Microbial systems for the generation and delivery of eukaryotically translatable mRNA to eukaryotes

Abstract

This invention provides a bacterial system for the production and delivery of eukaryotically translatable mRNA to eukaryotic cells. [Solution] The system utilizes invasive, non-pathogenic bacteria to produce functional mRNA cargo and deliver it to eukaryotic cells. In addition, the system utilizes bacteria to produce functional mRNA that can be extracted from bacterial cells for downstream application. The bacteria contain at least one prokaryotic expression cassette encoding mRNA; the mRNA contains a poly-A sequence transcribed in the bacteria and a 5' cap or pseudocap element, such as an internal ribosome entry site (IRES) element, which mediates translation within a eukaryotic host cell. Examples of therapeutic mRNA functions include, but are not limited to, providing gene material encoding antibodies, vaccine antigens, and deletion genes in the host.

Claims

1. A system for producing eukaryotically translatable mRNA, comprising a non-pathogenic bacterium engineered to have at least one expression cassette encoding eukaryotically translatable mRNA comprising at least one invasive factor and at least one expression cassette comprising an internal ribosome entry sequence (IRES), a nucleic acid sequence encoding a polypeptide, and a poly A tail, wherein the transcription of the eukaryotically translatable mRNA is under the control of a prokaryotic promoter.

2. A system for producing eukaryotic translateable mRNA according to claim 1, wherein the eukaryotic translateable mRNA is circularized within a bacterium during transcription.

3. A system for producing eukaryotic translateable mRNA according to claim 2, wherein the circularization of mRNA is promoted by the bacteriophage T4 rearrangement intron-exon (PIE) method.

4. A system for producing eukaryotic translateable mRNA according to any one of claims 1 to 3, wherein the IRES is an IRES selected from the group consisting of cricket paralysis virus (CrPV) IRES, foot-and-mouth disease virus (FMDV) IRES, and classical swine fever virus (CSFV) IRES.

5. A system for producing eukaryotic translateable mRNA according to any one of claims 1 to 4, wherein the invasive factor is encoded by the inv or hlyA gene.

6. A system for producing eukaryotic translateable mRNA according to any one of claims 1 to 4, wherein at least one invasive factor is encoded by the expression cassette, and at least one invasive factor is an invasin protein or a fragment or binding domain thereof and listeriolisin O (LLO).

7. A system for producing eukaryotically translatable mRNA according to any one of claims 1 to 6, wherein the expression cassette encoding the eukaryotically translatable mRNA is incorporated into the chromosome of the bacterium.

8. A system for producing eukaryotic translateable mRNA according to any one of claims 1 to 6, wherein the bacterium contains the at least one expression cassette encoding the eukaryotic translateable mRNA within a self-replicating vector independent of the chromosome.

9. A system for producing eukaryotic translateable mRNA according to any one of claims 1 to 8, wherein the polyA region contains 2 to about 500 adenine nucleotides.

10. A system for producing eukaryotically translateable mRNA according to any one of claims 1 to 9, wherein the mRNA encodes a viral polypeptide or a fragment thereof.

11. A system for producing eukaryotically translatable mRNA in eukaryotic cells according to any one of claims 1 to 10, wherein administration to a human patient or animal subject may be selected from the group consisting of intramuscular, parenteral, intravenous, intradermal, subcutaneous, intracardiac, intracerebral, intravitreous, intranasal, intrarectal, oral, intravaginal, inhalation, intraperitoneal, intraosseous, oral, and intraurethral administration.

12. A system for producing eukaryotic translatable mRNA, comprising a bacterium engineered to have at least one expression cassette encoding eukaryotic translatable mRNA comprising a eukaryotic promoter, an internal ribosome entry sequence (IRES), and a nucleic acid sequence encoding a polypeptide, wherein the transcription of the eukaryotic translatable mRNA is under the control of the eukaryotic promoter.

13. A system for producing eukaryotic translatable mRNA according to any one of claims 1 to 12, wherein the eukaryotic translatable mRNA encodes a protein having several functions selected from the group including enzymes, receptors, transporters, antibodies, structural proteins, and regulators, or a protein that is functional in itself.

14. A system for producing eukaryotically translatable mRNA, comprising a bacterium engineered to have at least one expression cassette encoding eukaryotically translatable mRNA comprising a 5' pseudocap element, a nucleic acid sequence encoding a polypeptide, and a poly A tail, wherein the transcription of the eukaryotically translatable mRNA is under the control of a prokaryotic promoter, and the bacterium is engineered to have an element that promotes the eukaryotically translatable mRNA to be circularized and covalently closed within the bacterium after transcription.

15. A system for producing eukaryotic translateable mRNA according to claim 14, wherein the element that promotes the formation of a covalently closed ring after transcription is a PIE sequence.

16. A system for producing eukaryotic translateable mRNA according to claim 15, wherein the PIE sequence comprises a bacteriophage T4 phage intron and an exon element.

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