978 Preclinical studies showed that β-thalassemia can be cured in mice by lentiviral-mediated transfer of the human β-globin gene. Based on these studies, clinical trials have been proposed or are underway. However, to date no study has addressed the efficacy of gene therapy in relationship to the different nature of the β-globin mutations. To address this question, we developed a new pre-clinical approach to predict the potential outcome of gene transfer in vivo, testing erythroid progenitor cells (ErPC) of a large group of β-thalassemic patients. The β0-39 and β+ IVS1-110 are two of the mutations represented in the majority of the patients enrolled in our study. Specifically, the β0-39 mutation modifies codon 39 into a premature termination codon, which, in the mutated mRNA, is easily identified and degraded by the non-sense mediated mRNA decay machinery. This and similar mutations leading to absent β-globin synthesis are defined as β0. The β+ IVS1-110 mutation, instead, activates an aberrant 3' cryptic splice site without completely abolishing normal splicing. For this reason some normal mRNA and protein is made and mutations such as this are classified as β+. We divided all patients, homozygous or compound heterozygous for β0 and β+ mutations, into three groups: β0/0, β+/+ or β+/0. Cells from patients (N=33) were infected with T9W, a lentiviral vector carrying the human β-globin gene and large elements from the human LCR, which was previously shown to cure thalassemic mice. Differentiated ErPCs of β0/0 patients (N=10), infected with T9W, increased their HbA levels in a vector-copy-number (VCN) dependent manner, reaching values similar to the expected normal ones. Conversely, ErPCs of β+/0 and β+/+ patients (N:23) responded only slightly or not at all, even though the transgenic β-globin mRNA was highly expressed and α-globin aggregates were observed. We observed that the β+ IVS1-110 mRNA appears more stable, compared to the β0-39 mRNA, representing, on average, 49% of the total β-globin mRNA. Similar results were observed with other alternative β+ splicing mutations analyzed. This led us to hypothesize that the aberrant mRNAs, generated by the alternative splicing, prevent the transgenic β-globin mRNA from being translated. We generated a second lentiviral vector in which a human ankyrin regulatory element flanks the transgenic human β-globin cassette (AnkT9W), after chromosomal integration. We compared T9W versus AnkT9 in murine erythroleukemia (MEL) cells with comparable VCN. AnkT9W expressed up to 3.5 times more human β-globin mRNA and produced nearly two times more absolute chimeric Hb (α-mouse:β-human). Furthermore, AnkT9W-RNA fractions in the cytoplasm had a net shift toward the highest multi-polysomal component, implying a translational advantage. In mice, compared to T9W, AnkT9W markedly improved the phenotype of thalassemic animals engrafted with lentiviral transduced thalassemic bone marrow cells. And finally, AnkT9W completely rescued HbA synthesis in cells of patients (N=5) carrying alternative splicing mutations, with HbA increasing according to VCN and simultaneously reducing the amount of α-globin aggregates. No major differences in HbA synthesis were observed in β0/0 cells infected with T9W or AnkT9W. We believe that the ankyrin element is responsible for increasing transcription of the β-globin transgenic mRNA during the early phases of erythroid differentiation, efficiently competing with stable aberrant mRNAs for translation. We are performing chromatin analyses and generating new vectors to further characterize the function of the ankyrin element. These new findings may have profound implications in designing gene therapy trials and in understanding the genotype/phenotype variability observed in β-thalassemia. Disclosures: No relevant conflicts of interest to declare.

B-cell linker (BLNK) protein is a non-redundant adaptor molecule in the signaling pathway activated by (pre) B-cell antigen receptor signals. We present two siblings with a homozygous deleterious frameshift mutation in BLNK, resulting in a block of B cell development in the bone marrow at the preB1 to preB2 stage, absence of circulating B cells and agammaglobulinemia. This is the first description of an enteroviral infection associated arthritis and dermatitis in a patient with BLNK deficiency.

A somatic point mutation in the JAK2 gene results in a constitutive activation of the JAK2 kinase in the erythropoietin (epo) signal transduction pathway. The mutation was found in Polycythemia Vera (PV) patients (>95%) as well as in other myeloproliferative disorders (MPD). In MPD the mutation was shown to occur in pluripotential hematopoietic stem cells and to be present in all blood cell lineages. We analyzed DNA samples from healthy individuals for JAK2V617F mutation by allele specific Real Time PCR. The mutation was detected in 9% of 114 samples with less than 0.001% of JAK2 mutant/wild-type (wt) DNA allelic ratio. The clonality of the mutation was studied by growing blood mononuclear cells from 8 healthy individuals in semi-solid culture in the presence of epo, allowing for erythroid differentiation. Individual erythroid colonies were picked up after 2 weeks. The mutation was detected in all 8 individuals at 5.6 – 24% (a median of 13.4%) of the colonies. The mutant/wt allelic ratio was 0.02% per single colony. This low frequency of positive mutant cells within a colony suggests that the mutation occurred at late stages of colony development concurrently with cell differentiation. In contrast, positive colonies derived from PV patients showed mutant/wt allelic ratio of either 50% (heterozygote) or 100% (homozygote), indicating the presence of the mutation at the early erythroid progenitor stage (BFUe). To further demonstrate that in cells from normal individuals the mutation occurs during erythroid differentiation, we studied 4 samples of CD34+ cells (purity >95%). Cells cultured in the presence of epo differentiated to erythroid precursors within 3 weeks. We found 2–4×10−5% of the JAK2V617F mutation in the CD34+ cells prior to culture, which increased more than 2 folds following cell differentiation. We hypothesized that maintenance of DNA fidelity is down graded with cell differentiation, rendering the cells susceptible for mutations in general and in JAK2 in particular. To prove this hypothesis we used mouse erythroleukemia cells (MEL) as a model for differentiation. MEL cells were transfected with plasmid containing human JAK2 wt or V617F DNA sequences and stimulated for differentiation by hemamethylene bisacetamide. After 5 days in culture human V617F sequence (~ 0.001%) was detected in cells transfected with JAK2 wt-containing plasmid and vice versa, indicating incorrect replication of the introduced plasmid. The changes occurred at 2 folds higher frequency in differentiated cells compared to undifferentiated cells. We speculated that accelerated erythropoiesis with increased erythroid differentiation will result in higher mutation rate. We therefore studied 2 conditions associated with increased hematopoiesis, Thalassemia major and smoking. We analyzed 54 samples from Thalassemic patients and detected the V617F mutation in 12 (22.2%) patients - a 2.4 -fold higher frequency compared to healthy individuals. We also screened peripheral blood of 79 heavy smokers and found the mutation in 20 (25%) individuals – a 2.8 fold higher than in non-smokers. As expected, the mutation was detected at very low frequency ~ 0.0024% of total DNA. Our results demonstrate that unlike in MPD where the JAK2V617F mutation occurs in pluripotential stem cells and expands extensively, in non-MPD individuals it arises in very low mutant/wt DNA allelic ratio during cell differentiation. These differences determine the lack of clinical significance of the JAK2V617F mutation in non-MPD individuals.