Telomerase inhibition for treating or inhibiting herpesvirus infections

Abstract

The present disclosure provides a method of inhibiting telomerase activity to reduce human herpesvirus (e.g., HMCV, EBV, HSV-1, and / or HHV-6 / 7 / 8) replication. The method comprises treating cells with a telomerase inhibitor prior to and after HCMV infection. The telomerase inhibitor may be a pharmaceutical inhibitor such as BIBR1532 or MST312, or an siRNA construct targeting the hTERT catalytic subunit of telomerase. Treatment with the telomerase inhibitor results in reduced viral titer, decreased viral gene expression, and diminished viral protein levels across different temporal phases of herpesvirus infection. The method provides a novel approach for inhibiting herpesvirus replication through targeting of host cell telomerase activity.

Description

TELOMERASE INHIBITION FOR TREATING OR INHIBITINGHERPESVIRUS INFECTIONSCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit ofU.S. Provisional Application No. 63 / 642,215, filed on May 3, 2024, which is incorporated herein by reference in its entirely.SEQUENCE LISTING

[0002] The instant application contains a Sequence Listing which has been submitted electronically in ST.26 format and is hereby incorporated by reference in its entirety. The ST.26 compliant Sequence Listing is submitted as a file named "PRIN-94176-SL.xml" created on May 2, 2025, and is 11,416 bytes in size. The Sequence Listing contains SEQ ID NOs: 1-12.FIELD OF INVENTION

[0003] The present disclosure relates to antiviral treatments for human herpesvirus infections, such as cytomegalovirus (HCMV), and more particularly to methods and compositions for inhibiting herpesvirus replication through telomerase inhibition or reduction ofhTERT expression.BACKGROUND

[0004] Current antiviral treatments for human herpesviruses, and human cytomegalovirus (HCMV) in particular, have limitations in efficacy and are associated with side effects. Existing therapies primarily target the viral DNA polymerase to inhibit viral replication. However, these drugs do not eliminate latent virus reservoirs and prolonged use can lead to the emergence of drug-resistant strains. Additionally, some antivirals have toxicity profiles that restrict their use in certain patient populations.

[0005] The persistence of latent herpesvirus infection and the potential for reactivation pose ongoing challenges for long-term management. Current treatments are unable to eradicate the virus from the host, leaving patients at risk for recurrent infections. Furthermore, there are limited options for treating drug-resistant strains that emerge during extended antiviral therapy.

[0006] It has been appreciated that a method is needed that overcomes one or more of these problems.SUMMARY

[0007] This Summary is provided to introduce a selection of concepts in a simplified form that are further described below in the Detailed Description.

[0008] In a first aspect, a method for inhibiting human herpesvirus replication in cells may be provided. In particular, this method may be utilized with any herpesvirus that upregulate human telomerase, such as human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), Herpes simplex virus type 1 (HSV-1), human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), or human herpesvirus 6 (HHV-8). The method may include contacting the cells with a telomerase inhibitor.

[0009] This method may effectively reduce herpesvirus replication by targeting telomerase activity, which plays a crucial role in the viral life cycle. Inhibiting telomerase may disrupt key processes required for successful herpesvirus infection and propagation. The telomerase inhibitor may be a pharmaceutical inhibitor. Using a pharmaceutical inhibitor may provide a targeted approach to telomerase inhibition, potentially offering greater specificity and efficacy in disrupting HCMV replication. The pharmaceutical inhibitor may be. e.g, BIBR1532 or MST312. BIBR1532 and MST312 are well-characterized telomerase inhibitors that may offer potent anti-herpesvirus activity through their specific mechanisms of action on telomerase function.

[0010] The telomerase inhibitor may be an siRNA construct targeting a human telomerase reverse transcriptase (hTERT) catalytic subunit of telomerase. Utilizing an siRNA construct may allow for highly specific inhibition of hTERT expression, potentially leading to more targeted disruption of telomerase activity and herpesvirus replication.

[0011] Contacting the cells with the telomerase inhibitor may include: contacting the cells with the telomerase inhibitor at a first time point prior to herpesvirus infection; contacting the cells with the telomerase inhibitor at a second time point after herpesvirus infection; and contacting the cells with the telomerase inhibitor at a third time point after herpesvirus infection.

[0012] This multi-timepoint administration strategy may ensure sustained telomerase inhibition throughout different stages of the herpesvirus infection process, potentially maximizing the anti-viral effect. The first time point may be between 1 hour and 4 hours prior to HCMV infection; the second time point may be between 1 hour and 4 hours after herpesvirus infection; and the third time point may be between 36 hours and 60 hours after herpesvirus infection. These specific time points may be optimized to target key stages in the herpesvirus replication cycle, potentially enhancing the overall inhibitory effect on viral propagation.

[0013] The method may result in at least one of: reduced viral titer; decreased viral gene expression: or diminished viral protein levels. These measurable outcomes may provide clear indicators of the method's effectiveness in inhibiting herpesvirus replication, demonstrating its potential as a therapeutic approach.

[0014] In a second aspect, a pharmaceutical composition for use in inhibiting human herpesvirus replication in cells according to the method of the first aspect may be provided. The pharmaceutical composition may include a telomerase inhibitor and a pharmaceutically acceptable carrier. This composition may offer a practical means of delivering the telomerase inhibitor for therapeutic use, potentially providing a new treatment option for herpesvirus infections. The telomerase inhibitor may be BIBR1532 or MST312. Including these specific inhibitors in the pharmaceutical composition may leverage their proven efficacy in telomerase inhibition for targeted anti-herpesvirus activity. The pharmaceutical composition may be formulated for administration to the cells at multiple time points relative to herpesvirus infection. This formulation strategy may allow for sustained telomerase inhibition throughout the herpesvirus infection process, potentially maximizing therapeutic efficacy.

[0015] The pharmaceutical composition may further include an additional antiviral agent. Combining a telomerase inhibitor with other antiviral agents may provide synergistic effects, potentially enhancing overall efficacy in combating herpesvirus infection.

[0016] In a third aspect, a delivery system for use in inhibiting human herpesvirus replication in cells according to the method of the first aspect may be provided. The delivery system may include a small interfering ribonucleic acid (siRNA) construct targeting a human telomerase reverse transcriptase (hTERT) catalytic subunit of telomerase. This delivery system may offer a highly specific approach to telomerase inhibition through targeted gene silencing, potentially providing effective and precise control over herpesvirus replication. The siRNA construct may be designed to reduce expression of the hTERT catalytic subunit. Specifically targeting hTERT expression may allow for precise modulation of telomerase activity, potentially leading to more effective inhibition of herpesvirus replication.

[0017] The delivery system may further include a transfection agent for delivering the siRNA construct to the cells. Including a transfection agent may enhance the efficiency of siRNA delivery to target cells, potentially improving the overall effectiveness of the telomerase inhibition strategy.

[0018] The delivery system may be configured to deliver the siRNA construct to the cells at multiple time points relative to herpesvirus infection. This multi-timepoint deliveryapproach may ensure sustained suppression of hTERT expression throughout different stages of herpesvirus infection, potentially maximizing the anti-viral effect.BRIEF DESCRIPTION OF FIGURES

[0019] Embodiments of the invention will be described, by way of example, with reference to the following drawings, in which:

[0020] FIG. I illustrates a flowchart for a method of inhibiting herpesvirus replication using telomerase inhibitor, according to aspects of the present disclosure.

[0021] FIG. 2A depicts a flowchart for administering a telomerase inhibitor in relation to herpesvirus infection, according to an embodiment.

[0022] FIG. 2B depicts a flowchart for administering a telomerase inhibitor in relation to herpesvirus infection, according to another embodiment.

[0023] FIG. 3A shows, for MRC5 cells infected with TB40E at MOI 3 and harvested at 12, 24, 72, and 96 HPI, a Telomerase repeat amplification protocol (TRAP) assay of protein extracts prepared from mock infected or HCMV TB40E infected MRC5 cells at 12, 24, 72, and 96 hpi. No Taq Control = Negative Control. HI = Heat Inactivated negative control, CHAPS = negative control, HEK293 = positive control, TSR8 = positive control.

[0024] FIG. 3B is a graph of TRAP-determined telomerase activity.

[0025] FIG. 3C shows, for MRC5 cells infected with TB40E at MOI 3 and harvested at 12. 24, 72, and 96 HPI, quantification of hTERT expression.

[0026] FIG. 3D shows, for MRC5 cells infected with HCMV strain TB40E at Multiplicity of Infection (MOI) 1 or 5 and harvested at 96 hpi, a TRAP assay of protein extracts prepared from mock-infected or HCMV TB40E infected MRC5 cells, 96 hpi. No Taq Control = Negative Control, HI= Heat Inactivated negative control, CHAPS = negative control, HEK293 = positive control, TSR8 = positive control.

[0027] FIG. 3E is a graph of TRAP-determined telomerase activity’.

[0028] FIG. 3F is a graph of of hTERT expression.

[0029] FIGS. 4A-4B are graphs showing, for infection of MRC5 cells with TB40E, or UV- irradiated TB40E, MOI 3, harvested 96 hpi. quantification of telomerase activity (4 A) and quantification of hTERT expression (4B), where P<0.0001. Calculations relative to Mock.

[0030] FIGS. 4C-4D are graphs showing, for treatment of TB40E-infected MRC5 cells, MOI 3, with 50 pM ganciclovir, quantification of telomerase activity, P=0.0189 (4C), and quantification of hTERT expression, P=0.0107 (4D), with calculations relative to Mock.

[0031]

[0032] FIG. 5 A shows, for post-translational telomerase inhibition with MST-312 rechuces viral titer of lab and clinical HCMN strains, where MRC5 or HFF cells were infected with HCMV strain TB40E or AD169, respectively, at MOI 3, treated with 0.5pM or 0.25 pM MST-312, respectively, and harvested at 96 hpi, a TRAP assay of protein extracts prepared from mock-infected, HCMV -infected, or HCMV-infected and MST-312 treated cells, 96 hpi. HI= Heat Inactivated negative control, CHAPS = negative control, HEK293 = positive control, TSR8 = positive control.

[0033] FIG. 5B is a graph of TRAP-determined telomerase activity from FIG. 5 A. Single replicate shown.

[0034] FIG. 5C is a graph of TB40E viral titer. P<0.0001.

[0035] FIG. 5D is a graph ofhTERT expression. P<0.0001.

[0036] FIG. 5E is a graph of hTERC expression. PO.OOOl.

[0037] FIG. 6A is a graph of a viral titer by fluorescent focus assay with Operetta imager following treatment of TB40-infected MRC5 cells with MST312. Differences between most conditions are significant (P<0.05).

[0038] FIG. 6B is a graph of hTERT expression (P<0.0001).

[0039] FIG. 6C is a graph of hTERC expression. Differences between most conditions are significant (P<0.05).

[0040] FIGS. 7A-7D show that hTERT knockdown severely reduces HCMV viral titer. Control = HFF cells. siRNA = HFF cells treated with siRNA targeting hTERT. Nonsense = HFF cells treated with non-targeting siRNA. Control + ADI 69 = AD1 9-infected HFF cells. hTERT + AD 169 = AD169-infected IIFF cells treated ith siRNA targeting hTERT. Nonsense + AD 169 = AD169-infected HFF cells treated with non-tar geting siRNA. All infected arms received AD169 MOI 3. The figures show quantification of viral titer (PO.OOOl) (7A), quantification of hTERT mRNA expression (7B), Non-radioactive TRAP assay of protein extracts; HI= Heat Inactivated negative control, CHAPS = negative control, HEK293 = positive control, TSR8 = positive control (7C), and quantification of relative telomerase activity. (P=0.0243) (7D).

[0041] FIGS. 8A-8G show, for MRC5 cells infected with TB40E at MOI 3, where cells were treated with 0.5pM MST-312, lysate harvested at 96 hpi, Viral gene expression following MST-312 treatment (8A), Western Blot of late protein pUL99 / pp28 with actin control and immediate early gene IE1 with actin control (8B), Western blot of Immediate Early protein IE2 and Early protein pUL26 (8C), Relative Quantification of Immediate Early protein IEI (8D),Relative Quantification of Immediate Early Protein IE2 (8E), Relative Quantification of Early protein pUL26 (8F), Relative Quantification of Late Protein pUL99 / pp28 (8G).

[0042] FIGS 8H-8I show, where all infected arms received AD 169 MOI 3, Relative Quantification of Immediate Early protein IE1 (8H), and Relative Quantification of Late Protein pUL99 / pp28 (81).

[0043] FIG. 9 is a plot showing changes in viral protein abundance following inhibition with MST-312. Following infection of MRC-5 cells (MOI 3) with TB40E clinical strainand treatment with pharmaceutical telomerase inhibitor MST312 (0.5 pM), lysates were analyzed by parallel reaction monitoring (PRM) to determine relative protein abundances of a pre-set protein panel, where the scale indicates fold changes of log2(TB40E+MST-312 / TB40E)

[0044] FIGS. 10A-10F are graphs showing knockdown of newly synthesized viral proteins with UV-irradiation of TB40E, four replicates, of Immediate Early (IE) genes (10A-10B), Early (E) genes (10C-10D), and Late (L) genes (10E-10F).

[0045] FIGS. 11A-11D are graphs showing non-cytotoxic dose determination of pharmaceutical telomerase inhibitors, where each graph represents average of three replicates, maximum non-cytotoxic dose cut-off at 10% cytotoxicity. Maximum non-cytotoxic dose of MST-312 in HFF cells was 0.25pM (11 A), MST-312 in MRC-5 cells was 0.5pM (1 IB), BIBR- 1532 in HFF cells was 15pM (11C), BIBR-1532 in MRC-5 cells was 20pM (1 ID).

[0046] FIGS. 12A shows post-translational telomerase inhibition with BIBR1532 reduces viral titer of lab and clinical HCMV strains, where MRC5 or HFF cells were infected with HCMV strain TB40E or AD 169, respectively, at MOI 3 , treated with 15pM or 20pMBIBRl 532, respectively, and harvested at 96 hpi, and specifically showing a TRAP assay of protein extracts prepared from mock-infected, HCMV -infected, or HCMV- infected and BIBRI 532-treated cells, 96 hpi. HI= Heat Inactivated negative control, CHAPS = negative control, HEK293 = positive control, TSR8 = positive control.

[0047] FIG. 12B-12H are graphs showing quantification of TRAP -determined telomerase activity (12B), quantification of viral titer (12C), quantification of hTERT expression (12D), quantification of hTERC expression (12E). quantification of viral titer by fluorescent focus assay with Operetta imager following dose titration of TB40-infected MRC5 cells with BIBRI 532. Values are averages of four experiments (12F), quantification of hTERT expression (12G), and quantification of hTERC expression (12H).

[0048] FIGS. 13A-13J show protein abundances of individual HCMV proteins following pharmaceutical telomerase inhibition, where, following infection of MRC-5 cells (MOI 3) with TB40E clinical strain and treatment with pharmaceutical telomerase inhibitor MST312(0.5pM), lysates were analyzed by Parallel Reaction Monitoring (PRM) to determine relative protein abundances of a pre-set protein panel, the scale indicates fold change of log2 (TB40E + MST-312 / TB40E) for abundance of pUL71 (13A) abundance of pp65 (13B), abundance of CEP2 / pUL94 (13C), Abundance of pUL96 (13D), abundance of pUL69 (13E), abundance of TERl / pUL89 (13F), abundance of TER2 / pUL56 (13G), abundance of gB (13H), abundance of gM (131), and abundance of gH (13J).

[0049] Common reference numerals are used throughout the figures to indicate similar features.DETAILED DESCRIPTION

[0050] The present disclosure relates to methods, compositions, and delivery systems for inhibiting human herpesvirus replication in cells. These methods, compositions, and delivery systems may be utilized with any herpesvirus that upregulate human telomerase, such as human cytomegalovirus (HCMV), Epstein-Barr virus (EBV), Herpes simplex virus type 1 (HSV-1), human herpesvirus 6 (HHV-6), human herpesvirus 7 (HHV-7), or human herpesvirus 6 (HHV- 8).

[0051] In particular, the disclosure provides approaches for contacting cells with telomerase inhibitors to reduce herpesvirus replication. The telomerase inhibitors may include pharmaceutical inhibitors or siRNA constructs targeting the human telomerase reverse transcriptase (hTERT) catalytic subunit of telomerase.

[0052] Pharmaceutical compositions comprising telomerase inhibitors and pharmaceutically acceptable carriers are described for inhibiting herpesvirus replication. Additionally, delivery systems containing siRNA constructs targeting the hTERT catalytic subunit are provided for reducing telomerase activity and herpesvirus replication in cells. The methods, compositions, and delivery systems disclosed herein offer novel approaches for interfering with herpesvirus replication through modulation of telomerase activity in host cells.

[0053] The method of inhibiting human herpesvirus replication using telomerase inhibitor is illustrated in FIG. 1. The process 100 may begin with providing 102 cells susceptible to the herpesvirus infection. In some examples, the cells may be from a human cell line, such as MRC-5, HFF, or HEK293 cells.

[0054] The next step involves preparing 104 a telomerase inhibitor. Initially, a decision point 106 is reached where the type of inhibitor is determined. If the inhibitor is a pharmaceutical, the method proceeds to preparing 108 a solution comprising the telomerase inhibitor compound. For example, a BIBR1532 or MST312 solution may be prepared, usingknown techniques. If the inhibitor is an siRNA, the method involves preparing 110 an siRNA construct targeting the human telomerase reverse transcriptase (hTERT) catalytic subunit.

[0055] Following the preparation of the chosen inhibitor, the method converges to a step where cells are contacted 112 with the telomerase inhibitor pre-infection. This represents contacting the cells with the telomerase inhibitor at a first time point prior to herpesvirus infection.

[0056] The subsequent step involves infecting 114 the cells with herpesvirus (or allowing the infection to occur). In some examples, the herpesvirus strain may be an HMCV strain, such as, e.g., TB40E or AD169.

[0057] After infection, there is a step of contacting 116 cells with the telomerase inhibitor post-infection. This step may involve contacting the cells with the telomerase inhibitor at a second time point after herpesvirus infection. In some examples, the method may include contacting the cells with the telomerase inhibitor at a third time point after herpesvirus infection.

[0058] The final step in the process is assessing 118 herpesvirus replication inhibition. This assessment may involve multiple approaches. In some examples, viral titer may be assessed using a fluorescent focus assay. The method may result in reduced viral titer. In other examples, viral gene expression may be assessed using qPCR. The method may result in decreased viral gene expression. Additionally, the method may result in diminished viral protein levels.

[0059] The flowchart in FIG. 1 depicts the sequence of steps and decision points in the method. The process includes both pre- and post-infection treatment with the inhibitor, followed by an assessment of the inhibition effect on herpesvirus replication.

[0060] FIG. 2A illustrates a flowchart for a method of administering a telomerase inhibitor in relation to human herpesvirus infection. The method begins with step 200, where cells and telomerase inhibitor are prepared. The telomerase inhibitor may be a pharmaceutical inhibitor such as BIBRI 532 or MST312, or an siRNA construct targeting the human telomerase reverse transcriptase (hTERT) catalytic subunit.

[0061] In step 202, the telomerase inhibitor is administered to the cells at a first time point prior to herpesvirus infection. This first administration occurs between 1 hour and 4 hours before infection. In some examples, the telomerase inhibitor is administered 2 hours prior to infection.

[0062] Step 204 involves infecting the cells with herpesvirus. For example, the herpesvirus may be a HCMV strain such as TB40E or AD 169.

[0063] Following infection, step 206 involves administering the telomerase inhibitor again at a second time point after herpesvirus infection. This second administration occurs between1 hour and 4 hours post-infection. In some examples, the telomerase inhibitor is administered2 hours after infection.

[0064] The process continues with step 208, where there is a waiting period of 36-60 hours. This waiting period allows for viral replication to progress.

[0065] After the waiting period, step 210 involves administering the telomerase inhibitor for a third time. This third administration occurs between 36 hours and 60 hours after infection. In some examples, the telomerase inhibitor is administered 48 hours after infection.

[0066] The final step 212 is to assess herpesvirus replication inhibition. This assessment may involve collecting and analyzing samples at specific timepoints, such as 24, 48, 72, and 96 hours post-infection. The assessment may include measuring viral titer, evaluating viral gene expression, or examining viral protein levels.

[0067] In some examples, when using an siRNA construct as the telomerase inhibitor, the delivery system may be configured to deliver the siRNA construct to the cells at multiple time points relative to herpesvirus infection. For instance, the siRNA construct may be delivered 24 hours prior to infection and again 24 hours after infection.

[0068] The method described in FIG. 2A provides a structured approach for studying the impact of telomerase inhibitor on herpesvirus replication. The multiple administrations of the inhibitor at specific time points before and after herpesvirus infection allow for a comprehensive evaluation of the inhibitor's effects throughout the viral replication cycle.

[0069] FIG. 2B illustrates a flowchart for a modified method of administering a telomerase inhibitor in relation to human herpesvirus infection. The method 250 begins with step 252, where telomerase inhibitor are prepared for a subject. The subject may already have been exposed to a herpesvirus. The telomerase inhibitor may be a pharmaceutical inhibitor such as BIBRI 532 or MST312, or an siRNA construct targeting the human telomerase reverse transcriptase (hTERT) catalytic subunit.

[0070] The method may then include administering 254 the telomerase inhibitor at a first time point. In some embodiments, this may be time up to, e.g., 1 day, 2 days, 5 days. 10 days, 30 days, 90 days, 180 days, or 365 days post-infection.

[0071] The method may then include waiting 256 some period of time. This waiting period may be, e.g., 12-48 hours. The method may then include administering 258 the telomerase inhibitor again. This cycle of waiting and administering additional telomerase inhibitors may be repeated multiple times.

[0072] The method may also include periodically (or at least, at the end of the proposed treatment), assessing 260 replication inhibition.

[0073] The pharmaceutical composition for inhibiting herpesvirus replication may further comprise an additional antiviral agent. The additional antiviral agent may be selected from nucleoside analogues, protease inhibitors, or other compounds known to interfere with viral replication. In some examples, the additional antiviral agent may be ganciclovir, valganciclovir. cidofovir, foscamet. or letermovir. The combination of a telomerase inhibitor with an additional antiviral agent may provide enhanced inhibition of herpesvirus replication compared to either agent alone.

[0074] The siRNA construct targeting the human telomerase reverse transcriptase (hTERT) catalytic subunit may be designed to reduce expression of hTERT. The siRNA construct may be complementary to a specific region of the hTERT mRNA sequence, leading to degradation of the mRNA and subsequent reduction in hTERT protein levels. In some examples, multiple siRNA constructs targeting different regions of the hTERT mRNA may be used in combination to enhance the knockdown effect.

[0075] The delivery system for the siRNA construct may further comprise a transfection agent for delivering the siRNA construct to the cells. The transfection agent may be a lipid- based agent, a polymer-based agent, or a nanoparticle-based agent. In some examples, the transfection agent may be a cationic liposome, a cationic polymer, or a cell-penetrating peptide. The transfection agent may facilitate the uptake of the siRNA construct by the target cells, improving the efficiency of hTERT knockdown.

[0076] To study the effect on telomerase activity, UV -inactivated herpesvirus virions may be used. UV inactivation renders the virus incapable of replication while maintaining the structural integrity of the viral particles. This approach allows for the examination of the effects of viral attachment and entry on telomerase activity, independent of viral gene expression and replication.

[0077] Ganciclovir may be used to study the effect of inhibiting late gene production on telomerase activity. Ganciclovir is a nucleoside analogue that interferes with viral DNA synthesis, primarily affecting the production of late viral genes. By comparing telomerase activity’ in herpesvirus-infected cells treated with ganciclovir to untreated infected cells, the contribution of late viral gene products to telomerase activation can be assessed.

[0078] Telomerase activity may be assessed using a TRAPeze Telomerase Detection Kit. This assay involves the amplification of telomere repeat sequences added by telomerase,followed by detection and quantification of the amplification products. The TRAPeze assay provides a sensitive and specific method for measuring telomerase activity in cell lysates.

[0079] Western blot analysis may be used to assess viral protein levels. This technique involves separating proteins by gel electrophoresis, transferring them to a membrane, and detecting specific viral proteins using antibodies. Western blot analysis allows for the quantification of individual viral proteins, providing insight into the effects of telomerase inhibition on different stages of the viral life cycle.

[0080] Parallel reaction monitoring (PRM) may be used to quantify herpesvirus protein abundances. PRM is a targeted mass spectrometry technique that allows for the precise quantification of specific proteins in complex biological samples. This method may be used to simultaneously measure the levels of multiple herpesvirus proteins, providing a comprehensive view of how telomerase inhibition affects the viral proteome throughout the infection cycle.

[0081] Example.

[0082] Treatment options remain limited for human herpesvirus such as cytomegalovirus (HCMV), a doublestranded DNA herpesvirus with acute and chronic pathogenic as well as potential oncomodulatory effects. While several anti-viral drugs attenuate active infection, efficacy is limited, viral resistance is significant, and there is no known existing method to eradicate the virus from latent infection of mononuclear cells. This means that once infected, humans are infected for life, and at risk of recrudescence as their immune competence fluctuates. HCMV seroprevalence and morbidity in immunocompromised patients and neonates infected in utero remains high in both developed and developing nations. Therefore, this virus remains a critical target for permanent effective antiviral therapy. The reverse transcriptase, telomerase, is a key component of the eukaryotic system.

[0083] HCMV infection increases telomerase activity and hTERT expression in a dose- and time-dependent manner.

[0084] To confirm the effect of HCMV infection on telomerase activity and provide new insight into hTERT expression following CMV infection, MRC5 cells were infected with clinically relevant HCMV strain TB40E. hTERT expression was measured with qPCR and telomerase activity was assessed with the TRAP assay. hTERT expression and telomerase activity increased throughout the HCMV replication cycle (see FIGS. 3A-3C), and these increases were dose-dependent (see FIGS. 3D-3F), consistent with the conclusion HCMV- mediated telomerase activity is at least in part mediated through hTERT expression.

[0085] The immediate early (IE) stage of the HCMV viral life cycle is important for HCMV-mediated telomerase activity.

[0086] To ascertain which viral life stages are required for HCMV -mediated telomerase activity, infection conditions were altered to focus on specific stages. Attempted infection of MRC5 cells with irradiated TB40E abrogated telomerase activity and hTERT expression (see FIGS. 4A-4B, 10A), suggesting newly expressed viral transcripts are important for telomerase activity, rather than non-specific interaction between viral structures and the host cell or effect of tegument proteins. Post-infection treatment with ganciclovir, a nucleoside analogue that affects late gene production and is used to attenuate active HCMV infection clinically, dramatically reduced viral titer, but did not affect telomerase activity or hTERT expression (see FIGS. 4C-4D, 10B), suggesting that genes expressed during the early stages of the life cycle are essential for moderating telomerase activity. Adjusting the time of first dose of MST-312 pharmaceutical telomerase inhibitor revealed that dosing started within the IE stage (0 and 6 HPI) led to significant reduction in viral titer (see FIG. 4C), suggesting a temporal importance of the IE stage in HCMV -mediated telomerase activity.

[0087] Post-translational telomerase inhibition reduces HCMV viral titer in a dosedependentfashion.

[0088] To assess the effect of telomerase inhibition on viral replication, TB40E-infected MRC5 cells, and AD169-infected HFF cells, were treated with respective non-cytotoxic doses (see FIGS. 11A-11D) of pharmaceutical inhibitor MST-312, a synthetic analogue of epigallocatechin gallate (EGCG) that competitively inhibits telomerase (Haberichter et al. 2015). MST-312 inhibition resulted in significant and dose-dependent reduction of viral titer as measured by fluorescent focus assay (FFA) (see FIGS. 5C, 6A) with significant reduction of telomerase activity (see FIGS. 5A-5B). hTERT expression was not affected by MST-312 inhibition (see FIGS. 5D, 6B), though, unexpectedly, hTERC expression was affected in a dosedependent fashion (see FIGS. 5E, 6C) indicating a potential mechanism of post- translational hTERC regulation not previously characterized. To establish that the viral inhibition by MST-312 was specific to telomerase inhibition, rather than an off-target effect of the compound, a second pharmaceutical telomerase inhibitor, BIBR-1532, was employed. BIBR-1532 non-competitively inhibits telomerase by interrupting enzyme processivity (Bryan et al. 2015). In both TB40E-infected MRC5 cells and AD169infected HFF cells, results were like the effect of MST-312 (see FIGS. 12A-12H). The observation that a second pharmaceutical telomerase inhibitor with different mechanism of action also reduces HCMV viral titer makes it unlikely that it is an off-target effect of the drugs.

[0089] siRNA knockdown against hTERT severely reduces viral titer.

[0090] To further demonstrate that the observed reduction in viral titer with pharmaceutical inhibition was due specifically to telomerase inhibition, and not to other potential effects of the pharmaceutical telomerase inhibitors, an siRNA construct targeting hTERT was optimized and applied 24 hours prior to, and again 24 hours following, AD 169 infection of HFF cells. At 48-72 HPI, viral titer was significantly reduced (see FIG. 7A), and both hTERT expression and telomerase activity were confirmed to be significantly diminished (see FIGS. 7B-7C). These findings suggest that abrogation of telomerase activity per se is likely to be responsible for the observed reduction in viral titer and supports the conclusion that hTERT activity is important for HCMV -mediated telomerase activity.

[0091] Telomerase Inhibition reduces HCMV gene expression and protein levels.

[0092] To characterize the effect of both post-translational and siRNA-mediated telomerase inhibition on HCMV gene expression, qPCR and western blot were used to assess viral mRNA and protein expression.

[0093] Telomerase inhibition significantly reduced expression from viral genes from each phase of the HCMV life cycle, (see FIG. 8A). Consistent with the effect on mRNA expression, western blot analysis reveal edsignifi cant reduction in the levels of representative viral proteins from each temporal class of the HCMV life cycle following treatment of infected cells with either pharmaceutical telomerase inhibition or siRNA knockdow n against hTERT (Figures 6B- K ).10094] Post-translational telomerase inhibition broadly affects HCMV protein expression.

[0095] To assess the effect of post-translational telomerase inhibition on HCMV protein abundance, A PRM assay of TB40E-infected, MST-312-treated MRC5 cell lysates at 24, 48, 72, and 96 HPI was employed. Pharmaceutical telomerase inhibition generally resulted in reductions in HCMV protein abundances throughout all phases of the viral life cycle (Figure 7). Notable proteins exhibiting significant reduction in abundance included pUL69, pUL71, pp65, pp28, pUL94 / CEP2, UL96, Tripartite Terminase components TERl / pUL89 and TER2 / pUL56, and envelope glycoproteins gM, gH, and gB (see FIGS. 13A-13J). Additionally, a few proteins showed notable increases in abundance, including pUL22A, IR11, and pUL95.

[0096] In this example, the follow ing methods and techniques were used.

[0097] Cells. Viruses, Viral Infection, Transfection, and Reagents.

[0098] Human lung fibroblasts (MRC-5), human foreskin fibroblasts (HFF), and human embryonic kidney (HEK293) cells were obtained from the American Type Culture Collection (ATCC). All cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) supplemented by 10% Fetal Bovine Serum (10% FBS; BenchMark), IX GlutaMAX (Gibco),IX Modified Eagle's Medium with Non-Essential Amino Acids (MEM NEAA; Gibco), IX sodium pyruvate (Gibco), and 1% Penicillin / Streptomycin (penstrep, ThermoFisher) (DMEM- FBS+++) at 37°C and 5%CO2. HFF cells were used at passages 912, MRC-5 cells at passages 18-22.

[0099] A GFP-tagged virus derived from clinical isolates, TB40 / E-GFP (TB40E), and GFP-tagged laboratory strain AD169-GFP (AD 169), were provided. P0 stock of AD 169 and TB40E were grown in HFF and MRC-5 cells, respectively, purified by centrifugation through a sorbitol cushion ( 20% sorbitol, 50 mM Tris- HC1, lmMMgC12, pH7.2), then concentrated and resuspended in DMEM. Viral titers were determined using a fluorescent focus assay (Zhu, Shen, and Shenk 1995; Koyuncu et al. 2013) on HFF or MRC-5 cells.

[0100] For experiments not utilizing siRNA constructs, cells were serum starved in Dulbecco's Modified Eagles Medium (DMEM), 1% Penicillin / Streptomycin (Pen / Strep, ThermoFisher) for 48 hours prior to infection. Cells w ere infected at a multiplicity of infection (MOI) of 3 unless otherwise specified, then incubated at 37°C for two hours, after which inoculum was removed, cells were washed with phosphate buffered saline (PBS, Gibco) and collected at indicated timepoints post-infection. UV-inactivation of TB40E virions was performed by UV irradiation of viral inoculum using Auto Cross Link settings (UV Stratalinker 2400; San Diego, CA). Ganciclovir (Sigma-Aldrich) was dissolved in DMSO (SigmaAldrich) and used at 50 / zM concentration. BIBR-1532 was dissolved in DMSO and used at 15 and 20 / zM in HFF and MRC- 5 cells, respectively. MST-312 was dissolved in DMSO and used at 0.25 and 0.5 / zM in HFF and MRC-5 cells, respectively. Non-cytotoxic doses of BIBR-1532 and MST-312 were determined for each cell type by dose titration and MTS absorbance assay (Promega). per manufacturer instructions. Unless otherwise specified, dosing for all drugs was done two hours prior to and post infection, with appropriate PBS washes, and at 48 hours post infection (hpi). Supernatant, RNA, and cell lysates were harvested at 96 hpi, unless otherwise specified.

[0101] For experiments using siRNA constructs, cells were transfected with siRNA against hTERT (Horizon Discovery, cat.#M-003547-02-00I0) or non-coding siRNA (Horizon Discovery, cat.#D-001206-14-20) in Lipofectamine RNAiMax Reagent (Thermo Fisher) and Opti-MEM media (Gibco) 24 hours prior to infection, in media as previously described excluding Pen / Strep. Infection proceeded as previously detailed, with appropriate PBS washes, and cells were transfected again at 24 hpi. Supernatant. RNA, and cell lysates were harvested at 48-72 hpi.

[0102] Fluorescent Focus Assay.

[0103] When indicated, experimental HCMV viral titers were determined by assaying for lEl-positive cells on reporter plates. Harvested supernatant was serially diluted 10 -fold and applied to serum-starved 96 -well reporter plates, which were fixed in methanol after 24 hours and stained with mouse anti-HCMV IE1 (1B12, 1 :40 dilution) and goat anti-mouse Alex Fluor- 488 conjugated secondary antibody (1: 1000 dilution; Invitrogen, cat.#A11029). Cells were visualized and the percentage of viral antigen-positive cells was calculated from at least 20 fields of view using the Operetta High Content Imaging System (PerkinElmer).

[0104] RNA Isolation, cDNA Synthesis, and qPCR Reaction.

[0105] RNA was harvested via Qiazol (Qiagen) lysis at 96 HPI, unless otherwise specified. RNA isolation was performed using miRNeasy Mini Kit (Qiagen, cat.#217004), per manufacturer's instructions. Isolated RNA samples were treated with TURBO DNA-free Kit (ThermoFisher), according to the manufacturer's protocol. RNA concentrations were measured by NanodropOne Spectrophotometer (Thermo Scientific).

[0106] cDNA synthesis was performed with Superscript III Reverse Transcriptase (ThermoFisher, cat.# 18080044), per manufacturer's instructions and using random hexamers (IDT), dNTPs (New England Biolabs), and RNaseOUT Recombinant Ribonuclease Inhibitor (ThermoFisher), and Mastercycler Nexus (Eppendorf). For each sample, a 17 uL solution of PCR-grade water, 1000 ng RNA, 1 zL random primers, and I uL I O / zM dNTP mix was made for each sample. Samples were heated at 65°C for five minutes and incubated on ice for 2 minutes before adding to each: 5 / / L of 5X First Strand Buffer (Invitrogen), 1 / zL of 0. 1 M dithiothreitol (DTT; Invitrogen), 1 / zL of SuperScript III RT, and 1 / zL of RNaseOUT Recombinant RNase Inhibitor (Invitrogen), for a final volume of 25 zL. For the negative controls, PCR-grade water was added instead of SuperScript III RT. Samples were then incubated at 25°C for 5 minutes and 50°C for one hour. The reaction was inactivated by heating the samples to 70°C for 15 minutes.

[0107] Host RNA transcripts were measured using Taqman Fast Advanced Master Mix (Thermo Fisher, cat.#4444556), per manufacturer's instructions. Primers were purchased from ThermoFisher (hTERT: Hs00972650_ml; hTERC: Hs03454202_sl; GAPDH: Hs02786624_g 1 ). qPCR performed in triplicate on Quantstudio 6 Flex (ThermoFisher) at thermocycling conditions of 50°C for 2 minutes, 95°C for 2 minutes and 40 cycles of 95°C for 1 second, and 60°C for 20 seconds.

[0108] Viral RNA transcripts were measured using PowerUp SYBR Green Master Mix (ThermoFisher, cat.#A25742). Primers were purchased from IDT (Table 1). qPCR performedin triplicate on Quantstudio 6 Flex (ThermoFisher) at thermocycling conditions of 50°C for 2 minutes, 95°C for 10 minutes, and 40 cycles of 95°C for 15 seconds and 60°C for 1 minute.

[0109] Table 1. Primer Sequences for HCMV viral gene expression assessments.

[0110] Assessment of Telomerase Activity.

[0111] Telomerase activity was assayed using a TRAPeze Telomerase Detection Kit (MilliporeSigma, cat.#S7700), per manufacturer instructions. Cells were harvested by scraping in cold PBS at 96 HPI, unless otherw ise specified. Cells were lysed in a solution of IX CHAPS Lysis Buffer (MilliporeSigma), resuspended Protease Inhibitor Cocktail Tablets (Roche), and RNaseOUT Recombinant RNase Inhibitor (Invitrogen) for 40 minutes. The lysate was next centrifuged at 4°C and 16,000xg for 20 minutes. Lysate protein concentration was measured with Pierce 660 nm Protein Assay (ThermoFisher) and SpectraMax GeminiXS (Molecular Devices Corporation) per manufacturer's instructions. A master mix of 18.8 L PCR-grade water, 2.5 / zL 10X TRAP Reaction Buffer (Sigma-Aldrich), 0.5 uL 50 X dNTP Mix (SigmaAldrich), 0.5 zL TS Primer (Sigma- Aldrich), 0.5 / L TRAP Primer Mix (Sigma- Aldrich), and 0.2 / zL OneTaq Hot Start DNA Polymerase (New England Biolabs) per normalized sample was prepared for final reaction volume of 25 / zL. PCR was performed with Mastercycler Nexus (Eppendorf) with thermocycling conditions of 30°C for 45 minutes, by 95°C for 5 minutes, 35 cycles of 95°C for 30 seconds, 52°C for 30 seconds, and 72°C for one minute, and finally 72°C for 3 minutes. Positive controls included HEK293 cells and TSR8TRAPeze internal positive control. Negative controls included CHAPs lysis buffer, control with no added polymerase, and heat inactivated samples. Heat inactivation for negative samples was performed by incubation at approximately 80°C - 90°C for 20 minutes. Samples were subsequently run on 10% non-denaturing polyacrylamide gel in 0.5 X Tris-Borate-EDTA (TBE) buffer. For polymerization, 0.5 / zL of N, N, N', TV '-Tetramethylethylenediamine (TEMED, VWR) and 10 zL of 10% ammonium persulfate (APS, Fisher Scientific) were added per mL of polyacrylamide gel solution. 25 kb DNA ladder was used for reference. Gels were run at 30 mA / 170 V for three hours, then stained with GelRed® Nucleic Acid Stain 10000X Water (Sigma-Aldrich) for 30 minutes before imaging with ChemiDoc MP Imaging System (BioRad). Quantification was performed on Image Lab Software (BioRad).

[0112] Western Blot.

[0113] Cell lysate was resuspended in 2X Laemmli Sample Buffer (BioRad) and PBS in a 1: 1 ratio and homogenized. Samples were incubated at 70°C for five minutes and protein concentration measured with Pierce 660 nm Protein Assay (ThermoFisher) with ionic detergent compatibility reagent (IDCR; ThermoFisher), according to manufacturer's instructions, using the SpectraMax GeminiXS (Molecular Devices Corporation). For gel electrophoresis, an SDS- PAGE gel (Any kD™ Mini-PROTEAN® TGX™ Precast Protein Gels; BioRad) was loaded with 8 / z L of Precision Plus Protein Standards (BioRad) and 10 / zL of normalized sample. Gels were run at 80 V for two hours at RT in IX Tris / Glycine / SDS Buffer (BioRad), then proteins w ere transferred onto a methanol-wetted Millipore Immobilon-P PVDF membrane at 120 mA at 4°C overnight using two black transfer pads and two pieces of Whatman paper soaked in Tris-glycine transfer buffer (BioRad). Membranes were rinsed with 10X Tris-buffered saline (TBS; BioRad) and blocked for one hour at RT with 5% nonfat dry milk and IX Tris-buffered Saline / Triton (TBST; BioRad).

[0114] Primary antibodies were mouse monoclonal antibodies anti-IEl (1B12; 1 :500 dilution), anti-IE2 (3A9; 1:500 dilution), anti-pUL26 (7H1-5; 1 : 100 dilution), anti-pUL69 (10E11; 1 : 100 dilution), anti pUL99 (10B4-29; 1: 100 dilution), all provided by T. Shenk (Princeton University), and anti- / ?-actin-HRP (1 : 100,000 dilution; Abeam; cat.# ab49900). Goat anti -mouse antibody (1 :10,000 dilution; Jackson ImmunoResearch Laboratories Inc.; cat.# 115-035 003) conjugated with horseradish peroxidase was used as secondary antibody. HyBlot CL autoradiography film (Thomas Scientific) was used to manually develop the blot before imaging with ChemiDoc MP Imaging System (BioRad). Quantification was performed on Image Lab Software (BioRad).

[0115] Parallel reaction monitoring (PRM)-based quantification of HCMV protein abundances.

[0116] Sample preparation for MS analysis. To analyze viral protein abundances during infection, cells were harvested 24, 48, 72, and 96 HPI by washing and scraping in cold PBS. Cells were collected by centrifugation, washed twice with cold PBS, snap frozen in liquid nitrogen, and stored at — 80°C. To lyse, frozen cell pellets were briefly thawed on ice and resuspended in 5% SDS, 25 mM TCEP (ThermoFisher, 77720), and 50 mM chloroacetamide. Lysis was accomplished using repeated rounds of boiling followed by cup hom sonication and proteins were recovered using methanol-chloroform precipitation (Kennedy et al. 2022). Samples were resuspended in 100 mM HEPES (pH 8.2) at a 0.5 pg / p protein concentration (as determined by BCA assay) and digested overnight at 37°C with MS-grade Pierce trypsin (ThermoFisher 90057) at a 1: 50 trypsin:protein mass ratio. Following overnight digestion, samples were acidified to 1% trifluoroacetic acid (TFA) and desalted using 3M Empore C18 Extraction Disks (ThermoFisher 14-386-2). Peptides were concentrated to near dryness by vacuum centrifugation and resuspended in 1%FA / 1%ACN for LCMS / MS analysis.

[0117] Targeted MS analysis by PRM. Targeted MS analysis was performed via LCMS / MS using a Dionex Ultimate 3000 nanoRSLC coupled to a Q Exactive HF mass spectrometer (ThermoFisher). Peptides were separated by reverse-phase chromatography on an EASY-Spray HPLC Column (ThermoFisher, 500 mm length, 2 pm particle size, 75 pm diameter) using a 60-minute gradient (3% to 35% B) with 250 nL / min flow rate. Mobile phase A consisted of 0.1% formic acid in water and mobile phase B consisted of 0.1% formic acid in 97% ACN. The PRM method consisted of targeted MS2 scans recorded in profile mode performed at a resolution of 30,000, with an AGC target of le5, maximum inject time of 200 ms, isolation window of 1.2, and normalized collision energy' of 27% controlled by a peptide inclusion list derived from the TRUSTED targeted MS assay (Kennedy et al. 2022). An MSI scan was performed at a resolution of 15,000 across a mass range of 400-2,000 with an automatic gain control (AGC) of 3e6, and max injection time of 15 ms.

[0118] Quantification and Analysis of HCMV protein abundances. Label -free quantitation was performed using previously described methods (Kennedy et al. 2022) and Skyline Daily software for targeted proteomics. A summed area under the curve of 3 transitions per peptide was used for quantitation. MSI intensity-based normalization, with peak intensity determined using Raw-Meat (Vast Scientific), was performed for normalization across samples and replicates. Individual peptides were normalized to their average abundance across all timepoints / sample conditions. Following normalization, if there were multiple peptides per protein, they were averaged. Average protein abundance was then calculated across replicates and proteins were organized by temporality (Kennedy et al. 2022).

[0119] Statistical Analyses. Statistical significance was analyzed using t-tests and oneway ANOVA, where appropriate, by the GraphPad Prism 10 software. P < 0.05 was considered significant.

[0120] HCMV remains a target for more effective anti-viral therapies. Exploration of the relationship between HCMV and host telomerase may be informative. That HCMV infection increases telomerase activity, in part through hTERT expression during the IE stage, paired with the finding that telomerase inhibition dramatically reduces viral titer across two HCMV strains in a dose-specific manner strongly suggests a biologically significant relationship between HCMV and host telomerase. It was found that posttranslational pharmaceutical telomerase inhibition, as well as genetic knockdown of telomerase activity through an siRNA construct targeting hTERT gene expression, are both effective virucidal agents against HCMV. There is currently no current therapy for any virus that utilizes either post-translational telomerase inhibition or hTERT knockdown.

[0121] Post-translational telomerase inhibition clearly reduced both viral gene expression, and viral protein levels, at all phases of the HCMV life cycle. The alteration in abundance of many viral proteins observed with PRM analysis identifies potential processes of the viral life cycle that may be impacted by the presence or absence of telomerase, investigation of which may provide mechanistic insights on how telomerase supports HCMV infection. For example, several proteins that were notably reduced following telomerase inhibition are considered important for proper formation and envelopment functioning of the viral assembly complex (vAC) (Edward Mocarski, Thomas Shenk. Paul Griffiths, Robert Pass 2014), including pUL71, pp65, pp28, pUL94 / CEP2, UL96, and gM (see FIGS. 8B, 8G, 14A-1D, 141) amongst other implicated proteins that were found to be more mildly reduced following telomerase inhibition. It is therefore possible that telomerase is important for proper vAC formation or function, potentially mediated through interactions with one or more of these proteins. However, additional examination of these proteins and vAC status under telomerase inhibition are required to ascertain whether telomerase inhibition directly interferes with proper vAC functioning or these changes in protein abundance are a secondary7effect of impaired viral replication. Though the reduction of pUL132 abundance was not as notable as other proteins, pUL132 has been known to interact with hTERT (Nobre et al. 2019) and has been found to be critical for proper vAC formation (Wu et al. 2020). It is possible that telomerase inhibitionreduced the function of pUL132, likewise casting a domino effect on other HCMV proteins. Interruption of the vAC could also explain the consistent reductions in envelope glycoproteins (see FIGS. 13H-13J).

[0122] Proteins important for the stabilization and translocation of nucleocapsids from the nucleus to the cytoplasmic vAC (Edward Mocarski, Thomas Shenk, Paul Griffiths, Robert Pass 2014) were also found to be affected by telomerase inhibition. TERl / pUL89 and TER2 / pUL56, two of the three subunits of tripartite terminase, were significantly reduced in abundance, while TER3 as well as both components of the nuclear egress complex, NECl / pUL50 and NEC2 / pUL53, also showed mild though consistent reduction in abundance (see FIGS. 13F- 13G). It is thus possible that telomerase supports encapsidation and nucleocapsid translocation. Some significantly reduced proteins, such as pp65 and pUL69, require phosphorylation by host CDK for proper localization to carry out their respective functions. Telomerase is known to interact with CDK as part of its canonical functioning (Frank, Hyde, and Greider 2006; Zhang et al. 2013). Telomerase-mediated interruption of CDK phosphorylation for such proteins could subsequently hinder encapsidation and thus impair new virion formation, consequently affecting numerous other viral proteins. Of the few proteins whose abundance noticeably increased following telomerase inhibition, pUL22A and IR11 represent anti-inflammatory and immuneevasive viral tactics, respectively. Such immunosuppressive measures may be rallied to aid viral success and persistence in the face of the stresses to replication resulting from telomerase inhibition. Further investigation into the proteins affected by telomerase inhibition will likely provide insights on the mechanism by which telomerase supports HCMV viral infection.

[0123] Overall, the present findings strongly indicate a biologically meaningful role for telomerase in HCMV active infection and suggest the potential for a novel clinical anti-viral treatment for active HCMV infection. These findings expand the more general phenomenon of telomerase significance in herpesvirus infections to soundly include HCMV and encourages continued exploration of the extent of telomerase importance across the herpesvirus family. Telomerase inhibition can be utilized to better study animal models of CMV as well as other herpesviruses, and to gain deeper insights into general HCMV and herpesvirus biology.

[0124] As disclosed herein, it has now been robustly demonstrated that infection with laboratory and clinical strains of HCMV across multiple cell lines significantly increases telomerase activity and hTERT expression, and that viral replication is then sharply reduced following treatment with a non-cytotoxic dose of pharmaceutical telomerase inhibitors or with treatment of an siRNA construct targeting the hTERT catalytic component of telomerase. Thedisclosed findings strongly support viricidal action against HCMV through either inhibition of telomerase action or indirect inhibition of telomerase hTERT expression. These findings represent a novel method of HCMV anti-viral treatment, and represents a potentially more effective method with which to treat patients suffering from HCMV.

[0125] The findings that genetic knockdown of telomerase activity' through an siRNA construct reducing gene expression of the hTERT catalytic subunit of telomerase significantly reduce HCMV viral titer has not been described in any publications or in any context.

[0126] The significance of the effect of hTERT genetic knockdown on HCMV viral titer provides new focus and purpose to the similar effects of the pharmaceutical inhibitors, and taken together strongly support a viricidal role for telomerase inhibition against HCMV infection. Inhibition of telomerase action or inhibition of hTERT expression is viricidal to HCMV.

[0127] More particularly, the synthesis of these findings represents a new use of telomerase inhibition, by either inhibition of telomerase activity or inhibition of hTERT expression, as a viricidal agent against HCMV.

[0128] Inter alia, the disclosed approach may be employed as a clinical anti-viral therapeutic for patients suffering from HCMV infection, in particular active HCMV infection; and may also be employed to better study animal models of HCMV and other herpesviruses, to gain insights into HCMV and herpesvirus biology.|0129| Human Cytomegalovirus remains a target for more effective anti-viral therapies. No vaccine is currently available to prevent HCMV disease, and while existing anti-viral therapies are effective at ameliorating active clinical infection, latent virus persists in the human host and can reactivate later in life and cause severe morbidity. There is currently no method to permanently eradicate HCMV from the human host.

[0130] There are no known therapies for Human Cytomegalovirus that utilize either direct telomerase inhibition or indirect telomerase knockdown via an siRNA construct reducing gene expression of the hTERT catalytic subunit of telomerase. The disclosed findings indicate that these two methods of telomerase inhibition present an anti-viral therapy again Human Cytomegalovirus.

[0131] The disclosed method of direct telomerase inhibition telomerase first involved ascertaining, through rigorous experimentation, the noncytotoxic doses of pharmaceutical inhibitors BIBR1532 and MST312 for the human cell lines used. A treatment regimen whereby cells were treated with one of these inhibitors at two hours prior to infection with HCMV, again two hours after infection with HCMV, and a third time at 48 hours after infection with HCMV,was conducted. Viral titer was assessed at 96 hours after infection. Overall, the process involves experimentation to ensure correct dosing of each drug, which are currently available for research purposes only. Neither of these pharmaceutical telomerase inhibitors are approved for clinical use.

[0132] The disclosed method of indirect telomerase knockdown via an siRNA construct reducing gene expression of the hTERT catalytic subunit of telomerase involves a rigorous optimization process of dosage and delivery conditions specific to cell type, for which HFF (Human Foreskin Fibroblast) cells were used. Once the dosage and delivery conditions were optimized, the process for successful abrogation of viral titer is: delivery of the siRNA construct 24 hours prior to infection with HCMV, followed by a repeated delivery of siRNA construct at 24 hours after infection with HCMV. Viral titer was assessed at 72 hours after infection with HCMV.

[0133] Neither the two pharmaceutical telomerase inhibitors used in direct telomerase inhibition for abrogation of viral titer, nor the siRNA construct used in indirect telomerase inhibition by reducing gene expression of the hTERT catalytic subunit of telomerase is currently available for use in humans, nor have they been known to have been tested in humans. Rigorous safety and efficacy testing, presumably first in an animal model such as mice, and then in human subjects, would be required to ascertain safe and effective usage for treatment of clinical HCMV infection.|0134] The disclosed approach may be used, inter alia, as a method of therapy for patients suffering from HCMV, and may represent a more effective treatment than currently available anti-viral therapies for the treatment of HCMV. Additionally, the disclosed approach may possess the advantage of being the first HCMV anti-viral therapy to eradicate HCMV from the human body, which would be a major breakthrough in treatment of HCMV and potentially other herpesviruses.

[0135] The current results implicate viral genome replication and viral entry / attachment as the stages of viral pathogenesis relevant to the mechanism of interaction betw een host telomerase and HCMV, as these processes are affected by telomerase inhibition.

[0136] Features of any of the examples or embodiments outlined above may be combined to create additional examples or embodiments without losing the intended effect. It should be understood that the description of an embodiment or example provided above is by way of example only, and various modifications could be made by one skilled in the art. Furthermore, one skilled in the art will recognize that numerous further modifications and combinations ofvarious aspects are possible. Accordingly, the described aspects are intended to encompass all such alterations, modifications, and variations that fall within the scope of the appended claims.

Claims

CLAIMS1 . A method for inhibiting human herpesvirus replication in cells, comprising contacting the cells with a telomerase inhibitor.

2. The method of claim 1, wherein the human herpesvirus is human cytomegalovirus (HCMV), Epstein-Barr virus (EBV). Herpes simplex virus type 1 (HSV-1). human herpesvirus 6 (HHV- 6), human herpesvirus 7 (HHV-7), or human herpesvirus 6 (HHV-8).

3. The method of claim 1, wherein the telomerase inhibitor is a pharmaceutical inhibitor.4 The method of claim 3, wherein the pharmaceutical inhibitor is BIBR1532 or MST312.

5. The method of claim 1, wherein the telomerase inhibitor is an siRNA construct targeting a human telomerase reverse transcriptase (hTERT) catalytic subunit of telomerase.

6. The method of claim 1, wherein contacting the cells with the telomerase inhibitor comprises: contacting the cells with the telomerase inhibitor at a first time point prior to herpesvirus infection; contacting the cells with the telomerase inhibitor at a second time point after herpesvirus infection; and contacting the cells with the telomerase inhibitor at a third time point after herpesvirus infection.

7. The method of claim 6, wherein: the first time point is between 1 hour and 4 hours prior to herpesvirus infection; the second time point is between 1 hour and 4 hours after herpesvirus infection; and the third time point is between 36 hours and 60 hours after herpesvirus infection.

8. The method of any of claims 1 to 7, wherein the method results in reduced viral titer, decreased viral gene expression, and / or diminished viral protein levels.

9. A pharmaceutical composition for use in inhibiting human herpesvirus replication in cells according to the method of any of claims 1 to 8, the pharmaceutical composition comprising:a telomerase inhibitor; and a pharmaceutically acceptable carrier.

10. The pharmaceutical composition of claim 9, wherein the telomerase inhibitor is BIBR1532 or MST312.

11. The pharmaceutical composition of claim 9 or 10, wherein the pharmaceutical composition is formulated for administration to the cells at multiple time points relative to herpesvirus infection.

12. The pharmaceutical composition of any of claims 9 to 11, further comprising an additional antiviral agent.

13. A delivery system for use in inhibiting human herpesvirus replication in cells according to the method of any of claims 1 to 8, the delivery system comprising an siRNA construct targeting a human telomerase reverse transcnptase (hTERT) catalytic subunit of telomerase.

14. The delivery’ system of claim 13, wherein the siRNA construct is designed to reduce expression of the hTERT catalytic subunit.

15. The delivery system of claim 13 or 14, further comprising a transfection agent for delivering the siRNA construct to the cells.

16. The delivery system of any of claims 13 to 15, wherein the delivery system is configured to deliver the siRNA construct to the cells at multiple time points relative to HCMV infection.

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