Team:LZU-CHINA/Implementation

Implementation

The main goal for our project is to help those people at high risk of infection prevent the infection of SARS-CoV-2 through knocking down the expression of ACE2 by CRISPR-RfxCas13d system temporarily.
From experiment results, our CRISPR-Cas13d system which is loaded in lentivirus can significantly degrade the infection of SARS-CoV-2 pseudovirus in cellular experiment. But we still have a lot of work to do in the future, in order to improve this therapy.
In our experiment, we verified the system at cellular level by using lentivirus. We originally intended to use rAAV instead of lentivirus to carry the CRISPR-Cas13d system in the future. Because of its low genotoxicity profile in humans and the lack of strong and direct evidence that recombinant AAVs (rAAVs) can cause vector genome-mediated host genotoxicity in humans[1]. But it is not suitable for development of therapy of SARS-CoV-2, due to AAV’s high cost of manufacturing and low yield.
Proprosed solution for this purpose that temporarily knocks down ACE2 on lung tissues surface would be delivering the synthetic mRNA of RfxCas13d with its crRNAs which are formulated with the new material called PBAEs (poly beta amino esters) through aerosol inhalation[2]. RfxCas13d is a bacterial protein, there will be immune responses to the protein itself, and this is more likely if it is expressed for long periods of time. So we plan to use synthetic mRNA of Cas13d. Synthetic mRNA expression is transient and has very little chance of integrating. In addition, innate immune responses can be mitigated through sequence design, modified nucleotides[3]. What’s more, in vitro transcribed mRNA has broad therapeutic applicability as it permits temporal and dose-dependent control of encoded protein expression.
Next we are going to seek cooperation with the labs that use PBAEs to develop nucleic-acid-based therapeutics.

Future Challenges

One of the main challenges we see with this project is the general people are cautious about RNA editing represented by CRISPR-Cas13 system. Further work is necessary in order to convince the population that RNA editing is nothing to be feared, but rather a useful tool that can benefit humanity in a variety of ways. Our current evidence suggests that knocking down ACE2 will not cause significant disfunction[4]. Moreover, compared with DNA editing like CRISPR-Cas9, RNA editing doesn’t cause permanent changes to the genome, and this reversible and easily regulated editing method may have security advantages.
RNA editing doesn’t face the same ethical challenges as genome editing.

References

[1] Wang D, Tai PWL, Gao G. Adeno-associated virus vector as a platform for gene therapy delivery. Nat Rev Drug Discov. 2019;18(5):358-378. doi:10.1038/s41573-019-0012-9
[2] Patel AK, Kaczmarek JC, Bose S, et al. Inhaled Nanoformulated mRNA Polyplexes for Protein Production in Lung Epithelium. Adv Mater. 2019;31(8):e1805116.
[3] Loomis KH, Lindsay KE, Zurla C, et al. In Vitro Transcribed mRNA Vaccines with Programmable Stimulation of Innate Immunity. Bioconjug Chem. 2018;29(9):3072-3083
[4] Gurley SB, Allred A, Le TH, et al. Altered blood pressure responses and normal cardiac phenotype in ACE2-null mice. J Clin Invest. 2006;116(8):2218-2225.