A protein transduction composition comprising a shuttle agent and a polypeptide cargo to be delivered intracellularly to mammalian target cells, wherein the shuttle agent is:
- (1) a peptide soluble in aqueous solution having an overall length of between 20 and 150 amino acids comprising
- (2) an amphipathic alpha-helical motif having
- (3) a positively-charged hydrophilic outer face comprising:
- (a) at least two adjacent positively-charged K and/or R residues upon helical wheel projection;
- and/or (b) a segment of six adjacent residues comprising three to five K and/or R residues upon helical wheel projection, based on an alpha helix having angle of rotation between consecutive amino acids of 100 degrees and/or an alpha-helix having 3.6 residues per turn;
- and
- (4) a hydrophobic outer face comprising a highly hydrophobic core consisting of spatially adjacent L, I, F, V, W, and/or M amino acids representing 12 to 50% of the amino acids of the peptide, based on an open cylindrical representation of the alpha-helix having 3.6 residues per turn;
- wherein at least six of the following parameters (5) to (15) are respected:
- (5) the peptide has a hydrophobic moment (μ) of 3.5 to 11;
- (6) the peptide has a predicted net charge of at least +4 at physiological pH, calculated from amino acid residues having charged side chains;
- (7) the peptide has an isoelectric point (pI) of 8 to 13;
- (8) the peptide is composed of 35% to 65% of any combination of the amino acids:
- A, C, G, I, L, M, F, P, W, Y, and V;
- (9) the peptide is composed of 0% to 30% of any combination of the amino acids:
- N, Q, S, and T;
- (10) the peptide is composed of 35% to 85% of any combination of the amino acids:
- A, L, K, or R;
- (11) the peptide is composed of 15% to 45% of any combination of the amino acids:
- A and L, provided there being at least 5% of L in the peptide;
- (12) the peptide is composed of 20% to 45% of any combination of the amino acids:
- K and R;
- (13) the peptide is composed of 0% to 10% of any combination of the amino acids:
- D and E;
- (14) the difference between the percentage of A and L residues in the peptide (% A+L), and the percentage of K and R residues in the peptide (% K+R), is less than or equal to 10%;
- and
- (15) the peptide is composed of 10% to 45% of any combination of the amino acids:
- Q, Y, W, P, I, S, G, V, F, E, D, C, M, N, T and H,
- wherein the concentration of the shuttle agent in said composition is at least 2.5 μM and is sufficient to increase transduction efficiency and cytosolic delivery of the polypeptide cargo, as compared to in the absence of the shuttle agent, upon contact of the composition with target eukaryotic cells,
- wherein the shuttle agent and the polypeptide cargo comprise independent polypeptide backbones or are not covalently bound, and
- wherein both the shuttle agent and the polypeptide cargo lack a cell penetrating domain.