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Gene therapy viral vector assessment on RPE cells

The ask

Our customer‘s goal was to confirm the dose-dependant ability of AAV constructs to transduce retinal tissue, more specifically the retinal pigment epithelium (RPE).

RPE

Our approach to developing a solution

We followed our standardised series of steps to develop a customised solution.

Step 1:

UNDERSTANDING THE PROBLEM

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 rapidly evaluate  if their AAV vector was able to transduce RPE cells and if the transduction efficiency was dose-dependant.

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 RPE with AAV vector
  • Step 2: Image quantification and flow cytometry to quantify transduction efficiency

Step 2:

DEVELOPING A CUSTOMISED EXPERIMENTAL PLAN

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

Our experimental plan involved using fully validated human iPSC-derived RPE.

For Step 1: RPE transduction

  • We transduced human RPE with AAV vector containing a GPF  reporter gene and encoding for AAV5 capsid and monitored transgene expression with live imaging up to 27 days post-transduction
  • The experiments aimed to evaluate the transduction efficiency of AAV constructs at two concentrations

For Step 2:   Imaging analysis

  • Quantification of the % of GFP-positive cells
  • Quantification of the transduction efficiency of AVV vectors in RPE by flow cytometry

Step 3:

PROJECT EXECUTION

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

 

Step 4:

RESULT DELIVERY

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

The study clearly showed :

  • Percentage of the area with GFP-positive cells over 28 days post transduction increased over time and  in dose- dependent manner
  • Flow cytometry analysis  allowed for accurate quantification and  confirmed  the transduction efficiency of RPE with AAV was dose- and time-dependant.

iPSC-derived RPE cells transduction study confirmed efficiency of AAV5 gene therapy vector to progress preclinical and in vivo studies.

Outcomes for the client

We delivered a robust dataset which clearly showed that human IPSC-derived RPE cells are a robust in vitro model for testing of AAV constructs. Our analysis gave confidence to our customer to proceed to preclinical studies with their AAV construct.

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