Recombinant factor VIII proteins

1. An isolated nucleic acid comprising a sequence encoding a recombinant FVIII protein, wherein the recombinant FVIII protein comprises:

a first polypeptide comprising Formula I: (A1)-a1-(A2)-a2-[B]; and a second polypeptide comprising Formula II: a3-(A3)-(C1);

wherein the first polypeptide and the second polypeptide are fused or exist as a heterodimer;

wherein, a) A1 is an A1 domain of FVIII; b) A2 is an A2 domain of FVIII; c) [B] is a B domain of FVIII, a fragment thereof, or is deleted; d) A3 is an A3 domain of FVIII; e) C1 is a C1 domain of FVIII; f) a1, a2, and a3 are acidic spacer regions;

wherein the A1 domain comprises a permissive loop-1 (A1-1) region and a permissive loop-2 (A1-2) region;

wherein the A2 domain comprises a permissive loop-1 (A2-1) region and a permissive loop-2 (A2-2) region;

wherein the A3 domain comprises a permissive loop-1 (A3-1) region and a permissive loop-2 (A3-2) region;

wherein a first half-life extending moiety and a second half-life extending moiety are inserted within the FVIII protein immediately downstream of amino acids which correspond to amino acids in mature native human FVIII selected from the group consisting of:

a) amino acids 18 and 1656 of SEQ ID NO:1;

b) amino acids 40 and 1656 of SEQ ID NO:1;

c) amino acids 399 and 1656 of SEQ ID NO:1;

d) amino acids 403 and 1656 of SEQ ID NO:1;

e) amino acids 1656 and 1720 of SEQ ID NO:1;

f) amino acids 1656 and 1900 of SEQ ID NO:1;

g) amino acids 18 and 399 of SEQ ID NO:1;

h) amino acids 26 and 399 of SEQ ID NO:1; and

i) amino acids 40 and 399 of SEQ ID NO:1;

wherein the first half-life extending moiety and the second half-life extending moiety are independently selected from the group consisting of:

(a) albumin;

(b) albumin-binding polypeptide;

(c) Fe;

(d) PAS;

(e) the C-terminal peptide (CTP) of the β subunit of human chorionic gonadotropin;

(f) polyethylene glycol (PEG);

(g) hydroxyethyl starch (HES);

(h) albumin-binding small molecules;

(i) a clearance receptor, or fragment thereof, wherein the clearance receptor blocks binding of the recombinant FVIII protein to FVIII clearance receptors;

(j) a low-density lipoprotein receptor-related protein 1 (LRP1) or a FVIII-binding fragment thereof;

(k) an XTEN; and

(l) any combination of (a)-(j); and

wherein the recombinant FVIII protein exhibits procoagulant activity.

2. The isolated nucleic acid of claim 1 , wherein the first half-life extending moiety is inserted immediately downstream of amino acid 399 of SEQ ID NO:1, corresponding to mature native human FVIII; and the second half-life extending moiety is inserted immediately downstream of amino acid 1656 of SEQ ID NO:1, corresponding to mature native human FVIII.

3. The isolated nucleic acid of claim 1 , wherein the first half-life extending moiety is inserted immediately downstream of amino acid 18 of SEQ ID NO:1, corresponding to mature native human FVIII; and the second half-life extending moiety is inserted immediately downstream of amino acid 399 of SEQ ID NO:1, corresponding to mature native human FVIII.

4. The isolated nucleic acid of claim 1 , wherein the first half-life extending moiety is inserted immediately downstream of amino acid 26 of SEQ ID NO:1, corresponding to mature native human FVIII; and the second half-life extending moiety is inserted immediately downstream of amino acid 399 of SEQ ID NO:1, corresponding to mature native human FVIII.

5. The isolated nucleic acid of claim 1 , wherein the first half-life extending moiety is inserted immediately downstream of amino acid 40 of SEQ ID NO:1, corresponding to mature native human FVIII; and the second half-life extending moiety is inserted immediately downstream of amino acid 399 of SEQ ID NO:1, corresponding to mature native human FVIII.

6. The isolated nucleic acid of claim 1 , wherein the first half-life extending moiety is inserted immediately downstream of amino acid 18 of SEQ ID NO:1, corresponding to mature native human FVIII; and the second half-life extending moiety is inserted immediately downstream of amino acid 1656 of SEQ ID NO:1, corresponding to mature native human FVIII.

7. The isolated nucleic acid of claim 1 , wherein the first half-life extending moiety is inserted immediately downstream of amino acid 40 of SEQ ID NO:1, corresponding to mature native human FVIII; and the second half-life extending moiety is inserted immediately downstream of amino acid 1656 of SEQ ID NO:1, corresponding to mature native human FVIII.

8. The isolated nucleic acid of claim 1 , wherein the first half-life extending moiety is inserted immediately downstream of amino acid 1656 of SEQ ID NO:1, corresponding to mature native human FVIII; and the second half-life extending moiety is inserted immediately downstream of amino acid 1720 of SEQ ID NO:1, corresponding to mature native human FVIII.

9. The isolated nucleic acid of claim 1 , wherein the first half-life extending moiety is inserted immediately downstream of amino acid 403 of SEQ ID NO:1, corresponding to mature native human FVIII; and the second half-life extending moiety is inserted immediately downstream of amino acid 1656 of SEQ ID NO:1, corresponding to mature native human FVIII.

10. The isolated nucleic acid of claim 1 , wherein the first half-life extending moiety is inserted immediately downstream of amino acid 1656 of SEQ ID NO:1, corresponding to mature native human FVIII; and the second half-life extending moiety is inserted immediately downstream of amino acid 1900 of SEQ ID NO:1, corresponding to mature native human FVIII.

11. The recombinant FVIII protein of claim 1 , wherein the first half-life extending moiety or the second half-life extending moiety is an albumin.

12. The recombinant FVIII protein of claim 1 , wherein the first half-life extending moiety or the second half-life extending moiety is an Fc.

13. The recombinant FVIII protein of claim 1 , wherein the first half-life extending moiety or the second half-life extending moiety is an XTEN.