Biología molecular de los vectores adenovirales / Molecular biology of adenoviral vectors

La terapia con genes postula el uso terapéutico del DNA como una nueva alternativa de la biomedicina para el tratamiento de las enfermedades humanas. Todas las proteínas están codificadas en el DNA, y muchas enfermedades resultan de: a) la ausencia o expresión aberrante de uno o más genes; b) la ausencia de formas funcionales; c) alteraciones en su proceso de regulación, transporte o degradación. Por lo tanto, tales enfermedades pueden ser potencialmente tratadas, restableciendo la expresión de la proteína involucrada en las células afectadas. Sin embargo, para lograr una transferencia exitosa del material genético al sitio blanco y evitar la destrucción del DNA o del vehículo seleccionado antes de llegar al sitio de interés, se han desarrollado varios sistemas virales. Entre los virus más conocidos están: el virus del herpes simple, adenovirus tipo 5, virus adenoasociado y algunos retrovirus complejos (lentivirus). En este artículo se exponen las características biológicas, la manipulación genética y propiedades de los adenovirus, así como su empleo en la medicina actual como vectores para transferir genes y su potencial implicación en la terapia génica.

Gene therapy is based on the use of DNA as a therapeutic material as an alternative therapeutic tool for treatment of human diseases. All proteins are codified into the DNA and several diseases result from the absence or aberrant expression of one or related genes, absence of expression of functional proteins, and alterations for regulation process in transport and degradation mechanisms. In this regard, several diseases could be potentially treated through the expression of the normal form of the involved protein. However, the main objective is to achieve a successful genetic material delivery into the target site and avoid the destruction of DNA or the selected vehicle before arrival at the final destination. Several efficient viral gene transfer systems have been developed. Viral-mediated gene delivery for experimental models has been designed from herpes virus (HV), adenovirus (adenovirous), adeno-associated virus (AAV) and retroviruses (lentiviral vectors). In this review we will discuss the specific biological and cloning properties of adenoviral vectors as a gene transfer tool and potential medical implications for gene therapy.

Construction of adenoviral vectors expressing F and G glycoproteins of human respiratory syncytial virus (HRSV)

Human Respiratory Syncytial Virus (HRSV) was first characterized in 1957 and has since been recognized as the most common viral cause of severe respiratory tract infection in young infants worldwide. Despite many years of research there is still no effective treatment or any immediate prospect of a vaccine. The HRSV genome is composed of single stranded negative sense RNA and the virion consists of a nucleocapsid packaged within a lipid envelope. The envelope contains spike-like projections, each being a homo-oligomer of one of three transmembrane viral envelope proteins: the attachment protein G, the fusion protein F involved in viral penetration and the small hydrofobic protein SH. The aim of this work was to construct two recombinant replication-defective adenoviruses carrying separately F and G genes from HRSV. This system was chosen because adenovirus delivers genes into target cells with high efficiency in a variety of cell lines and can be used in vitro and in vivo. In order to obtain the recombinant viruses, we did RT-PCR of RNA extracted from the HRSV A2 strain, the genes F and G were cloned in to pAdeno-X vectors. pAdeno-F and pAdeno-G were transfected in HEK-293 cells for the production of recombinant viruses, that expressed efficiently these two proteins and provide us the means for doing functional assays and immunization tests.

O Vírus Sincicial Respiratório Humano (HRSV) foi isolado e caracterizado pela primeira vez em 1957 e é considerado como o patógeno viral mais freqüente do trato respiratório de bebês e crianças. Apesar de muitos anos de pesquisa, não há ainda um tratamento específico ou uma vacina licenciada. Seu genoma é composto por uma fita simples de RNA polaridade negativa e o vírion consiste em um nucleocapsídeo empacotado por um envelope lipídico. O envelope contém projeções, chamadas espículas, constituídas de homoligômeros de uma das 3 glicoproteínas de membrana: a proteína de ligação G ("attachment"), a proteína de fusão F ("fusion") e a proteína SH ("small hydrofobic"). O objetivo deste trabalho foi construir dois adenovirus recombinantes defectivos em replicação expressando separadamente os genes F e G do HRSV. Este sistema foi escolhido porque os vetores adenovirais possuem a capacidade de inserir genes em uma grande variedade de linhagens celulares in vitro e in vivo. Para obtenção destes vetores adenovirais, um RT-PCR de RNA extraído do protótipo A2 de HRSV foi feito e os genes F e G clonados em vetores pAdeno-X. pAdeno-F e pAdeno-G foram transfectados em células HEK-293 para a produção do vírus recombinante, que expressaram corretamente essas duas proteínas constituem-se ferramentas para imunização e estudos funcionais.

Construction of HBsAg gene recombinant adenoviruses and packing in 293 cells / 中国免疫学杂志

Objective:After construction and identification of HBsAg gene recombinant backbone adenoviral vector,it is to prouduct HBsAg gene recombinant adenoviruses by packing PAd-Easy-1-HBs in 293 cells.Methods:The gene of interest was amplified from plasmid pEcob-6 PCR, the gene of interest which contained HBsAg gene was cloned into on adenoviral shuttle vector pAd-track-cmv. The pAd-track-cmv-HBs was linearized by digesting with restriction endonuclease Pme-1, and subsequently cotransformed into E.coli BJ-5183 cells with an adenoviral backbone vector pAd-Easy-1, Homologous recombinants were performed in bacterial cells. Finally, the linearized backbone adenoviral vector was transfected into adenoviruses packing cells lines,e.g. 293 cell by lipofectamine transfection. Transfections and viral productions can be minotored by green fluorescent protein(GFP). The expression of HBsAg in supermatant was investigated by ELISA. It was a certain HBsAg vaccine to amplify recombinant adenoviruses by repeating the infevtion cell to collect the viral supermatant.Results:GFP expression was visible by fluorescence microscopy after transfection. Adenoviral titer was monitored by GFP expression. GFP expression was visible after repeating the infection cell using the viral supernatant in more than 90 percent of the cells.The HBsAg also expressed in supermatant.Conclusion:HBsAg gene recombinant adenoviral backbone vector has been constructed successfully. HBsAg gene recombinant adenoviruses have been producted by packing in 293 cells. The study provides the possibility of further researches on the development of new anti-HBV vaccines.

Adenoviral-mediated high efficiency expression of enhanced green fluorescence protein gene in ex vivo expanded rat mesenchymal stem cells / 中国病理生理杂志

AIM: To investigate the feasibility and infection efficiency of MSCs with replication-deficient adenovirus containing delivered gene, and whether enhanced green fluorescence protein (EGFP) gene track the change during rMSCs differentiating neuron-like cells. METHODS: Rat marrow mesenchymal stem cells (rMSCs) were expanded in low density in vitro . Under the control of CMV promoter, pAd-EGFP-Vector was constructed by homologous recombination in E.coil BJ 5183, and the recombinant virus was produced in HEK 293 packaging cell line. rMSCs infected with Ad-EGFP were observed and analyzed with fluorescence microscope. Infection efficiency was assessed by microscopical scoring and flow cytometrics. After withdrawing serum and exposure to ?-mercaptoethanol medium, rMSCs infected with Ad-EGFP was induced to differentiate into neuron-like cells. As a control, the plasmid of pTrack-EGFP also was transfected into rMSCs to evaluate transfection efficiency.RESULTS: The results showed that Adenovirus vector (AdVec) delivered EGFP gene with high efficiency to marrow mesenchymal stem cells. Gene expression analysis showed that 36%?2 % of rMSCs infected with recombinant adenovirus expressed the transgene of EGFP at high levels. However, the transfection of plasmid pTrack-EGFP using routine method of lipofectamin mixed with plasmid DNA (pTrack-EGFP) was not easily successful and the transfection efficiency was much lower. rMSCs infected with Ad-EGFP in different passage could differentiate into typical morphology alike neural cells after withdrawing serum and exposure to ?-mercaptoethanol medium. Immuno-staining with neuron-specific enolase (NSE), a neuronal marker, was strong positive, which suggested that rMSCs infected with Ad-EGFP had the potential to differentiate into neurons or neuron-like cells. CONCLUSION: The AdVec system can deliver target gene into MSCs and EGFP gene carried by AdVec can track the change during rMSCs differentiating into neuron-like cells.

Adenovirus-mediated gene transfer of CD40Ig gene induced long-term survival of cardiac allograft in rats / 中国免疫学杂志

Objective:To induce long-term survival of cardiac allograft in rats by adenovirus-mediated gene transfer of CEMQIg gene, and to investigate the potential mechanisms involved in the induction of transplantation tolerance. Methods: The donor cardiac allograft from DA rats was heterotopically transplanted into the abdomen of LEW recipient rats, and recombinant adenoviruses containing EGFP gene or CD40Ig gene at a dose of 5 x ICf pfu were administered via portal vein, respectively, during the operation. The graft survival was monitored by daily palpation. The expression of CD4QIg fusion protein in the recipients was detected via EIISA. The tolerant mechanism was investigated via MLR, IL-2 reverse experiment and analyzing the expression of Thl/Th2 type cytokines in the recipients.Results: Compared with the untreated recipients, the mean survival time(MST) of the cardiac allograft was not prolonged in the recipients treated with AdEGFP adenovirus, whereas MST were prolonged significantly to 142.8 ?26.8 d in the recipients administered with AdCD40Ig adenovirus. The expression of CD40Ig fusion protein remained a long time but the levels gradually decreased. The results of MLR indicated that the induced tolerance in the recipients was donor-specific. The results of IL-2 reverse experiment demonstrated that the tolerance mechanisms were involved clonal anergy at the early stage of the established tolerance. The expression pattern of Thl/Th2 type cytokines did not indicate the polarization of Thl/Th2 type cytokines in the experimental models. Conclusion: A single injection of the defined dose of adenovirus containing CD40Ig gene via portal vein during operation is enough to induce long-term survival of cardiac allograft in rats.