Development of RNase P ribozyme-based therapy against oral herpesvirus infections by HSV-1 and KSHV

Abstract

Infections with human herpesviruses are known to be associated with oral lesions and other oral complications. For example, herpes simplex virus 1 (HSV-1) is the causative agent for cold sores. Kaposi’s sarcoma-associated herpesvirus (KSHV) causes oral Kaposi’s sarcoma (KS) and other oral complications among immunocompromised patients, such as HIV-positive individuals. Novel antiviral strategies are needed to effectively treat oral infections caused by HSV-1 and KSHV, given the limitations of current FDA-approved anti-HSV-1 and anti-KSHV therapies. Nucleic acid-based gene-interfering molecules, such as antisense oligonucleotides, ribozymes, and small interfering RNA (siRNA), are promising gene-targeting agents for therapeutic applications. Ribozymes derived from the catalytic RNA subunit of RNase P in Escherichia coli represent a novel class of RNA-based gene targeting molecules for degrading target mRNAs and modulating gene expression. Methods have been developed to engineer RNase P ribozymes that more effectively suppress gene expression. In this review, we summarize the recent advances in using RNase P ribozymes to block the expression of HSV-1 and KSHV genes and inhibit the infection of HSV-1 and KSHV. Moreover, we discuss the potential of engineered RNase P ribozymes as therapeutics to treat infections and associated diseases caused by HSV-1 and KSHV in the oral cavity.

Introduction

Ribonuclease P (RNase P) was first identified in the early 1970s by Sidney Altman as an endoribonuclease responsible for processing precursor transfer RNAs (pre-tRNAs) by cleaving 5′ leader sequences. ¹ It was also discovered that RNase P was not solely a protein enzyme but contained an RNA subunit, that possesses catalytic activity.^2,3 For example, M1 RNA, the RNA subunit of RNase P in Escherichia coli was demonstrated to have the catalytic ability to cleave the 5′ end of pre-tRNA in vitro independently. ² This discovery profoundly expanded our understanding of RNA’s functional capabilities, demonstrating that RNA itself can act as a catalyst. Building on this insight, subsequent investigations have been performed to understand how the RNase P catalytic RNAs recognize and cleave their RNA substrates.^4,5 These studies explored whether the catalytic M1 RNA subunit of RNase P could be engineered to target and cleave non-tRNA substrates in a sequence-specific manner.

In 1995, this concept was realized when M1 RNA, could be engineered to direct sequence-specific cleavage of a herpes simplex virus 1 (HSV-1) RNA by covalently attaching a guide sequence (GS) complementary to a target mRNA. ⁶ Later named M1GS, this ribozyme was designed to recognize and cleave the HSV-1 thymidine kinase (TK) mRNA. The resulting M1GS specifically cleaved the viral mRNA in vitro and in infected cells, leading to a ~80% reduction in both TK mRNA and protein levels. ⁶ These findings established the feasibility of M1 RNA as a programmable ribozyme capable of directing sequence-specific RNA degradation. Subsequent studies have demonstrated that M1GS ribozymes efficiently cleave numerous human and viral mRNAs in a sequence-specific manner in vitro and inhibit the expression of these target mRNAs and the replication of hepatitis B virus, HSV-1, human cytomegalovirus, human immunodeficiency virus (HIV), and KSHV in human cells.^7–13 Moreover, M1GS ribozymes have been demonstrated to effectively inhibit gene expression and replication of murine cytomegalovirus in mice in vivo.^14,15 Together, these studies defined the requirements and overall design principles required for M1GS, transforming RNase P from a pre-tRNA processing enzyme into a programmable platform for targeted RNA degradation.

Infections with human herpesviruses are known to be associated with oral lesions and other oral complications. HSV-1 is the causative agent for cold sores, and KSHV is the etiological agent of KS, a disease that occurs in several epidemiological forms that frequently manifests in the oral cavity. In this review, we summarize the recent advances in using RNase P ribozymes to block the expression of HSV-1 and KSHV genes and inhibit the infection of HSV-1 and KSHV. Moreover, we discuss the potential of engineered RNase P ribozymes as therapeutics to treat infections and associated diseases caused by HSV-1 and KSHV in the oral cavity.

Oral infections and diseases with HSV-1 and KSHV

The oral cavity is a critical site for infection, disease manifestation, and transmission of many herpesviruses, serving as both an initial point of entry and a reservoir for viral persistence and shedding.^16,17 Within this oral environment, distinct herpesviruses exploit oral tissues through unique biological strategies, such as HSV-1 and KSHV.^18,19

HSV-1 is a large, double-stranded DNA virus that enters through mucosal epithelial tissue, where it replicates productively before traveling to the sensory neurons of the trigeminal ganglia, where it establishes lifelong latency.^16,19,20 HSV-1 spreads through direct contact with contaminated saliva or other bodily fluids, replicates in mucocutaneous tissue, then travels to sensory neurons, where the virus can enter a non-infectious state before reactivation. Primary oral HSV-1 infection commonly manifests as herpetic gingivostomatitis and is characterized by widespread oral and perioral lesions, gingival inflammation, and fever.^16,19 After HSV-1 reactivation, it can cause recurrent herpes labialis (cold sores), often appearing on the lips and accompanied by symptoms such as tingling, itching, or burning, reflecting viral replication in sensory nerve endings and the adjacent epithelial tissues of the mucocutaneous surface. Importantly, viral shedding can occur even in the absence of visible lesions, thereby contributing to ongoing HSV-1 transmission. ²¹ Although the clinical symptoms of HSV-1 infection are considered mild, its recurrence, reactivation, and latency underscore the chronic nature of oral HSV-1 disease and the ongoing therapeutic challenges it poses.^16,19

KSHV, also known as human herpesvirus 8, ¹⁸ is an oncogenic gammaherpesvirus responsible for several malignancies and disorders, including KS, primary effusion lymphoma (PEL), and multicentric Castleman disease (MCD).^22,23 Among these, KS represents the most common clinical manifestation. ²³ KS occurs in several epidemiological forms, including AIDS-related (KS-HIV), classic (CKS), endemic African, classic Mediterranean, and rarer anaplastic variant (SKS). ²⁴ Among the different forms of KS, epidemic KS (KS-HIV) is the most strongly associated with the oral manifestation, with the oral cavity representing the first clinical site of disease in 22% of patients with KS.^24–27 After infecting endothelial cells, KSHV activates the mTOR pathways, altering the cells to have mesenchymal differentiation and promoting angiogenesis. ²⁸ Through immune suppression and inflammation, KSHV-infected cells can persist and proliferate. KSHV pathogenesis is driven by a highly regulated balance between latent and lytic viral expression. ²⁹ This process is tightly controlled by the latency-associated nuclear antigen (LANA) and the replication and transcription activator (RTA). ²⁹ LANA is responsible for maintaining the latent viral episome and persistent infection, while RTA functions as a regulator of lytic reactivation. ³⁰ Together, LANA and RTA initiate broad viral gene expression that drives oncogenic transformation, reactivation, and the development of KSHV-associated diseases. ³⁰ Although KSHV establishes latency in multiple cell types, its transmission and pathogenesis are closely linked to the oral cavity, serving as a significant reservoir for viral shedding.^22,31 Recent studies have demonstrated that KSHV infection is facilitated when basal epithelial cells are exposed to microtrauma or inflammation, thereby rendering the otherwise protected cell layers susceptible to viral access. ³² Oral lesions resulting from KSHV infection harbor high viral load, supporting the idea that the oral cavity functions as a hub for transmission and infection. ¹⁸ The oral cavity plays a role in KSHV disease manifestation, persistence, and transmission, underscoring that oral tissues are an optimal target for therapeutic intervention.

Current treatments against oral herpesvirus infections

Current therapeutic strategies for oral herpesvirus infection primarily focus on controlling active disease rather than eliminating persistent infection.^17–19 For HSV-1, treatment regimens commonly use antiviral agents that target viral replication during the lytic phase. ³³ This includes topical therapies such as acyclovir cream and docosanol, which reduce lesion duration and local symptoms when applied early during outbreaks. Unlike nucleoside analogs, docosanol blocks viral entry by inhibiting membrane fusion rather than targeting viral replication.^34,35 However, the clinical benefit of these treatments is limited by poor penetration into lesions, inability to affect latent HSV-1 reservoirs, and failure to significantly reduce recurrence frequency.^33,34,36 Systemic administration of nucleoside analog antivirals, including acyclovir, valacyclovir, and famciclovir, is used for more severe HSV-1 disease or frequent recurrence episodes. ³⁴ Since these antivirals inhibit HSV-1 DNA polymerase activity, they can only target the virus when it is lytically active, failing to eliminate latent HSV-1.^37,38 In addition, antiviral resistance can emerge from mutations in the viral DNA polymerase and thymidine kinase (TK), particularly in immunocompromised individuals.^34,37,38 There are currently no commercially licensed vaccines available for HSV-1, highlighting the limited therapeutic options for treating HSV-1 infection.

Treatment for KSHV-associated diseases presents distinct challenges due to the virus’s oncogenic nature and complex latency-lytic regulation, a process that remains incompletely understood. ¹⁸ Therapy for the KS, PEL, and MCD largely relies on surgery, chemotherapy, immunotherapy, and small-molecule inhibitors.^39,40 While antivirals targeting KSHV are available, they primarily inhibit KSHV DNA polymerase. These antivirals do not eliminate latent KSHV infection or directly treat the KSHV-driven malignancies. Although KSHV is required for the development of KS, nearly all lesions harbor KSHV viral DNA in its latent phase, rendering the lytically active antivirals largely ineffective. ²⁸ As a result, treatment for KSHV-driven diseases utilizes chemotherapy and immunologics that target tumor growth, cell signaling, and immune pathways rather than viral gene expression.^39,40 In KS, treatment may include immunomodulatory agents such as pomalidomide, which target angiogenic and inflammatory pathways that support tumor growth rather than directly suppressing KSHV.^39,40 In PEL and MCD, rituximab is used to target B cells, but, like pomalidomide, it does not directly inhibit KSHV gene expression; instead, it acts on downstream consequences of infection. ³⁹ Importantly, these approaches do not directly target KSHV persistence but act on its downstream consequences of infection at the level of KSHV-driven diseases. Emerging preclinical efforts have explored viral latency-associated proteins, such as LANA, as therapeutic targets due to their central roles in episome maintenance, transcriptional regulation, and oncogenic properties. ¹⁸ However, such strategies remain at the experimental stage, and no approved treatments target KSHV transcripts or proteins directly. ⁴¹ As a result, existing treatments address KSHV replication, tumor proliferation, or immune dysregulation in isolation, rather than a unified strategy that links KSHV persistence to disease manifestation.

Antiviral limitations are most pronounced in immunocompromised populations, where impaired immune surveillance, prolonged viral replication, and emergence of drug-resistant strains lead to more frequent and severe oral herpesvirus manifestations.^34,42 Prolonged exposure to nucleoside analogs such as acyclovir and ganciclovir leads to the emergence of resistant HSV-1 and KSHV strains through mutations in thymidine kinase or viral DNA polymerase. ³⁴ In these mutated strains, where TKs are mutated or absent, cross-resistance among nucleoside analogs can occur.^34,37 Consequently, increasing attention has been directed toward RNA-based therapeutics to overcome antiviral resistance and improve treatment options for immunocompromised patients. Approaches such as antisense oligonucleotides (ASOs), RNA interference (RNAi), and CRISPR-based systems utilize sequence-guided recognition to bind to viral RNA or DNA and inhibit gene expression through blocking translation, transcript degradation, or nuclease-mediated cleavage of viral genomes or RNA transcripts. ³⁴ Preclinical studies have demonstrated the potential of these RNA therapeutics as alternative antiviral strategies.^34,43–46 However, clinical application of these approaches remains limited due to challenges in efficient and targeted delivery, immunogenicity, and the unique oral anatomical barriers, such as rapid epithelial turnover. ³⁴

Ribonuclease P

RNase P is an enzyme present in all domains of life that processes precursor tRNAs and other small RNAs by cleaving the 5′ leader sequence.^47–49 While varying in composition, RNase P may consist of a catalytic RNA subunit and a protein subunit.^47–49 There is 1 protein component in bacteria, 4 in Archaea, and up to 10 protein subunits in Eukaryotes. ³ In Escherichia coli, from which the M1GS ribozyme is derived, RNase P is composed of a 377-nucleotide M1 RNA and a 14 kDa, 119-amino acid C5 protein. ³ M1 RNA is encoded by rnpB and forms a holoenzyme with a single protein cofactor, C5 in bacteria.^50,51 Structural analysis employing crystallography, cryo-EM, mutational, and phylogenetic studies of the M1 RNA-C5 interaction supports the current understanding of C5’s role in stabilizing and correctly folding M1 RNA into its catalytically active form.^{3,47,48,52–54} In vivo, both M1 RNA and C5 are required for cleavage; however, it has been demonstrated that M1 RNA can cleave pre-tRNA in the absence of C5 at high Mg²⁺ concentrations.^2,55 RNase P in humans consists of the catalytic H1 RNA, along with the 10 human RNase P protein (Rpp). ⁵⁶ In humans, H1 RNA is encoded by RPPH1 and associates with multiple Rpp subunits following transcription. ⁵⁷ H1 RNA is concentrated in the nucleoplasm, while several Rpp subunits localize to the nucleolus and coiled bodies, a more compartmentalized assembly pathway than observed in bacteria.^56,58,59 Rpp functions similarly to C5; however, the individual roles of these subunits remain a current area of investigation. It has been demonstrated that M1 RNA can function at a reduced level in human cells by interacting with Rpp.^6,60,61 Through in vitro selection procedures, future studies can investigate the optimization of M1 RNA to improve its functional compatibility with RPP, with the goal of enhancing M1GS activity in human cells.

RNase P substrate recognition and engineering of M1GS ribozymes from RNase P RNA

In contrast to sequence-specific endonucleases, RNase P relies on recognition of conserved RNA structural motifs, such as T-stem/loop, acceptor stem-like elements, and unpaired 5′ leader regions, and 3′ CCA sequence (Figure 1).^3–5,55,62 Recognition based on structure rather than primary sequence enables RNase P-mediated cleavage as long as the RNA substrate adopts analogous structural motifs. Building upon this property, external guide sequences (EGS) were developed as ASOs that bind to target RNAs and remodel them into pre-tRNA-like structures that are identified and cleaved by RNase P (Figure 1). A key advantage in RNase P-mediated cleavage is that RNase P is present across all phases of the cell cycle. ⁶³ EGS can also utilize established delivery strategies developed for ASOs and other short nucleic acid therapeutics, due to EGS’s relatively small size, enabling targeting of RNA substrates in both dividing and non-dividing cells. Moreover, various chemical modification, such as 2′-O-methylation, further improves EGS stability and resistance to degradation.^64,65 While EGS benefits from simple design, efficient delivery, and recruitment of endogenous RNase P, limited investigation of its pharmacokinetic and pharmacodynamic properties raises concerns that high EGS concentrations could saturate RNase P and disrupt normal RNA processing.

Figure 1.

Substrates for RNase P, EGS, and M1GS. (a) RNase P cleaves the precursor transfer RNA (pre-tRNA), which contains structural motifs such as the acceptor stem, D-stem loop, anticodon loop, variable loop, and T-stem loop. (b) External guide sequences (EGS) are antisense oligonucleotides that base-pair with the target mRNA, forming structures similar to RNase P’s natural target. Endogenous RNase P detects the mRNA:EGS complex and cleaves it. (c) M1GS RNA binds to a target mRNA substrate governed by its linked guide sequence (GS), allowing the attached M1 RNA to cleave the 5′ leader off the target substrate.

By covalently linking an EGS to M1 RNA, the catalytic RNA subunit of Escherichia coli RNase P, M1GS ribozymes unite substrate recognition and catalysis within a single RNA molecule (Figure 2). Linking M1 RNA to the GS allows M1GS to function independently of host RNase P. It has been demonstrated that M1GS outperforms EGS in low Mg²⁺ concentration in vivo.^6,66–68 M1GS was also found to cleave its target substrate more efficiently at 10 mM MgCl₂ compared to M1 RNA plus a separate guide sequence in a buffer at 100 mM MgCl₂. ⁶ In the presence of either the bacterial C5 protein or Rpp, M1GS remained catalytically active at 5 mM MgCl₂, demonstrating activity under physiological Mg²⁺ concentrations. ⁶ Although M1 RNA normally functions in complex with the bacterial RNase P protein C5, M1GS has been demonstrated to retain catalytic activity in the presence of Rpp. ⁶ In vitro assays comparing stimulation of M1GS activity using proteins demonstrated that C5 increased cleavage efficiency by a factor of 30 compared to Rpp’s fivefold increase in activity relative to M1GS alone.^6,60,61 Although M1GS activity supported by Rpp remains lower than that observed with C5, this reduced efficiency represents an important opportunity for improvement. Future studies may identify the specific Rpp proteins that bind and stabilize M1GS, enabling in vitro selection strategies to enrich for variants optimized for interaction with mammalian Rpp.

Figure 2.

Interaction between M1GS RNA and its target mRNA substrate.

In vitro selection strategies were developed to enhance catalytic efficiency and substrate binding beyond the natural capabilities of M1 RNA (Figure 3). ⁶⁹ In this approach, a large pool of M1GS variants was generated through mutagenesis and subjected to selection cycles using a biotinylated target RNA substrate, such as the HSV-1 TK mRNA, which can be immobilized on a streptavidin column. Variants capable of proper folding, high affinity, and efficient Mg²⁺ mediated cleavage were collected while inactive or weakly binding M1GS variants were eliminated. Repeated rounds of selection under increasingly stringent conditions (shortened annealing and incubation times) yield M1GS variants with substantially improved catalytic activity and binding affinity relative to the wildtype M1GS construct (Figure 3). In a previous study, ⁶⁹ some of the selected variants exhibited 20-fold increases in catalytic activity and 50-fold increases in binding affinity. When expressed in HSV-1-infected cells, these variants led to a 99% reduction in HSV-1 RNA levels and a 98% decrease in TK protein levels. ⁶⁹ Subsequent studies by Zou et al. ¹¹ examined whether the enhanced activity of the previously selected variant V41 resulted from general improvements in the M1 catalytic domain or was dependent on the original TK guide sequence. V41 variant retained superior cleavage activity compared to the wild-type M1 RNA when retargeted to a different mRNA through replacement of the guide sequence. These results indicated that the improved activity achieved by the in vitro selection strategy was encoded within the M1 catalytic domain itself, rather than being dependent on a specific target substrate. Collectively, these findings confirm that enhanced activity of M1GS ribozymes could be generated through this selection approach and that optimized variants observed not only retain their activity in cellular contexts but can also be retargeted to distinct mRNA substrates through guide sequence replacement. Together, these studies demonstrate not only that RNase P can be redirected to cleave non-pre-tRNA substrates, but that its catalytic performance can be engineered beyond its natural capabilities.

Figure 3.

In vitro selection of M1GS ribozymes.

Application for oral herpesvirus infections

HSV-1

Infected-cell polypeptide 4 (ICP4) is one of five immediate-early factors synthesized during productive infection of HSV-1 and is necessary for transcription of viral early and late genes that drive DNA replication and virion assembly. ⁷⁰ ICP4’s essential role in HSV-1 gene expression makes it an ideal upstream target for intervention, as loss or inhibition of ICP4 exerts broad downstream effects on HSV-1 replication and assembly. ⁷⁰ Notably, M1GS has already been found to inhibit downstream HSV-1 gene products in HSV-1 infected cells.^69,71 In vitro-selected M1GS variants directed against the HSV-1 TK mRNA achieved 99% reduction in TK transcripts and 98% reduction in protein expression in HSV-1 infected cells. ⁶⁹ In a study conducted by Trang et al., ⁷¹ in vitro-selected M1GS were designed to target ICP4 mRNA to evaluate whether M1GS constructs could effectively suppress HSV-1 expression in infected cells and induce downstream suppression of viral early and late genes.

M1GS targeting ICP4 was delivered to infected cells via a retroviral vector, resulting in 93% reduction in ICP4 mRNA levels and 92% reduction in protein expression. ⁷¹ Early gene products, TK, and late gene products, ICP35 and gB, were measured to assess the downstream effects of ICP4 knockdown, revealing 90% decreases in TK and ICP35, and a 91% decrease in gB mRNA levels. ⁷¹ Consistent with widespread inhibition of HSV-1 gene expression, viral growth was also observed with an approximately 4000-fold reduction in M1GS-expressing cells. ⁷¹ M1GS-mediated inhibition of an upstream regulatory transcript, as observed in this study, produced broad downstream suppression of viral gene expression. Targeting upstream transcripts through M1GS or related silencing strategies may provide a complementary approach to replication-dependent antiviral therapies that act downstream at the level of viral replication.

KSHV

A central regulator of the switch from latency to lytic reactivation in KSHV is controlled by the immediate-early transcription factor RTA (also known as ORF50).^29,31 RTA functions as the master lytic switch, initiating transcription of downstream viral lytic genes and modulating host pathways involved in apoptosis, immune signaling, and cell cycle control.^29,31 As a result, RTA represents an ideal upstream target for a therapeutic strategy aimed at suppressing KSHV reactivation and progression of KSHV-mediated diseases. In a study conducted by Liu et al., ⁷ M1GS constructs targeting RTA were designed to evaluate their ability to suppress KSHV lytic gene expression and replication in infected cells.

RTA-targeting M1GS constructs were evaluated in BCBL-1 cells, a KSHV-infected primary effusion lymphoma cell line. ⁷ Expression of M1GS resulted in a significant reduction in RTA expression, with approximately 94% and 92% decreases in RTA transcript and protein levels, respectively. ⁷ Consistent with RTA’s role as an upstream regulator of early and early/late viral gene expression, M1GS-mediated inhibition of RTA led to approximately 80%–85% reductions in early transcripts such as PAN RNA and in early/late genes such as ORF75, and multiple lytic proteins spanning early (ORF59), early/late (ORF26), and late (K8.1) gene products. ⁷ This widespread repression of lytic gene expression translated into a 250-fold reduction in viral production following TPA-mediated lytic reactivation. ⁷ Importantly, intracellular levels of KSHV latent genome DNA remained unchanged before induction, indicating that the observed reduction of viral output resulted from inhibition of lytic reactivation rather than the depletion of the latent KSHV reservoir. No cytotoxic effects were observed in M1GS-expressing cells for up to 30 days; however, further evaluation in primary KSHV-infected B cells will be necessary to assess translational relevance. ⁷ Together, these findings confirm that M1GS-mediated targeting of RTA effectively represses lytic reactivation. Further studies examining M1GS targeting of multiple viral transcripts may determine whether coordinated inhibition of both RTA and LANA can address both lytic reactivation and latent maintenance.

Advantages and disadvantages

M1GS ribozyme provides a highly specific and adaptable strategy for targeting viral transcripts, particularly upstream regulatory genes that govern downstream gene expression. M1GS activity relies on base pairing between a customizable guide sequence and the target RNA substrate to generate a pre-tRNA-like structure, that can be recognized and cleaved by the linked M1 RNA.^12,62 The structural requirements of M1GS confer high specificity and low off-target cleavage, distinguishing it from antisense approaches that tolerate mismatches or partial complementarity.^72–74 In vitro selection strategies have been employed to optimize M1GS catalytic efficiency and substrate binding further.^69,75 Importantly, M1GS ribozymes are highly adaptable, as guide sequences can be readily redesigned to target various viral transcripts, a feature demonstrated by their effectiveness against HSV-1 ICP4 or KSHV RTA, resulting in broad downstream suppression of early, early/late, and late genes.^7,71 Suppression of these upstream regulatory genes resulted in pronounced inhibition of viral replication and production. Unlike nucleoside analogs or disease-tailored therapies that target downstream manifestations of viral infection, M1GS-based targeting introduces a distinct approach that may broaden the therapeutic landscape of these oral viral infections.

Despite these advantages, several limitations currently constrain the clinical translation of M1GS. Much of M1GS research remains experimental, with the majority of supporting data derived from in vitro systems or transformed cell lines rather than primary cells or in vivo models. Although no cytotoxic effects were observed in KSHV-infected BCBl-1 cells following treatment, these findings have yet to be validated in primary B cells. ⁷ A comprehensive understanding of M1GS pharmacokinetics and pharmacodynamics is required to evaluate its clinical translation. Such studies could provide insight into the durability of transcript suppression, dosage, and cytotoxic effects, considerations that have informed therapeutic development in other nucleic acid-based platforms. In addition, M1GS activity depends on secondary and tertiary structural mimicry, limiting the applicability of common chemical modifications used to enhance RNA stability in other nucleic acid therapeutics.^72,76 While viral vectors have been effective for delivering M1GS to HSV-1 or KSHV-infected cells in experimental settings, their clinical applicability may be limited.^7,71 Lipid nanoparticle (LNP) based delivery represents a potential alternative; however, the comparatively large size of the M1GS ribozyme may pose challenges for efficient delivery. In addition, common LNP formulations that exhibit a strong tendency to accumulate in the liver may limit the applicability of M1GS LNP delivery for treating infections localized to non-hepatic tissues, including the oral cavity. ⁷⁷ Overcoming these delivery and translation challenges will be critical to advancing M1GS-based therapies toward clinical relevance.

Future direction and challenges

First, future studies should prioritize improving M1GS activity in human cells. A key limitation is the reduced efficiency of M1 RNA in M1GS ribozymes when supported by RPP, in comparison to the C5 protein.^6,60,61 Identifying which RPP proteins associate with M1GS in vivo and defining how these interactions improve substrate recognition and cleavage will be essential in future M1GS research. In vitro selection strategies provide a path forward in isolating M1GS variants optimized for compatibility with human RPP.^69,75 Such optimization could substantially enhance M1GS activity in physiological conditions and improve the clinical potential of M1GS-based therapies.

Second, future studies should prioritize identifying viral transcripts that more effectively link oral herpesvirus infection, latency, and disease manifestations. Current therapeutic approaches often target isolated aspects of oral herpesviruses, such as replication during lytic infection or disease-specific outcomes. M1GS provides a unique opportunity to connect these phases by targeting RNA transcripts that coordinate reactivation and downstream gene expression, such as in oral reservoirs where recurrent HSV-1 shedding and KSHV transmission occur. In cases where no single transcript fully bridges these phases, evaluating coordinated M1GS targeting multiple viral transcripts may inform whether synergistic suppression is possible.

Third, despite promising results in transformed cell lines, M1GS remains largely experimental, underscoring the need for M1GS research in primary cells and in vivo models. Pharmacokinetic and pharmacodynamic studies will be essential in evaluating the durability of transcript suppression, dose-response relationships, and potential cytotoxicity. These studies may inform whether M1GS therapies are feasible and safe in targeting oral herpesvirus infections. Importantly, these studies may reveal alternative therapeutic applications for M1GS that are better suited to address reactivation and long-term persistence that are characteristic of herpesvirus infections.

Finally, a major limitation in the clinical translation of M1GS is delivery. The relatively large size of M1GS RNA has necessitated that most experimental studies rely on plasmid-based expression or viral vectors to achieve M1GS activity in infected cells.^7,66,71 While these approaches have been successful in vitro, they present challenges for clinical application, particularly for targeting sites such as the oral cavity, where HSV-1 and KSHV infection occur, as well as other virus-infected tissues involved in disease manifestation. In addition, M1GS’s requirement for proper folding may limit the application of chemical modifications used in other RNA-based therapeutics. However, previous work with external guide sequences (EGS) has demonstrated that substituting nuclease-resistant 2′-O-methyl oligoribonucleotides into non-critical residues yields EGS that remain stable in 50% human serum for 24 h.^64,65 These same chemical modifications may apply to M1GS due to the shared characteristics with EGS. Chemical modifications widely used in other RNA therapeutics could also be investigated for M1GS constructs to improve delivery and stability. Among nonviral approaches, LNP delivery currently represents the most mature platform for RNA therapeutics. However, due to LNP’s tendency to accumulate in the liver, M1GS may be limited in their utility for infection localized to nonhepatic tissues such as the oral cavity or other infected sites. At present, the translational potential of M1GS remains incompletely defined due to its early-stage development. Future studies will be needed to focus on how improvements in delivery, stability, and activity under physiological conditions can support therapeutic development. As the broader field of RNA therapeutics continues to advance, these same developments may also be applied to M1GS to further support its future clinical translation. Looking forward, delivery strategies for M1GS could leverage virus-specific features rather than tissue-specific markers for HSV-1 and KSHV, given that the oral cavity is a key site of infection and transmission, and that disease manifestations arise in different tissues, such as endothelial and B cells.^21,23 The shared presence of viral genomes across these distinct tissues suggests that virus-directed delivery strategies could help bridge the currently isolated approaches to treating oral herpesvirus infection, latency, and disease.

Conclusion

In this review, we have discussed the history, design, mechanism, and therapeutic potential of M1GS as a gene targeting strategy for oral herpesvirus infections caused by HSV-1 and KSHV. We have summarized engineering principles underlying M1GS activation, including in vitro selection strategies that enhance catalytic efficiency and substrate recognition beyond native RNase P. We have discussed studies demonstrating that M1GS targeting of upstream regulatory transcripts such as HSV-1 ICP4 and KSHV RTA mRNAs results in broad downstream suppression of viral gene expression and replication. We further discussed how these findings distinguish M1GS from replication-dependent antivirals and disease-specific treatments. Finally, we examined key advantages, limitations, and future challenges of M1GS development, including optimization for human RNase P proteins, identification of viral targets relevant to oral infection and disease, the need for in vivo studies, and delivery constraints. Ultimately, these studies position M1GS as a potential new therapeutic approach that could help bridge currently isolated antiviral treatment strategies and inform new approaches to managing chronic and recurrent oral herpesvirus infections.