Gene therapy viral vector assessment on retinal organoids

We clearly defined the problem and questions to be answered through in-depth discussion between the customer and our technical experts.

The customer wished to compare the transduction efficiency of a series of AAV vectors with different capsids, promoters and transgenes.

Taking all project specific factors into account, we suggested a two-step approach as being the most appropriate for their situation:

• Step 1: Optimise the transduction of retinal organoids with AAV vectors
• Step 2:  Detailed imaging analysis and constructs comparison

We designed the specific experiments and presented the plan in our Statement of Work.

Our experimental plan involved using fully validated human retinal organoids.

For Step 1,  Organoids were transduced at day 150 of differentiation with AAV vectors and live imaging was performed from 5 to 27 days post-transduction.

• The experiments aimed to evaluate the transduction efficiency of the various AAV constructs.
• We also checked organoid viability was not negatively affected by AAV transduction.

For Step 2 we planned to carry out the following imaging analysis,:

• Quantification of the % of cells expressing the transgene
• Verify the nature of the transduced cells, namely photoreceptors by co-localisation studies

Our scientists carried out the agreed experiments within 3 months providing regular updates to the customer.

The study included the experimental phase, data processing, data analysis  and presentation of a data summary.

Example dataset

Imaging comparison of a series of AAV construct with different promotors, reporter genes and capsids.

We delivered a detailed report with reliable dataset which was shared digitally and discussed over a call upon request.

The study clearly showed :

• Transgene expression in photoreceptors post transduction was the highest when the transgene was under the control of the rhodopsin kinase GRK1 promotor reaching 5 % of cells with all capsid variants.
• Transduction of photoreceptors by confirming co-localisation of the transgene and recoverin by immunostaining.

This study expanded the options of AAV capsid and transgene to be used for preclinical testing of gene therapy to include CAG quad mutant variants and GRK1 into AAV vectors