Exosome packaging and targeted autophagy

Abstract

Provided herein are fusion constructs including a biologically active agent such as an antibody or derivative thereof which is functionally linked with a functional moiety such as an LC3 protein or portion thereof. Also provided are compositions and exosomes including such constructs, as well as methods for preparing exosomes containing such constructs, and methods for delivering such constructs to cells. Methods and uses of such constructs and exosomes for treating diseases or disorders are also provided, in which the constructs trigger autophagy of disease-related cellular or cytoplasmic targets such as misfolded/aggregated proteins in neurodegenerative diseases, for example.

1 . A fusion construct comprising a biologically active agent functionally linked with a functional moiety comprising at least one of an LC3 or GABARAP protein or a portion thereof; an LC3-interacting motif (LIM); an LC3-interacting region (LIR); Atg4 protein or an LC3-binding portion thereof; Atg7 protein or an LC3-binding portion thereof; Atg10 protein or an LC3-binding portion thereof; another LC3-binding sequence or domain; Atg5 protein or an autophagy-triggering portion thereof; Atg16L1 or an autophagy-triggering portion thereof; or another autophagy-triggering sequence or domain; or an amino acid sequence having at least 50% sequence identity therewith. 
     
     
         2 . The fusion construct of  claim 1 , wherein the biologically active agent comprises a targeting moiety for binding a cellular target, optionally wherein the biologically active agent comprises an antibody, an antigen-binding portion thereof, or an antibody derivative or mimic; or wherein the biologically active agent comprises a protein or peptide-based binder of the cellular target. 
     
     
         3 . (canceled) 
     
     
         4 . The fusion construct of  claim 2 , wherein the antibody derivative or mimic comprises a single-chain variable fragment (scFv), a nanobody, or an antibody-like molecule from a camel, llama, shark, or other animal, a centyrin, or other protein-based agent with target binding properties. 
     
     
         5 . The fusion construct of  claim 1 , wherein the biologically active agent comprises an antibody, antigen-binding fragment thereof, an antibody derivative or mimic, or a protein or peptide which specifically binds a cellular target comprising misfolded proteins, altered or post-translationally modified protein, proteins arranged in a specific conformation, protein inclusions, protein aggregates, lipid droplets, bacteria, virus, cytoplasmic DNA, or mitochondria or other cellular organelle or membraneless structure, or crystals or uric acid. 
     
     
         6 . The fusion construct of  claim 5 , wherein the cellular target is associated with a neurodegenerative disease, non-alcoholic steatohepatitis, inflammatory disease, autoimmune disease, or another disease or disorder relating to or treatable by autophagy; and/or wherein the misfolded proteins, altered protein, protein inclusions, and/or protein aggregates comprise TDP-43 inclusions found in ALS patients; Tau fibrils found in Alzheimer's, CTE, Corticobasal Degeneration, Progressive Supranuclear Palsy, and/or other Tauopathies patients; synuclein or Alpha-synuclein or Lewy bodies and/or damaged mitochondria found in Parkinson's patients; Htt repeats in Huntington's disease; dipeptide repeats produced by C9ORF72 intronic repeats; misfolded SOD1; and/or hyperphosphorylated or fibrillary Tau. 
     
     
         7 . (canceled) 
     
     
         8 . The fusion construct of  claim 1 , wherein the biologically active agent is functionally linked with the functional moiety directly, or indirectly via one or more linkers and/or intervening groups, optionally wherein the biologically active agent is functionally linked to an N-terminal portion or end or a C-terminal portion or end of the functional moiety. 
     
     
         9 . (canceled) 
     
     
         10 . (canceled) 
     
     
         11 . (canceled) 
     
     
         12 . The fusion construct of  claim 1 , wherein the LC3 protein comprises an LC3A, LC3B, LC3C, GABARAP, GABARAPL1, GABARAPL2, or GABARAPL3 homologue, or an amino acid sequence having at least 50% sequence identity therewith. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . The fusion construct of  claim 1 , wherein the biologically active agent comprises anti-TDP43 scFv clone VH7; anti-TDP-43 scFv 4aD1; anti-Tau scFv 4A3; anti-Tau scFv 4E4; adipophilin; perilipin2; a protein which binds to a lipid droplet surface; PINK1; Parkin; or a protein which binds mitochondria. 
     
     
         16 . A nucleic acid, expression vector, or host cell expressing the fusion construct as defined in  claim 1 . 
     
     
         17 . (canceled) 
     
     
         18 . (canceled) 
     
     
         19 . (canceled) 
     
     
         20 . The host cell of  claim 16 , wherein the host cell is Atg7 −/−  or has low or reduced levels of Atg7, or another protein required for autophagy such as Atg4, Atg14, Atg3, Atg14, or Atg12. 
     
     
         21 . (canceled) 
     
     
         22 . (canceled) 
     
     
         23 . The fusion construct of  claim 1  in an exosome. 
     
     
         24 . (canceled) 
     
     
         25 . The composition of  claim 1 , in combination with at least one autophagy-activating agent, an siRNA or other gene silencing agent, or both, optionally wherein the autophagy-activating agent comprises an mTOR inhibitor, rapamycin, sirolimus, eversolimus, tacrolimus, INK128, pp242, starvation, or other mTORC1 and/or mTORC inhibitor, Trehalose, or Beclin1 peptide, any combination thereof. 
     
     
         26 . (canceled) 
     
     
         27 . (canceled) 
     
     
         28 . (canceled) 
     
     
         29 . A method for packaging a fusion construct as defined in  claim 1  into an exosome, said method comprising:
 expressing the fusion construct in an exosome-producing cell or otherwise introducing the fusion construct into the exosome-producing cell; and
 culturing the exosome-producing cell in a cell media under conditions in which the exosome-producing cell generates and secretes exosomes comprising the fusion construct into the cell media; or 
 administering the exosome-producing cell into a subject in need thereof such that the exosome-producing cell generates and secretes exosomes comprising the fusion construct within the subject. 
 
 
     
     
         30 . (canceled) 
     
     
         31 . The method of  claim 29 , wherein the exosome-producing cell is Atg 7 −/−  or has low or reduced levels of Atg7. 
     
     
         32 . (canceled) 
     
     
         33 . (canceled) 
     
     
         34 . (canceled) 
     
     
         35 . (canceled) 
     
     
         36 . (canceled) 
     
     
         37 . The method of  claim 29 , wherein the method is for delivering the biologically active agent into a cell, said method comprising:
 administering the exosome-producing cell into a subject in need thereof such that the exosome-producing cell generates and secretes exosomes comprising the fusion construct within the subject so as to contact the cell with the secreted exosomes;   or   culturing the exosome-producing cell in a cell media under conditions in which the exosome-producing cell generates and secretes exosomes comprising the fusion construct into the cell media;   obtaining, isolating, or purifying the secreted exosomes comprising the fusion construct from the cell media; and   contacting the cell with the secreted exosomes.   
     
     
         38 . (canceled) 
     
     
         39 . (canceled) 
     
     
         40 . (canceled) 
     
     
         41 . (canceled) 
     
     
         42 . (canceled) 
     
     
         43 . The method of  claim 37 , wherein the exosome-producing cell is a 293 cell and the cell is a liver cell, a fibroblast cell, or a motor neuron;
 wherein the exosome-producing cell is a human neonatal fibroblast, and the cell is a liver cell, a brain cell, a spinal cord cell, or a kidney cell;   wherein the exosome-producing cell is an NSC-34 cell, and the cell is a liver cell, a small intestine cell, a brain cell, or a spinal cord cell; or   wherein the exosome-producing cell is a SH5Y cell, and the cell is a liver cell, a kidney cell, or a brain cell.   
     
     
         44 . (canceled) 
     
     
         45 . (canceled) 
     
     
         46 . (canceled) 
     
     
         47 . (canceled) 
     
     
         48 . (canceled) 
     
     
         49 . (canceled) 
     
     
         50 . (canceled) 
     
     
         51 . (canceled) 
     
     
         52 . (canceled) 
     
     
         53 . (canceled) 
     
     
         54 . A method for treating or preventing a disease or disorder in a subject in need thereof, said method comprising:
 administering the fusion construct of  claim 1  or an exosome comprising the fusion construct to the subject.   
     
     
         55 . The method of  claim 54 , wherein the step of administering results in the fusion construct being present in a cell of the subject which is causing, or affected by, the disease or disorder. 
     
     
         56 . The method of  claim 54 , wherein the disease or disorder is caused by, or associated with, any one or more of misfolded proteins, altered proteins, protein inclusions, protein aggregates, lipid droplets, bacteria, cytoplasmic DNA, or mitochondria;
 wherein the disease or disorder is a neurodegenerative disease or atherosclerosis or NASH or a viral infection; or   wherein the disease or disorder is Alzheimer's, CTE, and/or Tauopathy-related disease; Parkinson's; Frontal Temporal Dementia; and/or Amyotrophic Lateral Sclerosis (ALS).   
     
     
         57 . (canceled) 
     
     
         58 . (canceled) 
     
     
         59 . The method of  claim 54 , wherein the fusion construct is for administration in combination with at least one autophagy-activating agent, a gene silencing agent, or both, optionally wherein the autophagy-activating agent comprises an mTOR inhibitor, wherein the autophagy-activating agent comprises one or more of rapamycin, sirolimus, eversolimus, tacrolimus, INK128, pp242, starvation, or other mTORC1 and/or mTORC inhibitor, or wherein the autophagy-activating agent comprises Trehalose and/or Beclin1 peptide. 
     
     
         60 . (canceled) 
     
     
         61 . (canceled) 
     
     
         62 . (canceled) 
     
     
         63 . (canceled) 
     
     
         64 . (canceled) 
     
     
         65 . (canceled) 
     
     
         66 . (canceled) 
     
     
         67 . (canceled)