Plasmid constructs for heterologous protein expression and methods of use

Abstract

Provided are plasmid vector constructs encoding multiple immunomodulatory proteins where each protein or component thereof can be expressed utilizing appropriate promoters and/or translation modifiers. Additional immunomodulatory proteins and genetic adjuvants containing shared tumor antigens can be added to further therapeutic potential as well as allow tracking of therapeutic treatment. Also provides are methods of expressing the plasmid constructs.

1 . A method of reducing cancer growth in a lung in a subject comprising injecting at least one pharmaceutically effective dose of at least one nucleic acid encoding IL-12 into a tumor in the subject and administering at least one electroporation pulse to the tumor. 
     
     
         2 . The method of  claim 1 , wherein the nucleic acid comprises the formula:
   P-A-T-A′
   wherein:   a) P is an expression promoter;   b) A and A′ encode IL-12 subunits; and   c) T is a translation modulating element.   
     
     
         3 . The method of  claim 2 , wherein A encodes IL-12p35 and A′ encodes IL 12p40. 
     
     
         4 . The method of  claim 2 , wherein the translation modulating element is selected from the group consisting of: a 2A ribosomal skipping modulator and an internal ribosomal entry site (IRES). 
     
     
         5 . The method of  claim 2 , wherein the translation modulating element comprises a 2A ribosomal skipping modulator. 
     
     
         6 . The method of  claim 5 , wherein the 2A ribosomal skipping modulator comprises a P2A ribosomal skipping modulator. 
     
     
         7 . The method of  claim 2 , wherein the translation modulating element comprises an IRES. 
     
     
         8 . The method of  claim 2 , wherein A encodes IL-12p35, A′ encodes IL 12p40, and T encodes a P2A ribosomal skipping modulator. 
     
     
         9 . The method of  claim 2 , wherein A encodes IL-12p35, A′ encodes IL 12p40, and T encodes an IRES. 
     
     
         10 . The method of  claim 2 , wherein P is selected from the group consisting of a human CMV promoter, a simian CMV promoter, SV-40, mPGK, and β-Actin. 
     
     
         11 . The method of  claim 2 , wherein the nucleic acid comprises SEQ ID NO: 5, SEQ ID NO: 6, or SEQ ID NO: 7. 
     
     
         12 . The method of  claim 1 , wherein the electroporation pulse has a field strength of about 200 V/cm to about 1500 V/cm. 
     
     
         13 . The method of  claim 12 , wherein the electroporation pulse has a field strength selected from the group consisting of: 350 V/cm, 400 V/cm, and 1500 V/cm. 
     
     
         14 . The method of  claim 1 , wherein administering at least one electroporation pulse to the tumor comprises administering at least one electroporation pulse having a field strength of about 1500 V/cm and a pulse length of about 100 μseconds. 
     
     
         15 . The method of  claim 1 , wherein administering at least one electroporation pulse to the tumor comprises administering at least one electroporation pulse having a field strength of about 400 V/cm and a pulse length of about 10 mseconds. 
     
     
         16 . The method of  claim 1 , wherein administering at least one electroporation pulse to the tumor comprises administering at least one electroporation pulse having a field strength of about 350 V/cm and a pulse length of about 10 mseconds. 
     
     
         17 . The method of  claim 1 , wherein administering at least one electroporation pulse to the tumor comprises administering 8 electroporation pulses having a field strength of about 350 V/cm and a pulse length of about 10 mseconds. 
     
     
         18 . The method of  claim 1 , wherein the subject is a human.