Expression of human interferon in transgenic chloroplasts
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.