Host-Directed Therapeutics

Ideal Targets and in vivo Approaches

The spread of COVID-19 and its subsequent variants illustrate the urgent need to develop combative therapeutics that can quickly identify and attack emerging viral threats. Vaccines, such as mRNA-derived ones, represent the most effective method of viral control [1]. However, all current antivirals directly target viruses by inhibiting their protein functionality, exerting an intense selective pressure that encourages the fixation of drug-resistant mutations within the viral population [1]. Furthermore, viral-directed vaccines are specific, limiting their pan-viral efficacies and the possibility of repurposing for emerging diseases. Vaccine hesitancy, suboptimal efficacy, and the emergence of resistant viral strains exemplify the necessity for alternative antiviral treatments.

Introduction

A promising alternative to traditional vaccines are host-directed therapeutics (HDT). Viruses, like parasites, rely on host cell machinery to survive and replicate. HDTs utilize viruses’ host dependency to disrupt viral perturbation, targeting essential host factors for viral replication. These host factors are ideal targets for therapeutics because they will not mutate under selective pressure, lowering the possibility of viral resistance through escape mutants. Furthermore, many viruses share host-dependent factors for replication, so HDTs could potentially increase pan-viral treatment efficacies [1].

However, HDT’s implementation requires an extensive understanding of virus-host interactions and the identification of specific genetic regulators of viral infection and replication. Advancements in high-throughput genomic screening with CRISPR have identified various host-viral factors. Furthermore, safe regulation of these factors in vivo, remains a challenge. This paper explores promising host-pathogen interactions and the implementation of HDT in vivo.

Modulation of Sialic Acid

Viruses enter a host through cell surface receptors. Each specific receptor binding site determines its host range and pathogenicity. Sialic acid linked to glycoproteins is a primary receptor for pathogen binding. SLC35A1 and SLC35A2 are sialic acid transporters, and the complete knockout of these genes results in total surface sialic acid removal without increasing cell toxicity [2]. The transporter loss makes sialic acid unavailable for glycosyltransferases and prohibits viral cell entry.

Modulation of Transmembrane

After binding to sialic acid surface receptors, viruses travels through the cell’s transmembrane proteins. Transmembrane protein IGDCC4 plays a crucial role in influenza virus internalization into host cells [3]. In one such study, CRISPR/Cas9 gene knockout of transmembrane protein IGDCC4 in mice revealed reduced replication of N5H1 in lung epithelial cells without adverse effects on the mice [3].

Modulation of Endosome

After viral endocytosis through receptor-mediated transmembrane protein, acidification of the resulting endosome releases the viral genome into the cytoplasm. Accordingly, functional ATPase and endosome assembly components were shown to be essential host factors for viral release [2]. In one study, knockout of functional V-ATPase precludes viral endosomal escape [2]. The three proteins, WDR7, CCDC115, and TMEM199, were V-ATPases co-factors and facilitated V-ATPase assembly onto the endosome [2]. Functional loss of these factors led to lysosomal biogenesis and incoming endosome over-acidification, prohibiting viral entry, and increasing invading virions degradation [2].

In Vivo Modulation

Even with identification of promising host factors, safe modulations of these factors in vivo remain a major challenge. Modulating factors are often too broad and run the risk of off-targeting elsewhere in the host, leading to undesirable consequences. A recent study that used mRNA lipid nanoparticles to deliver CoV-2 spike genes as a COVID-19 vaccination strategy was shown to be safe and effective [4]. Although use of mRNA delivery was sufficient in some cases, generally mRNA delivery is highly transient, with maximum efficacy peaking and fading within 48 hours [4].

Self-amplifying RNAs (saRNAs) and CRISPR-mediated gene activation (CRISPRa) may provide a more lasting alternative [5*]. saRNA’s are RNA replicases that are derived from self-replicating alphaviruses. Upon delivery, translation of saRNA produces replicases which multiply copies of the original RNA strand [5*]. This replication mechanism induces an immune response at a lower dosage and is shown to have prolonged protein expression of up to 60 days [6*]. Long term expression can also be achieved through delivery of CRISPRa with associate viral vectors. Synthetic CRISPR transcription factors can modulate target genes in vivo and it has been shown that removal of VP64 from CRISPR notably decreases gene activation. Therefore, fusion of a transcriptional factor, such as VP64, to a nuclease-dead Cas9 should upregulate gene expression [7*].

CRISPR can also be used to down regulate genes through CRISPR interference and Cas13a. CRISPR interference is composed of a KRAB transcriptional repressor domain fused to a dead Cas9[7*,8]. CRISPRi’s are less toxic than Cas9 because they do not cause a double stranded break in the DNA. However, disruption of gene function is more sensitive to guide RNA selection than Cas9 and often results in incomplete gene silencing [7]. It has also been shown that Cas13a can successfully downregulate influenza and SARS-CoV-2 RNA in the respiratory epithelium of animal models [13]. In mice, Cas13a was shown to degrade influenza RNA while in hamsters, Cas13a reduced viral replication and symptoms [12,13*]. These findings suggest that RNA mediated targeting can be an effective and safe way to modulate host genome.

Conclusion

Host-directed therapies provide a promising alternative to traditional viral-directed vaccines. Genome-wide CRISPR screens and meta-analysis studies have identified various ideal host-dependent factors for antiviral targeting. Furthermore, these factors exhibit high efficacy in vivo with minimal host toxicity after being modulated by multiple strategies. Future studies will optimize these factors and identify other antiviral targeting candidates. A better understanding of viral-host interactions will provide molecular insight for broad-spectrum antiviral therapy development against viral infections.

References

1. Kumar N, Sharma S, Kumar R, Tripathi BN, Barua S, Ly H, Rouse BT: Host-Directed Antiviral Therapy. **Clin Microbiol Rev 2020, 33.

An extensive review on host-directed antivirals and FDA status on various pre-clinical therapeutics

2. Li B, Clohisey SM, Chia BS, Wang B, Cui A, Eisenhaure T, Schweitzer LD, Hoover P, Parkinson NJ, *Nachshon A, et al.: Genome-wide CRISPR screen identifies host dependency factors for influenza A virus infection. Nat Commun 2020, 11:164.

An extensive meta-analysis of high-throughput data from RNAi and CRISPR screens for identification of host-dependency factors.

3. Song Y, Huang H, Hu Y, Zhang J, Li F, Yin X, Shi J, Li Y, Li C, Zhao D, et al.: A genome-wide CRISPR/Cas9 gene knockout screen identifies immunoglobulin superfamily DCC subclass member 4 as a key host factor that promotes influenza virus endocytosis. PLoS Pathog 2021, 17:e1010141.

4. Chaudhary N, Weissman D, Whitehead KA: mRNA vaccines for infectious diseases: principles, delivery, and clinical translation. Nat Rev Drug Discov 2021, 20:817-838.

5. Bloom K, van den Berg F, Arbuthnot P: Self-amplifying RNA vaccines for infectious diseases. Gene *Ther 2021, 28:117-129. Explores the construction of self-amplifying RNA delivery vectors and their use as a vaccination strategy

6. Blakney AK, McKay PF, Yus BI, Aldon Y, Shattock RJ: Inside out: optimization of lipid nanoparticle formulations for exterior complexation and in vivo delivery of saRNA. Gene Ther 2019, 26:363372.

7. Pandelakis M, Delgado E, Ebrahimkhani MR: CRISPR-Based Synthetic Transcription Factors In Vivo: *The Future of Therapeutic Cellular Programming. Cell Syst 2020, 10:1-14. Explores the use of CRISPR-based gene targeting to modulate host gene expression.

8. Alerasool N, Segal D, Lee H, Taipale M: An efficient KRAB domain for CRISPRi applications in human cells. Nat Methods 2020, 17:1093-1096.