fda advance notice of proposed rulemaking for nicotine ... · nicotine metabolic pathway 11...
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FDA Advance Notice of Proposed Rulemaking for nicotine level of combusted cigarettes – Feasibility for compliance via tobacco plant genetics?
Ernie Hiatt, PhDRJ Reynolds Tobacco Company
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72ND Tobacco Science Research Conference16-19 September, 2018Memphis, TN, USA
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Objective: Obtain information for consideration in developing a tobacco product standard to set the maximum nicotine level for cigarettes
FDA ANPRM: Tobacco Product
Standard for Nicotine Level of Combusted
Cigarettes
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Assuming an average leaf nicotine content of 2.8%, a reduction of 98-99% is needed to achieve the 0.3, 0.4, 0.5 mg/g levels put forward in the FDA’s ANPRM
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FDA ANPRM Technical
Achievability(pp 11830-11832)
Tobacco Blending
Cross Breeding
Chemical Extraction
Genetic Engineering
Other Practices
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FDA ANPRM Technical
Achievability(pp 11830-11832)
Tobacco Blending
Cross Breeding
Chemical Extraction
Genetic Engineering
Other Practices
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Cross Breeding
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A & B loci (nic1/nic2)
• 1933 – University of KY received seven lots of Cuban cigar varieties with low nicotine content
• Cross breeding these with Burley and Flue-Cure types resulted in germplasm registrations of LA Burley 21 (1970) and LAFC 53 (1975)– Alkaloid content of both registrations listed as 0.20%
(2 mg/g)
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Recurrent Parent & Line
Total Alkaloids
(mg/g)Yield
(kg/ha)Grade Index
(1-100)
Hicks 27.0 2472 39
LA Lines (Hicks) 2.2 2284** 39
SC 58 38.6 2353 41
LA Lines (SC 58) 2.7 2206* 34**
NC 95 26.5 2561 45
LA Lines NC 95 1.8 2355** 35**
Coker 139 18.5 2941 46
LA Lines (Coker 139) 0.9 2891 38**
• Chaplin & Weeks (1976), Crop Science
• 2 years, 3 locations, 10 reps/yr, 100 plants/rep
• *,**Significantly different at 0.05 & 0.01 probability
A & B loci (nic1/nic2)
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A & B loci (nic1/nic2)
• Summers et al. (2017)• 2018 Georgia Tobacco Tour • 8 reps, 60 plants/rep
Treatment Total Alkaloids (mg/g) Yield
Grade Index
(1-100)Harvest 1 Harvest 2 Harvest 3 Harvest 4 lb/A kg/ha
NC 95 22 23 32 35 2416 2708 87.4
LAFC 53 1 2 2 3 2339 2622 86.9
% Reduction 95 91 94 91 N/A N/A N/A
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Genetic Engineering
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Nicotine metabolic pathway
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Pyrrolidine Ring Pyridine Ring
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Pyridine Ring – Quinolate phosphoribosyltransferase (QPT)Rxn. Quinolinic acid nicotinic acid mononucleotide (NAMN)
• Vector Tobacco 21-41 (Vector Tobacco Ltd. 2001) production of low nicotine commercial cigarettes– RNAi silencing (target root specific QPT2 promotor)– Field results
• 92.5% reduction in nicotine (1.4mg/g) • Reduction in tobacco specific nitrosamines (TSNAs)• Yields not significantly different from wild-type Burley 21 line
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Pyrrolidine Ring – Putrescine methyltransferase (PMT)Rxn. putrescine N-methylputrescine
0102030405060708090
100
Chintapakorn etal. 2003
Steppuhn et al.2004
Wang et al, 2008
Nicotine Percent Reduction
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• Chintapakorn and Hamill (2003)– Antisense technology – Increase in anatabine– Hairy roots
• Steppuhn et al. (2004)– RNAi Inverse repeat silencing– Increase in anatabine– Roots from GH plants
• Wang et al. (2008)– RNAi – 97% maximum nicotine reduction
• Wide range of nicotine reduction– GH plants
• No field trials for PMT knockouts
98-99%
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Pyrrolidine Ring – N-methylputrescine oxidase (MPO)Rxn. N-methylputrescine 4-methylaminobutanal
• Shoji & Hashimoto (2003)– RNAi technology – 93-99% nicotine reduction & 93-97% reduction in nornicotine– Increase in anatabine– Increase in anabasine– Hairy roots
• No field trials for MPO knockouts
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Nicotine metabolic pathway
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Pyrrolidine Ring Pyridine Ring
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Final steps in nicotine biosynthesis – A622(isoflavone reductase-like protein)
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60
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80
90
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DeBoer et al. 2009 Kajikawa et al. 2009
Nicotine Percent Reduction
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• DeBoer et al. (2009)– RNAi – 96.2% decrease in nicotine– 98.5% decrease in anabasine– Hairy roots
• Inhibited root growth• Kajikawa et al. (2009)
– 82.5% decrease in nicotine– 47% decrease in nornicotine– Hairy roots
• Inhibited root growth• No field trials for A622
knockouts
98-99%
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Final steps in nicotine biosynthesis – BBL(Berberine bridge enzyme-like protein)
0102030405060708090
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Kajikawa et al.2011
Lewis et al.2015
Lewis et al.2015
Nicotine Percent Reduction
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• Kajikawa et al. (2011)– RNAi – 85% decrease in nicotine– Hairy roots– GH plants
• Lewis et al. (2015)– RNAi– 72% decrease in nicotine– Field trials– 29% yield reduction
• Lewis et al. (2015)– Triple knockout EMS mutation– Not GMO– 94% decrease in nicotine– Field trials
Note: a new alkaloid formed: dihydrometanicotine (DMN)
98-99%
1.2 mg/g
6.8 mg/g
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Regulatory Genes
• Shoji et al. (2010) Silencing of ethylene response factor 189, ERF189– RNAi technology – 84-94% nicotine reduction– Nornicotine, anatabine and anabasine reduced 80-90%– Hairy roots– No field data
• Shoji and Hashimoto (2011) Silencing of basic helix-loop-helix (bHLH) – RNAi technology– Reduced gene expression of PMT, A622, BBL and ERF transcription factors– 88-97% nicotine reduction– Nornicotine, anatabine and anabasine also significantly reduced– No field trial data on plant performance
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Transporter Genes
• Kato et al. (2014) Silencing of Nicotine Uptake Permease (NUP1)– RNAi technology – NUP1 may control ERF189 and possibly PMT & QPT expression– 95% nicotine reduction– GH plants– Hairy roots– No field data
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Current state:
• From the published literature 0.3 – 0.5 mg/g is not currently achievable by cross breeding and genetic engineering alone
• The highest nicotine reduction was 99% by MPO RNAi silencing– In hairy root cultures– Increased toxicant levels– No data from commercial field grown plants
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Substantiating technical achievability
• Determine variability in nicotine, other compounds, yield & quality using very low nicotine tobacco lines
• Large scale replicated field trials• Multiple locations• Cured leaf values• Test nicotine lines using commercial production practices
• 2018 University Activity– Flue-Cured & Burley (2018)
• University of Georgia – LAFC 53• University of Kentucky – LA TN 90 • Note: Both studies with nic1/nic2 (total alkaloid values
shown in this presentation = 0.9 – 3.0 mg/g)
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Thank YouQuestions?
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