Development of a transient gene delivery vector to rejuvenate myofibroblasts for tissue engineering

Tissue engineering involves the use of synthetic or natural materials for the replacement of non-functional tissue or organs. One approach to tissue engineering involves harvesting the patients own cells, culturing them in vitro and then seeding the cells onto scaffolds. The use of autologous cells avoids problems associated with rejection, however a major limitation of this approach is the finite lifespan of primary cells in culture. This finite replicative lifespan is due to the shortening of telomeres, short repetitive sequences of DNA located at the ends of eukaryotic chromosomes. Ectopic expression of telomerase reverse transcriptase (hTERT) is able to reconstitute telomerase activity and maintain the length of telomeres. This study investigated an alternative gene delivery vector, the baculovirus, for the expression of hTERT in primary human cells. The aim was to rejuvenate cells for use in the tissue engineering of heart valves. The optimal conditions for the baculoviral transduction of primary fibroblasts were first determined. Transduction efficiencies of greater than 50% were routinely obtained, without the need for ultracentrifugation of the viral supernatant. A recombinant baculovirus was then used to efficiently deliver the hTERT gene to primary fibroblasts. The complete telomerase enzyme was found to be active, as evidenced by the Telomere Repeat Amplification Protocol. Although no increase in telomere length was detected, expression of hTERT in primary fibroblasts resulted in a significant extension of replicative lifespan. The transience of the gene delivery was confirmed with a baculoviral DNA half-life of approximately 10 days. To our knowledge this is a novel attempt to use a recombinant baculovirus for the extension of cellular lifespan by exogenous expression of telomerase

The awakening of the CDK10/Cyclin M protein kinase

Cyclin-dependent kinases (CDKs) play important roles in the control of fundamental cellular processes. Some of the most characterized CDKs are considered to be pertinent therapeutic targets for cancers and other diseases, and first clinical successes have recently been obtained with CDK inhibitors. Although discovered in the pre-genomic era, CDK10 attracted little attention until it was identified as a major determinant of resistance to endocrine therapy for breast cancer. In some studies, CDK10 has been shown to promote cell proliferation whereas other studies have revealed a tumor suppressor function. The recent discovery of Cyclin M as a CDK10 activating partner has allowed the unveiling of a protein kinase activity against the ETS2 oncoprotein, whose degradation is activated by CDK10/Cyclin M-mediated phosphorylation. CDK10/Cyclin M has also been shown to repress ciliogenesis and to maintain actin network architecture, through the phoshorylation of the PKN2 protein kinase and the control of RhoA stability. These findings shed light on the molecular mechanisms underlying STAR syndrome, a severe human developmental genetic disorder caused by mutations in the Cyclin M coding gene. They also pave the way to a better understanding of the role of CDK10/Cyclin M in cancer

Antiviral effect of ribonuclease conjugated oligodeoxynucleotides targeting the IRES RNA of the hepatitis C virus.

International audienceHepatitis C virus (HCV) translation initiation is mediated by a highly structured and conserved RNA, termed the Internal Ribosome Entry Site (IRES), located at the 5'-end of its single stranded RNA genome. It is a key target for the development of new antiviral compounds. Here we made use of the recently developed HCV cell culture system to test the antiviral activity of artificial ribonucleases consisting of imidazole(s) linked to antisense oligodeoxynucleotides targeting the HCV IRES. Results from the cell culture model indicate that the naked antisense oligodeoxynucleotide displayed an efficient antiviral activity. Despite the increased activity observed with the addition of imidazole moieties when tested with the cell-free system, it appears that these improvements were not reproduced in the cellular model

STAR syndrome-associated CDK10/Cyclin M regulates actin network architecture and ciliogenesis

<p>CDK10/CycM is a protein kinase deficient in STAR (toe Syndactyly, Telecanthus and Anogenital and Renal malformations) syndrome, which results from mutations in the X-linked <i>FAM58A</i> gene encoding Cyclin M. The biological functions of CDK10/CycM and etiology of STAR syndrome are poorly understood. Here, we report that deficiency of CDK10/Cyclin M promotes assembly and elongation of primary cilia. We establish that this reflects a key role for CDK10/Cyclin M in regulation of actin network organization, which is known to govern ciliogenesis. In an unbiased screen, we identified the RhoA-associated kinase PKN2 as a CDK10/CycM phosphorylation substrate. We establish that PKN2 is a bone fide regulator of ciliogenesis, acting in a similar manner to CDK10/CycM. We discovered that CDK10/Cyclin M binds and phosphorylates PKN2 on threonines 121 and 124, within PKN2′s core RhoA-binding domain. Furthermore, we demonstrate that deficiencies in CDK10/CycM or PKN2, or expression of a non-phosphorylatable version of PKN2, destabilize both the RhoA protein and the actin network architecture. Importantly, we established that ectopic expression of RhoA is sufficient to override the induction of ciliogenesis resulting from CDK10/CycM knockdown, indicating that RhoA regulation is critical for CDK10/CycM's negative effect on ciliogenesis. Finally, we show that kidney sections from a STAR patient display dilated renal tubules and abnormal, elongated cilia. Altogether, these results reveal CDK10/CycM as a key regulator of actin dynamics and a suppressor of ciliogenesis through phosphorylation of PKN2 and promotion of RhoA signaling. Moreover, they suggest that STAR syndrome is a ciliopathy.</p