Recombinant adeno-associated virus (AAV) is widely used for gene therapies and vaccines due to its broad tropism, prolonged transgene expression, and low immunogenicity. However, existing serotypes have limitations, including insufficient tissue specificity, low delivery efficiency with potential off-target transduction, pre-existing neutralizing antibodies, and manufacturing challenges. Our capsid engineering platform ensures the most effective and efficient development of novel AAV variants with enhanced properties for your therapeutic development goals.
Full Service Platform
The world’s only one-stop solution for all AAV preclinical and clinical needs, from vector design through GMP manufacturing.
Optimized Library Design
Our expertise allows us to design libraries with optimal complexity and custom-build diverse capsid libraries using versatile approaches.
High-Quality Library Packaging
We leverage our proprietary packaging platform to achieve high titers with each viral particle carrying its matching capsid variant.
Powerful Screening Capability
Robust in vitro and in vivo screening, including mouse, rat, and NHP studies conducted in AAALAC-accredited facilities.
Construction and screening of AAV capsid libraries allows for the identification of candidates with high therapeutic potential for 1) efficient transduction of target cells within specific tissues or organs, 2) binding of cell type-specific receptors with high affinity, or 3) evasion of neutralizing antibodies. A typical workflow of AAV capsid evolution is shown below:
Below are common strategies for creating diverse AAV capsid variants. The capsid library is then constructed by massively parallel cloning of the variants into AAV vectors, forming chimeric AAV genomes, each containing a rep gene and a capsid gene variant.
Random peptide display
Random peptide display involves the insertion of random peptide sequences of usually 7 to 9 amino acids into specific sites of the AAV capsid, aiming to alter the natural cellular interactions of the virus and retarget it to specific cell receptors. Peptides are typically inserted at locations of the AAV capsid that facilitate surface exposure of the peptide and are critical for virus-host interactions. For example, between positions 587 and 588 (within the variable region VRIII) of the AAV2 capsid is a preferred insertion site for most AAV2-based peptide display libraries, as peptide insertion here abolishes the heparan sulfate proteoglycan (HSPG, the primary AAV2 receptor) motif of AAV2 and enables the displayed peptides to interact efficiently with cell surface molecules.
Error-prone PCR
Error-prone PCR is the most straightforward approach for developing highly variable AAV capsid libraries, which involves the modification of standard PCR methods to mutagenize the AAV capsid gene. More specifically, error-prone PCR employs a combination of various sub-optimal PCR conditions, including low-fidelity polymerases, longer extension times, higher Mg2+ concentrations, addition of Mn2+, and varying dNTP concentrations for introducing random point mutations into the AAV capsid gene.
DNA family shuffling
DNA family shuffling is a highly efficient approach for generating chimeric AAV capsids by molecular interbreeding of parental capsid genes derived from different AAV serotypes. To accomplish this, the parental capsid genes of various AAV serotypes are fragmented, followed by their reassembly into novel full-length capsid variants by primer-less PCR, which recombines them based on partial sequence homology. As an alternative strategy, high complexity libraries can also be created by synthetic shuffling, which combines rational design (modifying the capsid based on prior knowledge of AAV biology) with directed evolution. In this approach, capsid locations suitable for mutagenesis are first identified and evaluated based on a detailed structural and sequence analysis of naturally occurring AAV serotypes. Fragments containing mutations are synthesized and assembled to form full-length novel capsid variants.
In silico design
In silico design of AAV capsid libraries utilizes a variety of approaches for computational prediction of capsid variant sequences with the potential to contribute to enhanced AAV performance. One commonly used approach is ancestral reconstruction, which involves in silico design of a putative ancestral AAV library followed by its experimental validation to identify highly potent ancestral capsid sequences with improved tropism. The rationale behind this approach is that evolutionary AAV intermediates that emerged by surviving the process of natural selection are highly likely to possess unique properties while maintaining virus structure and function. Machine learning is another commonly used in silico design approach that applies computational algorithms to predict the chances of viable virus production from hypothetical capsid variants. Machine learning algorithms heavily rely on available input data to learn structure-function relationships of proteins and apply that to predict the outcome of complex physiological processes such as viral capsid assembly.
Your novel AAV capsid will be thoroughly characterized and validated for its desired properties. This may include, for example, AAV biodistribution profiling across multiple species to assess tissue tropism, transduction efficiency, and potential off-target effects. We can also perform concurrent optimizations on both AAV transfer vectors and packaging vectors for the greatest potential in therapeutic development using our CliniVec™ consultation service.
Figure 1. Nucleotide composition of an (NNK)11 peptide display AAV capsid plasmid library. The library was constructed using the NNK degenerated codon strategy. Each bar represents a distinct nucleotide position along the DNA sequence, with colors indicating the observed nucleotides. All positions exhibited anticipated nucleotides with observed frequencies close to theoretical values (N = 25% A + 25% T + 25% G + 25% C, K = 50% T + 50% G).
Figure 2. Distribution of amino acids in the 7-mer variable region of a peptide display AAV capsid plasmid library. The library was constructed using the NNK degenerate codon strategy. Each bar represents the theoretical (teal) or actual (pink) percentage of specific amino acids or the stop codon. The observed frequencies of all 20 amino acids and the stop codon closely matched the theoretical frequencies.
Figure 3. Comparison of viral packaging reproducibility between two technical replicates of the same AAV capsid library using different platforms (Left: VB proprietary; Right: conventional). Strong correlation was observed between replicates when using the VB proprietary platform (r = 0.70), indicating high reproducibility. In contrast, the conventional packaging method showed poor correlation (r = 0.02), potentially due to stochastic events that occurred during packaging that reduced efficiency.
Figure 4. Strong correlation of in vivo AAV capsid library screening readouts across animals. (A) The AAV capsid library was injected subretinally, followed by RNA-seq. Read counts of variants in the retinas of different mice were compared, showing a strong correlation (r = 0.88). (B) The AAV capsid library was delivered via intravenous tail vein injection, followed by DNA-seq. Read counts in the livers of different mice were compared, demonstrating a strong correlation (r = 0.93). These results show that our in vivo screening platform is highly reliable for the discovery of novel AAV capsids.
VectorBuilder’s innovative capsid technology provides a powerful solution: engineered capsids with enhanced targeting specificity and maximum gene delivery performance, enabling safer, more effective, and more efficient AAV-based therapeutics.
Brain
Optimized for maximum efficiency and streamlined delivery across the blood-brain barrier.
Ocular
Our ocular capsid boasts excellent pan-retinal penetration and peripheral spread, with superior transduction efficiency in the retinal pigment epithelium (RPE).
Muscular
VectorBuilder’s novel capsids significantly enhance targeting and efficiency to skeletal, cardiac, and diaphragm muscles.
| Method | Description | Advantages | Limitations |
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| Random Peptide Display | Insertion of random peptide sequences of usually 7 to 9 amino acids into specific sites of the AAV capsid. |
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Potential immunogenicity concerns |
| Error-Prone PCR | Employs sub-optimal PCR conditions to introduce random point mutations into the AAV capsid gene. | Straightforward approach for developing highly variable AAV capsid libraries | Likelihood of functional diversity is low |
| DNA Family Shuffling | Genetic material from related AAV serotypes is shuffled to create chimeric capsids. |
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| In Silico Design | Computational methods used to predict and design AAV capsid variants. |
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| RNA-seq | DNA-seq | |
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| Advantages |
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| Limitations |
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