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2'-O-methoxyethyl splice-switching oligos
mm column (Agilent Technologies) and a gradient from 20% to 60% acetonitrile in 0.1% trifluoroacetic acid in 25 minutes, with UV detection at 215 nm. Standards of HbA, fetal hemoglobin (HbF), sickle hemoglobin (HbS), and hemoglobin C (HbC) were injected (Analytical Control Systems) and used to determine vari- ous hemoglobin peak types.23 For tetrameric analysis hemolysates were loaded into a System Gold 126 Solvent Module instrument (Beckman Coulter). Hemoglobin tetramers were separated on a weak cation-exchange PolyCAT A column (PolyLC), and detected at a wavelength of 415 nm. Hb were bound to the column with mobile phase A (20 mmol/L Bis-Tris, 2 mmol/L KCN, pH 6.96) and eluted with mobile phase B (20 mmoI/L Bis-Tris, 2 mmol/L KCN, 200 mmol/L NaCl, pH 6.55).
(RBPDB) at http://rbpdb.ccbr.utoronto.ca, are provided in the Online Supplementary Table S4.
We isolated hematopoietic stem cells (HSC) from peripheral blood mononuclear cells from patients’ blood using immunobeading.25 HSC were subsequently expand- ed and differentiated using a two-phase liquid culture sys- tem adapted from a previously described protocol26 and its formulation is summarized in the methods section. After 2 days of differentiation the 2'-MOE-SSO treatment occurred by syringe loading. The differentiated erythrob- lasts were collected at the conclusion of cell culture. Characterization of differentiating wild-type (WT) cells and b-thalassemic (BT) cells, with one allele affected by IVS2-745 mutation in combination with an IVS1-6 allele, at day 2, 5 and 8 by benzidine staining and flow cytome- try of the CD71, CD235a (GPA) along with other markers is depicted in the Online Supplementary Figure S3. While surface markers seem unaffected in WT and BT specimen, total HbA proportion and concentration per cell is dimin- ished in the BT specimen, as well as the amount of WT HBB mRNA, as shown in the Online Supplementary Figure S4. This reiterates the concept that the defect in BT cells is a quantitative one, completely dependent on the reduced level of HBB gene expression and translation.
Following treatment, we detected corrected splice rever- sal in differentiated IVS2-745/b0 erythroblasts at a dose as low as 5 mM of the IVS2-745 specific 2'-MOE-SSO. As expected, the samples treated with scramble 2'-MOE-SSO exhibited alternative splicing: both the longer 745 aberrant and the shorter WT mRNA forms were detected by elec- trophoresis (Online Supplementary Figure S5).
Amplicons of both WT and aberrant IVS2-745 comple- mentary DNA (cDNA) forms were extracted, purified and subcloned. The sequence of the amplicons subcloned matched the aberrant mRNA sequence with the extra 165 bp intronic segment retained after aberrant splicing of the IVS2-745 allele, which was not present in the correctly spliced HBB mRNA band (Online Supplementary Table S1). While both 2'-MOE-SSO 92 and 93 were effective, the most robust effect was observed in specimens treated with 2'-MOE-SSO 91. We tested 2'-MOE-SSO concentra- tion in steps and according to specimens’ accrual. The first attempted treatment with the 5 mM dose (Online Supplementary Figure S5A) showed a strong reduction of the aberrant IVS2-745 alternative splice variant in speci- men P1.
As patient sample collection requires both access to donors and trained researchers to isolate HSC, we escalat- ed the dose in subsequent patient samples. Accordingly, P2 was then treated with a 25 mM dose, which decreased the aberrant mRNA form further, indicating a dose-depen- dent response to the 2'-MOE-SSO (Online Supplementary Figure S5A). Given the high cell viability observed at 25 uM, all further specimens were treated increasing the dose to 50 mM (Online Supplementary Figure 5C and D). At this dose amplification of the aberrant IVS2-745 splicing is nearly undetected, while it remains unaltered in the spec- imens treated with the scramble 2'-MOE-SSO. The strong WT signal in untreated or scramble-treated specimens is likely due to the higher stability of the WT compared to the aberrant mRNA form27 and further justified by the nature of the reverse transcriptase PCR (RT-PCR) assay, which is a semi-quantitative method and does not neces- sarily reflect the absolute content of the two species in the samples. These results were obtained without cell loss, as
In vitro red blood cell sickling and morphological analysis
Treatment with 50 μM of either scramble or 91 2'-MOE-SSO occurred 2-3 days after start of differentiation. Cells were then cul- tured for 6 days in differentiation media before harvest. We assessed the degree of cell sickling in specimens under hypoxic conditions, using previously reported methodology.24,25 Briefly, 0.5-1 million cells were suspended in isotonic Hemox buffer (TCS Scientific Corp), pH 7.4, supplemented with 10 mM glucose and 0.2% bovine serum albumin, in individual wells of a Costar poly- styrene 96-well microplate (Corning). The microplate was then transferred to a Thermomixer R shaker-incubator (Eppendorf), and maintained under hypoxia (nitrogen gas), with continuous agitation at 500 rpm, at 37° C for 2 hours. At conclusion, aliquots of each sample were collected in 2% glutaraldehyde solution for immediate fixation without exposure to air. Subsequently, fixed cell suspensions were introduced into specialized glass microslides (Dawn Scientific) for acquisition of bright field images (at 40x magnification) of single layer cells on an Olympus BX40 micro- scope fitted with an Infinity Lite B camera (Olympus) and the cou- pled Image Capture software.
Results
2'-MOE-SSO induce splice switching, restoring adult hemoglobin production in cells from patients with b0/IVS2-745 genotype
In order to find the most efficacious oligonucleotides to block the aberrant splicing caused by the C>G mutation in IVS2-745, we designed SSO spanning the 165 basepair (bp) extra exon and the regions upstream and downstream of this exon. All the SSO are 18-mer oligonucleotides with uniform 2'-MOE modifications of each nucleotide sugar moiety, referred to herein as “2'-MOE-SSO”. These 2'- MOE-SSO were screened in mouse erythroleukemia (MEL) cells expressing the aberrant IVS2-745 HBB for splic- ing correction (Online Supplementary Figure S2). The top 3 2'-MOE-SSO, hereinafter referred to by identification numbers, 91, 92, 93, showed the most effective increase of wild-type HBB mRNA expression and were selected for further investigation. These 2'-MOE-SSO cluster in the 5'- end of the extra exon, within 50 nucleotides of the 3'-cryp- tic splicing site. A 2'-MOE-SSO not targeting any region was used as a scrambled control to evaluate any potential non-sequence specific effect. The sequences of the oligonucleotides and their binding sites on the HBB gene are provided in the Online Supplementary Tables S2 and S3, respectively. The binding areas of the three 2'-MOE-SSO and the predicted splicing factor binding sites, obtained through a search of the RNA-binding Protein DataBase
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