Regeneration of mammalian tissues using synthetic immodulins

Abstract

Methods and compositions involving synthetic immodulin peptides and helper molecules. The peptides exhibit new and surprising biological activities, such as co-stimulation of macrophage and myogenic cell differentiation markers. Methods are provided N by which peptide is contacted with one or more myeloid precursor cell populations, thereby increasing the abundance of CD169+, CCL22+, Clec4A, Clec9a+, and Clec 12a+ monocyte lineages which play important roles in cross presentation, post-apoptotic clearance, autoimmunity, and programmatic tissue regeneration, notably in contexts of tissue stress, insult and degeneration. The disclosed methods and compositions enable concurrent regeneration of diverse cellular elements in tissue where collaborating myeloid and non-myeloid lineages are located together in a living tissue following the contacting steps. Furthermore, the peptides of the invention can be delivered with inherently specific in vivo targeting, achieved through complexation to holotransferrin and/or size-specific glycosaminoglycans (e.g. high-molecular- weight hyaluronan) so as to discriminate between target environments in vivo or ex vivo.

What is claimed is: 
     
         1 . A method for stimulating levels of differentiation markers in myeloid and non-myeloid mammalian cell lineages in vivo or ex vivo, the method comprising:
 (i) contacting one or more mammalian cells of a myeloid lineage with a synthetic immodulin peptide about 20 to about 60 amino acids in length and comprising an amino acid sequence corresponding to any one of SEQ ID NOs:1-6; and   (ii) contacting one or more mammalian cells of a non-myeloid lineage with said synthetic immodulin peptide;   (iii) measuring a readout of differentiation for the myeloid mammalian cell lineage wherein said readout is accompanied by a significantly increased average level of at least one marker for myeloid lineage differentiation in the contacted cells of the myeloid lineage and wherein the at least one marker for myeloid lineage differentiation comprises CD169; and   (iv) measuring a readout of differentiation for the non-myeloid mammalian cell lineage wherein said readout is accompanied by a significantly increased average level of at least one marker for non-myeloid lineage differentiation in the contacted cells of the non-myeloid lineage;   wherein cells of said myeloid and non-myeloid lineages following the contacting steps are co-resident in a living tissue.   
     
     
         2 . The method according to  claim 1 , wherein the at least one marker for myeloid differentiation comprises a marker selected from the group consisting of: CCL22, Clec4A, Clec9a, Clec10a and Clec12a. 
     
     
         3 . The method according to  claim 1 , wherein the one or more mammalian cells of a non-myeloid lineage are from a neurogenic lineage, the readout is neurogenesis, and the at least one marker for non-myeloid differentiation is selected from the group consisting of: synaptophysin, synaptopodin, vimentin, NMDAR, AChR, and PSD95. 
     
     
         4 . The method according to  claim 1 , wherein the one or more mammalian cells of a non-myeloid lineage are from a myogenic lineage, the readout is myogenesis, and the at least one marker for non-myeloid differentiation is selected from the group consisting of: creatine kinase, myogenin, MYF-5 and actinin-2. 
     
     
         5 . The method according to  claim 1 , wherein the one or more mammalian cells of a non-myeloid lineage are from a osteogenic lineage, the readout is osteogenesis, and the at least one marker for non-myeloid differentiation is selected from the group consisting of alkaline phosphatase, calcified deposits, osteocalcin and BMP-2. 
     
     
         6 . The method according to  claim 1 , wherein the one or more mammalian cells of a non-myeloid lineage are from a fibroblastic lineage, the readout is dermal regeneration, and the at least one marker for non-myeloid differentiation is Col1A1. 
     
     
         7 . The method according to  claim 1 , wherein the one or more mammalian cells of a non-myeloid lineage are from a lymphocytic lineage, the readout is regulatory lymphocyte proliferation, and the at least one marker for non-myeloid differentiation is selected from the group consisting of: FoxP3 and IL-10. 
     
     
         8 . The method according to  claim 1 , wherein the synthetic immodulin peptide is covalently linked to at least one small non-amino acid molecule of molecular mass less than one thousand daltons. 
     
     
         9 . The method according to  claim 8 , wherein the small non-amino-acid molecule is selected from a group consisting of: PF-06409577, AICAR, D942, PT1, EX-229, GIT27, GW501516, GW3965, GW9578, RB394, MBX-8025, GW7647, ZLY032, GW590735, GW0742, Amorfrutin B, BMS195614, GW4064, BMS453, FTY720, artesunate, sobetirone, cilofexor, decanoic acid, eicosapentaenoic acid, docosahexanoic acid, lignoceric acid, TTNPB, adapalene, bexarotene, transcinnamic acid, fenofibric acid, ciprofibrate, chlorfibric acid, gemfibrozil, elafibrinor, pioglitazone, roziglitazone, valproic acid, 2-hexyl-4-pentynoic acid, and ibuprofen. 
     
     
         10 . The method according to  claim 8 , wherein the synthetic immodulin peptide is co-administered with a helper molecule selected from a group consisting of: ornithine, leucine, raffinose, trehalose, resveratrol, polydatin, ursolic acid, lactate, bile salt, metal, cyclodextrin and cyclic dinucleotide. 
     
     
         11 . The method according to  claim 8 , wherein the amino acid sequence of said synthetic immodulin peptide further comprises any of SEQ ID NOs: 7-14. 
     
     
         12 . The method according to  claim 8 , wherein the synthetic peptide is provided in a complex with non-covalently bound, purified holotransferrin at about 0.1 to about 10 molar equivalents. 
     
     
         13 . The method according to  claim 12 , wherein said synthetic peptide-holotransferrin complex further comprises about 0.01 to about 100 molar equivalents of purified high-molecular weight hyaluronan, wherein binding of said synthetic immodulin peptide-holotransferrin complex to high-molecular weight hyaluronan is significantly stronger at pH 5.2 than at pH 7.4. 
     
     
         14 . The method according to  claim 12 , wherein said synthetic peptide-holotransferrin complex further comprises about 0.01 to about 100 molar equivalents of purified high-molecular weight hyaluronan, wherein binding of said synthetic immodulin peptide-holotransferrin complex to high-molecular weight hyaluronan is significantly stronger than binding of said synthetic immodulin peptide-holotransferrin complex to low-molecular weight hyaluronan at pH 7.4. 
     
     
         15 . (canceled) 
     
     
         16 . A composition comprising a pH-sensitive non-covalent complex of:
 (i) a synthetic immodulin peptide about 20 to about 60 amino acids in length;   (ii) about 0.1 to about 10 molar equivalents of purified holotransferrin; and   (iii) about 0.01 to about 100 molar equivalents of purified high-molecular weight hyaluronan, wherein binding of said high-molecular weight hyaluronan within said pH-sensitive non-covalent complex is significantly stronger at pH than at pH 7.4.   
     
     
         17 . A composition comprising a size-sensitive non-covalent complex of:
 (i) a synthetic immodulin peptide about 20 to about 60 amino acids in length;   (ii) about 0.1 to about 10 molar equivalents of purified holotransferrin; and   (iii) about 0.01 to about 100 molar equivalents of purified high-molecular weight hyaluronan, wherein binding of said high-molecular weight hyaluronan within said size-sensitive non-covalent complex is significantly stronger than binding to low-molecular weight hyaluronan at pH 7.4.