Nanoparticles comprising protein-polynucleotide complexes and for delivering protein based complexes

Abstract

This invention provides nanoparticles containing protein-polynucleotide complexes and methods of manufacture and methods of their use. These particles, when administered to a subject in need, are capable of delivering these complexes to target cells and target intracellular locations where they can perform a therapeutic function. In some embodiments, this therapeutic function includes gene editing, induction of gene skipping, and regulation of gene expression. The instant nanoparticles are generally formed by designing and synthesizing the polynucleotide to according to its intended function, combining it with a protein selected for its substrate specificity and enzymatic function in a manner to form a polynucleotide-protein complex, encapsulating the complexes by dispersion into a water-insoluble surfactant system, optionally adding a targeting ligand, and stabilizing the nanoparticles by crystallization of the ligand to the surface of the nanoparticles.

Having described our invention, we claim: 
     
         1 . A composition comprising nanoparticles comprising a polynucleotide component, a protein component, a surfactant having an HLB value of less than 6.0 units, optionally a hydrophilic polymer, and optionally Li +  and Cs + , wherein:
 a) the protein component and the polynucleotide component are capable of functioning together in a biologic system as a biologic agent; 
 b) the protein component and the polynucleotide component form a complex; 
 c) the complex and the surfactant form a surfactant micelle core; 
 d) the optionally hydrophilic polymer, if present, forms a shell around the micelle core; 
 e) the nanoparticles have an average diameter of less than about 50 nanometers, and 
 f) the biologic agent is optionally a therapeutic agent. 
 
     
     
         2 . The composition of  claim 1  wherein the biologic agent is a RISC or a sgRNA-Cas complex. 
     
     
         3 . The composition of  claim 1  wherein the biologic agent is a PNA, or an SGN. 
     
     
         4 . The composition of  claim 2  wherein the protein component is a RISC associated protein and the polynucleotide component is a guide strand and wherein the guide strand is a member selected from the group consisting of ssRNA, siRNA, and miRNA. 
     
     
         5 . The composition of  claim 4  wherein the RISC-associated protein is an argonaute protein. 
     
     
         6 . The composition of  claim 5  wherein the argonuate protein is AGO-2. 
     
     
         7 . The composition of  claim 1  wherein the protein component is a CRISPR associated protein and the polynucleotide component is sgRNA. 
     
     
         8 . The composition of  claim 7  wherein the CRISPR associated protein is Cas-9. 
     
     
         9 . The composition of  claim 7  wherein the CRISPR associated protein is dCas- 
     
     
         10 . The composition of  claim 1 , further comprising a donor template DNA. 
     
     
         11 . A method of treating a subject in need comprising administering the composition of  claim 1 , wherein the subject has a genetic mutation and wherein the polynucleotide is configured to correct the genetic disease. 
     
     
         12 . A method of treating a subject in need comprising administering the composition of  claim 10 , wherein the subject has a genetic mutation and wherein the polynucleotide and the donor template DNA of the composition are configured to correct the genetic disease. 
     
     
         13 . A method of treating a subject in need comprising administering a composition of  claim 1 , wherein the polynucleotide is configured to inhibiting synthesis of a gene wherein the subject is affected by an autosomal dominant heritable disease resulting from a mutation in the gene. 
     
     
         14 . The composition of  claim 1 , wherein the polynucleotide component is configured to target the biologic agent to a gene that is expressed in muscle, lung, or liver. 
     
     
         15 . A composition comprising nanoparticles comprising a first protein component, a second protein component different from the first, a surfactant having an HLB value of less than 6.0 units, optionally a hydrophilic polymer, and optionally Li+ and Cs+, wherein:
 a) the first protein component and the second protein component form a complex;   b) the complex and the surfactant form a surfactant micelle core;   c) the optionally hydrophilic polymer, if present, forms a shell around the micelle core;   d) the nanoparticles have an average diameter of less than about 50 nanometers, and   e) the biologic agent is optionally a therapeutic agent.   
     
     
         16 . A method of treating a subject in need comprising administering the composition of  claim 12 , wherein the polynucleotide and the donor template DNA of the composition are configured to correct the genetic disease. 
     
     
         17 . A method of treating a subject in need comprising administering a composition of  claim 1 , wherein the polynucleotide is configured to inhibiting synthesis of a gene wherein the subject is affected by an autosomal dominant heritable disease resulting from a mutation in the gene. 
     
     
         18 . The method of  claim 12 , wherein the genetic mutation is one affecting one or more of lung function, liver function, and muscle function. 
     
     
         19 . A method of making a nanoparticle comprising:
 a) designing a polynucleotide component and a protein component, wherein the polynucleotide component targets a gene of interest and comprises structural features necessary to interact functionally with the protein component and wherein the polynucleotide component and the protein component make a bioactive agent;   b) combining the polynucleotide component and the protein component in solution;   c) providing sufficient time for the protein component and the polynucleotide component to form a complex;   d) encapsulating the complex by dispersion into a biocompatible, water-miscible solvent to form inverted micelles or reverse micelles;   e) optionally coating the micelles with a ligand by mixing the ligand with the micelles in an aqueous buffered-solution; and   f) optionally, stabilizing the ligand-coated micelles by mixing metal ions into the aqueous buffered-solution, wherein the metal ions comprise lithium pretreated with Cesium.