Granted Patent B2

US 12,209,262 · App. 17/269,488 · Granted Jan 28, 2025

Inventors: Amit Nathwani (London, GB); Jenny Mcintosh (London, GB)

Assignee: UCL Business LTD

C12N9/644A61K31/573C12N7/00G01N33/4905A61K38/00C12N2750/14142C12N2750/14143C12N2830/008C12N2830/50C12Y304/21022G01N2333/9645

Abstract

Claims (42)

1. A polynucleotide comprising a Factor IX nucleotide sequence, wherein the Factor IX nucleotide sequence encodes a Factor IX protein and has at least 90% identity to SEQ ID NO. 5 and wherein the Factor IX nucleotide sequence comprises a codon that encodes leucine at a position corresponding to position 384 of wild type Factor IX, wherein the wild type Factor IX is encoded by SEQ ID NO: 9.

2. The polynucleotide of claim 1 , wherein the Factor IX nucleotide sequence encodes a Factor IX protein and has at least 95% identity to SEQ ID NO. 5.

3. The polynucleotide of claim 1 , wherein the Factor IX nucleotide sequence encodes a Factor IX protein and has at least 99% identity to SEQ ID NO. 5.

4. The polynucleotide of claim 1 , wherein the Factor IX nucleotide sequence encodes a Factor IX protein and has 100% identity to SEQ ID NO. 5.

5. The polynucleotide of claim 1 , wherein the polynucleotide further comprises a transcription regulatory element comprising: (i) an A1AT promoter or a fragment thereof, and/or (ii) an HCR enhancer or a fragment thereof.

6. The polynucleotide of claim 5 , wherein the HCR enhancer or a fragment thereof comprises a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO. 13.

7. The polynucleotide of claim 5 , wherein the A1AT promoter or a fragment thereof comprises a polynucleotide sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO. 14.

8. The polynucleotide of claim 5 , wherein the transcription regulatory element is at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, or 100% identical to SEQ ID NO. 6.

9. The polynucleotide of claim 1 , wherein the Factor IX protein comprises a sequence which has 100% identity to SEQ ID NO. 8.

10. A composition comprising the polynucleotide of claim 1 and a pharmaceutically acceptable excipient.

11. A viral particle comprising a recombinant genome comprising the polynucleotide of claim 1 .

12. The viral particle of claim 11 :

(i) which is an adenoviral, or lentiviral viral particle; or

(ii) which is an AAV viral particle; or

(iii) which is an AAV viral particle wherein the recombinant genome further comprises:

a) AAV2 ITRs; and/or

b) a poly A sequence; and/or

c) two resolvable ITRs; or

(iv) wherein the recombinant genome is single-stranded.

13. The viral particle of claim 11 , wherein on transduction into Huh7 cells the viral particle expresses Factor IX protein or a fragment thereof having a Factor IX activity greater than the activity of Factor IX expressed from a viral particle comprising a Factor IX nucleotide sequence of SEQ ID NO: 12 and a transcription regulatory element of SEQ ID NO. 7.

14. A composition comprising the viral particle of claim 11 and a pharmaceutically acceptable excipient.

15. A method of treating haemophilia in a patient in need thereof comprising administering an effective amount of the polynucleotide of claim 1 to the patient.

16. The method of claim 15 , wherein the haemophilia is haemophilia B and/or the patient has antibodies or inhibitors to Factor IX.

17. The method of claim 15 , wherein the administering comprises administering an AAV viral particle comprising a recombinant genome comprising the polynucleotide.

18. A method of achieving a stable Factor IX activity level of at least 20% in a patient having haemophilia B comprising administering an effective amount of the polynucleotide of claim 1 to the patient.

19. The method of claim 18 , wherein the administering comprises administering an AAV viral particle comprising a recombinant genome comprising the polynucleotide.

20. The method of claim 18 , wherein:

(a) the Factor IX activity level is stable after at least 10 weeks, at least 15 weeks, at least 20 weeks, at least 30 weeks, at least 40 weeks, or at least 50 weeks from administration of the polynucleotide, viral particle or composition;

(b) the patient is a patient who is refractory to treatment with Factor IX having an activity lower than the activity of Factor IX expressed from a viral particle comprising (i) a Factor IX nucleotide sequence of SEQ ID NO. 4 or 5 and (ii) a transcription regulatory element of SEQ ID NO. 6;

(c) the patient is a patient who is refractory to treatment with a Factor IX polypeptide that is expressed at a level lower than that encoded by a nucleotide sequence comprising (i) a Factor IX nucleotide sequence of SEQ ID NO. 4 or 5 and (ii) a transcription regulatory sequence of SEQ ID NO. 6;

(d) the polynucleotide is administered at a dose effective to achieve a stable Factor IX activity level of at least 20% in a patient having haemophilia B;

(e) the polynucleotide administered at a dose of at least 4.5×10 11 vg/kg, or at a dose of less than 5×10 11 vg/kg, or at a dose of between 4.5×10 11 and 1×10 12 vg/kg, or at a dose of between 4.5×10 11 vg/kg and 4.9×10 11 vg/kg, optionally wherein the dose is determined by quantifying the number of vector genomes using qPCR, optionally wherein the qPCR uses primers which bind to a promoter region, optionally wherein the promoter region is the promoter region of a transgene cassette;

(f) the polynucleotide that is administered is produced from a mammalian cell and/or possesses characteristics which result from use of mammalian viral vector production cells and distinguish from vectors produced in insect viral vector production cells (e.g. baculovirus system);

(g) the stable Factor IX activity level is a stable Factor IX activity level of at least 25% or of at least 30%, optionally wherein the stable Factor IX activity level is a stable Factor IX activity level of at least 35%, optionally wherein the stable Factor IX activity level is of at least or around 40%;

(h) the Factor IX activity level is at least 20% at a time point at least 50 weeks or at around 52 weeks after administration of the polynucleotide, optionally wherein the Factor IX activity level maintains at at least 20% for a continuous period of at least 40 weeks immediately preceding the time point;

(i) the Factor IX activity level is at least 25% at a time point at least 50 weeks or at around 52 weeks after administration of the polynucleotide, optionally wherein the Factor IX activity level maintains at at least 25% for a continuous period of at least 40 weeks immediately preceding the time point;

(j) the Factor IX activity is at least or around 40% at a time point at least 50 weeks or at around 52 weeks after administration of the polynucleotide, optionally wherein the Factor IX activity level maintains at at least 40% for a continuous period of at least 40 weeks immediately preceding the time point;

(k) the Factor IX activity is measured using a clotting assay to determine the Factor IX activity in a blood sample from the patient;

(l) the Factor IX activity is measured by taking a blood sample from the patient and using a clotting assay to determine the Factor IX activity in the blood sample;

(m) the clotting assay is a one-stage clotting assay;

(n) the method comprises co-administration of a steroid; or

(o) the method comprises co-administration of prednisolone.

Assignments (2)

Priority Claims (1)

Continuity (1)

References Cited (288)