Expression of human interferon in transgenic chloroplasts

Abstract

A plastid transformation vector for a stably transforming a plastid genome is provided. The vector includes, as operably-linked components, a first flanking sequence, a DNA sequence coding for a therapeutic human IFN, which is capable of expression in the plastid and a second flanking sequence. The invention also provides isolated and purified IFN, wherein the IFN is configured in a monomeric or multimeric form and is a structural equivalent to orally administered human IFN. Also provided are methods for variable-expressing biopharmaceutical proteins in plants suitable for mammal consumption. The method includes integrating a plastid transformation vector into a plastid genome of a plant cell; growing the plant cell to express a biopharmaceutical protein, such as therapeutic human interferon IFN. Also disclosed are plants transformed with the aforementioned vectors, and the progeny thereof. Also, disclosed is the IFN, which is IFNα2b.

1 . A plastid transformation vector for stably transforming a plastid, comprising, as operably-linked components, a first flanking sequence, a DNA sequence coding for a therapeutic human interferon (1N), which is capable of expression in a plastid, and a second flanking sequence.  
     
     
         2 . The vector of  claim 1 , wherein said therapeutic human IFN further comprises a polyhistidine purification tag and a thrombin cleavage site.  
     
     
         3 . The vector of  claim 1  further comprising a regulatory sequence.  
     
     
         4 . The vector of  claim 3 , wherein said regulatory sequence comprises a promoter operative in said plastid genome.  
     
     
         5 . The vector of  claim 4 , wherein said promoter is 16srRNA.  
     
     
         6 . The vector of  claim 3 , wherein said regulatory sequence comprises light regulated psbA 5′, and psbA 3′ elements.  
     
     
         7 . The vector of  claim 1 , stably integrated into a plastid genome of an edible plant.  
     
     
         8 . The vector of  claim 7 , wherein the edible plant is a low-nicotine tobacco plant or carrot plant.  
     
     
         9 . The vector of  claim 8 , wherein the low-nicotine tobacco plant is LAMD-609.  
     
     
         10 . The vector of  claim 1 , wherein the vector is competent for stabling integrating into a plastid genome of a plant cell and wherein the flanking DNA sequences are substantially homologous to sequences in a spacer region of said plastid genome.  
     
     
         11 . The vector of  claim 10 , wherein said spacer region is a transcriptionally active spacer region.  
     
     
         12 . The vector of  claim 1 , wherein the plastid is selected from the group consisting of chloroplast, chromoplast, amyloplast, proplastide, leucoplast and etioplast.  
     
     
         13 . The vector of  claim 3 , wherein said regulatory sequence further comprises a 5′ untranslated region (5′UTR) capable of providing transcription and translation enhancement of said DNA sequence coding for therapeutic human interferon (IFN).  
     
     
         14 . The vector of  claim 3 , wherein said regulatory sequences further comprises a 3′ untranslated region (3′UTR) capable of conferring transcript stability to said therapeutic human interferon (IFN).  
     
     
         15 . The vector of  claim 1 , wherein said first flanking sequence is trnI, and wherein said second flanking sequence is trnA.  
     
     
         16 . The vector of  claim 15 , wherein trnI and trnA provide for homologous recombination to insert an IFN containing cassette into the spacer region in an inverted repeat region of a chloroplast genome.  
     
     
         17 . The vector of  claim 1 , wherein said DNA sequence coding for therapeutic human interferon IFN is located in a single copy region of said plastid genome.  
     
     
         18 . The vector of  claim 13 , wherein said 5′ UTR is a 5′UTR of psbA.  
     
     
         19 . The vector of  claim 14 , wherein said 3′UTR is a 3′UTR of psbA.  
     
     
         20 . The vector of  claim 1 , further comprising a DNA sequence encoding a selectable marker.  
     
     
         21 . The vector of  claim 20 , wherein said selectable marker is an antibiotic-free selectable marker.  
     
     
         22 . The vector of  claim 21 , wherein said antibiotic-free selectable marker is Betaine aldehyde dehydrogenase (BADH).  
     
     
         23 . The vector of  claim 20 , wherein said DNA sequence encoding a selectable marker encodes an antibiotic resistant selectable marker.  
     
     
         24 . The vector of  claim 23 , wherein said antibiotic resistant selectable marker is aadA.  
     
     
         25 . A method for producing IFN comprising: 
 integrating the plastid transformation vector of  claim 1  into the plastid genome of a plant cell; and    growing said plant cell to thereby express said IFN.    
     
     
         26 . The method of  claim 25 , wherein said IFN is competent to produce an immunogenic response in a mammal.  
     
     
         27 . The method of  claim 26 , wherein said immunogenic response is substantially free of negative side effects associated with injected IFN.  
     
     
         28 . An isolated and purified IFN, competent to produce and immunogenic response in a mammal.  
     
     
         29 . The isolated and purified IFN of  claim 28 , wherein said IFN is configured in a monomeric form.  
     
     
         30 . The IFN of  claim 29 , wherein said IFN is configured in a multimeric form.  
     
     
         31 . The IFN of  claim 29 , wherein said IFN is a structural equivalent to natural human IFN.  
     
     
         32 . An orally administerable therapeutic human interferon IFN, suitable for oral administration to a mammal.  
     
     
         33 . A method for variable-expressing IFN comprising: 
 integrating a plastid transformation vector according to  claim 1  into a plastid genome of a plant cell; and    growing said plant cell to express said recombinant therapeutic human interferon IFN.    
     
     
         34 . The method of  claim 33 , further comprising: 
 extracting IFN from leaves of a stably transformed plant isolating IFNα2b from other plant proteins.    
     
     
         35 . A plant stably transformed with the transformation vector of  claim 1 .  
     
     
         36 . A progeny of the plant of  claim 35 .  
     
     
         37 . A seed of the plant of  claim 35 .  
     
     
         38 . A part of the plant of  claim 35 , comprising a plastid including said DNA sequence coding for therapeutic human interferon IFN.  
     
     
         39 . The plant of  claim 35 , wherein said plant is an edible plant suitable for mammal consumption.  
     
     
         40 . The plant of  claim 39 , wherein said edible plant is LAMD-609.  
     
     
         41 . The plant of  claim 35 , wherein said plant further comprises at least one chloroplast transformed with the vector of  claim 1 .  
     
     
         42 . The plant of  claim 35 , wherein said plant further comprises mature leaves transformed with the vector of  claim 1 .  
     
     
         43 . The plant of  claim 35 , wherein said plant further comprises young leaves transformed with the vector of  claim 1 .  
     
     
         44 . The plant of  claim 35 , wherein said plant further comprises old leaves transformed with the vector of  claim 1 .  
     
     
         45 . The plant of  claim 40 , wherein the expression of IFN is at least about 6.0 percent total soluble protein.  
     
     
         46 . The plant of  claim 40 , wherein said expression of IFN in said edible plant is about 12.5 percent total soluble protein.  
     
     
         47 . The plant of  claim 35 , wherein said plant is  Nicotiana tabacum  cv. Petit Havana.  
     
     
         48 . The plant of  claim 47 , wherein the expression of IFN in said  Nicotiana tabacum  cv. Petit Havana is at least 4.0 percent total soluble protein.  
     
     
         49 . The plant of  claim 47 , wherein the expression of IFN in said  Nicotiana tabacum  cv. Petit Havana is about 18.5 percent total soluble protein.  
     
     
         50 . A method of producing a biopharmaceutical protein of interest in a low-nicotine tobacco plant comprising: 
 obtaining low-nicotine tobacco plant plastid,    transforming said plastid with an expression vector comprising a nucleic acid that encodes said biopharmaceutical protein of interest,    expressing said biopharmaceutical protein in said plastid, and    recovering said biopharmaceutical protein of interest.    
     
     
         52 . A plastid transformation vector for a stably transforming a plastid genome, comprising, as operably-linked components, a first flanking sequence, a DNA sequence coding for a therapeutic human interferon IFN or a substantially homologous DNA sequence of therapeutic human interferon IFN, wherein the therapeutic human interferon IFN is operably linked to a polyhistidine purification tag and a thrombin cleavage site, and a second flanking sequence.  
     
     
         53 . The plastid transformation vector of  claim 1 , wherein said IFN is IFNα2b.