Genetic instability constitutes the key driving force underlying tumoral evolution. The finding that multiple tumor suppressor genes function in DNA damage checkpoint to prevent mutagenesis confirmed this view. Therapeutically, the defect in checkpoint allowing genetic instability also represents vulnerability as it could be exploited to trigger genome disintegration, resulting in tumor cell death. This point has been successfully demonstrated in the case of a tumor suppressor gene defect (see below). Here, in a similar vein, we explored whether genetic instability underlying viral evolution could be exploited for the therapeutic purpose of suppressing infectivity.

Within cells, progression through the G1 phase is governed by the prototypic tumor suppressor retinoblastoma (RB) protein, a component of the DNA damage checkpoint [1]. RB also controls cell cycle progression across the S phase; after the cloning of the human RB gene by F. Hong (a.k.a. Frank Un, Frank D. Hong) et al. [2, 3] at the University of California in San Diego (United States), RB protein was found to be phosphorylated in vivo. His determination of the RB gene sequence was pivotal in identifying the cyclin-dependent kinase (CDK) complex as the enzyme catalyzing RB phosphorylation [2, 3]. The cyclic phosphorylation pattern of RB is maintained by distinct cyclin-CDK complexes, with the least phosphorylated species found in the G1 phase [4]. It led to the development of various inhibitors, which include palbociclib (Pfizer Pharmaceutical, United States), ribociclib (Novartis Pharmaceutical, Switzerland), and abermaciclib (Eli Lilly Pharmaceutical, United States) approved by the U.S. Food and Drug Administration (FDA) for treating metastatic breast cancer.

RB protein’s role in regulating transcription was inferred from F. Hong et al. [3] finding of its ability to bind to DNA, which was subsequently confirmed [5]. This property was corroborated via observing the RB-DNA complex formed by purified RB protein and double-stranded DNA [6]. Its atypical electrophoretic migration pattern led to his subsequent finding of RB’s oligomerizing property [7–9]. RB protein’s interaction with E2F confirmed RB gene’s role in transcriptional regulation. Partial proteolysis of RB by C. E. Hensey (University College Dublin, Ireland) et al. [7] revealed the existence of distinct domains for interaction with associating proteins and a separate module at the N-terminus. The prototypic antimetabolite drug hydroxyurea is FDA-approved for treating chronic myelogenous leukemia, and head and neck squamous cell carcinoma. [10]. Through the analysis of RB-associating protein, F. Hong discovered that the sensitivity to hydroxyurea could be restored to resistant human cancer cells at the City of Hope National Medical Center and Beckman Research Institute (United States) in collaboration with C. Bronner (Institut national de la santé et de la recherche médicale, France) et al. [11].

RB gene’s role in chromosome organization and architecture was indicated by F. Hong and W. H. Lee found [12]; that RB protein exhibits similarity in sequence with neurofilament L-type subunit, which was corroborated by R. F. Doolittle (University of California at San Diego, United States) [12]. Consistently, he observed RB oligomers and detected the polymerization of RB protein [7]. The association of RB with nuclear matrix was documented by S. Penman (University of Texas Health Science Center at San Antonio) et al. [13]. The finding that RB protein may self-interact to form higher-ordered structures was instrumental in extending RB gene’s function to DNA replication, repair, recombination, condensation, heterochromatinization, epigenetics, etc. [1]. The prototypic DNA crosslinking drug cisplatin is FDA-approved for treating ovarian cancer, testicular cancer, and bladder cancer. F. Hong [14] discovered that G1 arrest mediated by RB represents a critical determinant of cisplatin cytotoxicity in RB-positive human cancers retaining DNA damage checkpoint.

The inactivation of RB through mutation causes genetic instability [1, 15]. F. Hong observed that ectopic expression of RB driven by SV40 large T promoter causes transfected cells to arrest as enlarged cells (unpublished data). Retrovirally expressed RB suppressed the growth of human RB, prostate cancer, and breast cancer [16–18]. Further, purified human recombinant RB protein induced G1 arrest in microinjected cells [19]. These findings revealed RB gene’s function in DNA damage checkpoint in the G1 or S phase [20]. The prototypic antimicrotubule drug Taxol is FDA-approved for treating breast cancer, non-small cell lung cancer, and ovarian cancer. F. Hong [15] discovered that by exploiting the defective checkpoint rendered by inactive RB (or p53), Taxol (paclitaxel) induces lethality via triggering chromosome fragmentation, i.e., ‘mitotic catastrophe’. It led to the elucidation of the tumor-specific lytic path ‘hyperploid progression mediated death’ targeting RB or p53-mutant human cancer cells at the University of Texas Southwestern Medical Center (United States), establishing a framework for developing cytotoxic cancer therapy devoid of side effects [15].

RB gene’s role as a therapeutic biomarker originates from F. Hong et al. [21] discovery of an RB mutant affecting its promoter activity [22]. Earlier, he identified and characterized the promoter of the human RB gene [23]. It represents the earliest RB mutant to be identified in human prostate cancer [21]. The mutational status of RB dictates the optimal strategy for treating advanced-stage cancer patients, e.g., hormone therapy [example (ex.), Enzalutamide] versus antimicrotubule chemotherapy (ex., Taxol) for prostate cancer.

Increasingly, modern medicine relies on targeting upstream genetic components such as mRNA or chromosomal DNA. Notable among them is genetic medicine using oligonucleotides to suppress translation by hybridizing to mRNA or degrading mRNA through RNA interference or RNase H1. The first clinical trial of siRNA employed siRNA targeting human ribonucleotide reductase M2 subunit contained in nanoparticles designed by M. E. Davis (California Institute of Technology, United States) et al. [24] to treat various types of human cancer. Nevertheless, oligonucleotide drugs, by themselves, cannot translocate across the cell membrane for delivery in a targeted manner, contributing to side effects. To facilitate delivery, the siRNA was conjugated to the tumor specifically internalizing peptide HN-1 for targeted delivery into the head and neck or solid breast tumor. HN-1 peptide was isolated via biopanning of M13 bacteriophage-based random peptide display library using human head and neck squamous carcinoma cells by F. Hong and G. L. Clayman [25] at the University of Texas M. D. Anderson Cancer Center (United States). HN-1 has been conjugated to various agents for cancer therapy (ex., siRNA oligonucleotide, Taxol, protein kinase C inhibitory peptide, diphtheria toxin, doxorubicin) or tumor imaging (ex., near-infrared dye, radioisotope) [26–34]. His delineation of discoidin domain tyrosine kinase receptor 1 (DDR1) as the putative receptor for HN-1 and its therapeutic application to breast, lung, and other cancer types was recently reviewed [35]. Its ability to penetrate tumor mass may be useful in delivering immunotherapeutics to the interior of solid human cancers. For prostate cancer, he described the role of monoamine oxidase A in modulating anticancer immunity at the University of Southern California (United States) [36, 37]. FDA has approved immunotherapeutics targeting hypermutated tumors [38].

The recent coronavirus disease 2019 (COVID-19) pandemic has affected many with underlying conditions [39, 40]. Among the impacted were those who have cancer, whose fatality rate increased markedly following the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection. Various preventive measures have been developed, including mRNA vaccines expressing the modified spike protein [41]. These countermeasures significantly reduced COVID-19-associated mortality. Nevertheless, subsequently emerged variants were able to impact host immunity via acquiring a mutation in epitopes [42]. Adaptation by SARS-CoV-2 was not unexpected, given its high level of mutability [43, 44]. The genetic instability of SARS-CoV-2 gives rise to point mutation, insertion, deletion, and complex mutation [45]. The virally encoded RNA-dependent RNA polymerase (RDRP) incurs a high error rate during nucleotide incorporation despite the proofreading capacity (3’-to-5’ exoribonuclease) of nonstructural protein 14 (NSP14). Furthermore, genetic recombination may occur due to template switching between homologous RNA strands by RDRP [45].

Modulating the cell cycle via targeting its regulators represents a key replicative strategy amongst viruses [46]. Virally encoded oncogenic proteins that interact with RB protein include large T antigen (simian vacuolating virus SV40), E1A (adenovirus), and E7 (human papillomavirus) [47]. For SARS-CoV-1, structural protein 3a inhibits RB phosphorylation, causing G1 arrest [48]. In the case of SARS-CoV-2, NSP1 was shown to suppress host cell protein synthesis, resulting in G1 arrest, which is regulated by RB [49]. SARS-CoV-2 was also shown to cause S arrest (which is controlled by RB) via depleting deoxynucleotides [50].

The acquisition of S1/S2 furin cleavage site in spike protein significantly expanded the tropism of SARS-CoV-2 (see below). The increased dissemination despite reduced virulence is a distinguishing feature amongst subsequently arisen variants. Here, to address this issue, the possibility of exploiting the hypermutability of SARS-CoV-2 to suppress its infectivity is explored. To apply virolytic pressure, oligonucleotides targeting the S1/S2 site-encoding sequence may be deployed to trigger genomic RNA degradation. A potential consequence of the above approach is that resistant variants (if they emerge) may carry a mutation in S1/S2 site-encoding sequence to block oligonucleotide hybridization, which may encode defective substrate for furin by default. Hence, the prospect of applying targeting oligonucleotides to direct the devolution of the S1/S2 site, delimiting tropism to attenuate the infectivity of SARS-CoV-2, is described. The precedent for the above comes from previous reports documenting reduced viral fitness conferred by resistance mutations. The approach is reminiscent, albeit distinct from the ‘directed evolution’ described by P. A. Romero and F. H. Arnold (California Institute of Technology, United States; Nobel Prize 2018) [51].

SARS-CoV-2 is a positive-stranded RNA virus that belongs to the subgenus Sarbecoviruses (order Nidovirales; family Coronaviridae; subfamily Orthocoronavirinae; genus Betacoronavirus) [101, 102]. SARS-CoV-2 is internalized following the interaction of its spike protein with the surfacially expressed ACE2, which hydrolyzes the vasoconstrictor angiotensin II [103]. Internalization of SARS-CoV-2 may occur via distinct pathways [98]. Its spike protein contains two proteolytic cleavage sites, i.e., S1/S2 located at the junction between S1 and S2 subunits and S2’ located within the S2 subunit [98, 104]. Despite the cleavage at the S1/S2 site (priming), the S1 and S2 subunits are held together via noncovalent interactions whereas the cleavage at the S2’ site triggers structural rearrangement to expose the ‘fusion peptide’ to engage cell membrane for internalization [105]. The cleavage at the S1/S2 by furin may occur during the virus maturation in SARS-CoV-2-producing cells [98]. One entry route involves cleaving SARS-CoV-2 spike protein (internalized via clathrin-mediated endocytosis after binding to ACE2) at the S2’ site by cathepsin to enable membrane fusion necessary for cytosol entry. For direct entry at the cell surface, the S2’ site is cleaved by transmembrane protease, serine 2 (TMPRSS2) to allow fusion of the viral envelope with the cell membrane. Cleavage of the S2’ site by furin was also reported [106].

Multiple coronavirus species, whose genomic RNA sequences align closely with that of SARS-CoV-2, have been identified [86]. SARS-CoV-2 spike protein contains S2’ furin cleavage site (PSKPSKRSFIEDLLFNKV: furin recognition motif in italics), which includes Arg-815 (GenBank entry MN908947.3, ‘Severe acute respiratory syndrome coronavirus 2 isolate Wuhan-Hu-1’) [107]. Likewise, the S2’ site is also present in the spike protein of closely related coronaviruses: bat coronavirus RaTG13 (PSKPSKRSFIEDLLFNKV, GenBank entry MN996532.2, ‘Bat coronavirus RaTG13’), human SARS-1 coronavirus (PLKPTKRSFIEDLLFNKV, GenBank entry AY278741.1, ‘SARS coronavirus Urbani’), bat coronavirus Rs4231 (PLKPTKRSFIEDLLFNKV, GenBank entry KY417146, ‘Bat SARS-like coronavirus isolate Rs4231’), and bat coronavirus BtRs-BetaCoV/YN2018B (PLKPTKRSFIEDLLFNKV, GenBank entry MK211376.1, ‘Coronavirus BtRs-BetaCoV/YN2018B’). Intriguingly, most proximally related coronaviruses lack the S1/S2 site despite the presence of the S2’ site.

The acquisition of the S1/S2 furin cleavage site in spike protein is critical for the expanded tropism (plus human transmission) of SARS-CoV-2 [108]. The presence of both the S1/S2 site and the S2’ site in spike protein of SARS-CoV-2 was confirmed using furin inhibitors [105]. Consistently, the absence of the S1/S2 site reduced the pathogenicity of SARS-CoV-2 [109]. Further, ectopically introducing the S1/S2 furin cleavage site to the SARS-CoV-1 spike protein (which lacks the S1/S2 site) increased fusogenicity [110, 111]. Thus, the S1/S2 site may represent a critical determinant for SARS-CoV-2 infectivity. Subsequent SARS-CoV-2 variants such as Omicron exhibited increased dissemination despite reduced virulence [73].

To delimit tropism, an optimal strategy for disabling the S1/S2 site was sought, which could exploit the hypermutability of SARS-CoV-2. Inhibiting furin may cause adverse side effects as the enzyme participates in various normal physiological functions. The structural analysis of furin revealed that its active site for catalysis consists of a groove for binding substrates, which is lined with numerous negatively charged amino acids—i.e., Asp and Glu. Consistently, peptides containing positively charged polyarginine represent potent inhibitors of furin. As prior screenings may have relied on assays that monitor the inhibition of furin-like activity in cell lysate, inhibitors with higher selectivity for furin were sought. The molecular structure of proprotein convertase furin bound to the hexapeptide Arg-Arg-Arg-Val-Arg-Amba is shown (Figure 3B).

Due to its random-coil secondary structure, sterically blocking access to furin by using antibodies to mask the S1/S2 site of spike protein may be difficult [112]. An alternative strategy is to utilize oligonucleotides to target the S1/S2 site for pharmacological inhibition at the nucleic acid level: (1) as the S1/S2 furin cleavage site is retained by SARS-CoV-2 for propagation, it represents a valid therapeutic target; (2) targeting RNA than protein is more efficacious as it lies upstream in genetic information flow; (3) oligonucleotides may confer targeting specificity at the nucleotide sequence level; (4) oligonucleotides could trigger genomic RNA degradation via RNA interference (ex., siRNA) or RNAse H (ex., gapmer); (5) antisense oligonucleotides (ex., morpholino) can interfere with translation or RNA processing; (6) antisense oligonucleotides targeting SARS-CoV-2 genome have been reported, indicative of its feasibility; (7) oligonucleotide drugs may provide virus-specificity at the genetic level; (8) unlike conventional structure-activity relationship-based chemical drug development, only mRNA sequence is necessary to design oligonucleotide drugs.

Briefly, developing the chemical method underlying oligonucleotide synthesis entailed several major advances. In 1955, A. Todd (University of Cambridge, England; Nobel Prize 1957, nucleotide, nucleotide coenzyme) and A. M. Michelson reported the synthesis of dithymidine dinucleotide, which involved condensing of thymidine 3’-(benzyl phosphorochloridate) 5’-(di-benzyl phosphate) with 3’-O-acetylthymidine [113]. In 1958, H. G Khorana and P. T. Gilham (British Columbia Research Council, Canada) [114] introduced the phosphodiester method for oligonucleotide synthesis, which utilized N,N’-dicyclohexylcarbodiimide as activating agent; however, branching at internucleosidic phosphate remained problematic. In 1969, R. L. Letsinger and K. K. Ogilvie (Northwestern University, United States) [115] described a phosphotriester approach to resolve the above issue by protecting the phosphate moiety and subsequently implemented a solid-phase synthesis method. In 1975, W. B. Lunsford (a former colleague of W. Letsinger; Baylor University, United States) and colleagues [116] reported phosphite triester methodology, which uses 3’-O-chlorophosphite containing more reactive phosphorus(III) to form internucleosidic linkage, with whom M. Castro and F. Hong worked on oligonucleotide synthesis. In 1981, F. Hong worked on cloning of E. coli genes encoding the phosphate transferase system with S. Roseman (discoverer of phosphate transferase system, Johns Hopkins University, United States; a former colleague of H. G. Khorana) [117]. In 1981, M. H. Caruther (a former colleague of W. Letsinger; University of Colorado, United States) and M. D. Matteucci [118] used nucleoside phosphoramidites as a precursor for oligonucleotide synthesis, which utilizes 2-cyanoethyl phosphite protecting group. Critical to current solid-phase oligonucleotide synthesis using deoxynucleoside phosphoramidites was the solid support-based peptide synthesis methodology developed by R. B. Merrifield (Rockefeller Institute, United States; Nobel Prize 1984) [119] in 1963.

Synthetic oligonucleotides have been used extensively in various molecular biological applications, i.e., gene cloning, sequencing, site-directed mutagenesis, analytical assay (ex., microarray), gene expression analysis (ex., fluorescent in situ hybridization), gene synthesis, etc. These include diagnostic assays (e.g., polymerase chain reaction to identify genetic mutants, DNA paternity testing). The use of oligonucleotides has also been extended to therapy. In 1978, M. L. Stephenson and P. C. Zamecnik (Harvard University, United States) [120] described suppressing the translation of Rous sarcoma virus 35S RNA using oligonucleotides. Rous sarcoma virus, which causes chicken cancer, was discovered by P. Rous (Rockefeller Institute for Medical Research, United States; Nobel Prize 1966) [121] in 1911. Following the discovery of RNA interference by A. Fire (Carnegie Institution of Washington, United States; Nobel Prize 2006) et al. [122], the potential utility of siRNA as a pharmaceutical agent was investigated. Its advantages include targeting mRNA rather than protein, exploiting the host cell’s RNA interference complex, its repeated use for mRNA degradation, etc. For clinical application, multiple issues need to be addressed, i.e., inciting of innate immunity, degradation by ribonucleases, off-target suppression, selective delivery to intracellular target, etc. Critical to resolving these issues is the ability to chemically modify oligonucleotides. For instance, to protect from nucleases, internucleosidic phosphodiester linkage may be modified to contain phosphorothioate. Alternatively, sugar moiety may be replaced with locked nucleic acid (i.e., bridged nucleic acid) or phosphoramidate morpholino oligomer [123]. Modified nucleobases (ex., pseudouridine) may be incorporated to attenuate immunogenicity [124]. To reduce side effects caused by off-targeting, specific modifications may be introduced to the sugar moiety [125].

Various oligonucleotide-based therapeutics (ex., siRNA, gapmer, antisense oligonucleotides, aptamer, morpholino oligonucleotide) have been approved by FDA for clinical use. The siRNA drugs approved by FDA include patisiran (hereditary transthyretin-mediated amyloidosis), givosiran (acute hepatic porphyria), lumasiran (primary hyperoxaluria type 1). Gapmers represent single-stranded oligonucleotides composed of DNA and RNA, which rely on the intracellular enzyme RNase H to degrade target mRNA. Gapmers that have been FDA-approved include mipomersen (homozygous familial hypercholesterolemia), and inotersen (familial amyloid neuropathies). For targeted delivery to the liver, siRNA drugs were conjugated to N-acetylgalactosamine (GalNAc) [126]. For tumor-specific delivery, siRNA therapeutics targeting ribonucleotide reductase were conjugated to tumor-specifically internalizing peptide HN-1 [26, 35]. Several phosphoramidate morpholino oligomers have been FDA-approved to modulate dystrophin exon splicing defects associated with Duchenne’s X-linked genetic disorder’s muscular dystrophy [127].

Multiple oligonucleotide therapeutics targeting viral RNA have been reported. For SARS-CoV-1, siRNAs have been developed that target the genomic RNA sequence encoding ORF1b (NSP12) or spike protein [128]. To inhibit the replication of SARS-CoV-1, its ‘leader sequence’ has been targeted using siRNA [129]. For SARS-CoV-2, modified antisense oligonucleotides (ex., morpholino-based oligonucleotides, gapmers) have been devised to suppress translation or promote degradation [130, 131]. Knockdown of TPPRSS2 expression by using ‘cell penetrating peptide-conjugated phosphorodiamidate morpholino oligomer (PPMO)’ reduced the cytopathic effect of SARS-CoV-2 in vitro [78].

A potential consequence of applying the above-described virolytic pressure is that resistant variants (if emerge) may carry mutation in the S1/S2 site-encoding sequence to abrogate hybridizing to the oligonucleotides, which is facilitated by its hypermutability. The altered sequence, by default, may encode a degenerated S1/S2 site that is no longer recognizable to furin for proteolytic cleavage, thus delimiting the tropism of SARS-CoV-2. Previous reports documenting reduced viral fitness conferred by resistance mutations set the precedent for the above. Firstly, in the case of cancer, drug treatment may induce evolution in treated cells toward acquiring resistance [132]. For RNA viruses, resistance to siRNA drugs may be acquired via incurring mutation in the target sequence to yield a mismatched template that prevents hybridization. In the case of the human immunodeficiency virus (HIV), viral escape variants that arose following the treatment with Nef-targeting siRNA accumulated mutation in target sequence, i.e., partial or complete deletion, nucleotide substitution [133, 134]. Secondly, resistance mutation may reduce viral fitness. In the case of the influenza virus, mutations that caused resistance to Oseltamivir, which inhibits neuraminidase, reduced replicative fitness [135]. Decreased fitness may occur when mutations occur in highly conserved sequences in the viral genome [134]. The high mutation rate may cause RNA viruses to exist as a population of ‘quasi-species’, i.e., closely related mutants [135]. It, in turn, may elevate the extent of genetic variability, increasing the likelihood of yielding variants that are less competent in replication. Consistently, mutations occurring in functionally significant regions were detrimental to hepatitis C virus [136]. For highly evolving RNA viruses, antiviral drugs inhibiting critical viral proteins may induce mutations that impede viral replication or pathogenesis.

Significant advances have been made in our understanding of biological organisms at the genetic level. Nonetheless, the genetic information gained may need to be interpreted in the context of their evolving nature given its fluidity. Earlier, molecular evolution was examined via the analysis of fibrinogen [137]. Yet, our understanding of the nature of the forces driving evolution appears incomplete—as was apparent from the inability to predict the emergence of SARS-CoV-2 despite years of research. Presented in this report is a therapeutic strategy through which the genetic hypermutability of SARS-CoV-2 may be exploited to attenuate infectivity. ‘Targeting oligonucleotide directed devolution’ entails applying virolytic pressure by inducing genomic RNA degradation to drive molecular evolution towards selecting degenerative S1/S2 site mutations in naturally occurring resistant variants. The possibility of resistance mutations occurring at a distant site interfering with oligonucleotide hybridization at the S1/S2 site structurally cannot be excluded. Conceivably, this approach may target multiple critical domains within a single viral gene, may target multiple essential viral genes, or may be applied repeatedly to ensuing variants. As an alternative approach, oligonucleotides may be used for ‘therapeutic RNA editing’ to inactivate the S1/S2 site. Adenosine deaminases acting on RNA (ADAR) represent intracellular enzymes which catalyze the hydrolytic deamination of adenosine to inosine. To treat genetic disorders caused by base substitution, Woolf (Ribozyme Pharmaceuticals, Inc., United States) et al. [138] used complementary RNA oligonucleotide to hybridize to mutant dystrophin mRNA encoding a premature UAG stop codon. The endogenously expressed double-stranded RNA adenosine deaminase recognized the duplex and converted to inosine, allowing translation to resume [138]. All in all, we hope these novel approaches may lead to the development of a potential deterrence against rapidly evolving pathogens like SARS-CoV-2 that exacerbate the disease burden caused by cancer [139].