Viral nucleic acid molecules, compositions and methods of use thereof

Abstract

The present disclosure relates to defective interfering genes and viruses thereof. It has been discovered that defective interfering genes designed according to influenza and coronavirus species can inhibit infection or replication of the parental virus. In vivo, DIGs induce rapid onset prophylactic protection in infected animals against lethal viral doses. Accordingly, nucleic acids containing DIGs, pharmaceutical compositions thereof, and related methods of use are disclosed herein. For example, described herein are isolated polynucleotides containing one or more defective interfering genes, wherein each of the one or more defective interfering genes contains a nucleotide sequence corresponding to one or more portions of an influenza or coronavirus gene or genome, wherein the nucleotide sequence comprises a deletion in the gene. The DIGs can be in the form of a plasmid. Pharmaceutical compositions of the plasmids can be used to limit viral replication, as well as to prevent or treat influenza or coronavirus associated diseases.

Description

[0001] Cross-references to related applications

[0002] This application claims priority and interest in U.S. Provisional Application No. 63 / 283,101, filed November 24, 2021, the entire contents of which are incorporated herein by reference.

[0003] sequence list reference

[0004] A sequence list text file named "UHK_01130_US_ST26.xml" created on October 18, 2022, and having a size of 89,128 bytes, is incorporated herein by reference. Technical Field

[0005] The disclosed invention generally relates to antiviral agents, and more specifically to compositions and methods for preventing and / or treating viral infections and diseases. Background Technology

[0006] Clinical studies have shown that early treatment or preventative treatment may be a more effective way to control these diseases. In addition to therapeutic antiviral agents under development, prophylactic antiviral agents may play an important role in controlling infectious diseases. Vaccination is currently the most effective strategy for preventing viral infection, but vaccine efficacy is strain-dependent, and historically, vaccination protection against influenza has ranged from 10% to 60% over the past few decades [Centers for Disease Control and Prevention, Update: drug susceptibility of swine-origin influenzaA (H1N1) viruses, MMWR Morb Mortal Wkly Rep, 58, 433-435 (2009)]. Given the ongoing spread of SARS-CoV-2 variants, this may also apply to COVID-19 vaccines. Effective vaccine protection requires at least two weeks to develop sufficient titers of neutralizing antibodies. Zanamivir and oseltamivir, neuraminidase inhibitors targeting influenza viruses, are approved for prevention, but both require daily administration, and drug resistance can arise with frequent use. Antiviral drugs directly targeting SARS-CoV-2 have shown benefit for patients with mild to moderate symptoms, but their benefit for patients presenting with severe symptoms is unclear [Beigel, JH]. et al. , The New England Journal of Medicine,383, 1813-1826, Doi: 10.1056 / NEJMoa2007764, (2020); Spinner, CD et al ., JAMA , 324(11), 1048-1057, Doi: 10.1001 / jama.2020.16349, (2020); Thiruchelvam, K. et al. , Expert Rev Anti Infect Ther [1-19, Doi: 10.1080 / 14787210.2021.1949984, (2021)]. This may suggest that early treatment or preventative treatment should be considered for COVID-19 cases.

[0007] Defective interference gene (DIG) is a viral gene with an internal deletion [Huang, AS & Baltimore, D]. Nature 226, 325-327, Doi: 10.1038 / 226325a0, (1970)]. Influenza virus DIG can inhibit the replication of homologous full-length viral RNA, and its antiviral activity is affected by the length and source of DIG [Meng, B]. et al ., Virology journal 14, 138, Doi: 10.1186 / s12985-017-0805-6 (2017); Duhaut, SD& Dimmock, NJ J Gen Virol 83, 403-411, Doi: 10.1099 / 0022-1317-83-2-403,(2002); Dimmock, NJ, et al ., J Virol 82 (17), 8570-8578, Doi: 10.1128 / JVI.00743-08 (2008); Bdeir, N. et al . PLoS One, 14, e0212757, Doi: 10.1371 / journal.pone.0212757, (2019); Zhao, H. et al . Nat Commun[9, 2358, Doi:10.1038 / s41467-018-04792-7, (2018)]. Analysis of subgenomic RNA of coronaviruses indicates that coronaviruses produce defective genes during viral replication. However, it is unclear which DIG (digestive enzyme) plays the best role in anti-influenza activity, and whether any DIG can significantly inhibit coronaviruses in vitro and in vivo.

[0008] For emerging influenza viruses and coronaviruses, especially for people at high risk of infection and / or with severe illness, there is a need to develop prophylactic antiviral agents with novel antiviral mechanisms and broad-spectrum antiviral activity.

[0009] One object of the present invention is to provide an antiviral composition and a method of using the same.

[0010] Another object of the present invention is to provide compositions and methods for delivering nucleic acids encoding defect interference genes.

[0011] Another object of the present invention is to provide compositions and methods for the prevention and / or treatment of viral diseases, including individual illnesses caused by influenza viruses and coronaviruses, including SARS-CoV-2.

[0012] Any discussion of the documents, actions, materials, devices, articles of manufacture, etc. contained in this specification should not be construed as an admission that any or all of these contents form part of the prior art, or as being considered common general knowledge in the field related to this invention because they existed prior to the priority date of each claim of this application. Summary of the Invention

[0013] Effective antiviral agents against pathogenic viral species and their variants have been developed. Working examples demonstrate that the development of defective interference genes (DIGs) designed based on influenza and coronavirus species can significantly inhibit infection and replication of homologous viruses. In in vivo testing, DIGs derived from influenza viruses or coronaviruses induced rapidly onset prophylactic protection in infected animals, thus providing an alternative to current clinical strategies for the prevention of influenza viruses and coronaviruses. Therefore, this document discloses nucleic acids containing DIGs, pharmaceutical compositions thereof, and related methods of use.

[0014] Specifically, this invention discloses an isolated polynucleotide containing one or more defective interfering genes, wherein each of the one or more defective interfering genes contains a nucleotide sequence corresponding to one or more portions of a viral gene or genome, wherein the nucleotide sequence contains deletions in the gene. More specifically, the portion of the viral gene contains deletions relative to the viral gene. Preferably, the virus is an influenza virus (e.g., influenza A virus, influenza B virus, or influenza C virus) or a coronavirus (e.g., SARS-CoV1, SARS-CoV2, MERS-CoV, and other coronaviruses).

[0015] In some forms, when the virus is an influenza virus, the gene encodes an RNA polymerase or a subunit thereof, such as, but not limited to, PA, PB1, and PB2. In some forms, the nucleotide sequence may contain an internal (central) deletion in the gene. More specifically, the portion of the viral gene contained in the nucleotide sequence contains an internal deletion relative to the viral gene. In some forms, the nucleotide sequence comprises about 150 to 600 nucleotides from the 5' end of the gene, about 150 to 600 nucleotides from the 3' end of the gene, or a combination thereof. Preferably, the nucleotide sequence comprises about 450 nucleotides from the 5' end of the gene and about 450 nucleotides from the 3' end of the gene.

[0016] In certain forms, the one or more defective interfering genes comprise the nucleotide sequence of any one of SEQ ID NO: 7-11, 14-16, or 18, or a nucleotide sequence having 75% or higher sequence identity with any one of SEQ ID NO: 7-11, 14-16, or 18. In some forms, the polynucleotide comprises the nucleotide sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0017] In some forms, when the virus is a coronavirus, such as a beta-coronavirus, more preferably SARS-CoV-2, the gene encodes a structural, non-structural, or accessory protein selected from ORF1a, ORF1b, S, M, ORF3a, ORF6, ORF7a, ORF8, and ORF10, or fragments thereof. In some forms, the nucleotide sequence comprises approximately 600 to 1200 nucleotides from the 5' end of the coronavirus genome, approximately 600 to 1200 nucleotides from the 3' end of the coronavirus genome, approximately 600 to 1200 nucleotides of the gene encoding ORF1b, or combinations thereof. In some forms, the one or more defective interfering genes comprise the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a nucleotide sequence having 75% or higher sequence identity with SEQ ID NO: 5 or SEQ ID NO: 6.

[0018] In any of the foregoing embodiments, the deletion may comprise approximately 400 to 2000 nucleotides or approximately 3000 to 27000 nucleotides relative to the virus upon which DIG is based. In some forms, the deletion comprises consecutive nucleotides. In some forms, the deletion comprises discontinuous nucleotides.

[0019] The present invention also provides a vector. In some forms, the vector contains the polynucleotides disclosed in this invention. The vector may also contain one or more promoters and / or polyadenylation signals operatively linked to one or more defective interfering genes contained in the polynucleotide. Preferably, the vector is an expression vector, such as a plasmid.

[0020] The present invention also describes compositions containing the polynucleotides or carriers disclosed herein. In some forms, the composition further comprises a peptide. Suitable peptides include TAT-P1 (YGRKKRRQRRRCWGPCPTAFRQIGNCGRFRVRCCRIR; SEQ ID NO: 3), TAT2-P1 (RKKRRQRRRCWGPCPTAFRQIGNCGRFRVRCCRIR; SEQ ID NO: 4), LAH4:KKALLAHALHLLALLALHLAHALKKA-NH2 (SEQ ID NO: 83), or combinations thereof. In some forms, the weight ratio of the peptide to the polynucleotide in the composition is in the range of about 2:1 to 4:1. Preferably, the weight ratio is 4:1.

[0021] In some forms, the composition may also comprise one or more additional disclosed polynucleotides or one or more additional disclosed vectors. In some forms, the composition comprises three of the disclosed vectors, wherein the first vector comprises the nucleotide sequence of SEQ ID NO: 12, wherein the second vector comprises the nucleotide sequence of SEQ ID NO: 13, and wherein the third vector comprises the nucleotide sequence of SEQ ID NO: 14.

[0022] In some forms, peptides are complexed with polynucleotides to form nanoparticles. Therefore, the disclosed compositions may comprise peptide-polynucleotide nanoparticles. In some forms, the average diameter of the nanoparticles is less than 200 nm or less than 150 nm. In some forms, the average diameter of the nanoparticles is about 135 nm.

[0023] The present invention also provides pharmaceutical compositions. In some forms, the pharmaceutical composition comprises any of the aforementioned compositions in combination with a pharmaceutically acceptable carrier or excipient.

[0024] This invention describes methods of using the disclosed nucleic acids and compositions thereof. This invention discloses a method for generating one or more defective interference genes, the method comprising introducing the disclosed vector into a host cell and incubating the host cell under conditions sufficient for polynucleotide expression, thereby generating the one or more defective interference genes.

[0025] The present invention also provides a method for reducing the replication of influenza virus or coronavirus in cells, the method comprising introducing a disclosed vector into cells under conditions suitable for the cells to produce defective viruses containing one or more RNAs transcribed from polynucleotides, thereby reducing viral replication.

[0026] This invention discloses a method for treating a subject with influenza virus or coronavirus infection by administering an effective amount of the disclosed pharmaceutical composition to the subject. The invention also describes a method for preventing or treating influenza virus or coronavirus-related illness in a subject, the method comprising administering an effective amount of the pharmaceutical composition to the subject. In some forms, the subject has been exposed to, is infected with, or is at risk of infection with, influenza virus or coronavirus. In some forms, the subject is immunocompromised.

[0027] The influenza virus can be selected from either influenza A or influenza B viruses. Suitable influenza virus strains include H1N1, H2N2, H3N2, H3N8, H5N1, or H7N9. In some forms, the coronavirus is either SARS-CoV-1 or SARS-CoV-2. Suitable SARS-CoV-2 virus strains or variants include: SARS-CoV-2 HKU-001a, SARS-CoV-2 B.1.1.7 (Alpha variant), SARS-CoV-2 B.1.351 (Beta variant), SARS-CoV-2 B.1.617.1 (Kappa variant), SARS-CoV-2 B.1.617.2 (Delta variant), and SARS-CoV-2 B.1.617.3.

[0028] In any of the aforementioned methods, the composition can be administered orally, intranasally, or intratracheally. Preferably, the subject is a human being.

[0029] Other advantages of the disclosed methods and compositions will be set forth in part in the description which follows, and in part will be understood from some of the description, or may be learned by practice of the disclosed methods and compositions. The advantages of the disclosed methods and compositions will be realized and obtained by means of the elements and combinations particularly pointed out in the appended claims. It should be understood that the foregoing general overview and the following detailed description are merely exemplary and explanatory, and do not limit the claimed invention. Attached Figure Description

[0030] The accompanying drawings, which are included in and form part of this specification, illustrate several embodiments of the disclosed methods and compositions and, together with the specification, serve to explain the principles of the disclosed methods and compositions.

[0031] Figure 1A This is a schematic diagram illustrating the construction of the defective interference gene (DIG). DIGs with internal deletions DI-PB2, DI-PB1, and DI-PA were generated via fusion PCR. Dashed lines represent internal deletions in the wild-type viral polymerase genes. The solid-line sequences of the shortened viral polymerase genes PB2, PB1, and PA shown are inserted into the PHW2000 vector. Figure 1B This is a bar chart showing the antiviral activity of various DIGs against A(H7N7) virus in A549 cells. Figure 1C This is a bar chart showing the dose-dependent antiviral efficiencies of single PAD4 and combinations of DIG-3 (including PAD3, PB2D3, and PB1D3) and DI-PAD4 (including PAD4, PB2D3, and PB1D3). A549 cells were transfected with plasmid DIG or the empty vector (PHW) at the indicated concentrations, and then infected with H7N7 virus 24 hours post-transfection. Viral titers in the cell supernatant were measured by plaque assay 48 hours post-infection. Figure 1D Survival curves show the protective effects of two doses of DIG against A(H1N1)-infected mice. Mice were intratracheally inoculated with DI-PAD4, PAD4, DIG-3, or an empty vector (PHW) packaged in TAT1-P1 at 48 and 24 hours prior to viral inoculation. Survival curves were generated from 5 mice in each group. Figure 1E This illustrates the survival curves showing the protective effect of a single dose of DIG against A(H1N1)-infected mice. Mice were intratracheally inoculated with DI-PAD4, PAD4, DIG-3, or PHW packaged in TAT1-P1 24 hours prior to viral inoculation. Survival curves were generated from groups of 10 mice.

[0032] Figure 2A The construction of coronavirus DIG is illustrated. Coronavirus DIG (CD2100 and CD3600) was synthesized based on the parental sequences of HKU-001a and inserted into the vector PHW2000. Dashed lines represent internal deletions in wild-type HKU001a (SARS-CoV-2). The three solid-line sequences linked together were inserted into the PHW2000 vector to generate CD2100 and CD3600. Figure 2BThis is a bar chart showing the expression of DIG RNA in 293T, Calu-3, and HK-2 cells. RNA expression in cells was measured by RT-qPCR using specific CD2100 and CD3600 primers 24 hours after plasmid transfection. Figure 2C This is a bar graph showing the antiviral activity of DIG in HK-2 cells. SARS-CoV-2 (B.1.1.63) was added to DIG-transfected cells for viral replication. Viral titers in cell supernatants were measured using a plaque assay 48 hours post-infection. Figure 2D This is a bar graph showing the dose-dependent antiviral activity of CD3600. One day prior to viral infection, HK-2 cells were transfected with either CD3600 or PHW (0.5 µg, 0.25 µg, or 0.125 µg / well). Viral titers in the cell supernatant were measured 48 hours post-infection. Figure 2E This is a bar chart showing the antiviral activity of CD3600 during multiple viral replication cycles after viral passage. Virus supernatant collected from HK-2 cells (transfected with PHW or CD3600 and infected with SARS-CoV-2) was passaged in Vero-E6 cells, and the viral titer in the supernatant was determined 24 hours post-infection. Figure 2F This is a bar chart showing the broad-spectrum antiviral activity of CD3600 against five SARS-CoV-2 variants.

[0033] Figure 3A This is a bar chart showing the transfection efficiency of TAT-P1, TAT2-P1, and jetPEI in mouse lungs. pCMV-Luc was packaged in the vector shown with a peptide:DNA weight ratio of 4:1. Luciferase expression in mouse lungs was measured 24 hours post-transfection. Luciferase expression in mouse lungs was normalized to 1 mg of protein, and TAT-P1 to 1000. Mock represents mice treated with 5% glucose without pCMV-Luc. Figure 3B This is a bar graph showing the hydrodynamic diameter of the pDI-PAD4 nanoparticles packaged in the carrier shown. Dimensions were measured using a DynaPro Plate Reader dynamic laser particle size analyzer. Figure 3C This demonstrates the use of nanoparticles TAT2-P1 / pCMV-Luc (1 mg / ml) respectively. -1 / 0.25 mg ml -1 ) and TAT2-P1 / pCMV-Luc (2 mg ml) -1 / 0.5 mg ml -1 A bar graph showing luciferase expression in transfected mouse lungs. Mock represents mouse lungs inoculated with TAT2-P1 without DNA.

[0034] Figure 4A Survival curves show the protective efficacy of antiviral agents administered to the lungs of mice 3 days prior to viral challenge. Three days prior to A(H1N1)pdm09 virus challenge, mice were intratracheally inoculated with TAT2-P1 / DI-PAD4 (20 µg / 5 µg), zanamivir (40 µg), or TAT2-P1 / PHW (20 µg / 5 µg). For vaccine treatment, mice were intratracheally inoculated with TAT2-P1 / PHW and intramuscularly injected with 480 ng of vaccine (Vaccine-480) 3 days prior to A(H1N1)pdm09 virus challenge. Figure 4B It shows the corresponding Figure 4A Changes in the body weight of infected mice. Figure 4C Survival curves show the protective efficacy of antiviral agents administered to the lungs of mice 5 days prior to viral challenge. Mice were intratracheally inoculated with TAT2-P1 / PHW, TAT2-P1 / DI-PAD4, or vaccine-480 5 days prior to A(H1N1)pdm09 virus challenge. Figure 4D It shows the corresponding Figure 4C Changes in body weight of infected mice. Survival curves were generated from groups of 5 to 10 mice. Figure 4E and 4F This is a bar graph showing the viral titer in hamster lungs 2 days post-infection. TAT2-P1 / CD3600 (50 µg / 12.5 µg) was intranasally inoculated into the hamster lungs one day (CD3600-D1) or 3 days (CD3600-D3) before SARS-CoV-2 (Delta) challenge. TAT2-P1 / PHW was administered to the hamster lungs one day before viral challenge. Lung tissue was collected on day 2 post-infection for viral load measurement using plaque assay and RT-qPCR.

[0035] Figures 5A to 5G This is a bar chart confirming that TAT2-P1 & LAH4 enhance gene expression and inhibit SARS-CoV-2 variants in vivo. Figure 5A Luciferase expression in 293T cells is shown. pCMV-Luc was packaged in the vectors shown (TAT2-P1, LAH4, and TAT2-P1:LAH4 = 3:2, 4:1 or 9:1) and transfected into 293T cells (n = 4). Figure 5B Luciferase expression in mouse lungs is shown (n = 4). pCMV-Luc was packaged in the vector shown and inoculated into mouse lungs. Luciferase expression in mouse lungs was detected 24 hours post-transfection. Figure 5C The peptide nanoparticle size (n = 4) of the plasmid packaged by the vector shown is shown. express P< 0.05, and express P <0.01. Figure 5D This study demonstrates that TAT2-P1 & LAH4 deliver CD3600 to significantly inhibit the replication of the Omicron (n = 3) variant in hamster lungs. One day prior to viral challenge, a single dose of CD3600 was intratracheally inoculated into the hamster lungs. Viral load in the hamster lungs was measured on day 2 post-infection. Figure 5E As shown, TAT2-P1 significantly inhibited SARS-CoV-2 infection in VeroE6 cells (n = 4). SARS-CoV-2 treated with the peptide shown was used in a plaque reduction assay. PFU (plaque forming units, %) is the number of plaques from the peptide-treated virus, normalized relative to the number of plaques from the untreated virus. express P <0.01. Figure 5F The study showed that hamster lungs were treated with two doses of PBS (n = 4), TAT2-P1 (n = 3), or TAT2-P1 & LAH4 with CD3600 (n = 4) or PHW (n = 3) 1 day before and 8 hours after SARS-CoV-2 infection in Omeprone. Figure 5G The study showed that PBS (n = 4) or TAT2-P1 & LAH4 with CD3600 was administered to the lungs of hamsters 1 day before and 8 hours after infection with Delta SARS-CoV-2. Viral load was measured on day 2 post-infection. express P < 0.05, and express P <0.01. P The values ​​were calculated using a two-tailed Student's t-test.

[0036] Figure 6 This is a bar chart showing the cytotoxicity of TAT2-P1 and LAH4 in 293T cells. The specified concentrations of peptide were incubated in 293T cells for 24 hours. Cell viability (%) is the OD value of peptide-treated cells, normalized relative to the OD value of untreated cells. Cytotoxicity was determined by the MTT assay. Data are expressed as mean ± SD of four biological samples.

[0037] Figure 7 This is a bar chart showing the stability of nanoparticles used for cell transfection. Luciferase plasmids packaged in TAT2-P1 & LAH4 (4:1) were prepared in 293T cells at 72 h, 24 h, and 15 min before transfection. Luciferase expression in cells was measured at 24 h post-transfection. Data are presented as mean ± SD of at least four biological samples. Detailed Implementation

[0038] The disclosed methods and compositions can be more readily understood by referring to the following detailed description of the specific embodiments and examples included therein, as well as the accompanying drawings and their preceding and following descriptions.

[0039] It has been found that a defective interfering gene of influenza virus delivered to the lungs of mice via TAT-P1 can protect mice from infection with influenza A virus [Zhao, H]. et al . Nat Commun , 9, 2358, Doi: 10.1038 / s41467-018-04792-7, (2018)]. Practical examples show that the antiviral activity of DIG in the lungs is related to the antiviral activity of DIG in lung tissue and the transfection efficiency of DIG. TAT-mediated transfection mainly occurs through endocytosis, including clathrin-mediated endocytosis [Richard, JP et al. , J Biol Chem 280, 15300-15306, Doi:10.1074 / jbc.M401604200, (2005)], caveolae / lipid-raft-mediated endocytosis[Ferrari, A. et al ., Mol Ther 8, 284-294; Doi: 10.1016 / s1525-0016(03)00122-9, (2003)], micropinocytosis [Wadia, JS, et al., Nat Med 10, 310-315; Doi:10.1038 / nm996, (2004)], and endocytosis independent pathways [Duchardt, F. et al ., Traffic 8, 848-866, Doi: 10.1111 / j.1600-0854.2007.00572.x. (2007)]. In the airways and lungs, passive cellular uptake of nanoparticles is primarily mediated by endocytosis and is influenced by nanoparticle size due to the barrier effect of airway mucus [Wang, Z]. et al. , ACS Nano 3, 4110-4116, Doi:10.1021 / nn9012274, (2009); Zhao, F. et al . Small7, 1322-1337, Doi: 10.1002 / smll.201100001, (2011); Duncan, GA, et al ., Mol Ther 24, 2043-2053, Doi:10.1038 / mt.2016.182 (2016); Foroozandeh, P. & Aziz, AA Nanoscale Res Lett 13, 339, Doi: 10.1186 / s11671-018-2728-6 (2018)]. One feasible way to increase uptake efficiency in the airways and lungs is to optimize nanoparticle size to reduce the barrier effect of mucus, thereby overcoming the limitations imposed by the average pore size (100 to 200 nm) in airway mucus [Duncan, GA, et al ., Mol Ther 24, 2043-2053, Doi:10.1038 / mt.2016.182 (2016); Mastorakos P. et al ., Proc Natl Acad SciU SA 112, 8720-8725, Doi: 10.1073 / pnas.1502281112 (2015)].

[0040] The studies described in the working examples demonstrate that the defective interfering influenza PA gene (PAD4) exhibits the best antiviral activity in human A549 cells compared to 12 other DIGs from the polymerase gene of segmented RNA influenza viruses. Treatment of mice with a combination of three defective interfering genes, DIG-4 (including PAD4, PB1D3, and PB2D3), conferred the best protection against A(H1N1)pdm09 virus compared to mice treated with PAD4 or DIG-3 alone. It was also found that the TAT2-P1 vector transfected the plasmid more efficiently in vivo and was less toxic than the TAT-P1 vector. Therefore, when CD3600 was administered to the lungs of hamsters one day prior to viral challenge, a single dose of TAT2-P1 / CD3600 inhibited SARS-CoV-2 replication in hamsters. It was also observed that when mice were administered DIG-4 3 days and 5 days before viral challenge, a single dose of TAT2-P1 / DIG-4 effectively protected 90% and 50% of mice from lethal influenza virus infection, respectively.

[0041] These experimental results demonstrate that a single dose of DIG for coronavirus or influenza virus can provide rapidly onset prophylactic protection in infected animals, thus offering an alternative to current clinical strategies for prophylactic treatment of coronavirus and influenza viruses. This is because DIG has a low likelihood of inducing drug-resistant viruses [Dimmock, NJ & Easton, AJ]. Journal of virology [88, 5217-5227, Doi: 10.1128 / JVI.03193-13, (2014)], so DIG can circumvent the drug resistance challenges associated with current methods.

[0042] It should be understood that, unless otherwise stated, the disclosed methods and compositions are not limited to specific synthetic methods, specific analytical techniques, or specific reagents, and therefore can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting.

[0043] I. Definition

[0044] In the context of genes or genomes, "defective interference" refers to genes derived from or associated with the parent virus. These genes are called "defective" because they lack the ability to encode all the viral proteins required for independent replication, and are therefore defective in the absence of the parent virus (also known as a helper virus). Thus, a helper virus is needed to trans-provide the missing replication proteins. DIGs are called "interferences" because they can attenuate symptoms caused by the helper virus. Typically, a DIG has one or more deletions relative to the parent viral gene or genome. Therefore, a DIG may have discontinuous portions of its corresponding helper viral gene or genome.

[0045] The terms "SARS-CoV-2" and "Severe Acute Respiratory Syndrome Coronavirus Type 2" refer to coronaviruses derived from beta coronaviruses. Sarbecovirus SARS-CoV-2 is a subgenus of pathogenic coronavirus strains that are the pathogens of the COVID-19 pandemic. The high rate of genetic mutation in the SARS-CoV-2 genome has led to the rapid development of multiple variants of the SARS-CoV-2 virus. During this pandemic, multiple variants of the virus causing COVID-19 have been recorded globally, including variants of B.1.1.7, B.1.351, and P.1.

[0046] The terms "influenza virus" and "influenza" are used interchangeably and refer to influenza A, influenza B, influenza C, and influenza D viruses. In the United States, human influenza A and B viruses cause seasonal epidemics (known as "flu season") in humans almost every winter. A global pandemic of influenza is called an "influenza pandemic" and often occurs when a new and very different influenza A virus emerges that can both infect humans and spread effectively between them. Based on the proteins involved, influenza A viruses can be classified into hemagglutinin subtypes or neuraminidase subtypes. There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes. Influenza A viruses are the primary cause of influenza pandemics.

[0047] In the context of nucleic acids, "correspondence" refers to the relationship where the first nucleotide sequence and the second nucleotide sequence encode the same product. These two nucleotide sequences do not need to be identical for correspondence, but they can be identical. One of these two nucleotide sequences can be designed based on the sequence of the other nucleotide sequence.

[0048] As used herein, “introduction” means contact. “Contact” refers to a state that allows or facilitates direct proximity or association between at least two elements. For example, introducing a composition (e.g., a disclosed polynucleotide or vector containing a defective interfering gene) into a cell is to establish contact between the cell and the composition. This term includes the penetration of the contacted composition into the cell interior by any suitable means, such as transfection, electroporation, transduction, gene gun, nanoparticle delivery, etc.

[0049] The term "operably linked" refers to a functional connection between a regulatory sequence and a heterologous nucleic acid sequence that allows them to function in the intended manner (e.g., causing the latter to be expressed). This term encompasses the localization of the sequence to be transcribed in the regulatory region and the nucleic acid in order to influence the transcription or translation of that sequence. For example, to bring the coding sequence under the control of a promoter, the translation initiation site of a polypeptide's translation reading frame is typically located 1 to approximately 50 nucleotides downstream of the promoter. However, the promoter can be located up to approximately 5000 nucleotides upstream of the translation initiation site or approximately 2000 nucleotides upstream of the transcription initiation site.

[0050] "Isolated" means altered or removed from its natural state. Isolated nucleic acids may exist in a substantially purified form or may exist in a non-natural environment (e.g., host cells). "Isolated" nucleic acids include nucleic acid segments or fragments that have been separated from sequences flanking them in their natural state, such as DNA fragments that have been removed from sequences adjacent to segments normally present in their natural genome. The term also applies to nucleic acids that have been substantially purified from other components naturally accompanying the nucleic acid (e.g., RNA, DNA, or proteins naturally accompanying the nucleic acid in cells). Thus, the term includes, for example, mRNA or recombinant DNA that has been integrated into a vector, integrated into an autonomously replicating plasmid or virus, integrated into the genomic DNA of a prokaryote or eukaryote, or existing as a separate molecule independent of other sequences (e.g., as cDNA or a fragment of genomic or cDNA produced by PCR or restriction enzyme digestion). Isolation does not require absolute purity and may include at least 50% isolated, for example, at least 75%, 80%, 90%, 95%, 98%, 99%, or even 99.9% isolated proteins, peptides, nucleic acids, or viral molecules.

[0051] The terms "inhibition" or "reduction" refer to a reduction, such as in activity, expression, or level. This can be a complete or partial inhibition of activity or expression. Inhibition can be compared to a control or standard level. Inhibition can be 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, or 51%. 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100%.

[0052] The term “prevention” as used in this article refers to the administration of a compound before the onset of clinical symptoms of a disease or condition in order to prevent abnormal physical symptoms associated with the disease or condition.

[0053] A “vector” is a composition of substances containing isolated nucleic acids and capable of delivering those isolated nucleic acids into the cell. Examples of vectors include, but are not limited to, linear polynucleotides, polynucleotides associated with ionic or amphiphilic compounds, plasmids, and viruses. Therefore, the term “vector” includes autonomously replicating plasmids or viruses. The term is also interpreted to include non-plasmid and non-viral compounds that facilitate the transfer of nucleic acids into cells, such as polylysine compounds, liposomes, etc. Examples of viral vectors include, but are not limited to, adenovirus vectors, adeno-associated virus vectors, retrovirus vectors, etc. An “expression vector” is a vector containing a polynucleotide having an expression control sequence operatively linked to the nucleotide sequence to be expressed. The expression vector contains sufficient cis-acting elements for expression; other elements for expression may be provided by the host cell or in an in vitro expression system. Expression vectors include all vectors known in the art, such as granules, plasmids (e.g., naked or contained in liposomes), phage particles, BACs, YACs, and viral vectors containing recombinant polynucleotides (e.g., vectors derived from lentiviruses, retroviruses, adenoviruses, and adeno-associated viruses).

[0054] The term "sequence identity percentage (%)" describes the percentage of nucleotides or amino acids in a candidate sequence that are identical to those in a reference nucleic acid sequence after aligning the sequences and introducing gaps (if necessary) to obtain the maximum sequence identity percentage. Alignments used to determine the sequence identity percentage can be performed in various ways known to those skilled in the art, such as using publicly available computer software like BLAST, BLAST-2, ALIGN, ALIGN-2, or Megalign (DNASTAR) software. Appropriate parameters used to measure the alignment, including any algorithms required to achieve maximum alignment across the full length of the sequences being compared, can be determined by known methods.

[0055] The percentage of sequence identity between a given nucleic acid or amino acid sequence C and a given nucleic acid or amino acid sequence D (which can be alternatively expressed as a percentage of specific sequence identity between a given sequence C and a given sequence D) is calculated as follows: 100 multiplied by the fraction W / Z, Where W is the number of nucleotides or amino acids that are considered identical matches in the sequence alignment program of C and D, and Z is the total number of nucleotides or amino acids in D. It should be understood that when the length of sequence C is not equal to the length of sequence D, the sequence identity % of C and D will not be equal to the sequence identity % of D and C.

[0056] The term "effective amount" refers to an amount sufficient to provide the desired pharmacological and / or physiological effect. The exact amount required can vary from subject to subject, depending on the subject's species, age and general condition, the severity of the disease being treated, the specific compound used, and its administration method, etc. Therefore, it is impossible to specify an exact "effective amount." However, those skilled in the art can determine a suitable effective amount using only routine experiments.

[0057] "Treatment" refers to the medical administration of a subject with the aim of curing, improving, stabilizing, or preventing a disease, pathological condition, or symptom. This term includes active treatment, i.e., treatment specifically aimed at improving a disease, pathological condition, or symptom, and also includes etiological treatment, i.e., treatment aimed at eliminating the cause of the related disease, pathological condition, or symptom. Furthermore, this term includes palliative treatment, i.e., treatment designed to relieve symptoms rather than cure a disease, pathological condition, or symptom; preventive treatment, i.e., treatment involving minimizing or partially or completely suppressing the development of a related disease, pathological condition, or symptom; and supportive treatment, i.e., treatment used to supplement another specific therapy aimed at improving a related disease, pathological condition, or symptom. It should be understood that while treatment aims to cure, improve, stabilize, or prevent a disease, pathological condition, or symptom, it is not actually necessary to result in a cure, improvement, stabilization, or prevention. The effectiveness of treatment can be measured or assessed in a manner as described herein and as known in the art suitable for the disease, pathological condition, or symptom involved. Such measurement and assessment can be performed in a qualitative and / or quantitative manner. Therefore, for example, the characteristics or features of a disease, pathological condition or symptom and / or the symptoms of a disease, pathological condition or symptom can be reduced to any effect or any amount.

[0058] "Pharmaceutical acceptable" means that the material is not biologically or otherwise undesirable, meaning that the material can be administered to a subject together with the selected compound without causing any undesirable biological effects or interacting in a harmful manner with any other component of the pharmaceutical composition in which the material is contained.

[0059] As used herein, the term "subject" refers to any individual, organism, or entity. Typical subjects include animals (e.g., mammals such as mice, rats, rabbits, goats, pigs, chimpanzees or horses, non-human primates, and humans) and / or plants. The term does not indicate a specific age or sex. Therefore, it is expected to encompass adult and newborn subjects, as well as fetuses, whether male or female. Subjects may be healthy or have or be susceptible to diseases, conditions, or symptoms.

[0060] Unless otherwise stated herein, the enumeration of numerical ranges is intended only as a shorthand for individually referring to each individual value falling within the range, and each individual value is incorporated into this specification as if it were individually enumerated herein.

[0061] The term “about” is used to describe values ​​that are about + / - 10% above or below the stated value; in other forms, these values ​​may be about + / - 5% above or below the stated value; in other forms, these values ​​may be about + / - 2% above or below the stated value; in other forms, these values ​​may be about + / - 1% above or below the stated value; the foregoing ranges are intended to clarify the context and do not imply any further limitations.

[0062] II. Composition

[0063] 1. Nucleic acid

[0064] This invention discloses a nucleic acid containing a defective interference gene (DIG), which is designed based on influenza virus and coronavirus species and can significantly inhibit the infection and replication of homologous viruses.

[0065] Specifically, the present invention discloses isolated polynucleotides containing one or more defective interfering genes, wherein each of the one or more defective interfering genes contains a nucleotide sequence corresponding to one or more portions of a viral gene or genome, wherein the nucleotide sequence contains deletions in the gene. More specifically, the portion of the viral gene contains deletions relative to the viral gene. Preferably, the virus is an influenza virus (e.g., influenza A virus, influenza B virus, or influenza C virus) or a coronavirus.

[0066] In some forms, when the virus is an influenza virus, the gene encodes an RNA polymerase or its subunits, such as, but not limited to, PA, PB1, and PB2. In some forms, the nucleotide sequence may contain an internal (central) deletion in the gene. More specifically, the viral gene portion contained in the nucleotide sequence contains a deletion relative to the source viral gene. In some forms, the nucleotide sequence contains about 150 to 600 nucleotides from the 5' end of the gene, about 150 to 600 nucleotides from the 3' end of the gene, or a combination thereof. Preferably, an internal deletion is present that prevents the gene fragment from producing a functional protein, but is not large enough to prevent the viral gene fragment from being packaged into a viral particle. The 150 nucleotides from the 5' and 3' ends are essential for viral replication and packaging and are therefore retained. Even more preferably, the nucleotide sequence contains about 450 nucleotides from the 5' end of the gene and about 450 nucleotides from the 3' end of the gene.

[0067] In certain forms, the one or more defective interfering genes comprise the nucleotide sequence of any one of SEQ ID NO: 7-11, 14-16, or 18, or a nucleotide sequence having 75% or higher sequence identity with any one of SEQ ID NO: 7-11, 14-16, or 18. In some forms, the polynucleotide comprises the nucleotide sequences of SEQ ID NO: 14, SEQ ID NO: 15, and SEQ ID NO: 16.

[0068] In some forms, when the virus is a coronavirus, such as a beta-coronavirus, more preferably SARS-CoV-2, the gene encodes a structural, non-structural, or accessory protein selected from ORF1a, ORF1b, S, and M, or fragments thereof. In some forms, the nucleotide sequence comprises approximately 600 to 1200 nucleotides from the 5' end of the coronavirus genome, approximately 600 to 1200 nucleotides from the 3' end of the coronavirus genome, approximately 600 to 1200 nucleotides of the gene encoding ORF1b, or combinations thereof. In some forms, the one or more defective interfering genes comprise the nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a nucleotide sequence having 75% or higher sequence identity with SEQ ID NO: 5 or SEQ ID NO: 6.

[0069] In any of the foregoing embodiments, the deletion may comprise approximately 400 to 2000 nucleotides or approximately 3000 to 27000 nucleotides relative to the virus upon which DIG is based. In some forms, the deletion comprises consecutive nucleotides. In some forms, the deletion comprises discontinuous nucleotides.

[0070] The present invention also describes compositions containing the polynucleotides or carriers disclosed herein. In some forms, the composition further comprises a peptide. Suitable peptides include TAT-P1 (YGRKKRRQRRRCWGPCPTAFRQIGNCGRFRVRCCRIR; SEQ ID NO: 3), TAT2-P1 (RKKRRQRRRCWGPCPTAFRQIGNCGRFRVRCCRIR; SEQ ID NO: 4), LAH4 (KKALLAHALHLLALLALHLAHALKKA-NH2; SEQ ID NO: 83), or combinations thereof. In some forms, the weight ratio of the peptide to the polynucleotide in the composition is in the range of about 2:1 to 4:1. Preferably, the weight ratio is 4:1.

[0071] In some forms, peptides are complexed with polynucleotides to form nanoparticles. Therefore, the disclosed compositions may comprise peptide-polynucleotide nanoparticles. In some forms, the average diameter of the nanoparticles is less than 200 nm or less than 150 nm. In some forms, the average diameter of the nanoparticles is about 135 nm.

[0072] Influenza viruses can be selected from either influenza A or influenza B viruses. Suitable influenza virus strains include H1N1, H2N2, H3N2, H3N8, H5N1, or H7N9. In some forms, the coronavirus is SARS-CoV-2. Suitable SARS-CoV-2 virus strains or variants include: SARS-CoV-2 HKU-001a, SARS-CoV-2 B.1.1.7 (Alpha variant), SARS-CoV-2 B.1.351 (Beta variant), SARS-CoV-2 B.1.617.1 (Kappa variant), SARS-CoV-2 B.1.617.2 (Delta variant), and SARS-CoV-2 B.1.617.3.

[0073] a. Coronavirus DIG

[0074] In some implementations, DIG is derived from one or more coronaviruses. Coronaviruses (order Nidovirales, family Coronaviridae, and genus Coronaviruses) are a variety of large, enveloped, positive-sense RNA viruses that cause respiratory and intestinal diseases in humans and other animals.

[0075] Coronaviruses typically have narrow host specificity and can cause severe disease in many animals, and several viruses, including infectious bronchitis virus, feline infectious peritonitis virus, and porcine transmissible gastroenteritis virus, are important veterinary pathogens. Human coronavirus (HCoV) has been found in both group 1 (HCoV-229E) and group 2 (HCoV-OC43) and has historically caused approximately 30% of mild upper respiratory tract illnesses.

[0076] At approximately 30,000 nucleotides, their genomes are the largest found among any RNA virus. Three groups of coronaviruses exist; groups 1 and 2 contain mammalian viruses, while group 3 contains only avian viruses. Within each group, coronaviruses are further classified into different species based on host range, antigenic relationships, and genomic organization. The genomic organization is typical of coronaviruses, characterized by characteristic gene sequences at both ends (5'-replicaase [rep], spike [S], envelope [E], membrane [M], nucleocapsid [N]-3') and short untranslated regions. The SARS-CoV replicase gene (comprising approximately two-thirds of the genome) encodes two polyproteins (encoded by ORF1a and ORF1b) that undergo co-translational proteolytic processing. Downstream of rep are four developing reading frames (ORFs), which are predicted to encode the structural proteins S, E, M, and N common to all known coronaviruses.

[0077] In some implementations, DIG is derived from the SARS-CoV-2 β-coronavirus, a subgenus of Sarbecovirus. The SARS-CoV-2 virus shares approximately 75% genomic sequence identity with the SARS-CoV virus identified in 2003. An exemplary nucleic acid sequence of the SARS-CoV-2 ORF1a / b gene is listed in GenBank accession number MN908947.3. The genomic organization of the SARS-CoV-2 virus is similar to that of other β-coronaviruses; the six functional open reading frames (ORFs) are arranged from 5' to 3' as follows: replicase (ORF1a / ORF1b), spike (S), envelope (E), membrane (M), and nucleocapsid (N). Furthermore, seven putative ORFs encoding accessory proteins are scattered among the structural genes.

[0078] In some preferred embodiments, the DIG comprises one or more SARS-CoV-2 nucleic acid sequences from one or more genes encoding structural (S, E, M, N) or non-structural (NSP) SARS-CoV-2 proteins. In some embodiments, the DIG comprises one or more SARS-CoV-2 genes, or gene expression products having selected epitopes in the SARS-CoV-2 genome conserved in a variety of different coronaviruses. In some embodiments, the SARS-CoV-2 variant is selected from SARS-CoV-2B.1.1.7 (Alpha variant), SARS-CoV-2 B.1.351 (Beta variant), SARS-CoV-2 B.1.617, SARS-CoV-2 B.1.617.1 (Kappa variant), SARS-CoV-2 B.1.617.2 (Delta variant), and SARS-CoV-2B.1.617.3.

[0079] b. Influenza virus DIG

[0080] In some implementations, DIG is a defective influenza virus particle. Influenza virus DIG can be derived from a specific branch or strain of influenza, or it can be a synthetic DIG designed to correspond to highly conserved genes in a variety of different influenza virus strains.

[0081] There are four types of influenza viruses: A, B, C, and D. Human influenza A and B viruses cause seasonal epidemics. Influenza A virus is the only known influenza virus to cause a pandemic (i.e., a global influenza epidemic). Influenza C virus infection usually causes mild illness and is not considered a potential cause of a human influenza pandemic. Influenza D virus primarily affects cattle, and it is unclear whether it can infect or cause disease in humans (see www.cdc.gov / flu / about / viruses / types.htm).

[0082] Influenza A virus particles are covered with glycoprotein spikes consisting of hemagglutinin (HA) and neuraminidase (NA) in a ratio of approximately four to one, protruding from a host cell-derived lipid membrane. A smaller number of matrix (M2) ion channels traverse the lipid envelope, with an M2:HA ratio of approximately one M2 channel for every 10¹–10² HA molecules. The envelope and its three intact membrane proteins, HA, NA, and M2, cover the matrix surrounding the M1 protein core of the virion. Within the M1 matrix lies a nuclear export protein (NEP; also known as non-structural protein 2, NS2) and ribonucleoprotein (RNP) complex, which comprises a viral RNA fragment coated with nucleoprotein (NP) and a heterotrimeric RNA-dependent RNA polymerase composed of two "basic polymerase" and one "acidic polymerase" subunit (PB1, PB2, and PA). Influenza B virus has a similar organizational structure and four envelope proteins: HA, NA, NB (instead of M2), and BM2. Therefore, in some embodiments, DIG is derived from one or more of the HA, NA, M2, NS2, NB, PB1, PB2, PA, or NP genes of any influenza A or B virus. In a specific embodiment, DIG is derived from the PA gene of an influenza A or B virus (Bouvier and Palese P, Vaccine. 2008;26 Suppl 4(Suppl 4):D49-D53. doi:10.1016 / j.vaccine.2008.07.039).

[0083] The genomes of influenza A and B viruses each consist of eight negative-sense single-stranded viral RNA (vRNA) segments, while the genome of influenza C virus has seven segments. The eight segments of influenza A and B viruses (and the seven segments of influenza C virus) are numbered in descending order of length. In influenza A and B viruses, segments 1, 3, 4, and 5 each encode only one protein: PB2, PA, hemagglutinin (HA) protein, and nucleoprotein (NP). All influenza viruses encode the polymerase subunit PB1 on segment 2; in some influenza A virus strains, this segment also encodes the accessory protein PB1-F2 in the +1 alternation reading frame. PB1-F2 is a small 87-amino acid protein with pro-apoptotic activity. No analogues of PB1-F2 have been found in influenza B or C viruses. Conversely, segment 6 of the influenza A virus encodes only the NA protein, while segment 6 of the influenza B virus encodes both the NA protein and, within the -1 alternating reading frame, the NB matrix protein, which is the integrated membrane protein corresponding to the M2 protein in the influenza A virus. Segment 7 of both influenza A and B viruses encodes the M1 matrix protein. In the influenza A virus genome, the M2 ion channel is also expressed from segment 7 via RNA splicing, while in influenza B virus it encodes its BM2 membrane protein within the +2 alternating reading frame. Finally, both influenza A and B viruses possess a single RNA segment (segment 8) from which they express the interferon antagonist NS1 protein and, via mRNA splicing, express NEP / NS2, which is involved in the export of viral RNPs from the host cell nucleus. Influenza A viruses are classified into different subtypes based on the hemagglutinin (H) and neuraminidase (N) proteins on the viral surface. There are 18 different hemagglutinin subtypes and 11 different neuraminidase subtypes (H1 to H18 and N1 to N11, respectively). Therefore, in some embodiments, DIG is derived from the PA gene of the influenza virus. In other embodiments, DIG is derived from the PB1 and / or PB2 genes of the influenza virus. Although there may be 198 different combinations of influenza A virus subtypes, only 131 subtypes have actually been detected. Currently, the influenza A virus subtypes that routinely circulate in humans include A(H1N1) and A(H3N2). Therefore, in some embodiments, DIG is derived from either the A(H1N1) or A(H3N2) influenza virus, or provides immunity against either the A(H1N1) or A(H3N2) influenza virus. In some embodiments, DIG is conserved among all A(H1N1) influenza viruses and / or provides immunity against all A(H1N1) influenza viruses. In some embodiments, DIG is conserved among all A(H3N2) influenza viruses and / or provides immunity against all A(H3N2) influenza viruses. In a preferred embodiment, the antigen is conserved among all A(H1N1) influenza viruses and / or provides immunity against all A(H1N1) influenza viruses.

[0084] Influenza A viruses are further classified into several subtypes (e.g., H1N1 or H3N2), while influenza B viruses are classified into one of two lineages: B / Yamagata and B / Victoria. Both influenza A and B viruses can be further classified into specific branches and subbranches. Alternatively, branches and subbranches can be referred to as “groups” and “subgroups,” respectively. Influenza branches or groups are further subdivisions of influenza viruses (in addition to subtypes or lineages) based on the similarity of their HA gene sequences. Branches and subbranches appear as a group of viruses on a phylogenetic tree that typically have similar genetic changes (i.e., nucleotide or amino acid changes) and share a common ancestor represented as a node in the tree. Branches and subbranches that are genetically different from other branches or subbranches are not necessarily antigenically different (i.e., viruses from a particular branch or subbranch may not have changes that affect host immunity compared to other branches or subbranches). In some embodiments, DIG is conserved among two or more influenza viruses in the same subtype and / or subclade, and / or provides immunity against two or more influenza viruses in the same subtype and / or subclade. In a preferred embodiment, DIG is conserved among two or more influenza viruses in different subtypes and / or subclades, and / or provides immunity against two or more influenza viruses in different subtypes and / or subclades. In some embodiments, DIG is conserved among all influenza viruses in the same subtype and / or subclade, and / or provides immunity against all influenza viruses in the same subtype and / or subclade. In a preferred embodiment, DIG is conserved among multiple influenza viruses in different subtypes and / or subclades, and / or provides immunity against multiple influenza viruses in different subtypes and / or subclades.

[0085] The currently circulating influenza A(H1N1) virus is linked to the 2009 H1N1 pandemic that occurred in the spring of 2009 and caused the influenza pandemic (see www.cdc.gov / flu / about / viruses / types.htm). This virus is called “A(H1N1)pdm09 virus” or “2009 H1N1”, and it has been circulating seasonally from 2009 to 2021. Over time, these H1N1 viruses have undergone relatively minor genetic and antigenic changes.

[0086] Among influenza viruses that circulate in humans and cause human disease, the A(H3N2) influenza virus changes more rapidly genetically and antigenically, forming many separate, genetically distinct branches that continue to co-circulate. Therefore, in some embodiments, DIG is derived from all currently circulating H1N1 influenza viruses and / or provides immunity against all currently circulating H1N1 influenza viruses. In some embodiments, DIG is derived from all currently circulating H3N2 influenza viruses and / or provides immunity against all currently circulating H3N2 influenza viruses. In a preferred embodiment, DIG is derived from both currently circulating H1N1 and H3N2 influenza viruses and / or provides immunity against both currently circulating H1N1 and H3N2 influenza viruses. In some embodiments, DIG is derived from the PA gene of influenza A virus or the expression product of the PA gene of influenza A virus.

[0087] Influenza B virus is classified into two lineages: B / Yamagata and B / Victoria. Influenza B virus is also classified into specific branches and sub-branches. Compared to influenza A virus, influenza B virus changes more slowly in terms of genetic and antigenic characteristics. Recent surveillance data show that influenza B viruses from both lineages co-circulate in the United States and globally. Therefore, in some implementations, DIG is derived from influenza B virus and / or provides immunity against influenza B virus. In some implementations, DIG is derived from all currently circulating influenza B viruses and / or provides immunity against all currently circulating influenza B viruses. In some implementations, DIG is derived from the expression product of the influenza B virus NP gene or the influenza B virus PA gene.

[0088] In some embodiments, DIG is derived from B / Yamagata and B / Victoria influenza viruses, and / or provides immunity against B / Yamagata and B / Victoria influenza viruses. In other embodiments, DIG is derived from one or more H1N1 influenza viruses and one or more influenza B viruses, and / or provides immunity against one or more H1N1 influenza viruses and one or more influenza B viruses. In other embodiments, DIG is derived from one or more H3N2 influenza viruses and one or more influenza B viruses, and / or provides immunity against one or more H3N2 influenza viruses and one or more influenza B viruses. In other embodiments, DIG is derived from one or more H1N1 influenza viruses, one or more H3N2 influenza viruses, and one or more influenza B viruses, and / or provides immunity against one or more H1N1 influenza viruses, one or more H3N2 influenza viruses, and one or more influenza B viruses.

[0089] c. Other viruses DIG

[0090] In some implementations, DIG is isolated from viruses including, but not limited to, those from any of the following viral families: Arenaviridae ( Arenaviridae Arteritis virus genus ( Arterivirus ), Astroviridae ( Astroviridae Baculoviridae ( Baculoviridae ), Baculoviruses ( Badnavirus ), Bacteroidetes ( Barnaviridae ), Dual RNA Virology ( Birnaviridae ), Broccoli mosaic virus family ( Bromoviridae Amazon pneumonia virus ( Bunyaviridae ), Caliciviridae ( Caliciviridae ), Genus Phytovirus ( Capillovirus ), Carnation Latent Virus ( Carlavirus ), Cauliflower mosaic virus (Cauliflower mosaic virus) Caulimovirus ), Porconiidae ( Circoviridae ), genus of linear viruses ( Closterovirus ), Cowpea mosaic virus family ( Comoviridae ), genus *Gnaphalidae* ( Corticoviridae ), Cysticophages ( Cystoviridae Hepatitis D virus ( Deltavirus ), Carnation ringspot virus genus ( Dianthovirus ), Ototomyc virus genus ( Enamovirus Filoviridae ( Filoviridae For example, Marburg virus and Ebola virus (such as Zaire, Reston, Ivory Coast, or Sudan strains), Flaviviridae ( Flaviviridae For example, hepatitis C virus, dengue virus type 1, dengue virus type 2, dengue virus type 3, and dengue virus type 4), and hepatotropic DNA virus family ( Hepadnaviridae Herpesviridae ( Herpesviridae For example, herpesviruses 1, 3, 4, 5, and 6, as well as cytomegaloviruses and attenuated viruses (VVVs). Hypoviridae ), Iridoviridae ( Iridoviridae ), smooth phage family ( Leviviridae ), Lipophageae ( Lipothrixviridae Microphages ( Microviridae Orthomyxoviridae ( Orthomyxoviridae ), Papillomavirus Family ( Papovaviridae ), Paramyxoviridae ( Paramyxoviridae For example, measles, mumps, and human respiratory syncytial virus), parvovirus family ( Parvoviridae), Picornaviridae ( Picornaviridae For example, poliovirus, rhinovirus, hepatitis virus, and foot-and-mouth disease virus), poxvirus family ( Poxviridae For example, vaccinia virus and smallpox virus), Reoviridae ( Reoviridae For example, rotaviruses), retroviruses ( Retroviridae For example, lentiviruses, such as human immunodeficiency virus (HIV) type 1 and HIV type 2, and rhabdoviridae ( Rhabdoviridae For example, rabies virus, measles virus, respiratory syncytial virus, etc.), and the family Phaeoviridae ( Togaviridae For example, rubella virus, dengue virus, etc.) and monoviridae ( Totiviridae ).

[0091] Viral DIG can be derived from specific viral strains, such as human papillomavirus (HPV), herpesviruses (e.g., herpes simplex virus types 1 and 2); hepatitis viruses, such as hepatitis A virus (HAV), hepatitis B virus (HBV), hepatitis C virus (HCV), hepatitis D virus (HDV), hepatitis E virus (HEV), and hepatitis G virus (HGV)); tick-borne encephalitis virus; parainfluenza virus, varicella-zoster virus, cytomegalovirus, Epstein-Barr virus, rotavirus, rhinovirus, adenovirus, Coxsackievirus, Venezuelan equine encephalitis virus, Japanese encephalitis virus, yellow fever virus, Rift Valley fever virus, and lymphocytic choriomeningitis virus.

[0092] 2. Expression vehicle

[0093] In some implementations, polynucleotides are integrated into the vector or a portion thereof. Methods for constructing expression vectors containing genetic sequences and suitable transcriptional and translational control elements are well known in the art. These methods include in vitro recombinant DNA techniques, synthetic techniques, and in vivo genetic recombination. Expression vectors typically contain regulatory sequences and elements necessary for the translation and / or transcription of an inserted coding sequence, which may be, for example, a target polynucleotide. The coding sequence can be operatively linked to promoters and / or enhancers to help control the expression of the desired gene product. Promoters used in biotechnology vary depending on the intended type of gene expression control. They are generally classified as constitutive promoters, tissue-specific or developmental stage-specific promoters, inducible promoters, and synthetic promoters.

[0094] For example, in some embodiments, the nucleic acid encoding the defective interfering particle is operatively linked to a promoter or other regulatory element known in the art. Thus, the polynucleotide can be a vector, such as an expression vector. The engineering of polynucleotides for expression in prokaryotic or eukaryotic systems can be performed using techniques generally known to those skilled in the art of recombinant expression. Expression vectors typically comprise one of a composition under the control of one or more promoters. To bring the coding sequence “under” the control of a promoter, the 5’ end of the translation start site of the reading frame is typically positioned “downstream” from the selected promoter (i.e., the 3’) by about 1 to 50 nucleotides. The “upstream” promoter stimulates transcription of the inserted DNA and promotes the expression of the encoded recombinant protein. This is the meaning of “recombinant expression” in the context used herein.

[0095] Many standard techniques can be used to construct expression vectors containing appropriate nucleic acids and transcription / translation control sequences for the expression of proteins or peptides in a variety of host expression systems. Cell types that can be used for expression include, but are not limited to, mammalian cells and bacteria transformed with recombinant phage DNA, plasmid DNA, or coliform DNA expression vectors. It should be understood that any of these vectors can be used for packaging and delivery of DIG.

[0096] Expression vectors used in mammalian cells typically include an origin of replication (if needed), a promoter preceding the gene to be expressed, and any necessary ribosome binding sites, RNA splicing sites, polyadenylation sites, and transcription terminator sequences. The origin of replication can be provided by constructing a vector containing a foreign origin, for example, derived from SV40 or other viruses (e.g., polyomaviruses, adenoviruses, VSVs, BPVs), or it can be provided through the host cell's chromosomal replication mechanism. If the vector integrates into the host cell's chromosome, the latter is usually sufficient.

[0097] Promoters are derived from the genome of mammalian cells (e.g., metallothionein promoters) or mammalian viruses (e.g., adenovirus late promoters; vaccinia virus 7.5K promoters). Furthermore, it is possible and likely desirable to use promoters or control sequences that are typically associated with the desired gene sequence, provided that the control sequence is compatible with the host cell system.

[0098] Many virus-based expression systems can be used; for example, commonly used promoters are derived from polyomaviruses, adenovirus type 2, cytomegaloviruses, and simian virus 40 (SV40). Early and late promoters of SV40 are useful because both are readily available from the virus as fragments containing the SV40 viral origin of replication. Smaller or larger SV40 fragments can also be used, provided they contain approximately 250 bp of sequence extending from the HindIII site to the BglI site located at the viral origin of replication.

[0099] When using adenovirus as an expression vector, the coding sequence can be linked to the adenoviral transcription / translation control complex, such as a late promoter and a triple leader sequence. This chimeric gene can then be inserted into the adenoviral genome via in vitro or in vivo recombination. Insertion into a non-essential region of the viral genome (e.g., region E1 or E3) produces a live recombinant virus capable of expressing the protein in an infected host.

[0100] Efficient translation of the composition may also require specific initiation signals. These signals include the ATG start codon and adjacent sequences. Exogenous translation control signals, including the ATG start codon, may also be required. Those skilled in the art can readily determine this requirement and provide the necessary signals. It is well known that the start codon must be in-frame (or in phase) with the reading frame of the desired coding sequence to ensure translation of the entire insert. These exogenous translation control signals and start codons can be derived from a variety of natural and synthetic sources. Expression efficiency can be improved by including appropriate transcriptional enhancer elements or transcription terminators.

[0101] In eukaryotic expression, if the original clone does not contain a suitable polyadenylation site, it is generally desirable to incorporate a suitable polyadenylation site into the transcription unit. Typically, the poly A addition site is located approximately 30 to 2000 nucleotides downstream of the protein termination site, pre-transcriptional termination.

[0102] Stable expression is preferred for long-term, high-yield production of recombinant proteins. For example, cell lines that stably express constructs encoding proteins can be engineered. Unlike expression vectors containing viral replication sources, host cells can be transformed using vectors controlled by appropriate expression control elements (e.g., promoters, enhancers, sequences, transcription terminators, polyadenylate sites, etc.) and optional markers. After introducing exogenous DNA, engineered cells can be grown in enrichment media for 1 to 2 days and then transferred to selective media. Optional markers in recombinant plasmids provide resistance to selection and allow cells to stably integrate the plasmid into the chromosome, grow into aggregates, and subsequently clone and expand into cell lines.

[0103] In a preferred embodiment, the polynucleotide cargo is RNA, such as mRNA. The mRNA may encode the target polypeptide. In some embodiments, the mRNA has a cap at the 5' and / or 3' poly(A) tail, which can regulate ribosome binding, translation initiation, and mRNA stability in the cell.

[0104] 3. Pharmaceutical preparations

[0105] The present invention also provides pharmaceutical formulations comprising any of the aforementioned compositions in combination with a pharmaceutically acceptable carrier or excipient.

[0106] A pharmaceutical formulation is prepared using a pharmaceutically acceptable "carrier" composed of materials considered safe and effective, and can be administered to an individual without causing undesirable biological side effects or undesirable interactions. A "carrier" is any component present in a pharmaceutical formulation other than the active ingredient. The term "carrier" includes, but is not limited to, diluents, binders, lubricants, disintegrants, fillers, and coating compositions. For detailed information on materials, equipment, and processes used to prepare tablets and delayed-release dosage forms, see Pharmaceutical Dosage Forms: Tablets, eds. Lieberman et al. (New York: Marcel Dekker, Inc., 1989), and Ansel et al., Pharmaceutical Dosage Forms and Drug Delivery Systems, 6.sup.th Ed. (Media, PA: Williams & Wilkins, 1995).

[0107] III. Preparation Method

[0108] This document describes methods for preparing the disclosed nucleic acid compositions containing defective interfering genes and pharmaceutical formulations thereof. Examples of methods for preparing and validating the disclosed nucleic acid DIG are described in the examples. Defective interfering viral RNA for integration into viruses is typically generated via recombinant methods. In some forms, the vector contains the disclosed polynucleotide. The vector may also contain one or more promoters and / or polyadenylation signals operatively linked to one or more defective interfering genes contained in the polynucleotide. Preferably, the vector is an expression vector, such as a plasmid. Many techniques and methods for preparing the disclosed DIG, polynucleotides, vectors, and compositions are known and suitable for this use.

[0109] The present invention discloses a method for generating one or more defective interference genes, the method comprising introducing a disclosed vector into a host cell and incubating the host cell under conditions sufficient to express the polynucleotide, thereby generating the one or more defective interference genes.

[0110] IV. Instructions for Use

[0111] The present invention also provides methods of using the disclosed nucleic acid composition containing a defective interfering gene and the pharmaceutical formulation thereof. The present invention provides a method for reducing the replication of influenza virus or coronavirus in cells, the method comprising introducing the disclosed vector into the cell under conditions suitable for the cell to produce a defective virus comprising one or more RNAs transcribed from the said polynucleotide, thereby reducing viral replication.

[0112] In some forms, the method can be used to treat a subject with influenza virus or coronavirus infection by administering an effective amount of the disclosed pharmaceutical composition to the subject. In some forms, the method can be used to prevent or treat a subject with influenza virus or coronavirus-related illness by administering an effective amount of the pharmaceutical composition to the subject. In some forms, the subject has been exposed to, infected with, or is at risk of infection with influenza virus or coronavirus. In some forms, the subject is immunocompromised.

[0113] In some forms, the method involves using a defective interfering gene as an antiviral agent. The influenza virus can be selected from either influenza A or influenza B viruses. Suitable influenza virus strains include H1N1, H2N2, H3N2, H3N8, H5N1, or H7N9. In some forms, the coronavirus is either SARS-CoV-1 or SARS-CoV-2. Suitable SARS-CoV-2 virus strains or variants include: SARS-CoV-2 HKU-001a, SARS-CoV-2 B.1.1.7 (Alpha variant), SARS-CoV-2 B.1.351 (Beta variant), SARS-CoV-2 B.1.617.1 (Kappa variant), SARS-CoV-2 B.1.617.2 (Delta variant), and SARS-CoV-2 B.1.617.3. In all cases, the sequence is positive from 5' to 3' (reverse genomic significance). This sequence is also represented as DNA.

[0114] The disclosed defective interfering virus gene can also be used to induce a host immune response. For example, the disclosed defective interfering virus gene can be used in vaccine compositions. The disclosed defective interfering virus gene can also be used for broad-spectrum antiviral activity by inducing a host immune response.

[0115] In any of the aforementioned methods, the composition can be administered orally, intranasally, or intratracheally. Preferably, the subject is a human being.

[0116] V. Reagent Kit

[0117] The disclosed polynucleotides, reagents, compositions, and other materials can be packaged together in any suitable combination as a kit for carrying out or assisting in the carrying out of the method. It is useful if the components in a given kit are designed and suitable for use in the method.

[0118] For example, a kit comprising a vaccine or other composition for administration to a subject may include a predetermined dose of the composition in a sterile needle, ampoule, tube, container, or other suitable container. The kit may include instructions for dosage and administration. In some forms, the composition is lyophilized. The kit may also include reagents (e.g., saline, buffer solution) and instructions for forming the formulation for administration. The instructions may specify suitable storage conditions for the kit and its components.

[0119] Kits for producing proteins are also provided. Such kits may include the disclosed polynucleotides (e.g., plasmids or other expression vectors), viruses, and / or instructions for use. The kit may also include reagents and instructions for transfecting or transducing recipient cells.

[0120] The disclosed compositions and methods can be further understood through the following numbered paragraphs.

[0121] 1. A separated polynucleotide comprising one or more defective interference genes, wherein each of the one or more defective interference genes comprises a nucleotide sequence corresponding to one or more portions of a viral gene, wherein the portion of the viral gene comprises a deletion relative to the viral gene, wherein the virus is an influenza virus or a coronavirus.

[0122] 2. The polynucleotide as described in paragraph 1, wherein the virus is an influenza A virus, an influenza B virus, or an influenza C virus.

[0123] 3. The polynucleotide as described in paragraph 2, wherein the gene encodes an RNA polymerase or a subunit thereof selected from PA, PB1, and PB2.

[0124] 4. The polynucleotide as described in any one of paragraphs 1 to 3, wherein a portion of the viral gene contains an internal deletion relative to the viral gene.

[0125] 5. The polynucleotide as described in any one of paragraphs 1 to 4, wherein the nucleotide sequence comprises about 150 to 600 nucleotides from the 5' end of the gene and about 150 to 600 nucleotides from the 3' end of the gene.

[0126] 6. The polynucleotide as described in paragraph 5, wherein the nucleotide sequence comprises approximately 450 nucleotides from the 5' end of the gene and approximately 450 nucleotides from the 3' end of the gene.

[0127] 7. The polynucleotide as described in any one of paragraphs 1 to 6, wherein at least one of the one or more defective interference genes comprises a nucleotide sequence of SEQ ID NO: 5-10, 13-15 or 17, or a nucleotide sequence of SEQ ID NO: 5-10, 13-15 or 17 having 75% or higher sequence identity.

[0128] 8. The polynucleotide as described in any one of paragraphs 1 to 7, wherein the polynucleotide collectively comprises the nucleotide sequences of SEQ ID NO:12, SEQ ID NO:13 and SEQ ID NO:14.

[0129] 9. The polynucleotide as described in paragraph 1, wherein the virus is a coronavirus, preferably a β-coronavirus, more preferably SARS-CoV-2.

[0130] 10. The polynucleotide as described in paragraph 9, wherein the gene encodes a structural, non-structural, accessory protein or fragment thereof selected from ORF1a, ORF1b, S, M, ORF3a, ORF6, ORF7a, ORF8 and ORF10.

[0131] 11. The polynucleotide as described in paragraph 9 or 10, wherein the nucleotide sequence comprises about 600 to 1200 nucleotides from the 5' end of the coronavirus genome, about 600 to 1200 nucleotides from the 3' end of the coronavirus genome, about 600 to 1200 nucleotides of a gene encoding ORF1b, or a combination thereof.

[0132] 12. The polynucleotide as described in any one of paragraphs 9 to 11, wherein at least one of the one or more defective interference genes comprises a nucleotide sequence of SEQ ID NO: 5 or SEQ ID NO: 6, or a nucleotide sequence having 75% or higher sequence identity with SEQ ID NO: 5 or SEQ ID NO: 6.

[0133] 13. The polynucleotide as described in any one of paragraphs 1 to 12, wherein the deletion comprises about 400 to 2000 nucleotides or about 3000 to 27000 nucleotides, optionally wherein the nucleotide is a continuous or discontinuous polynucleotide.

[0134] 14. A vector comprising the polynucleotide described in any one of paragraphs 1 to 13, wherein the vector comprises one or more promoters and / or polyadenylation signals operatively linked to the one or more defective interference genes.

[0135] 15. The vector as described in paragraph 14, wherein the vector is an expression vector, preferably a plasmid.

[0136] 16. A method for generating one or more defective interference genes, the method comprising introducing the vector described in paragraph 14 or 15 into a host cell and incubating the host cell under conditions sufficient for polynucleotide expression, thereby generating the one or more defective interference genes.

[0137] 17. A method for reducing the replication of influenza virus or coronavirus in a cell, the method comprising introducing a vector as described in paragraph 14 or 15 into the cell under conditions suitable for the cell to produce a defective virus comprising one or more RNAs transcribed from a polynucleotide, thereby reducing viral replication.

[0138] 18. A composition comprising a polynucleotide as described in any one of paragraphs 1 to 4 or a carrier as described in paragraphs 14 or 15.

[0139] 19. The composition as described in paragraph 18 further comprises a peptide selected from the group consisting of: TAT-P1 (YGRKKRRQRRRCWGPCPTAFRQIGNCGRFRVRCCRIR; SEQ ID NO: 3), TAT2-P1 (RKKRRQRRRCWGPCPTAFRQIGNCGRFRVRCCRIR; SEQ ID NO: 4), LAH4 (KKALLAHALHLLALLALHLAHALKKA-NH2; SEQ ID NO: 83), or a combination thereof.

[0140] 20. The composition as described in paragraph 19, wherein the weight ratio of peptide to polynucleotide is in the range of about 2:1 to 4:1, preferably 4:1.

[0141] 21. The composition as described in paragraph 19 or 20, wherein the peptide is complexed with a polynucleotide to form a plurality of nanoparticles.

[0142] 22. The composition as described in paragraph 21, wherein the nanoparticles have an average diameter of less than 200 nm, preferably less than 150 nm, more preferably about 135 nm.

[0143] 23. The composition of any one of paragraphs 18 to 22, further comprising one or more additional polynucleotides as described in any one of paragraphs 1 to 4 or one or more additional vectors as described in paragraphs 14 or 15.

[0144] 24. The composition as described in paragraph 23, wherein the composition comprises three of the carriers, wherein the first carrier comprises the nucleotide sequence of SEQ ID NO: 12, wherein the second carrier comprises the nucleotide sequence of SEQ ID NO: 13, and wherein the third carrier comprises the nucleotide sequence of SEQ ID NO: 14.

[0145] 25. A pharmaceutical composition comprising the composition as described in any one of paragraphs 17 to 24 and a pharmaceutically acceptable carrier or excipient.

[0146] 26. A method of treating a subject with an influenza virus or coronavirus infection, the method comprising administering to the subject an effective amount of the pharmaceutical composition as described in paragraph 25.

[0147] 27. A method for preventing or treating a subject with an influenza virus or coronavirus-related illness, the method comprising administering to the subject an effective amount of the pharmaceutical composition as described in paragraph 25.

[0148] 28. The method as described in paragraph 26 or 27, wherein the subject has been exposed to, infected with, or is at risk of infection with the influenza virus or coronavirus.

[0149] 29. The method of any one of paragraphs 26 to 28, wherein the subject is immunocompromised.

[0150] 30. The method as described in any one of paragraphs 26 to 29, wherein the influenza virus is an influenza A virus or an influenza B virus, or wherein the coronavirus is SARS-CoV-2.

[0151] 31. The method as described in paragraph 30, wherein the influenza A virus is selected from H1N1, H2N2, H3N2, H3N8, H5N1 and H7N9.

[0152] 32. The method as described in paragraph 30, wherein SARS-CoV-2 is selected from: SARS-CoV-2 HKU-001a, SARS-CoV-2 B.1.1.7 (Alpha variant), SARS-CoV-2 B.1.351 (Beta variant), SARS-CoV-2 B.1.617.1 (Kappa variant), SARS-CoV-2 B.1.617.2 (Delta variant), and SARS-CoV-2 B.1.617.3.

[0153] 33. The method of any one of paragraphs 26 to 32, wherein the composition is administered orally, intranasally, or intratracheally.

[0154] 34. The method as described in any one of paragraphs 26 to 33, wherein the subject is a human being.

[0155] Example

[0156] Example 1: Generation and characterization of antiviral DIG exhibiting effective activity against influenza virus and SARS-CoV-2 virus

[0157] Materials and methods

[0158] Cell and virus cultures

[0159] Martin Darby canine kidney (MDCK, ATCC CCL-34), 293T (ATCC CRL-3216), A549 (ATCC CCL-185), Vero-E6 (ATCC CRL-1586), VeroE6-TMPRSS2 (VeroE6-T), Calu-3 (ATCC HTB-55) [Zhao] et al. , Nat Commun 12, 1517, doi: 10.1038 / s41467-021-21825-w (2021)] and HK-2 cells [Yeung et al. , Cell [184, 2212-2228.e2212, Doi: 10.1016 / j.cell.2021.02.053(2021)] Supplemented with 10% fetal bovine serum (FBS), 100 IU / ml -1 Penicillin and 100 μg ml -1 Streptomycin was cultured in Dulbecco Minimal Medium (DMEM) or DMEM-F12K. The viral strains used in this study included A / HongKong / 415742Md / 2009 (H1N1) [Zhao, H]. et al ., Sci Rep 6, 22008, Doi: 10.1038 / srep22008 (2016)], A / Netherlands / 219 / 2003 (H7N7) [Zhao, H. et al., Virology 498,1-8, 10.1016 / j.virol.2016.08.004 (2016)], and SARS-CoV-2 variants [Zhao, H]. et al. , Nat Commun 11, 4252, Doi: 10.1038 / s41467-020-17986-9 (2020) and Chen,LL et al., Clin Infect Dis[Doi: 10.1093 / cid / ciab656 (2021)]. Influenza virus was cultured in MDCK cells, while SARS-CoV-2 was cultured in VeroE6 or VeroE6-T cells, and viral titers were determined by plaque assay.

[0160] Construction and Expression of Coronavirus DIG

[0161] Based on previous research that identified possible packaging genes for coronaviruses [Qin, L]. et al ., Acta Pharmacol Sin , 24, 489-496 (2003); Masters, PS Virology 537, 198-207, Doi:10.1016 / j.virol.2019.08.031 (2019); Seim, I., Roden, CA & Gladfelter, AS bioRxiv [Doi: 10.1016 / j.bpj.2021.06.018 (2021)], the 5' end sequence (approximately 700 and 1200 bp), the internal sequence in ORF 1b, and the 3' end sequence of SARS-CoV-2 in HKU-001a were synthesized (GenBank: MT230904.1, see Table 1). The synthesized gene was inserted into the PHW2000 vector to generate plasmids CD2100 and CD3600 expressing the defective gene of SARS-CoV-2. The DNA sequence of the constructed plasmid with CoV-DIG was verified by Sanger sequencing. Twenty-four hours after transfection, the expression of CD2100 and CD3600 in 293T, A549, and HK-2 cells was measured by RT-qPCR. To determine DIG RNA expression, all RNA samples were treated with DNase I (QIAGEN, Cat# 79254) according to the manufacturer's instructions and further purified using the RNeasy mini kit (Qiagen, Cat# 74106). RNA expression levels were detected by RT-qPCR using DIG primers (see Table 2).

[0162] Table 1: Sequence of Coronavirus DIG

[0163]

[0164]

[0165]

[0166]

[0167] Table 2: Primers for RT-qPCR

[0168] Influenza virus DIG construction

[0169] Based on previous studies of DIGs with approximately 300 nucleotides at the 3' and 5' ends, DIGs with approximately 150 nucleotides at the 3' and 450 nucleotides at the 5' ends were constructed [Zhao, H]. et al . Dual-functional peptide with defective interfering genes effectively protects mice against avian and seasonal influenza. Nat Commun 9, 2358 (2018), see Table 3]. Based on its antiviral activity in A549 cells, DIG was further constructed with approximately 225 nucleotides at the 3' and 600 nucleotides at the 5' ends. The full-length sequences of the wild-type A / WSN / 1933 PA, PB1, and PB2 genes were used as templates to generate defective interfering PA, PB1, and PB2 genes (DI-PA, DI-PB1, and DI-PB2). Short gene fragments at the 3' and 5' ends of DIG were amplified using gene-specific primers designed using Primer Premier 5.0 (see Table 3). The amplified short gene fragments at the 3' and 5' ends were fused by fusion PCR [Heckman, KL & Pease, LR Gene splicing and mutagenesis by PCR-driven overlap extension]. Nat Protoc 2 [924-932 (2007)], using two pairs of primers for each gene to generate DI-PA, DI-PB1, and DI-PB2 genes (see Table 4). The fused DI-PA, DI-PB1, and DI-PB2 genes (Table 3) were inserted into the BsmBI / BsaI site of the PHW2000 vector to generate plasmids for DI-PA, DI-PB1, and DI-PB2, respectively. The DNA sequences of the construct plasmids containing DIG were verified by Sanger sequencing.

[0170] Table 3: Influenza virus DIG sequence

[0171]

[0172]

[0173]

[0174]

[0175]

[0176]

[0177]

[0178]

[0179] Table 4: Primers used for DIG construction

[0180]

[0181] In vitro DIG transfection and antiviral assay

[0182] For in vitro antiviral experiments, plasmids containing DI-PA, DI-PB1, DI-PB2, CD1200, CD3600, and the empty vector PHW2000 were transfected into A549 or HK-2 cells using Lipofectamine 3000 reagent (Invitrogen, Cat# 1857483) or GeneJuice (Sigma, Cat# 70967), according to the manufacturer's instructions. Twenty-four hours after transfection, the cell transfection medium was replaced with fresh DMEM containing 0.005 MOI A(H7N7) virus or 0.01 MOI SARS-CoV-2 for infection in A549 cells (influenza virus) or HK-2 cells (SARS-CoV-2). Supernatant was collected 48 hours post-infection. Viral titers were determined using the aforementioned plaque assay [Zhao, H]. et al ., Sci Rep 6, 22008, Doi:10.1038 / srep22008 (2016)]. The antiviral activity of DIG was demonstrated by comparing the viral titers in the supernatant of cells transfected with DIG plasmid or empty vector PHW.

[0183] Toxicity analysis

[0184] TAT-P1 and TAT2-P1 were synthesized by China Peptide (Shanghai, China, see Table 5). Cytotoxicity was determined by detecting the 50% cytotoxic concentration (CC50) using a tetrazolium-based colorimetric MTT assay as described above [Zhao, H]. et al ., Sci Rep 6, 22008, Doi: 10.1038 / srep22008 (2016)]. 293T cells were cultured at 3 × 10⁻⁶. 4 Initially, cells were seeded at a density of 10% FBS in 96-well cell culture plates using DMEM supplemented with 10% FBS. After overnight culture, the cell culture medium was replaced with fresh DMEM supplemented with various concentrations of peptides and 1% FBS. After incubation at 37°C for 24 hours, MTT solution (5 mg / ml) was added to each well. －1 The nanoparticles were incubated at 37°C for 4 hours (10 μl / well) and then 100 μl of 0.01 M HCl solution containing 10% SDS was added to each well. After further incubation at room temperature and shaking overnight, the plate was read at OD570 using a Victor™ X3 Multilabel Reader (PerkinElmer, USA). Cell culture wells without peptides were used as experimental controls, and the culture medium itself was used as a blank control. The in vivo toxicity of the nanoparticles was measured by testing the weight loss in mice intratracheally inoculated with TAT-P1 / DNA (20 μg / 5 μg), TAT2-P1 / DNA (20 μg / 5 μg), and in vivo jetPEI / DNA (0.7 μl / 5 μg).

[0185] Table 5: Peptide Sequences

[0186] Gel retardation analysis

[0187] Based on previous research [Zhao, H.] et al.Nat Commun [9, 2358, Doi: 10.1038 / s41467-018-04792-7, (2018)], peptides and DNA were premixed with 0.5 μg plasmid DNA in 4 μl distilled water at room temperature for 15 min at various weight ratios. The samples were loaded into 1% agarose gels containing ethidium bromide nucleic acid dye. Gel electrophoresis was performed in TBE buffer at 120 V for 20 min, and the agarose gels were then observed under UV irradiation.

[0188] Fluid dynamics particle size measurement

[0189] Based on previous research [Zhao, H.] et al. Nat Commun [9, 2358, Doi: 10.1038 / s41467-018-04792-7, (2018)], peptides and DNA were prepared in various weight ratios. Peptide and plasmid DNA solutions were prepared separately in distilled water. Equal volumes of peptide and plasmid DNA solutions were mixed together to obtain a final volume of 4 μl containing 0.5 μg of plasmid DNA. After incubating the complex at room temperature for 15 minutes, the 4 μl complex was diluted to 50 μl in distilled water, and the particle size was measured using a DynaPro® Plate Reader dynamic laser particle size analyzer (Wyatt, USA).

[0190] Transmission electron microscopy analysis

[0191] To determine the size and shape of the virus or nanoparticles, peptide / DNA nanoparticles premixed at a peptide:DNA weight ratio of (4:1) were applied to a continuous carbon grid, and excess solution was removed. The grid was transferred to 4% uranium acetate and incubated for 1 minute. After removing the solution, the grid was air-dried at room temperature. [Zhao, H.] et al ., Nat Commun [12,1517, Doi: 10.1038 / s41467-021-21825-w (2021)]. For each peptide / DNA nanoparticle, TEM images were acquired using a FEITecnal G2-20 TEM, and two independent experiments were performed.

[0192] In vitro luminescence assay

[0193] Peptides and plasmid DNA were mixed with 0.5 μg of plasmid DNA in 4 μl of distilled water at various weight ratios. After incubation at room temperature for 15 minutes, 293T cells in 24-well plates were transfected with the peptide / DNA complex, which contained the peptide and 0.1 μg of each pHW2000 plasmid encoding the small genomes (pLuciferase, firefly luciferase reporter) of PA, PB1, PB2, NP, and pPoLI-fluc-RT [Zhao, H]. et al . Virology 498, 1-8, Doi: 10.1016 / j.virol.2016.08.004 (2016)]. 24 hours after transfection, the assay was performed using a Victor X3 Multilabel reader (PerkinElmer, USA) and a luciferase assay system (Promega, Cat). # (E1910) Measure luminescence. Normalize the luminescence readings to 1 mg of protein.

[0194] In vivo bioluminescence analysis

[0195] A peptide complex containing pCMV-Cypridina Luc (pCMV-Luc, ThermoFisher, Cat# RF233236) was prepared in 40 μl of distilled water with 5 μg of plasmid DNA at a peptide:DNA weight ratio of 4:1. After incubating the complex at room temperature for 15 minutes, it was intratracheally inoculated into mouse lungs, and luciferase expression in the lung tissue was measured 24 hours later. A jetPEI / pCMV-Luc complex (0.7 μl / 5.0 μg) was prepared as a positive control according to the manufacturer's protocol (Polyplus Transfection, Cat# 201-10G). Mouse lungs inoculated with either the peptide or jetPEI alone were used as negative controls. To detect bioluminescent signals, mouse lung tissue was homogenized and centrifuged at 15,000 × g for 10 minutes. The supernatant was used to analyze luciferase protein expression using the Cypridina luciferase rapid assay kit (ThermoFisher, Cat# 16168). The expression level of luciferase in mouse lungs was normalized to 1 mg of protein, and TAT-P1 was set to 1000 as the normalization reference value for analysis. For in vivo bioluminescence imaging, mouse lungs were removed, and the substrate was added to the lungs for imaging using the IVIS® Spectrum in vivo optical imaging system (PerkinElmer, USA).

[0196] RT-qPCR assay

[0197] Real-time RT-qPCR was performed as described above [Zhao, H.]. et al ., Sci Rep 6, 22008, Doi:10.1038 / srep22008 (2016)]. RNA was reverse transcribed into cDNA using the PrimeScript II 1st Strand cDNA Synthesis Kit (Takara, Cat# 6210A) with primers Uni-12 or random primers on a GeneAmp® PCR system 9700 (Applied Biosystems, USA). cDNA was then amplified using LightCycle® 480 SYBR GreenI Master (Roach, USA, see Table 3) with primers specific to DI-PA, DI-PB1, DI-PB2, cytokines, and chemokines. For quantitative analysis, 10 μL of each reaction was prepared. 1 Up to 10 6Ten-fold serial dilutions of 1 copy of the standard plasmid were used to generate calibration curves. Real-time qPCR experiments were performed using the LightCycler® 96 system (Roche, USA).

[0198] In vivo antiviral assay

[0199] Antiviral analysis of nanoparticles TAT-P1 / DIG and TAT2-P1 / DIG was performed in BALB / c mice and hamsters [Zhao, H.]. et al ., Nat Commun 12, 1517, Doi: 10.1038 / s41467-021-21825-w(2021)]. Female BALB / c mice (for H1N1 virus, 10–12 weeks old) were obtained from the University of Hong Kong Centre for Comparative Medicine Research, and female hamsters (for SARS-CoV-2 virus, 4–6 weeks old) were obtained from the Laboratory Animal Service Centre of the Chinese University of Hong Kong through the University of Hong Kong Centre for Comparative Medicine Research. Animals were housed in a biosafety level 2 / 3 laboratory (housed at 22–25°C, dark / light cycle) with free access to standard pelleted feed and water. All experimental protocols followed approved standard operating procedures for animal facilities at biosafety level 2 and were approved by the Live Animal Use Committee for Teaching and Research of the University of Hong Kong [Zheng, BJ]. et al ., Proceedings of the National Academy of Sciences of the United States of America [105, 8091-8096, Doi: 10.1073 / pnas.0711942105(2008)]. Mouse-adapted A(H1N1)pdm09 virus was used for lethal challenge in mice. To evaluate prophylactic efficacy, mice were intratracheally inoculated with 40 μl of TAT-P1 / DIG (20.0 μg / 5.0 μg), TAT2-P1 / DIG (20.0 μg / 5.0 μg), or zanamivir (40.0 μg / mouse, in PBS) 1 to 10 days prior to viral challenge. Influenza vaccine-480 (480.0 ng, dissolved in PBS) was intramuscularly injected into mice. Subsequently, 3 LD 50The virus was intranasally inoculated into mice. Survival and overall condition were monitored for 16 days or until death. For DIG, an anti-SARS-CoV-2 (B.1.617.2, Delta) conjugate in hamsters, TAT2-P1 / CD3600 or TAT2-P1 / PHW (50.0 μg / 12.5 μg) was inoculated into the lungs of hamsters 1 or 3 days before viral inoculation (500 PFU). Experimental animals were randomly assigned to each group. Viral load and histopathological changes in the lungs of hamsters were tested on day 2 post-infection.

[0200] result

[0201] Coronavirus DIG inhibits SARS-CoV-2 replication in human HK-2 cells.

[0202] To date, research demonstrating whether defective genes in coronaviruses significantly interfere with coronavirus replication is very limited [Vignuzzi, M. & López, CB]. Nat Microbiol 4, 1075-1087, Doi: 0.1038 / s41564-019-0465-y (2019)]. To identify the DIG against coronaviruses, based on the possible packaging sequence of SARS-CoV [Qin, L]. et al ., Acta Pharmacol Sin 24, 489-496, (2003); Masters, PS Virology [537, 198-207, Doi: 10.1016 / j.virol.2019.08.031 (2019)], constructed coronavirus DIG (CD2100 and CD3600), which includes the 5' end sequence of SARS-CoV-2, the internal sequence in ORF 1b, and the ' end sequence ( Figure 2A Gene expression was first tested in human cell lines including 293T, Calu-3, and HK-2. High RNA levels of CD2100 and CD3600 were confirmed in the transfected HK-2 cell line, similar to RNA expression in 293T cells. Figure 2B Therefore, HK-2 was selected for the antiviral assay of CD2100 and CD3600. After transfecting HK-2 cells with DIG, SARS-CoV-2 was added to the cells for infection. Compared with the empty vector PHW, CD3600 and CD2100 significantly inhibited SARS-CoV-2 replication. Figure 2C CD3600 exhibited significantly better antiviral activity than CD2100. To further confirm the antiviral effect of CD3600 in cells, it was demonstrated that CD3600 significantly inhibited SARS-CoV-2 replication in a dose-dependent manner compared to PHW. Figure 2D ).

[0203] The ability of CoV-DIG to exert antiviral activity across multiple growth cycles was tested, which may contribute to the expansion of DIG's antiviral activity. It was observed that, compared to the non-DIG vector (PHW) control, at 24 hours post-infection, the virus in the supernatant of CD3600-containing cells showed significantly less viral replication during viral passage in VeroE6 / TMPRSS2 cells. Figure 2E This indicates that transfected CD3600 exerts extended anti-SARS-CoV-2 replication activity even during multiple growth cycles of viral passage in VeroE6 / TMPRSS2 cells. To confirm the presence of CD3600-deficient virus in the passage supernatant of VeroE6 / TMPRSS2 cells, PHW-treated and CD3600-treated viruses with the same PFU were cultured, and the supernatant was collected 72 hours post-infection for ultracentrifugation to recover the virus for TEM assays. Compared with PHW-treated cell culture supernatant (100–150 nm), CD3600-treated cell culture supernatant showed an increase in small spherical particles (< 40 nm). This result indicates that treatment with CD3600 leads to the packaging of smaller defective virus particles. Furthermore, RT-qPCR data showed a higher CD3600 RNA copy number but a lower PFU titer in the supernatant compared to PHW-treated virus. Furthermore, CD3600 demonstrated broad antiviral activity against five other SARS-CoV-2 variants, consistent with the broad antiviral mechanism of DIG, which involves the consumption of viral replication and its packaging components, competitively inhibiting viral packaging without targeting specific viral proteins. In summary, these results indicate that CoV-DIG, possessing the 5' end sequence of SARS-CoV-2, the internal sequence in ORF 1b, and the 3' end sequence, can broadly inhibit SARS-CoV-2 replication by maintaining its self-sustaining DIG production across multiple growth cycles.

[0204] The optimal defective interference gene against influenza virus in A549 cells and mice

[0205] To identify effective antiviral DIGs against influenza virus, 10 DIGs of different gene lengths, ranging from 150 nt to 600 nt, with 3' and 5' ends of the polymerase gene, were designed and constructed. Figure 1A To identify DIGs with more potent antiviral activity in vitro and in vivo, each DIG was transfected into A549 cells, followed by infection with A(H7N7) virus 24 hours post-transfection, without the addition of trypsin to the cell culture. Figure 1BPAD4, containing 450 nt at the 3' and 5' ends of the PA gene fragment, showed the strongest antiviral activity, reducing viral load by 3-4 log, which was 4 to 10 times lower than that of PAD3, PB1D3, and PB2D4 used in previous studies [Zhao, H]. et al . Nat Commun , 9, 2358, Doi:10.1038 / s41467-018-04792-7, (2018)]. PB2D3, PB1D3, PAD2, and PAD3 showed very strong antiviral activity, inhibiting viral replication by 2-3 log. PB2D2, PB2D4, PB1D2, PB1D4, and PAD5 showed weak antiviral activity, with or without significant differences ( ). Figure 1B PB2D1, PB1D1, and PAD1, with approximately 150 nt at both the 3' and 5' ends, did not show antiviral activity, consistent with previous studies demonstrating that polymerase genes require at least 150 nt at the 5' end for viral gene packaging [Duhaut, SD & Dimmock, NJ]. J Gen Virol 83, 403-411, Doi: 10.1099 / 0022-1317-83-2-403. (2002); Duhaut, SD & Dimmock, NJ Virology [248, 241-253, Doi: 10.1006 / viro.1998.9267 (1998)]. These data indicate that PAD4 with 450 nt of both 3' and 5' ends exhibits the best antiviral activity. PB2D3 with 300 nt of both 3' and 5' ends and PB1D3 with 300 nt of both 3' and 5' ends showed better antiviral activity than DIG that was too short or too long, respectively. Next, the antiviral activity of PAD4 alone was further tested, along with different concentrations of two combinations of DIG (…). Figure 1C A comparison was made. PAD4 alone showed stronger antiviral activity in a dose-dependent manner, with viral load > 5 times lower than that of DIG-3 (a combination of PAD3, PB2D3, and PB1D3) or DI-PAD4 (a combination of PAD4, PB2D3, and PB1D3).

[0206] Considering that multiple DIGs would have a greater chance of interfering with multiple homologous full-length genes in vivo, DI-PAD4, single PAD4, and DIG-3 were chosen to evaluate their antiviral efficacy in mice. Theoretically, the three DIGs could produce seven types of defective interfering influenza viruses, which, compared to a single DIG, could increase the chance of inhibiting wild-type virus replication in mouse lungs. For in vivo transfection of DIGs, DIGs packaged in a TAT-P1 vector were intratracheally injected into the lungs of mice. Administration of two doses of DI-PAD4, single PAD4, or DIG-3 at 48 hours and 24 hours before infection provided 100%, 80%, and 80% protection against lethal challenge with A(H1N1)pdm09 virus, respectively. Figure 1D This was significantly better than the empty plasmid PHW (0%). For a regimen involving a single intratracheal administration 24 hours prior to infection ( Figure 1E Mice receiving DI-PAD4 (90%) had significantly higher survival rates than mice receiving either PAD4 (50%) or DIG-3 (50%) alone. Survival data indicate that the combination of DI-PAD4 provides better protection in mice than either PAD4 or DIG-3 alone. Therefore, DI-PAD4 was chosen for further in vivo studies.

[0207] TAT2-P1 showed gene delivery efficiency in lung tissue.

[0208] To demonstrate the superior antiviral activity of DIG in vivo, the in vivo delivery efficiency of TAT-peptide-based vectors was investigated. Previous studies have shown that TAT2 (RKKRRQRRR; SEQ ID NO: 1) is a shorter peptide form that exhibits better transduction efficiency in vitro than TAT (YGRKKRRQRRR; SEQ ID NO: 2) [Park, J]. et al . J Gen Virol 83, 1173-1181, doi: 10.1099 / 0022-1317-83-5-1173 (2002); Vives, E.,Brodin, P. & Lebleu, B. J Biol Chem [272, 16010-16017, doi: 10.1074 / jbc.272.25.16010 (1997)]. Therefore, the TAT2-P1 vector was constructed to evaluate its in vitro and in vivo transfection efficiency compared to TAT-P1.

[0209] First, the DNA-binding capacity of these peptide-based vectors was determined using gel retardation analysis. When the peptide to plasmid DNA weight ratio was greater than 2, the plasmid DNA could be completely packaged by these vectors. Transfection efficiency indicated that a peptide:DNA weight ratio (4:1) showed effective transfection in cells. Next, to evaluate the DNA transfection efficiency of these vectors in vivo, pCMV-Luc packaged with TAT-P1, TAT2-P1, or in vivo jetPEI was intratracheally injected into mice, and luciferase expression was measured 24 hours post-transfection. TAT2-P1 showed significantly better transfection efficiency in mouse lungs than TAT-P1. Figure 3A TAT2-P1 is less toxic than TAT-P1 in cells and in mice.

[0210] To identify the mechanism by which TAT2-P1 achieves higher in vivo transfection efficiency, the particle size of the peptide / pCMV-Luc particles was measured. pCMV-Luc particles packaged with TAT-P1 and TAT2-P1 formed particles with average diameters of 148 nm and 134 nm, respectively. Figure 3B TEM images of these nanoparticles show that the particles formed by TAT2-P1 are uniform spherical nanoparticles and smaller than TAT-P1 nanoparticles. These data suggest that the high transfection efficiency of TAT2-P1 in mouse lungs may be related to the smaller particle size of TAT2-P1, but lack statistical significance.

[0211] To confirm that particle size affects TAT2-P1 gene delivery in the lungs, TAT2-P1 / pCMV-Luc of different sizes were prepared by mixing TAT2-P1 with different concentrations of DNA, but using the same amount of peptide / DNA and a ratio (4:1). TAT2-P1 / pCMV-Luc (2 mg / ml) -1 / 0.5 mg ml -1 The particle size (approximately 190 nm) is larger than that of TAT2-P1 / pCMV-Luc (1 mg / ml). -1 / 0.25 mg ml -1 The particle size (approximately 130 nm) is larger. Furthermore, in mouse lungs, pCMV-Luc packaged with TAT2-P1 (2 mg ml-1) showed significantly lower transfection efficiency compared to pCMV-Luc packaged with TAT2-P1 (1 mg ml-1). Figure 3C However, in 293T cells, compared with TAT2-P1 (1 mg ml) -1 Compared to TAT2-P1 (2 mg / ml), -1 The transfection efficiency of TAT2-P1 (2 mg / ml) was not affected. This result in 293T cells indicates that the transfection efficiency of TAT2-P1 (2 mg / ml) was unaffected.-1 The transfection efficiency of the plasmid DNA packaged in the mouse lungs was lower than that of TAT2-P1 (1 mg / ml). -1 The difference in DNA packaging efficiency under these two conditions is not due to variations in the two conditions. Therefore, only the particle size difference of the TAT2-P1 / DNA nanoparticles significantly affects gene delivery of TAT2-P1 in mouse lungs. These data indicate that TAT2-P1 / DNA can form uniform spherical nanoparticles smaller than 140 nm, which can penetrate the barrier of average pore size (100 to 200 nm) in airway mucus, thereby increasing DNA uptake in the lungs.

[0212] Digitized Iodine (DIG) Prophylactic Activity Against Influenza Virus and SARS-CoV-2

[0213] To investigate the prophylactic protection of DIG in vivo, a single dose of TAT2-P1 / DIG-4 was used to evaluate its prophylactic protection against influenza virus infection in mice compared with the vaccine (Influsplit Tetra) and zanamivir [Zhao, H.]. et al . Nat Commun 9, 2358, doi: Nat Commun (2018)]. When using body weight as a reference, the vaccine dose (480 ng) is almost 80 times higher than the vaccine dose used in humans [Groves, HT]. et al.. Front Immunol 9,126, doi: 10.3389 / fimmu.2018.00126 (2018)]. Mice administered TAT2-P1 / DI-PAD4 3 days before infection had a significantly higher survival rate (90%) than mice administered vaccine-480 (20%), zanamivir (20%), or TAT2-P1 / PHW (0%, negative control) 3 days before infection. Figure 4A Compared with the TAT2-P1 / PHW group, mice treated with TAT2-P1 / DI-PAD4 showed significantly less weight loss from day 2 to day 8. Figure 4B Mice treated with zanamivir one day before viral infection had a 90% survival rate. Zanamivir provided effective protection one day before infection, but no significant protection was provided three days before infection, consistent with zanamivir's short half-life. For mice treated with TAT2-P1 / DIG-4 five days before viral challenge (… Figure 4C The survival rate (50%) was significantly higher than that of mice treated with vaccine-480 (10%) or TAT2-P1 / PHW (0%). Mice treated with TAT2-P1 / DIG-4 showed less weight loss from day 4 to day 8 compared to mice treated with PHW. Figure 4DWhen administered to mice 10 days before viral challenge, vaccine-480 showed a 40% survival rate, consistent with the fact that the vaccine requires more than 1 to 2 weeks to induce immune protection.

[0214] To investigate the antiviral activity of coronavirus CD3600 against SARS-CoV-2 in vivo, TAT-P1 / CD3600 was inoculated into the lungs of hamsters 1 or 3 days before SARS-CoV-2 challenge. The viral titer in the lungs of hamsters reached its peak 2 days post-infection, indicating a non-lethal SARS-CoV-2 model. [Chan, JF] et al . Clin Infect Dis , 71(9):2428-2446. doi: 10.1093 / cid / ciaa325 (2020); Kaptein, SJF et al . Proc Natl Acad Sci USA 117, 26955-26965 doi: 10.1073 / pnas.2014441117 (2020)], therefore, plaque assay was performed on the second day after infection ( Figure 4E ) and RT-qPCR ( Figure 4F The viral load in hamster lungs was measured. When administered to hamster lungs one day prior to viral challenge, CD3600 significantly inhibited SARS-CoV-2 replication in the lungs. Compared to PHW-treated lungs, reduced inflammatory changes were detected in the lungs of hamsters treated with CD3600. In summary, these in vivo data suggest that a single dose of TAT2-P1 / DIG, administered to the lungs of hamsters one day prior to viral challenge, provides rapid-onset (3 or 5 days) prophylactic protection against influenza A virus in mice and significantly inhibits SARS-CoV-2 replication in hamsters.

[0215] TAT2-P1 & LAH4 showed highly efficient gene transfection in lung tissue.

[0216] To further investigate gene delivery in the lung airways using peptide-based vectors, the peptide LAH4 (Lam, JK) was demonstrated. et al. Effective endogenous gene silencing mediated by pH-responsive peptides proceeds via multiple pathways. J Control Release 158, 293-303 (2012)) showed significantly higher transfection efficiency in cells than TAT2-P1 ( Figure 5A ), of which TC 50 Higher than 125 μg / ml -1 ( Figure 6 However, its transfection efficiency in lung tissue was significantly lower than that of TAT2-P ( ), Figure 5B The high transfection efficiency in cells indicates that the plasmid packaged by LAH4 is effective. 35 High endosome escape capacity. However, low transfection efficiency in lung tissue may be due to the large size of LAH4 nanoparticles. Figure 5C ), which is not spherical in TEM images. pCMV is composed of LAH4 (1 mg ml) －1 TAT2-P1 & LAH4 (3:2), TAT2-P1 & LAH4 (4:1), and TAT2-P1 & LAH4 (9:1) were packaged. The nanoparticles were negatively stained for transmission electron microscopy analysis. Analysis of the TEM images showed that TAT2-P1 and LAH4 can form small spherical nanoparticles. Since size and shape are potential barriers to penetration through the mucosal layer in lung tissue (Zhao, F.), this approach is necessary. et al. Cellular uptake, intracellular trafficking, and cytotoxicity of nanomaterials. Small 7, 1322-1337 (2011); Duncan, GA, et al . The Mucus Barrier to Inhaled Gene Therapy. Mol The r 24, 2043-2053 (2016);Wang, Z., et al . Size and dynamics of caveolae studied using nanoparticles inliving endothelial cells. ACS Nano (3, 4110-4116), it is speculated that using TAT2-P1 & LAH4, which can form small spherical nanoparticles with high endosome escape ability, can improve transfection efficiency. The TAT2-P1:LAH4 ratio is 4:1 ( Figure 5C Under these conditions, TAT2-P1 & LAH4-pCMV can form spherical nanoparticles (approximately 140 nm). The transfection efficiency in cells (4:1) is significantly higher than that of TAT2-P1. Figure 5A Furthermore, TAT2-P1 & LAH4 nanoparticles remain stable at room temperature for over 72 hours. Figure 7 Importantly, TAT2-P1 & LAH4 showed significantly higher transfection efficiency in mouse lungs than TAT2-P1 alone or LAH4 alone. Figure 5BThese results further demonstrate that small nanoparticle size (TAT2-P1 & LAH4) is important for penetrating airway mucus to increase gene expression in the lungs.

[0217] TAT2-P1 & LAH4 / CD3600 inhibits SARS-CoV-2 variants in hamsters.

[0218] The results showed that administering a single dose of TAT2-P1&LAH4 / CD3600 to hamsters the day before viral challenge inhibited SARS-CoV-2 replication in the hamster lungs. Figure 4D The results showed that the avidin-derived peptide P9R could inhibit SARS-CoV-2 (Zhao, H). et al A broad-spectrum virus- and host-targeting peptide against respiratory viruses, including influenza virus and SARS-CoV-2. NatCommun 11, 4252 (2020). P1 is derived from P9R (Table 4). This further demonstrates that P1 and TAT2-P1 significantly inhibit SARS-CoV-2 replication ( Figure 5E When two doses of TAT2-P1&LAH4 / CD3600 were administered to the lungs of hamsters one day before and eight hours after SARS-CoV-2 challenge, TAT2-P1&LAH4 / CD3600 more effectively inhibited SARS-CoV-2 omeprazole replication in the lungs of hamsters. Figure 5F TAT2-P1 & LAH4-PHW also inhibited the replication of SARS-CoV-2. Figure 5G This can be attributed to the antiviral activity of TAT2-P1 against SARS-CoV-2. Figure 5E and 5F The dual function of TAT2-P1 (gene delivery and antiviral) enabled TAT2-P1 & LAH4 / CD3600 to exhibit stronger antiviral activity against SARS-CoV-2 in vivo. Furthermore, two doses of TAT2-P1 & LAH4 / CD3600 were demonstrated to significantly inhibit Delta SARS-CoV-2 replication in hamster lungs. Figure 5FNanoparticle staining was performed on hamster lungs. Hamsters were infected with either the Delta or Omeprone variants and treated with two doses of PBS or TAT2-P1&LAH4 / CD3600. Infected lungs were collected on day 2 post-infection for NP staining. Staining of uninfected hamster lung tissue served as a control. Nanoparticle staining further demonstrated that CD3600 reduced the replication of both Omeprone and Delta variants in hamster lungs. These results indicate that the peptide TAT2-P1&LAH4 nanoparticles, with their small spherical size and efficient endosome escape capability, can effectively penetrate airway mucus to express genes and deliver DIG in the lung airways, thereby significantly inhibiting the replication of SARS-CoV-2 variants in hamster lungs.

[0219] discuss

[0220] This study confirmed that defective interfering genes (DIGs) can be used to inhibit influenza viruses and coronaviruses in vitro and in vivo. The CoV-DIG identified in this study significantly inhibited SARS-CoV-2 replication, and the inhibition of influenza viruses by DIGs suggests its potential as a broad-spectrum antiviral agent with a high resistance barrier. It is anticipated that more DIGs targeting other viruses, including segmented and non-segmented RNA viruses, will be discovered. With the development of in vivo gene transfection vectors, DIG antiviral agents will play an increasingly important role in antiviral development.

[0221] For respiratory viral diseases, vaccination is the most effective prevention strategy. In the absence of vaccination, early initiation of antiviral treatment is crucial for good patient outcomes, as clearly demonstrated in COVID-19 and influenza virus-related illnesses. Currently, the World Health Organization (WHO) recommends daily administration of prophylactic medications for pre- and post-exposure prophylaxis against influenza for 10 days, but drug resistance is a concern. There are currently no widely available and effective medications recommended for the prevention of coronaviruses [Principi, N]. et al . Front Med (Lausanne) 6, 109 doi:10.3389 / fmed.2019.00109 (2019); Anand, U. et al . Front Immunol12, 658519 doi: 10.3389 / fimmu.2021.658519. (2021)]. Therefore, it is important to identify antiviral agents with novel antiviral mechanisms for prophylactic use, which should be characterized by rapid onset of action, long-lasting activity, and low likelihood of inducing drug-resistant viruses. For influenza virus, a single dose of DIG-4 was significantly more effective than a single dose of vaccine within 5 days in vivo. The weak prophylactic protection of vaccines in mice within one week is due to the slow onset of vaccine-induced immunity, which typically takes about 2 weeks [Groves, HT]. et al. Front Immunol 9, 126 doi: 10.3389 / fimmu.2018.00126 (2018); Mohn,KG et al . Hum Vaccin Immunother [14, 571-578 doi: 10.1080 / 21645515.2017.1377376. (2018)]. The prophylactic effect of zanamivir showed that a single dose of prophylactic treatment administered one day before viral challenge effectively protected mice from infection. However, in the current experiment, a single dose of zanamivir did not provide the same protection as long-acting DIG-4.

[0222] SARS-CoV-2 vaccines have been found to be promising in controlling COVID-19 in many countries. However, due to hesitation regarding vaccines and concerns about variant strains, antiviral agents against SARS-CoV-2 are still needed as an alternative approach to better control coronavirus disease and reduce mortality. This study reports the development of CoV-DIG, an antiviral agent that can complement a different class of current anticoronavirus drug candidates. A single dose of coronavirus CD3600 showed rapidly onset preventative activity while significantly inhibiting SARS-CoV-2 replication in hamsters. The shorter CD2100 showed lower antiviral activity against SARS-CoV-2 than CD3600, while PAD4 showed the strongest antiviral activity against influenza virus in vitro, suggesting that the length of DIG can determine the effectiveness of blocking wild-type virus replication [Marriott, AC & Dimmock, NJ]. Rev Med Virol 20, 51-62 doi: 10.1002 / rmv.641. (2010)]. The antiviral activity of DIG does not depend on specific inhibition of specific proteins or genes, but rather on the inhibition of viral genome packaging into new viral particles [Meng, B]. et al . Virology journal 14, 138 doi: 10.1186 / s12985-017-0805-6. (2017)]. When used as a prophylactic antiviral agent, this inhibition is unlikely to lead to drug-resistant viruses.

[0223] Previous studies have shown that influenza-deficient interferon virus can protect mice challenged with influenza virus without relying on interferon responses, and can also protect aged mice [Scott, PD, et al. Virology journal 8, 212 (2011); Easton, AJ et al . Vaccine 29, 2777-2784 doi: 10.1016 / j.vaccine.2011.01.102.(2011)]. The rapid onset of action of TAT2-P1 / DIG in prophylactic antiviral activity in susceptible mice challenged with influenza A virus or hamsters challenged with SARS-CoV-2 also suggests that antiviral activity may be independent of host immune response. This could provide additional or potential advantages for the effective protection of DIG in elderly or immunocompromised patients. The broad-spectrum antiviral activity of influenza DIG against different influenza viruses and the broad-spectrum antiviral activity of CoV-DIG against SARS-CoV-2 variants suggest that DIG regimens may be less likely to induce viral resistance [Zhao, F]. et al.Small 7, 1322-1337 doi: 10.1002 / smll.201100001. (2011); Dimmock, NJ & Easton, AJ Journal of virology [88,5217-5227 doi: 10.1128 / JVI.03193-13 (2014)]. Although influenza viruses and COVID-19 vaccines are less effective in immunocompromised patients, TAT2-P1 / DIG nanoparticles may be a potential candidate to provide rapidly onset prophylactic protection with a low likelihood of inducing resistance to coronaviruses and influenza viruses.

[0224] It should be understood that the disclosed methods and compositions are not limited to the specific methods, schemes, and reagents described, as these can vary. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of the invention, which is defined only by the appended claims.

[0225] This invention discloses materials, compositions, and components that can be used, combined with, or used in the preparation of the disclosed methods and compositions or products thereof. These and other materials are disclosed herein, and it should be understood that while specific references to every different individual and collective combination and arrangement of these compounds may not be explicitly disclosed when disclosing combinations, subsets, interactions, groups, etc., of these materials, each case is specifically considered and described herein. For example, if DIG is disclosed and discussed, and various modifications that can be made to a variety of molecules including DIG are discussed, then every combination and arrangement of DIG, and possible modifications, is specifically considered unless explicitly stated otherwise. Thus, if a class of molecules A, B, and C and a class of molecules D, E, and F are disclosed, and examples of combinations of molecules A through D are disclosed, then each is considered individually and collectively, even if not individually enumerated. Therefore, in this example, each of the combinations AE, AF, BD, BE, BF, CD, CE, and CF is specifically considered and should be considered as disclosed from exemplary combinations of A, B, and C; D, E, and F; and AD. Similarly, any subsets or combinations of these are also specifically considered and disclosed. Therefore, for example, subgroups AE, BF, and CE are specifically considered and should be considered as disclosed from exemplary combinations of A, B, and C; D, E, and F; and AD. Furthermore, each material, composition, component, etc., as contemplated and disclosed above may also be specifically and independently included or excluded from any group, subgroup, list, collection, etc., of such materials. These concepts apply to all aspects of this application, including but not limited to steps in methods of preparing and using the disclosed compositions. Therefore, if various additional steps are available, it should be understood that each of these additional steps can be performed using any particular embodiment or combination of embodiments of the disclosed methods, and each such combination is specifically considered and should be considered as disclosed.

[0226] It is important to note that, as used herein and in the appended claims, the singular forms “a,” “an,” and “the” also include plural references unless the context clearly indicates otherwise. Thus, for example, a reference to “a DIG” includes multiple such DIGs, a reference to “the DIG” is a reference to one or more DIGs and their equivalents known to those skilled in the art, and so on.

[0227] In the specific embodiments and claims of this specification, the word “comprising” and its variations, such as “including” and “containing”, means “including but not limited to” and is not intended to exclude, for example, other additives, components, integers or steps.

[0228] "Optional" or "optionally" means that the event, situation, or material described below may or may not occur or not exist, and the description includes instances where the event, situation, or material occurs or exists, as well as instances where it does not occur or does not appear.

[0229] Unless the context clearly indicates otherwise, the use of the word "can" indicates an option or capability of the object or condition in question. Generally, using "can" in this way implies an affirmative statement of the option or capability, without excluding the possibility that such an option or capability may not exist in other forms or implementations of the object or condition referred to. Unless the context clearly indicates otherwise, the use of the word "may" indicates an option or capability of the object or condition referred to. Generally, using "may" in this way implies an affirmative statement of the option or capability, without excluding the possibility that such an option or capability may not exist in other forms or implementations of the object or condition referred to. Unless the context clearly indicates otherwise, "may" as used herein does not refer to unknown or questionable characteristics of the object or condition.

[0230] A range may be expressed herein as from “about” a particular value and / or to “about” another particular value. When expressing such a range, it is also specifically considered that what is disclosed is a range from one particular value and / or to another particular value, unless the context specifically indicates otherwise. Similarly, when a value is expressed as an approximation using the antecedent “about,” it is understood that the particular value forms another particularly anticipated implementation, which should be considered disclosed unless the context specifically indicates otherwise. It should also be understood that each endpoint of a range is significant relative to and independent of the other endpoint, unless the context explicitly states otherwise. It should be understood that all individual values ​​and subranges of values ​​contained within explicitly disclosed ranges are also specifically considered and should be considered disclosed unless the context explicitly states otherwise. Finally, it should be understood that all ranges refer to the enumerated ranges and the set of individual numbers from (including) a first endpoint to (including) a second endpoint. In the latter case, it should be understood that any individual number may be chosen as a form of the quantity, value, or characteristic referred to by the range. In this way, a range describes a set of numbers or values ​​from a first endpoint to a second endpoint and including both the first and second endpoints, whereby a single member (i.e., a single number) can be selected as the quantity, value, or characteristic referred to by the range. The foregoing applies regardless of whether some or all of these embodiments are explicitly disclosed in a particular context.

[0231] Unless otherwise stated, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the disclosed methods and compositions pertain. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of these methods and compositions, particularly useful methods, apparatus, and materials are those described above. Nothing herein should be construed as an admission that the invention was not entitled to prior disclosure due to prior inventions. No references are acknowledged as prior art. The discussion of the references states the claims made by their authors, and the applicant reserves the right to question the accuracy and relevance of the cited references. It should be clearly understood that although numerous publications are cited herein, such citations do not constitute an admission that any of these documents constitutes common general knowledge in the art.

[0232] While descriptions of materials, compositions, components, steps, techniques, etc., may include many options and alternatives, this should not be construed as, or an admission, that these options and alternatives are equivalent to each other, or in particular, obvious alternatives. Therefore, for example, a list of different DIGs does not imply that the listed DIGs are obvious to each other, nor does it acknowledge equivalence or obviousness.

[0233] Each polynucleotide disclosed herein is intended and should be considered as specifically disclosed herein. Furthermore, each subgroup that can be identified within this disclosure is intended and should be considered as specifically disclosed herein. Therefore, it is specifically considered that any polynucleotide, or subgroup of polynucleotides, may be explicitly included or excluded from the use of polynucleotides, or included or excluded from the list of polynucleotides or outside of it.

[0234] Those skilled in the art will recognize or be able to identify numerous equivalents of specific embodiments of the methods and compositions described herein, which can be determined solely through routine experiments. These equivalents are intended to be included in the following claims.

Claims

1. A composition comprising (i) An isolated polynucleotide containing one or more defective interference genes or a vector containing said polynucleotide; in, Each of the one or more defective interference genes comprises a nucleotide sequence corresponding to one or more portions of a viral gene, wherein the portion of the viral gene comprises a deletion relative to the viral gene, wherein the virus is SARS-CoV-2, and at least one of the one or more defective interference genes consists of the nucleotide sequence of SEQ ID NO:

6. The vector contains one or more promoters and / or polyadenylation signals operatively linked to the one or more defective interference genes; as well as (ii) Peptides selected from the following: TAT2-P1, or a combination of TAT2-P1 and LAH4; wherein the amino acid sequence of TAT2-P1 is as shown in RKKRRQRRRCWGPCPTAFRQIGNCGRFRVRCCRIR, and the amino acid sequence of LAH4 is as shown in KKALLAHALHLLALLALHLAHALKKA-NH2.

2. The composition of claim 1, wherein, The weight ratio of the peptide to polynucleotide is in the range of 2:1 to 4:

1.

3. The composition of claim 1, wherein, The weight ratio of peptide to polynucleotide is 4:

1.

4. The composition according to any one of claims 1 to 3, wherein, The peptide and the polynucleotide combine to form multiple nanoparticles.

5. The composition of claim 4, wherein, The nanoparticles have an average diameter of less than 200 nm.

6. The composition of claim 4, wherein, The nanoparticles have an average diameter of less than 150 nm.

7. The composition of claim 4, wherein, The nanoparticles have an average diameter of less than 135 nm.

8. The composition of claim 1, wherein, The carrier is an expression carrier.

9. The composition of claim 1, wherein, The vector is a plasmid.

10. The composition of any one of claims 1 to 3, further comprising one or more additional polynucleotides or one or more additional vectors.

11. A pharmaceutical composition comprising the composition as described in any one of claims 1 to 10 and a pharmaceutically acceptable carrier or excipient.

12. Use of an effective amount of the pharmaceutical composition of claim 11 in the preparation of a medicament for treating a subject with a coronavirus infection; wherein the coronavirus is SARS-CoV-2.

13. The use as described in claim 12, wherein, The subjects had been exposed to, infected with, or at risk of contracting the coronavirus.

14. The use as described in claim 12 or 13, wherein, The subjects were immunocompromised.

15. The use as described in claim 12 or 13, wherein, The SARS-CoV-2 strains were selected from: SARS-CoV-2 HKU-001a, SARS-CoV-2 B.1.1.7 (Alpha variant), SARS-CoV-2 B.1.351 (Beta variant), SARS-CoV-2 B.1.617.1 (Kappa variant), SARS-CoV-2 B.1.617.2 (Delta variant), and SARS-CoV-2 B.1.617.

3.

16. The use as described in claim 12 or 13, wherein, The composition is administered orally, intranasally, or intratracheally.

17. The use as described in claim 12 or 13, wherein, The subjects were human.

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