Stable Gene Transfer to Proliferating Cells

Abstract

Provided herein are methods for facilitating or inducing stable transgene integration and expression in a proliferating cell, comprising administering to the cell (i) a recombinant AAV (rAAV) vector comprising the transgene flanked by transposon-derived inverted terminal repeat sequences, which sequences are in turn flanked by AAV-derived inverted terminal repeat regions, and (ii) a source of a transposase that recognises said transposon-derived inverted terminal repeat sequences and directs the genomic integration of the transgene into the genome of the proliferating cell. Also provided are methods and transgene delivery systems for the treatment or prevention of diseases affecting, associated with or characterised by proliferating cells, including paediatric liver diseases, bone marrow diseases and cancer.

1 . A method for stably integrating a transgene into the genome of a proliferating cell and/or inducing stable transgene expression in a proliferating cell, the method comprising administering to the cell: (i) a recombinant AAV (rAAV) vector comprising the transgene flanked by transposon-derived inverted terminal repeat sequences, which sequences are in turn flanked by AAV-derived inverted terminal repeat regions; and (ii) a source of a transposase that recognises said transposon-derived inverted terminal repeat sequences and directs the genomic integration of the transgene into the genome of the proliferating cell. 
     
     
         2 . A method according to  claim 1 , wherein the genomic integration of the transgene into the genome of the proliferating cell facilitates or induces the stable transgene expression. 
     
     
         3 - 4 . (canceled) 
     
     
         5 . A method according to  claim 1 , wherein the transgene and flanking transposon-derived inverted terminal repeat sequences form a transposon-transgene cassette, optionally comprising one or more further sequences or genetic elements including, for example, a promoter, enhancer, post-regulatory element and/or polyadenylation signal sequence. 
     
     
         6 . A method according to  claim 1 , wherein the transgene is operably linked to a promoter. 
     
     
         7 . A method according to  claim 6 , wherein the promoter is a tissue-specific promoter. 
     
     
         8 . (canceled) 
     
     
         9 . A method according to  claim 1 , wherein the transposase is provided to the proliferating cell in a form so as to allow transient expression of the transposase in the cell. 
     
     
         10 . A method according to  claim 9 , wherein the transposase is administered to the proliferating cell in the form of mRNA. 
     
     
         11 . A method according to  claim 1 , wherein the transposase is administered to the proliferating cell via a second rAAV vector comprising a polynucleotide encoding the transposase, optionally operably linked to a suitable promoter, and optionally flanked by AAV-derived inverted terminal repeat regions. 
     
     
         12 . A method according to  claim 11 , wherein the promoter is a tissue-specific promoter. 
     
     
         13 . (canceled) 
     
     
         14 . A method according to  claim 1 , wherein the AAV sequences used in the rAAV vector(s) are derived from AAV2 or AAV8. 
     
     
         15 . (canceled) 
     
     
         16 . A method according to  claim 1 , wherein the transposase is the piggyBac transposase, and the transposon-derived inverted terminal repeat sequences are derived from the piggyBac transposon. 
     
     
         17 . A method according to  claim 1 , wherein the proliferating cell is a rapidly proliferating cell. 
     
     
         18 . (canceled) 
     
     
         19 . A method according to  claim 17 , wherein the proliferating cell is from a neonatal or juvenile liver. 
     
     
         20 . A method according to  claim 1 , wherein the proliferating cell is a disease cell. 
     
     
         21 . (canceled) 
     
     
         22 . A method according to  claim 1 , for the treatment of paediatric liver diseases. 
     
     
         23 . A method for treating a disease of, affecting, or associated with, a proliferating cell, comprising administering to a subject in need thereof (i) a recombinant AAV (rAAV) vector comprising a transgene flanked by transposon-derived inverted terminal repeat sequences, which sequences are in turn flanked by AAV-derived inverted terminal repeat regions; and (ii) a source of a transposase that recognises said transposon-derived inverted terminal repeat sequences and directs the genomic integration of the transgene into the genome of the proliferating cell, wherein said administration results in the stable integration and expression of the transgene to thereby treat the disease. 
     
     
         24 . (canceled) 
     
     
         25 . A method according to  claim 23 , wherein the disease is associated with the deficiency of one or more gene products in the proliferating cell, and wherein expression of the transgene normalises production and activity of the deficient gene product. 
     
     
         26 . A method according to  claim 23 , wherein the disease is a paediatric liver disease, a bone marrow disease or a cancer. 
     
     
         27 . A method according to  claim 26 , wherein the paediatric liver disease is selected from OTC deficiency, ASS deficiency and progressive familial intrahepatic cholestasis. 
     
     
         28 . A method according to  claim 27 , wherein the progressive familial intrahepatic cholestasis is progressive familial intrahepatic cholestasis type 3. 
     
     
         29 . A method according to  claim 27 , wherein: (i) when the disease is OTC deficiency the transgene comprises a polynucleotide encoding ornithine transcarbamylase (OTC); (ii) when the disease is ASS deficiency the transgene comprises a polynucleotide encoding argininosuccinate synthetase (ASS); or (iii) when the disease is progressive familial intrahepatic cholestasis the transgene comprises a polynucleotide encoding ATP-binding cassette subfamily B member 4 (ABCB4). 
     
     
         30 - 31 . (canceled) 
     
     
         32 . A transgene delivery and expression system for inducing stable transgene expression in a proliferating cell or for treating a disease of, affecting, or associated with, a proliterating cell, wherein the system comprises (i) a recombinant AAV (rAAV) vector comprising the transgene flanked by transposon-derived inverted terminal repeat sequences, which sequences are in turn flanked by AAV-derived inverted terminal repeat regions; and (ii) a source of a transposase that recognises said transposon-derived inverted terminal repeat sequences and directs the genomic integration of the transgene into the genome of the proliferating cell. 
     
     
         33 - 35 . (canceled)