A role for adeno-associated viral vectors in gene therapy: [review]

Gene therapy constitutes a therapeutic intervention based on modification of the genetic material of living cells, by correcting genetic defects or overexpressing therapeutic proteins. The success of gene therapy protocols depends on the availability of therapeutically suitable genes, appropriate gene delivery systems and proof of safety and efficacy. Recent advances on the development of gene delivery systems, particularly on viral vectors engineering and improved gene regulatory systems, have led to marked progress in this field. Although the available vector systems can successfully transfer genes into cells, the ideal delivery vehicle has not been found. In this context, adeno-associated virus vectors (AAV) are arising as a promising tool for a wide range of applications, due to a combination of characteristics such as lack of pathogenicity and immunogenicity, wide range of cell tropism and long-term gene expression. Since its isolation, the biological properties of the adeno-associated virus have been increasingly understood, improving our ability to manipulate and use it as a safe and efficient gene therapy vector of wide spectrum. In this work, we review the bases of gene therapy, main types of gene transfer systems and basic properties and use of AAV vectors.

rAAV:a Promising Vehicle for Gene Therapy / 生物化学与生物物理进展

Adeno-associated virus(AAV)is a member of the parvovirus family,single-stranded DNA-containing nonenveloped icosahedral viruses.AAVs have been regarded as promising vectors for human gene therapy as they have the capacity to establish long-term latency within human cells without any apparent pathogenicity.However,a lot of obvious defects in their applications have been revealed recently,including the paucity of cell surface receptors on some cells,the lack of site-specific integration by recombinant AAV vectors,and the host immune responses to AAV capsid components and transgene products and so forth.Driven by these defects,increasing efforts are being made to study biological properties and infectious pathway of AAVs.It is consequently optimized by the modification of the AAV vectors to produce new generation of recombinant AAV vectors with more security,efficiency and site-specific targeting,allowing AAVs to move forward into broader clinical application.

Construction and identification of the small interfering RNA eukaryotic expression vectors of human ClC-2 gene / 医学研究生学报

Objective :To construct the small interfering RNA(siRNA) eukaryotic expression vectors of human ClC-2 gene. Methods: According to the program and principles of DEQOR about designing siRNA,two pairs ClC-2 mRNA-targeted hairpin siRNA were devised,and the two pairs complemantary oligonucleotide strands of DNA fragments that encoded the above siRNA were synthesized by chemosynthesis.After annealing of the complementary strands,the DNA fragments were connected to the polyclone sites of plasmid eukaryotic expression vector pSUPER.puro that was cut by restriction endonuclease BglⅡ and HindⅢ,followed by transformation,amplification and plasmid extraction in E.coli,and finally,the two recombinant plasmids were identified by agarose gel electrophoresis by means of cutting with EcoRⅠand HindⅢ and by DNA sequence analysis.The plasmids were transfected transiently into human glioma cell line BT-325 cells by Lipofectamine~(TM)2000.The mRNA expression of ClC-2 gene was detected by(RT-PCR.) Results: The connections between the DNA fragments encoding ClC-2-targeted siRNA and the pSUPER.puro plasmid were correct,as confirmed by agarose gel electrophoresis and DNA sequencing(analysis.) ClC-2 mRNA expression of the BT-325 cells transfected two recombinant vectors was(significantly) decreased.Conclusion: The two RNAi recombinant vectors of human ClC-2 gene were successfully constructed.They were named pSUPER.puro-siClC-21 and pSUPER.puro-siClC-22,respectively.This laid the groundwork for future research about ClC-2 gene affecting invasion and migration of human glioma cells.