Silencing and position-effect (PE) variegation (PEV), which is due to integration of viral vectors in heterochromatin regions, are considered significant obstacles to obtaining a consistent level of transgene expression in gene therapy. The inclusion of chromatin insulators into vectors has been proposed to counteract this position-dependent variegation of transgene expression. Here, we show that the sea urchin chromatin insulator, sns5, protects a recombinant gamma-retroviral vector from the negative influence of chromatin in erythroid milieu. This element increases the probability of vector expression at different chromosomal integration sites, which reduces both silencing and PEV. By chromatin immunoprecipitation (ChIP) analysis, we demonstrated the specific binding of GATA1 and OCT1 transcription factors and the enrichment of hyperacetylated nucleosomes to sns5 sequences. The results suggest that this new insulator is able to maintain a euchromatin state inside the provirus locus with mechanisms that are common to other characterized insulators. On the basis of its ability to function as barrier element in erythroid milieu and to bind the erythroid specific factor GATA1, the inclusion of sns5 insulator in viral vectors may be of practical benefit in gene transfer applications and, in particular, for gene therapy of erythroid disorders.

Wharton's jelly mesenchymal stromal cells (WJ-MSCs) have been recently exploited as a feeder layer in coculture systems to expand umbilical cord blood-hematopoietic stem/progenitor cells (UCB-HSPCs). Here, we investigated the role of WJ-MSCs in supporting ex vivo UCB-HSPC expansion either when cultured in direct contact (DC) with WJ-MSCs or separated by a transwell system or in the presence of WJ-MSC-conditioned medium. We found, in short-term culture, a greater degree of expansion of UCB-CD34 cells in a DC system (15.7 ± 4.1-fold increase) with respect to the other conditions. Moreover, in DC, we evidenced two different CD34 cell populations (one floating and one adherent to WJ-MSCs) with different phenotypic and functional characteristics. Both multipotent CD34/CD38 and lineage-committed CD34/CD38 hematopoietic progenitors were expanded in a DC system. The former were significantly more represented in the adherent cell fraction than in the floating one (18.7 ± 11.2% vs. 9.7 ± 7.9% over the total CD34 cells). Short-term colony forming unit (CFU) assays showed that HSPCs adherent to the stromal layer were able to generate a higher frequency of immature colonies (CFU-granulocyte/macrophage and burst-forming unit erythroid/large colonies) with respect to the floating cells. In the attempt to identify molecules that may play a role in supporting the observed ex vivo HSPC growth, we performed secretome analyses. We found a number of proteins involved in the HSPC homing, self-renewal, and differentiation in all tested conditions. It is important to note that a set of sixteen proteins, which are only in part reported to be expressed in any hematopoietic niche, were exclusively found in the DC system secretome. In conclusion, WJ-MSCs allowed a significant ex vivo expansion of multipotent as well as committed HSPCs. This may be relevant for future clinical applications.

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Lentiviruses have a number of molecular features in common, starting with the ability to integrate their genetic material into the genome of non-dividing infected cells. A peculiar property of non-primate lentiviruses consists in their incapability to infect and induce diseases in humans, thus providing the main rationale for deriving biologically safe lentiviral vectors for gene therapy applications. In this review, we first give an overview of non-primate lentiviruses, highlighting their common and distinctive molecular characteristics together with key concepts in the molecular biology of lentiviruses. We next examine the bioengineering strategies leading to the conversion of lentiviruses into recombinant lentiviral vectors, discussing their potential clinical applications in ophthalmological research. Finally, we highlight the invaluable role of animal organisms, including the emerging zebrafish model, in ocular gene therapy based on non-primate lentiviral vectors and in ophthalmology research and vision science in general.

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