Sustained and promoter dependent bone morphogenetic protein expression by rat mesenchymal stem cells after BMP-2 transgene electrotransfer.

Transplantation of mesenchymal stem cells (MSCs) with electrotransferred bone morphogenetic protein-2 (BMP-2) transgene is an attractive therapeutic modality for the treatment of large bone defects: it provides both stem cells with the ability to form bone and an effective bone inducer while avoiding viral gene transfer. The objective of the present study was to determine the influence of the promoter driving the human BMP-2 gene on the level and duration of BMP-2 expression after transgene electrotransfer into rat MSCs. Cytomegalovirus, elongation factor-1α, glyceraldehyde 3-phosphate dehydrogenase, and beta-actin promoters resulted in a BMP-2 secretion rate increase of 11-, 78-, 66- and 36-fold over respective controls, respectively. In contrast, the osteocalcin promoter had predictable weak activity in undifferentiated MSCs but induced the strongest BMP-2 secretion rates in osteoblastically-differentiated MSCs. Regardless of the promoter driving the transgene, a plateau of maximal BMP-2 secretion persisted for at least 21 d after the hBMP-2 gene electrotransfer. The present study demonstrates the feasibility of gene electrotransfer for efficient BMP-2 transgene delivery into MSCs and for a three-week sustained BMP-2 expression. It also provides the first in vitro evidence for a safe alternative to viral methods that permit efficient BMP-2 gene delivery and expression in MSCs but raise safety concerns that are critical when considering clinical applications.

[Alphavirus derived vector, still a relevant system through new mobilisation procedure]. / Les vecteurs dérivés des alphavirus, un système toujours pertinent grâce à de nouveaux modes de vectorisation.

Among eukaryotic cell-expression-systems, the one derived from alphaviruses, including Semliki forest virus (SFV), offers an efficient method for protein production in mammalian cells. Despite this efficacy, twenty years after their discovery alphaviruses vectors remain poorly used. Alphavirus vectors exist as naked RNA vectors or as recombinant particles. The use of costly RNA-based replicons, and the fact that production of recombinant particles is a complex process to carry out, have hampered the attractiveness of the methods. Lastly, the apoptotic signals induced by alphavirus vectors replication leads to a rapid death of the producing cells. This feature, which can be detrimental in vitro, is advantageously exploited for in vivo applications. Besides laboratory applications, alphavirus vectors have been explored in rare phase I clinical trials, for vaccine development and cancer gene therapy, therefore, alphavirus vector will benefit from the advent of new, biosafety-efficient, methods for particles production. Most of the recent advances in the field proposed an heterologous mobilisation of alphavirus replicon. While increasing biosafety aspects, new methods are also simpler regarding the genesis of recombinant particles. In the present review, we overview the alphavirus life cycle with a special attention to the features influencing vector design and utility.

Semliki Forest virus-derived virus-like particles: characterization of their production and transduction pathways.

A procedure for the mobilization of Semliki Forest virus (SFV)-derived replicons using virus-like particles (VLPs) has been recently proposed. VLPs were obtained from 293T cells co-expressing the vesicular stomatitis virus glycoprotein (VSV-G) and a modified SFV replicon. Advantages of SFV VLPs include improved safety with a lack of sequence homology between components and reducing the risk of recombination events that could lead to the formation of autonomous particles. Characterization of SFV VLPs reveals a discrepancy in their ability to infect cells reported to be permissive. Furthermore, it was noted that not all viral envelopes were able to promote VLP release equally from transfected cells. These observations encouraged the examination of the molecular mechanisms supporting the different steps of VLP assembly and transduction. The use of a VSV-G related pathway for VLP entry into target cells was demonstrated; it was also observed that an internal ribosome entry site may not be adapted to control transgene expression in all cells. Finally, the need for a membrane-binding domain to obtain a fully active SFV replication complex and VLP formation was documented.

Cellular factors influencing Semliki Forest Virus vector biology.

Viral vectors are currently the best tools for gene delivery in a therapeutic setting, especially for in vivo use. Alphaviruses, a family of positive singlestranded RNA viruses, have been engineered to allow the formation of a highly efficient replicon. Using these replicons, it is possible to generate recombinant particles. Parental viruses and recombinant vectors share certain pathways while interacting with their target cells. In this review, we describe the consecutive events leading to transduction, and transgene expression, in view of the cellular factors that affect each individual step. Classical virology will benefit from the knowledge accumulated studying vectors, and such work will shed light on crosstalk between intruding viruses and their hosts. Ultimately, these data should help the design of vectors adapted to specific target cells.

Maintenance of a large pericentric inversion generated by the hobo transposable element in a transgenic line of Drosophila melanogaster.

The impact of the hobo transposable element in the global reorganization of the Drosophila melanogaster genome has been investigated in transgenic lines generated by the injection of hobo elements into the Hikone strain, which lacked them previously. Extensive surveys of transgenic lines followed for 250 generations have identified 13 inversions with hobo inserts at most breakpoints. One of these inversions is pericentric on chromosome 2. It has been maintained in the line where it was discovered and in several sublines at frequencies from 0.19 to 0.45, generating stable chromosomal polymorphisms, similar to cosmopolitan paracentric inversions in natural populations. Individuals homozygous for this inversion were viable and fertile, allowing the creation of a new homozygous strain.

The Drosophila wing differentiation factor vestigial-scalloped is required for cell proliferation and cell survival at the dorso-ventral boundary of the wing imaginal disc.

Links between genes involved in development, proliferation and apoptosis have been difficult to establish. In the Drosophila wing disc, the vestigial (vg) and the scalloped (sd) gene products dimerize to form a functional transcription factor. Ectopic expression of vg in other imaginal discs induces outgrowth and wing tissue specification. We investigated the role of the VG-SD dimer in proliferation and showed that vg antagonizes the effect of dacapo, the cyclin-cdk inhibitor. Moreover, ectopic vg drives cell cycle progression and in HeLa cultured cells, the VG-SD dimer induces cell proliferation per se. In Drosophila, ectopic vg induces expression of dE2F1 and its targets dRNR2 and string. In addition vg, but not dE2F1, interacts with and induces expression of dihydrofolate reductase (DHFR). Moreover, a decrease in VG or addition of aminopterin, a specific DHFR inhibitor, shift the dorso-ventral boundary cells of the disc to a cell death sensitive state that is correlated with reaper induction and DIAP1 downregulation. This indicates that vg in interaction with dE2F1 and DHFR is a critical player for both cell proliferation and cell survival in the presumptive wing margin area.

TONDU (TDU), a novel human protein related to the product of vestigial (vg) gene of Drosophila melanogaster interacts with vertebrate TEF factors and substitutes for Vg function in wing formation.

The mammalian TEF and the Drosophila scalloped genes belong to a conserved family of transcriptional factors that possesses a TEA/ATTS DNA-binding domain. Transcriptional activation by these proteins likely requires interactions with specific coactivators. In Drosophila, Scalloped (Sd) interacts with Vestigial (Vg) to form a complex, which binds DNA through the Sd TEA/ATTS domain. The Sd-Vg heterodimer is a key regulator of wing development, which directly controls several target genes and is able to induce wing outgrowth when ectopically expressed. Here we show that Vg contains two distinct transcriptional activation domains, suggesting that the function of Vg is to mediate transcriptional activation by Sd. By expressing a chimeric GAL4-Sd protein in Drosophila, we found that the transcriptional activity of the Vg-Sd heterodimer is negatively regulated at the AP and DV boundary of the wing disc. We also identify a novel human protein, TONDU, which contains a short domain homologous to the domain of Vg required for interaction with Sd. We show that TONDU specifically interacts with a domain conserved in all the mammalian TEF factors. Expression of TDU in Drosophila by means of the UAS-GAL4 system shows that this human protein can substitute for Vg in wing formation. We propose that TDU is a specific coactivator for the mammalian TEFs.

Specific interactions between vestigial and scalloped are required to promote wing tissue proliferation in Drosophila melanogaster.

The two genes vestigial (vg) and scalloped (sd) are required for wing development in Drosophila melanogaster. They present similar patterns of expression in second and third instar wing discs and similar wing mutant phenotypes. vg encodes a nuclear protein without any recognized nucleic acid-binding motif. Sd is a transcription factor homologous to the human TEF-1 factor whose promoter activity depends on cell-specific cofactors. We postulate that Vg could be a cofactor of Sd in the wing morphogenetic process and that, together, they could constitute a functional transcription complex. We investigated genetic interactions between the two genes. We show here that vg and sd co-operate in vivo in a manner dependent on the structure of the Vg protein. We ectopically expressed vg in the patch (ptc) domains. We show evidence that wing-like outgrowths induced by ectopic expression of vg are severely reduced in vg or sd mutant backgrounds. Accordingly, we demonstrate that ptc-GAL4-driven expression of vg induces both expressions of the endogenous vg and sd genes and that the two Vg and Sd proteins have to be produced together to promote wing proliferation. Furthermore, we show an interaction between the two proteins by double hybrid experiments in yeast. Our results therefore support the hypothesis that Sd and Vg directly interact in vivo to form a complex regulating the proliferation of wing tissue.