Methods for delivering genes

Abstract

Methods of delivering transgenes to target cells using plasmids comprising viral inverted terminal repeat (ITR) sequences are described. Such plasmids are capable of directing sustained transgene expression in target cells in rats provided that at least one adeno-associated virus (AAV) ITR sequence is present in the plasmid, regardless of whether that ITR is located upstream or downstream of the transgene. In a particular embodiment, plasmids comprising one or more AAV ITR sequence and an IL-10 transgene are shown to be effective in sustained reversal of pain in an animal model of neuropathic pain.

We claim: 
     
         1 . A plasmid DNA molecule comprising a polynucleotide encoding at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) and also encoding an IL-10 operably linked to expression control elements that control expression of said IL-10 in vivo. 
     
     
         2 . The plasmid DNA molecule of  claim 1 , wherein the IL-10 comprises a sequence of amino acids with at least 90% sequence identity to the contiguous sequence of amino acids of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. 
     
     
         3 . The plasmid DNA molecule of  claim 2 , wherein the IL-10 comprises a sequence of amino acids with at least 95% sequence identity to the contiguous sequence of amino acids of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. 
     
     
         4 . The plasmid DNA molecule of  claim 1 , wherein the plasmid DNA molecule is encapsulated by, adsorbed to, or associated with a particulate carrier, 
     
     
         5 . The plasmid DNA molecule of  claim 4 , wherein the particulate carrier is a microparticle derived from polymethyl methacrylate polymers, poly(lactides) or poly(lactide-co-glycolides). 
     
     
         6 . The plasmid DNA molecule of  claim 5 , wherein the particulate carrier is a microparticle derived from poly(lactide-co-glycolides). 
     
     
         7 . The plasmid DNA molecule of  claim 4 , wherein the plasmid DNA molecule is encapsulated by said microparticle. 
     
     
         8 . The plasmid DNA molecule of  claim 1 , wherein the polynucleotide encodes two AAV ITRs. 
     
     
         9 . The plasmid DNA molecule of  claim 1 , wherein a single intrathecal administration of said plasmid DNA molecule to a mammalian subject is capable of providing expression of IL-10 in vivo for at least 10 days. 
     
     
         10 . A plasmid DNA molecule comprising a polynucleotide encoding at least one adeno-associated virus (AAV) inverted terminal repeat (ITR) and also encoding an IL-10, operably linked to expression control elements, wherein the plasmid DNA molecule is encapsulated by a non-liposomal particulate carrier, and further wherein a single intrathecal administration of said encapsulated plasmid DNA molecule to a mammalian subject is capable of providing expression of IL-10 in vivo for at least 10 days. 
     
     
         11 . The plasmid DNA molecule of  claim 10 , wherein the IL-10 comprises a sequence of amino acids with at least 90% sequence identity to the contiguous sequence of amino acids of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. 
     
     
         12 . The plasmid DNA molecule of  claim 11 , wherein the IL-10 comprises a sequence of amino acids with at least 95% sequence identity to the contiguous sequence of amino acids of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3. 
     
     
         13 . The plasmid DNA molecule of  claim 10 , wherein the particulate carrier is a microparticle derived from polymethyl methacrylate polymers, poly(lactides) or poly(lactide-co-glycolides). 
     
     
         14 . The plasmid DNA molecule of  claim 13 , wherein the particulate carrier is a microparticle derived from poly(lactide-co-glycolides). 
     
     
         15 . The plasmid DNA molecule of  claim 10 , wherein the polynucleotide encodes two AAV ITRs. 
     
     
         16 . A plasmid DNA molecule comprising a polynucleotide encoding two adeno-associated virus (AAV) inverted terminal repeat (ITR) and also encoding an IL-10 comprising a sequence of amino acids with at least 90% sequence identity to the contiguous sequence of amino acids of SEQ ID NO:1, SEQ ID NO:2 or SEQ ID NO:3, operably linked to expression control elements, wherein the plasmid DNA molecule is encapsulated by a microparticle derived from poly(lactide-co-glycolides), and further wherein a single intrathecal administration of said encapsulated plasmid DNA molecule to a mammalian subject is capable of providing expression of IL-10 in vivo for at least 10 days.