Non-Viral Episomal Vector Mediates Efficient Gene Transfer of the β-Globin Gene into K562 and Human Haematopoietic Progenitor Cells.

β-Thalassemia is a subgroup of inherited blood disorders associated with mild to severe anemia with few and limited conventional therapy options. Lately, lentiviral vector-based gene therapy has been successfully applied for disease treatment. However, the current development of non-viral episomal vectors (EV), non-integrating and non-coding for viral proteins, may be helpful in generating valid alternatives to viral vectors.

Protein network and pathway analysis in a pharmacogenetic study of cyclosporine treatment response in Greek patients with psoriasis.

Although cyclosporine comprises a well-established systemic therapy for psoriasis, patients show important heterogeneity in their treatment response. The aim of our study was the pharmacogenetic analysis of 200 Greek patients with psoriasis based on the cyclosporine pathway related protein-protein interaction (PPI) network, reconstructed through the PICKLE meta-database. We genotyped 27 single nucleotide polymorphisms, mapped to 22 key protein nodes of the cyclosporine pathway, via the utilization of the iPLEX®GOLD panel of the MassARRAY® System.

Advances in the Development and the Applications of Nonviral, Episomal Vectors for Gene Therapy.

Nonviral and nonintegrating episomal vectors are reemerging as a valid, alternative technology to integrating viral vectors for gene therapy, due to their more favorable safety profile, significantly lower risk for insertional mutagenesis, and a lesser potential for innate immune reactions, in addition to their low production cost. Over the past few years, attempts have been made to generate highly functional nonviral vectors that display long-term maintenance within cells and promote more sustained gene expression relative to conventional plasmids. Extensive research into the parameters that stabilize the episomal DNA within dividing and nondividing cells has shed light into the genetic and epigenetic mechanisms that govern replication and transcription of episomal DNA within a mammalian nucleus in long-term cell culture.

Exploring pharmacogenetic variants for predicting response to anti-TNF therapy in autoimmune diseases: a meta-analysis.

The aim of this study is to explore how SNPs may affect the response to anti-TNF-α therapy in the major autoimmune diseases, such as psoriasis, rheumatoid arthritis, inflammatory bowel diseases and Spondyloarthritis. We conducted a systematic overview on the field, by assessing all studies that examined the association between polymorphisms and response to anti-TNF-α therapy in participants of European descent. In total, six independent SNPs located in , , and genes were significantly associated with response to TNF-α blockers, found mainly in disease-subgroup analyses.

Episomal vectors based on S/MAR and the β-globin Replicator, encoding a synthetic transcriptional activator, mediate efficient γ-globin activation in haematopoietic cells.

We report the development of episomal vectors for the specific γ-globin transcription activation in its native position by activator Zif-VP64, based on the Scaffold/Matrix Attachment Region (S/MAR) for episomal retention and the β-globin Replicator, the DNA replication-Initiation Region from the β-globin locus. Vector Zif-VP64-Ep1 containing transcription cassettes CMV- Zif-VP64 and CMV-eGFP-S/MAR transfected a)K562 cells; b)murine β-YAC bone marrow cells (BMC); c)human haematopoietic progenitor CD34 cells, with transfection efficiencies of 46.3 ± 5.

Impact of ZBTB7A hypomethylation and expression patterns on treatment response to hydroxyurea.

Background: We aimed to clarify the emerging epigenetic landscape in a group of genes classified as "modifier genes" of the β-type globin genes (HBB cluster), known to operate in trans to accomplish the two natural developmental switches in globin expression, from embryonic to fetal during the first trimester of conception and from fetal to adult around the time of birth. The epigenetic alterations were determined in adult sickle cell anemia (SCA) homozygotes and SCA/β-thalassemia compound heterozygotes of Greek origin, who are under hydroxyurea (HU) treatment. Patients were distinguished in HU responders and HU non-responders (those not benefited from the HU) and both, and in vivo and in vitro approaches were implemented.

Improved molecular platform for the gene therapy of rare diseases by liver protein secretion.

Many rare monogenic diseases are treated by protein replacement therapy, in which the missing protein is repetitively administered to the patient. However, in several cases, the missing protein is required at a high and sustained level, which renders protein therapy far from being adequate. As an alternative, a gene therapy treatment ensuring a sustained effectiveness would be particularly valuable.

The β-globin Replicator greatly enhances the potential of S/MAR based episomal vectors for gene transfer into human haematopoietic progenitor cells.

Specific human chromosomal elements enhance the performance of episomal gene-transfer vectors. S/MAR-based episomal vector pEPI-eGFP transfects CD34 haematopoietic cells, but only transiently. To address this issue we reinforced (1) transgene transcription by replacing the CMV promoter driving eGFP with the EF1/HTLV or SFFV promoters to produce vectors pEPI-EF1/HTLV and pEPI-SFFV, respectively; and (2) plasmid replication by inserting the replication-Initiation Region (IR) from the β-globin locus into vector pEPI-SFFV to produce vector pEP-IR.

Surfactant Protein A and B Gene Polymorphisms and Risk of Respiratory Distress Syndrome in Late-Preterm Neonates.

Background And Objectives: Newborns delivered late-preterm (between 340/7 and 366/7 weeks of gestation) are at increased risk of respiratory distress syndrome (RDS). Polymorphisms within the surfactant protein (SP) A and B gene have been shown to predispose to RDS in preterm neonates. The aim of this study was to investigate whether specific SP-A and/or SP-B genetic variants are also associated with RDS in infants born late-preterm.

Cytochrome P450 2C19 polymorphism and antiplatelet therapy. Who should really be genotyped?

CYP2C19 is one of the principal enzymes involved in the metabolism of clopidogrel. The genes encoding CYP enzymes are polymorphic, with common alleles conferring reduced function. A loss-of-function allele, CYP2C19*2, is associated with an increased risk of major adverse cardiovascular events, particularly stent thrombosis, in patients with acute coronary syndromes who are receiving clopidogrel, especially among those undergoing percutaneous coronary intervention.

KLF10 gene expression is associated with high fetal hemoglobin levels and with response to hydroxyurea treatment in β-hemoglobinopathy patients.

Aim: In humans, fetal hemoglobin (HbF) production is controlled by many intricate mechanisms that, to date, remain only partly understood.

Patients & Methods: Pharmacogenomic analysis of the effects of hydroxyurea (HU) on HbF production was undertaken in a collection of Hellenic β-thalassemia and sickle cell disease (SCD) compound heterozygotes and a collection of healthy and KLF1-haploinsufficient Maltese adults, to identify genomic signatures that follow high HbF patterns.

Results: KLF10 emerged as a top candidate.

A single nucleotide polymorphism in the HBBP1 gene in the human β-globin locus is associated with a mild β-thalassemia disease phenotype.

The rs2071348 (g.5264146A>C) polymorphism on the HBB pseudogene, namely HBBP1, previously emerged as a variant significantly associated with a milder disease phenotype in Asian β(0)-thalassemia/hemoglobin (Hb) E (β(0)-thal/Hb E [β26(B8)Glu→Lys, GAG>AAG]) patients. In this study, we aimed to explore the possible association of rs2071348 with β-thalassemia (β-thal) disease severity in a group of β-thal major (β-TM) patients (severe phenotype) and β-thal intermedia (β-TI) patients (mild phenotype) of Hellenic origin and compare the results with normal (non thalassemic) individuals of the same origin.

Santorini mutation detection meeting 2011: rapid advance in sequencing technology poses challenges for interpretation of genetic variations.

The 11th International Symposium on Mutations in the Genome was held on 6-10 June, 2011, in Santorini, Greece. Meeting participants described novel detection technologies, rapid advances in whole genome and whole-exome sequencing, but also highlighted the urgent need for the development of sequence variation databases and the clinical interpretation of the genomic data. This report summarizes some of the major themes presented during the meeting.

Resistance to high-maintenance dose of prasugrel treated by ticagrelor: a case report.

We describe a case of a 34-year-old woman with chronic renal failure under haemodialysis. The patient exhibited high on-treatment platelet reactivity to gradually stronger thienopyridine regimens, including standard and high maintenance doses of prasugrel. Platelet function was monitored by VerifyNow assay and genotyping for various single-nucleotide polymorphisms was performed.

CYP2C19*2 and other genetic variants affecting platelet response to clopidogrel in patients undergoing percutaneous coronary intervention.

Introduction: Information regarding any possible additional effect of genetic variants other than CYP2C19*2 on platelet reactivity in patients undergoing percutaneous coronary intervention (PCI), while on dual antiplatelet therapy, is sparse.

Materials And Methods: Genotyping for CYP2C19*2, CYP2C19*17, CYP2C9*3, CYP2B6*5, ABCB1 and P2RY12 (c.-217+2739T>C) variants was performed in 146 consecutive PCI patients receiving clopidogrel.

Prasugrel overcomes high on-clopidogrel platelet reactivity post-stenting more effectively than high-dose (150-mg) clopidogrel: the importance of CYP2C19*2 genotyping.

Objectives: The primary aim of the study was to determine the antiplatelet effects of prasugrel versus high-dose clopidogrel in patients with high on-treatment platelet reactivity (HTPR) after percutaneous coronary intervention (PCI) and, secondarily, their relation to cytochrome (CYP) 2C19*2 carriage.

Background: High on-treatment platelet reactivity after clopidogrel administration after PCI is linked to the loss-of-function CYP2C19*2 allele and accompanied by an increased risk of adverse events.

Methods: We performed a prospective, randomized, single-blind, crossover study of platelet inhibition by prasugrel 10 mg/day versus high-dose 150 mg/day clopidogrel in 71 (of 210 screened; 33.

Angiotensin-converting enzyme gene polymorphism and respiratory muscle function in infants.

Objective: Angiotensin-converting enzyme (ACE) gene contains a polymorphism consisting of either the presence (I) or absence (D) of a 287-bp fragment. Recent studies have suggested that the I-allele may be associated with superior exercise endurance; respiratory muscle function may be similarly influenced. The pressure-time index of inspiratory muscles (PTImus) is a measure of the load-capacity ratio of the inspiratory muscles.

Region-specific genetic heterogeneity of HBB mutation distribution in South-Western Greece.

beta-Thalassemia (beta-thal), is caused by reduced or absent synthesis of beta-globin chains resulting in impaired erythropoiesis. It is the most common single gene defect disease in Greece, with heterozygous rates reaching, on average, 8% in the general population. Here, we performed molecular analyses on 199 unrelated beta-thal and compound beta-thal/sickle cell disease patients, of whom 157 originated from three prefectures of South-Western Greece, namely Achaia, Ilia and Etoloakarnania.

Association of circulating angiotensin converting enzyme activity with respiratory muscle function in infants.

Background: Angiotensin converting enzyme (ACE) gene contains a polymorphism, consisting of either the presence (I) or absence (D) of a 287 base pair fragment. Deletion (D) is associated with increased circulating ACE (cACE) activity. It has been suggested that the D-allele of ACE genotype is associated with power-oriented performance and that cACE activity is correlated with muscle strength.

Allelic imbalance of expression and epigenetic regulation within the alpha-synuclein wild-type and p.Ala53Thr alleles in Parkinson disease.

Genetic alterations in the alpha-synuclein (SNCA) gene have been implicated in Parkinson Disease (PD), including point mutations, gene multiplications, and sequence variations within the promoter. Such alterations may be involved in pathology through structural changes or overexpression of the protein leading to protein aggregation, as well as through impaired gene expression. It is, therefore, of importance to specify the parameters that regulate SNCA expression in its normal and mutated state.

The functional role of S/MARs in episomal vectors as defined by the stress-induced destabilization profile of the vector sequences.

The scaffold/matrix attachment regions (S/MARs) are chromosomal elements that participate in the formation of chromatin domains and have origin of replication support functions. Because of all these functions, in recent years, they have been used as part of episomal vectors for gene transfer. The S/MAR of the human beta-interferon gene has been shown to support efficient episome retention and transgene expression in various mammalian cells.