Adeno-Associated Virus Production and Modification of AAV

 Adeno-Associated Adeno-Associated (AAV) was first discovered in the 1960s. Since then it has evolved into an innovative viral vector for the field of gene therapy and delivery (1). AAV naturally does not display pathogenicity, and it triggers a minimal immune response in your body(2). To serve as a recombinant virus expression vector wild-type AAV is extensively modified to include only the necessary genes necessary for viral packaging. AAV capsid proteins are the AAV capsid proteins that are responsible for the viral phenotype. Researchers have taken advantage of this to design Recombinant AAV targeting specific types in mammalian cells (2). For more information regarding the origins, biology, and functions of AAV check out at Adeno-Associated virus a brief introduction to the Knowledge Base. The methods and techniques required to create the recombinant AAV will be described in this article along with the various modifications that can be made to AAV. AAV platform.

The generation of rAAV vectors needs four essential components that include plasmids that act in-trans and the transgene operating inside-cis. 

The components are: 1.) A plasmid that contains all the AAV Rep as well as Cap gene needed to form capsids and replicate 2.) One that has essential adenovirus aider proteins and 3) an RNA cassette that contains the transgene, which is enclosed by two terminal repeats inverted (ITR) as well as four) an adenovirus manufacturing cell. The preparation of these components typically requires some days of work prior to preparation. Because AAV is extremely infectious and is naturally found in a significant proportion of humans it is highly recommended that the cells, as well as all other components, are examined for transient wild-type AAV infection prior to use. Additionally, there are limitations on size that must be taken into consideration when selecting the gene that will be introduced into the vector rAAV.

Others Vector Modifications

As previously mentioned other modifications have been made by researchers on rAAV vectors to enhance their effectiveness including capsid modification to alter its preference toward certain types of tissue. The current modifications for the AAV capsid serotypes follow:

Transcapsidation

Transcapsidation involves the packing of the ITR from one particular serotype in AAV into the capsids of a different kind of serotype. The majority of cases are based on the extensively studied AAV cell line genome that is wrapped into different AAV serotypes to study the effectiveness. The disadvantage of using transpeptidation for modifying AAV tropism is the possibility of a lower titer, depending on the serotypes employed. Furthermore, due to an interaction of the C-terminus, the coat of protein on the capsid and the genome of the virus results in fewer yields from AAV particles are likely when the virus is produced.

Absorption by Receptor Ligands

Adsorption of Receptor Ligands onto the AAV Capsid Surface is the introduction of foreign peptides onto the capsid's surface. The primary objective is to target specific cells that do not have a specific AAV serotype that has an affinity which greatly increases the applications of AAV as a tool for gene therapy. Initial approaches utilized AAV-specific anti-idiotypic connected to another antibody that is specifically linked to receptors of the cell on targeted cells. These modifications have proven to be effective in modifying the vector tendencies towards the targeted cells.

Mosaic Capsid

Mosaic Capsid modification involves packing in the AAV genome/rAAV transgene to form an AAV capsid that is composed of capsid proteins that have not been modified by two Serotypes. The purpose is to use mosaic capsids to extend the resemblance that is associated with AAV to extend the resemblance of the AAV vector to cover a diverse variety of cells. To achieve this the two plasmids having genes from a distinct serotype are merged when packaged which can result in the creation of a virus that has approximately 50 per cent of the viral proteins from one type and 50 per cent in the second. While it's impossible to determine precisely the percentage of capsid proteins that are from one type of serotype as compared with the others, outcomes of the experiments with mosaic capsids demonstrate that the tropism is indeed expanded to both serotypes. Additionally, the results of experiments show that, although the original serotypes are unable to transmit a specific type of tissue separately, however, the altered mosaic capsids might be able to transmit more effectively, leading to speculation that the mechanism for trafficking of mosaic capsids is distinct.