Engineered parasites for delivering protein to the central nervous system (CNS)

Patent metadata

Field	Value
Publication number	US-11260081-B2
Application number	US-201716313060-A
Country	US
Kind code	B2
Filing date	Jun 29, 2017
Priority date	Jun 29, 2016
Publication date	Mar 1, 2022
Grant date	Mar 1, 2022

Abstract

Provided are nucleic acid constructs, Toxoplasma comprising same, pharmaceutical compositions comprising same and methods using same for delivering a protein-of-interest to a tissue-of-interest of a subject, such as the CNS and further treating a pathology which is treatable by administration of a therapeutic polypeptide in a central nervous system of the subject.

First claim

What is claimed is:

1. A method of administering a protein-of-interest into a central nervous system of a subject, the method comprising:
administering to the subject a Toxoplasma transformed with a nucleic acid construct comprising a heterologous polynucleotide comprising a first nucleic acid sequence encoding a Toxoplasma secreted protein in frame fused upstream to a second nucleic acid sequence encoding a pharmaceutical polypeptide, wherein said heterologous polynucleotide is operably linked to a promoter for directing transcription of said heterologous polynucleotide in a Toxoplasma , wherein said promoter is selected from the group consisting of: a constitutive promoter, an inducible promoter, a latent period-specific promoter, and a Toxoplasma endogenous promoter with the proviso that said promoter is not a Toxofilin promoter, wherein said toxoplasma secreted protein is secreted to a host cell, and wherein said toxoplasma secreted protein is selected from the group consisting of a rhoptry secreted protein, a microneme protein, a dense granule protein and a Toxoplasma gondii macrophage migration inhibitory factor (TgMIF), and wherein said dense granule protein is selected from the group consisting of GRA16 and GRA24,
thereby administering the protein-of-interest to the central nervous system of the subject.

2. A method of treating a subject in need thereof, comprising administering to the subject a Toxoplasma transformed with a nucleic acid construct comprising a heterologous polynucleotide comprising a first nucleic acid sequence encoding a Toxoplasma secreted protein in frame fused upstream to a second nucleic acid sequence encoding a pharmaceutical polypeptide, wherein said heterologous polynucleotide is operably linked to a promoter for directing transcription of said heterologous polynucleotide in a Toxoplasma , wherein said promoter is selected from the group consisting of: a constitutive promoter, an inducible promoter, a latent period-specific promoter, and a Toxoplasma endogenous promoter with the proviso that said promoter is not a Toxofilin promoter, wherein said toxoplasma secreted protein is secreted to a host cell, wherein said toxoplasma secreted protein is selected from the group consisting of a rhoptry secreted protein, a microneme protein, a dense granule protein and a Toxoplasma gondii macrophage migration inhibitory factor (TgMIF), and wherein said dense granule protein is selected from the group consisting of GRA16 and GRA24, wherein the toxoplasma being devoid of virulence genes which are not necessary for delivery of the protein-of-interest into a central nervous system (CNS) of a subject, and wherein said subject is diagnosed with a pathology treatable by administration of said pharmaceutical polypeptide in a central nervous system of the subject, thereby treating the subject in need thereof.

3. A method of treating a subject in need thereof, comprising administering to the subject a Toxoplasma comprising a nucleic acid construct which comprises a heterologous polynucleotide comprising a first nucleic acid sequence encoding a Toxoplasma secreted protein in frame fused upstream to a second nucleic acid sequence encoding a pharmaceutical polypeptide, wherein said heterologous polynucleotide is operably linked to a promoter for directing transcription of said heterologous polynucleotide in a Toxoplasma , wherein said toxoplasma secreted protein is selected from the group consisting of a rhoptry secreted protein, a microneme protein, a dense granule protein and a Toxoplasma gondii macrophage migration inhibitory factor (TgMIF), and wherein said dense granule protein is selected from the group consisting of GRA16 and GRA24, and wherein said subject is diagnosed with a pathology treatable by administration of said pharmaceutical polypeptide in a central nervous system of the subject, thereby treating the subject in need thereof.

4. A method of administering a polypeptide-of-interest to a subject, comprising administering to the subject a Toxoplasma comprising a nucleic acid construct which comprises a heterologous polynucleotide comprising a first nucleic acid sequence encoding a Toxoplasma secreted protein in frame fused upstream to a second nucleic acid sequence encoding the polypeptide-of-interest, wherein said heterologous polynucleotide is operably linked to a promoter for directing transcription of said heterologous polynucleotide in a Toxoplasma , wherein said promoter is selected from the group consisting of: a constitutive promoter, an inducible promoter, a latent period-specific promoter, and a Toxoplasma endogenous promoter with the proviso that said promoter is not a toxofilin promoter, and wherein said toxoplasma secreted protein is selected from the group consisting of a rhoptry secreted protein, a microneme protein, a dense granule protein and a Toxoplasma gondii macrophage migration inhibitory factor (TgMIF), and wherein said dense granule protein is selected from the group consisting of GRA16 and GRA24, thereby administering the polypeptide-of-interest to the subject.

5. The method of claim 1 , further comprising administering to the subject an immunosuppression drug prior to administration of said Toxoplasma and/or subsequent to administration of said Toxoplasma and/or concomitantly with administration of said Toxoplasma to the subject.

6. The method of claim 1 , wherein said pharmaceutical polypeptide comprises a wild type amino acid sequence corresponding to said endogenous protein capable of treating the pathology.

7. The method of claim 1 , wherein said pharmaceutical polypeptide comprises an antibody capable of treating the pathology.

8. The method of claim 1 , wherein said pharmaceutical polypeptide comprises a toxin capable of treating the pathology.

9. The method of claim 1 , further comprising administering to the subject a drug capable of inducing a self-destruction element.

10. The method of claim 1 , wherein said toxoplasma persists in said infect host cell.

11. The method of claim 1 , wherein said Toxoplasma secreted protein is secreted continuously.

12. The method of claim 1 , wherein said pharmaceutical polypeptide reaches a nucleus of a host cell.

13. The method of claim 1 , wherein said Toxoplasma secreted protein is selected from the group consisting of a microneme protein and a dense granule protein.

14. A method of administering a protein-of-interest into a central nervous system of a subject, the method comprising:
administering to the subject a Toxoplasma transformed with a nucleic acid construct comprising a heterologous polynucleotide comprising a first nucleic acid sequence encoding a Toxoplasma secreted protein in frame fused upstream to a second nucleic acid sequence encoding a pharmaceutical polypeptide, wherein said heterologous polynucleotide is operably linked to a promoter for directing transcription of said heterologous polynucleotide in a Toxoplasma , wherein said promoter is selected from the group consisting of: a constitutive promoter, an inducible promoter, a latent period-specific promoter, and a Toxoplasma endogenous promoter with the proviso that said promoter is not a Toxofilin promoter, wherein said toxoplasma secreted protein is secreted to a host cell, wherein said Toxoplasma secreted protein is secreted from a dense granule of said Toxoplasma and is selected from the group consisting of GRA16 and GRA24, thereby administering the protein-of-interest to the central nervous system of the subject.

15. The method of claim 1 , with the proviso that said nucleic acid construct does not compri

Assignees

• Univ Ramot
• Univ Glasgow Court

Inventors

• Rechavi Oded
• Bracha Shahar
• Sheiner Lilach

Classifications

• C07K14/45 Primary

  Toxoplasma · CPC title

• C12N15/79

  Vectors or expression systems specially adapted for eukaryotic hosts · CPC title

• C07K2319/10

  containing a tag for extracellular membrane crossing, e.g. TAT or VP22 · CPC title

• C07K2319/42

  containing a HA(hemagglutinin)-tag · CPC title

• C12N9/93

  Ligases (6) · CPC title