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A. Dong et al.
content in erythroid cells combines to form insoluble hemichromes that damage cell membranes, while their heme component leads to the formation of toxic reactive oxygen species (ROS) and increased oxidative stress. In combination, these factors result in ineffective erythro- poiesis and apoptosis in the erythroid lineage.4,5
Methods
Human Ethics
Patients were recruited and samples obtained according to the Declaration of Helsinki, following approvals by the (i) Institutional Ethics Committee of the Fondazione Istituto di Ricovero e Cura a Carattere Scientifico Ca’ Granda Ospedale Maggiore Policlinico, Milan, #391/2012 for P1, P2, and P4, (ii) Cyprus National Bioethics Committee, National Grant EEBK/ΕΠ/2012/05, E.U. Grant EEBK/ΕΠ/2013/23 for P3 and P5 and (iii) Children’s Hospital of Philadelphia, Institutional Review Board (IRB) #15-012123 for S1. S2 was obtained during automated red cell exchange as part of routine clinical care; as S2 was unlinked and de-identified medical waste, the Montefiore Medical Center IRB deemed it IRB-exempt. All subjects gave their informed consent prior to their inclusion in the study.
Human erythroblast culture and treatment
Whole blood underwent CD34+ selection using immunomag-
+
netic separation (Miltenyi Biotec). CD34 cells were kept undiffer-
entiated in expansion media with bi- or tri-weekly media changes at a cell density <0.5x106 cells/mL. After 12 days in expansion, cells were transferred into differentiation media.
2'-MOE-SSO treatment via syringe loading occurred on day 14. Cells were resuspended at a concentration of 1x106/100 mL in a solution with the 2'-MOE-SSO (at a starting dose of 5 mM) and were passed ten times through a 25 gauge needle,21 followed by a 1 hour incubation at 37oC. Following 2'-MOE-SSO treatment,
6
cells were plated at 1x10 cells/mL in fresh differentiation media.
Transduction with lentiviral vectors (AnkCT9W or AnkCT9W- 745) were performed within the expansion phase (around day 10) at low MOI (<10) to reach low integration rate (below three copies). After transduction, cells were seeded for a few more days in fresh expansion media. Both transduced or 2'-MOE-SSO treat- ed cells were put in differentiation media for up to 6 days. Vector copy number (VCN) in transduced cells was determined by quan- titative polymerase chain reaction (Q-PCR) (see the Online Supplementary Appendix). Toxicity was assessed by trypan blue staining22 and level of differentiation was assessed by benzidine staining.23
Media for CD34+ cells and erythroid culture
CD34+ expansion media: serum-free StemSpan supplemented with 10 mL/mL CC-100 (both Stemcell Technologies), 2 U/mL ery- thropoietin (Amgen), 10−6 M dexamethasone (Sigma) and 100 U/ml penicillin/streptomycin (GIBCO, ThermoFisher Scientific). Differentiation media: Iscove’s Modified DMEM (Cellgro) with 3% AB serum (Atlanta Biologicals), 2% human plasma (Stemcell Tech), 10 mg/mL insulin (Sigma), 3 U/mL heparin, 200 ug/mL transferrin (Athens Research & Technology), 10 ng/mL stem cell factor (Peprotech), 3 U/mL erythropoietin. Freeze media: 50% characterized fetal bovine serum (Hyclone), 10% dimethyl sulfox- ide (Sigma), and 40% Iscove’s Modified DMEM. Thaw media: Iscove’s Modified DMEM supplemented with 5% characterized fetal bovine serum.
Globins single-chain and tetrameric hemoglobin analysis by direct- and reverse-phase high performance liquid chromatography
Cell pellets were disrupted with Cytobuster (EMD Millipore) for single-chain analysis and with water for tetramer analysis. Single-chain quantification was assessed by reverse-phase high performance liquid chromatography (HPLC). Hemolysates were injected into a Hitachi D-7000 HSM Series apparatus (Hitachi Instruments) using a Zorbax 5 mm 300SB-C8 300 Å, LC 150x2.1
Some b-thalassemia mutations create new cryptic splice sites which, even though the original splice sites are intact, impair normal splicing. Such mutations activate aberrant splice sites and change the splicing pathway.6
The IVS2-745 HBB mutation creates an aberrant 5' splice site at nucleotide 745 of intron 2 and activates a common cryptic 3' splice site at nucleotide 579 within the same intron (Online Supplementary Figure S1). Portions of the intronic sequence between the newly activated splice sites are recognized by the splicing machinery as exons and are incorrectly retained in the spliced mRNA. The retained intronic sequence of 165 nucleotides carries a stop codon that prevents proper translation of the mRNA and causes a deficiency in b‐globin leading to b‐tha- lassemia.7 The IVS2-745 HBB is a b+ allele, whose correct splice sites remain potentially functional and produce a significantly reduced amount of correctly spliced HBB messenger RNA (mRNA) and consequently adult hemo- globin (HbA).8 Despite the fact that some HbA is made, in homozygosity, the IVS2-745 mutation leads to severe transfusion-dependent thalassemia major (Hb VAR data- base: https://globin.bx.psu.edu/hbvar/menu.html).
Since defective b‐globin genes like IVS2-745 HBB pre- serve correct splice sites, approaches based on interference between the spliceosome machinery and the aberrant splice site interaction, have been developed and success- fully shown to restore the normal b-globin splicing pat- tern.9
The 2'-O-methoxyethyl modification (2'-MOE) chem- istry is currently the most advanced of the 2'-modified series of antisense oligonucleotides. Uniformly distributed 2'-MOE-splice switching oligos (SSO) (2'-MOE-SSO) are not subject to RNase H degradation when they bind their targets, which may be due to the steric hindrance con- ferred throughout the oligo by the methoxyethyl group.10,11 Over thirty 2'-MOE based compounds are cur- rently being tested in clinical trials for cancer and cardio- vascular, metabolic, and neurological diseases through assessment of safety, tolerability and pharmacokinet- ics.12,13 Safety studies of 2'-MOE SSO show they are well tolerated in multiple species from rodents to non-human primates.14,15 Thus, they make an attractive candidate for clinical applications, including at least one approved treat- ment and multiple ongoing clinical trials.16,19
Here, we demonstrate that uniform 2'-MOE-SSO tar- geting the IVS2-745 HBB mutation are effective in treating erythroid cells from thalassemic patients. The 2'-MOE- SSO exert physical obstruction on the alternative splicing site, preventing recognition from the spliceosome20 and favoring the correct splicing of the pre-mRNA. By prevent- ing incorrect splicing and therefore avoiding alternative splicing patterns of pre-mRNA, 2'-MOE-SSO increase HbA production. Furthermore, 2'-MOE-SSO alleviate other previously unstudied thalassemic cell parameters, such as a recovery of the stoichiometry of α- and b-globin chains, a reduction of toxic α aggregates, and the correc- tion of erythrocyte deformities in cells derived from patients heterozygous for the IVS2-745 HBB and sickle cell anemia mutation.
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haematologica | 2021; 106(5)