Gain-of-function FVIII variants
| FVIII variant . | Gain of function . | Protein change . | Fold change . | Reference . |
|---|---|---|---|---|
| SQ | High expression | B domain replaced by 14 amino acid linker (Figure 1) with increased messenger RNA levels but decreased secretion efficiency | 2 | 21 |
| N6 | Increase secretion | B domain replaced by 226 amino acid linker from the N-terminal of the B domain | 4 | 29 |
| V3 | Increase secretion | Incorporation of 6 N-glycosylation sites within SQ- linker (Figure 1) | 2-3 | 31 |
| ΔF∗ | Increase secretion | Removal of furin recognition motif from SQ-linker (Figure 1) | 3 | 34 |
| Δ3-SP/DE∗ | Increase secretion | Partial removal of furin recognition motif from SQ-linker and elimination of secondary cleavage site in a3 (Figure 1) | 2-5 | 35 |
| F309S | Increase secretion | Polar amino acid substitution in A1 hydrophobic patch | 2.3 | 36 |
| L303E/F309S | Increase secretion | Polar amino acid substitutions in A1 hydrophobic patch | 3 | 36 |
| ET3∗ | Increase secretion | Human/porcine hybrid | 10 | 40 |
| X5∗ | Increase secretion | 5 porcine amino acid substitutions in A1 (V86I, S108A, K132G, T147M, and P152L) | 8 | 41 |
| C1899G/C1903G | Increase secretion | 2 substitutions within A3 to eliminate a disulfide bond | 1.5 | 65 |
| P290T | High specific activity | 1 amino acid substitution in A1 | 1.5 | 66 |
| K1813A | High specific activity | 1 amino acid substitution in A3 | 2 | 67 |
| K12∗ | High specific activity | 10 canine amino acid substitutions (V1857I, H1859R, M1907K, M1926K, L1975V, A1993V, H2007Q, D2066E, K2085M, and Q2113H) and 2 additional substitutions (S2157N and R2159H) in the light chain | 6 | 68 |
| IR8 | Inactivation resistant | B domain replaced by 54 amino acid linker from the N-terminal; a3 removed; R336I and R562K to limit APC inactivation; R740A to limit thrombin cleavage | 5 | 69 |
| CC | Inactivation resistant | 2 substitutions (Y664C/T1826C) or (M662C/D1828C) to introduce a disulfide bond between A2 and A3 | 2 | 70 |
| VV | Inactivation resistant | 2 hydrophobic substitutions for negative charge amino acids in the A2-domain (D519V and E665) | 2 | 71 |
| Inactivation resistant | Elimination of both APC cleavage sites (R336Q and R562Q) | 5 | 72 |
| FVIII variant . | Gain of function . | Protein change . | Fold change . | Reference . |
|---|---|---|---|---|
| SQ | High expression | B domain replaced by 14 amino acid linker (Figure 1) with increased messenger RNA levels but decreased secretion efficiency | 2 | 21 |
| N6 | Increase secretion | B domain replaced by 226 amino acid linker from the N-terminal of the B domain | 4 | 29 |
| V3 | Increase secretion | Incorporation of 6 N-glycosylation sites within SQ- linker (Figure 1) | 2-3 | 31 |
| ΔF∗ | Increase secretion | Removal of furin recognition motif from SQ-linker (Figure 1) | 3 | 34 |
| Δ3-SP/DE∗ | Increase secretion | Partial removal of furin recognition motif from SQ-linker and elimination of secondary cleavage site in a3 (Figure 1) | 2-5 | 35 |
| F309S | Increase secretion | Polar amino acid substitution in A1 hydrophobic patch | 2.3 | 36 |
| L303E/F309S | Increase secretion | Polar amino acid substitutions in A1 hydrophobic patch | 3 | 36 |
| ET3∗ | Increase secretion | Human/porcine hybrid | 10 | 40 |
| X5∗ | Increase secretion | 5 porcine amino acid substitutions in A1 (V86I, S108A, K132G, T147M, and P152L) | 8 | 41 |
| C1899G/C1903G | Increase secretion | 2 substitutions within A3 to eliminate a disulfide bond | 1.5 | 65 |
| P290T | High specific activity | 1 amino acid substitution in A1 | 1.5 | 66 |
| K1813A | High specific activity | 1 amino acid substitution in A3 | 2 | 67 |
| K12∗ | High specific activity | 10 canine amino acid substitutions (V1857I, H1859R, M1907K, M1926K, L1975V, A1993V, H2007Q, D2066E, K2085M, and Q2113H) and 2 additional substitutions (S2157N and R2159H) in the light chain | 6 | 68 |
| IR8 | Inactivation resistant | B domain replaced by 54 amino acid linker from the N-terminal; a3 removed; R336I and R562K to limit APC inactivation; R740A to limit thrombin cleavage | 5 | 69 |
| CC | Inactivation resistant | 2 substitutions (Y664C/T1826C) or (M662C/D1828C) to introduce a disulfide bond between A2 and A3 | 2 | 70 |
| VV | Inactivation resistant | 2 hydrophobic substitutions for negative charge amino acids in the A2-domain (D519V and E665) | 2 | 71 |
| Inactivation resistant | Elimination of both APC cleavage sites (R336Q and R562Q) | 5 | 72 |
Variants highlighted in bold are included in rAAV vectors approved or in clinical trials.
Variants engineered based on FVIII orthologs