The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has proved to evade neutralization by antibodies elicited by vaccination and natural infection with the ancestral strains by the emergence of new variants. New therapeutic measures are essential to achieve better control of viral spread and disease severity.
Study: Lung-selective Cas13d-based nanotherapy inhibits lethal SARS-CoV-2 infection by targeting host protease Ctsl. Image Credit: alphaspirit.it/ Shutterstock
A new preprint shows the potential role of a host protease cathepsin L (Ctsl) inhibitor, which mediates viral entry into the host cell by its effect on the viral spike protein.
A preprint version of the study is available on the bioRxiv* server while the article undergoes peer review.
SARS-CoV-2 entry into host cells is a complex process that depends on the actions of specific host proteases, including TMPRSS2 and Cathepsin L (Ctsl). Even though Ctsl is an entry factor for SARS-CoV-2, attempts to use Ctsl inhibitors to achieve the in vivo suppression of viral entry into the host cell have not been met with much success in the presence of the alternative entry protease, TMPRSS2.
For this reason, the current study aimed to knock out the Ctsl gene using gene therapy, and cause impaired function of the host protease more efficiently than the use of molecular inhibitors of the enzyme. This increase in efficacy maybe because of the ability to block the host protease’s catalytic and non-catalytic domains.
A challenge faced by researchers in this area is the known importance of the cathepsins in a host of physiological processes, including the immune response and development.
To overcome this obstacle, the authors of this paper focused on the transient knockdown of Ctsl mRNA transcripts using CRISPR/CasRx. The advantage of this approach over the older and powerful CRISPR/Cas9 system is that it does not cause permanent deletion of the Ctsl gene.
It has been shown that CasRx can knock down Ctsl mRNA while allowing other cathepsins to be expressed in a pristine fashion, thus ensuring that the processes regulated by them continue to function unimpaired. For instance, Ctsl has a major role in antigen processing, antigen presentation, and antibody generation, all of which are crucial components of humoral and cellular immunity.
What did the study show?
An important finding was that this system protected mice expressing the human ACE2 receptor and exposed to SARS-CoV-2 at a lethal dose after pretreatment with the LNP-CasRx-pre-gCtsl system.
The mechanism of action of this therapy may be via a reduction of the viral load, with a corresponding decline in the concentrations of cytokines and chemokines, both of which prevent severe lung pathology.
In vitro, this knockdown is found to suppress the entry of SARS-CoV-2 into cells whether or not they express the other host protease implicated in viral entry – TMPRSS2. This finding emphasizes the need for Ctsl for viral entry by several different pathways.
The Delta variant of the virus has rapidly become dominant over most of the globe, being far more transmissible than earlier variants. Part of the reason for this is the massive increase in viral entry caused by the increased rate of cleavage of the viral spike protein that is essential to accomplish virus-cell membrane fusion and endocytosis of the virus.
Ctsl knockdown inhibits the entry of the Delta variant into cells expressing TMPRSS2, indicating that Ctsl-mediated cleavage of this spike variant may be a prerequisite for infection. This could mean that Ctsl is a virulence-enhancing factor for the Delta variant.
With fears of future pandemics caused by other coronaviruses, much attention is being paid to the development of pan-coronavirus therapies and prophylactics. In this context, it is gratifying that the LNP-CasRx-pre-gCtsl system also prevents the entry of pseudoviruses expressing the SARS-CoV spike into TMPRSS2 cells.
What are the implications?
The researchers developed an mRNA-directed approach to suppress lung Ctsl activity specifically and block lethal SARS-CoV-2 infection in a mouse model using a CRISPR/Cas13d-based nanoparticle therapy.
The nanotherapy successfully reduced lung Ctsl expression efficiently and safely, avoiding off-target effects by its specificity. The pretreated mice survived the lethal dose of the virus as the therapy reduced the viral load in the lungs, prevented a cytokine storm in these organs, and prevented severe interstitial pneumonia.
Ctsl is implicated in the entry of several wild-type and mutant coronaviruses into the host cell. Most of these viruses do not mutate at a high rate in the Ctsl target site – the cleavage site between the S1/S2 interface and the S2 position that gives rise to the fusion peptide. As such, a Ctsl knockdown strategy could offer a viable and effective approach to treating and preventing such viral infections.
The CasRx RNA editor is also versatile, allowing genomic RNAs to be designed to order within the CasRx-based LNP system so that they can knock down any host factor as required to inhibit coronavirus infection.
The broad spectrum of in vitro activity, covering SARS-CoV and SARS-CoV-2 spike proteins and the Delta variant, independent of TMPRSS2 expression, is an encouraging finding. These results should be investigated further in clinical trials.
bioRxiv publishes preliminary scientific reports that are not peer-reviewed and, therefore, should not be regarded as conclusive, guide clinical practice/health-related behavior, or treated as established information.
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