Chloroplasts Engineering to Express Pharmaceutical Proteins

Abstract

Vaccines for conferring immunity in mammals to infective pathogens are provided, as well as vectors and methods for plastid transformation of plants to produce protective antigens and vaccines for oral delivery. The vaccines are operative by parenteral administration as well. The invention also extends to the transformed plants, plant parts, and seeds and progeny thereof. The invention is applicable to monocot and dicot plants.

1 - 11 . (canceled) 
     
     
         12 . A process for obtaining a PA polypeptide comprising: integrating a plastid transformation vector, which comprises an expression cassette comprising, as operably linked components in the 5′ to the 3′ direction of translation, a promoter operative in said plastid, a selectable marker sequence. a heterologous polynucleotide sequence coding for comprising at least 70% identity to a PA polypeptide, transcription termination functional in said plastid, and flanking each side of the expression cassette, flanking DNA sequences which are homologous to a DNA sequence of the target plastid genome, whereby stable integration of the heterologous coding sequence into the plastid genome of the target plant is facilitated through homologous recombination of the flanking sequence with the homologous sequences in the target plastid genome, into the plastid genome of a plant cell, wherein said plastid transformation vector that further comprises a polynucleotide encoding a histidine tag such that said histidine tag is conjugated with said PA polypeptide upon expression; growing a plant comprising said plant cell to thereby express said protective antigen; obtaining crude plant extract from said plant; and subjecting said crude plant extract to a nickel column. 
     
     
         13 . (canceled) 
     
     
         14 . (canceled) 
     
     
         15 . A method for vaccinating a human against Hepatitis C infection, comprising administering to the human an immunizing amount of a composition comprising a Hepatitis C NS3 antigen polypeptide, wherein said Hepatitis C NS3 antigen polypeptide is derived from a plant transformed to express said Hepatitis C NS3 antigen polypeptide. 
     
     
         16 . (canceled) 
     
     
         17 . A stable plastid transformation and expression vector which comprises an expression cassette comprising, as operably linked components in the 5′ to the 3′ direction of translation, a promoter operative in said plastid, a selectable marker sequence, a heterologous polynucleotide sequence coding for comprising at least 70% identity to a Hepatitis C NS3 antigen protein, transcription termination functional in said plastid, and flanking each side of the expression cassette, flanking DNA sequences which are homologous to a DNA sequence of the target plastid genome, whereby stable integration of the heterologous coding sequence into the plastid genome of the target plant is facilitated through homologous recombination of the flanking sequence with the homologous sequences in the target plastid genome. 
     
     
         18 . A vector of  claim 17 , wherein the plastid is selected from the group consisting of chloroplasts, chromoplasts, amyloplasts, proplastide, leucoplasts and etioplasts. 
     
     
         19 . A vector of  claim 17 , wherein the selectable marker sequence is an antibiotic-free selectable marker. 
     
     
         20 . A stably transformed plant which comprises plastid stably transformed with the vector of  claim 17  or the progeny thereof, including seeds. 
     
     
         21 . A stably transformed plant of  claim 20  which is a monocotyledonous or dicotyledonous plant. 
     
     
         22 . A stably transformed plant of  claim 20  which is maize, rice, grass, rye, barley, oat, carrot, wheat, soybean, peanut, grape, potato, sweet potato, pea, canola, tobacco, tomato or cotton. 
     
     
         23 . A stably transformed plant of  claim 20  which is edible for mammals and humans. 
     
     
         24 . A stably transformed plant of  claim 20  in which all the chloroplasts are uniformly transformed. 
     
     
         25 . A process for producing a Hepatitis C NS3 antigen polypeptide comprising: integrating a plastid transformation vector according to  claim 17  into the plastid genome of a plant cell; growing said plant cell to thereby express said protective antigen. 
     
     
         26 - 37 . (canceled) 
     
     
         38 . A method of retarding the development of diabetes in a subject in need thereof comprising obtaining from a plant a composition comprising a CTB-Pris polypeptide and administering to said subject said composition. 
     
     
         39 . A composition for retarding the development of diabetes comprising a therapeutically effective amount of a CTB-Pris polypeptide and a plant remnant. 
     
     
         40 . A stable plastid transformation and expression vector which comprises an expression cassette comprising, as operably linked components in the 5′ to the 3′ direction of translation, a promoter operative in said plastid, a selectable marker sequence, a heterologous polynucleotide sequence coding for comprising at least 70% identity to a CTB-Pris protein, transcription termination functional in said plastid, and flanking each side of the expression cassette, flanking DNA sequences which are homologous to a DNA sequence of the target plastid genome, whereby stable integration of the heterologous coding sequence into the plastid genome of the target plant is facilitated through homologous recombination of the flanking sequence with the homologous sequences in the target plastid genome. 
     
     
         41 - 42 . (canceled) 
     
     
         43 . A stably transformed plant which comprises plastid stably transformed with the vector of  claim 40  or the progeny thereof, including seeds. 
     
     
         44 . A stably transformed plant of  claim 43  which is a monocotyledonous or dicotyledonous plant. 
     
     
         45 . A stably transformed plant of  claim 43  which is maize, rice, carrot, grass, rye, barley, oat, wheat, soybean, peanut, grape, potato, sweet potato, pea, canola, tobacco, tomato or cotton. 
     
     
         46 . A stably transformed plant of  claim 43  which is edible for mammals and humans. 
     
     
         47 . A stably transformed plant of  claim 43  in which all the chloroplasts are uniformly transformed. 
     
     
         48 . A process for producing a CTB-Pris polypeptide comprising: integrating a plastid transformation vector according to  claim 40  into the plastid genome of a plant cell; growing said plant cell to thereby express said CTB-Pris polypeptide. 
     
     
         49 . A plastid genome transformed to contain a CTB-Pris polynucleotide configured so as to express CTB-Pris protein. 
     
     
         50 - 78 . (canceled)