SKELETAL MUSCLE DELIVERY PLATFORMS AND METHODS OF USE THEREOF
The present disclosure relates to delivery vehicles that specifically and efficiently direct payloads to skeletal muscle cells in a subject, in vivo. The delivery vehicles disclosed herein include targeting ligands (such as compounds that have affinity for integrins, including alpha-v-beta-6) and pharmacokinetic/pharmacodynamic (PK/PD) modulators, to facilitate the delivery of payloads to cells, including to skeletal muscle cells. Suitable payloads for use in the delivery vehicles disclosed herein include RNAi agents, which can be linked or conjugated to the delivery vehicles, and when delivered in vivo, provide for the inhibition of gene expression in skeletal muscle cells. Pharmaceutical compositions that include the skeletal muscle cell delivery vehicle are also described, as well as methods of use for the treatment of various diseases and disorders where delivery of a therapeutic payload to a skeletal muscle cell is desirable.
1 . A delivery vehicle for inhibiting expression of a gene expressed in skeletal muscle cells comprising:
(a) an RNAi agent comprising:
(i) an antisense strand comprising 17-49 nucleotides wherein at least 15 nucleotides are complementary to the mRNA sequence of a gene that is expressed in skeletal muscle cells
(ii) a sense strand that is 16-49 nucleotides in length that is at least partially complementary to the antisense strand;
(b) a targeting ligand with affinity for a receptor present on the surface of a skeletal muscle cell, wherein the targeting ligand is a polypeptide; and
(c) a PK/PD modulator;
wherein the RNAi agent is covalently linked to the targeting ligand and to the PK/PD modulator.
2 . The delivery vehicle of claim 1 , wherein the targeting ligand has affinity for an integrin receptor.
3 . The delivery vehicle of claim 1 , wherein the targeting ligand has affinity for the αvβ6 integrin receptor.
4 . The delivery vehicle of claim 1 , wherein the polypeptide of the targeting ligand is a polypeptide of Formula (P):
or a pharmaceutically acceptable salt thereof, wherein
Xaa 1 is L-arginine optionally having an N-terminal cap,
wherein each indicates a point of connection to G′;
G′ is L-glycine or N-methyl-L-glycine;
D is L-aspartic acid (L-aspartate);
L is L-leucine;
Xaa 2 is an L-α amino acid, an L-β amino acid, or an α,α-disubstituted amino acid;
Xaa 3 is an L-α amino acid, an L-β amino acid, or an α,α-disubstituted amino acid;
Xaa 4 is an L-α amino acid, an L-β amino acid, or an α,α-disubstituted amino acid;
Xaa 5 is an L-α amino acid, an L-β amino acid, or an α,α-disubstituted amino acid; and
indicates a point of connection to the RNAi agent.
5 - 16 . (canceled)
17 . The delivery vehicle of claim 1 , wherein the targeting ligand has the formula:
or a pharmaceutically acceptable salt thereof, wherein indicates a point of connection to the remainder of the delivery vehicle.
18 . The delivery vehicle of claim 1 , wherein the targeting ligand has the formula:
or a pharmaceutically acceptable salt thereof, wherein indicates a point of connection to the remainder of the delivery vehicle.
19 - 22 . (canceled)
23 . The delivery vehicle of claim 1 , wherein the PK/PD modulator comprises at least one polyethylene glycol (PEG) unit.
24 . The delivery vehicle of claim 1 , wherein the PK/PD modulator comprises at least ten PEG units.
25 . The delivery vehicle of claim 24 , wherein the PK/PD modulator is:
PEG40K (2 × 2-arm),
wherein n and m are each independently integers, and the molecular weight of the sum of all
PEG units is about 40 kilodaltons
PEG40K (4-arm),
wherein n is an integer, and the molecular weight of the sum of all PEG units is about 40
kilodaltons
PEG40K (2-arm),
wherein n is an integer, and the molecular weight of the sum of all PEG units is about 40
kilodaltons
PEG40K,
wherein n is an integer, and the molecular weight of the sum of all PEG units is about 40
kilodaltons
PEG10K,
wherein n is an integer, and the molecular weight of the sum of all PEG units is about 10
kilodaltons
PEG5K,
wherein n is an integer, and the molecular weight of the sum of all PEG units is about 5
kilodaltons
DSPE-PEG5K-NHS
wherein n is an integer, and the molecular weight of the sum of all PEG units is about 5
kilodaltons
DSPE-PEG5K-MAL
Wherein n is an integer, and the molecular weight of the sum of all PEG units is about 5
kilodaltons
DSPE-PEG5K-N3
wherein n is an integer, and the molecular weight of the sum of all PEG units is about 5
kilodaltons
PEG47 + C22
PEG47 + CLS (cholesterol)
PEG23 + C22
Bis(PEG23 + C14)
Bis(PEG23 + C22)
Bis(PEG47 + C22)
PEG48 + C22
PEG71 + C22
PEG95 + C22
PEG71 + CLS
PEG95 + CLS
Bis(PEG23 + C18)
Tris(PEG23 + C22)
Tris(PEG23 + CLS)
Bis(PEG23 + CLS)
PEG5K + C22
wherein n is an integer, and the molecular weight of the sum of all PEG units is about 5
kilodaltons
C18
(NHS)-PEG1K + C18
wherein n is an integer, and the molecular weight of the sum of all PEG units is about 1
kilodalton
(NHS)-PEG2K + C18
wherein n is an integer, and the molecular weight of the sum of all PEG units is about 2
kilodaltons
(NHS)-PEG5K + C18
wherein n is an integer, and the molecular weight of the sum of all PEG units is about 5
kilodaltons
(MAL)-PEG5K + C18
wherein n is an integer, and the molecular weight of the sum of all PEG units is about 5
kilodaltons
PEG48 + C18
or a pharmaceutically acceptable salt of any of these PK/PD modulators, wherein indicates a point of connection to the RNAi agent.
26 . The delivery vehicle of claim 1 , wherein the PK/PD modulator is a PK/PD modulator of Formula (I):
or a pharmaceutically acceptable salt thereof, wherein
L A is a bond or a bivalent moiety connecting Z to the RNAi agent;
Z is CH, phenyl, or N;
L 1 and L 2 are each independently linkers comprising at least about 5 PEG units;
X and Y are each independently lipids comprising from about 10 to about 50 carbon atoms; and
indicates a point of connection to the RNAi agent.
27 - 33 . (canceled)
34 . The delivery vehicle of claim 26 , wherein the PK/PD modulator of Formula (I) is a PK/PD modulator of Formula (Ia):
or a pharmaceutically acceptable salt thereof.
35 . The delivery vehicle of claim 26 , wherein the PK/PD modulator of Formula (I) is a PK/PD modulator of Formula (Ib):
or a pharmaceutically acceptable salt thereof.
36 . The delivery vehicle of claim 26 , wherein the PK/PD modulator of Formula (I) is a PK/PD modulator of Formula (Ic):
or a pharmaceutically acceptable salt thereof.
37 - 42 . (canceled)
43 . The delivery vehicle of claim 26 , wherein at least one of X and Y is selected from the group consisting of:
Name
Structure
Lipid 1
Lipid 2
Lipid 3
Lipid 4
Lipid 5
Lipid 6
Lipid 7
Lipid 8
Lipid 9
Lipid 10
Lipid 11
Lipid 12
Lipid 14
Lipid 15
Lipid 16
Lipid 17
Lipid 18
Lipid 19
Lipid 20
Lipid 21
Lipid 22
Lipid 23
Lipid 24
wherein indicates a point of connection to L 1 or L 2 .
44 . The delivery vehicle of claim 26 , wherein both X and Y are each independently selected from the group consisting of:
Name
Structure
Lipid 1
Lipid 2
Lipid 3
Lipid 4
Lipid 5
Lipid 6
Lipid 7
Lipid 8
Lipid 9
Lipid 10
Lipid 11
Lipid 12
Lipid 14
Lipid 15
Lipid 16
Lipid 17
Lipid 18
Lipid 19
Lipid 20
Lipid 21
Lipid 22
Lipid 23
Lipid 24
wherein indicates a point of connection to L 1 or L 2 .
45 - 46 . (canceled)
47 . The delivery vehicle of claim 1 , wherein the PK/PD modulator is selected from the group consisting of:
or a pharmaceutically acceptable salt of any of these PK/PD modulators, wherein each indicates a point of connection to the RNAi agent.
48 . The delivery vehicle of claim 1 , wherein the RNAi agent inhibits expression of the mRNA of a human gene in a skeletal muscle cell.
49 . The delivery vehicle of claim 4 , wherein the pharmaceutically acceptable salt is a sodium salt.
50 . (canceled)
51 . A composition comprising the delivery vehicle of claim 1 .
52 . A pharmaceutical composition comprising the composition of claim 51 and a pharmaceutical excipient.
53 . The pharmaceutical composition of claim 52 , wherein the pharmaceutical excipient is selected form water for injection and saline solution.
54 . The pharmaceutical composition of claim 53 , wherein the pharmaceutical excipient is saline solution.
55 . A method of treating a disease or disorder of a skeletal muscle cell comprising administering to a subject in need thereof a composition of claim 51 .
56 . The method of claim 55 , wherein the disease or disorder is muscular dystrophy.
57 . The method of claim 56 , wherein the muscular dystrophy is selected from the group consisting of: Duchenne muscular dystrophy, myotonic muscular dystrophy, Becker muscular dystrophy, limb-girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, and Emery-Dreifuss muscular dystrophy.
58 - 65 . (canceled)
66 . A method of making the delivery vehicle of claim 1 , the method comprising:
(i) synthesizing the sense strand;
(ii) synthesizing the antisense strand;
(iii) annealing the sense strand and the antisense strand;
(iv) before or after annealing the sense strand and the antisense strand, conjugating the targeting ligand to the sense strand or the antisense strand; and
(v) before or after annealing the sense strand and the antisense strand, and before or after conjugating the targeting ligand to the sense strand or the antisense strand, conjugating the PK/PD modulator to the sense strand or the antisense strand.