Vector for the selective silencing of a gene in astrocytes

Patent metadata

Field	Value
Publication number	US-9695443-B2
Application number	US-201314403722-A
Country	US
Kind code	B2
Filing date	May 24, 2013
Priority date	May 25, 2012
Publication date	Jul 4, 2017
Grant date	Jul 4, 2017

Abstract

The present invention relates to a viral vector for silencing a gene specifically in astrocytes comprising: —an astrocyte-specific viral envelope protein, —a first nucleic acid sequence encoding a transcription activator and at least one target sequence of a neuron-specific miR under the control of an astrocyte-specific promoter, and—a second nucleic acid sequence encoding a RNA for silencing the gene under the control of a promoter inducible by the transcription activator.

First claim

The invention claimed is:

1. A viral vector for silencing a gene specifically in astrocytes comprising:
an astrocyte-specific viral envelope protein,
a first nucleic acid sequence encoding a transcription activator and at least one target sequence of a neuron-specific microRNA (miR) under the control of an astrocyte-specific promoter, and
a second nucleic acid sequence encoding a RNA for silencing the gene under the control of a promoter inducible by the transcription activator,
wherein the first and second nucleic acid sequences are on the same nucleic acid molecule.

2. The viral vector according to claim 1 , wherein the astrocyte-specific viral envelope protein is Mokola virus G protein (G-MOK).

3. The viral vector according to claim 1 , wherein the transcription activator is the tetracycline transactivator (tTA) and the promoter inducible by the transcription activator is the tetracycline response element (TRE).

4. The viral vector according to claim 3 , wherein the astrocyte specific promoter is the promoter of rat GS gene.

5. The viral vector according to claim 1 , wherein the astrocyte specific promoter is the promoter of the glutamine synthase (GS) gene.

6. The viral vector according to claim 1 , wherein the target sequence of the neuron-specific miR is a target sequence of miR124 (miR124T).

7. The viral vector according to claim 1 , wherein the nucleic acid sequence encoding a RNA for silencing the gene encodes a siRNA or a shRNA targeting the gene mRNA.

8. The viral vector according to claim 7 , wherein the shRNA is embedded in a miR sequence.

9. The viral vector according to claim 8 , wherein the miR sequence is a miR30 sequence.

10. The viral vector according to claim 1 , wherein the viral vector is a lentiviral vector.

11. The viral vector according to claim 10 , wherein the lentiviral vector is an HIV-1 vector.

12. The viral vector according to claim 9 , wherein the nucleic acid molecule comprises, from 5′ to 3′:
a 5′ long terminal repeat (LTR) sequence,
a sequence encoding a shRNA targeting the gene embedded in a miR30 sequence,
the GS rat promoter,
a sequence encoding the tetracycline transactivator
the woodchuck hepatitis post-transcriptional regulatory element (WPRE)
a sequence encoding 4 copies of a miR124 target sequence,
a 3′ long terminal repeat (LTR) sequence comprising the tetracycline response element (TRE).

13. The viral vector according to claim 1 , wherein the gene is the huntingtin gene.

14. The viral vector according to claim 1 , for use in the prevention or treatment of astrocyte-mediated diseases.

15. A pharmaceutical composition comprising a viral vector according to claim 1 as an active ingredient and a pharmaceutically acceptable vehicle or excipient.

16. An in vitro method, of silencing a gene in astrocytes, comprising contacting the viral vector according to claim 1 with at least one astrocyte.

17. A nucleic acid molecule comprising from 5′ to 3′:
a 5′ long terminal repeat (LTR) sequence,
a sequence comprising from 4 to 1000 nucleotides,
the GS rat promoter,
a sequence encoding the tetracycline transactivator,
the woodchuck hepatitis post-transcriptional regulatory element (WPRE),
a sequence encoding 4 copies of a miR124 target sequence,
a 3′ long terminal repeat (LTR) sequence comprising the tetracycline response element (TRE).

18. The nucleic acid sequence according to claim 17 , wherein the sequence comprising from 4 to 1000 nucleotides is a multiple cloning site.

19. The nucleic acid sequence according to claim 17 , wherein the sequence comprising from 4 to 1000 nucleotides is sequence encoding a siRNA or a shRNA.

Assignees

• Commissariat A I'Energie Atomique Et Aux Energies Alternatives
• Commissariat Energie Atomique

Inventors

• Deglon Nicole

Classifications

• C12N2810/6081

  rhabdoviridae, e.g. VSV · CPC title

• C12N2740/15043

  viral genome or elements thereof as genetic vector · CPC title

• C12N2830/48

  regulating transport or export of RNA, e.g. RRE, PRE, WPRE, CTE · CPC title

• C12N2740/15033

  Use of viral protein as therapeutic agent other than vaccine, e.g. apoptosis inducing or anti-inflammatory · CPC title

• C12N2830/205

  bidirectional · CPC title