Materials and complexes for the delivery of biologically-active materials to cells

1. A non-viral delivery complex, for delivering genetic material when combined with a nucleic acid, that comprises:

(i) a peptide of formula A-B-C wherein

A is a polycationic nucleic acid-binding component,

B is a spacer element peptide that is susceptible to cleavage within a cell, and

C is a cell surface receptor binding component,

wherein B, the spacer element peptide comprises amino acid sequences susceptible to cleavage by furin selected from RX 1 KR [SEQ ID NO: 2] and RX 2 RR [SEQ ID NO: 3] in which X 1 and X 2 which may be the same or different and each represent any amino acid residue, or comprises amino acid sequences susceptible to cleavage by cathepsin selected from X 3 X 4 in which X 3 is selected from Tyrosine (Tyr, Y), Phenylalanine (Phe, F), Leucine (Leu, L), Valine (Val, V) and Isoleucine (Ile, I) and X 4 is selected from Glycine (Gly, G), Alanine (Ala, A) and Glutamic acid (Glu, E);

and wherein C, the cell surface receptor binding component, comprises a peptide sequence selected from:

RGD;

RRETAWA [SEQ ID NO: 5];

LDV;

X 5 SM in which X 5 is a basic amino acid residue;

LX 6 HK [SEQ ID NO: 6] in which X 6 is Q or P;

PSGX 7 ARA [SEQ ID NO: 7] in which X 7 is A or T;

SX 8 RSMNF [SEQ ID NO: 8] in which X 8 is an acidic amino acid residue;

LX 9 HKSMP [SEQ ID NO: 9] in which X 9 is P or Q;

PX 10 X 11 X 12 T [SEQ ID NO: 19] in which X 10 , X 11 and X 12 , which may be the same or different, each represents an amino acid residue;

PSX 13 S [SEQ ID NO: 20] in which X 13 represents an amino acid residue;

QX 14 X 15 X 16 Q [SEQ ID NO: 21] in which X 14 and X 16 , which may be the same or different, each represents an amino acid residue, and X 15 represents an amino acid residue having an amide side chain; and

SX 17 S in which X 17 represents an amino acid residue having an aliphatic side chain; and

(ii) a lipid of formula (III):

wherein:

each Y is the same or different and is selected from —O—, and —O—C(O)—;

each R 4 is the same or different and is selected from a straight or branched, saturated or unsaturated C 7-24 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR′, wherein R′ is a C 1-6 hydrocarbyl group;

each R 5 is the same or different and is selected from a straight or branched, saturated or unsaturated C 1-10 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR′, —C(O)OH, —CN, —N(R′) 2 , and —C(O)R′, wherein each R′ is the same or different and is a C 1-6 hydrocarbyl group;

Sp is a C 1-8 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR′, wherein R′ is a C 1-6 hydrocarbyl group;

W is selected from —O—C(O)—, —C(O)O—, and the group of formula (IV):

n denotes the number of chains, and n=1 if the linker W is —O—C(O)— or —C(O)O—, and n=2 if the linker W is the group of formula (IV);

each B is the same or different and is a C 1-6 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR 1 , —C(O)OH, —CN, —NR 1 R 2 , —C(O)OR 1 , —OC(O)R 1 and —C(O)R 1 ;

R 1 and R 2 are the same or different and are C 1-4 hydrocarbyl;

m is an integer from 1 to 100; and

Q is selected from —N + (R 3 ) 3 , —OH, and —OR 3 wherein R 3 is an unsubstituted C 1-4 alkyl group or a trifluoromethyl group.

2. A non-viral delivery complex, for delivering genetic material when combined with a nucleic acid, that comprises:

(i) a peptide of formula A-B-C wherein

A is a polycationic nucleic acid-binding component,

B is a spacer element peptide that is susceptible to cleavage within a cell, and

C is a cell surface receptor binding component,

wherein B, the spacer element peptide comprises amino acid sequences susceptible to cleavage by furin selected from RX 1 KR [SEQ ID NO: 2] and RX 2 RR [SEQ ID NO: 3] in which X 1 and X 2 which may be the same or different and each represent any amino acid residue, or comprises amino acid sequences susceptible to cleavage by cathepsin selected from X 3 X 4 in which X 3 is selected from Tyrosine (Tyr, Y), Phenylalanine (Phe, F), Leucine (Leu, L), Valine (Val, V) and Isoleucine (Ile, I) and X 4 is selected from Glycine (Gly, G), Alanine (Ala, A) and Glutamic acid (Glu, E);

and wherein C, the cell surface receptor binding component, comprises a peptide sequence selected from:

RGD;

RRETAWA [SEQ ID NO: 5];

LDV;

X 5 SM in which X 5 is a basic amino acid residue;

LX 6 HK [SEQ ID NO: 6] in which X 6 is Q or P;

PSGX 7 ARA [SEQ ID NO: 7] in which X 7 is A or T;

SX 8 RSMNF [SEQ ID NO: 8] in which X 8 is an acidic amino acid residue;

LX 9 HKSMP [SEQ ID NO: 9] in which X 9 is P or Q;

PX 10 X 11 X 12 T [SEQ ID NO: 19] in which X 10 , X 11 and X 12 , which may be the same or different, each represents an amino acid residue;

PSX 13 S [SEQ ID NO: 20] in which X 13 represents an amino acid residue;

QX 14 X 15 X 16 Q [SEQ ID NO: 21] in which X 14 and X 16 , which may be the same or different, each represents an amino acid residue, and X 15 represents an amino acid residue having an amide side chain; and

SX 17 S in which X 17 represents an amino acid residue having an aliphatic side chain; and

(ii) a lipid of formula (VI):

wherein:

each Y is the same or different and is selected from —O—, and —O—C(O)—;

each R 4 is the same or different and is selected from a straight or branched, saturated or unsaturated C 7-24 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR′, wherein R′ is a C 1-6 hydrocarbyl group;

each R 5 is the same or different and is selected from a straight or branched, saturated or unsaturated C 1-10 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR′, —C(O)OH, —CN, —N(R′) 2 , and —C(O)R′, wherein each R′ is the same or different and is a C 1-6 hydrocarbyl group;

Sp is a C 1-8 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR′, wherein R′ is a C 1-6 hydrocarbyl group;

W is a group susceptible to cleavage within a cell;

q denotes the number of P-G-chains;

each B is the same or different and is a C 1-6 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR 1 , —C(O)OH, —CN, —NR 1 R 2 , —C(O)OR 1 , —OC(O)R 1 and —C(O)R 1 ;

R 1 and R 2 are the same or different and are C 1-4 hydrocarbyl;

p is an integer from 1 to 8; and

Q is selected from —N + (R 3 ) 3 , —OH, and —OR 3 wherein R 3 is an unsubstituted C 1-4 alkyl group or a trifluoromethyl group.

3. A non-viral transfection complex comprising the non-viral delivery complex as claimed in claim 1 or claim 2 and a nucleic acid.

4. A pharmaceutical composition which comprises a transfection complex as claimed in claim 3 in admixture with a pharmaceutically suitable carrier.

5. A method for the treatment of a cancer in a human or in a non-human animal which comprises administering a transfection complex as claimed in claim 3 to the human or to the non-human animal by direct injection into protected tissues or general parenteral administration for non-protected tissues, wherein a target cell is transfected with the nucleic acid.

6. A peptide of formula A-B-C wherein:

A is a polycationic nucleic acid-binding component,

B is a spacer element peptide that is susceptible to cleavage by furin, and

C is a cell surface receptor binding component,

and wherein A, the polycationic nucleic acid-binding component, is an oligo-lysine molecule, an oligo-histidine molecule, an oligo-arginine molecule, an oligo-ornithine molecule, an oligo-diaminopropionic acid molecule or an oligo-diaminobutyric acid molecule or a combined oligomer comprising any combination of histidine, arginine, lysine, ornithine, diaminopropionic acid, and diaminobutyric acid residues or polyethylenimine (PEI);

B, the spacer element peptide, comprises the amino acid sequence RX 1 KR [SEQ ID NO: 2] or RX 2 RR [SEQ ID NO: 3] in which X 1 and X 2 which may be the same or different and each represent any amino acid residue;

and wherein C, the cell surface receptor binding component, comprises a peptide sequence selected from:

a) CRGDCLG [SEQ ID NO: 22];

b) CRGDCLG [SEQ ID NO: 22];

c) ACDCRGDCFCG [SEQ ID NO: 23];

d) CRGDMFGCA [SEQ ID NO: 24];

e) CRRETAWACG [SEQ ID NO: 25];

f) CRGEMFGCA [SEQ ID NO: 26];

g) CSERSMNFCG [SEQ ID NO: 27];

h) CYGLPHKFCG [SEQ ID NO: 28]; and

i) CLPHKSMPCG [SEQ ID NO: 29].

7. The peptide of claim 6 wherein A, the polycationic nucleic acid-binding component, is an oligo-lysine molecule.

8. The peptide of claim 6 wherein B, the spacer element peptide, comprises the amino acid sequence RVRR [SEQ ID NO: 3].

9. The peptide of claim 6 wherein C, cell surface receptor binding component, comprises the amino acid sequence CRGDCLG [SEQ ID NO: 22].

10. The peptide of claim 6 , which is (K) 16 RVRRGACRGDCLG [SEQ ID NO: 30].

11. A non-viral delivery complex, for delivering genetic material when combined with a nucleic acid, that comprises:

(i) a peptide of formula A-B-C wherein

A is a polycationic nucleic acid-binding component,

B is a spacer element peptide that is susceptible to cleavage by furin, and

C is a cell surface receptor binding component,

and wherein A, the polycationic nucleic acid-binding component, is an oligo-lysine molecule, an oligo-histidine molecule, an oligo-arginine molecule, an oligo-ornithine molecule, an oligo-diaminopropionic acid molecule or an oligo-diaminobutyric acid molecule or a combined oligomer comprising any combination of histidine, arginine, lysine, ornithine, diaminopropionic acid, and diaminobutyric acid residues or polyethylenimine (PEI);

B, the spacer element peptide, comprises the amino acid sequence RX 1 KR [SEQ ID NO: 2] or RX 2 RR [SEQ ID NO: 3] in which X 1 and X 2 which may be the same or different and each represent any amino acid residue;

and wherein C, the cell surface receptor binding component, comprises a peptide sequence selected from:

a) CRGDCLG [SEQ ID NO: 22];

b) CRGDCLG [SEQ ID NO: 22];

c) ACDCRGDCFCG [SEQ ID NO: 23];

d) CRGDMFGCA [SEQ ID NO: 24];

e) CRRETAWACG [SEQ ID NO: 25];

f) CRGEMFGCA [SEQ ID NO: 26];

g) CSERSMNFCG [SEQ ID NO: 27];

h) CYGLPHKFCG [SEQ ID NO: 28]; and

i) CLPHKSMPCG [SEQ ID NO: 29];

and

(ii) a lipid of formula (III):

wherein:

each Y is the same or different and is selected from —O—, and —O—C(O)—;

each R 4 is the same or different and is selected from a straight or branched, saturated or unsaturated C 7-24 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR′, wherein R′ is a C 1-6 hydrocarbyl group;

each R 5 is the same or different and is selected from a straight or branched, saturated or unsaturated C 1-10 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR′, —C(O)OH, —CN, —N(R′) 2 , and —C(O)R′, wherein each R′ is the same or different and is a C 1-6 hydrocarbyl group;

Sp is a C 1-8 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR′, wherein R′ is a C 1-6 hydrocarbyl group;

W is selected from —O—C(O)—, —C(O)O—, and the group of formula (IV):

n denotes the number of chains, and n=1 if the linker W is —O—C(O)— or —C(O)O—, and n=2 if the linker W is the group of formula (IV);

each B is the same or different and is a C 1-6 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR 1 , —C(O)OH, —CN, —NR 1 R 2 , —C(O)OR 1 , —OC(O)R 1 and —C(O)R 1 ;

R 1 and R 2 are the same or different and are C 1-4 hydrocarbyl;

m is an integer from 1 to 100; and

Q is selected from —N + (R 3 ) 3 , —OH, and —OR 3 wherein R 3 is an unsubstituted C 1-4 alkyl group or a trifluoromethyl group.

12. A non-viral transfection complex comprising the non-viral delivery complex as claimed in claim 11 and a nucleic acid.

13. A non-viral delivery complex, for delivering genetic material when combined with a nucleic acid, that comprises:

(i) a peptide of formula A-B-C wherein

A is a polycationic nucleic acid-binding component,

B is a spacer element peptide that is susceptible to cleavage by furin, and

C is a cell surface receptor binding component,

and wherein A, the polycationic nucleic acid-binding component, is an oligo-lysine molecule, an oligo-histidine molecule, an oligo-arginine molecule, an oligo-ornithine molecule, an oligo-diaminopropionic acid molecule or an oligo-diaminobutyric acid molecule or a combined oligomer comprising any combination of histidine, arginine, lysine, ornithine, diaminopropionic acid, and diaminobutyric acid residues or polyethylenimine (PEI);

B, the spacer element peptide, comprises the amino acid sequence RX 1 KR [SEQ ID NO: 2] or RX 2 RR [SEQ ID NO: 3] in which X 1 and X 2 which may be the same or different and each represent any amino acid residue;

and wherein C, the cell surface receptor binding component, comprises a peptide sequence selected from:

a) CRGDCLG [SEQ ID NO: 22];

b) CRGDCLG [SEQ ID NO: 22];

c) ACDCRGDCFCG [SEQ ID NO: 23];

d) CRGDMFGCA [SEQ ID NO: 24];

e) CRRETAWACG [SEQ ID NO: 25];

f) CRGEMFGCA [SEQ ID NO: 26];

g) CSERSMNFCG [SEQ ID NO: 27];

h) CYGLPHKFCG [SEQ ID NO: 28]; and

i) CLPHKSMPCG [SEQ ID NO: 29];

and

(ii) a lipid of formula (VI):

wherein:

each Y is the same or different and is selected from —O—, and —O—C(O)—;

each R 4 is the same or different and is selected from a straight or branched, saturated or unsaturated C 7-24 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR′, wherein R′ is a C 1-6 hydrocarbyl group;

each R 5 is the same or different and is selected from a straight or branched, saturated or unsaturated C 1-10 hydrocarbyl group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR′, —C(O)OH, —CN, —N(R′) 2 , and —C(O)R′, wherein each R′ is the same or different and is a C 1-6 hydrocarbyl group;

Sp is a C 1-8 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen and OR′, wherein R′ is a C 1-6 hydrocarbyl group;

W is a group susceptible to cleavage within a cell;

q denotes the number of chains;

each B is the same or different and is a C 1-6 alkylene group which is unsubstituted or substituted by one or more substituents selected from hydroxy, halogen, —OR 1 , —C(O)OH, —CN, —NR 1 R 2 , —C(O)OR 1 , —OC(O)R 1 and —C(O)R 1 ;

R 1 and R 2 are the same or different and are C 1-4 hydrocarbyl;

p is an integer from 1 to 8; and

Q is selected from —N + (R 3 ) 3 , —OH, and —OR 3 wherein R 3 is an unsubstituted C 1-4 alkyl group or a trifluoromethyl group.

14. A non-viral transfection complex comprising the non-viral delivery complex as claimed in claim 13 and a nucleic acid.