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

Patent metadata

Field	Value
Publication number	US-2020121731-A1
Application number	US-201716313060-A
Country	US
Kind code	A1
Filing date	Jun 29, 2017
Priority date	Jun 29, 2016
Publication date	Apr 23, 2020
Grant date	—

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

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 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.

2 - 4 . (canceled)

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

6 . The nucleic acid construct of claim 51 , wherein said Toxoplasma secreted protein is selected from the group consisting of a microneme protein and a dense granule protein.

7 . The nucleic acid construct of claim 1 , 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.

8 - 9 . (canceled)

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

11 . (canceled)

12 . The nucleic acid construct of claim 1 , wherein said Toxoplasma secreted protein comprises Toxoplasma gondii macrophage migration inhibitory factor (TgMIF).

13 . (canceled)

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

15 . The nucleic acid construct of claim 1 , with the proviso that said nucleic acid construct does not comprise a Cre-recombinase coding sequence.

16 . (canceled)

17 . The nucleic acid construct of claim 14 , wherein said inducible self-destruction element is active in response to a drug.

18 . (canceled)

19 . 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.

20 - 22 . (canceled)

23 . A toxoplasma transformed with the nucleic acid construct of claim 1 .

24 . The Toxoplasma of claim 23 , being not attenuated.

25 . The Toxoplasma of claim 23 , being devoid of Toxoplasma elements which facilitate propagation of said Toxoplasma in a host cell.

26 . The Toxoplasma of claim 25 , 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.

27 . A pharmaceutical composition comprising the Toxoplasma of claim 23 , and a pharmaceutically acceptable carrier.

28 . The pharmaceutical composition of claim 27 , for treating a subject diagnosed with a pathology characterized by a deficient endogenous protein in a central nervous system of a subject.

29 . The pharmaceutical composition of claim 27 , for treating a subject diagnosed with a pathology which is treatable by administration of said pharmaceutical polypeptide in a central nervous system of a subject.

30 . (canceled)

31 . A method of administering a protein-of-interest into a central nervous system of a subject, the method comprising:
administering to the subject the Toxoplasma of claim 23 , thereby administering the protein-of-interest to the central nervous system of the subject.

32 . A method of treating a subject in need thereof, comprising administering to the subject the Toxoplasma of claim 24 , 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.

33 . 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 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.

34 . (canceled)

35 . 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, thereby administering the polypeptide-of-interest to the subject.

36 . The method of claim 31 , 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.

37 . The method of claim 31 , wherein said pharmaceutical polypeptide comprises a wild type amino acid sequence corresponding to said endogenous protein capable of treating the pathology.

38 . The method of claim 31 , wherein said pharmaceutical polypeptide comprises an antibody capable of treating the pathology.

39 . The method of claim 31 , wherein said pharmaceutical polypeptide comprises a toxin capable of treating the pathology.

40 . The method of claim 31 , further comprising administering to the subject a drug capable of inducing said self-destruction element.

41 . The nucleic acid construct of claim 1 , wherein said toxoplasma persists in said infect host cell.

42 . The nucleic acid construct of claim 1 , 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.

43 . The nucleic acid construct of claim 1 , wherein said Toxoplasma secreted protein is secreted continuously.

44 . The nucleic acid construct of claim 1 , wherein said pharmaceutical polypeptide reaches a nucleus of a host cell.

Assignees

• Ramat At Tel Aviv Univ Ltd
• Univ Glasgow Court

Inventors

• Rechavi Oded
• Bracha Shahar
• Sheiner Lilach

Classifications

• A61P25/28

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• C12N15/65

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

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  acting on amide bonds in linear amides {(3.5.1)} · CPC title

• C12Y302/01046

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