Compositions and methods for treating viral infections
The disclosure provides methods and compositions utilizing recombinant nucleic acid constructs or a replication incompetent virus-like particle encoding a chemokine, cytokine, or apoptosis inducing protein (e.g. Caspase 9 (Casp9)), or other toxins in a form which can only be transcribed in the presence of a viral polymerase. These methods can be adapted to target many viral infections and reduce or eliminate viral load, and provide a fundamentally different treatment for viral infections.
1. A recombinant nucleic acid molecule comprising:
a negative strand nucleic acid molecule or a pregenomic RNA (pgRNA) nucleic acid molecule encoding a chemokine, a cytokine, an apoptosis inducing protein, a diphtheria toxin A, a diphtheria toxin A fragment, or any combination thereof, flanked by a first viral transcription recognition signal and a second viral transcription recognition signal, wherein the negative strand nucleic acid molecule or pgRNA nucleic acid molecule is from a virus selected from the group consisting of hepatitis B virus, influenza virus and coronavirus;
a first promoter upstream (5′) of the first viral transcription recognition signal and the 3′ end of the negative strand nucleic acid molecule or pgRNA nucleic acid molecule, and
a second promoter located between the second viral transcription recognition signal and the 5′ end of the negative strand nucleic acid molecule or pgRNA nucleic acid molecule encoding a chemokine, a cytokine, an apoptosis inducing protein, a diphtheria toxin A, a diphtheria toxin A fragment, or any combination thereof,
wherein
(i) the first promoter and the second promoter are oriented in opposite direction and toward each other;
(ii) the second promoter and the second viral transcription recognition signal are upstream (5′) of the 5′ end of the negative strand nucleic acid molecule or pgRNA nucleic acid molecule;
(iii) the first and second viral transcription recognition signals are oriented in the same direction; and
(iv) wherein
a. if the negative strand nucleic acid molecule or pgRNA nucleic acid molecule is from a hepatitis B virus, each of the first and second viral transcription recognition signals comprises the epsilon recognition signal sequence of SEQ ID NO: 1;
b. if the negative strand nucleic acid molecule or pgRNA nucleic acid molecule is from an influenza virus, the second viral transcription recognition signal comprises the sequence of SEQ ID NO: 22 and the first viral transcription recognition signal comprises the sequence of SEQ ID NO: 23; or
c. if the negative strand nucleic acid molecule or pgRNA nucleic acid molecule is from a coronavirus, each of the first and second viral transcription recognition signals comprises the sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30.
2. The recombinant nucleic acid molecule of claim 1 further comprising a poly A tail downstream (3′) of the negative strand nucleic acid molecule or pgRNA nucleic acid molecule encoding a chemokine, a cytokine, an apoptosis inducing protein, or combination thereof.
3. The recombinant nucleic acid molecule of claim 1 wherein the apoptosis inducing protein is selected from the group consisting of BAX, BID, BAK, BAD, caspase 2, caspase 8, caspase 9, caspase 10, caspase 11, caspase 12, cytochrome C, SMAC, and apoptosis-inducing factor, and any combination thereof.
4. The recombinant nucleic acid molecule of claim 1 wherein the first promoter comprises a ubiquitous promoter or a liver-tissue specific promoter, selected from the group consisting of TBG (Thyroxine Binding Globulin) promoter, albumin promoter and enhancing element, AFP (alpha-fetoprotein) promoter, AAT (Alpha-1-antitrypsin) promoter, ApoE (Apolipoprotein E) promoter and PEPCK (Phosphoenolpyruvate carboxykinase) promoter.
5. The recombinant nucleic acid molecule of claim 1 wherein the second promoter comprises elongation factor 1alpha binding sequence (EFS).
6. The recombinant nucleic acid molecule of claim 1 wherein the chemokine is selected from the group consisting of CCL1, CCL2, CCL3, CCL4, CCL5, CCL6, CCL7, CCL8, CCL9, CCL10, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CXCL1, CXCL2, CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCL17, XCL1, XCL2, and CX3CL1.
7. The recombinant nucleic acid molecule of claim 1 wherein the cytokine is selected from the group consisting of IL-15, IL-2, IL-8, IL-10, IL-12, IL-6, IFN-α, IFN-, IFN-γ, TNF-a, CD40L, Mig, and Crg-2.
8. A vector comprising the recombinant nucleic acid molecule of claim 1 .
9. The vector of claim 8 , wherein the vector comprises a delivery vector or vehicle for delivery to a mammalian cell.
10. The vector of claim 9 , wherein the delivery vector or vehicle comprises a virus-like particle (VLP), an adeno-associated virus (AAV), a liposome, a nanoparticle, a micelle, a polymeric vesicle, or a polymersome.
11. A pharmaceutical composition comprising the vector of claim 9 .
12. A replication incompetent virus-like particle (VLP) comprising:
an optional translocation motif (TLM) fused to a capsid protein from a hepatitis B viral capsid or an influenza viral capsid or coronavirus fusion protein;
a negative strand nucleic acid molecule or a pregenomic RNA (pgRNA) nucleic acid molecule encoding a chemokine, a cytokine, an apoptosis inducing protein, a diphtheria toxin A, a diphtheria toxin A fragment, or any combination thereof, flanked by a first and second viral transcription recognition signals, wherein the negative strand nucleic acid molecule or pgRNA nucleic acid molecule is from a virus selected from the group consisting of hepatitis B virus, influenza virus and coronavirus;
a first promoter upstream (5′) of the first viral transcription recognition signal and the 3′ end of the negative strand nucleic acid molecule or pgRNA nucleic acid molecule, and
a second promoter located between the second viral transcription recognition signal and the 5′ end of the negative strand nucleic acid molecule or pgRNA nucleic acid molecule encoding a chemokine, a cytokine, an apoptosis inducing protein, a diphtheria toxin A, a diphtheria toxin A fragment, or any combination thereof,
wherein
(i) the first promoter and the second promoter are oriented in opposite direction and toward each other;
(ii) the second promoter and the second viral transcription recognition signal are upstream (5′) of the 5′ end of the negative strand nucleic acid molecule or pgRNA nucleic acid molecule;
(iii) the first and second viral transcription recognition signals are oriented in the same direction; and
(iv) wherein
a. if the negative strand nucleic acid molecule or pgRNA nucleic acid molecule is from a hepatitis B virus, each of the first and second viral transcription recognition signals comprises the epsilon recognition signal sequence of SEQ ID NO: 1;
b. if the negative strand nucleic acid molecule or pgRNA nucleic acid molecule is from an influenza virus, the second viral transcription recognition signal comprises the sequence of SEQ ID NO: 22 and the first viral transcription recognition signal comprises the sequence of SEQ ID NO: 23; or
c. if the negative strand nucleic acid molecule or pgRNA nucleic acid molecule is from a coronavirus, each of the first and second viral transcription recognition signals comprises the sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30.
13. A replication incompetent virus-like particle (VLP) comprising:
a negative strand nucleic acid molecule or a pregenomic RNA (pgRNA) nucleic acid molecule encoding a chemokine, a cytokine, an apoptosis inducing protein, a diphtheria toxin A, a diphtheria toxin A fragment, or any combination thereof, flanked by a first and second viral transcription recognition signals, wherein the negative strand nucleic acid molecule or pgRNA nucleic acid molecule is from a virus selected from the group consisting of hepatitis B virus, influenza virus and coronavirus;
a first promoter upstream (5′) of the first viral transcription recognition signal and the 3′ end of the negative strand nucleic acid molecule or pgRNA nucleic acid molecule; and
a second promoter located between the second viral transcription recognition signal and the 5′ end of the negative strand nucleic acid molecule or pgRNA nucleic acid molecule encoding a chemokine, a cytokine, an apoptosis inducing protein, a diphtheria toxin A, a diphtheria toxin A fragment, or any combination thereof,
wherein
(i) the first promoter and the second promoter are oriented in opposite direction and toward each other;
(ii) the second promoter and the second viral transcription recognition signal are upstream (5′) of the 5′ end of the negative strand nucleic acid molecule or pgRNA nucleic acid molecule;
(iii) the first and second viral transcription recognition signals are oriented in the same direction; and
(iv) wherein
a. if the negative strand nucleic acid molecule or pgRNA nucleic acid molecule is from a hepatitis B virus, each of the first and second viral transcription recognition signals comprises the epsilon recognition signal sequence of SEQ ID NO: 1;
b. if the negative strand nucleic acid molecule or pgRNA nucleic acid molecule is from an influenza virus, the second viral transcription recognition signal comprises the sequence of SEQ ID NO: 22 and the first viral transcription recognition signal comprises the sequence of SEQ ID NO: 23; or
c. if the negative strand nucleic acid molecule or pgRNA nucleic acid molecule is from a coronavirus, each of the first and second viral transcription recognition signals comprises the sequence of SEQ ID NO: 25, SEQ ID NO: 26, SEQ ID NO: 28, or SEQ ID NO: 30.
14. A pharmaceutical composition comprising the replication incompetent virus-like particle of claim 12 .
15. A method of inducing an apoptotic response against a cell infected with a virus selected from the group consisting of hepatitis B virus, influenza virus and coronavirus, in a mammalian subject in need thereof, the method comprising contacting the subject with the pharmaceutical composition of claim 11 or the pharmaceutical composition of claim 14 .
16. A method of treating a hepatitis B, influenza, or coronavirus infection in a mammalian subject, the method comprising administering to the subject the pharmaceutical composition of claim 11 or the pharmaceutical composition of claim 14 .
17. A method of inducing an apoptotic response against a cell infected with a virus selected from the group consisting of hepatitis B virus, influenza virus and coronavirus, in a mammalian subject in need thereof, the method comprising contacting the subject with the pharmaceutical composition of claim 14 .
18. A method of treating a hepatitis B, influenza, or coronavirus infection in a mammalian subject, the method comprising administering to the subject the pharmaceutical composition of claim 14 .