Molecular Design and Production of AAV Viral Vectors for Gene Therapy
Adeno-associated virus (AAV) is a helper-dependent single-stranded DNA parvovirus. Over the years, AAV has become the vector of choice in the gene therapy field due to its safety profile and low immunogenicity. With a carrying capacity of 4.2 kbp, these v
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Introduction Adeno-associated virus (AAV) is a small parvovirus measuring approximately 20 nm diameter. This helper-dependent virus was discovered in 1965 as a contaminant to viral cultures of adenovirus [1]. AAV has single-stranded DNA genome with two inverted terminal repeats (TRs). The wild-type AAV genome encodes two genes, capsid structure and viral replication regulation genes (Cap and Rep). The capsid structure of AAV is composed of the 60 protein subunits composed of three subunits (VP1, VP2, and VP3) that form an icosahedral shell. These capsid proteins determine the interaction of the viral particles with the host cell. AAV vectors have low immunogenicity when used as viral vectors [2]. However, AAV transduction is known to cause an initial inflammatory response due to the presence of capsid proteins [3, 4]. Wild-type AAV integrates into the host genome at AAVS1, a specific site in human chromosome 19 [5, 6]. AAV cloning plasmids only retain the TR sequences of the wild-type AAV, with the gene of interest (i.e., promoter-open reading frame and polyA signal) cloned between both TRs. The AAV vector genome remains as episomal concatemers in the nucleus of the host cell (see Fig. 1)
Alexandra R. Lucas (ed.), Viruses as Therapeutics: Methods and Protocols, Methods in Molecular Biology, vol. 2225, https://doi.org/10.1007/978-1-0716-1012-1_5, © Springer Science+Business Media, LLC, part of Springer Nature 2021
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Fig. 1 Recombinant adeno-associated virus (AAV) as a gene therapy vector. A gene of interest, including its promoter and poly-A signal, is cloned between the two terminal repeat (TRs) sequences of an AAV. The TRs and the gene of interest are packaged into the AAV viral capsid using a co-transfection method in vitro. Viral particles are concentrated and purified before use. Once inside a cell, the AAV genome is delivered to the nucleus, where it becomes an episomal double-stranded DNA concatemer
[7–9]. Because of its safety profile, AAV has become the platform of choice in the field of gene therapy. AAV vectors have several advantages over other viral vectors, such as lentivirus and adenovirus. AAV vectors infect a diverse range of cell types and have the ability to transduce postmitotic cells. AAV also exhibit long-term expression in postmitotic cells. However, AAV vectors have limitations that determine which diseases can benefit from using this platform. A significant limitation of AAV is its relatively small genetic carrying capacity. This restricts the length of the transgene that can be inserted into an AAV vector genome to about 4.5 kilobases (kb) for single-stranded AAV. For selfcomplementary AAV (scAAV), the size of the transgene is further limited to 2.4 kb. Considering that the median length of a human protein-coding mRNA is 2.787 kb [10], AAV can still be used in a significant number of gene transfer methods. Although it has an even more significant size disadvantage, scAAV eliminates the need for second-strand AAV DNA synthesis since the viral particles co
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