Fusogenic lipid nanoparticles for target cell-specific production of a therapeutic protein fusogenic lipid nanoparticles and methods of manufacture and use thereof for the target cell-specific production of a therapeutic protein and for the treatment of a disease

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.

1 - 35 . (canceled) 
     
     
         36 . A lipid-based nanoparticle (LNP) formulation for targeted production of a therapeutic protein within target cells, the LNP formulation comprises:
 a. a lipid nanoparticle vector comprising 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) at a molar ratio of 22.5-37.5 mole %; and   b. an expression construct, wherein the expression construct is configured for preferential production of the therapeutic protein within the target cells, wherein the expression construct comprises:
 i. a transcriptional promoter that is activated in response to one or more factors that are preferentially produced within the target cells as compared to non-target cells; and 
 ii. a nucleic acid that is operably linked to and under regulatory control of the transcriptional promoter, wherein the nucleic acid encodes the therapeutic protein, wherein the therapeutic protein is capable of reducing growth or survival of the target cells, wherein the therapeutic protein is produced within the target cells but is substantially not produced in the non-target cells. 
   
     
     
         37 . The LNP formulation of  claim 36 , wherein the lipid nanoparticle vector further comprises 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). 
     
     
         38 . The LNP formulation of  claim 37 , wherein the lipid nanoparticle vector comprises the DODAP at a molar ratio of at least 35 mole %. 
     
     
         39 . The LNP formulation of  claim 36 , wherein the lipid nanoparticle vector comprises the DOPE at a molar ratio of about 30 mole %. 
     
     
         40 . The LNP formulation of  claim 36 , wherein the expression construct is present in the LNP formulation at a concentration ranging from 20 μg/mL to 1.5 mg/mL. 
     
     
         41 . The LNP formulation of  claim 36 , wherein the transcriptional promoter is a p16 transcriptional promoter or a p53 transcriptional promoter. 
     
     
         42 . The LNP formulation of  claim 36 , wherein the transcriptional promoter is a p16 transcriptional promoter. 
     
     
         43 . The LNP formulation of  claim 36 , wherein the therapeutic protein is selected from the group consisting of a caspase (Casp), an inducible caspase (iCasp), a self-activating caspase (saCasp), BAX, DFF40, HSV-TK, and cytosine deaminase. 
     
     
         44 . The LNP formulation of  claim 36 , wherein the therapeutic protein is a caspase. 
     
     
         45 . The LNP formulation of  claim 36 , wherein the therapeutic protein is a Casp3, a Casp8, or a Casp9. 
     
     
         46 . The LNP formulation of  claim 36 , wherein the therapeutic protein is Casp9. 
     
     
         47 . The LNP formulation of  claim 36 , wherein the therapeutic protein is an inducible Casp9 (iCasp9). 
     
     
         48 . The LNP formulation of  claim 36 , wherein the therapeutic protein is a self-activating Casp9 (saCasp9). 
     
     
         49 . The LNP formulation of  claim 36 , wherein the lipid nanoparticle vector further comprises a fusogenic protein. 
     
     
         50 . The LNP formulation of  claim 49 , wherein the fusogenic protein comprises a fusion-associated small transmembrane (FAST) protein. 
     
     
         51 . The LNP formulation of  claim 49 , wherein the fusogenic protein comprises an ectodomain amino acid sequence from a first reovirus FAST protein and an endodomain amino acid sequence from a second reovirus FAST protein. 
     
     
         52 . The LNP formulation of  claim 49 , wherein the fusogenic protein comprises an ectodomain amino acid sequence with at least 80% sequence identity to an endodomain of a p14 FAST protein and an endodomain amino acid sequence with at least 80% sequence identity to a p15 FAST protein. 
     
     
         53 . The LNP formulation of  claim 49 , wherein the fusogenic protein comprises the amino acid sequence of SEQ ID NO: 17. 
     
     
         54 . A lipid-based nanoparticle (LNP) formulation for targeted production of a therapeutic protein within target cells, the LNP formulation comprising:
 a. a lipid nanoparticle vector comprising 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol (DMG-PEG) at a molar ratio of 3-5 mole %; and   b. an expression construct, wherein the expression construct is configured for preferential production of the therapeutic protein within the target cells, wherein the expression construct comprises:
 i. a transcriptional promoter that is activated in response to one or more factors that are preferentially produced within the target cells as compared to non-target cells; and 
 ii. a nucleic acid that is operably linked to and under regulatory control of the transcriptional promoter, wherein the nucleic acid encodes the therapeutic protein, wherein the therapeutic protein is capable of reducing growth or survival of the target cells, wherein the therapeutic protein is produced within the target cells but is substantially not produced in the non-target cells. 
   
     
     
         55 . The LNP formulation of  claim 54 , wherein the lipid nanoparticle vector further comprises 1,2-dioleoyl-3-dimethylammonium-propane (DODAP). 
     
     
         56 . The LNP formulation of  claim 55 , wherein the lipid nanoparticle vector comprises the DODAP at a molar ratio of at least 35 mole %. 
     
     
         57 . The LNP formulation of  claim 54 , wherein the lipid nanoparticle vector comprises the DMG-PEG at a molar concentration of about 4 mole %. 
     
     
         58 . The LNP formulation of  claim 54 , wherein the expression construct is present in the LNP formulation at a concentration ranging from 20 μg/mL to 1.5 mg/mL. 
     
     
         59 . The LNP formulation of  claim 54 , wherein the transcriptional promoter is a p16 transcriptional promoter or a p53 transcriptional promoter. 
     
     
         60 . The LNP formulation of  claim 54 , wherein the transcriptional promoter is a p16 transcriptional promoter. 
     
     
         61 . The LNP formulation of  claim 54 , wherein the therapeutic protein is selected from the group consisting of a caspase (Casp), an inducible caspase (iCasp), a self-activating caspase (saCasp), BAX, DFF40, HSV-TK, and cytosine deaminase. 
     
     
         62 . The LNP formulation of  claim 54 , wherein the therapeutic protein is a caspase. 
     
     
         63 . The LNP formulation of  claim 54 , wherein the therapeutic protein is a Casp3, a Casp8, or a Casp9. 
     
     
         64 . The LNP formulation of  claim 54 , wherein the therapeutic protein is Casp9. 
     
     
         65 . The LNP formulation of  claim 54 , wherein the therapeutic protein is an inducible Casp9 (iCasp9). 
     
     
         66 . The LNP formulation of  claim 54 , wherein the therapeutic protein is a self-activating Casp9 (saCasp9). 
     
     
         67 . The LNP formulation of  claim 54 , wherein the lipid nanoparticle vector further comprises a fusogenic protein. 
     
     
         68 . The LNP formulation of  claim 67 , wherein the fusogenic protein comprises a fusion-associated small transmembrane (FAST) protein. 
     
     
         69 . The LNP formulation of  claim 67 , wherein the fusogenic protein comprises an ectodomain amino acid sequence from a first reovirus FAST protein and an endodomain amino acid sequence from a second reovirus FAST protein. 
     
     
         70 . The LNP formulation of  claim 67 , wherein the fusogenic protein comprises an ectodomain amino acid sequence with at least 80% sequence identity to an endodomain of a p14 FAST protein and an endodomain amino acid sequence with at least 80% sequence identity to a p15 FAST protein.