Recombinant adeno-associated virus 8-mediated inhibition of microRNA let-7a ameliorates sclerosing cholangitis in a clinically relevant mouse model

BACKGROUND Primary sclerosing cholangitis(PSC)is characterized by chronic inflammation and it predisposes to cholangiocarcinoma due to lack of effective treatment options.Recombinant adeno-associated virus(rAAV)provides a promising platform for gene therapy on such kinds of diseases.A microRNA(miRNA)let-7a has been reported to be associated with the progress of PSC but the potential therapeutic implication of inhibition of let-7a on PSC has not been evaluated.AIM To investigate the therapeutic effects of inhibition of a miRNA let-7a transferred by recombinant adeno-associated virus 8(rAAV8)on a xenobiotic-induced mouse model of sclerosing cholangitis.METHODS A xenobiotic-induced mouse model of sclerosing cholangitis was induced by 0.1% 3,5-Diethoxycarbonyl-1,4-Dihydrocollidine(DDC)feeding for 2 wk or 6 wk.A single dose of rAAV8-mediated anti-let-7a-5p sponges or scramble control was injected in vivo into mice onset of DDC feeding.Upon sacrifice,the liver and the serum were collected from each mouse.The hepatobiliary injuries,hepatic inflammation and fibrosis were evaluated.The targets of let-7a-5p and downstream molecule NF-κB were detected using Western blot.RESULTS rAAV8-mediated anti-let-7a-5p sponges can depress the expression of let-7a-5p in mice after DDC feeding for 2 wk or 6 wk.The reduced expression of let-7a-5p can alleviate hepato-biliary injuries indicated by serum markers,and prevent the proliferation of cholangiocytes and biliary fibrosis.Furthermore,inhibition of let-7a mediated by rAAV8 can increase the expression of potential target molecules such as suppressor of cytokine signaling 1 and Dectin1,which consequently inhibit of NF-κB-mediated hepatic inflammation.CONCLUSION Our study demonstrates that a rAAV8 vector designed for liver-specific inhibition of let-7a-5p can potently ameliorate symptoms in a xenobiotic-induced mouse model of sclerosing cholangitis,which provides a possible clinical translation of PSC of human.

Packaging and Functional Identification of Recombinant Adeno-associated Virus Encoding cdc2-siRNA

Cyclin dependent kinases （cdks） play an important role in the pathogenesis of multiple neurodegenerative diseases. To explore the possibility of cdks-related gene therapy for neurodegen-erative diseases, we packed recombinant adeno-associated virus （rAAV） encoding cdc2-siRNA. The expressing plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA was constructed by using molecular biological techniques. The rAAV encoding cdc2-siRNA （rAAV-EGFP-U6-cdc2-siRNA） was packed by calcium phosphate mediated co-transfection of the plasmid pAAV-MCS-EGFP-U6-cdc2-siRNA, p-RC and p-Helper into AAV-293 cells. DNA sequencing proved the successful construction of U6-cdc2-siRNA in pAAV-MCS-EGFP. Seventy-two h after packaging, the expression of EGFP could be detected in AAV-293 cells. Western blotting revealed that cdc2 gene expression in AAV-293 cells was down-regulated markedly after transfection with rAAV-EGFP-U6-cdc2-siRNA, which evidenced the satisfactory silencing effect of this virus. It was concluded that the packaging of rAAV encoding cdc2-siRNA was successful. rAAV encoding cdc2-siRNA could silence cdc2 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.

Construction of Recombinant Fowlpox Virus(rFpv) Expressing HIV-1 Gag-pol Protein

The induction of human immunodeficiency virus（HIV）-specific T-cell response is generally considered as critical to the development of effective immunity to HIV type 1 （ HIV-1 ）. Recombinant Avipoxvirus vectors are used widely for vaccination against HIV-1, where the induction of a cytotoxic CD8 ＋ T-cell（CTL） response seems to be an important component of protective immunity. A recombinant fowlpox virus（rFPV/Gag-pol） expressing the Gag-pol protein of HIV was constructed and characterized. The specific expression protein in CEF cells infected by recombinant fowlpox and the specific antibody in the sera of mice immunized with rFPV were analyzed via Western-blot.

Immune Efficacy of a Recombinant Fowlpox Virus Co-Expressing HA and NA Genes of Avian Influenza Virus in SPF Chickens

A recombinant fowlpox virus co-expressing Haemagglutinin (HA) and Neuraminidase (NA)named as rFPV-HA-NA was produced by HA and NA gene of A/Goose/Guangdong/3/96(H5N1)isolate of avian influenza virus recombined into the genome of fowlpox virus. In thisstudy, to evaluate its ability of protecting chickens against challenge with a lethaldose of highly pathogenic isolates of avian influenza virus, eight-week-old specific-pathogenic-free (SPF) chickens were vaccinated with recombinant virus or the wildtypefowlpox virus by wing-web puncture. After challenge 4 weeks with 10 LD50 highly pathogenicavian influenza virus H5N1 and H7N1 isolate, all chickens vaccinated with recombinantvirus were protected, while the chickens vaccinated with the wildtype fowlpox virus orunvaccinated controls experienced 100% mortality respectively following challenge. Thiscomplete protection was accompanied by the high levels of specific antibody response tothe respective components of the recombinant virus.

STRUCTURE AND EXPRESSION OF EPSTEIN-BARR VIRUS MEMBRANE ANTIGEN IN RECOMBINANT VACCINIA VIRUS

The Epstein-Barr virus membrane antigen was constructed and inserted into vaccinia virus, Tian-tan strain in order to study the effect of this virus on EB infection and tumorogenesis. The EBV-derived membrane antigen was expressed under the control of a 7.5 K promoter of vaccinia virus. The antibody against the membrane antigen of EB virus was produced on rabbits vaccinated with recombinant vaccinia virus.

CONSECUTIVE IMMUNIZATION WITH RECOMBINANT FOWLPOX VIRUS AND PLASMID DNA FOR ENHANCING CELLULAR AND HUMORAL IMMUNITY

Objective: To investigate the influence of consecutive immunization on cellular and humoral immunity in mice. Methods: We evaluated a consecutive immunization strategy of priming with recombinant fowlpox virus vUTALG and boosting with plasmid DNA pcDNAG encoding HIV-1 capsid protein Gag. Results: In immunized mice, the number of CD 4 + T cells from splenic lymphocytes increased significantly and the proliferation response of splenocytes to ConA and LPS elevated markedly and HIV-1-specific antibody response could be induced. Conclusion: Consecutive immunization could increase cellular and humoral immunity responses in mice.

The Helper Activities of Different Avian Viruses for Propagation of Recombinant Avian Adeno-Associated Virus

To compare the helper activities of different avian viruses for propagation of recombinant avian adeno-associated virus （rAAAV）, AAV-293 cells were cotransfected with the AAAV vector pAITR-GFP containing green fluorescent protein （GFP） gene, the AAAV helper vector pcDNA-ARC expressing the rep and cap genes, and the adenovirus helper vector pHelper expressing Ad5 E2A, E4, and VA-RNA genes. Chicken embryonic fibroblast （CEF） or chicken embryonic liver （CEL） cells were cotransfected with the AAAV vector and the AAAV helper vector, followed by infection with Marek＇s disease virus （MDV）, avian adenovirus, chicken embryo lethal orphan （CELO） virus or infectious bursal disease virus （IBDV）. Infectious rAAAV particles generated by the two strategies were harvested and titrated on CEF and CEL cells. A significantly higher viral titer was obtained with the helper activity provided by the pHelper vector than by MDV or CELO virus. Further experiments showed that rAAAV-mediated green fluorescent protein （gfp） expression was overtly enhanced by MDV or CELO virus super infection or treatment with sodium butyric acid, but not by IBDV super infection. These data demonstrated that MDV and CELO viruses could provide weak helper activity for propagation of rAAAV, and rAAAV- mediated transgene expression could be enhanced by super infection with the helper viruses.

Recombinant adeno-associated virus delivered human thioredoxin-PR39 prevents hypoxia-induced apoptosis of ECV304 cells

Human thioredoxin and antibacterial peptide, PR39, have been shown to have potent antioxidant effects that may prolong survival of cells during hypoxia. The pSSCMV/human thioredoxin-PR39 vector was successfully constructed in this study and used to infect ECV304 cells. Transfected ECV304 cells were incubated at 1%, 5% hypoxic, and normal oxygen conditions. We found that the number of apoptotic cells after transfection with recombinant adeno-associated virus-human thioredoxin -PR39 was significantly lower than controls, suggesting a protective effect of the recombinant human thioredoxin-PR39 protein on hypoxic cells.

Melittin analog p5RHH enhances recombinant adeno-associated virus transduction efficiency

Objective Melittin and its derivatives have been characterized to establish effective gene delivery systems.Their capability of facilitating endosomal release enhances the nanoparticles-based gene delivery.Nevertheless,little investigation has been conducted to explore its potential application in the context of viral vectors.Methods Various melittin-derived peptides were inserted into the loop VIII of the capsid proteins of recombinant adeno-associated virus vectors.These vectors carrying either gfp or fluc genes were subjected to qPCR assays and transduction assays of HEK293T cells to investigate the efficiency of vector production and gene delivery.In addition,the ability of a specific p5RHH-rAAV vector to deliver genes was examined through in vitro transduction of different cultured cells and in vivo tail vein administration to C57BL/6 mice.Finally,the intricate details of the vector-mediated transduction mechanisms were revealed by specific pharmacological inhibitors of every stage of the rAAV2 intracellular life cycle.Results A total of 76 melittin-related peptides were compiled from existing literature.Among them,cMA2,Melt13,p5RHH and aAR3 were found to significantly enhance the gene delivery efficiency of rAAV2 vectors.The p5RHH-rAAV2 vectors efficiently transduced not only rAAV-potent cell lines but also cell lines previously considered resistant to rAAV.Mechanistically,bafilomycin A1,a vacuolar endosome acidification inhibitor,completely inhibited the transgene expression mediated by the p5RHH-rAAV2 vectors.Most importantly,p5RHH-rAAV8 vectors also demonstrated increased hepatic transduction in vivo in C57BL/6 mice.Conclusion The incorporation of melittin analogues into the rAAV capsids results in a significant improvement in rAAV-mediated transgene expression.While further modifications remain an area of interest,our studies have substantially broadened the pharmacological prospects of melittin in the context of viral vector-mediated gene delivery.

Design of live-attenuated animal vaccines based on pseudorabies virus platform

Pseudorabies virus(PRV)is a double-stranded DNA virus with a genome approximating 150 kb in size.PRV contains many non-essential genes that can be replaced with genes encoding heterogenous antigens without affecting viral propagation.With the ability to induce cellular,humoral and mucosal immune responses in the host,PRV is considered to be an ideal and potential live vector for generation of animal vaccines.In this review,we summarize the advances in attenuated recombinant PRVs and design of PRV-based live vaccines as well as the challenge of vaccine application.

R5 to X4 coreceptor switch of human immunodeficiency virus type 1B' and B'/C recombinant subtype isolates in China

The chemokine receptors CCR5 and CXCR4 play an important role as coreceptors for human immunodeficiency virus type 1 （HIV-1） entring into cells. HIV-1 isolates can be distinguished by the chemokine coreceptors. Nonsyncytium inducing （NSI）, macrophage tropic viruses utilizing CCR5, are called R5 viruses; syncytium inducing （SI） isolates use CXCR4 and known as X4 viruses. R5 viruses generally are associated with latent stage of infection and X4 viruses with later,

Comparative study of the transfection efficiency of commonly used viral vectors in rhesus monkey (Macaca mulatta) brains

Viral vector transfection systems are among the simplest of biological agents with the ability to transfer genes into the central nervous system. In brain research, a series of powerful and novel gene editing technologies are based on these systems. Although many viral vectors are used in rodents, their full application has been limited in non-human primates. To identify viral vectors that can stably and effectively express exogenous genes within non- human primates, eleven commonly used recombinant adeno-associated viral and lentiviral vectors, each carrying a gene to express green or red fluorescence, were injected into the parietal cortex of four rhesus monkeys. The expression of fluorescent cells was used to quantify transfection efficiency. Histological results revealed that recombinant adeno-associated viral vectors, especially the serotype 2/9 coupled with the cytomegalovirus, human synapsin I, or Ca2~/calmodulin-dependent protein kinase II promoters, and lentiviral vector coupled with the human ubiquitin C promoter, induced higher expression of fluorescent cells, representing high transfection efficiency. This is the first comparison of transfection efficiencies of different viral vectors carrying different promoters and serotypes in non-human primates （NHPs）. These results can be used as an aid to select optimal vectors to transfer exogenous genes into the central nervous system of non-human primates.

Improving Cross-Protection against Influenza Virus Using Recombinant Vaccinia Vaccine Expressing NP and M2 Ectodomain Tandem Repeats

Conventional influenza vaccines need to be designed and manufactured yearly.However,they occasionally provide poor protection owing to antigenic mismatch.Hence,there is an urgent need to develop universal vaccines against influenza virus.Using nucleoprotein(NP)and extracellular domain of matrix protein 2(M2e)genes from the influenza A virus A/Beijing/30/95(H3N2),we constructed four recombinant vaccinia virus-based influenza vaccines carrying NP fused with one or four copies of M2e genes in different orders.The recombinant vaccinia viruses were used to immunize BALB/C mice.Humoral and cellular responses were measured,and then the immunized mice were challenged with the influenza A virus A/Puerto Rico/8/34(PR8).NP-specific humoral response was elicited in mice immunized with recombinant vaccinia viruses carrying full-length NP,while robust M2e-specific humoral response was elicited only in the mice immunized with recombinant vaccinia viruses carrying multiple copies of M2e.All recombinant viruses elicited NP-and M2e-specific cellular immune responses in mice.Only immunization with RVJ-4M2eNP induced remarkably higher levels of IL-2 and IL-10 cytokines specific to M2e.Furthermore,RVJ-4M2eNP immunization provided the highest cross-protection in mice challenged with 20 MLD5〇of PR8.Therefore,the cross-protection potentially correlates with both NP and M2e-specific humoral and cellular immune responses induced by RVJ-4M2eNP,which expresses a fusion antigen of full-length NP preceded by four M2e repeats.These results suggest that the rational fusion of NP and multiple M2e antigens is critical toward inducing protective immune responses,and the 4M2eNP fusion antigen may be employed to develop a universal influenza vaccine.

The Neuroprotective Effects of Cyclin-dependent Kinase-5 Inhibition in Mice with Niemann-Pick Disease Type C

In order to investigate the neuroprotective effects of cyclin-dependent kinase-5 （cdk-5） inhibition in mice with Niemarm-Pick disease type C （NPC） （npc^-/-）, recombinant adeno-associated virus （rAAV） carrying the small interfering RNA （siRNA） specific for cdk-5 gene was injected into 3-day-old npc^-/- mice intracerebroventricularly. The rAAV-GFP-injected age-matched npc^-/- mice and non-surgery age-matched npc^-/- mice were employed as controls （n=6-10/group）. From the 4th to 8th week after the treatment, mice were weighed, and evaluated for limb motor activity by using the coat hanger test once a week. Eight-week-old npc^-/- mice were sacrificed by decapitation, and brains were quickly dissected and halved sagittally. Immunohistochemistry, Western blotting, and HE staining were used to evaluate the neuropathology in npc^-/- mice. The results showed that rAAV-cdk-5-siRNA-GFP significantly reduced the number of axonal spheroids, delayed the death of Purkinje neurons, ameliorated motor defects in npc^-/- mice, and significantly attenuated the hyperphosphorylation oftau proteins. These data suggested that inhibition of cdk-5 activity has neuroprotective effect on neurons in NPC mice.

Construction and immunogenicity of recombinant pseudorabies virus expressing the modified GP5m protein of porcine reproduction and respiratory syndrome virus

Pseudorabies virus(PRV),an alpha-herpesvirus,has been developed as a live viral vector for animal vaccines.However,the PRV recombinant virus TK^(-)/gE^(-)/GP5^(+)expressing GP5 of porcine reproductive and respiratory syn-drome virus(PRRSV),based on the PRV genetically depleted vaccine strain TK^(-)/gE^(-)/LacZ^(+),scarcely stimulated the vaccinated animals to produce neutralizing antibodies against PRRSV.To develop a booster-specific immune response of such PRV recombinants,the ORF5m gene(the modified ORF5 gene having better immune responses)was substituted for the ORF5 gene and introduced into PRV TK^(-)/gE^(-)/LacZ^(+),resulting in a PRV recombinant named TK^(-)/gE^(-)/GP5m^(+),which expressed the modified GP5m protein.The recombinant virus was confirmed using PCR,Southern blotting and Western blotting.TK^(-)/gE^(-)/GP5m^(+)and TK^(-)/gE^(-)/GP5^(+)expressing the authentic GP5 protein were inoculated into Balb/c mice to evaluate their immune responses.The results indicated that the protecting neutralization antibodies(the 3/6 vaccinated mice obtained 1:16)and cell immune responses induced by TK^(-)/gE^(-)/GP5m^(+)against PRRSV were higher than that induced by TK^(-)/gE^(-)/GP5^(+).Thus,the development of the new PRV recombinant expressing the modified GP5m protein as a candidate vaccine established the basis for the study of bivalent genetic engineering vaccines against PRRSV and PRV.

3H-31, A Non-structural Protein of Heliothis virescens ascovirus 3h,Inhibits the Host Larval Cathepsin and Chitinase Activities

3h-31 of Heliothis virescens ascovirus 3h(Hv AV-3h)is a highly conserved gene of ascoviruses.As an early gene of Hv AV-3h,3h-31 codes for a non-structural protein(3H-31)of Hv AV-3h.In the study,3h-31 was initially transcribed and expressed at 3 h post-infection(hpi)in the infected Spodoptera exigua fat body cells(Se FB).3h-31 was further inserted into the bacmid of Autographa californica nucleopolyhedrovirus(Ac MNPV)to generate an infectious baculovirus(Ac MNPV-31).In vivo experiments showed that budded virus production and viral DNA replication decreased with the expression of 3H-31,and lucent tubular structures were found around the virogenic stroma in the Ac MNPV-31-infected Se FB cells.In vivo,both LD50and LD90values of Ac MNPV-31 were significantly higher than those of the wild-type Ac MNPV(Ac MNPV-wt)in third instar S.exigua larvae.An interesting finding was that the liquefaction of the larvae killed by the infection of Ac MNPV-31 was delayed.Chitinase and cathepsin activities of Ac MNPV-31-infected larvae were significantly lower than those of Ac MNPV-wt-infected larvae.The possible regulatory function of the chitinase and cathepsin for 3H-31 was further confirmed by RNAi,which showed that larval cathepsin activity was significantly upregulated,but chitinase activity was not significantly changed due to the RNAi of 3h-31.Based on the obtained results,we assumed that the function of 3H-31 was associated with the inhibition of host larval chitinase and cathepsin activities,so as to restrain the hosts in their larval stages.

The use of miR122 and its target sequence in adeno-associated virus-mediated trichosanthin gene therapy

Objective:Plant-derived cytotoxic transgene expression,such as trichosanthin(tcs),regulated by recombinant adeno-associated virus(r AAV)vector is a promising cancer gene therapy.However,the cytotoxic transgene can hamper the vector production in the r AAV producer cell line,human embryonic kidney(HEK293)cells.Here,we explored micro RNA-122(miR122)and its target sequence to limit the expression of the cytotoxic gene in the r AAV producer cells.Methods:A miR122 target(122 T)sequence was incorporated into the 30 untranslated region of the tcs c DNA sequence.The firefly luciferase(fluc)transgene was used as an appropriate control.Cell line HEK293-mir122 was generated by the lentiviral vector-mediated genome integration of the mir122 gene in parental HEK293 cells.The effects of miR122 overexpression on cell growth,transgene expression,and r AAV production were determined.Results:The presence of 122 T sequence significantly reduced transgene expression in the miR122-enriched Huh7 cell line(in vitro),fresh human hepatocytes(ex vivo),and mouse liver(in vivo).Also,the normal liver physiology was unaffected by delivery of 122 T sequence by r AAV vectors.Compared with the parental cells,the miR122-overexpressing HEK293-mir122 cell line showed similar cell growth rate and expression of transgene without 122 T,as well as the ability to produce liver-targeting r AAV vectors.Fascinatingly,the yield of r AAV vectors carrying the tcs-122 T gene was increased by 77.7-fold in HEK293-mir122 cells.Moreover,the tcs-122 T-containing r AAV vectors significantly reduced the proliferation of hepatocellular carcinoma cells without affecting the normal liver cells.Conclusion:HEK293-mir122 cells along with the 122 T sequence provide a potential tool to attenuate the cytotoxic transgene expression,such as tcs,during r AAV vector production.

Prevalence of neutralizing antibodies against liver-tropic adeno-associated virus serotype vectors in 100 healthy Chinese and its potential relation to body constitutions

Recombinant adeno-associated virus （rAAV） serotype 2, 3 and 8 vectors are the most promising liver- tropic AAV serotype vectors. Liver diseases are significant problems in China. However, to date, few studies on AAV neutralizing antibodies （Nabs） were working with the Chinese population or with the rAAV3 vectors. The present study aimed to determine the prevalence of Nabs in Chinese population against wild-type AAV2, AAV3 and AAV8 capsids as well as additional two AAV3 variants. In addition, we performed a preliminary analysis to investigate the potential influence of traditional Chinese medicine body constitutions on AAV Nabs. Our work demonstrated that the majority of healthy Chinese subjects were positive for AAV Nabs, with the order of AAV2 〉 AAV3 = AAVLK03 〉 AAV8. There was no difference between： 1）AAV3 and its variants; 2） male and female subjects; and 3） different age cohorts （〈 35, 36- 50, and 〉 51 years old）. People in the Qi-deficiency constitution had significantly increased AAV8 Nabs than people in the Gentleness constitution. Our studies may have impact on the future clinical design of AAV-based gene therapy in the Chinese population.