Systems and methods of delivery of bioactive agents using bacterial toxin-derived transport sequences

Abstract

The field of the present invention relates, in part, to a strategy for novel pharmaceutical applications. More specifically, the present invention relates to a non-toxic mutant form of the Vibrio cholera Cholix gene (ntCholix), a variant of Cholix truncated at amino acid A386 (Cholix386) and the use of other various Cholix-derived polypeptide sequences to enhance intestinal delivery of biologically-active therapeutics. Importantly, the systems and methods described herein provide for the following: the ability to deliver macromolecule doses without injections; the ability to deliver cargo, such as (but not limited to) siRNA or antisense molecules into intracellular compartments where their activity is required; and the delivery of nanoparticles and dendrimer-based carriers across biological membranes, which otherwise would have been impeded due to the barrier properties of most such membranes.

What is claimed is: 
     
         1 . An isolated delivery construct comprising: a modified Cholix toxin, wherein the Cholix toxin has a mutation in domain III that renders the Cholix toxin non-toxic; and a therapeutic cargo, wherein the isolated delivery construct is suitable for delivering the therapeutic cargo via transcytosis through a polarized gut epithelial cell. 
     
     
         2 . The isolated delivery construct according to  claim 1 , further comprising one or more cleavable linkers. 
     
     
         3 . The isolated delivery construct according to  claim 1 , wherein the mutation in domain III comprises a deletion of the amino acid residue Glu 581 . 
     
     
         4 . The isolated delivery construct of  claim 1 , wherein the delivery construct is suitable for preferentially transporting the therapeutic cargo across a polarized gut epithelial cell versus a cell comprising a CD91 cell receptor. 
     
     
         5 . The isolated delivery construct of  claim 1 , wherein the non-toxic Cholix is suitable for delivering the therapeutic cargo via binding a cell receptor not bound by a non-toxic ExoA. 
     
     
         6 . The isolated delivery construct according to  claim 1 , wherein the therapeutic cargo is a macromolecule, small molecule, siRNA, PNA, miRNA, DNA, plasmid, antisense molecule, nanoparticle, or dendrimer-based carrier. 
     
     
         7 . The isolated delivery construct of  claim 1 , wherein the therapeutic cargo is an antineoplastic compound, methylhydrazines, steroid hormone, immunosuppressive, immunostimulant, antimicrobial compound, antifungal, antiviral, blood clotting protein, antihalmintic, radiopharmaceutic, gastrointestinal drug, hematologic compound, immunoglobulin, anticoagulant, fibrolysin inhibitor, peripheral anti-adrenergic drug, centrally acting antihypertensive drug, antihypertensive direct vasodilator, drug affecting renin-angiotensin system, calcium entry blocker, neuromuscular blocking drug, centrally acting muscle relaxant, neurotransmitter, neurotransmitter agent, diuretic drug, or antimigraine drug. 
     
     
         8 . A pharmaceutical composition comprising: a modified Cholix toxin, wherein the Cholix toxin has a mutation in domain III that renders the Cholix toxin non-toxic; a therapeutic cargo; and a pharmaceutically acceptable carrier; wherein the pharmaceutical composition is formulated for administration to a subject in need thereof via a non-injectable route and is suitable for delivery via transcytosis through a polarized gut epithelial cell. 
     
     
         9 . The pharmaceutical composition according to  claim 8 , wherein the pharmaceutical composition is formulated for oral administration. 
     
     
         10 . The pharmaceutical composition according to  claim 8 , wherein the pharmaceutical composition is formulated in a capsule or tablet. 
     
     
         11 . The pharmaceutical composition according to  claim 8 , wherein the pharmaceutical composition is formulated in a dosage form. 
     
     
         12 . The pharmaceutical composition according to  claim 8 , comprising about 1 μg to about 1 μg of the isolated delivery construct, from about 10 μg to about 100 mg of the isolated delivery construct, or from about 10 μg to about 1000 μg of the isolated delivery construct. 
     
     
         13 . A method for delivering a therapeutic cargo to a subject, the method comprising orally administering to the subject a pharmaceutical composition comprising a non-toxic Cholix toxin coupled to the therapeutic cargo, wherein the non-toxic Cholix toxin does not have a functional domain III. 
     
     
         14 . The method of  claim 13 , wherein the non-toxic Cholix toxin has a mutation in domain III. 
     
     
         15 . The method of  claim 14 , wherein the non-toxic Cholix toxin has a mutation at amino acid residue Glu 581 . 
     
     
         16 . The method of  claim 13 , wherein the therapeutic cargo is delivered at a dose from about 1 μg to 1 g, 10 μg to about 100 mg, or 10 μg to about 1000 μg. 
     
     
         17 . The method of  claim 13 , wherein the therapeutic cargo is transported preferentially across a polarized epithelial cell versus a cell comprising a CD91 cell receptor. 
     
     
         18 . The method of  claim 13 , wherein the non-toxic Cholix delivers the therapeutic cargo via binding a cell receptor not bound by a non-toxic ExoA.