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

Patent metadata

Field	Value
Publication number	US-12070476-B2
Application number	US-202217584508-A
Country	US
Kind code	B2
Filing date	Jan 26, 2022
Priority date	Jun 29, 2016
Publication date	Aug 27, 2024
Grant date	Aug 27, 2024

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 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 Toxoplasma secreted protein is secreted to a host cell when infected by the Toxoplasma , 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.

2. The nucleic acid construct of claim 1 , wherein said endogenous promoter is not an endogenous promoter of a rhoptry protein.

3. The nucleic acid construct of claim 1 , wherein said Toxoplasma secreted protein is secreted from a rhoptry of said Toxoplasma.

4. The nucleic acid construct of claim 1 , wherein said Toxoplasma secreted protein is a non-rhoptry protein.

5. The nucleic acid construct of claim 4 , wherein said Toxoplasma secreted protein is selected from the group consisting of a microneme protein and a dense granule protein.

6. The nucleic acid construct of claim 1 , wherein said Toxoplasma secreted protein is secreted from a dense granule of said Toxoplasma.

7. The nucleic acid construct of claim 1 , wherein said Toxoplasma secreted protein is secreted from a microneme of said Toxoplasma.

8. A nucleic acid construct comprising 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, and wherein said Toxoplasma secreted protein comprises Toxoplasma gondii macrophage migration inhibitory factor (TgMIF).

9. The nucleic acid construct of claim 1 , further comprises a third nucleic acid sequence encoding an inducible self-destruction element.

10. The nucleic acid construct of claim 9 , wherein said inducible self-destruction element is active in response to a drug.

11. The nucleic acid construct of claim 1 , further comprises at least one in frame cleavage site which allows detachment of said pharmaceutical polypeptide from said Toxoplasma secreted protein.

12. A nucleic acid construct system comprising at least two nucleic acid constructs, wherein a first nucleic acid construct of said at least two nucleic acid constructs is the nucleic acid construct of claim 1 , and a second nucleic construct of said at least two nucleic acid constructs comprises a polynucleotide encoding a selectable marker.

13. A toxoplama transformed with the nucleic acid construct of claim 1 .

14. A toxoplama transformed with the nucleic acid construct system of claim 12 .

15. The Toxoplasma of claim 13 , being not attenuated.

16. A pharmaceutical composition comprising the Toxoplasma of claim 13 , and a pharmaceutically acceptable carrier.

17. The pharmaceutical composition of claim 16 , further comprises an immunosuppression agent.

18. The nucleic acid construct of claim 6 , wherein said dense granule protein is GRA16 or GRA24.

Assignees

• Univ Ramot
• Univ Glasgow Court

Inventors

• Rechavi Oded
• Bracha Shahar
• Sheiner Lilach

Classifications

• C12Y603/05005

  Carbamoyl-phosphate synthase (glutamine-hydrolysing) (6.3.5.5) · CPC title

• C12N15/79

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

• C12N15/65

  using markers (enzymes used as markers C12N15/52) · CPC title

• C12N9/93

  Ligases (6) · CPC title

• C07K2319/10

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