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1014 Adult Hemoglobin Production, Chain Rebalance, and Splice Correction in IVS2-745 Beta-Thalassemia Patient Cells Using 2’-O-Methoxyethyl Splice-Switching Oligos

Thalassemia and Globin Gene Regulation
Program: Oral and Poster Abstracts
Type: Oral
Session: 112. Thalassemia and Globin Gene Regulation: Molecular Mechanisms of Thalassemia
Monday, December 5, 2016: 5:45 PM
Room 7AB (San Diego Convention Center)

Alisa Cheung Dong1,2*, Valentina Ghiaccio1*, Irene Motta, MD3*, Shuling Guo, PhD4*, Raechel Peralta, MS4*, Coralea Stephanou, PhD5*, Paola Delbini, PhD6*, Connie Chen7*, Soteroula Christou8*, Marina Kleanthous, PhD9*, Maria Domenica Cappellini, MD10, Osheiza Abdulmalik, DVM, MS11, Laura Breda, PhD12* and Stefano Rivella, PhD12

1Hematology, Children's Hospital of Philadelphia, Philadelphia, PA
2Graduate School of Medical Sciences, Weill Cornell Medical College of Cornell University, New York, NY
3Rare Diseases Center, Department of Medicine and Medical Specialities, Ca’ Granda IRCCS Foundation Ospedale Maggiore Policlinico, Milan, Italy
4Ionis Pharmaceuticals, Inc., Carlsbad, CA
5Molecular Genetics of Thalassaemia Department, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
6Rare Diseases Center, Department of Medicine and Medical Specialities, Ca’ Granda IRCCS Foundation Ospedale Maggiore Policlinico, Milan, Italy
7Weill Cornell Medical College, New York
8Thalassaemia Center, Archbishop Makarios III Hospital, Nicosia, Cyprus
9The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
10Department of Medicine and Medical Specialities, Department of Clinical Science and Community Health, Rare Diseases Center, Fondazione IRCCS “Ca’ Granda”, Ospedale Maggiore Policlinico, University of Milan, Milan, Italy
11Division of Hematology, The Children's Hospital of Philadelphia, Philadelphia, PA
12Children's Hospital of Philadelphia, Philadelphia, PA

Hemoglobinopathies are the most common inherited blood disorders. World Health Organization statistics show that in the Mediterranean, Eastern European, and Middle Eastern regions, frequencies range from 0.1 to 4.9/1000 of live births. The mutation known as IVS2-745 is relatively common in the regions of Spain, Jordan, Romania, and Serbia (Ithanet Database, http://www.ithanet.eu/db/ithamaps), reaching as high as 15-20% of beta-thalassemia mutations in these regions. The IVS2-745 is a splicing mutation that occurs in intron 2 of the beta-globin gene and results in an aberrantly spliced mRNA that incorporates an extra exon and premature stop codon. Here we report novel uniform 2’-O-methoxyethyl (2’-MOE) splice switching oligos (SSOs) that reverse the aberrant splicing and restore up to 80% adult hemoglobin (HbA) production in vitro.

Uniform 2’-MOE SSOs do not mediate RNase H degradation when they bind their targets; therefore, they can be used to redirect the splicing machinery and restore WT splicing. After generating mouse erythroleukemia cells that carry the human IVS2-745 mutated beta-globin gene, lead 2’-MOE SSOs targeting the 745 mRNA were raised against these cell lines. With these lead SSOs we have demonstrated aberrant 745 to WT splice switching in 5 patient samples. CD34+ cells were isolated from the blood of four 745/β0 compound heterozygotes and one 745/ 745 homozygote (Breda et al, PloS One 2012). After CD34+ expansion, cells were differentiated to the red cell lineage and treated via syringe loading or lipofectamine transfection with 2’-MOE SSOs. Up to 80% HbA protein production was restored with 2’MOE-SSO treatment in the 745 homozygote patient sample (Figure 1), and up to 60% HbA in multiple 745 compound heterozygote specimens. Compared to 3-6% HbA in scramble treated controls, this represents up to a 20-fold increase in HbA with treatment.

In addition to HbA production, we have shown improvement in other parameters characteristic in beta-thalassemia, such as the imbalance of alpha and beta chains and the accumulation of toxic alpha-only homotetramers. 2’MOE SSOs are able to reinstate balance of beta- to alpha-like chains, which resulted in a near elimination of toxic alpha-only homotetramers in the homozygote cell lysate as detected by HPLC (Figure 1). We further proved the benefit of 2’MOE SSOs in a 745/Sickle model system, where in vitro sickling was significantly reduced as a result of increased levels of HbA. To create this model system, we transduced CD34+ cells from a homozygous sickle patient specimen with a lentivirus expressing human IVS2-745 beta-globin. With vector copy numbers ~2, this system replicates what a single allele would do, as the 2 endogenous sickle alleles are equally matched. Upon differentiation and exposure to hypoxia, in vitro sickling was reduced by 50% in 2’MOE-SSO treated samples as compared to scramble controls (Figure 2).

In summary, 2’MOE-SSOs are a promising therapy for certain splicing forms of beta-thalassemia. Their ability to correct the underlying splicing cause offers a pharmacological treatment that is both direct and specific. As such, this therapy could help patients reduce their transfusion dependence or even reach transfusion independence.


Disclosures: Guo: Ionis Pharmaceuticals: Employment, Equity Ownership. Peralta: Ionis Pharmacueticals: Employment. Cappellini: Celgene: Membership on an entity's Board of Directors or advisory committees; Genzyme-Sanofi: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees.

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