Reduction of integrin alpha 4 activity through splice modulating antisense oligonucleotides

Abstract

With recent approvals of antisense oligonucleotides as therapeutics, there is an increasing interest in expanding the application of these compounds to many other diseases. Our laboratory focuses on developing therapeutic splice modulating antisense oligonucleotides to treat diseases potentially amendable to intervention during pre-mRNA processing, and here we report the use of oligomers to down-regulate integrin alpha 4 protein levels. Over one hundred antisense oligonucleotides were designed to induce skipping of individual exons of the ITGA4 transcript and thereby reducing protein expression. Integrin alpha 4-mediated activities were evaluated in human dermal fibroblasts and Jurkat cells, an immortalised human T lymphocyte cell line. Peptide conjugated phosphorodiamidate morpholino antisense oligomers targeting ITGA4 were also assessed for their effect in delaying disease progression in the experimental autoimmune encephalomyelitis mouse model of multiple sclerosis. With the promising results in ameliorating disease progression, we are optimistic that the candidate oligomer may also be applicable to many other diseases associated with integrin alpha 4 mediated inflammation. This highly specific strategy to down-regulate protein expression through interfering with normal exon selection during pre-mRNA processing should be applicable to many other gene targets that undergo splicing during expression.

Figure 1

Design and experimental evaluation of ITGA4-targeting AOs. AOs designed to induce exon 3 skipping of the ITGA4 transcript are shown. (a) Splice enhancer motifs predicted by the Human splicing finder tool for exon 3 (grey box) and partial intron 2 and 3 (solid black lines outside the grey box). The height of the peaks represents the collective strength of the motifs. Purple bars show the location and strength of each predicted motif. (b) AO designed for exon 3 based on the analysis in (a). The negative numbers in red indicate the nucleotide positions of the intron relative to exon and the numbers in black represent the nucleotide positions of the exon relative to the acceptor site. Short black lines represent AOs designed for initial screen that cover the sequences around the peaks, acceptor and donor sites. The short red lines represent microwalking AOs designed to refine AO H3A(+41 +65) to achieve maximum exon skipping efficiency with minimal AO length. Only selective microwalking AOs for H3A(+41 +65) are shown as examples for illustration. Gel fractionation of RT-PCR products of ITGA4 amplicons from healthy fibroblasts transfected with 2OMe PS AOs at various concentrations (100, 50 and 5 nM from left to right) for 24 hr is shown above each AO. The percentage of exon skipping determined as described in Materials and Methods and the AO names are also shown. (c) The top three AOs for exon 3, 4 and 19 selected after microwalking to perform further studies. RT-PCR products of ITGA4 amplicons from healthy fibroblasts after transfection with the corresponding 2OMe PS AOs at indicated dosages for 24 hr and confirmation of exon skipping by Sanger sequencing are shown. Ctrl: control AO, UT: untreated. The gels were cropped for presentation and full-size gels are presented in Supplementary Fig. S5.

Figure 2

Evaluation of the best performing exon skipping 2OMe PS AOs in healthy dermal fibroblasts. The ITGA4 transcript, ITGA4 protein expression and activity of healthy dermal fibroblasts after treatment with the top three exon skipping AOs targeting exon 3, 4 and 19 for 48 hr were analysed. (a) Gel fractionation of RT-PCR products of ITGA4 amplicons amplified from healthy dermal fibroblasts transfected with the 2OMe PS AOs at 100 nM for 48 hr. Ctrl: control AO, UT: untreated. CCND1 transcript encoding cyclin D protein was used as a loading control. Transcripts with exon(s) missing are labelled. (b) Western blot analysis of ITGA4 protein expression, (c) analysis of fibroblast migration using an established wound healing assay, and (d) relative fibroblast adhesion to extracellular matrix fibronectin, laminin and VCAM-1 using the treated and untreated healthy dermal fibroblasts from (a). Beta tubulin was used as a reference protein for western blot analysis. Densitometric analysis of western blots and relative migration and cell adhesion analysed as described in Materials and Methods for three biological replicates are shown as bar graphs. The gels and blots were cropped for presentation and full-size gels and blots are presented in Supplementary Fig. S5. Error bar; SEM. Scale bar 100 µm.

Figure 3

Evaluation of the best performing exon skipping PMOs in Jurkat cells. The ITGA4 transcript, ITGA4 protein expression and activity in Jurkat cells treated with indicated PMOs for three days were analysed. (a) Gel fractionation of RT-PCR products of the ITGA4 amplicons and (b) western blot analysis of ITGA4 protein from Jurkat cells nucleofected with PMOs, as indicated above the gel, at 50 µM for three days. Beta tubulin was used as a reference protein. Densitometric analysis of three biological replicates for western blots is shown on the right. Error bar; SEM. (c) Flow cytometry analysis of surface receptor ITGA4 on Jurkat cells treated with 50 µM of PMO for three days. (d) Immunolabeling of ITGA4 protein in Jurkat cells treated with 50 µM of PMO for three days. Arrow head shows cell with intracellular accumulation of ITGA4 protein. Refer to Supplementary Fig. S3 for the display lookup tables. Green: ITGA4, blue: nucleus. Scale bar 25 µm. (e) Migration of Jurkat cells, treated with 50 µM of indicated PMO for three days, via interaction with VCAM-1, was assessed as shown in the illustration on the left and the percentages of cells migrated after 5 hr are shown as a bar graph. The experiment was performed in duplicate. Error bar; SD. GTC: Gene Tools control AO, UT: untreated. The gels and blots were cropped for presentation and full-size gels and blots are presented in Supplementary Fig. S5.

Figure 4

In vivo validation of peptide-conjugated PMOs (PPMOs) in the mouse EAE model of MS. (a) Gel fractionation of RT-PCR products of murine Itga4 amplicons and (b) western blot analysis of ITGA4 protein expression from primary murine splenocytes nucleofected with PMOs, as indicated, at 50 µM for 48 hr. Beta tubulin was used as a reference protein. The transcripts with missing exons missing are shown. GTC: Gene Tools control AO, UT: untreated. (c) The disease course of EAE mice intraperitoneally injected with PPMOs targeting Itga4 exons 3, 4 and 27 at 10 mg/kg on alternate days, as indicated by arrows. The clinical scores were determined over 21 days as described in Supplementary Table S5. (d) Flow cytometry analysis of infiltrating myeloid cells, and subsets of T cells in brain and secondary lymphoid organs (SLO: spleen/Inguinal lymph nodes pooled). ●: m4A(+51 +75) treated mice, ■: ant-ITGA4 antibody treatment, ▲: m27A(+18 +42) treatment, ▼: m3D(+5 −20) treatment, ◆: PBS treatment and ○: GTC treatment. Raw data is provided in Supplementary Tables S6 and S7. (e) Representative haematoxylin and eosin staining of the spinal cords isolated from EAE mouse treated with m4A(+51 +75) or GTC PMO (top panel). A box plot for the percentage of area in spinal cords, marked by inflammation (arrow heads), observed in m4A(+51 +75) and GTC treated groups (n = 5 per group). Scale bar: 200 µm. Error bars: SEM. GTC: Gene Tools control AO. The gels and blots were cropped for presentation and full-size gels and blots are presented in Supplementary Fig. S5.