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The DJ serotype also shows efficient transfection of many cultured cells, making DJ especially suitable for cell culture and in vivo applications. AAV2 is still the basis for most AAV systems, but now, engineered capsids including DJ and DJ8 with tissue-specific tropisms or higher infectivity are available 10. This AAV production system is referred to as ‘helper-free’ and consists of three different plasmids encoding essential viral and helper genes: pHelper, AAV trans-plasmid comprising AAV replication ( Rep) and capsid ( Cap) genes, and AAV cis-plasmid encoding the gene of interest, promoter, and inverse terminal repeats (ITRs) 1.Įarly work used the capsid and viral machinery derived from AAV serotype 2 (AAV2). Human embryonic kidney cells (HEK) 293T cells, which express SV40 large T antigen, supply additional necessary proteins 9. Current AAV expression systems avoid using helper viruses and include the plasmid pHelper instead 8, containing essential genes such as E2A and E4. Helper viruses are difficult to remove and may induce undesired effects such as inflammation in the host. AAV production requires cytopathogenic effects, which only occur after co-infection with a helper adenovirus or herpesvirus 8, 9.
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Thus post-mitotic tissues, such as neurons and cardiomyocytes, may express transgenes over several months 8.ĪAVs are ideal for gene therapy due to their low immunogenicity, restricted generation of neutralizing antibodies, and replication defectiveness. These head-to-tail circular concatemers remain intact in non-dividing cells but are lost during mitosis. AAVs engineered for research or gene therapy, however, do not incorporate into the genome and instead form episomal concatemers in the host cell nucleus 7. Wild-type AAVs, as part of their lysogenic cycle, can integrate into the AAVS1 site of human chromosome 19 or rarely at random locations 6.
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Furthermore, several recent clinical trials demonstrated the full potential of AAVs for human gene therapy 1, 2, 3, 4.ĪAVs belong to the Parvoviridae family and are small non-enveloped viruses containing a linear single-stranded (ss) DNA 5. In recent years, adeno-associated viruses (AAVs) have been used for many in vitro and in vivo applications due to their high transduction efficiency, safety, and extended stable gene expression. Our study provides an improved protocol for a more economical and efficient purified AAV preparation.ĭelivering a gene of interest to cells or animals has become an essential technique in biomedical research. AAVs coding for glutaredoxin-1 (Glrx) shRNA successfully inhibited Glrx expression by ~66% in the liver and skeletal muscle. For proof of concept, we verified in vivo transduction via Western blot, qPCR, luminescence, and immunohistochemistry. Of note, we achieved titers of 10 10–10 11 viral genome copies per µl with a typical production volume of up to 1 ml while requiring five times less than the usual number of HEK293T cells used in standard protocols. Furthermore, we then implemented an iodixanol gradient purification, which resulted in preparations with purities adequate for in vivo use. Using a helper-free AAV system, we purified AAVs from HEK293T cell lysates and medium by polyethylene glycol precipitation with subsequent aqueous two-phase partitioning. Here, we report an improved protocol to produce serotype-independent purified AAVs economically. However, major obstacles remain for widespread AAV utilization, such as impractical purification strategies and low viral quantities. Combined with modern gene technologies, such as cell-specific promoters, the Cre/lox system, and genome editing, AAVs represent a practical, rapid, and economical alternative to conditional knockout and transgenic mouse models. Among viral delivery systems, adeno-associated viruses (AAVs) are relatively safe and demonstrate high gene transfer efficiency, low immunogenicity, stable long-term expression, and selective tissue tropism.
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Delivering and expressing a gene of interest in cells or living animals has become a pivotal technique in biomedical research and gene therapy.