mice since previous work demonstrated that Notch3 is preferentially expressed in cells of this lineage including osteocytes [12, 17, 54]. Five of the 12 ASOs tested decreased Notch3^6691-TAATGA mRNA by ~60% to 100% 72 h after the addition of the ASO at 20 μM to the culture medium of osteoblast- enriched cells from Notch3^em1Ecan mice (Table 2). The effect of three ASOs (ASO14; ASO17; ASO18) was specific to the mutant Notch3 mRNA since these ASOs did not downregulate Notch3 wild type mRNA (Figure 3). These three ASOs were studied further, and ASO18 was tested at multiple doses to confirm the downregulation of Notch3^6691-TAATGA mutant transcripts. Notch3 mutant ASO18 at 1 to 20 μM for 72 h downregulated Notch3^6691-TAATGA mRNA 80% to 100% in Notch3^em1Ecan osteoblasts and did not alter the expression of Notch1, Notch2, wild type Notch3 or Notch 4 mRNA (Figure 4, Panels A and B). In addition, this Notch3 mutant ASO downregulated Notch3^6691TAATGA transcripts by 65% without affecting Notch3 mRNA in cultures of bone marrow stromal cells (Figure 4, Panel C) confirming the effect observed in calvarial osteoblasts. As expected, the Notch3^6691TAATGA transcript was not detected in control wild type cells.

Figure 2.

The safety profile of 24 mutant Notch3 ASOs was evaluated in mice following subcutaneous administration once a week for 4.5 weeks at 50 mg/Kg. At the end of the study, body, liver, kidney, and spleen weights were assessed, and plasma levels of alanine aminotransferase, aspartate aminotransferase, total bilirubin, albumin, and blood urea nitrogen were measured. Individual values are shown, and bars and ranges represent means ± SD; n = 4 mice for each ASO tested. Twelve ASOs considered safe were selected for subsequent activity studies.

Table 2.

Screening of Notch3 mutant ASOs for activity in vitro.

ASO	Notch3 Downregulation	Notch36691TAATGA Downregulation
07	0%	0%
08	0%	0%
10	0%	0%
11	0%	0%
13	0%	0%
14	~15%	100%
16	~40%	100%
17	0%	70%
18	0%	60%
19	~40%	0%
20	~25%	0%
21	100%	100%

Twelve ASOs considered safe were tested at 20 μM for their effect on Notch3 wild type or Notch3^6691TAAGTAmutant transcripts in osteoblast-enriched cells from Notch3^em1Ecan mice. Notch3 mutant ASOs that were not safe or borderline safe in vivo were not tested in vitro.

Figure 3.

Effect of control or Notch3 mutant ASOs (14, 17 and 18) on Notch3 and Notch3^6691-TAATGA mRNA expression in calvarial osteoblast-enriched cells from Notch3^em1Ecan mice. Notch3 and Notch3^6691-TAATGA mRNA levels were obtained 72 h after the addition of Notch3 mutant or control ASO at 20 μM to the culture medium. Transcript levels are expressed as copy number following correction for Rpl38. Individual values are shown, and bars and ranges represent means ± SD; n = 3 technical replicates. *Significantly different between Notch3 mutant ASO and control ASO by ANOVA with post-hoc analysis by Holm Šidák, p <0.05.

Figure 4.

Effect of control or Notch3 mutant ASO 18 on Notch3^6691-TAATGA mRNA expression in calvarial osteoblast-enriched (A and B) and bone marrow stromal (C) cells from Notch3^em1Ecan mice. In Panel A, Notch3^6691-TAATGA mRNA levels were obtained 72 h after the addition of Notch3 mutant or control ASO at the indicated doses. In Panel B, Notch1, Notch2, Notch3, Notch3^6691-TAATGA and Notch4 mRNA were detected 72 h after the addition of Notch3 mutant or control ASO at 20μM. In Panel C, Notch3 and Notch3^6691TAATGA mRNA levels were obtained after the addition of Notch 3 mutant (closed circles grey bars,) or control ASO (open circles, white bars,) at 20μM for 72 h. Transcript levels are expressed as copy number following correction for Rpl38. In Panels A and B, values are means ± SD. In Panel C, individual values are shown, and bars and ranges represent mean ± SD; n = 3 or 4 technical replicates. *Significantly different between Notch3 mutant ASO and control ASO by ANOVA with post-hoc analysis by Holm Šidák (Panels A and C) or unpaired t-test (Panel B), p <0.05.

3.2. Effect of Notch3 Mutant ASO on General Characteristics and Femoral Microarchitecture of Notch3^em1Ecan Mice

Heterozygous Notch3^em1Ecan mutant male mice were compared to wild type sex-matched littermate controls in a C57BL/6 genetic background. Male mice were studied since they have an osteopenic phenotype that is sustained for up to 3 to 4 months of age whereas female Notch3^em1Ecan mutants tend to have a more transient osteopenia [17]. Homozygous Notch3^em1Ecan mice were not studied since the homozygous mutation is developmentally lethal [17]. Three of the Notch3 mutant ASOs that suppressed Notch3^6691-TAATGA transcripts in vitro specifically (Figure 3) were tested for their effects in vivo and were tolerated well without overt signs of toxicity. Two of 3 Notch3 mutant ASOs had no effect or inconsistent effects on the cancellous or cortical bone osteopenia of Notch3^em1Ecan mice and were not studied further (data not shown).

Mouse Notch3 mutant ASO18 or control ASO were administered once a week at 50 mg/Kg for 4 weeks. Notch3 mutant ASO18 was administered to 26 Notch3^em1Ecan mice and to 32 wild type littermates, and Notch3 control ASO was administered to 23 Notch3^em1Ecan mice and 34 control littermates. The weight of Notch3^em1Ecan heterozygous male mice was not different from those of control wild type mice and the femoral length was slightly decreased in Notch3^em1Ecan mice. These parameters were not altered by the administration of the Notch3 mutant ASO compared to control ASO. The administration of the Notch3 mutant ASO for a 4-week period decreased Notch3^6691-TAATGA mRNA expression by ~50% in tibiae from Notch3^em1Ecan mutant mice without affecting Notch3 mRNA (Figure 5). In accordance with previous observations, μCT of the femoral mid-diaphysis revealed that 2 month old Notch3^em1Ecan male mice had decreased cortical bone volume/total volume (BV/TV) associated with a 26% increase in porosity, a 18% decrease in cortical thickness and a 9% decrease in polar moment of inertia (Figure 6) [17]. The administration of the Notch3 mutant ASO increased cortical BV/TV and resulted in a 8% increase in cortical thickness and a 6% decrease in cortical porosity in Notch3^em1Ecan mice. In addition, when compared to control ASO, the Notch3 mutant ASO increased polar moment of inertia in Notch3^em1Ecan mice by 16% (p 0.07 by ANOVA; p < 0.05 by unpaired t-test), indicating an amelioration of skeletal fragility. No effect was noted in wild type mice by the administration of Notch3 ASO since the ASO was designed to target the Notch3 mutation. Cancellous bone microarchitecture demonstrated that Notch3^em1Ecan mice had a 37% decrease in cancellous BV/TV and in trabecular number when compared to wild type littermates (Figure 6). Administration of Notch3 mutant ASO did not increase femoral BV/TV significantly in Notch3^em1Ecan (16%) mice and had no effect on control littermates (Figure 7). Therefore, the effect of the Notch3 mutant ASO was restricted to the cortical bone osteopenia of Notch3^em1Ecan mice.

Figure 5.

Body weight, femoral length and Notch3 and Notch3^6691-TAATGA transcripts in tibiae of 2-month-old male Notch3^em1Ecan mutant mice and littermate controls treated with Notch3 mutant ASO 18 (closed circles, grey bars) or control ASO (open circles, white bars) at 50 mg/Kg subcutaneously, once a week for 4 weeks. Individual values are shown, and bars and ranges represent means ± SD; n = 34 wild type mice treated with control ASO, n = 32 wild type mice treated with Notch3 ASO and n = 23 Notch3^em1Ecan mice treated with control ASO and n = 26 Notch3^em1Ecan mice treated with Notch3 mutant ASO, except for Notch3^6691-TAATGA mRNA values n = 5 biological replicates. *Significantly different between Notch3 ASO and control, p < 0.05. #Significantly different between Notch3^em1Ecan mutant and control mice, p < 0.05, by ANOVA with post-hoc analysis by Holm Šidák or unpaired t-test for Notch3^6691-TAATGA mRNA.

Figure 6.

Cortical bone microarchitecture assessed by μCT of the mid-diaphyseal femur from 2 month old Notch3^em1Ecan mutant male mice and sex-matched littermate controls treated with Notch3 mutant ASO 18 (closed circles, grey bars; n = 32 for wild type mice, n = 26 for Notch3^em1Ecan) or control ASO (open circles, white bars; n = 34 for wild type mice, n = 23 for Notch3^em1Ecan) both at 50 mg/Kg subcutaneously, once a week for 4 weeks prior to sacrifice. Parameters shown are cortical bone volume/total volume (BV/TV, %), cortical porosity (%), cortical thickness (mm) and polar moment of inertia (pMOI). Individual values are shown, and bars and ranges represent means ± SD of biological replicates. *Significantly different between Notch3 mutant and control ASO, p < 0.05. #Significantly different between Notch3^em1Ecan and control, p < 0.05, by ANOVA with post-hoc analysis by Holm Šidák. Representative images show cortical bone osteopenia in Notch3^em1Ecan mutant mice and its amelioration by Notch3 mutant ASOs. Scale bars in the right corner represent 100 μm. Arrow on scout μCT images point to the area analyzed.

Figure 7.

Cancellous bone microarchitecture assessed by μCT of the distal femur from 2 month old Notch3^em1Ecan mutant male mice and sex-matched littermate controls treated with Notch3 mutant ASO 18 (closed circles, grey bars; n = 32 for wild type mice, n = 26 for Notch3^em1Ecan) or control ASO (open circles, white bars; n = 34 for wild type mice, n = 23 for Notch3^em1Ecan) both at 50 mg/Kg subcutaneously, once a week for 4 weeks prior to sacrifice. Parameters shown are trabecular bone volume/total volume (BV/TV, %); trabecular number (1/mm) and thickness (um). Individual values are shown, and bars and ranges represent means ± SD of biological replicates. #Significantly different between Notch3^em1Ecan and control, p < 0.05, by ANOVA with post-hoc analysis by Holm Šidák. Representative images show cancellous bone osteopenia in Notch3^em1Ecan mutant mice and no effect by Notch3 mutant ASO. Scale bars in the right corner represent 100 μm.

4. DISCUSSION

The current work was undertaken to determine whether ASOs could be developed to target and downregulate a Notch3 mutation in a mouse model (Notch3^em1Ecan) replicating the genetic variant found in LMS. Because Notch3^em1Ecan mice present with osteopenia, as a therapeutic end point we tested whether an improvement in genetic outcome would translate in amelioration of this aspect of the Notch3^em1Ecan phenotype. About 50% of the Notch3 mutant specific ASOs designed were found not to be toxic in vivo and were studied further for their activity in skeletal cells. Three of the 12 ASOs considered safe downregulated the Notch3^6691-TAATGA mutation specifically without an effect on wild type Notch3 transcripts in osteoblast cultures. One of the ASOs was pursued in detail and found to inhibit Notch3 mutant transcripts in vivo and ameliorate phenotypic manifestations of Notch3^em1Ecan mice.

Although a variety of approaches to downregulate Notch signaling have been reported, they often are not specific to Notch activity or to a specific Notch receptor. Anti-Notch antibodies targeting the NRR are an exception and we have demonstrated that anti-NOTCH2 NRR and anti-NOTCH3 NRR antibodies are effective in reversing the skeletal phenotype of Notch2^tm1.1Ecan and of Notch3^em1Ecan mice, models of Hajdu Cheney Syndrome and LMS, respectively [25, 26]. Although anti-Notch NRR antibodies are effective in their ability to downregulate a specific Notch receptor, they do not discriminate between the wild type and mutant forms of the Notch receptor. They induce substantial downregulation of Notch signaling with a potential of gastrointestinal toxicity. Because of these reasons, we search for other alternatives to reverse/ameliorate skeletal phenotypes associated with Notch2 or Notch3 mutations.

We demonstrated that Notch2 ASOs downregulate Notch2 expression in vitro and in vivo and ameliorate the osteopenia of mice harboring a Notch2 mutation replicating the one found in HCS [36]. Similarly, we found that Notch3 ASOs targeting Notch3 downregulate Notch3 wild type and mutant transcripts and ameliorate the cortical osteopenia of Notch3^em1Ecan mice [39].

The current approach was designed to target the Notch3^6691-TAATGA mutation specifically in a heterozygous mouse model that replicates the genetic variant of LMS with the intent to preserve the activity of the wild type allele and create a state of functional normality. This approach would seem ideal for the targeting of mutations found in monogenic disorders with dominant inheritance, such as LMS. The downregulation of the Notch3 mutant transcript by the Notch3 ASO was effective in in vitro models and in vivo it ameliorated the cortical osteopenia of Notch3^em1Ecan mice. However, Notch3 mutant ASOs did not modify significantly the cancellous bone osteopenia of Notch3^em1Ecan mice. The present study limited the administration of the Notch3 mutant specific ASO to 50 mg/Kg for a 4-week period, and it is possible that a longer period of administration could have a greater beneficial effect on the bone microarchitecture of Notch3^em1Ecan mice. The effect of Notch3 mutant ASOs in vivo was similar to the one that we reported recently on the effect of Notch3 ASOs. It is possible that Notch3 ASOs are more effective in the cortical compartment which is rich in osteocytes because Notch3 is preferentially expressed in this cellular environment [12, 16, 54, 55]. It is also possible that ASOs are transported more efficiently to cortical than to trabecular bone since the blood vessel network differs between these two skeletal compartments. A central nutrient artery and periosteal arteries irrigate cortical long bones, whereas epiphyseal arteries irrigate metaphyseal, cancellous-rich bone [56, 57]. However, perfusion efficiency studies do not reveal apparent differences between epiphyseal and diaphyseal bone vascularization [58].

Although the subcutaneous administration of Notch3 ASOs is practical from a possible therapeutic point of view, the present and prior studies suggest that ASOs are partially effective in the downregulation of the Notch3 mutant transcripts in skeletal tissue and the reversal of skeletal phenotypes. Greater efficacy might be attained by enhancing the transport of the ASO to the target cell [59]. This has been achieved for skeletal muscle by conjugating ASOs to antigen binding fragments to the transferrin receptor 1 [60]. Although our study demonstrates amelioration of the cortical osteopenia in a mouse model that harbors a genetic variant found in LMS, there was not complete reversal of the phenotype. Future work developing strategies to target ASOs to specific skeletal cells may serve to improve their efficacy and potential therapeutic outcomes.

The present findings confirm that a mouse model replicating a mutation found in LMS displays femoral cancellous and cortical bone osteopenia [17]. There are some limitations of this study. The phenotype of the Notch3^em1Ecan mutant mouse recapitulates aspects of LMS including osteopenia, but not the neurological manifestations of the disease [16, 17]. Because only male mice were reported in the present study, it is important not to extrapolate the results observed to female mice. Phenotypic alterations of experimental and control mice were assessed by μCT, and analyses required the ex vivo exam of bone following the sacrifice of mice. Therefore, the same animal could not be analyzed before and after the administration of Notch3 ASOs. Cells were obtained from mice of both sexes for in vitro experiments since sex cell specific culture is at times impractical. The effects of Notch3 ASOs were assessed in cells of the osteoblast lineage and not in the myeloid/osteoclast lineage because Notch3 is not expressed in this lineage and NOTCH3 does not have direct effects on osteoclastogenesis [3, 16, 17].

5. CONCLUSIONS

In conclusion, a Notch3 mutant ASO downregulated Notch3 mutant expression specifically and ameliorated the cortical osteopenia of a mouse model that replicates the genetic variant found in LMS.

Highlights.

• Lateral Meningocele Syndrome (LMS) is associated with NOTCH3 mutations

• A mouse model of LMS (Notch3^em1Ecan) harbors a Notch3 mutation and exhibits cortical and trabecular osteopenia

• Antisense oligonucleotides targeting the Notch3 mutation in the mouse environment ameliorate the cortical osteopenia of Notch3^em1Ecan mice

ABBREVIATIONS

α-MEM

α-minimum essential medium

ASO

antisense oligonucleotide

bp

base pair

BV/TV

bone volume/total volume

CADASIL

cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy

cEt

constrained ethyl

DMEM

Dulbecco’s modified Eagle’s medium

FBS

fetal bovine serum

HES

Hairy Enhancer of Split

HEY

HES-related with YRPM

LMS

Lehman Syndrome or Lateral Meningocele Syndrome

μCT

microcomputed tomography

NRR

negative regulatory region

NICD

NOTCH intracellular domain

PS

phosphorothiate

pMOI

polar moment of inertia

PEST

proline (P), glutamic acid (E), serine (S) and threonine (T)

qRT-PCR

quantitative reverse transcript-polymerase chain reaction