Evolution and Screening Methods
Our proprietary episomal DNA extraction method screens only infectious variants and effectively removes background noise. With powerful data analysis, candidate variants can be identified after just a few rounds of in vitro or in vivo selection. After thorough evaluation and characterization, capsid variants with improved properties are then validated.
Workflow
Library Design and Construction- Library QC and Refinement-Screening-Variant Refinement- Data Analysis- Variant Characterization
Library Design and Construction
Most AAV capsid libraries use a combination of 3 approaches: error-prone PCR, DNA shuffling, and random peptide insertion. These approaches are prone to disadvantages such as low complexity, likeliness to not evade preexisting human antibodies, and inability to improve other properties.
The approach that Porton Advanced uses consists of diversifying selected surface positions in an iterative manner to successfully address these limitations.
Rational Design
Apply knowledge of epitopes, cell surface receptors, binding peptides, etc. to design new capsids with desired properties.
Directed Evolution
- Harness the power of evolution to engineer improved capsids using capsid libraries.
- Design strategies for library design, complexity, and selection.
Combination of Rational Design and Directed Evolution
- Use directed evolution to improve rationally designed capsids.
- Refine evolved capsids using rational design.
- Rationally design capsid libraries for directed evolution.
Advantages of our Rationally Designed Capsid Libraries
- Designed based on 3D structure and bioinformatics
- Precise modifications at carefully selected positions
- Incremental construction to maximize compatibility with capsid assembly
- Diversification covering a wide range of surface regions
- High complexity
- Heightened probability of generating novel tropism due to the large number of simultaneously mutated positions
- Better evasion of preexisting neutralizing antibodies than any other approach
Library QC and Refinement
- Each region of each library and sub-library, at both the plasmid and viral levels, is analyzed by Illumina sequencing using our own bioinformatics software.
- If needed, changes are made to the design or construction strategy to improve the library’s complexity.
Example of amino acid distribution and enrichment analysis. The y-axis shows variable amino acid positions. Each color on the left 2 panels represents a different amino acid. The x-axis for the 2 left panels shows the distribution of amino acids at each variable position. The right panel is a heat map showing the positive (in shades of blue) or negative (in shades of red) amino acid enrichment at each variable position. The x-axis on the right side lists the 20 amino acids. Ideally, as in this example, the amino acid distribution should not vary too much between the plasmid (left) and viral (center) libraries, resulting in a mostly white heat map.
Example of sequence cluster cardinality distribution analysis. The x-axis shows the sequence copy number while the y-axis shows the fraction of distinct sequences existing at that copy number. An ideal library would show as an almost vertical line, with plasmid and viral libraries very close to each other, as seen in the blue and green examples above. Three capsid libraries are shown here, each at both plasmid (-P) and viral (-V) level.
The average number of mutations per sequence is another library (or variable region) characteristic. Ideally, at the plasmid level, it should be close to the library design, and, as in this example, it should not vary too much from plasmid to viral library.
Screening
We choose between non-human primates, or humanized/xenografted mice with 3D cultures of human tissues. A combination of both in vivo and in vitro selections will be utilized in the multi-stage evolution of selected variants.
Based on the patent-protected Episomal DNA extraction method, we screen infectious viruses rather than genomes, which greatly improves our screening efficiency. Lastly, weuse the new PacBio NGS platform to sequence the AAV capsid genes through our own software code.
Screening platform
- In vivo selection is more appropriate when the intended gene therapy is supposed to be delivered systemically. Due to interspecies differences in tropism, non-human primates and mice xenografted with human tissue are the models of choice.
- In other cases, in vitro selection can be advantageous when relevant models are available, as it is much faster. In general, 3D cultures such as spheroids, organoids, explants, or organs on a chip, are necessary.
Amplification type
- Co-infection with human adenovirus (mostly applicable to in vitroselection) can replicate AAV without the need for PCR and enriched library preparation, saving time and resources.
- In most cases, however, PCR and enriched library generationare necessary to give better results.
Selection level
- Infection:DNA from capsids that successfully infected the target cells is amplified.
- Transcription: mRNA generated from the library rAAV genome is amplified, selecting variants capable of functional transduction.
- Protein Expression: capsid sequences from cells displaying a particular phenotype are amplified (mostly applicable in vitro). Ex: GFP expression mediated by the library genome.
Variant Refinement
Capsid candidates identified from initial selection rarely exhibit all the desired properties in terms of transduction efficiency, tissue specificity, immune evasion, or manufacturability, and therefore often need to be further improved.
Rational Design Approach
Using knowledge both from relevant scientific literature and prior experience, amino acid substitutions are performed in order to increase neutralizing antibodies evasion (by altering known epitopes) or increase transduction efficiency (by preventing ubiquitination and proteasomal degradation). Domain swaps between top capsid variant candidates may improve tissue specificity.
Directed Evolution Approach
Several types of capsid libraries based on top candidates can be designed: DNA shuffling might combine properties of different capsids; random peptide insertion can increase tissue specificity; diversification of unmutated variable regions can improve immune evasion, tissue specificity and transduction efficiency.
Data Analysis
Sequence distribution and enrichment are monitored throughout the selection process using Pacbio sequencing analyzed using our own bioinformatics pipeline. Promising candidates can thus be identified early. Alternatively, the analysis helps decide whether to continue selection or to modify the directed evolution strategy.
Example of enrichment analysis in several target tissues (x-axis) after the first round of selection. Distinct sequences, sorted by overall frequency, are stacked on the y-axis. Shades of color indicate positive (blue) and negative (red) enrichment.
Enrichment scores are calculated by 3 different methods, based on complete sequence (left), variable region (middle), and amino acid enrichment (right).
Example of sequence frequency (y-axis) evolution during 5 rounds of selection (x-axis). Only the 25 most frequent sequences are represented in individual colors.
Example of sequence frequency (y-axis) distribution in various tissue samples (z-axis). Individual distinct sequences are shown on the x axis.
Example of detailed enrichment analysis. Top enriched sequences in one particular target tissue are shown, sorted by overall enrichment score (total of sequence, variable region, and amino acid-based enrichment scores). The enrichment factor of each variable region is displayed in shades of blue (positive enrichment) and red (negative enrichment). Amino acid enrichment is displayed in regular font (slightly enriched), underlined (more enriched), and bold + underlined (extremely enriched). The right side shows enrichment scores in other tissue samples, with shades of green illustrating tissue specificity (lower scores in all other tissues means higher specificity).
Variant Characterization
Each novel capsid variant is characterized by being compared to the most relevant capsids, including its parental serotype and the best performing variants from up-to-date scientific literature. Analyzed properties include production yield, thermal stability, transduction efficiency, immune evasion and tissue specificity.
Highlights
- Based on the platform, Porton Advanced provides ready to use capsid variants and capsid variants. Click here to explore how to collaborate with us.