Alpha-like Chains Research Articles

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

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. [Display omitted] [Display omitted] DisclosuresGuo: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.

T-cell receptors in ectothermic vertebrates.

The structure and expression of genes encoding molecules homologous to mammalian T-cell receptors (TCR) have been recently studied in ectothermic vertebrate species representative of chondrychthians, teleosts, and amphibians. The overall TCR chain structure is well conserved in phylogeny: TCR beta- and TCR alpha-like chains were detected in all the species analyzed; TCR gamma- and TCR delta-like chains were also present in a chondrychthian species. The diversity potential of the variable (V) and joining (J) segments is rather large and, as in mammals, conserved diversity (D) segments are associated to the TCR beta and TCR delta chains. An important level of junctional diversity occurred at the V-(D)-J junctions, with the potential addition of N- and P-nucleotides. Thus, the conservation of the structure and of the potential of diversity of TCR molecules have been under a permanent selective pressure during vertebrate evolution. The structure of MHC class I and class II molecules was also well conserved in jawed vertebrates. TCR and MHC molecules are strongly functionally linked and play a determinant role in the initiation and the regulation of the specific immune responses; thus, it is not surprising that their structures have been reciprocally frozen during evolution.

Characterization of the embryonic globin chains of the marsupial Tammar wallaby, Macropus eugenii.

The embryonic hemoglobins of the marsupial Tammar wallaby (Macropus eugenii) are known to aggregate, which was shown by the finding that the Hill coefficient, h, was greater than 4.0 in the upper part of the oxygen equilibrium curve. Here, we have undertaken a detailed primary structure analysis of the Tammar wallaby pouch young hemoglobin complement, which we hoped might provide clues into the residues that cause aggregation and a high embryonic h. The Tammar wallaby embryonic hemoglobin complement is principally four major hemoglobins each with a different isoelectric point. Two early expressed hemoglobins contain the same embryonic beta-like chain, epsilon (epsilon), but two separate alpha-like chains, termed zeta and zeta prime (zeta and zeta') both of which are N-terminally blocked. The later two expressed hemoglobins contain the same adult alpha-chain, but different beta-like chains. The latest expressed hemoglobin contains the same beta-like chain, epsilon, as the two early expressed forms, but the third expressed hemoglobin contains a unique beta-like chain which we have termed omega (omega). A protein database similarity search using the first 54 N-terminal amino acids of the omega-chain showed a range of sequence identities of 57-72% to all known mammalian beta-like chains, including the other marsupial epsilon-chains. The closest identity, reflected by both the highest percentage identity and Smith-Waterman score, was with the embryonic beta-chains of the aves. While the primary structures of the hemoglobins reported here do not explain the low hemoglobin-oxygen affinity in embryonic marsupial blood, the finding of the similarity with the bird globin-like sequence with one of the marsupial chains has implications on mammalian globin evolution. How many other marsupials and placental mammals are harboring a bird-like globin in their embryos?

Koelliker haemoglobins in developing chick embryo.

1. Three Koelliker haemoglobins, HbKE, HbKA and HbKH, derived from a post-translational loss of alpha-Arg-141, were isolated from red cells of chicken embryos. HbKE is typical of embryos up to 7 days of incubation, HbKA and HbKH are found in mature embryos. 2. All the precursor haemoglobins contain alpha A chains. HbKA derives from adult haemoglobin A whose globin composition is alpha A2 beta 2, HbKH from embryonic haemoglobin H with a globin composition alpha A2 beta H2 and HbKE from embryonic haemoglobin E with globin composition alpha A2 epsilon 2. 3. No Koelliker derivatives of haemoglobins with alpha-like chains other than alpha A were observed.

Minor early embryonic chick hemoglobin M. Amino acid sequences of the epsilon and alpha D chains.

Erythrocytes of the early chick embryo contain four hemoglobins, two major and two minor. In this paper, we present amino acid sequences for the beta-like and alpha-like chains of HbM, the least abundant of the four early chick hemoglobins. The complete amino acid sequence of the beta-like chain of HbM is identical with that of the epsilon chain of HbE, the other minor early embryonic hemoglobin in the domestic chicken. Analysis of the alpha-like chain of HbM (92 of 141 residues) reveals a globin sequence closely related to the minor adult alpha D chain. Comparison of our sequence data with the nucleotide sequence of the alpha D globin gene suggests that a single gene encodes both the embryonic and adult alpha D globin polypeptides. We discuss the structure, possible function, and evolution of the HbM globin chains.

Linkage of genes for adult alpha-globin and embryonic alpha-like globin chains.

In alpha-thalassemia, the genetic locus for the alpha chains of adult hemoglobin is not expressed. We have examined the hemoglobins of a number of individual mouse embryos heterozygous for a particular alpha-thalassemia (Hbath-J) and find no decrease in the proportion of hemoglobins containing the alpha chain as compared to the hemoglobin containing the alpha-like embryonic globin chain. This result suggests that the locus for this embryonic alpha-like chain is inactivated or deleted in these embryos as well. Because a single mutational event inactivated adult and embryonic loci, we conclude that they are probably closely linked to one another on the same chromosome. We also present evidence that an unusual hemoglobin in the blood of these embryos is composed only of an embryonic beta-like chain, and is thus analogous to the hemoglobin H (beta 4 tetramer) of adult alpha-thalassemics.

Amino acid composition of some tryptic peptides of the aminoethylated alpha-like chain of [formula omitted] hemoglobin

Alpha-like chain of the main hemoglobin fraction was digested with trypsin and the liberated peptides were isolated by column chromatography on Dowex 50×8. Amino acid composition of several peptides, including the C-terminal was determined after acid hydrolysis.