AAV Vector Design

AAV vectors have been widely used for gene delivery and gene therapy. The first step to your success for using AAV vectors is to design an efficient shuttle plasmid that can be  packaged into AAV capsid.It’s important to carefully plan and optimize each of Transgene expression strategy, tag methods and AAV packaging capacity, to ensure efficient packaging, high transgene expression, and targeted delivery of the AAV vector. 

Here are the key elements typically found in an AAV vector:

• Inverted Terminal Repeats (ITRs): ITRs are short DNA sequences located at both ends of the AAV genome. They are essential for AAV replication, packaging into capsids, and integration into the host cell genome. ITRs serve as the starting point for viral DNA replication and guide the encapsidation of the viral genome into the AAV capsid. Typical ITR is 145bp. Some versions lost the 11bp-15 bp of  3’OH or 11 bp of C-domain. It is important to make sure your ITRs are functional before staring AAV packaging. 
• Promoter: The promoter is a regulatory DNA sequence that controls the initiation of transcription of the gene of interest. It dictates when and where the gene will be expressed in the target cells. Selecting the appropriate promoter allows researchers to achieve the desired level and specificity of gene expression.
• Intron. Intron can increase transcript levels by affecting the rate of transcription, nuclear export, and transcript stability. Moreover, introns can also increase the efficiency of mRNA translation.
• Gene of Interest (Transgene): This is the DNA sequence encoding the protein or RNA of interest that you want to introduce into the target cells. It can include therapeutic genes, reporter genes, or any other functional genetic element. It is critical to ensure that the size of the transgene or gene of interest is compatible with the packaging capacity AAV vectors, as it only  can accommodate payloads of around 4.7 kilobases (kb) or slightly less.
• Polyadenylation (PolyA) Signal: The polyA signal is a sequence that signals the termination of transcription and facilitates the addition of a polyadenine tail to the RNA molecule. This step is crucial for generating mature mRNA and ensuring proper gene expression.
• Enhancers and Regulatory Elements: These elements are responsible for fine-tuning the level and specificity of gene expression. Enhancers, such as WPRE or WPRE3, can increase the activity of promoters in a tissue-specific or inducible manner, allowing for precise control of gene expression.
• Selectable Marker: For plasmid production and selection in host cells, a selectable marker such as Amp or Kana is often included. This allows for the identification of cells that have taken up the AAV vector.
• Origin of Replication: If the vector is used as a plasmid during production, it will require an origin of replication to replicate within host cells during bacterial culture.