Adeno-associated Virus (AAV) as Vaccine-vectors

Due to its unique biology, simple structure, and no known disease association, AAV has become the preferred carrier for most gene therapy applications. Gene therapy using AAV has been demonstrated to be safe and well tolerated in almost all clinical settings, which reveals that this vector has important application value in many respects. Creative Biolabs designs and manufactures safe and efficient rAVV vaccine vectors through a variety of techniques and alternative methods. As a service provider, our goal is to provide the highest level of service, confidentiality and customer support.

Introduction of Adeno-associated Virus Structure

Adeno-associated virus (AAV) is a very small (20-26 nm) icosahedral and non-enveloped virus belonging to the Parvoviridae family. The AAV particles contain a single-stranded DNA genome consisting of about 4.7 kb, which includes two major open reading frames, Rep and Cap. Rep encodes four regulatory proteins (Rep78, Rep68, Rep52, and Rep40) that play key roles in viral DNA replication and encapsidation. Cap encodes three capsid proteins (VP1, VP2, and VP3) and assembly-activated proteins (AAP) that promote capsid formation. The genome is flanked by an inverted terminal repeat (ITR) containing a Rep recognition sequence important for AAV DNA replication and packaging.

Adeno-associated Viruses as Vaccine Vectors

The propagation of AAV in tissue culture requires the help of another virus in which adenovirus (AdV) and herpes virus (HSV) are traditionally used as AAV helper viruses. The AAV vector is a recombinant variant of the wild-type AAV virus in which the native coding region and the non-coding region have been replaced by the gene of interest. The vector genetic construct retains the lateral ITR, which is the only cis-acting element required for AAV DNA replication and encapsidation. Through these modifications, recombinant adeno-associated viruses (rAAV) becomes a replication-defective entity that can only infect cells and deliver DNA to the nucleus.

Fig.2 The three components must be delivered to the host cell line by transfection or viral infection: the vector AAV DNA containing the gene of interest, the Rep and Cap genes and the helper genes from adenovirus. Rep78 and 68 promote AAV DNA rescue and subsequent replication. Cap protein is synthesized in the cytoplasm and shuttles to the nucleus for assembly. AAP supports the assembly and maturity of AAV capsids. Rep52 and 40 interact with single-stranded DNA and capsids to promote viral DNA encapsidation. P, promoter; pA, polyadenylation sequence; E1a and E1b: adenovirus proteins. (Aponteubillus, et al. 2018)

The Production of Adeno-associated Vectors

Key aspects of rAAV vector production include host cell lines that produce rAAV as well as the design of AAV genes, promoters, and regulatory elements. Adjusting the expression of these elements not only contributes to increase productivity but also improves process robustness and product quality. Currently, four commonly used rAAV expression systems include adenovirus, herpesvirus, baculovirus complementary systems, and yeast expression systems.

• Adenovirus complementation systems
  The production of rAVV in the adenoviral complementary system has traditionally been carried out by plasmid transfection processes in which the AAV Rep/Cap gene, the ITR-flanked gene of interest (GOI), and the AdV helper gene are supplied to the HEK293 cell line containing E1a/E1b. (Fig 3a)
• HSV complementation systems
  The HSV complementation system uses two recombinant herpesvirus strains to provide AAV Rep/Cap genes, GOI and HSV helper elements to mammalian cell lines such as BHK. (Fig 3b)
• Baculovirus expression systems
  The baculovirus expression system requires two recombinant baculovirus strains to provide insect cells with the ability to produce AAV. AAV protein expression is controlled by the insect cell Sf9 native promoter. (Fig 3c)
• Yeast expression system
  The yeast-based system is transformed with a set of extrachromosomal plasmids containing six AAV expression cassettes and GOI. AAV protein expression is controlled by the yeast natural promoter. (Fig 3d)

Fig.3 The rAAV production system. (Aponteubillus, et al. 2018)

Advantages of Adeno-associated Virus Vectors

• Non-pathogenicity
• Low inflammatory potential
• Availability of viral serotypes with different tissue tropisms
• Long-lasting gene expression

Empowered by leading technology and years of experience in the viral vaccine, Creative Biolabs provides the world's first-class AVV vaccine vector design services to customers around the world by considering the critical parameters of AVV including vector design, capsid selection, desired target cells and tissue types, and route of administration. If you have any needs, we are your best choice.

Reference

1. Aponteubillus J J, et al. (2018). Molecular design for recombinant adeno-associated virus (rAAV) vector production. Applied Microbiology & Biotechnology, 102(3):1045-1054.

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