Formulation and delivery of modified nucleoside, nucleotide, and nucleic acid compositions

Abstract

The present disclosure provides, inter alia, formulation compositions comprising modified nucleic acid molecules which may encode a protein, a protein precursor, or a partially or fully processed form of the protein or a protein precursor. The formulation composition may further include a modified nucleic acid molecule and a delivery agent. The present invention further provides nucleic acids useful for encoding polypeptides capable of modulating a cell's function and/or activity.

1 . A method of increasing the duration of protein expression from an mRNA in a mammalian cell or tissue of a mammal comprising administering said mRNA via electroporation. 
     
     
         2 . The method of  claim 1 , wherein administration comprises the steps of
 (a) injecting the mammal with mRNA, and   (b) delivering one or more electric pulses at or near the site of injection.   
     
     
         3 . The method of  claim 2 , wherein injection is selected from the group consisting of intramuscular injection and intradermal injection. 
     
     
         4 . The method of  claim 3 , wherein injection is intramuscular injection. 
     
     
         5 . The method of  claim 2 , wherein the one or more electric pulses are delivered in a first stage, a second stage and a third stage. 
     
     
         6 . The method of  claim 5 , wherein
 the first stage is a single pulse at an amplitude of approximately 450V for a pulse duration of approximately 0.05 ms and a pause interval of approximately 0.2 ms;   the second stage is a single pulse at an amplitude of approximately 450V for a pulse duration of approximately 0.05 ms and a pause interval of approximately 50 ms; and   the third stage is a pulse repeated eight times at an amplitude of approximately 110V for a pulse duration of approximately 10 ms and a pause interval of approximately 20 ms.   
     
     
         7 . The method of  claim 1 , wherein the duration is increased for at least 2 hours as compared to administration of mRNA without electroporation. 
     
     
         8 . The method of  claim 1 , wherein the duration is increased for at least 8 hours as compared to administration of mRNA without electroporation. 
     
     
         9 . The method of  claim 1 , wherein the duration is increased for at least 24 hours as compared to administration of mRNA without electroporation. 
     
     
         10 . The method of  claim 1 , wherein the duration is increased for at least 1 week as compared to administration of mRNA without electroporation. 
     
     
         11 . The method of  claim 1 , wherein the duration is increased for at least 2 weeks as compared to administration of mRNA without electroporation. 
     
     
         12 . The method of  claim 1 , wherein the duration is increased for at least 3 weeks as compared to administration of mRNA without electroporation. 
     
     
         13 . The method of  claim 1 , wherein the mRNA comprises at least one chemical modification. 
     
     
         14 . The method of  claim 13 , wherein the at least one chemical modification is selected from the group consisting of pseudouridine, 1-methylpseudouridine and 5-methylcytosine. 
     
     
         15 . The method of  claim 1 , wherein the mRNA is administered at a dose selected from the group consisting of 0.025 mg/kg, 0.25 mg/kg, 2.5 mg/kg and 5 mg/kg.