DESC:DESCRIPTION:
Field of the invention:
[0001] The present disclosure generally relates to the technical field of a treatment of viral infections, and in specific relates to a method for inhibiting dengue virus replication using siRNA (small interfering RNA) technology to achieve the desired therapeutic effect and manipulate gene expression.
Background of the invention:
[0002] Dengue virus is a member of the Flaviviridae family, characterized by a single-stranded RNA genome and a single envelope with a diameter of 30 nm and positive polarity. It is one of the most common diseases affecting people worldwide. Dengue fever is transmitted through mosquito bites or contact with infected individuals, resulting in a high fever, rash, muscle and joint pains, and, in some cases, hemorrhagic manifestations. Individuals with dengue hemorrhagic fever are at a heightened risk of mortality. Moreover, while people develop permanent immunity against the specific dengue viral type they are infected with, they remain susceptible to other viral types. Consequently, individuals residing in endemic regions often experience infections with all four dengue virus serotypes.

[0003] Dengue fever is caused by any of four related virus serotypes (DENV-1, DENV-2, DENV-3, and DENV-4) transmitted by mosquitoes. It is crucial that once dengue infection is detected, rapid supportive treatment be provided. Infection can be asymptomatic or manifest a range of clinical symptoms, including self-limiting febrile fever and potentially life-threatening acute dengue disease. Clinicians report hundreds of thousands of clinical dengue disease cases to the WHO (World Health Organization) annually, with a fatality rate ranging between 0.5% and 5.0%. Any of the four serotypes can cause severe disease in humans, and all four are spread internationally.

[0004] The siRNA therapeutic technology demonstrates promising leads and can be applied in various contexts. However, for siRNA to be transformed into therapeutic agents, it’s in vivo stability and intracellular delivery efficiency must be enhanced to ensure its effective delivery to target cells. To address these challenges, research has focused on methods that involve either modifying specific nucleotides or the backbone of siRNA to improve in vivo stability and resistance to nucleases, or utilizing carriers such as viral vectors, liposomes, or nanoparticles.

[0005] Existing technology features a novel dengue virus-specific silencing RNA (siRNA) delivery system employing a double-stranded oligo RNA structure. This structure consists of a dengue virus-specific siRNA conjugated to hydrophilic and hydrophobic moieties at both ends. The conjugation facilitates efficient cellular uptake of the siRNA and its assembly into nanoparticles in aqueous solutions. The double-stranded oligo RNA structure includes methods for its preparation and a pharmaceutical composition for preventing or treating dengue virus infection. However, there is a potential risk of the double-stranded oligo RNA structure silencing unintended genes with similar sequences, leading to inadvertent side effects.

[0006] Therefore, there is a need for a method for inhibiting dengue virus replication with siRNA (small interfering RNA) technology to achieve the desired therapeutic effect and manipulating gene expression. There is also a need for a method that DensiT-4S to target conserved regions of the dengue virus genome, making it effective against all four serotypes (DENV-1, DENV-2, DENV-3, DENV-4) of the dengue virus. Further, there is also a need for a method that minimal off-target binding to human RNA, which reduces the potential for side effects and increases the safety profile of the therapeutic.
Objectives of the invention:
[0007] The primary objective of the invention is to design, synthesize and evaluate a single siRNA molecule (DensiT-4S) for inhibiting all four serotypes of dengue viral replication with siRNA (small interfering RNA) technology to achieve the desired therapeutic effect by manipulating gene expression.

[0008] Another objective of the invention is to provide a method that utilizes the DensiT-4S to target conserved regions of all four dengue viral serotypes (DENV-1, DENV-2, DENV-3, DENV-4), thereby minimizing the likelihood of viral mutations escaping the therapeutic effect and reducing the chances of the virus developing resistance to the treatment.

[0009] Another objective of the invention is to provide a method that interrupts the infection cycle early, potentially reducing disease severity and preventing the progression to more severe forms of dengue such as dengue haemorrhagic fever or dengue shock syndrome.

[0010] Another objective of the invention is to provide a method that prevents viral replication upon infection and is beneficial in areas experiencing dengue outbreaks, where high-risk populations could be treated early to prevent disease onset.

[0011] Another objective of the invention is to provide a method that offers an additional line of defence, particularly for individuals who do not respond adequately to vaccines or who are already infected with the dengue virus.

[0012] Yet another objective of the invention is to provide a method that potentially delivers the siRNA therapeutic using various platforms, including lipid nanoparticles, viral vectors, or direct injection, thereby making it adaptable to different clinical situations and patient needs.

[0013] Further objective of the invention is to provide a method that utilizes RNA interference to treat dengue infections represents a cutting-edge approach in antiviral therapy, utilizing the host's natural cellular machinery to degrade viral RNA, which could be adapted to target other RNA viruses as well.
Summary of the invention:
[0014] The present disclosure proposes a method for inhibiting dengue virus replication using Densit-4S siRNA therapeutics. The following presents a simplified summary in order to provide a basic understanding of some aspects of the claimed subject matter. This summary is not an extensive overview. It is not intended to identify key/critical elements or to delineate the scope of the claimed subject matter. Its sole purpose is to present some concepts in a simplified form as a prelude to the more detailed description that is presented later.

[0015] In order to overcome the above deficiencies of the prior art, the present disclosure is to solve the technical problem to provide a method for inhibiting dengue virus replication with siRNA (small interfering RNA) technology to achieve the desired therapeutic effect by manipulating gene expression.

[0016] According to one aspect, the invention provides the method for inhibiting dengue virus replication. At one step, a small interfering RNA (siRNA) molecule (DensiT-4S) is synthesized and delivered to human embryonic kidney 293 (HEK293) cells. At another step, the RNA-induced silencing complex (RISC) is activated that separates the antisense strand of the DensiT-4S siRNA. The antisense strand is complementary to a conserved region of the dengue viral mRNA.

[0017] At another step, the antisense strand of DensiT-4S siRNA binds to the conserved region of dengue viral mRNA across all four serotypes. At another step, induce degradation of the dengue viral mRNA by the RISC complex, thereby inhibiting viral replication within the infected human embryonic kidney 293 (HEK293) cells. Further, at another step, administers a therapeutically effect of the DensiT-4S siRNA formulation to the human embryonic kidney 293 (HEK293) cells to prevent further dengue virus propagation and infection, thereby leading to the degradation of the viral mRNA and inhibition of viral replication.

[0018] In one embodiment, the DensiT-4S siRNA is chemically synthesized and formulated for intravenous administration using lipid nanoparticles. In one embodiment, the degradation of dengue viral mRNA in the human embryonic kidney 293 (HEK293) cells reduces viral load, thereby preventing and mitigating the onset of dengue fever symptoms.

[0019] In one embodiment, the DensiT-4S siRNA is configured to target a single conserved region within the dengue viral RNA genome of all four serotypes, thereby enhancing inhibitory effect on viral replication. In one embodiment, the multiple conserved regions are selected from the 3' untranslated region essential for viral replication.

[0020] The efficacy of DensiT-4S was tested using the eGFP reporter gene assay. In this assay, the natural fluorescence of eGFP was decreased at least 3-fold by the DensiT-4S thus confirming its proposed function.

[0021] In one embodiment, the DensiT-4S siRNA is modified by PEGylation to increase its stability and circulation time within the patient’s bloodstream, thereby improving therapeutic efficacy. In one embodiment, the DensiT-4S siRNA formulation is used in combination with other antiviral agents, such as nucleoside analogues or protease inhibitors, to achieve a multi-targeted approach against dengue virus.

[0022] Further, objects and advantages of the present invention will be apparent from a study of the following portion of the specification, the claims, and the attached drawings.
Detailed description of drawings:
[0023] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate an embodiment of the invention, and, together with the description, explain the principles of the invention.

[0024] FIG. 1 illustrates a flowchart of a method for inhibiting dengue virus replication, in accordance to an exemplary embodiment of the invention.

[0025] FIG. 2A illustrates a schematic view of the method for inhibiting dengue virus replication, in accordance to an exemplary embodiment of the invention.

[0026] FIG. 2B illustrates a schematic view of multiple sequence alignment (MSA) results comparing the genomic RNA sequences of the four dengue virus serotypes, in accordance to an exemplary embodiment of the invention.

[0027] FIG. 2C illustrates a schematic view of a recombinant plasmid, in accordance to an exemplary embodiment of the invention.

[0028] FIG. 3A illustrates a pictorial representation of a differential interference contrast (DIC) microscopy image of cells, in accordance to an exemplary embodiment of the invention.

[0029] FIG. 3B illustrates a pictorial representation of an enhanced Green Fluorescent Protein (eGFP) microscopic image of cells in the absence of DensiT-4S, in accordance to an exemplary embodiment of the invention.

[0030] FIG. 3C illustrates a pictorial representation of the eGFP microscopic image of cells in the presence of DensiT-4S, in accordance to an exemplary embodiment of the invention.

[0031] FIG. 4 illustrates a graphical representation of quantification of fluorescence levels in cells transfected with the eGFP in the presence and absence of DensiT-4S siRNA, in accordance to an exemplary embodiment of the invention.
Detailed invention disclosure:
[0032] Various embodiments of the present invention will be described in reference to the accompanying drawings. Wherever possible, same or similar reference numerals are used in the drawings and the description to refer to the same or like parts or steps.

[0033] The present disclosure has been made with a view towards solving the problem with the prior art described above, and it is an object of the present invention to provide a method for inhibiting dengue virus replication with siRNA (small interfering RNA) technology to achieve the desired therapeutic effect and manipulating gene expression.

[0034] According to one exemplary embodiment of the invention, FIG. 1 refers to a flowchart 100 of the method for inhibiting dengue virus replication. The method utilizes DensiT-4S to target conserved regions of the dengue virus genome, making it effective against all four serotypes (DENV-1, DENV-2, DENV-3, DENV-4) of the dengue virus. The method interrupts the infection cycle early, potentially reducing disease severity and preventing the progression to more severe forms of dengue such as dengue haemorrhagic fever or dengue shock syndrome.

[0035] The DensiT-4S is an siRNA-based therapeutic specifically designed to inhibit dengue virus replication by targeting and binding to the viral genomic RNA, independent of the viral serotype. This precise binding triggers the degradation of the viral RNA by the human RISC (RNA-induced silencing complex) proteins, effectively halting the virus's ability to replicate, spread, and cause further infection by destroying its genetic material.

[0036] At step 102, a small interfering RNA (siRNA) molecule is synthesized as DensiT-4S siRNA and is delivered to human embryonic kidney 293 (HEK293) cells. At step 104, the RNA-induced silencing complex (RISC) is activated that separates the antisense strand of the DensiT-4S siRNA, which is complementary to a conserved region of the dengue viral mRNA.

[0037] At step 106, the antisense strand of DensiT-4S siRNA binds to the conserved region of the dengue viral mRNA across all four serotypes. At step 108, the RISC complex induces the degradation of the dengue viral mRNA, thereby inhibiting viral replication within the infected human embryonic kidney 293 (HEK293) cells. At step 110, a therapeutically effective dose of the DensiT-4S siRNA formulation is administered to human embryonic kidney 293 (HEK293) cells to prevent further dengue virus propagation and infection, thereby leading to the degradation of viral mRNA and inhibition of viral replication.

[0038] In one embodiment, the DensiT-4S siRNA is chemically synthesized and formulated for intravenous administration using lipid nanoparticles. In another embodiment, the degradation of dengue viral mRNA in human embryonic kidney 293 (HEK293) cells reduces the viral load, thereby preventing and mitigating the onset of dengue fever symptoms.

[0039] In one embodiment, the DensiT-4S siRNA is configured to target a single conserved region within the dengue viral RNA genome, thereby enhancing inhibitory effect on viral replication. In one embodiment, the multiple conserved regions are selected from the 3' untranslated regions essential for viral replication.

[0040] In one embodiment, the DensiT-4S siRNA is modified by PEGylation to increase its stability and circulation time within the patient’s bloodstream, thereby improving therapeutic efficacy. In one embodiment, the DensiT-4S siRNA formulation is used in combination with other antiviral agents, such as nucleoside analogues or protease inhibitors, to achieve a multi-targeted approach against dengue virus.

[0041] According to another exemplary embodiment of the invention, FIG. 2A refers to a schematic view 200 of the method for inhibiting dengue virus replication. In one example embodiment, the DensiT-4S siRNA is a double-stranded RNA molecule designed to target the viral genome of the dengue virus. The DensiT-4S siRNA is engineered to bind to conserved regions of the viral RNA, making it effective across all four dengue serotypes. The RNA-Induced Silencing Complex (RISC) is assembled, which is crucial for the RNA interference process. The RISC complex unwinds the double-stranded DensiT-4S siRNA, retaining only the antisense strand (blue strand) that is complementary to the target viral mRNA.

[0042] The RISC complex is activated by the integration of the antisense strand of DensiT-4S siRNA, while the sense strand (red) is discarded. The activated RISC, containing the antisense strand, is now primed to recognize and bind to the viral mRNA. Once the dengue virus infects the human embryonic kidney 293 (HEK293) cell, it releases its genomic RNA, also known as viral mRNA. The antisense strand of DensiT-4S siRNA within the activated RISC complex recognizes and binds to the complementary sequence of the viral mRNA (green strand).

[0043] Upon recognition, the RISC complex triggers the degradation of the viral mRNA. The binding of siRNA to the viral mRNA induces RNA interference (RNAi), which results in the cleavage and degradation of the viral mRNA, preventing its translation into viral proteins. The viral mRNA is fragmented, inhibiting viral replication. Without functional viral proteins, the virus cannot propagate further.

[0044] According to another exemplary embodiment of the invention, FIG. 2B refers to a schematic view 202 of multiple sequence alignment (MSA) results compared to the four dengue virus serotypes. In one embodiment, the method utilizes a multiple sequence alignment (MSA) to compare the viral genome sequences of the four dengue virus serotypes. Each row represents a specific serotype (S1, S2, S3, S4), and the letters represent nucleotide bases (A, T, G, C). The "*" symbols below each column indicate positions where the nucleotides are fully conserved across all four serotypes. Gaps ("-") in the sequences indicate regions where one or more serotypes may have deletions or insertions.

[0045] Based on these conserved sequences, five siRNA molecules were designed for these conserved sequences listed as follows: siRNA1: 5'-UUCCUGAUCUCCAAUCUCCUCUGGGGG-3' (Complementary to green highlight), siRNA2: 5'-UCUGGUCUCUAGGACGACAGAG-3' (Complementary to cyan highlight), siRNA3: 5'-UUUGUCGUAUAACUGCGACCCU-3' (Complementary to magenta highlight), siRNA4: 5'-CCAAUCUCCUCUGGGGAGGG-3' (Complementary to yellow highlight), and siRNA5: 5'-CUGAUCUCCAAUCUCCUCUGGGGGG-3' (Complementary to green highlight).

[0046] The yellow highlight is a conserved sequence region common to all four dengue serotypes, making it a key target for siRNA design. These bases likely represent an important functional site in the viral genome that the DensiT-4S siRNA aims to bind and degrade. Green highlight is a conserved region shared by all serotypes, marked for siRNA targeting. This region is crucial for ensuring that DensiT-4S works against all dengue virus types.

[0047] Magenta highlight is conserved region and also serves as a target for DensiT-4S, reinforcing the broad-spectrum capability of the therapeutic across different serotypes. Cyan highlight is a critical conserved region located at the 3' untranslated region (UTR), which is an ideal target for disrupting viral RNA stability or replication. The MSA diagram directly supports the design and selection of the DensiT-4S siRNA strands. The sequences highlighted in the MSA were used to design siRNA molecules that complement these conserved regions. Once the siRNA enters the host cell, it becomes part of the RISC complex, where it binds to the viral genomic RNA, thereby initiating viral mRNA degradation and halting viral replication.

[0048] In one embodiment herein, the siRNA1 is complementary to the green highlighted region in the Dengue viral mRNA. Ten hits were found in the human genome that code for human proteins that are critical for human physiology. Hence, siRNA1 may have side effects, (as depicted in Table. 1).

[0049] Table. 1:

[0050] In one embodiment herein, the siRNA 2 is complementary to the cyan highlighted region in the Dengue viral mRNA. Ten hits were found in the human genome, out of which 3 hits code for the same human protein. Hence, siRNA2 may not have side effects. The siRNA2 is a relatively low risk of off-target effects. Off-target effects can lead to unwanted cellular responses, toxicities, and other adverse effects during treatment. Since, the siRNA2 appears to target a conserved region in the dengue virus while having minimal overlap with human gene targets, it presents a promising therapeutic profile (as depicted in Table. 2).

[0051] Table. 2:

[0052] In one embodiment herein, the siRNA3 highlights a crucial trade-off in developing RNA-based therapies. Although, the siRNA3 targets a specific region of the dengue viral mRNA, its substantial interaction with the human genome and the associated risk of side effects warrant caution. The importance of selecting siRNA sequences that maximize antiviral efficacy while minimizing potential off-target effects in the human embryonic kidney 293 (HEK293) cells, which is critical for the safety and effectiveness of any therapeutic intervention against dengue virus infections.

[0053] Table. 3:

[0054] In one embodiment herein, the siRNA 4 is the complementary to the yellow highlighted region in the Dengue viral mRNA. Ten hits were found in the human genome, out of which 7 hits code for human proteins. Hence, siRNA4 is potential off-target interactions with human proteins raise concerns about its safety and efficacy as a therapeutic agent. This necessitates careful consideration and further evaluation of siRNA4’s suitability for use in treating dengue virus infections, particularly in ensuring that it minimizes off-target effects and preserves normal human cellular function.

[0055] Table. 4:

[0056] In one embodiment herein, the analysis of siRNA5 indicates that, although it is designed to target a specific region of the dengue virus, its significant overlap with human mRNA sequences raises concerns regarding its safety and efficacy as a therapeutic agent. The potential for off-target effects necessitates a thorough examination of siRNA5’s interactions with human genes, ensuring that it minimizes unintended consequences while effectively combating dengue virus infections.

[0057] Table. 5:

[0058] According to another exemplary embodiment of the invention, FIG. 2C refers to a schematic view 204 of a recombinant plasmid. In one embodiment herein, the recombinant pcDNA3.1(+)-C-eGFP plasmid is a widely used eukaryotic expression vector. Additionally, the recombinant plasmid is employed as a cloning vehicle to insert the conserved viral sequence (highlighted with green color in Fig 2B) into its multiple cloning site (MCS). The plasmid incorporates a robust cytomegalovirus (CMV) promoter to drive the expression of the enhanced green fluorescent protein (eGFP), a highly sensitive reporter gene that emits green fluorescence under blue light excitation. This eGFP serves as a reliable indicator of transfection efficiency and can be used to assess the impact of the DensiT-4S siRNA on gene silencing.

[0059] According to another exemplary embodiment of the invention, FIG. 3A refers to a pictorial representation 300 of a differential interference contrast (DIC) of cells. In one embodiment herein, the differential interference contrast (DIC) is provided by the microscopy to demonstrate the cellular structures without fluorescence, thereby providing a clear picture of the cell morphology and the presence of cells. The DIC confirms the cells are intact and viable under the microscopy conditions, thereby establishing a baseline for comparison with the eGFP images.

[0060] According to another exemplary embodiment of the invention, FIG. 3B refers to a pictorial representation 302 of an enhanced Green Fluorescent Protein (eGFP) (-DensiT-4S) of cells. The eGFP demonstrates the cells expressing enhanced Green Fluorescent Protein (eGFP) in the absence of DensiT-4S siRNA treatment. The strong green fluorescence signal indicates active expression of eGFP in the cells. In one example, the bright eGFP fluorescence demonstrates that the cells are actively expressing the reporter gene (eGFP), as the viral mRNA has not been inhibited by DensiT-4S.

[0061] According to another exemplary embodiment of the invention, FIG. 3C refers to a pictorial representation 304 of the eGFP(+DensiT-4S) of cells. In one embodiment herein, the eGFP(+DensiT-4S) demonstrates the cells after treatment with the DensiT-4S siRNA. The fluorescence signal is significantly reduced compared to the previous image, thereby indicating that the DensiT-4S siRNA has been effective in silencing the eGFP mRNA. The reduction in eGFP fluorescence suggests that DensiT-4S is actively targeting and degrading the eGFP mRNA, which results in less eGFP being produced.

[0062] According to another exemplary embodiment of the invention, FIG. 4 refers to a graphical representation 400 of quantification of fluorescence levels in cells transfected with the eGFP in the presence and absence of DensiT-4S siRNA. In one embodiment herein, the graph with y-axis represents Relative Fluorescence Units (RFU), which measures the intensity of eGFP fluorescence. The x-axis shows two conditions such as eGFP (-DensiT-4S) and eGFP (+DensiT-4S), where the "+" indicates the treatment with DensiT-4S siRNA and the "-" indicates no treatment. In another embodiment herein, the eGFP (-DensiT-4S) represented in a blue bar with RFU value of 0.215. additionally, the RFU value indicates the eGFP fluorescence in the absence of DensiT-4S. The high fluorescence signal indicates that eGFP mRNA is being fully expressed and translated into the fluorescent protein in the cells.

[0063] In one embodiment herein, the eGFP (+DensiT-4S) represented in a red bar with the RFU value of 0.075. Additionally, the RFU value represents the eGFP fluorescence in the presence of DensiT-4S siRNA. The significantly lower RFU value compared to the control indicates that DensiT-4S has successfully targeted and degraded the eGFP mRNA, reducing the amount of eGFP protein produced. The fluorescence level has dropped substantially (3-fold decrease), demonstrating that DensiT-4S is effectively inhibiting eGFP expression. In one embodiment herein, the both bars of the graph are accompanied by error bars, which indicate the variability of the data. These error bars represent the standard deviation (SD), showing the consistency and reliability of the measurements. The small size of the error bars suggests that the data points are closely clustered around the mean, implying low variability and high precision in the experimental results.

[0064] Numerous advantages of the present disclosure may be apparent from the discussion above. In accordance with the present disclosure, the method is disclosed. The proposed method utilizes DensiT-4S to target a single conserved region of the dengue virus genome, making it effective against all four serotypes (DENV-1, DENV-2, DENV-3, DENV-4) of the dengue virus. The proposed method interrupts the infection cycle early, potentially reducing disease severity and preventing the progression to more severe forms of dengue, such as dengue hemorrhagic fever or dengue shock syndrome. The proposed method is minimal off-target binding to human RNA, which reduces the potential for side effects and increases the safety profile of the therapeutic.

[0065] The proposed method prevents viral replication upon infection and is beneficial in areas experiencing dengue outbreaks, where high-risk populations could be treated early to prevent disease onset. The proposed method offers an additional line of defence, particularly for individuals who do not respond adequately to vaccines or who are already infected with the dengue virus. The proposed method utilizes RNA interference to treat dengue infections and represents a cutting-edge approach in antiviral therapy, utilizing the host's natural cellular machinery to degrade viral RNA, which could be adapted to target other RNA viruses as well.

[0066] The proposed method utilizes the siRNA that is extensively analysed for off-target interactions with the human genome, minimal interaction with human genes, thereby reducing the likelihood of side effects to human proteins. The proposed method potentially delivers the siRNA therapeutic using various platforms, including lipid nanoparticles, viral vectors, or direct injection, thereby making it adaptable to different clinical situations and patient needs.

[0067] It will readily be apparent that numerous modifications and alterations can be made to the processes described in the foregoing examples without departing from the principles underlying the invention, and all such modifications and alterations are intended to be embraced by this application.
,CLAIMS:CLAIMS:
I / We Claim:
1. A method for inhibiting dengue virus replication, comprising:
synthesizing a small interfering RNA (siRNA) molecule as DensiT-4S siRNA and delivering it to human embryonic kidney 293 (HEK293) cells;
activating RNA-induced silencing complex (RISC) that separates the antisense strand of the DensiT-4S siRNA, wherein the antisense strand is complementary to a conserved region of a dengue viral mRNA;
binding the antisense strand of the DensiT-4S siRNA to the conserved region of the dengue viral mRNA across all four serotypes;
inducing degradation of the dengue viral mRNA by the RISC complex, thereby inhibiting viral replication within the infected human embryonic kidney 293 (HEK293) cells; and
administering a therapeutic effect of the DensiT-4S siRNA to the human embryonic kidney 293 (HEK293) cells to prevent further dengue virus propagation and infection, thereby leading to the degradation of the viral mRNA and inhibition of viral replication.
2. The method as claimed in claim 1, wherein the DensiT-4S siRNA is chemically synthesized and formulated for intravenous administration using lipid nanoparticles.
3. The method as claimed in claim 1, wherein the degradation of dengue viral mRNA in the human embryonic kidney 293 (HEK293) cells reduces viral load, thereby preventing and mitigating the onset of dengue fever symptoms.
4. The method as claimed in claim 1, wherein the DensiT-4S siRNA is configured to target a single conserved region within the dengue viral RNA genome, thereby enhancing inhibitory effect on viral replication.
5. The method as claimed in claim 4, wherein the multiple conserved regions are selected from 3' untranslated region essential for viral replication.
6. The method as claimed in claim 1, wherein the DensiT-4S siRNA is modified by PEGylation to increase stability and circulation time within the patient's bloodstream, thereby improving therapeutic efficacy.
7. The method as claimed in claim 1, wherein the DensiT-4S siRNA is used in combination with antiviral agents, such as nucleoside analogues and protease inhibitors, to achieve a multi-targeted approach against dengue virus.