Structure, manufacturing and uses of human-derived cell-permeable peptides conjugated with specific biologically active cargo peptides

Abstract

Embodiments disclosed herein provide compositions for conjugates, including fusion proteins, and methods of using them to treat a variety of conditions. In some embodiments, the conjugates and/or fusion proteins incorporate a 60-amino acid human homeodomain (e.g., peptides derived from human HOX genes), to translocate functional and regulatory peptides and proteins or other biologically active molecules such as nucleic acids, which are not naturally associated with the human homeodomain, across cell and nuclear membranes to intended sites of action without provoking an unwanted immune response that may reduce exposure to the conjugate and/or result in a clinical adverse event. In further embodiments, disclosed conjugates and fusion proteins can pass through the blood-brain barrier to allow entry into the CNS. In various embodiments, the disclosed compositions are suitable for delivery into a cell (i) the expression product of a gene of interest and/or (ii) novel peptides or polynucleotides to regulate gene function.

We claim: 
     
         1 . A conjugate comprising:
 a first region comprising a homeodomain structure and having a polypeptide sequence as described in SEQ ID No. 2 or a variant thereof; and   a second region comprising a functional or regulatory polypeptide or protein not naturally associated with the first region.   
     
     
         2 . A conjugate of  claim 1  wherein the second region includes at least one of a NEMO binding domain, a PC1 CTT-derived fusion protein, an enzyme that restores enzymatic function in subjects with inherited enzyme deficiencies, a gene product of RPE65, a zinc finger peptide, an Ag85A 99-118 epitope peptide, or an Ag85A 70-78 epitope peptide. 
     
     
         3 . The conjugate of  claim 1  wherein the second region is conjugated to the C-terminus of the first region. 
     
     
         4 . The conjugate of  claim 1  wherein the second region is conjugated to the N-terminus of the first region. 
     
     
         5 . The conjugate of  claim 1  in the form of a fusion protein. 
     
     
         6 . The conjugate of  claim 1  wherein the second region interacts with an intracellular target and/or an intranuclear target. 
     
     
         7 . The conjugate of  claim 6  wherein the interaction of the second region with the intracellular target and/or intranuclear target affects one or more of the following: NF-κB activation, uveitis, PC1 transcription factor activity in renal tubular cells with polycystic kidney disease mutations, glucocerebrosidase substrate in cells with Gaucher Disease mutations of GCase, glycosaminoglycans in subjects with Hurler Syndrome or Hunter Syndrome, retinal membrane guanylyl cyclase substrate in retinal cells deficient in retinal membrane guanylyl cyclase, regulation of Huntingtin mutant gene transcription in cells with Huntington's Disease mutations, and delivery of antigenic epitopes into cytoplasm of antigen-presenting cells for provoking an effective immune response in a vaccine embodiment such as for a tuberculosis vaccine to prevent tuberculosis in subjects exposed to the infection. 
     
     
         8 . The conjugate of  claim 1  wherein the first region comprises a polypeptide sequence derived from a human gene. 
     
     
         9 . The conjugate of  claim 1  wherein the second region includes a polypeptide sequence derived from a human gene or variant thereof. 
     
     
         10 . The conjugate of  claim 1 , further comprising a linker sequence between the first and second regions. 
     
     
         11 . A composition comprising the conjugate of  claim 1  and a pharmaceutically acceptable carrier. 
     
     
         12 . The composition of  claim 11  wherein the composition is in the form of an inhalable composition, an eye drop formulation other ophthalmic composition for local or injectable use, an enema, a topical composition, or an injectable composition including injectable implants for sustained release. 
     
     
         13 . A conjugate prepared by a method comprising the steps:
 culturing a host cell transformed with an expression vector comprising a nucleic acid encoding a conjugate according to  claim 1  under conditions which provide for the expression of the conjugate within the host cell; and   recovering the conjugate by affinity purification.   
     
     
         14 . A conjugate comprising:
 a first region comprising a homeodomain structure; and   a second region comprising a functional or regulatory nucleic acid or peptide nucleic acid not naturally associated with the first region.   
     
     
         15 . The conjugate of  claim 14  wherein the first region is a HOX D12 homeodomain. 
     
     
         16 . The conjugate of  claim 14  wherein the second region comprises DNA, RNA, LNA, PNA, γPNA, or a combination of these components that has intracellular biological activity. 
     
     
         17 . A method of treating dry eye, autoimmune and inflammation-related hair loss, cancer, metabolic syndrome, treating an inflammatory airway disease, colo-rectal inflammatory bowel disease, ischemia reperfusion injury following transient ischemia, polycystic kidney disease, a lysosomal storage disease, Leber Congenital Amaurosis type 1, Huntington's Disease, tuberculosis in a subject comprising administering a formulation comprising the conjugate of  claim 1  to the subject. 
     
     
         18 . The method of  claim 18  wherein the subject is a human. 
     
     
         19 . A method of treating a condition in a subject comprising administering a systemic formulation comprising the conjugate of  claim 1  to the subject. 
     
     
         20 . The method of  claim 18  wherein the subject is a human.