CaPRiCoRN: CRISPR/Cas9 ribonucleoprotein containing retroviral nanoparticles for safe gene therapies
Dr Florian Gatzke, Goethe University Frankfurt am Main; Dr Frank Schnuetgen, Goethe University Frankfurt am Main; Prof. Hubert Serve, Goethe University Frankfurt am Main
CRISPR / Cas9 gene editing has revolutionized the modification of genomic DNA through its unsurpassed ease of use. Despite its success in preclinical research, the system to date has not been successfully applied in the clinic therapeutically. In the first Quarter of 2019 there were only 17 clinical studies worldwide that investigate the therapeutic use and potential of a lentiviral inserted CRISPR / Cas9 system. One problem with the route of delivery via integrating lentiviruses is the low efficiency, which is a consequence of inefficient transduction and expression of CRISPR / Cas9 protein due to the size of the vector construct.
To address the issues of applying gene editing approaches efficiently in clinical practise, we sought a method to combine the benefits of a time-limited application of CRISPR / Cas9 ribonucleoproteins with those of non-toxic biological administration via retroviral particles offering broad therapeutic applicability to a variety of diseases. As a result of the observation that CRISPR / Cas9 protein can be detected in virions during a regular generation of lentiviruses we surprisingly found that the presence of CRISPR / Cas9 protein is not dependent on the presence of a lentiviral genome. This circumstance allowed for an adapterless incorporation of CRISPR / Cas9 protein into retroviral particles in the total absence of viral genetic information so that integration into a host genome is NOT impossible. Compared with an application of CRISPR / Cas9 via integrating lentiviruses, a transient presence of CRISPR / Cas9 RNP in the recipient cells is demonstrated 12 hours after transduction with the CaPRiCoRN system the inventor’s developed. CaPRiCoRN transduced CRISPR / Cas9 RNP are no longer detectable in the target cells after 10 days.
The CaPRiCoRN transduction system provides a scalable solution for therapeutic use aiming at in-vivo gene correction therapy at an unsurpassed safety profile. Especially in situations where the extraction and reinfusion of the intent-to-treat-cell population is not possible, the CaPRiCoRN transduction system might provide a suitable delivery solution for an unaddressed need. The unprecedented modularity of the CaPRiCoRN System builds on decades of experience with lentiviruses: As such, tropism can be altered by using alterative enevelopes and the system is optimized for but not limited to CRISPR/Cas9 as incorporated effector protein. Furthermore existing manufacturing pipelines for GMP-grade production of lentiviruses for therapeutic applications facilitate the implementation in existing industrial environments.
The CaPRiCoRN System is currently undergoing testing in primary human cell cultures to test for the ability to modify endogenous disease loci. The testing will further encompass the modular exchange oft he viral envelope to modify the tropism and increase the specificity of transduction to further improve the safety profile of CaPRiCoRN transduction system.
Priority EP patent application filed.