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.
At AAVnerGene, we provide One-Stop Services of Design, Construction and Cloning of Customized AAV Vectors. Send your requirements to email@example.com or submit the request form on website. Our molecular and AAV experts will take care of your projects and perform the following steps:
- Consultation: Comprehensive support in the AAV vector design:
- Preparation of DNA fragments:
- Digestion fragments from plasmids.
- Amplification of requested sequences.
- Synthesis of any size of DNA fragments with low cost.
- Molecular cloning
- Cloning with standard cloning techniques or Gibson assembly.
- Multiple fragments can be assembled together at once.
- Quality control: Each construct undergoes strict quality control.
- Restriction enzyme digestion to identify correct clones.
- SmaI digestion to confirm two ITRs.
- DNA sequencing to verify inserts.
- Whole plasmid sequencing on request.
- Delivery and documentation. The experimental strategy, raw data and final results are summarized in a detailed report.
- The plasmid DNA (mini scale preparation) is delivered to customer.
- Higher scales are available on request.
AAV plasmid backbones
The typical size of an AAV plasmid backbone is about 3 kb. This backbone contains crucial elements for various functions, including plasmid propagation, AAV replication, and packaging. However, its small size makes it susceptible to reverse packaging, which occurs when it is inadvertently enclosed within the AAV capsid during vector production.
Efforts have been made to prevent reverse genome backbone packaging, which can compromise the functionality of AAV vectors. One approach involves using an oversized plasmid backbone that exceeds the typical AAV packaging capacity. By doing so, the aim is to reduce the chances of the entire plasmid backbone being reverse-packaged into the AAV capsid.
AAVnerGene has developed two types of oversized AAV plasmid backbones: pAAVone and mini-pHelper-AAV.
The pAAVone plasmids are oversized AAV plasmid backbones that go beyond the typical AAV packaging capacity. They not only contain the essential ITRs, but also include both Ad helper genes(E2A, E4orf6 and VA RNA) and AAV helper genes(Rep and Cap). This backbone is designed to produce AAV with our unique AAVone system, which only use one plasmid.
The mini-pHelper-AAV plasmids are designed to contain Ad helper genes located outside the inverted terminal repeats (ITRs). This backbone is designed to produce AAV with our dual-V2 production system.
Type of AAV Backbone
Amp or Kana
Amp or Kana
- mini-pHelper based AAV backbones(including all backbones for Dual-V2 and AAVone systems.) are available for academic use, and industry entities should contact us for access.
- AAVnerGene offers different versions of ITRs on request, such as 11 bp deletion in C domain, 15 bp deletion at terminal, or a combination of both.
|Typical gene synthesis||GS-STD||Regular gene synthesis||$0.20/bp||1-2 weeks|
|Difficulty gene synthesis||GS-HD||GC-rich, high secondary structure||$0.35/bp||2-3 weeks|
|Plasmid Clone||CLONE||Cloning any fragments into AAV backbone||$300||1-2 weeks|
|Mini Prep||AMP-10||10 ug plasmid endotoxin free plasmid||$100||3 days|
|Midi Prep||AMP-100||100 ug of endotoxin free plasmid||$150||3-5 days|
|Maxi Prep||AMP-400||400 ug of endotoxin free plasmid||$200||3-5 days|
|Mega Prep||AMP-2000||2000 ug of endotoxin free plasmid||$500||3-5 days|
|Giga Prep||AMP-10000||10000 ug of endotoxin free plasmid||$1,000||5-7 days|
- Customer needs to provide templates for subclone.
- For complicated clones, additional charge may be added.
- We also provide oligo pool synthesis for library generation.
- All plasmids are prepared with NEB stable strain to minimize ITR lost or deletion.
AAVnerGene provides AAV vector optimization services with our Barcode technology. Customer can provide us the list of enhancers, promoters, introns, transgenes and capsids, we’ll clone them into DNA barcoded backbone. The AAV viruses can be produced by individual or pooled way. By using NGS analysis the DNA/RNA barcodes, customers can select the best expression cassettes and capsids.