FIELD

The present disclosure relates generally to the field of virotherapy. More particularly, the present disclosure relates to RNA viruses as vectors for delivery of at least one of DNA-dependent activator of interferon-regulatory factors (DAI) and receptor-interacting serine/threonine-protein kinase 3 (RIPK3) to infected cells or tumor cells to facilitate death of such cells as well as to enhance an immune response against the underlying infection or tumor.

BACKGROUND

Various publications, including patents, published applications, accession numbers, technical articles and scholarly articles are cited throughout the specification. Each of these cited publications is incorporated by reference, in its entirety and for all purposes, in this document.

Influenza A virus (IAV), a member of the family Orthomyxoviridae and primary causative agent of influenza in birds and mammals, is an enveloped virus with a negative-sense, single-stranded, segmented RNA genome. Acute IAV infection is accompanied by lysis of infected primary epithelial cells and fibroblasts in culture, and by destruction of airway epithelia in vivo, indicative of an integral role for cell death in the virus life cycle, the immune response to this virus, or both. It is believed that cell death may represent a host defense mechanism that limits both virus spread and host immunopathology early in an infection.

In murine fibroblasts and airway epithelial cells, IAV lysis is driven by receptor-interacting serine/threonine-protein kinase 3 (RIPK3). Upon IAV infection, RIPK3 nucleates a necrosome complex, that minimally also includes receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and mixed lineage kinase domain-like (MLKL), which then activates both apoptosis, via a RIPK1+Fas-Associated protein with Death Domain (FADD)+Caspase 8 axis, and necroptosis, mediated by MLKL. Formation of the RIPK3 necrosome and consequent activation of cell death both require active IAV replication. DNA-dependent activator of interferon-regulatory factors (DAI) senses IAV genomic RNA and links replicating IAV to RIPK3 activation. It is believed that DAI recognizes IAV RNA by a mechanism requiring the second of its Zα domains, and nucleates a RHIM-dependent RIPK3-containing necrosome. DAI also mediates IAV-induced RIPK3-independent apoptosis. Consequently, cells lacking DAI are remarkably resistant to IAV-triggered lysis, and DAI-deficient mice are hyper-susceptible to lethal infection by this virus.

SUMMARY

The present disclosure provides RNA virus vectors. These RNA virus vectors may be used for treating tumors, and also for treating virus infections, particularly for human patients. These RNA virus vectors may be used in the manufacture of a medicament, including a medicament for treating tumors as well as a medicament for treating viral infections.

In general, an RNA virus vector comprises an RNA virus that comprises at least a gene encoding the human DNA-dependent activator of interferon-regulatory factors (DAI) protein. The RNA virus may further comprise a gene encoding the human receptor-interacting serine/threonine-protein kinase 3 (RIPK3) protein. In some embodiments, the RNA virus is a positive sense RNA virus, non-limiting examples of which include an attenuated Kunjin virus, Polio virus, Semliki Forest virus, Venezuelan Equine Encephalitis virus, or Sinbis virus. In some embodiments, the RNA virus is a negative sense RNA virus, non-limiting examples of which include an attenuated Rabies virus, Influenza virus, Vesicular Stomatitis virus, Respiratory Syncytial virus, Sendai virus, Measles virus, New Castle Disease virus, or Simian Virus 5 (SV5) virus. In some embodiments, the RNA virus is Influenza virus. In some embodiments, the RNA virus is a retrovirus, such as an attenuated Lentivirus.

The gene encoding the human DAI protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:13. The gene may comprise DNA or RNA, and RNA may comprise a negative sense RNA. The gene encoding the human DAI protein may comprise the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:4, or comprise the complement thereof.

The gene encoding the human RIPK3 protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:14. The gene may comprise DNA or RNA, and RNA may comprise a negative sense RNA. The gene encoding the human RIPK3 protein may comprise the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:5, or comprise the complement thereof.

In some embodiments, the RNA virus further comprises a gene encoding the human mixed lineage kinase domain-like (MLKL) protein. The gene encoding the human MLKL protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:15. The gene may comprise DNA or RNA, and RNA may comprise a negative sense RNA. The gene encoding the human MLKL protein may comprise the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:6, or comprise the complement thereof.

The RNA virus vectors may be used in a method for treating a tumor in a subject in need thereof. In general, such methods comprise comprising infecting cells of the tumor with the RNA virus vector that comprises a gene encoding the human DAI protein and, optionally, a gene encoding the human RIPK3 protein and, optionally, a gene encoding the human MLKL protein. Such genes may comprise DNA or RNA, and RNA may comprise a negative sense RNA. The gene encoding the human DAI protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:13, for example, the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:4. The gene encoding the human RIPK3 protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:14, for example, the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:5. The gene encoding the human MLKL protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:15, for example, the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:6. The methods may be used to treat any tumor. In some embodiments, the tumor is a tumor of the head and neck, esophagus, lung, breast, pancreas, kidney, liver, stomach, colon, ovary, uterus, prostate gland, bladder, or blood.

As part of the tumor-treating method, the step of infecting cells of the tumor may comprise administering the RNA virus vector to the subject. The RNA virus vector may be actively targeted to the tumor, or may travel via the blood or other biologic fluid to the tumor location.

The RNA virus vectors may be used in methods for treating a viral infection in a subject in need thereof. In general, such methods comprise comprising infecting cells infected with the viral infection with the RNA virus vector that comprises a gene encoding the human DAI protein and, optionally, a gene encoding the human RIPK3 protein and, optionally, a gene encoding the human MLKL protein. Such genes may comprise DNA or RNA, and RNA may comprise a negative sense RNA. The gene encoding the human DAI protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:13, for example, the nucleic acid sequence of SEQ ID NO:1 or SEQ ID NO:4. The gene encoding the human RIPK3 protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:14, for example, the nucleic acid sequence of SEQ ID NO:2 or SEQ ID NO:5. The gene encoding the human MLKL protein may comprise a nucleic acid sequence encoding the amino acid sequence of SEQ ID NO:15, for example, the nucleic acid sequence of SEQ ID NO:3 or SEQ ID NO:6. The methods may be used to treat any viral infection, including a negative sense viral infection. The methods may be used to treat an influenza virus infection, including influenza Type A, Type B, and Type C infections.

As part of the infection-treating methods, the step of infecting cells infected with a virus infection may comprise administering the RNA virus vector to the subject. The RNA virus vector may be actively targeted to infected cells, or may travel via the blood or other biologic fluid to the site of the infection.

An RNA virus vector may be used for the treatment of a tumor. The RNA virus vector may be used for the treatment of a tumor of the head and neck. The RNA virus vector may be used for the treatment of a tumor of the esophagus. The RNA virus vector may be used for the treatment of a tumor of the lung. The RNA virus vector may be used for the treatment of a tumor of the breast. The RNA virus vector may be used for the treatment of a tumor of the pancreas. The RNA virus vector may be used for the treatment of a tumor of the kidney. The RNA virus vector may be used for the treatment of a tumor of the liver. The RNA virus vector may be used for the treatment of a tumor of the stomach. The RNA virus vector may be used for the treatment of a tumor of the colon. The RNA virus vector may be used for the treatment of a tumor of the ovary. The RNA virus vector may be used for the treatment of a tumor of the uterus. The RNA virus vector may be used for the treatment of a tumor of the prostate gland. The RNA virus vector may be used for the treatment of a tumor of the bladder. The RNA virus vector may be used for the treatment of a tumor of the blood.

An RNA virus vector may be used for the treatment of a viral infection. The RNA virus vector may be used for the treatment of a negative sense RNA virus infection. The RNA virus vector may be used for the treatment of an influenza Type A infection. The RNA virus vector may be used for the treatment of an influenza Type B infection. The RNA virus vector may be used for the treatment of an influenza Type C infection.

