Methods and compositions for nucleic acid and protein payload delivery

Abstract

Provided are methods and compositions for delivering a nucleic acid, protein, and/or ribonucleoprotein payload to a cell. Also provided are delivery molecules that include a peptide targeting ligand conjugated to a protein or nucleic acid payload (e.g., an siRNA molecule), or conjugated to a charged polymer polypeptide domain (e.g., poly-arginine such as 9R or a poly-histidine such as 6H, and the like). The targeting ligand provides for (i) targeted binding to a cell surface protein, and (ii) engagement of a long endosomal recycling pathway. As such, when the targeting ligand engages the intended cell surface protein, the delivery molecule enters the cell (e.g., via endocytosis) but is preferentially directed away from the lysosomal degradation pathway.

I/We claim: 
     
         1 . A delivery molecule, comprising:
 a targeting ligand comprising a dimeric signal peptide domain that orthosterically binds to cKit and is conjugated to a protein payload, a nucleic acid payload, and/or a charged polymer polypeptide domain, wherein   conjugation of the protein payload, the nucleic acid payload, and/or the charged polymer polypeptide domain does not interfere with the targeting ligand binding interface with cKit, relative to the targeting ligand binding interface with cKit without conjugation to the protein payload, the nucleic acid payload, and/or the charged polymer polypeptide domain.   
     
     
         2 . The delivery molecule of  claim 1 , wherein the targeting ligand is a stem cell factor (SCF)-mimetic peptide. 
     
     
         3 . The delivery molecule of  claim 2 , wherein the SCF-mimetic peptide comprises an amino acid sequence that has 85% or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 162, SEQ ID NO. 170, SEQ ID NO: 171, and EKFILKVRPAFKAV. 
     
     
         4 . The delivery molecule of  claim 1 , wherein the targeting ligand comprises an amino acid sequence that has 85% or more sequence identity to SNYSIIDKLVNIVDDLVECVKENS or Ac-SNYSAibADKAibANAibADDAibAEAibAKENS. 
     
     
         5 . The delivery molecule of  claim 1 , wherein the targeting ligand is conjugated to the protein payload, nucleic acid payload, and/or charged polymer polypeptide domain via a linker. 
     
     
         6 . The delivery molecule of  claim 5 , wherein the linker comprises the amino acid sequence selected from the group consisting of SEQ ID NO:17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45. 
     
     
         7 . The delivery molecule of  claim 6 , wherein the targeting ligand is conjugated to a charged polymer polypeptide domain via the linker, and wherein the charged polymer polypeptide domain comprises:
 (i) one or more cationic polymers selected from the group consisting of poly-arginine, poly-lysine, poly-histidine, poly-ornithine, and poly-citrulline; or   (ii) one or more anionic polymers comprising a molecule selected from the group consisting of poly(glutamic acid), poly(aspartic acid), glycosaminoglycan, a glycoprotein, a polysaccharide, poly(mannuronic acid), poly(guluronic acid), heparin, heparin sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, aggrecan, and poly(glucosamine).   
     
     
         8 . The delivery molecule of  claim 7 , wherein the charged polymer polypeptide domain comprises a cysteine residue for conjugation to the linker, a nuclear localization signal, or a cationic polypeptide. 
     
     
         9 . The delivery molecule of  claim 1 , wherein the nucleic acid payload is an RNAi agent;
 optionally, wherein the RNAi agent is an siRNA molecule.   
     
     
         10 . The delivery molecule of  claim 1 , wherein the targeting ligand is configured to engage the long endosomal recycling pathway upon binding of the targeting ligand to a cell surface protein and subsequent endocytosis of the delivery molecule. 
     
     
         11 . A delivery molecule, comprising:
 a targeting ligand comprising a heparin domain that binds to a heparin-affinity domain of cKit, the targeting ligand:   conjugated to a protein payload, a nucleic acid payload, and/or a charged polymer polypeptide domain, and configured to engage the long endosomal recycling pathway upon binding of the targeting ligand to a cell surface protein and subsequent endocytosis of the delivery molecule.   
     
     
         12 . The delivery molecule of  claim 11 , wherein the targeting ligand is a stem cell factor (SCF)-mimetic peptide. 
     
     
         13 . The delivery molecule of  claim 12 , wherein the SCF-mimetic peptide comprises an amino acid sequence that has 85% or more sequence identity to an amino acid sequence selected from the group consisting of SEQ ID NO. 162, SEQ ID NO. 170, SEQ ID NO: 171, and EKFILKVRPAFKAV. 
     
     
         14 . The delivery molecule of  claim 13 , wherein the targeting ligand comprises an amino acid sequence that has 85% or more sequence identity to SNYSIIDKLVNIVDDLVECVKENS or Ac-SNYSAibADKAibANAibADDAibAEAibAKENS. 
     
     
         15 . The delivery molecule of  claim 13 , wherein the targeting ligand is conjugated to the protein payload, nucleic acid payload, and/or charged polymer polypeptide domain via a linker. 
     
     
         16 . The delivery molecule of  claim 15 , wherein the linker comprises the amino acid sequence selected from the group consisting of SEQ ID NO:17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, and 45. 
     
     
         17 . The delivery molecule of  claim 16 , wherein the targeting ligand is conjugated to a charged polymer polypeptide domain via the linker, and wherein the charged polymer polypeptide domain comprises:
 (i) one or more cationic polymers selected from the group consisting of poly-arginine, poly-lysine, poly-histidine, poly-ornithine, and poly-citrulline; or   (ii) one or more anionic polymers comprising a molecule selected from the group consisting of poly(glutamic acid), poly(aspartic acid), glycosaminoglycan, a glycoprotein, a polysaccharide, poly(mannuronic acid), poly(guluronic acid), heparin, heparin sulfate, chondroitin, chondroitin sulfate, keratan, keratan sulfate, aggrecan, and poly(glucosamine).   
     
     
         18 . The delivery molecule of  claim 17 , wherein the charged polymer polypeptide domain comprises a cysteine residue for conjugation to the linker, a nuclear localization signal, or a cationic polypeptide. 
     
     
         19 . The delivery molecule of  claim 11 , wherein the nucleic acid payload is an RNAi agent; optionally, wherein the RNAi agent is an siRNA molecule. 
     
     
         20 . The delivery molecule of  claim 11 , wherein the heparin affinity domain is at the orthosteric site of cKit.