AAV Vector Design
AAV vectors are extensively utilized in gene delivery and gene therapy. The initial step towards successful use of AAV vectors involves designing an efficient shuttle plasmid for packaging into the AAV capsid. Meticulous planning and optimization of transgene expression strategies, tagging methods, AAV genome sizes, and plasmid backbones are essential to ensure efficient packaging, high transgene expression, and targeted delivery of the AAV vector. We provide AAVdesigner tools for pAAVone, pAAVdual, and pAAVtri backbones, tailored to support the AAVone®, AAVdual®, and AAVtri production systems respectively.
Design your AAV vectors with our AAVdesginer tool, or send your requirements to customer@aavnergene.com. or submit a Request Form. Our team of molecular and AAV experts will manage your project and provide the following support:
Create Your Ideal AAV Vectors Today! Accelerate Your Journey In Gene Therapy and Beyond.
AAV vector design at AAVnerGene
We provide comprehensive One-Stop Services of Design, Construction and Cloning of Customized AAV Vectors.
- AAV Plasmid Backbones: Selection the best AAV production systems fit your purposes:
- AAV Vector Genome Types:
- Cis Elements Selection: Tailored strategies for optimal gene expression and AAV packaging capacity.
- Expression and Labeling Strategies: Flexible options for detection.
- Small tags such as HA, His, Flag……
- Large fluorescent tags: such as EGFP, mCherry, TdTomato, Fluc, Rluc, Gluc, Cluc……..
- Multiple gene expression: Multiple expression cassettes, IRES, T2A, P2A, fusion protein…..
- AAV Genome Optimization:
Maximal transduction efficiency and high specificity
Optimized packaging capacity
High full-to-empty capsid ratio
Minimal partial genomes
Reduced impurities
- AAV Serotype Selection: Guidance on the best serotypes from >1000 capsids for your application.
- Common Serotypes: AAV1, AAV2, AAV3B, AAV5, AAV6, AAV7, AAV8, AAV9, AAV11, AAV12, AAV13, AAV-DJ, AAVrh.10, AAVrh.74, AAV2-Retro
- Tissue specific capsids: Liver, Brain, CNS, Lung, Heart, Muscle, Retina……
- Capsid Optimization: Comprehensive AAV capsid evaluation, screening and optimization services.
- Novel AAV Capsids: AAVnerGene is continually developing new serotypes through research efforts, such as AAV-ShD.
- AAV Packaging Services: High quality, short turnaround, low cost AAV packaging services.
- AAV Experimental Design: Expert design with extensive experience.
- AAV-shRNA design/screening and AAV-shRNA library evolution
- AAV-sgRNA design/screening and AAV-sgRNA library evolution
- Troubleshooting: Expert advice throughout your projects.
Processes to generate AAV plasmids
- Design AAV vectors:
- Use our AAVdesginer tool.
- Sent requests or maps to us.
- Preparation of DNA fragments:
- Digestion fragments from plasmids (Free).
- Amplification of requested sequences from ORF collections or genomes .
- Synthesis of DNA fragments with low cost ($0.2/bp).
- Molecular cloning:
- Cloning with standard cloning techniques, Gibson assembly or Gateway cloning.
- Multiple fragments can be assembled together at once.
- Strict quality control:
- Restriction enzyme digestion to identify correct clones.
- SmaI/AdhI digestion to confirm two intact ITRs.
- Sanger sequencing to verify inserts.
- Whole plasmid sequencing on request ($30/plasmid).
- Delivery and documentation:
- The plasmid DNA (mini scale preparation) is delivered to customer. Higher plasmid scales are available on request.
- Corresponding AAV vectors with any capsids can be produced within two weeks under our AAV Packaging Services.
Price and turnaround
| Services | Description | Price | Turnaround |
| Regular Gene Synthesis | Regular gene/DNA fragment synthesis | $0.20/bp | 1-2 weeks |
| Difficulty Gene Synthesis | GC-rich, high secondary structure DNAs | $0.35/bp | 2-3 weeks |
| Plasmid Clone | Cloning any fragments into AAV backbones | $300 | 1-2 weeks |
| Mini Prep | 10 ug plasmid endotoxin free plasmid | $100 | 2-3 days |
| Midi Prep | 100 ug of endotoxin free plasmid | $150 | 3-5 days |
| Maxi Prep | 400 ug of endotoxin free plasmid | $200 | 3-5 days |
| Mega Prep | 2,000 ug of endotoxin free plasmid | $500 | 3-5 days |
| Giga Prep | 10,000 ug of endotoxin free plasmid | $1,000 | 5-7 days |
| Whole Plasmid Sequencing | Nanopore sequencing | $30 | 2-3 days |
Key elements in AAV plasmids
- Inverted Terminal Repeats (ITRs): ITRs are short DNA sequences located at both ends of the AAV genome, essential for AAV replication, packaging into capsids, and integration into the host cell genome. They serve as the starting point for viral DNA replication and guide the encapsidation of the viral genome. The wild-type AAV2 ITR is 145 nucleotides long but is relatively unstable. A commonly used variant is the 130 bp ITR (ITR130), which omits the 15 nucleotides from the 3′ OH end. Additionally, an 11 bp deletion in the C-domain (ITR119) is often seen in AAV plasmids. These mutated ITRs are repaired during AAV replication, resulting in final vectors containing wild-type ITRs. It’s crucial to ensure that your ITRs are functional before initiating AAV packaging.
- Promoters: The promoter is a regulatory DNA sequence that initiates transcription of the gene of interest, dictating the timing and location of gene expression in target cells. Choosing the appropriate promoter enables researchers to achieve desired expression levels and specificity. Commonly used promoters like CMV, CAG, uBC, and EF1a drive strong, consistent expression across various cell types. Tissue-specific promoters allow for precise control of gene expression in targeted biological contexts.
- Introns. Introns can enhance transcript levels by affecting transcription rates, nuclear export, and transcript stability. They also contribute to the efficiency of mRNA translation, making them valuable additions to AAV vectors.
- Gene of Interest (GOI): The GOI is the DNA sequence encoding the desired protein or RNA to be introduced into target cells. This can include therapeutic genes, reporter genes, or functional genetic elements such as sgRNA, shRNA, or lncRNA. It’s critical to ensure that the size of the transgene is compatible with packaging capacity of AAV vectors, which typically accommodate payloads of around 4.7 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: Incorporating enhancers into AAV vectors can significantly boost gene expression. Enhancers can be placed either before the promoter or after the transgene, depending on the desired regulatory effect and the specific experimental setup.
- The CMV (Cytomegalovirus) enhancer is often placed upstream of a promoter to increase transcriptional activity.
The WPRE (Woodchuck Hepatitis Virus Post-Transcriptional Regulatory Element) is typically placed downstream of the transgene, within the 3′ untranslated region (3′ UTR)..
- Regulatory Elements: These elements are responsible for fine-tuning the level and specificity of gene expression. Several additional regulatory elements can be incorporated to fine-tune gene expression:
- Insulator Sequences: Prevent the spread of heterochromatin, ensuring that the inserted gene is not silenced by neighboring DNA regions.
- MicroRNA Target Sites: Can be added to the 3′ UTR to post-transcriptionally regulate gene expression, allowing for cell-specific silencing of the therapeutic gene.
- 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.
AAV plasmid backbones
The AAV plasmid includes essential backbone elements for plasmid propagation, such as the origin of replication and a selection marker.
- pAAVtri: The regular pAAV backbone carrying two ITRs. The typical size of an AAV plasmid backbone is approximately 3 kb. The compact size makes it vulnerable to reverse packaging or read-through, where the backbone is inadvertently enclosed within the AAV capsid during vector production.
To mitigate the risk of reverse genome backbone packaging, one effective strategy is to utilize oversized plasmid backbones that exceed the typical AAV packaging capacity. This approach reduces the likelihood of the entire plasmid backbone being read-through or reverse-packaged into the AAV capsid. AAVnerGene has developed two types of oversized AAV plasmid backbones: pAAVone and pAAVdual.
- pAAVone：The pAAVone plasmids are oversized AAV plasmid backbones, measuring 13 kb, which exceed the typical AAV packaging capacity of approximately 5.0 kb. In addition to essential elements for plasmid propagation, pAAVone includes both adenoviral helper genes (E2A, E4orf6, and VA RNA) and AAV helper genes (Rep and Cap). This backbone is specifically designed for AAV production using our unique AAVone® system, which utilizes a single plasmid. The AAVone system significantly increases AAV yield while dramatically reducing the number of plasmids and the overall plasmid amounts required.
- pAAVdual：The pAAVdual plasmids are engineered to position adenoviral helper genes externally to the ITRs. These plasmids are designed for generating AAV vectors using our innovative AAVdual® system. The incorporation of Ad helper genes into the AAV backbone not only reduces the likelihood of reverse packaging but also minimizes the number of plasmids needed, thereby simplifying the workflow. Importantly, this design retains the flexibility to produce AAV vectors with a variety of serotypes.
Type of Backbone | Backbone Size | Selection Marker | Ori | Ad Genes | Rep/Cap | Genome | Production System |
pAAVtri | 2.6 kb | Kana | pUC/F1 | No | No | ssAAV | AAVtri |
pAAVtri-sc | 2.6 kb | Kana | pUC/F1 | No | No | scAAV | AAVtri |
pAAVdual | 8.4 kb | Kana | pUC/F1 | Yes | No | ssAAV | AAVdual |
pAAVdual-sc | 8.4 kb | Kana | pUC/F1 | Yes | No | scAAV | AAVdual |
pAAVone | 13 kb | Kana | pUC/F1 | Yes | Yes | ssAAV | AAVone |
pAAVone-sc | 13 kb | Kana | pUC/F1 | Yes | Yes | scAAV | AAVone |
Notes:
AAVnerGene offers different versions of ITRs on request, such as ITR130 and ITR119.
Both pAAVone and pAAVdual plasmids are available for research use only. pAAVone plasmids with different AAV serotypes are available.
Workflow of different AAV production systems