Co-delivery of nucleic acids for simultaneous suppression and expression of target genes

Abstract

Nanoparticulate pharmaceutical formulations and methods for co-delivery of two or more species of nucleic acids for simultaneous suppression and expression of target genes in a cell, are provided. The nanoparticles encapsulate two or more nucleic acid species. The first nucleic acid suppresses expression of a gene or product thereof, e.g., inhibitory nucleic acid, such as antisense, siRNA, miRNA, Dicer siRNA, piRNA, etc. The second nucleic acid increases expression of, or encodes, an endogenous or exogenous protein or polypeptide, e.g., an mRNA. The first and second nucleic acid species simultaneously target or affect the same or different cellular processes within a cell including communication, senescence, DNA repair, gene expression, metabolism, necrosis, and apoptosis.

1 . A nanoparticle formulation comprising
 polymeric or inorganic nanoparticles, liposomes or micelles encapsulating   an inhibitory functional nucleic acid or an expression construct encoding an inhibitory functional nucleic acid, wherein the inhibitory functional nucleic acid inhibits an activity in a cell, and   a stimulatory nucleic acid species which enhances or increases an activity in a cell or encodes a protein or peptide.   
     
     
         2 . The nanoparticle formulation of  claim 1 ,
 wherein the inhibitory functional nucleic acid specifically inhibits or reduces expression of a target gene or product thereof in a cell, and the stimulatory functional nucleic acid increases or induces expression of an endogenous or heterologous protein or polypeptide.   
     
     
         3 . The nanoparticle formulation of  claim 1  wherein the inhibitory functional nucleic acid is an inhibitory RNA. 
     
     
         4 . The nanoparticle formulation of  claim 3 , wherein the inhibitory RNA is antisense, siRNA, miRNA, piRNA, Dicer siRNA or shRNA. 
     
     
         5 . The nanoparticle formulation of  claim 1 , wherein the stimulatory functional nucleic acid is an mRNA or a deoxyribonucleic acid (DNA). 
     
     
         6 . The nanoparticle formulation of  claim 4 , wherein the inhibitory functional nucleic acid reduces or suppresses the expression of an immune costimulatory molecule or signal. 
     
     
         7 . The nanoparticle formulation of  claim 6 , wherein the costimulatory molecule or signal is selected from the group consisting of B7/CD28 family members, Butyrophilins, LAIR Family members, Nectin and Nectin-like Ligand/Receptor co-signaling molecules, ILT/CD85 family proteins, TNF superfamily members, SLAM family members, and TIM family co-Signaling molecules. 
     
     
         8 . The nanoparticle formulation of  claim 6 , wherein the costimulatory molecule or signal is selected from the group consisting of B7-1/CD80, B7-2/CD86, B7-H2, B7-H3, B7-H4, B7-H6, B7-H7/HHLA2, BTLA, CD28, CD30L, CTLA-4, ICOS, PD-1, PD-L1/B7-H1, PD-L2/B7-DC, PDCD6, TMIGD2/CD28H, VISTA/B7-H5/PD-1H, BTN1A1/Butyrophilin, NTB-A/SLAMF6, and SLAM/CD150, TIM-1/KIM-1/HAVCR, TIM-3, TIM-4, CD7, CD160, CD200, CD300a/LMIR1, CD300d/LMIR4, CLECL1/DCAL-1, DAP12, Dectin-1/CLEC7A, DPPIV/CD26, EphB6, Integrin alpha 4 beta 1, Integrin alpha 4 beta 7/LPAM-1, LAG-3, and TSLP R. 
     
     
         9 . The nanoparticle formulation of  claim 1 ,
 wherein the stimulatory nucleic acid encodes an antigen, and   wherein the inhibitory functional nucleic acid inhibits one or more co-stimulatory molecules of an antigen presenting cell to induce immunological tolerance to the antigen encoded by the stimulatory nucleic acid.   
     
     
         10 . The nanoparticle formulation of  claim 9 , wherein the stimulatory nucleic acid expresses an antigen selected from the group consisting of an antigen to which tolerance is desired. 
     
     
         11 . The nanoparticle formulation of  claim 4 ,
 wherein the inhibitory nucleic acid reduces or suppresses the expression of or inhibits the function of a tumorigenic driver or oncogene, and   wherein the stimulatory nucleic acid encodes or enhances the function of a tumor repressor.   
     
     
         12 . The nanoparticle formulation of  claim 10 , wherein the antigen is a viral capsid protein from a virus selected from the group consisting of an Adeno-Associated Virus (AAV), a Herpesvirus, a retrovirus and a lentivirus. 
     
     
         13 . The nanoparticle formulation of  claim 12 , wherein the viral capsid protein is a VP1, VP2, or VP3 capsid protein from an Adeno-Associated Virus (AAV) subtype selected from the group consisting of AAV1, AAV2, AAV3, AAV4, AAV5, AAV6, AAV7, AAV8, AAV9, and AAV10. 
     
     
         14 . The nanoparticle formulation of  claim 1 , wherein the inhibitory functional nucleic acid, and/or the stimulatory functional nucleic acid is modified to acquire one or more properties selected from the group consisting of increase nuclease resistance, enhanced membrane permeability, and reduced immunogenicity. 
     
     
         15 . The nanoparticle formulation of  claim 1 , wherein the first and stimulatory functional nucleic acids can affect the same, or different cellular process(es). 
     
     
         16 . The nanoparticle formulation of  claim 15 , wherein the cellular process is selected from the group consisting of cell communication, cellular senescence, DNA repair, gene expression, metabolism, necrosis, and programmed cell death (apoptosis), immune stimulatory, or immune co-stimulatory signal. 
     
     
         17 . The nanoparticle formulation of  claim 1  comprising an excipient for administration to an individual in need thereof. 
     
     
         18 . The nanoparticle formulation of  claim 17 , wherein the nanoparticles have a diameter of between about 10 nm and about 500 nm, inclusive. 
     
     
         19 . The nanoparticle formulation of  claim 17 , wherein the nanoparticles have a diameter of between 20 nm and about 500 nm, inclusive, between about 25 nm and about 250 nm, between about 40 nm and about 150 nm, between about 50 nm and about 150 nm, or between about 50 nm and about 100 nm, inclusive. 
     
     
         20 . The nanoparticle formulation of  claim 17 , wherein the nanoparticles are in a form selected from the group consisting of polymeric nanoparticles, lipid nanoparticles, metallic or ceramic nanoparticles, and combinations thereof. 
     
     
         21 . The nanoparticle formulation of  claim 1  wherein the nanoparticles comprises one or more polymer. 
     
     
         22 . The nanoparticle formulation of  claim 21 , wherein the nanoparticles comprise a polymer selected from the group consisting of polyesters, polyanhydrides, polycaprolactone, polyorthoesters, polyhydroxyalkanoates, polyalkylene oxides, copolymers thereof, and blends thereof. 
     
     
         23 . The nanoparticle formulation of  claim 22 , comprising a blend of polyesters selected from the group consisting of polyglycolic acid, polylactic acid, polyglycolic-lactic acid, copolymers of polyglycolic acid, polylactic acid, polyglycolic-lactic acid and polyalkylene glycol or copolymers thereof. 
     
     
         24 . The nanoparticle formulation of  claim 1 , wherein the nanoparticles comprise one or more lipids, alone or in combination with polymer. 
     
     
         25 . The nanoparticle formulation of  claim 24 , wherein the nanoparticles are in a form selected from the group consisting of liposomes, micelles, and combinations thereof. 
     
     
         26 . The nanoparticle formulation of  claim 24 , wherein the lipid-conjugated polymer is 1,2 distearoyl-sn-glycero-3-phosphoethanolamine (DSPE)-terminated polyethylene glycol (PEG). 
     
     
         27 . The nanoparticle formulation of  claim 1 , wherein the nanoparticles further comprise a ligand. 
     
     
         28 . The nanoparticle formulation of  claim 27 , wherein the ligand is selected from the group consisting of a targeting ligand, an adhesion ligand, a cell-penetrating ligand, an endosomal-penetrating ligand, and combinations thereof. 
     
     
         29 . The nanoparticle formulation of  claim 30 , wherein the ligand is covalently, or non-covalently attached to the surface of the nanoparticle. 
     
     
         30 . The nanoparticle formulation of  claim 1  formed by emulsion with a non-aqueous solvent, solvent extraction, or nanoprecipitation. 
     
     
         31 . The nanoparticle formulation of  claim 30 , wherein the nanoparticle is formed by self-assembly of amphiphilic polymer optionally in combination with hydrophobic polymer. 
     
     
         32 . The nanoparticle formulation of  claim 31  comprising a blend of hydrophobic polymer and amphiphilic polymer. 
     
     
         33 . The nanoparticle formulation of  claim 31  wherein the hydrophilic portion of the amphiphilic polymers is a polyalkylene oxide or derivative thereof. 
     
     
         34 . The nanoparticle formulation of  claim 1  comprising an additional therapeutic, prophylactic or diagnostic agent selected from the group consisting of proteins or peptides, nucleic acids, lipids, sugars or polysaccharides, small molecules, or combinations thereof. 
     
     
         35 . The nanoparticle formulation of  claim 34 , wherein the additional agent is a chemotherapeutic or antiinfective for treatment of a disorder characterized by a stimuli effecting release or which can be exposed to a stimuli. 
     
     
         36 . The nanoparticle formulation of  claim 1  comprising between about 1% and about 70% weight/weight, between about 5% and about 50% weight/weight, or between about 10% and about 30% weight/weight of the first and stimulatory functional nucleic acid, alone or in combination with an additional therapeutic agent, prophylactic agent, diagnostic agent, or combination thereof. 
     
     
         37 . The nanoparticle formulation of  claim 1 , wherein the particles release the inhibitory and stimulatory functional nucleic acid primarily within certain target cells. 
     
     
         38 . A method of simultaneously delivering two nucleic acid species to cells comprising administering the nanoparticle formulation of  claim 1  to an individual in need thereof or claims therefrom. 
     
     
         39 . The method of  claim 38 , wherein the subject has a disease or disorder, or is at risk of developing a disease or disorder selected from the group consisting of cancer, infection, inflammation, and autoimmune disease or disorder. 
     
     
         40 . The method of  claim 39  for treating cancer comprising
 administering to a subject with cancer the nanoparticles of  claim 1 , wherein the inhibitory and stimulatory nucleic acids affect the same or different cellular processes in cancer cells in an effective amount to reduce one or more symptoms of the cancer. 
 
     
     
         41 . The method of  claim 40 , wherein the cellular process or processes is selected from the group consisting of apoptosis, cell survival signaling, proliferation, sensitivity to anticancer agents, a DNA damage and repair pathway or signaling. 
     
     
         42 . The method of  claim 40  for modulating an immunological response towards an antigen comprising administering to a subject in need thereof the nanoparticle formulation of  claim 1 , wherein the inhibitory nucleic acid inhibits or reduces a target gene or product thereof which is directly or indirectly involved in the immunological response, and the stimulatory nucleic acid encodes the antigen. 
     
     
         43 . The method of  claim 42 , wherein the target gene or product thereof is a molecule associated with the mammalian target of rapamycin (mTOR) pathway. 
     
     
         44 . The method of  claim 43 , wherein the target gene or product thereof inhibits the mTOR complex 1 (mTORC1), and/or any of the downstream effector molecules thereof. 
     
     
         45 . The method of  claim 42 , wherein the target gene or product thereof is a costimulatory molecule of a professional antigen presenting cell. 
     
     
         46 . The method of  claim 45 , wherein the costimulatory molecule is selected from the group consisting of B7-1, B7-2, B7-H3, B7-H4, CD40, OX40L, ICOS-L, PD-L1, PD-L2, LIGHT, CD70, 4-1BBL, CD30L, SLAM, and combinations thereof. 
     
     
         47 . The method of  claim 38 , wherein the effect of the inhibitory nucleic acid in the cells is greater when delivered by the nanoparticles, than when the inhibitory nucleic acid is delivered in the absence of the stimulatory nucleic acid. 
     
     
         48 . The method of  claim 38 , wherein the effect of the stimulatory nucleic acid in the cells is greater when delivered by the nanoparticles, than when the stimulatory nucleic acid is delivered in the absence of the inhibitory nucleic acid. 
     
     
         49 . A nanoparticular formulation for inducing immunological tolerance to an antigen in a subject, comprising
 nanoparticles having an average diameter of between 40 and 100 nm,   the nanoparticles having encapsulated therein one or more tolerance-inducing nucleic acids that silence or down-regulate one or more costimulatory molecules,   wherein the nanoparticles are preferentially taken up by dendritic cells, subcapsular macrophages, or antigen-presenting cells, and the nucleic acid is expressed in the cells in an amount effective to induce tolerance to a co-administered antigen.   
     
     
         50 . The nanoparticular formulation of  claim 49  for inducing tolerance wherein the nanoparticles are selected from the group consisting of polymeric nanoparticles, metal or ceramic nanoparticles, liposomes, lipid micelles, and polymeric-lipid nanoparticles. 
     
     
         51 . The nanoparticular formulation of  claim 49  for inducing tolerance wherein the nanoparticles are formulated in a suspension with antigen to which tolerance is to be induced, the antigen in solution or in particles.