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AAV vectors

AAV vectors have been widely used as gene delivery vehicles for both basic scientific research, and human gene therapy. They have been successfully used in clinical trials, and are approved as drugs for human use. 

Advantages

AAV is often the preferred method for delivering genes to target cells due to its high titer, mild immune response, ability to infect a broad range of cells, and overall safety. 

  • Non-pathogenic. AAV has increasingly become an important gene therapy vector, which is largely due to the fact that wild-type AAV (wtAAV) is not related to any known human diseases.

  • Low immunogenicity. AAV safely transduces postmitotic tissues with relatively low immunogenicity compared with other vectors like adenovirus and HSV.

  • Broad  tropism range. AAV vectors have a broad host and cell type tropism range and transduce both dividing and nondividing cells in vitro and in vivo. Furthermore, the recent discovery of novel AAV serotypes will expand even more the universe of potential target organs, tissues and cells.

  • Long term and high expression. AAV vectors maintain (over several years) high levels of gene expression in vivo, in the absence of a significant immune response to the transgene product .

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Disadvantages

AAV is often the preferred method for delivering genes to target cells due to its high titer, mild immune response, ability to infect a broad range of cells, and overall safety. 

  • Small package capacity. The cloning capacity of the vector is relatively limited and most therapeutic genes require the complete replacement of the virus's 4.8 kilobase genome. Large genes are, therefore, not suitable for use in a standard AAV vector. Options are currently being explored to overcome the limited coding capacity.

  • Slow gene expression. Gene expression is generally of slow onset, due to the requirement of conversion of the single-stranded AAV DNA into double-stranded DNA before gene expression can be initiated. Researchers have created an altered version of AAV termed self-complementary adeno-associated virus (scAAV). Whereas AAV packages a single strand DNA and must wait for its second strand to be synthesized, scAAV packages two shorter strands that are complementary to each other. By avoiding second-strand synthesis, scAAV can express more quickly, although as a caveat, scAAV can only encode half of the already limited capacity of AAV.

  • Pre-existing immunity. As a result of natural infections, antibodies to AAV can be found in many animals including humans. The pre-existing immunity in individuals may result in low levels of transgene delivery and expression.

AAV vector design

For wtAAV, this genome comprises of two open reading frames (ORFs), rep and cap, flanked by two inverted terminal repeats(ITRs). To generate AAV vector, the rep and cap genes are replaced with transgene-expression cassettes. These ITRs form hairpins at the end of the sequence to serve as primers to initiate synthesis of the second strand before subsequent steps of infection can begin.The second strand synthesis is considered to be one of several blocks to efficient infection. To generate the scAAV vectors,  one of ITRs is mutated and it prevents Rep-mediated nicking and force. Thus, both the coding and complementary sequences of the transgene expression cassette are present on each plus- and minus-strand genome. 

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Elements for AAV production

  • Two ITRs: ITRs are the only cis element required for rAAV replication and packaging. To generate recombinant AAV (rAAV) vectors, the two ORFs are replaced by a DNA molecule encoding the therapeutic protein.

  • Rep Proteins: Although four non-structural proteins are encoded in the AAV genome, Rep78, Rep68, Rep52 and Rep40, only Rep78 and Rep52 are required as trans elements for rAAV production.

  • Cap proteins: The capsid proteins, VP1, VP2 and VP3, are essential for AAV capsid assembly, and are therefore required trans elements. 

  • The AAV helper viruses, such as adenovirus (Ad) and herpes simplex virus (HSV), provide the additional trans-acting factors required for rAAV production, such as  E1a, E1b, E2a, VA RNA and E4orf6.

  • Host cells: AAV is a defective virus and its replication relies on host cells to provide different factors.

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AAV vector production systems

Triple-plasmid transfection system

The most common method to produce AAV vector by transient transfection is the triple plasmid system in combination with E1-expressing cells (Such as HEK-293T).

  • Transgene plasmid: This plasmid contains the vector genome, which is an expression cassette with promoter, gene of interest and polyA signal , flanked by two ITRs (mostly from AAV2).

  • AAV helper plamsid: This plasmid encodes Rep proteins, commonly from AAV2, and Cap proteins that are specific for the desired serotypes.

  • Ad helper plamsid: The third plasmid carries the minimal adenoviral genes required to support AAV replication (E2a, E4 and VA). The E1 gene is integrated into HEK-293T cells. 

Notably, genes coding for AAV and Ad helper functions can be cloned in a single plasmid and therefore a double plasmid transfection  is also able to generate AAV vector.  

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Vaccinia-Adenovirus system(VV-Ad system)

This system uses a single vaccinia viral(VV) vector to provide the necessary Rep and Cap proteins, and a single adenovirus AAV hybrid (Ad/AAV) carrying the rAAV genome. AAV vector can be produced by simply infecting the E1a/E1b expressing QW158-7 suspension cells with these two viral vectors.

  • VW22 vector: The AAV trans factors, Rep78, Rep52, VP1 and VP2 were stably integrated into a single vaccinia virus carrier by maximizing the use of alternative codons. Rep52 (Rep52*) and VP1 (VP1*) genes are codon-optimized to avoid recombination with Rep78 and VP2. Since VP2 is initiated by an ACG start codon, the expression of VP2 is less robust compared to VP3 in this system.  

  • Ad/AAV hybrid: The cis AAV genome was carried by an E1/E3 gene-deleted adenovirus. 

  • QW158-7 cells: The host cell line was generated by introducing the E1a/E1b genes into Hela-S3 cells.

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Baculovirus/insect cell production system

Recombinant baculovirus derived from the Autographa californica nuclear polyhedrosis virus has been widely employed for large-scale production of heterologous proteins in cultured insect cells. To produce AAV vectors, genetic constructions of the recombinant baculoviruses must be produced first.

rBac-Rep: The two-split rep orf are driven by two insect promoters, the Polhydrine promoter (pPol) of AcMNPV and a truncated form of the immediate-early 1 gene promoter (pEI1) of Orgyia pseudotsugata nuclear polyhedrosis virus. The difference of the promoter strength allows high expression of the small Rep and a reduced expression of the toxic large Rep.

rBac-Cap express capsid proteins. The three proteins are directly translated from one transcript.

 

rBac-GFP carries a rAAV-GFP genome. The presence of a CMV and p10 promoter allows GFP expression in both mammalian and insect cell. 

Co-infection of those recombinant baculoviruses into Sf9 will generate AAV vectors in cell culture. Notably, rep and cap genes can be cloned in a single baculovirus and therefore a double baculovirues system  is also able to generate AAV vector.  

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Herpesvirus-based systems

Recombinant HSV (rHSV) are replication-deficient vectors that have been deleted for TK and ICP27 genes, which encode for toxic proteins, but still provide helper functions for AAV replication. AAV vector genome or rep/cap genes can be cloned in TK site of the HSV genome by homologous recombination. rHSV stocks are usually propagated in Vero or baby hamster kidney (BHK) cells (adherent or suspension culture). For AAV production, 293 or BHK cells are coinfected with rHSV carrying vector genome and rHSV with rep/cap genes. The use of infectious rHSV vectors avoids any transfection steps being a suitable scalable method for rAAV production.  

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