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1. WO2020193434 - VECTEURS BIFONCTIONNELS PERMETTANT LE SILENÇAGE ET L'EXPRESSION DE BCL11A D'UN HBB ANTI-FALCIFORMATION ET LEURS UTILISATIONS POUR LA THÉRAPIE GÉNIQUE DE B-HÉMOGLOBINOPATHIES

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CLAIMS:

1. A nucleic acid molecule having the sequence as set forth in SEQ ID NO: l wherein a sequence encoding for an artificial microRNA (amiR) suitable for reducing the expression of BCL11 A, in particular of the BCL11 A-XL isoform, is inserted i) between the nucleotide at position 85 and the nucleotide 86 at position in SEQ ID NO: l and/or ii) between the nucleotide at position 146 and the nucleotide 147 at position in SEQ ID NO: l .

2. The nucleic acid molecule of claim 1 wherein the amiR consists of a shRNA that is embedded into a miRNA backbone.

3. The nucleic acid molecule of claim 2 wherein the miRNA backbone is derived from miR-142, miR-155, miR-181 and miR-223.

4. The nucleic acid molecule of claim 2 wherein the shRNA of the present invention adopts a stem-loop structure wherein the stem region is a region formed by a guide strand and a passenger strand.

5. The nucleic acid molecule of claim 4 wherein the sequence encoding for the guide strand consists of the sequence as set forth in SEQ ID NO: 2.

6. The nucleic acid molecule of claim 4 wherein the loop segment is encoded by the sequence as set forth in SEQ ID NO:3.

7. The nucleic acid molecule of claim 2 wherein the sequence encoding for the shRNA consists of the sequence as set forth in SEQ ID NO:4.

8. The nucleic acid molecule of claim 1 wherein the sequence encoding for the amiR consists of the sequence as set forth in SEQ ID NO:5.

9. The nucleic acid molecule of claim 1 that has a sequence as set forth in SEQ ID NO:6 or SEQ ID NO:7.

10. A transgene encoding for an anti-sickling HBB, wherein said transgene comprises the nucleic acid molecule of claim 1.

11. The transgene of claim 10 which comprises the sequence as set forth in SEQ ID NO:9 or SEQ ID NO: 10.

12. The transgene of claim 10 which is placed under the transcriptional control of the HBB promoter and key regulatory elements from the 16-kb human b-locus control region (PLCR), wherein the key regulatory elements consist of the 2 DNase I hypersensitive sites HS2 and HS3.

13. A viral vector comprising the transgene of claim 10.

14. The viral vector of claim 13 which is a lentiviral vector.

15. A method of obtaining a population of host cells transduced with the transgene of claim 10, which comprises the step of transducing a population of host cells in vitro or ex vivo with the viral vector of claim 13.

16. The method of claim 15 wherein the host cell is selected from the group consisting of hematopoietic stem/progenitor cells, hematopoietic progenitor cells, hematopoietic stem cells (HSCs), pluripotent cells (i.e. embryonic stem cells (ES) and induced pluripotent stem cells (iPS)).

17. A method of treating a hemoglobinopathy in a subject in need thereof, the method comprising transplanting a therapeutically effective amount of the population of host cells obtained by the method of claim 16.