Engineering synthetic brain penetrating gene vectors

Abstract

A synthetic gene delivery platform with a dense surface coating of hydrophilic and neutrally charged PEG, capable of rapid diffusion and widespread distribution in brain tissue, and highly effective gene delivery to target cells therein has been developed. Nanoparticles including nucleic acids, are formed of a blend of biocompatible hydrophilic cationic polymers and they hydrophilic cationic polymer conjugated to hydrophilic neutrally charged polymers such as polyethylene glycol. The nanoparticles are coated with polyethylene glycol at a density that imparts a near neutral charge and optimizes rapid diffusion through the brain parenchyma. Methods of treating a disease or disorder of the brain including administering a therapeutically effective amount of nanoparticles densely coated with polyethylene glycol are also provided.

We claim: 
     
         1 . A nanoparticle formulation for delivery of nucleic acid to tissue including brain, comprising
 nucleic acids;   a first hydrophilic cationic polymer; and   a second hydrophilic, neutrally charged linear or branched polymer selected from the group consisting of polyethylene glycol, polyethylene oxide, and copolymers thereof,   wherein between 90% and 75% of the cationic polymer is conjugated to the hydrophilic polymer or wherein at least 50% of the cationic polymer is conjugated to a branched hydrophilic polymer, wherein the nucleic acid is encapsulated within the nanoparticle or associated with the surface of the nanoparticle and wherein the nanoparticle is coated with the hydrophilic polymer at a density that imparts a near neutral charge and enhances the diffusivity through the tissue.   
     
     
         2 . The nanoparticle formulation of  claim 1  for delivery into the brain parenchyma. 
     
     
         3 . The nanoparticle formulation of  claim 1  with a diameter of less than or equal to 114 nm, 100 nm or 50 nm. 
     
     
         4 . The nanoparticle formulation of  claim 1  wherein the first cationic polymer or second neutrally charged hydrophilic polymer is branched. 
     
     
         5 . The nanoparticle formulation of  claim 1  wherein the second hydrophilic polymer is polyethylene glycol having a molecular weight between 1,000 Daltons and 10,000 Daltons. 
     
     
         6 . The nanoparticle formulation of  claim 5  wherein the polyethylene glycol has a molecular weight of 5,000 Daltons. 
     
     
         7 . The nanoparticle formulation of  claim 1 , wherein the first cationic polymer is branched polyethyleneimine with a molecular weight between 10,000 daltons and 50,000 Daltons. 
     
     
         8 . The nanoparticle formulation of  claim 7  wherein the molar ratio of second neutrally charged hydrophilic polymer to first cationic polymer is greater than 8. 
     
     
         9 . The nanoparticle formulation of  claim 8 , wherein the N to P ratio of nucleic acid to polymer is at least 2. 
     
     
         10 . The nanoparticle formulation of  claim 1 , wherein the first cationic polymer is poly-L lysine. 
     
     
         11 . The nanoparticle formulation of  claim 10 , wherein the second neutrally charged hydrophilic polymer is branched polyethylene glycol with a molecular weight of the individual branches of 5,000 Daltons. 
     
     
         12 . The nanoparticle formulation of  claim 11 , wherein at least 50% of the poly-L lysine is conjugated with polyethylene glycol. 
     
     
         13 . The nanoparticle formulation of  claim 1 , wherein the mass of the second neutrally charged hydrophilic polymer is at least 1/10,000, 1/7500, 1/5000, 1/4000, 1/3400, 1/2500, 1/2000, 1/1500, 1/1000, 1/500, 1/250, 1/200, 1/150, 1/100, 1/75, 1/50, 1/25, 1/20, ⅕, ½, or 9/10 of the mass of the particle. 
     
     
         14 . The nanoparticle formulation of  claim 1 , wherein the weight percent of the second hydrophilic polymer relative to total nanoparticle is at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, or greater. 
     
     
         15 . The nanoparticle formulation of  claim 1 , wherein the nanoparticles are formulated in an osmotic vehicle like mannitol to enhance uptake into the brain. 
     
     
         16 . A dosage formulation for delivery of a therapeutic agent to the brain consisting of
 a therapeutically effective amount of the nanoparticles of any one of  claims 1 - 15  for administration to the brain; and   a pharmaceutically acceptable excipient for delivery into the brain.   
     
     
         17 . The formulation of  claim 16 , wherein the nanoparticles are formulated for direct administration to the brain using convection enhanced delivery. 
     
     
         18 . The formulation of  claim 16 , wherein the nanoparticles are formulated for systemic or intranasal administration to the brain. 
     
     
         19 . The formulation of  claim 16 , wherein the nanoparticle releases an effective amount of the nucleic acids over a period of at least 10 minutes, 20 minutes, 30 minutes, one hour, two hours, hour hours, six hours, ten hours, one day, three days, seven days, ten days, two weeks, one month, or longer. 
     
     
         20 . A method of making nanoparticles densely coated with hydrophilic polymer for the delivery of nucleic acids to the brain, comprising
 preparing a blended polymer by mixing a first cationic hydrophilic polymer with the first polymer conjugated to a second neutrally charged hydrophilic polymer;   adding the nucleic acid to the blended polymer; and   purifying the nanoparticles, to produce the nanoparticles of any one of  claims 1 - 15 .   
     
     
         21 . A method for treating one or more symptoms of a disease or disorder of the brain, comprising administering to the brain a formulation comprising
 a therapeutically effective amount of the nanoparticle formulation of any one of  claims 1 - 19 .   
     
     
         22 . The method of  claim 21 , wherein the formulation is administered directly to the brain. 
     
     
         23 . The method of  claim 21 , wherein the formulation is administered systemically or intransally. 
     
     
         24 . The method of  claim 21 , wherein the particles are administered in combination with one or more techniques to facilitate passage of the particles through the blood brain barrier. 
     
     
         25 . The method of  claim 24 , wherein the technique is selected from the group consisting of topical injection, direct implantation, convection enhanced delivery, electron paramagnetic resonance, ultrasound sonication with or without microbubbles, and use of osmotic agents. 
     
     
         26 . The method of  claim 21 , wherein the disease or disorder is selected from the group consisting of tumors, neurological disorders, and brain injury or trauma.