Yan Ming Anson Lau
Conference 2022 Poster Presentation
The Clearance of Latent Cytomegalovirus in Donor Lungs Using Multiplexed Gene Editing Nanoparticles Before Transplantation.
Authors and Affiliations
Anson Lau1, Omar F. Khan1,2
1. Institute of Biomedical Engineering, University of Toronto, Toronto, Canada
2. Department of Immunology, University of Toronto, Toronto, Canada
Human Cytomegalovirus (HCMV) infection is among the most common cause of morbidity and mortality in lung transplant recipients. It is estimated that up to 50–90% of the adult population is infected by HCMV, making donor organs likely to harbour the incurable virus. Gene editing technology where CRISPR/Cas9 cleaves regions of viral DNA offers a promising solution to eliminate HCMV in the donor lungs. Traditionally, gene-editing payloads are delivered virally. However, viral delivery could risk permanent alteration of the host genome, trigger inflammatory reactions and has a relatively small payload capacity. Contrastingly, we hypothesize that our synthetic, high-capacity nanoparticles can transiently co-deliver a controlled dosage of multiple viral gene-targeting single guide RNAs (sgRNAs) and Cas9 mRNA (multiplexing) to eliminate latent HCMV in infected donor lungs without inducing immunogenic responses.
SgRNAs are designed to target 5 HCMV genes essential for reactivation and replication, they are UL44, UL57, UL70, and exon 2 and 5 of UL122/123. After, nanoparticles are made using michael addition and ring open epoxide reaction and subsequently mixed with sgRNAs and Cas9 mRNA to self-assemble into the multiplexed nanoparticles. In collaboration with the Toronto Lung Transplant Program, procured human donor lungs with high HCMV load are perfused with the multiplexed nanoparticles using ex vivo lung perfusion (EVLP). During the perfusion, various physical lung function parameters, cytokines level and cytotoxicity markers are continuously monitored. After the perfusion, editing events and HCMV load are quantified to determine the therapeutic efficacy of the treatment.
The delivery of CRISPR/Cas9 nucleic acids using our nanoparticles in EVLP is expected to target latent HCMV genomes residing in the lungs with minimal immunogenic response and cytotoxicity. Viral genome sequencing is expected to observe indels at the 5 targeted site. The viral load within the lung tissue is expected to decrease. Due to the decrease in viral load, HCMV-related lung transplant complications are expected to decrease along with an increase in life expectancy.
By eliminating latent HCMV within donor lungs before transplantation, the pool of viable donor lungs can be drastically increased to meet the growing need for donor organs. Furthermore, HCMV-related transplant complications can also be greatly reduced which can improve the transplantation prospect and recipient life-expectancy.