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US-12624368-B2 - Nucleic acid molecules and dual-functional peptides having antiviral activity and delivery activity, compositions and methods thereof

US12624368B2US 12624368 B2US12624368 B2US 12624368B2US-12624368-B2

Abstract

Disclosed are delivery and expression systems of multiple antiviral therapeutic molecules. The therapeutic molecules include a novel class of dual-functional peptide and defective interfering genes of a virus. Also disclosed are compositions comprising the therapeutic molecules that are useful for the treatment and prevention of viral infections. Also disclosed herein are the method of making and using a vector that expresses the therapeutic molecules. Therapeutic molecules include cellular components such as RNA, DNA, peptide, proteins or combination thereof.

Inventors

  • Hanjun Zhao
  • Kai Wang Kelvin TO
  • Kwok Yung Yuen

Assignees

  • THE UNIVERSITY OF HONG KONG

Dates

Publication Date
20260512
Application Date
20190426

Claims (10)

  1. 1 . A vector comprising one or more viral genes wherein each of the viral genes comprises a deletion to form a detective interfering gene (DIG), the vector expresses one or more nucleic acid molecules that interfere with expression of one or more wild-type viral genes that do not comprise the deletion, the DIG consists of one or more defective genes, that express detective interfering RNAs without any full-length viral RNA and do not generate self-replicable reassortants, wherein the vector further comprises a dual-functional peptide comprising an HIV-1 Tat Peptide (TAT) and a cationic peptide comprising SEQ ID NO: 4 (P1); the DIG is a defective viral polymerase gene; the viral polymerase genes are PB2, PB1 and PA; the DIG comprises defective PB2, PB1 and PA; and the DIG comprises SEQ ID NO: 38 (DI-PA), SEQ ID NO: 39 (DI-PB1) and SEQ ID NO: 40 (DI-PB2).
  2. 2 . The vector of claim 1 , wherein the deletion is an internal deletion.
  3. 3 . The vector of claim 1 , wherein the nucleic acid molecule suppresses replication of a wild-type virus when transfected into cells, animals or humans.
  4. 4 . The vector of claim 3 , wherein the replication of the wild-type virus treated with the vector is reduced by about 10-20%, about 20-30%, about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-100% as compared to a wild-type virus that is not treated with the vector.
  5. 5 . The vector of claim 1 , wherein the vector exerts antiviral activity by preventing endosomal acidification.
  6. 6 . A vector comprising one or more viral genes wherein each of the viral genes comprises a deletion to form a detective interfering gene (DIG), said vector expresses one or more nucleic acid molecules that interfere with expression of one or more wild-type viral genes that do not comprise the deletion, the DIG consists of one or more defective genes, that express detective interfering RNAs without any full-length viral RNA and do not generate self-replicable reassortants, wherein the vector further comprises a dual-functional peptide comprising an HIV-1 Tat Peptide (TAT) and a cationic peptide, comprising SEQ ID NO: 4 (P1); the DIG is a defective viral polymerase gene; the viral polymerase genes are PB2, PB1 and PA; the DIG comprises defective PB2, PB1 and PA; and the DIG comprises SEQ ID NO: 38 (DI-PA), SEQ ID NO: 39 (DI-PB1) and SEQ ID NO: 40 (DI-PB2), wherein the deletion is about 50-100 base pair, 100-150 base pair, 150-200 base pair, 200-250 base pair, 250-300 base pair, 300-350 base pair, 350-400 base pair, 400-450 base pair, 450-500 base pair, 500-550 base pair, 550-600 base pair, 600-650 base pair, 650-700 base pair, 700-750 base pair, 750-800 base pair, 800-850 base pair, 850-900 base pair, 900-950 base pair, 950-1000 base pair, 1000-1200 base pair, 1200-1500 base pair, 1500-1800 base pair, or 1800-2100 base pair in length.
  7. 7 . The vector of claim 6 , wherein the deletion is an internal deletion.
  8. 8 . The vector of claim 6 , wherein the nucleic acid molecule suppresses replication of a wild-type virus when transfected into cells, animals or humans.
  9. 9 . The vector of claim 8 , wherein the replication of the wild-type virus treated with the vector is reduced by about 10-20%, about 20-30%, about 30-40%, about 40-50%, about 50-60%, about 60-70%, about 70-80%, about 80-90%, or about 90-100% as compared to a wild-type virus that is not treated with the vector.
  10. 10 . The vector of claim 6 , wherein the vector exerts antiviral activity by preventing endosomal acidification.

Description

1. FIELD OF THE INVENTION Disclosed are delivery and expression systems of multiple antiviral therapeutic molecules. The therapeutic molecules include a novel class of dual-functional peptide and defective interfering genes of a virus. Also disclosed are compositions comprising the therapeutic molecules that are useful for the treatment and prevention of viral infections. Also disclosed herein are the method of making and using a vector that expresses the therapeutic molecules. 2. BACKGROUND OF THE INVENTION Seasonal influenza virus annually causes over 3 to 5 million cases of severe illness with about 0.25 million deaths globally. Antigenically-shifted zoonotic influenza viruses pose threats of another pandemic1-3. Neuraminidase inhibitors, such as oseltamivir and zanamivir, have been used clinically for many years. However, human isolates of A(H1N1)pdm09, A(H3N2), A(H5N1) and A(H7N9) resistant to neuraminidase inhibitors have been found4-7. Convalescent blood products with high titer of specific neutralizing antibody have been shown to improve survival, but are not readily available8. Thus, broad spectrum antivirals with low possibility to induce resistance are urgently needed for controlling influenza virus infections. Defective interfering (DI) viruses, which are arisen naturally during viral replication with internal deletions in viral genes9, 10, can compete with the growth of wild-type virus and therefore suppress the replication of wild-type virus by interfering with the expression of the cognate full-length RNAs9, 11, 12. Though influenza DI virus (DIV) has been shown to be effective in vivo as a potential broad-spectrum antiviral with low risk for inducing resistance13-16, there are several concerns of influenza DIV used as therapeutic agents. Firstly, influenza DIV may reassort with wild-type influenza A virus to generate novel reassortants17. Secondly, neutralizing antibody may develop against the DIV and affect the antiviral effect in subsequent use. Delivering defective interfering genes (DIG) as an antiviral may avoid the risk of generating new reassortant virus and the problem of unwanted immunogenicity. There is limited efficacy of current antivirals and antiviral-resistant mutations impair anti-influenza treatment. Thus, there is a strong need for high efficiency antiviral molecules to prevent and treat influenza. It is in regard to these issues and others that the present disclosure is provided. 3. SUMMARY Provided herein are defective interfering viruses with internal deletions in viral genes that compete with the growth of wild-type, non-detective virus. Also provided are defective interfering viruses that suppress the replication of wild-type virus by interfering with the expression of cognate full-length RNAs. Provided herein is a vector comprising one or more viral genes wherein each of the viral gene comprises a deletion to form a detective interfering gene (DIG), said vector expresses one or more nucleic acid molecules that interfere with expression of one or more wild-type viral genes that do not comprise the deletion. Provided herein is a composition comprising the disclosed vector and a pharmaceutical carrier. Provided herein is a composition comprising the one or more nucleic acid molecules expressed from the disclosed vector and a TAT-P1 peptide. Also provided herein is a cell comprising the disclosed vector. Provided herein is a fusion protein comprising HIV-1 Tat peptide (TAT) and a cationic peptide P1 (derived from P9). In one embodiment, provided herein is a fusion protein comprising TAT and P2. In one embodiment, provided herein is a fusion protein comprising TAT and P3. Provided herein is a method of preventing or treating a subject against avian or seasonal influenza virus, said method comprises administering to the subject an effective amount of the vector disclosed herein. In certain embodiments, viral replication of wild-type virus is significantly reduced when treated with defective interfering virus. In certain embodiments, the replication of viruses that are treated with DIG is 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 90-100% reduced as compared to wild-type viruses that are not treated with DIG. In certain embodiments, 293T cells or A549 cells are transfected with DIG. Also disclosed is a kit, a medical device, or an inhaler, comprising the disclosed vector. 4. BRIEF DESCRIPTION OF THE DRAWINGS The invention is illustrated in the figures of the accompanying drawings which are meant to be exemplary and not limiting, in which like references are intended to refer to like or corresponding parts, and in which: FIG. 1 Construction and antiviral activity of defective interfering genes (DIG). (a) The plasmid construction of DI-PB2, DI-PB1 and DI-PA. The indicated sequences of shortened viral polymerase gene PB2, PB1 and PA were inserted into phw2000, respectively. Dotted lines indicate the internal deletion of wild-type (WT) viral polymerase genes