Aiming to CRISPR modify B cells for CAR B cell therapies

This project is focused on developing a strategy to create therapeutic B cells, capable of antigen-induced secretion of any antibody, including selected monoclonal antibodies. To do this we aim to use gene engineering techniques to replace the endogenous antibody cassette within B cells with a chosen antibody cassette. This antibody should then be expressed and routed to the cell membrane. Where it will act as a signaling-coupled BCR ( Cell Receptor) and will also be secreted as free antibody when the BCR is activate by antigen binding.

1. Identify AAV capable of targeting B cells selectively. Using the new technology developed jointly by Oxgene (OG) and the University to create a vast AAV library, this would be the first demonstration of the library utility and could itself give a high profile publication.
(This new technology uses a TESSA adenovirus to package a library of plasmids expressing different AAV genomes into AAV2-pseudotyped library of different AAV genomes. That library will then be infected into host cells at multiplicity of infection (MOI)<1, so that the progeny AAV viruses produced by each cell express the capsid protein that is encoded by their genome. The technology should avoid the production of mosaic AAV and should also avoid unwanted contamination with adenovirus.)

2. New, smaller CRISPR system that can fit inside an AAV. Demonstrate this is feasible using a boiler plate target.
(A new, smaller, CRISPR system has just been published that may be suitable for incorporation into AAV. We need to assess whether this agent is capable of gene editing B cells when applied using AAV).

3. Use a library approach to define guide that can target the new CRISPR approach to the BCR and allow it to be replaced with a therapeutic antibody cassette.

4. Assess AAV-based delivery strategies for BCR gene editing in vitro and in vivo..

(This would involve consideration of whether all of the components can be incorporated into a single AAV or whether two AAVs are required to infect target B cells simultaneously. The system could be structured to maximise efficiency and also to minimise unwanted effects. WE may then move to an in vivo model to assess likely utility.)