Fusogenic lipid nanoparticles and methods for the manufacture and use thereof for the target cell-specific production of a therapeutic protein and for the treatment of a disease, condition, or disorder associated with a target cell

Abstract

Provided nucleic acid-based expression construct for the target cell-specific production of a therapeutic protein, such as a pro-apoptotic protein, within a target cell, including a target cell that is associated with aging, disease, or other condition, in particular a target cell that is a senescent cell or a cancer cell. Also provided are formulations and systems, including fusogenic lipid nanoparticle (LNP) formulations and systems, for the delivery of nucleic acid-based expression constructs as well as methods for making and using such nucleic acid-based expression constructs, formulations, and systems for reducing, preventing, and/or eliminating the growth and/or survival of a cell, such as a senescent cell and/or a cancer cell, which is associated with aging, disease, or other condition as well as methods for the treatment of aging, disease, or other conditions by the in vivo administration of a formulation, such as a fusogenic LPN formulation, comprising an expression construct for the target cell-specific production of a therapeutic protein, such as a pro-apoptotic protein, in a target cell that is associated with aging, disease, or other condition, in particular a target cell that is a senescent cell or a cancer cell.

What is claimed is: 
     
         1 . An expression construct for targeted production of a therapeutic protein within a target cell, said expression construct comprising:
 a. a transcriptional promoter that is activated in response to one or more factors each of which is produced within a target cell; and   b. a nucleic acid that is operably linked to and under regulatory control of said transcriptional promoter, wherein said nucleic acid encodes a therapeutic protein that can reduce, prevent, and/or eliminate the growth and/or survival of a cell, including said target cell.   
     
     
         2 . The expression construct of  claim 1  wherein said transcriptional promoter is activated in said target cell but is not activated in a normal mammalian cell that is not associated with said disease. 
     
     
         3 . The expression construct of  claim 2  wherein at least one of said factors is not produced in said normal mammalian cell that is not associated with said disease. 
     
     
         4 . The expression construct of  claim 3  wherein said normal mammalian cell is a normal human cell. 
     
     
         5 . The expression construct of  claim 4  wherein said normal human cell is selected from the group consisting of a normal skeletal myoblast, a normal adipose cell, a normal cell of the eye, a normal brain cell, a normal liver cell, a normal colon cell, a normal lung cell, a normal pancreas cell, and/or a normal heart cell, which normal cell is not associated with disease, condition, or aging. 
     
     
         6 . The expression construct of  claim 1  wherein said target cell is selected from the group consisting of a mammalian cell and a bacterial cell. 
     
     
         7 . The expression construct of  claim 6  wherein said target cell is a mammalian cell. 
     
     
         8 . The expression construct of  claim 7  wherein said mammalian target cell is a human cell selected from the group consisting of a senescent cell, a cancer cell, and a cell that is infected with an infectious disease agent. 
     
     
         9 . The expression construct of  claim 8  wherein said human target cell is a senescent cell. 
     
     
         10 . The expression construct of  claim 9  wherein said transcriptional promoter is selected from the group consisting of a p16INK4a/CDKN2A transcriptional promoter and a p21/CDKN1A transcriptional promoter. 
     
     
         11 . The expression construct of  claim 9  wherein said transcriptional promoter is responsive to a factor selected from the group consisting of SP1, ETS1, ETS2, and p53/TP53. 
     
     
         12 . The expression construct of  claim 9  wherein said nucleic acid encodes a therapeutic protein selected from the group consisting of CASP3, CASP8, CASP9, BAX, DFF40, HSV-TK, and cytosine deaminase. 
     
     
         13 . The expression construct of  claim 9  wherein said therapeutic protein induces cell death in said target cell. 
     
     
         14 . The expression construct of  claim 13  wherein said induced cell death occurs via a cellular process selected from the group consisting of apoptosis, necrosis/necroptosis, autophagic cell death, endoplasmic reticulum-stress associated cytotoxicity, mitotic catastrophe, paraptosis, pyroptosis, pyronecrosis, and entosifs. 
     
     
         15 . The expression construct of  claim 8  wherein said human mammalian target cell is a cancer cell. 
     
     
         16 . The expression construct of  claim 13  wherein said cancer cell is selected from the group consisting of a brain cancer cell, a prostate cancer cell, a lung cancer cell, a colorectal cancer cell, a breast cancer cell, a liver cancer cell, a hematologic cancer cell, and a bone cancer cell. 
     
     
         17 . The expression construct of  claim 13  wherein said transcriptional promoter is selected from the group consisting of the p21 cip1/waf1  promoter, the p27 kip1  promoter, the p57 kip2  promoter, the TdT promoter, the Rag-1 promoter, the B29 promoter, the Blk promoter, the CD19 promoter, the BLNK promoter, and the λ5 promoter. 
     
     
         18 . The expression construct of  claim 13  wherein said transcriptional promoter is responsive to a factor selected from the group consisting of an EBF3, O/E-1, Pax-5, E2A, p53, VP16, MLL, HSF1, NF-IL6, NFAT1, AP-1, AP-2, HOX, E2F3, and/or NF-κB transcription factor. 
     
     
         19 . The expression construct of  claim 13  wherein said nucleic acid encodes a therapeutic protein selected from the group consisting of CASP3, CASP8, CASP9, BAX, DFF40, HSV-TK, and cytosine deaminase. 
     
     
         20 . The expression construct of  claim 8  wherein said target cell is a human cell that is infected with an infectious disease agent or a bacterial cell. 
     
     
         21 . The expression construct of  claim 20  wherein said infectious agent is a virus selected from the group consisting of a herpes virus, a polio virus, a hepatitis virus, a retrovirus virus, an influenza virus, and a rhino virus. 
     
     
         22 . The expression construct of  claim 20  wherein said nucleic acid encodes a therapeutic protein selected from the group consisting of CASP3, CASP8, CASP9, BAX, DFF40, HSV-TK, and cytosine deaminase. 
     
     
         23 . A system for the targeted production of a therapeutic protein within a target cell, said system comprising:
 a. a vector that is capable of delivering a nucleic acid to a cell, said vector comprising an expression construct; and   b. an expression construct for the targeted production of a therapeutic protein within a target cell, said expression construct comprising:
 i. a transcriptional promoter that is activated in response to one or more factors each of which is produced within said target cell; and 
 ii. a nucleic acid that is operably linked to and under regulatory control of said transcriptional promoter, wherein said nucleic acid encodes a therapeutic protein that can reduce, prevent, and/or eliminate the growth and/or survival of a cell, including said target cell. 
   
     
     
         24 . The system of  claim 23  wherein said vector is selected from the group consisting of a liposome, a viral vector, a nanoparticle, a polyplex, and a dendrimer. 
     
     
         25 . The system of  claim 23  wherein said vector is a liposome wherein said liposome comprises a fusogenic peptide 
     
     
         26 . The system of  claim 23  wherein said vector is a viral vector wherein said viral vector comprises is selected from the group consisting of a herpes simplex viral vector, a lentiviral vector, an adenoviral vector, and an adeno-associated viral vector. 
     
     
         27 . The system of  claim 23  wherein said vector is a nanoparticle wherein said nanoparticle is selected from the group consisting of a including a gold nanoparticle, a silica nanoparticle, an iron oxide nanoparticle, a titanium nanoparticle, a hydrogel nanoparticle, and a calcium phosphate nanoparticle. 
     
     
         28 . The system of  claim 23  wherein said transcriptional promoter is activated in said target cell but is not activated in a normal mammalian cell that is not associated with said disease. 
     
     
         29 . The system of  claim 23  wherein at least one of said factors is not produced in said normal mammalian cell that is not associated with said disease. 
     
     
         30 . The system of  claim 23  wherein said mammalian target cell is a human cell selected from the group consisting of a senescent cell, a cancer cell, and a cell that is infected with an infectious disease agent. 
     
     
         31 . The system of  claim 30  wherein said human target cell is a senescent cell. 
     
     
         32 . The system of  claim 30  wherein said transcriptional promoter is selected from the group consisting of a p16INK4a/CDKN2A transcriptional promoter and a p21/CDKN1A transcriptional promoter. 
     
     
         33 . The system of  claim 32  wherein said transcriptional promoter is responsive to a factor selected from the group consisting of SP1, ETS1, ETS2, and p53/TP53. 
     
     
         34 . The system of  claim 30  wherein said nucleic acid encodes a therapeutic protein selected from the group consisting of CASP3, CASP8, CASP9, BAX, DFF40, HSV-TK, and cytosine deaminase. 
     
     
         35 . The system of  claim 34  wherein said therapeutic protein induces cell death in said target cell. 
     
     
         36 . The system of  claim 35  wherein said induced cell death occurs via a cellular process selected from the group consisting of apoptosis, necrosis/necroptosis, autophagic cell death, endoplasmic reticulum-stress associated cytotoxicity, mitotic catastrophe, paraptosis, pyroptosis, pyronecrosis, and entosifs. 
     
     
         37 . The system of  claim 30  wherein said human mammalian target cell is a cancer cell. 
     
     
         38 . The system of  claim 37  wherein said cancer cell is selected from the group consisting of a brain cancer cell, a prostate cancer cell, a lung cancer cell, a colorectal cancer cell, a breast cancer cell, a liver cancer cell, a hematologic cancer cell, and a bone cancer cell. 
     
     
         39 . The system of  claim 37  wherein said transcriptional promoter is selected from the group consisting of the p21 cip1/waf1  promoter, the p27 kip1  promoter, the p57 kip2  promoter, the TdT promoter, the Rag-1 promoter, the B29 promoter, the Blk promoter, the CD19 promoter, the BLNK promoter, and the λ5 promoter. 
     
     
         40 . The system of  claim 37  wherein said transcriptional promoter is responsive to a factor selected from the group consisting of EBF3, O/E-1, Pax-5, E2A, p53, VP16, MLL, HSF1, NF-IL6, NFAT1, and NF-κB. 
     
     
         41 . The system of  claim 37  wherein said nucleic acid encodes a therapeutic protein selected from the group consisting of CASP3, CASP8, CASP9, BAX, DFF40, HSV-TK, and cytosine deaminase. 
     
     
         42 . The system of  claim 30  wherein said target cell is a human cell that is infected with an infectious disease agent. 
     
     
         43 . The system of  claim 42  wherein said infectious agent is a virus selected from the group consisting of a herpes virus, a polio virus, a hepatitis virus, a retrovirus virus, an influenza virus, and a rhino virus. 
     
     
         44 . The system of  claim 42  wherein said nucleic acid encodes a therapeutic protein selected from the group consisting of CASP3, CASP8, CASP9, BAX, DFF40, HSV-TK, and cytosine deaminase. 
     
     
         45 . A method for reducing, preventing, and/or eliminating the growth of a target cell, said method comprising contacting a target cell with a system for the targeted production of a therapeutic protein within said target cell, said system comprising:
 a. a vector that is capable of delivering a nucleic acid to a cell, said vector comprising an expression construct; and   b. an expression construct for the targeted production of a therapeutic protein within a target cell, said expression construct comprising (i) a transcriptional promoter that is activated in response to one or more factors each of which is produced within said target cell; and (ii) a nucleic acid that is operably linked to and under regulatory control of said transcriptional promoter, wherein said nucleic acid encodes a therapeutic protein,
 wherein production of said therapeutic protein in said target cell reduces, prevents, and/or eliminates the growth and/or survival of said target cell. 
   
     
     
         46 . A method for the treatment of a disease or condition in a patient having a target cell, said method comprising: administering to said patient a system for the targeted production of a therapeutic protein within a target cell, said system comprising
 a. a vector that is capable of delivering a nucleic acid to a cell, said vector comprising an expression construct; and   b. an expression construct for the targeted production of a therapeutic protein within a target cell, said expression construct comprising (i) a transcriptional promoter that is activated in response to one or more factors each of which is produced within said target cell; and (ii) a nucleic acid that is operably linked to and under regulatory control of said transcriptional promoter, wherein said nucleic acid encodes a therapeutic protein, wherein production of said therapeutic protein in said target cell reduces, prevents, and/or eliminates the growth and/or survival of said target cell thereby slowing, reversing, and/or eliminating said disease or condition in said patient.