Construction of the recombinant adenovirus vector carrying antisense multidrug resistance gene

BACKGROUND: Multidrug resistance proteins serve as transporters for chemical drugs in human malignancies. The objective of this study was to construct a homologous recombinant adenovirus carrying a reversal fragment of multidrug resistance gene 1 (mdr1) gene cDNA sequence. METHODS: The fragment of the mdr1 gene from the plasmid pHaMDRI-1 carrying the whole human mdr1 cDNA sequence was inserted reversely into the shuttle plasmid pAdTrack-CMV of adenoviral vector system AdEasy. The homologous recombination process was taken place in E. coli BJ5183 with the backbone plasmid pAdEasy-1. After packaging in 293 cells, recombinant adenoviral plasmid was generated. The recombinant adenoviral plasmid was identified by polymerase chain reaction (PCR), restriction endonucleases digest, DNA sequence analysis and fluorescence microscopic photograph, respectively. RESULTS: The recombinant adenovirus pAdEasy-GFPASmdr1 was successfully constructed and identified by PCR, restriction digest, and sequencing with strong green fluorescence expression in fluorescence microscopic photograph. CONCLUSIONS: The recombinant adenoviral mdr1 vector would introduce the antisense mdr1 gene into the human multidrug resistance hepatocellular cell fine effectively, which would provide an experimental basis to study the multidrug resistance in human hepatocellular carcinoma.

Construction of Rat Calcineurin A α cDNA Recombinant Adenovirus Vector and Its Identification

Rat calcineurin （CAN） A a isoform （Ppp3ca） cDNA recombinant adenovirus vector was constructed in order to explore the effect of CaN on the myocardium apoptosis induced by ischemia-reperfusion injury. Total RNA was isolated from the heart of the adult Wistar rht, and Ppp3ca CDS segment of approximate 1.59 kb size was amplified by reverse transcriptional PCR method. Ppp3ca cDNA segment was cloned into pMD18-T Simple vector for sequencing, and the right clone was named T-Ppp3ca. Ppp3ca cDNA segment obtained from T-Ppp3ca was ligated with pShuttle2-IRES-EGFP to construct a recombinant plasmid pShuttle2-Ppp3ca-IRES-EGFP. Ppp3ca-IRES-EG- FP expression cassette containing CMV, Ppp3ca-IRES-EGFP and SV40 polyA DNA fragment （3.97 kb） obtained from pShuttle2-Ppp3ca-IRES-EGFP was connected with pAdeno-X backbone sequence to construct a recombinant plasmid pAdeno Ppp3ca. After being identified by PCR and enzyme digestion, recombinant plasmid pAdeno-Ppp3ca was packaged in HEK293 cells. Supernatant of adenovirus from HEK293 cells was collected after a visible cytopathic effect （CPE） appeared. The DNA of the recombinant adenovirus was extracted with the standard method. The presence of the recombinant adenovirus was verified by PCR. The results showed that sequencing results verified that the PCR product of Ppp3ca gene was identical to GenBank. Agarose electrophoresis showed the bands of recombined plasmid pAdeno-Ppp3ca and the recombinant adenovirus identified by enzyme digestion and PCR were in the right range corresponding with expectation. It was concluded that the recombinant adenovirus carrying rat calcineurin A a （Ppp3ca） cDNA as well as a report gene-enhancer green fluorescent protein gene was successfully constructed in this experiment.

Generation and Immunogenicity of a Recombinant Adenovirus Co-Expressing the E2 Protein of Classical Swine Fever Virus and the GP5 Protein of Porcine Reproduction and Respiratory Syndrome Virus

Classical swine fever （CSF） and porcine reproduction and respiratory syndrome （PRRS） are both economically important, highly contagious diseases of swine worldwide. To develop an effective vaccine to control these two diseases, we constructed a recombinant adenovirus rAdV-GP52AE2, using a replication-defective human adenovirus serotype 5 as a delivery vector, to co-express the GP5 protein of highly pathogenic porcine reproduction and respiratory syndrome virus （PRRSV） and the E2 protein of classical swine fever virus （CSFV）. Foot-and-mouth disease virus （FMDV） 2A peptide was used as a linker between the GP5 and E2 proteins to allow automatic self-cleavage of the polyprotein. The GP5 and E2 genes were expressed as demonstrated by immunofluorescence assay and Western blotting. Immunization of mice resulted in a CSFV-neutralizing antibody titer of 1：128 and a PRRSV-neutralizing antibody titer of 1：16. The lymphoproliferative responses were detected by Cell Counting Kit-8 assay and the stimulation index of CFSV-specific and PRRSV-specific lymphocytes in the rAdV-GP52AE2 group was significantly higher than that in the negative control group. The results show that rAdV-GP52AE2 can induce both effective humoral and cell-mediated immune responses in mice. The protective efficacy of the recombinant virus against CSF was evaluated in immunized rabbits, which were protected from fever induced by challenge with C-strain. Our study provides supporting evidence for the use of FMDV 2A to develop a bivalent genetically-engineered vaccine.

Recombinant adenovirus-mediated overexpression of 3β-hydroxysteroid-Δ24 reductase

3β-Hydroxysteroid-△24 reductase （DHCR24） is a multifunctional enzyme that localizes to the endoplasmic reticulum and has neuroprotective and cholesterol-synthesizing activities. DHCR24 overexpression confers neuroprotection against apoptosis caused by amyloid β deposition. The present study aimed to construct two recombinant adenoviruses driving DHCR24 expression specifically in neurons. Two SYN1 promoter DNA fragments were obtained from human （h） and rat （r）. Recombinant Ad-r（h）SYN1-DHCR24 was transfected into AD-293, N2A （mouse neuroblastoma）, and MIN6 （mouse pancreatic carcinoma） cells. Western blot analysis showed DHCR24 was specially expressed in 293 and N2A cells, but no specific band was found in MIN6 cells. This demonstrates that the recombinant adenoviruses successfully express DHCR24, and no expression is observed in non-neuronal cells. TUNEL assay results showed apoptosis was inhibited in adenovirus-transfected neurons. Detecting reactive oxygen species by immunoflu- orescence, we found that adenovirus transfection inhibits apoptosis through scavenging excess reactive oxygen species. Our findings show that the recombinant DHCR24 adenoviruses induce neuron-specific DHCR24 expression, and thereby lay the foundation for further studies on DHCR24 gene therapy for Alzheimer＇s disease.

Rapid construction of phosphatase and tensin homolog-deleted on chromosome ten gene recombinant adenovirus using the AdEasy system

Recent studies have shown that phosphatase and tensin homolog-deleted on chromosome ten （PTEN） gene plays an important role in ischemic brain damage and synaptic plasticity. The AdEasy system, which has been widely used, greatly simplifies preparation of recombinant adenovirus. Therefore, recombinant defective adenovirus vector carrying human PTEN tumor suppressor gene （Ad-PTEN） was constructed using the AdEasy-1 system and was transfected into HEK293 cells for packaging and amplification. Infection efficiency and expression intensity were observed in primary cultured rat hippocampal neurons infected with Ad-PTEN in vitro. Results revealed a cytopathic effect in green fluorescent protein expression, which increased with prolonged time. After three cycles of amplification, the adenovirus titer was increased to an adequate titer for infecting hippocampal neurons. The entire process typically requires 4-5 weeks for completion. Results suggested that recombinant defective adenovirus vector carrying the PTEN gene was successfully and rapidly constructed using the AdEasy system.

Optical control after transfection of channelrhodopsin-2 recombinant adenovirus in visual cortical cells

Channelrhodopsin-2 ectopically expressed in the retina can recover the response to blue light in genetically blind mice and rats, but is unable to restore visual function due to optic nerve or optic tract lesions. Long Evans rats at postnatal day 1 were used for primary culture of visual cortical cells and 24 hours later, cells were transfected with recombinant adenovirus carrying channelrhodopsin-2 and green fluorescent protein genes. After 2 4 days of transfection, green fluorescence was visible in the cultured cells. Cells were stimulated with blue light （470 nm）, and light-induced action potentials were recorded in patch-clamp experiments. Our findings indicate that channelrhodopsin-2-recombinant adenovirus transfection of primary cultured visual cortical cells can control the production of action potentials via blue light stimulation.

Construction and Immunogenicity of a Recombinant Adenovirus Expressing the HA Gene of H5N1 Subtype Swine Influenza Virus

To construct a recombinant adenovirus shuttle plasmid pDC315-H5HA-EGFP,the HA gene of A/Swine/Fujian/1/2001(H5N1) was amplified by RT-PCR and then inserted into adenovirus shuttle plasmid pDC315.A replication-defective recombinant adenovirus expressing the HA gene(rAd-H5HA-EGFP) was generated by co-transfecting the recombinant shuttle plasmid pDC315-H5HA-EGFP and the genomic plasmid pBHGlox△E1,E3Cre in HEK293 cells.The recombinant adenovirus was confirmed by PCR,RT-PCR and Western blot assay.These results demonstrated that HA protein was properly expressed by the rAd-H5HA-EGFP in HEK293 cells and had natural biological activities.The TCID50 of the rAd-H5HA- EGFP was assessed to be 2.26×1010/mL after propagation and purification.Immunization of BALB/ c mice indicated that rAd-H5HA-EGFP induced HI antibodies and protected mice from replication of the challenge virus in their lungs.

Construction of Recombinant Adenovirus Carrying GRIM19 and Its Effect on SW480 Cells

In order to examine the effect of GRIM19 on colon cancer cell SW480, the recombinant adenovirus carrying GRIM19 gene was constructed and transfected into SW480 cells. GRIM19 cDNA was amplified by PCR with the template pcxn2-GRIM19 and cloned into the shuttle plasmid pAdTrack-CMV. The plasmid pAdTrack-CMV-GRIM19 was linearized by PmeⅠ and homologously recombined with bone plasmid pAdEasy-1 in BJ5183, followed by identification by enzyme digestion. After transfection of linearized pAd-GRIM19 with PacⅠ into HEK293 cells, Ad-GRIM19 was obtained and amplified by 3 circles. SW480 cells were infected with Ad-GRIM19. The apoptosis rate was detected by flow cytometry. Agarose electrophoresis revealed the bands of recombinant plasmids identified by enzyme digestion were in the right range corresponding with expectation. Under the fluorescent microscopy, the package of Ad-GRIM19 in HEK293 cells and the expression of Ad-GRIM19 in SW480 cells were observed. The transfection of Ad-GRIM19 into SW480 cells increased the apoptosis rate of SW480 cells as compared with controls, It was concluded that Ad-GRIM19 was successfully constructed and the overexpression of GRIM19 in colon cancer cell lines could promote the apoptotic cell death.

Inhibition of Dual Specific Oncolytic Adenovirus on Esophageal Cancer via Activation of Caspases by a Mitochondrial-dependent Pathway

We investigated the anti-tumor effects of dual cancer specific-oncolytic adenovirus Ad-VP on esophageal cancer（EC）. The anti-tumor activity of Ad-VP was compared with that of the control recombinant adenoviruses （Ad-GP, Ad-Apoptin, Ad-EGFP） in human esophageal cancer cell EC-109 and human normal liver cell L02 in vitro. In 3-（4,5-dimethylthiazol-2-yl）-2,5-diphenyltetrazolium bromide（MTT） assays, the growth of EC-109 cells was slightly inhibited by Ad-GP, Ad-Apoptin and Ad-EGFE However, Ad-VP induced a significant cytotoxic effect. Infection of EC-109 cells with Ad-VP resulted in a significant induction of apoptosis of them in vitro, detected by 4＇,6-diamidino-2-phenylindole（DAPI） or acridine orange and ethidium bromide staining. The results of Western blot and flow cytometric assay indicate the loss of mitochondrial membrane potential（Aψm）, the release of eytochrome c and the activation of caspase-3, 6 and 7 in Ad-VP infected EC-109 cells. In contrast, all these assays show almost no effects of the recombinant adenoviruses on L02 cells. These results demonstrate that the treatment of tumors with Ad-VP selectively inhibits tumor growth and induces apoptosis of esophageal cancer cells. Ad-VP may provide a novel and powerful strategy for cancer gene therapy.

Transfection of the glial cell line-derived neurotrophic factor gene promotes neuronal differentiation

Glial cell line-derived neurotrophic factor recombinant adenovirus vector-transfected bone marrow mesenchymal stem cells were induced to differentiate into neuron-like cells using inductive medium containing retinoic acid and epidermal growth factor. Cell viability, micro- tubule-associated protein 2-positive cell ratio, and the expression levels of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43 protein in the su- pernatant were significantly higher in glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells compared with empty virus plasmid-transfected bone marrow mes- enchymal stem cells. Furthermore, microtubule-associated protein 2, glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein743 mRNA levels in cell pellets were statistically higher in glial cell line-derived neurotrophic factor/bone marrow mesen- chymal stem cells compared with empty virus plasmid-transfected bone marrow mesenchymal stem cells. These results suggest that glial cell line-derived neurotrophic factor/bone marrow mesenchymal stem cells have a higher rate of induction into neuron-like cells, and this enhanced differentiation into neuron-like cells may be associated with up-regulated expression of glial cell line-derived neurotrophic factor, nerve growth factor and growth-associated protein-43.

Anti-tumor Immune Response Mediated by Newcastle Disease Virus HN Gene

Hemagglutinin-neuramidinase（HN） is one of the most important surface structure proteins of the Newcastle disease virus（NDV）. HN not only mediates receptor recognition but also possesses neuraminidase（NA） activity, which gives it the ability to cleave a component of those receptors, NAcneu. Previous studies have demonstrated that HN has interesting anti-neoplastic and immune-stimulating properties in mammalian species, including humans. To explore the application of the HN gene in cancer gene therapy, we constructed a Lewis lung carcinoma（LLC） solid tumor model using C57BL/6 mice. Mice were injected intratumorally with the recombinant adenovirus expressing HN gene（Ad-HN）, and the effect of HN was explored by natural killer cell activity assay, cytotoxic lymphocyte activity assay, T cell subtype evaluation, and Th1/Th2 cytokines analysis. The results demonstrate that HN not only can elicit clonal expansion of both CD4＋ and CD8＋ T cell populations and cytotoxic T lymphocyte（CTL） and killer cell response, but also skews the immune response toward Th1. Thus, vaccination with Ad-HN may be a potential strategy for cancer gene therapy.

Adenovirus Mediated BIMS Transfer Induces Growth Supression and Apoptosis in Raji Lymphoma Cells

Objective To transfer pro-apoptotic BIM directly into tumor cells bypass the complicated biologica processes of BIM activation so as to reverse the chemoresistance of cancer cells. Methods BIMS was specifically amplified from HL-60 cells by RT-PCR, confirmed to be correct by sequencing and cloned into shuttle vector pAdTrack-CMV carrying a green fluorescence protein gene to generate a recombinant plasmid pAdTrack-CMV-BIMS. This plasmid and adenovirus backbone plasmid pAdEasy-1 were linearized and electroporated into E.coli BJ5183 host bacteria to mediate homologous recombination. The positive clone was identified by restrict endonuclease digestion. The recombinant pAdEasy-CMV-BIMS was transferred into HEK293 cells for packaging and amplification. The successful construction of recombinant human BIMS adenovirus （Ad-BIMS） was demonstrated by Western blot. To test whether Ad-BIMS has the capability of inducing apoptosis of tumor cells, Ad-BIMS was used to infect GC resistant Burkitt lymphoma Raji cells. Results After infected for 2-5 days, BIMS expression in Raji cells was detected by RT-PCR and Western blot. The significant growth retardation and apoptosis of Raji cells were also observed by MTI- and flow cytometry. Conclusion These results indicated that BIMS might be a potential candidate of gene therapy for chemoresistant tumor cells.

Twenty years of Gendicine?rAd-p53 cancer gene therapy:The first-in-class human cancer gene therapy in the era of personalized oncology

Genetic mutations in TP53 contribute to human malignancies through various means.To date,there have been a variety of therapeutic strategies targeting p53,including gene therapy to restore normal p53 function,mutant p53 rescue,inhibiting the MDM2-p53 interaction,p53-based vaccines,and a number of other approaches.This review focuses on the functions of TP53 and discusses the aberrant roles of mutant p53 in various types of cancer.Recombinant human p53 adenovirus,trademarked as Gendicine,which is the first anti-tumor gene therapy drug,has made tremendous progress in cancer gene therapy.We herein discuss the biological mechanisms by which Gendicine exerts its effects and describe the clinical re-sponses reported in clinical trials.Notably,the clinical studies suggest that the combination of Gendicine with chemotherapy and/or radiotherapy may produce more pronounced efficacy in slowing tumor growth and progression than gene therapy/chemotherapy alone.Finally,we summarize the methods of administration of recombinant human p53 adenovirus for different cancer types to provide a reference for future clinical trials.

Antiapoptotic Effect of Gene Therapy with Recombinant Adenovirus Vector Containing Hypoxia-inducible Factor-1α after Cerebral Ischemia and Reperfusion in Rats

Background：Mounting evidence has demonstrated that hypoxia-inducible factor-1α （HIF-1α） could attenuate brain injuries after cerebral ischemia and reperfusion （CIR）.However,few reports have addressed the therapeutic efficacies of a recombinant adenovirus vector containing HIF-1o （AdHIF-1o） gene after ischemia and reperfusion.The aim of this study was to examine the antiapoptotic and neuroprotective effects ofAdHIF-1o gene for cerebral injuries after ischemia and reperfusion in rats.Methods：From February to December 2016,male Sprague-Dawley rats were randomly divided into normal,sham,CIR,AdHIF-1α,and recombinant adenovirus （Ad） groups.Middle cerebral artery occlusion model was established by Longa＇s method and reperfusion resumed at 2 h postocclusion.AdHIF-1α solution,Ad solution,and phosphate-buffered saline were injected into the right lateral ventricle of rats in AdHIF-lα,Ad,and CIR groups.Brain tissue sections were observed under fluorescent microscope to confirm the definite expression of recombinant adenovirus in Ad and AdHIF-1o groups.The expressions of HIF-lα protein were analyzed by immunohistochemical staining at 6 h,24 h,and 72 h postreperfusion.Brain water content and neurological deficit scores were evaluated at 6 h,24 h,and 72 h postreperfusion.Pathological brain injuries were examined after hematoxylin and eosin stain and nerve cell apoptosis was measured after terminal-deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL） stain at 72 h postreperfusion.Comparisons were conducted with one-way analysis of variance by post hoc Scheffe＇s test among different experimental groups.Results：Green fluorescent protein was successfully expressed in brain tissue ofAd andAdHIF-1α groups from 24 h to 21 days postinjection.As detected by immunohistochemical staining,the expressions of HIF-lα protein were obviously enhanced in AdHIF-1o group than those in CIR and Ad groups at 24 h and 72 h postreperfusion,respectively.There were significant reductions of brain water content （78.83% ± 0.34% vs.83.21% ± 0.50% and 83.35% ± 0.32%;84.13% ± 0.24% vs.89.76% ± 0.34% and 89.70% ± 0.18%;respectively;all P 〈 0.05） and neurological deficit scores （2.90 ± 0.74 vs.3.50 ± 0.52 and 3.60 ± 0.53 at 24 h;2.40 ± 0.84 vs.3.60 ± 0.52 and 3.50 ± 0.53 at 72 h;respectively;all P 〈 0.05） in AdHIF-1 α group versus CIR and Ad groups at 24 h and 72 h postreperfusion,respectively.The pathologic changes ofAdHIF-1 α group were milder than those in CIR and Ad groups at 72 h postreperfusion.The percentage of TUNEL-positive cells in cerebral subcortex decreased significantly in AdHIF-1α group versus CIR and Ad groups at 72 h postreperfusion （P 〈 0.05）.Conclusion：AdHIF-1α has an obvious neuroprotective effect on ischemia and reperfusion in rat brains possibly through inhibiting the apoptosis of nerve cells.

Recombinant adenovirus with human indoleamine-2,3- dioxygenase and hepatitis B virus preS was constructed and expressed in HepG2 cells

Background Indoleamine-2,3-dioxygenase （IDO） is proven to suppress hepatitis B virus （HBV） specific immune response and depletion of IDO may be a useful approach for HBV therapy. To test this concept, we constructed recombinant adenovirus with human IDO and HBV preS, which would form the basis for future in vivo experiments.Methods The fragment of human IDO and HBV preS cDNA were subcloned into multiple cloning sites in an adenoviral vector system containing two cytomegalovirus （CMV） promoters. Recombination was conducted in the Escherichia coli BJ5183. The recombinant adenovirus containing hlDO gene and HBVpreS gene was packaged and amplified in 293 cells.Integration was confirmed by polymerase chain reaction as well as the quantification of viral titers. HepG2 cells were infected with the recombinant adenovirus and mRNA and protein specific for hlDO and HBVpreS was detected by RT-PCR and Western blotting respectively.Results The recombinant adenovirus was produced successfully. Its titer was 2.5x109 efu/ml. IDO and HBVpreS mRNA as well as the encoded proteins could be found in transfected HepG2 cells, but not in control HepG2 cells.Conclusion The transfer of hlDO-HBVpreS with double-promoter adenoviral vector was efficient. The recombinant adenovirus with hlDO and HBVpreS would provide the experimental basis for future studies.

Inhibition of vascular smooth muscle cell proliferation by in troduction of retinoblastoma gene via a recombinant adenovirus vector

This study was supported in part by grant from National Natural Science Foundation of China (No. 39570775). Objective To investigate the vascular smooth muscle cell (SMC) growth suppression by recombinant adenovirus vector expressing a retinoblastoma (Rb) protein and to explore a gene therapy approach for vascular proliferative disorders including atherosclerosis and artery restenosis. Methods A replication deficient adenovirus vector encoding a wild type Rb and AdCMVRb, was constructed and transfected into cultured rabbit aortic SMC. The efficiency of gene transfection and expression was detected by immunochemical staining and polymerase chain reaction. The role of Rb in regulating vascular SMC proliferation was observed by cell counting, thymidine incorporation, and flow cytometry. Results Wild type Rb gene transfected effectively into the cultured SMC with AdCMVRb can suppress growth factor stimulated cell proliferation through regulation of DNA synthesis and cell cycle progression. Conclusion The results demonstrate the potential of adenovirus mediated Rb gene therapy for atherosclerosis and artery restenosis after balloon angioplasty.

Expression and function of DMT1 without IRE in C6 cells mediated by recombinant adenovirus

Divalent metal transporter 1(DMT1)is a ferrous iron import protein.The improper expression of DMT1 is involved in neurodegenerative diseases.In the present study,we constructed a recombinant adenovirus containing the gene of DMT1 without the iron response element(DMT1-IRE)and investigated its expression and function in the C6 glioma cell line.The DMT1-IRE gene,obtained by RT-PCR,was cloned into the shuttle plasmid-ing pAdTrack-CMV containing greenfluorescent protein(GFP)reporter gene.Linearized plasmid pAdTrack-CMV-DMT1-IRE was subsequently co-transformed into Escher-ichia coli(E.coli)BJ5183 cells along with an adenoviral backbone plasmid pAdEasy-1 after digestion with Pme I.Pac I-digested pAdEasy1-DMT1-IRE was then transfected into E1-transformed human embryonic kidney cells(HEK293 cells),in which recombinant adenoviruses were generated within 7 to 10 days.The results demon-strated that we obtained the DMT1-IRE gene.pAdEasy1-DMT1-IRE yielded a large fragment,plus a smaller fragment of 4.5 kb after digestion with Pac I.PCR confirmed pAdEasy1-DMT1-IRE contained gene DMT1-IRE,indicating the successful construction of recombi-nant adenovirus plasmid containing DMT1-IRE.GFPfluorescence further confirmed the generation of recombi-nant AdDMT1-IRE adenovirus.AdDMT1-IRE could efficiently infect C6 glioma cells.And cell viability decreased in AdDMT1-IRE infected cells after iron overload compared to the control.These results suggest that the over expressed DMT1-IRE can aggravate the iron induced cell death due to its iron influx function.

Adenovirus-mediated overexpression of novel mutated IκBα inhibits nuclear factor κB activation in endothelial cells

Nuclear factor κB （NF-κB） overactivation, requiring phosphorylation and degradation of its inhibitor IκBα, is the basis for chronicity of airway inflammation in asthma. Based on our previous plasmid pShuttle-IκBα, carrying an IκBα gene from human placenta, we optimized a novel IκBα mutant （IκBα） gene, constructed and characterized its replication-deficient recombinant adenovirus （AdIκBαM）, and tested whether AdIκBαM-mediated overexpression of IκBαM could inhibit the NF-κB activation in endothelial cells.

Characterization of the essential role of bone morphogenetic protein 9 (BMP9) in osteogenic differentiation of mesenchymal stem cells (MSCs) through RNA interference

Mesenchymal stem cells(MSCs)are multipotent stem cells and capable of differentiating into multiple cell types including osteoblastic,chondrogenic and adipogenic lineages.We previously identified BMP9 as one of the most potent BMPs that induce osteoblastic differentiation of MSCs although exact molecular mechanism through which BMP9 regulates osteogenic differentiation remains to be fully understood.Here,we seek to develop a recombinant adenovirus system to optimally silence mouse BMP9 and then characterize the important role of BMP9 in osteogenic differentiation of MSCs.Using two different siRNA bioinformatic prediction programs,we design five siRNAs targeting mouse BMP9(or simB9),which are expressed under the control of the converging H1 and U6 promoters in recombinant adenovirus vectors.We demonstrate that two of the five siRNAs,simB9-4 and simB9-7,exhibit the highest efficiency on silencing exogenous mouse BMP9 in MSCs.Furthermore,simB9-4 and simB9-7 act synergistically in inhibiting BMP9-induced expression of osteogenic markers,matrix mineralization and ectopic bone formation from MSCs.Thus,our findings demonstrate the important role of BMP9 in osteogenic differentiation of MSCs.The characterized simB9 siRNAs may be used as an important tool to investigate the molecular mechanism behind BMP9 osteogenic signaling.Our results also indicate that recombinant adenovirus-mediated expression of siRNAs is efficient and sustained,and thus may be used as an effective delivery vehicle of siRNA therapeutics.