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

We report the development of episomal vectors for the specific gamma-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 beta-globin Replicator, the DNA replication-Initiation Region from the beta-globin locus. Vector Zif-VP64-Ep1 containing transcription cassettes CMV-Zif-VP64 and CMV-eGFP-S/MAR transfected a)K562 cells; b)murine beta-YAC bone marrow cells (BMC); c)human haematopoietic progenitor CD34(+) cells, with transfection efficiencies of 46.3 +/- 5.2%, 23.0 +/- 2.1% and 24.2 +/- 2.4% respectively. K562 transfections generated stable cell lines running for 28 weeks with and without selection, with increased levels of gamma-globin mRNA by 3.3 +/- 0.13, of gamma-globin protein by 6.75 +/- 3.25 and HbF protein by 2 +/- 0.2 fold, while the vector remained episomal and non integrated. In murine beta-YAC BMCs the vector mediated the activation of the silent human gamma-globin gene and in CD34+ cells, increased gamma-globin mRNA, albeit only transiently. A second vector Zif-VP64-Ep2, with both transcription cassettes carrying promoter SFFV instead of CMV and the addition of beta-globin Replicator, transferred into CD34+ cells, produced CD34(+) eGFP(+) cells, that generated colonies in colony forming cell cultures. Importantly, these were 100% fluorescent, with 2.11 +/- 0.13 fold increased gamma-globin mRNA, compared to non-transfected cells. We consider these episomal vectors valid, safer alternatives to viral vectors

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. We constructed a non-viral, episomal vector pEPβ-globin for the physiological β-globin gene based on two human chromosomal elements: the scaffold or matrix attachment region (S/MAR), allowing for long nuclear retention and non-integration and the β-globin replication initiation region (IR), allowing for enhancement of replication and establishment. After nucleofections into K562 cells with a transfection efficiency of 24.62 ± 7.7%, the vector induces stable transfection and is detected in long-term cultures as a non-integrating, circular episome expressing the β-globin gene efficiently. Transfections into CD34+ cells demonstrate an average efficiency of 15.57 ± 11.64%. In the colony-forming cell assay, fluorescent colonies are 92.21%, which is comparable to those transfected with vector pEP-IR at 92.68%. Additionally, fluorescent colonies produce β-globin mRNA at a physiologically 3-fold higher level than the corresponding non-transfected cells. Vector pEPβ-globin provides the basis for the development of therapeutic EV for gene therapy of β-thalassemias

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

<div><p>Background and Objectives</p><p>Newborns delivered late-preterm (between 34^0/7 and 36^6/7 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.</p><p>Methods</p><p>This prospective cross-sectional study included 56 late-preterm infants with and 60 without RDS. Specific SP-A1/SP-A2 haplotypes and SP-B Ile131Thr polymorphic alleles were determined in blood specimens using polymerase-chain-reaction and DNA sequencing.</p><p>Results</p><p>The SP-A1 6A⁴ and the SP-A2 1A⁵ haplotypes were significantly overrepresented in newborns with RDS compared to controls (OR 2.86, 95%CI 1.20–6.83 and OR 4.68, 95%CI 1.28–17.1, respectively). The distribution of the SP-B Ile131Thr genotypes was similar between the two late-preterm groups. Overall, the SP-A1 6A⁴ or/and SP-A2 1A⁵ haplotype was present in 20 newborns with RDS (35.7%), resulting in a 4.2-fold (1.60–11.0) higher probability of RDS in carriers. Multivariable regression analysis revealed that the effect of SP-A1 6A⁴ and SP-A2 1A⁵ haplotypes was preserved when adjusting for known risk or protective factors, such as male gender, smaller gestational age, smaller weight, complications of pregnancy, and administration of antenatal corticosteroids.</p><p>Conclusions</p><p>Specific SP-A genetic variants may influence the susceptibility to RDS in late-preterm infants, independently of the effect of other perinatal factors.</p></div

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. Liver is an optimal organ for the secretion and systemic distribution of a therapeutic transgene product. Cutting edge non-viral gene therapy tools were tested in order to produce a high and sustained level of therapeutic protein secretion by the liver using the hydrodynamic delivery technique. The use of S/MAR matrix attachment region provided a slight, however not statistically significant, increase in the expression of a reporter gene in the liver. We have selected the von Willebrand Factor (vWF) gene as a particularly challenging large gene (8.4 kb) for liver delivery and expression, and also because a high vWF blood concentration is required for disease correction. By using the optimized miniplasmid pFAR free of antibiotic resistance gene together with the Sleeping Beauty transposon and the hyperactive SB100X transposase, we have obtained a sustainable level of vWFblood secretion by the liver, at 65% of physiological level. Our results point to the general use of this plasmid platform using the liver as a protein factory to treat numerous rare disorders by gene therapy.status: publishe

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

International audienceMany 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. Liver is an optimal organ for the secretion and systemic distribution of a therapeutic transgene product. Cutting edge non-viral gene therapy tools were tested in order to produce a high and sustained level of therapeutic protein secretion by the liver using the hydrodynamic delivery technique. The use of S/MAR matrix attachment region provided a slight, however not statistically significant, increase in the expression of a reporter gene in the liver. We have selected the von Willebrand Factor (vWF) gene as a particularly challenging large gene (8.4 kb) for liver delivery and expression, and also because a high vWF blood concentration is required for disease correction. By using the optimized miniplasmid pFAR free of antibiotic resistance gene together with the Sleeping Beauty transposon and the hyperactive SB100X transposase, we have obtained a sustainable level of vWFblood secretion by the liver, at 65% of physiological level. Our results point to the general use of this plasmid platform using the liver as a protein factory to treat numerous rare disorders by gene therapy

Distribution of SP-A1 and SP-A2 haplotypes in the two late-preterm study groups.

<p>Distribution of SP-A1 and SP-A2 haplotypes in the two late-preterm study groups.</p