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.
- 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.
- 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.
- 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.
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.
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.
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).
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.