Reduced risk tobacco products and methods of making same

Abstract

Embodiments provided herein concern tobacco and tobacco products having a reduced amount of a harmful compound. More specifically, several embodiments concern approaches to modify the expression of a gene that is involved in the production of a harmful compound in tobacco, tobacco products made using these approaches and methods of determining whether the removal of said compounds using said approaches yields a tobacco and/or a tobacco product that has a reduced potential to contribute to a tobacco-related disease.

1 . A method of making a tobacco product with a reduced potential to contribute to a tobacco related disease comprising:
 (a) providing a genetically modified tobacco configured to deliver a reduced amount of a compound that contributes to a tobacco related disease, as compared to a reference tobacco or a conventional tobacco;   (b) contacting a mammalian cell with smoke, or a smoke condensate obtained from said genetically modified tobacco;   (c) identifying a modulation of homeostasis of said cell, as compared to a control cell, which has been contacted with smoke, or a smoke condensate obtained from said reference tobacco or said conventional tobacco, wherein a decreased modulation of homeostasis in said cell compared to modulation of homeostasis in said control cell indicates a reduction in the potential to contribute to a tobacco related disease; and   (d) incorporating said identified genetically modified tobacco into a tobacco product.   
     
     
         2 . The method of  claim 1 , wherein modulation of homeostasis in the cell is identified by determining the presence, absence or level of a molecular marker in the cell. 
     
     
         3 . The method of  claim 1 , wherein the mammalian cell is a lung cell or a cell of the oral cavity. 
     
     
         4 . The method of  claim 1 , wherein the genetically modified tobacco is identified as producing a reduced amount of a compound that contributes to a tobacco related disease, as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class. 
     
     
         5 . The method of  claim 1 , wherein the genetically modified tobacco is incorporated into a tobacco product that contains a filter, which retains an increased amount of a compound that contributes to a tobacco related disease, as compared to a reference filter or a conventional filter. 
     
     
         6 . The method of  claim 1 , wherein the genetically modified tobacco comprises a heterologous nucleic acid that inhibits expression of an enzyme in the nicotine biosynthetic pathway. 
     
     
         7 . The method of  claim 6 , wherein said heterologous nucleic acid inhibits expression of at least two enzymes in the nicotine biosynthetic pathway. 
     
     
         8 . The method of  claim 1 , wherein the genetically modified tobacco comprises a heterologous nucleic acid that inhibits expression of an enzyme in the sterol biosynthetic pathway. 
     
     
         9 . The method of  claim 8 , wherein said heterologous nucleic acid inhibits expression of at least two enzymes in the sterol biosynthetic pathway. 
     
     
         10 . The method of  claim 1 , wherein said genetically modified tobacco comprises a heterologous nucleic acid that inhibits expression of an enzyme in the nicotine biosynthetic pathway and an enzyme in the sterol biosynthetic pathway. 
     
     
         11 . The method of  claim 1 , wherein said genetically modified tobacco has a reduced amount of nornicotine and a conventional amount of nicotine. 
     
     
         12 . The method of  claim 1 , wherein said genetically modified tobacco comprises a nucleic acid construct selected from the group consisting of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50. 
     
     
         13 . A tobacco product made by the method of  claim 1 . 
     
     
         14 . A tobacco product comprising a genetically modified tobacco that comprises a reduced amount of nicotine as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class and a heterologous nucleic acid that inhibits expression of at least two enzymes involved in nicotine biosynthesis. 
     
     
         15 . A tobacco product comprising a genetically modified tobacco that comprises a reduced amount of a sterol as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class and a heterologous nucleic acid that inhibits expression of an enzyme involved in sterol biosynthesis. 
     
     
         16 . The tobacco product of  claim 14 , wherein said genetically modified tobacco comprises a nucleic acid construct as described herein. 
     
     
         17 . The tobacco product of  claim 14  or  15 , wherein said genetically modified tobacco comprises a nucleic acid construct selected from the group consisting of SEQ. ID. NOs.: 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49 and 50. 
     
     
         18 . The method of  claim 1 , wherein said genetically modified tobacco comprises a reduced activity of a gene selected from the group consisting of arginine decarboxylase (ADC), methylputrescine oxidase (MPO), NADH dehydrogenase, ornithine decarboxylase (ODC), phosphoribosylanthranilate isomerase (PRAI), putrescine N-methyltransferase (PMT), quinolate phosphoribosyl transferase (QPT), S-adenosyl-methionine synthetase (SAMS), or A622 or comprises an inhibition of a gene that regulates the production of sterol biosynthesis include HMG-CoA reductase, 14alpha demethylase, squalene synthase, SMT2, SMT1, C14 sterol reductase, A8-A7-isomerase, and C4-demethylase. 
     
     
         19 . The method of  claim 1 , wherein said genetically modified tobacco has reduced production of a compound that contributes to a tobacco related disease which is stable over at least 2, 3, 4, 5, 6, 8, 10, 12, 15, 20, 25, 30, 40 or 50 generations. 
     
     
         20 . The method of  claim 1 , wherein said genetically modified tobacco has agronomic characteristics suitable for commercial production. 
     
     
         21 . The method of  claim 20 , wherein said agronomic characteristics are phenotypically different from conventional tobacco, and said agronomic characteristics can be compensated for by conventional agronomic methods. 
     
     
         22 . The method of  claim 21 , wherein said conventional agronomic methods are selected from the group consisting of irrigation, administration of fertilizer, and administration of nutrients. 
     
     
         23 . A genetically modified tobacco that produces a reduced amount of a compound that contributes to a tobacco related disease, as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class, comprising a heterologous nucleic acid that inhibits expression of an enzyme in the biosynthetic pathway of a compound that contributes to a tobacco related disease. 
     
     
         24 . A reduced risk tobacco product comprising a genetically modified tobacco that produces a reduced amount of a compound that contributes to a tobacco related disease, as compared to a conventional tobacco product of the same class or a reference tobacco product of the same class. 
     
     
         25 . The tobacco or tobacco product of  claim 23  or  claim 24 , wherein said modified tobacco comprises a nucleic acid construct as described herein. 
     
     
         26 . The tobacco of  claim 23 , wherein said modified tobacco comprises a heterologous nucleic acid that inhibits expression of at least two enzymes in the nicotine biosynthetic pathway. 
     
     
         27 . The tobacco of  claim 23 , wherein said modified tobacco comprises a heterologous nucleic acid that inhibits expression of at least two enzymes in the sterol biosynthetic pathway. 
     
     
         28 . A method of making a reduced risk tobacco product comprising:
 (a) providing a modified tobacco or modified tobacco product configured to deliver to a user a reduced amount of a compound that contributes to a tobacco related disease, as compared to a reference tobacco or tobacco product or a conventional tobacco or tobacco product;   (b) contacting smoke or smoke condensate obtained from said modified tobacco or modified tobacco product with a cell;   (c) identifying a modulation of homeostasis of said cell, as compared to a control cell, which has been contacted with smoke or a smoke condensate obtained from said reference tobacco or tobacco product or said conventional tobacco or tobacco product, wherein a decreased modulation of homeostasis in said cell compared to modulation of homeostasis in said control cell indicates a reduction in the potential to contribute to a tobacco related disease; and   (d) incorporating said modified tobacco or modified tobacco product into said reduced risk tobacco product.   
     
     
         29 . The method of  claim 28 , wherein modulation of homeostasis in the cell is identified by determining the presence, absence or level of a molecular marker in the cell. 
     
     
         30 . The method of  claim 28 , wherein the modified tobacco is genetically modified tobacco. 
     
     
         31 . The method of  claim 30 , wherein the genetically modified tobacco is modified according to  claim 6 . 
     
     
         32 . A reduced risk tobacco substantially described herein. 
     
     
         33 . A reduced risk tobacco product substantially described herein. 
     
     
         34 . Use of a tobacco or tobacco product of  claim 25 . 
     
     
         35 . An isolated nucleic acid substantially as described herein. 
     
     
         36 . An isolated inhibition cassette substantially as described herein. 
     
     
         37 . A genetically modified tobacco having a reduced amount of nicotine as compared to conventional tobacco, further comprising a heterologous nucleic acid that encodes a gene that produces a composition selected from the group consisting of a medicinal compound, industrial oil, or dietary supplement, wherein said composition is substantially not present in conventional or wild-type tobacco. 
     
     
         38 . The genetically modified tobacco of  claim 37 , wherein said medicinal compound is an antibody or fragment thereof or an immunogenic preparation. 
     
     
         39 . The genetically modified tobacco of  claim 37 , wherein said medicinal compound is a vaccine preparation. 
     
     
         40 . The genetically modified tobacco of  claim 37 , wherein said medicinal compound is a veterinary product. 
     
     
         41 . A method of producing a reduced-nicotine tobacco plant, comprising genetically engineering A622 suppression in said plant. 
     
     
         42 . The reduced-nicotine tobacco plant of  claim 41 . 
     
     
         43 . A tobacco product comprising said tobacco plant of  claim 42 . 
     
     
         44 . A cigarette comprising said tobacco plant of  claim 42 . 
     
     
         45 . The method of  claim 41 , wherein said engineering comprises introducing into a plant cell of said plant a nucleic acid construct that expresses a short interfering RNA that suppresses A622 expression. 
     
     
         46 . The method of  claim 41 , wherein said engineering comprises introducing into a plant cell of said plant an enzymatic RNA molecule which cleaves the A622 mRNA transcript. 
     
     
         47 . The method of  claim 41 , wherein said engineering comprises introducing into a plant cell of said plant a nucleic acid construct, comprising, in the 5′ to 3′ direction, a promoter operably linked to a heterologous nucleic acid encoding at least a portion of A622 in the sense or antisense orientation, and a terminator. 
     
     
         48 . An isolated nucleic acid molecule comprising a nucleotide sequence selected from the group consisting of:
 (a) a nucleotide sequence set forth in SEQ ID NO: 5;   (b) a nucleotide sequence comprising at least 15 consecutive nucleotides of the nucleotide sequence of SEQ ID NO: 5; and   (c) a nucleotide sequence that differs from the nucleotide sequences of (a) or (b) due to degeneracy of the genetic code and encodes a polypeptide with A622 expression, wherein said nucleotide sequence is operatively linked to a heterologous promoter.   
     
     
         49 . A genetically engineered tobacco plant comprising a chimeric nucleic acid construct comprising said nucleic acid molecule of  claim 48  linked to a heterologous nucleic acid. 
     
     
         50 . Seeds from said plant of  claim 49 . 
     
     
         51 . A reduced-nicotine tobacco product comprising said plant of  claim 49 . 
     
     
         52 . A method for reducing an alkaloid in a tobacco plant, comprising suppressing A622. 
     
     
         53 . The method of  claim 52 , wherein said alkaloid is anatabine. 
     
     
         54 . A genetically engineered tobacco plant having A622 suppression, wherein said plant exhibits reduced A622 expression compared to a non-transformed control plant and comprises plant cells containing:
 (a) a nucleic acid construct comprising in the 5′ to 3′ direction, a promoter operable in said plant cell and a heterologous DNA operably associated with said promoter; wherein said heterologous DNA is selected from the group consisting of:
 (i) the DNA sequence of SEQ ID NO: 5; and 
 (ii) a DNA sequence comprising at least 15 consecutive nucleotides of the DNA sequence of SEQ ID NO: 5. 
   
     
     
         55 . The genetically engineered plant of  claim 54 , wherein said heterologous DNA is in antisense orientation. 
     
     
         56 . The genetically engineered plant of  claim 54 , wherein said heterologous DNA is in the sense orientation. 
     
     
         57 . Progeny of the plant according to  claim 54 , wherein said progeny have A622 suppression. 
     
     
         58 . Seeds of the plant according to  claim 54 . 
     
     
         59 . A method of making a genetically engineered reduced-nicotine tobacco cell having A622 suppression, said method comprising: providing a plant cell of a type known to express A622; providing a nucleic acid construct, comprising, in the 5′ to 3′ direction, a promoter operable in a plant cell and nucleic acid comprising a portion of a sequence encoding A622 mRNA, said nucleic acid operably associated with said promoter; and transforming said plant cell with said nucleic acid construct to produce transformed cells, wherein said cell has A622 suppression compared to an untransformed cell. 
     
     
         60 . A plant cell containing said nucleic acid construct according to  claim 45 ,  47 ,  49  or  59 . 
     
     
         61 . A method for reducing an alkaloid in a tobacco plant, comprising
 (a) suppressing A622; and   (b) suppressing an additional nicotine biosynthesis enzyme selected from the group consisting of quinolate phosphoribosyl transferase, ornithine decarboxylase, and putrescine N-methyltransferase.   
     
     
         62 . A method for reducing total alkaloid content in a tobacco plant, comprising
 (a) suppressing A622; and   (b) suppressing an additional nicotine biosynthesis enzyme selected from the group consisting of quinolate phosphoribosyl transferase, ornithine decarboxylase, and putrescine N-methyltransferase.   
     
     
         63 . The method of  claim 62 , wherein the method comprises suppressing A622 and quinolate phosphoribosyl transferase. 
     
     
         64 . A genetically engineered tobacco plant produced by the method of  claim 63 , wherein said plant is characterized by decreased total alkaloid content, and suppressed A622 and quinolate phosphoribosyl transferase. 
     
     
         65 . A smoking cessation product comprising a portion of said tobacco plant of  claim 64 . 
     
     
         66 . A tobacco plant having A622 suppression and reduced total alkaloid content. 
     
     
         67 . A reduced-nicotine tobacco product that is characterized by a reduced collective amount of N′-nitrosonornicotine (NNN), 4-methylnitrosoamino-1-(3-pyridyl)-1butanone (NNK), N′-nitrosoanatabine (NAT) and N′-nitrosoanabasine (NAB) and that is prepared from a tobacco plant genetically engineered for A622 suppression, said reduced collective amount of NNN, NNK, NAT and NAB relative to a highly similar tobacco product prepared from a non-transformed control tobacco plant. 
     
     
         68 . The tobacco product of  claim 67 , wherein said tobacco product is a cigarette. 
     
     
         69 . A tobacco product having a reduced amount of nitrosoanatabine (NAT) prepared from a tobacco plant having genetically engineered A622 suppression, said amount of NAT being reduced relative to the level in a similar tobacco product prepared from a non-transformed control tobacco plant. 
     
     
         70 . The tobacco product of  claim 69 , wherein said tobacco product is a cigarette. 
     
     
         71 . A reduced-anabasine tobacco product prepared from a tobacco plant having genetically engineered A622 suppression. 
     
     
         72 . The tobacco product of  claim 71 , wherein said tobacco product is a cigarette. 
     
     
         73 . A method for determining the risk potential of a tobacco or tobacco product comprising:
 (a) providing a tobacco or a tobacco product;   (b) obtaining smoke or a smoke condensate from said tobacco or tobacco product;   (c) contacting a human cell with smoke or smoke condensate obtained from said tobacco or said tobacco product; and   (d) identifying a tobacco-induced cell change in said human cell contacted with said smoke or smoke condensate.   
     
     
         74 . The method of  claim 73 , wherein said smoke or smoke condensate is obtained from a cigarette after passing through a filter attached to said cigarette. 
     
     
         75 . The method of  claim 73 , wherein two or more tobaccos or tobacco products are compared for their impact on cell homeostasis, gene expression, protein expression, or damage to genetic material. 
     
     
         76 . A method of making a cigarette comprising:
 (a) providing a modified tobacco;   (b) contacting a human cell with smoke or smoke condensate obtained from said modified tobacco;   (c) identifying a change in homeostasis of said human cell that was contacted with smoke or smoke condensate obtained from said modified tobacco, as compared to a control human cell, which was contacted with smoke or a smoke condensate obtained from a reference tobacco or a conventional tobacco; and   (d) incorporating said modified tobacco into a cigarette when a reduced change in homeostasis in said human cell compared to the change in homeostasis in said control human cell is identified.   
     
     
         77 . The method of  claim 76 , wherein said change in homeostasis in the cell is identified by determining the presence, absence or level of a molecular marker in the cell. 
     
     
         78 . The method of  claim 76 , wherein the modified tobacco is a reconstituted tobacco. 
     
     
         79 . The method of  claim 76 , wherein said modified tobacco in step (a) is incorporated into a cigarette containing a filter; the smoke or smoke condensate that is contacted with said human cell in step (b) is obtained from said cigarette containing said filter; the reference tobacco or conventional tobacco is incorporated into a reference cigarette or a conventional cigarette having a reference filter or a conventional filter; and the smoke or smoke condensate that is contacted with the control human cell in step (c) is obtained from said reference cigarette or conventional cigarette containing said reference filter or conventional filter. 
     
     
         80 . The method of  claim 79 , wherein said filter contains antioxidants, copper, carbon, activated charcoal or a radical scavenger. 
     
     
         81 . The method of  claim 76 , wherein said filter reduces tobacco smoke-induced modulation of cell homeostasis. 
     
     
         82 . A cigarette configured to reduce the risk of using said cigarette comprising a reduced risk tobacco and a filter that is designed to reduce the risk associated with cigarette smoke, wherein the reduced risk cigarette configuration and said reduced risk tobacco act synergistically to reduce the change in cell homeostasis. 
     
     
         83 . The cigarette of  claim 82 , wherein said cigarette contains conventional tobacco. 
     
     
         84 . The cigarette of  claim 82 , wherein said cigarette contains modified tobacco. 
     
     
         85 . The cigarette of  claim 84 , wherein said modified tobacco is reconstituted tobacco. 
     
     
         86 . The cigarette of  claim 82 , wherein said filter contains antioxidants, copper, carbon, activated charcoal or a radical scavenger.