Note: Descriptions are shown in the official language in which they were submitted.
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METHOD OF REDUCING THE HARMFUL EFFECTS OF ORALLY OR
TRANSDERMALLY DELIVERED NICOTINE
FIELD OF THE INVENTION
The present invention generally relates to the reduction of the harmful
effects df
orally or transdermally delivered nicotine in conventional tobacco-use
cessation programs.
More specifically, embodiments concern methods of reducing the harmful effects
of
nicotine intake, associated with conventional tobacco-use cessation programs,
by providing
tobacco products, which contain a reduced amount of nicotine and tobacco
specific
nitrosamines (TSNAs).
BACKGROUND OF THE INVENTION
The addictive properties of tobacco products are largely attributable to the
presence
of nicotine and the habitual use of the delivery system (e.g., the oral
fixation associated
with the act of smolcing or chewing tobacco, smoke intake, and taste). Many
tobacco-use
cessation programs involve the use of nicotine replacement therapy (NRT),
wherein various
amounts of nicotine are given to the individual as a replacement for tobacco
use. Several
types of tobacco-use cessation products, which involve NRT, are currently
available. For
example, nicotine patches, gums, capsules, inhalers, nasal sprays, and
lozenges are
conventional products of NRT. Although these conventional products of NRT may
help
tobacco users by suppressing the symptoms of nicotine withdrawal, they do
little to satisfy
a tobacco user's cravings for the habitual use of the delivery system.
(Dotinga, Study
Bursts Nicotine Gum's Bubble, Health - Health Scout News, September 20, 2002).
The
factors involved with the habitual use of the delivery system are hereinafter
referred to as
"secondary factors of addiction." These secondary factors of addiction are
largely
psychological factors that have only an incidental relationship to the
chemical dependence
on nicotine.
In addition to the fact that conventional NRT does little to quell the
secondary
factors of addiction, NRT has had only limited success in enabling people to
quit tobacco
use. For example, among over-the-counter NRT gum users, abstinence rates were
16.1% at
6 weeks and 8.4% at 6 months; whereas, for prescription NRT gum users
abstinence rates
were 7.7% at 6 weeks and 7.7% at 6 months. (Shiffinan et al., Addiction 97:505-
516,
2002). Users of the of the NRT patch experienced only slightly better results;
over-the
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counter patch users were reported to have 19.0% abstinence at 6 weeks and 9.2%
at 6
months; whereas, prescription NRT patch users experienced 16.0% abstinence at
6 weeks
and 3.0% abstinence at 6 months. Id. Others report slightly better results in
that smoking
cessation with patch or gum show verified abstinence rates at 12 months in the
range of
20%. (O'Brien, Lecture given to medical studerats at tlae University of
Pennsylvania on
Septernbe~ 2~, 1995). One study, however, goes so far as to say that NRT is no
longer
effective in increasing long-term successful cessation in California smokers.
(Pierce and
Gilpin, .Ianaa, 288:1260-1264 (2002)). Clearly, it appears that tobacco
addiction is a
complex web of psychological factors (i.e., the secondary factors) coupled
with nicotine
dependence and existing NRT is largely ineffective.
By design, conventional NRT relies on tobacco users to gradually reduce their
daily
nicotine intake, while they mentally curb their cravings for the secondary
factors of
addiction. In practice, however, many program participants only replace the
addiction for
tobacco with a far more expensive addiction to the NRT product. In some cases,
program
participants ingest far more nicotine than they would from conventional
tobacco use to
compensate for lack of fulfillment of the secondary factors of addiction. hl
other cases,
program participants continue using the NRT product for long periods after the
initial
program has been completed and eventually return to tobacco products.
The intake of large amounts of nicotine and long-term use of NRT raises
serious
health concerns. In some cases, nicotine overdose may occur with overzealous
use of NRT
products. Symptoms of nicotine overdose include nausea and/or vomiting,
increased
watering of mouth (severe), abdominal or stomach pain (severe), diarrhea
(severe), pale
slcin, cold sweat, headache (severe), dizziness (severe), disturbed hearing
and vision,
tremor, confusion, wealeness (severe), extreme exhaustion, fainting, low blood
pressure,
difficulty in breathing (severe), irregular heartbeat, or convulsions
(seizures).
Psychological stress may also occur in individuals using NRT for long periods
of time
because nicotine releases epinephrine, a hormone that stimulates a stress
response in the
body. The psychological effects of nicotine include irritability, anxiety,
sleep disturbances,
nervousness, poor mood and temperament, headaches, fatigue, nausea, and a long-
term
craving for tobacco.
Furthermore, recent research has established that nicotine stimulates the
growth of
blood vessels during periods of inflammation and promotes angiogenesis,
atherosclerosis
and tumor growth. (Heeschen, et al., Nature Medicine 7:833, 2001). Nicotine
may also be
a precursor for the endogenous formation of carcinogenic substances such as 4-
2
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(methynitrosamino)-1-(3-pyridyl)-1-butanone (NNI~) by the body's owri
metabolic system.
(Hecht et al., Proc. Nat. Acad. Sci. 97:12493-12497, 2000). There remains a
need for
nicotine reduction and/or tobacco-use cessation programs that utilize tobacco
products that
that contain reduced amounts of nicotine and TSNAs.
SUMMARY OF THE INVENTION
The present invention relates to methods of reducing the harmful effects of
nicotine
intake associated with conventional tobacco-use cessation programs. More
specifically,
tobacco products with reduced levels of tobacco specific nitrosamines (TSNAs)
are
provided. Additionally, methods of making or blending these reduced TSNA
tobacco
products are provided.
In some embodiments of the present invention, methods of making a blended
reduced nicotine tobacco are provided, by providing a first tobacco; a second
tobacco
produced from a genetically modified tobacco plant having a reduced level of
QPTase (as
compared to an unmodified tobacco plant of the same variety); and blending the
first and
second tobacco to obtain a reduced nicotine tobacco. A tobacco product having
the
blended reduced nicotine tobacco produced by this method is also provided.
In further embodiments, methods of making a blended reduced TSNA tobacco are
provided, by providing a first tobacco; a second tobacco produced from a
genetically
modified tobacco plant having a reduced level of QPTase (as compared to an
unmodified
tobacco plant of the same variety); and blending the first and second tobacco
to obtain the
reduced TSNA tobacco. A tobacco product having the blended reduced TSNA
tobacco
produced by this method is also provided.
In yet further embodiments, methods of making a reduced nicotine tobacco
product
with a desired amount of nicotine are provided, by providing a first tobacco
which has a
measured amount of nicotine; a second tobacco with a measured amount of
nicotine,
produced from a genetically modified tobacco plant having a reduced level of
QPTase (as
compared to an umnodified tobacco plant of the same variety); and blending the
first and
second tobacco to produce a reduced nicotine tobacco product with a desired
amount of
nicotine. The reduced nicotine tobacco product can be, for example, a blended
cigarette.
The blended cigarette can contain, for example, 0.6 mg or less nicotine, 0.3
mg or less
nicotine, or 0.05 mg or less nicotine. Tobacco products having the blended
reduced
nicotine tobacco produced by these methods are also provided. Additionally,
tobacco-use
cessation bits having tobacco products produced by these methods are provided.
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Additional embodiments of the present invention provide methods of making a
reduced TSNA tobacco product with a desired amount of TSNA by providing a
first
tobacco with a measured amount of TSNA; providing a second tobacco with a
measured
amount of TSNA, produced from a genetically modified tobacco plant having a
reduced
level of QPTase (as compared to an unmodified tobacco plant of the same
variety); and
blending the first and second tobacco to produce a reduced TSNA tobacco
product with a
desired amount of TSNA.
Further embodiments of the present invention provide methods of reducing the
nicotine consumption of a tobacco user by providing to the tobacco user a
first tobacco
product having tobacco produced from a modified tobacco plant having a reduced
level of
QPTase (as compared to an unmodified tobacco plant of the same variety); and a
second
tobacco product having tobacco produced from a modified tobacco plant having a
reduced
level of QPTase (as compared to an unmodified tobacco plant of the same
variety), where
the second tobacco product has less nicotine than the first tobacco product;
providing the
tobacco user with additional tobacco products having tobacco produced from a
modified
tobacco plant having a reduced level of QPTase (as compared to an unmodified
tobacco
plant of the same variety), where subsequent tobacco products have
sequentially reduced
amounts of nicotine, starting with a third product that has less nicotine than
the first or
second tobacco product.
Yet further embodiments of the present invention provide methods of reducing
the
TSNA consumption of a tobacco user by providing the tobacco user with a first
tobacco
product having tobacco produced from a modified tobacco plant having a reduced
level of
QPTase (as compared to an munodified tobacco plant of the same variety); and a
second
tobacco product having tobacco produced from a modified tobacco plant having a
reduced
level of QPTase (as compared to an unmodified tobacco plant of the same
variety), where
the second tobacco product has less TSNA than the first tobacco product; and
further
providing the tobacco user with additional tobacco products having tobacco
produced from
a modified tobacco plant having a reduced level of QPTase, as compared to an
unmodified
tobacco plant of the same variety, wherein the subsequent tobacco products
have
sequentially reduced amounts of TSNA, starting with a third product that has
less TSNA
than the first or second tobacco product.
Additional embodiments of the present invention provide for the use of a
genetically modified tobacco produced from a tobacco plant having a reduced
amount of
QTPase to prepare a blended tobacco product that has a selected amount of
nicotine. The
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blended tobacco product can be, for example, a blended cigarette. The blended
tobacco
product can be, for example, a blended cigarette having 0.6 mg nicotine or
less, 0.3 mg
nicotine or less, or 0.05 mg nicotine or less.
Further embodiments of the present invention provide for the use of a
genetically
modified tobacco produced from a tobacco plant that has a reduced amount of
QTPase to
prepare a .blended tobacco product that has a selected amount of TSNA. The
blended ,
tobacco product can be, for example, a blended cigarette. The blended tobacco
product can
be, for example, a blended cigarette having 0.6 mg nicotine or less, 0.3 mg
nicotine or less,
or 0.05 mg nicotine or less.
DETAILED DESCRIPTION OF THE INVENTION
The present invention concerns nicotine reduction and/or tobacco-use cessation
programs, which involve the use of modified tobacco products that contain
reduced
amounts of nicotine and TSNAs. While most tobacco cessation programs rely
heavily on
nicotine replacement therapy (NRT), many of the embodiments described herein
focus less
on nicotine replacement and more on replacing the secondary factors of
addiction such as
smoke intake, oral fixation, and taste. A copending application entitled
"Modifying
Nicotine and Nitrosamine levels in Tobacco" (W002100199), which was published
in
English designating the United States of America and claiming priority to U.S.
Provisional
Application No. 60/371635. Also incorporated by reference in their entireties
are related
U.S. Patent Nos. 6,586,661 and 6,423,520.
Some embodiments of the invention concern the use of low nicotine and TSNA
tobacco products that have burning and taste characteristics that are
virtually
indistinguishable from conventional tobacco products. While there are many
ways to
create such reduced nicotine and/or TSNA products, the preferred methods use
techniques
in plant genetic engineering to reduce or eliminate enzymes involved in
nicotine
biosynthesis. Preferably, techniques in plant genetic engineering are used to
selectively
reduce the amount of the enzyme quinolate phosphoribosyl transferase (QPTase),
which is
involved in the production of nicotine at the root cortex. There may be many
ways to
reduce levels of QPTase in tobacco plants, given the teachings described
herein and the
level of slcilh in the art, however, the preferred methods involve the use of
antisense
technology or molecular decoy technology.
Several approaches to create tobacco and tobacco products that have a reduced
amount of nicotine and/or TSNAs have been discovered. Interestingly, it was
discovered
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that TSNA content in a tobacco plant can be lowered by reducing the nicotine
content in
the tobacco plant. In some embodiments, antisense technology is used to lower
nicotine
and TSNA levels in tobacco plants. (See PCT/US98/11893). In other embodiments,
molecular decoy technology is used to lower nicotine and/or TSNA levels in
tobacco plants
(See U.S. patent application serial number 09/941,042).
By one approach, for example, a DNA construct encoding an antisense RNA that
complements at least a portion of the QPTase gene (SEQ. ID. No. 1) is prepared
such that
transcription of the complementary strand of RNA reduces expression of the
endogenous
quinolate phosphoribosyl gene, which, in turn, reduces the amount of nicotine
and,
concomitantly, the amount of TSNA in the tobacco plant. By another approach,
transcription factor molecular decoys for the QPTase gene, which are nucleic
acid
fragments that correspond to the 5' upstream regulatory elements (e.g., Nic 1
and Nic 2
transcription factor binding sites) are inserted into the plant cell. The
transcription factors
bind to the decoy fragments rather than the endogenous transcription factor
binding sites
and a reduction in the level of transcription of QPTase is obtained.
Once the transgenic tobacco plants having reduced nicotine and/or TSNA are
made,
the tobacco is harvested and cured by conventional methods and is incorporated
into a
variety of tobacco products. Preferably, the transgenic tobacco is blended
such that
specific amounts of nicotine and/or TSNA are obtained in specific products.
That is, the
blending is conducted so that tobacco products of varying amounts of nicotine
and/or
TSNAs are made. ~ In this manner, a step-wise tobacco-use cessation program
can be
established, wherein a program participant begins the program at step 1 with a
tobacco
product having only slightly less nicotine; at step 2 the program participant
begins using a
tobacco product with less nicotine than the products used in step l; at step
3, the program
participant begins using a tobacco product with less nicotine than the
products in step 2;
and so on, for as many steps as desired for a particular tobacco-use cessation
program.
Ultimately, the tobacco product used by the program participant can have an
amount of
nicotine that is less than that which is required to become addictive or
maintain an
addiction. In this manner, a nicotine reduction and/or tobacco-use cessation
program is
provided that limits the exposure of a program participant to nicotine yet
retains the
secondary factors of addiction, including but not limited to, smolce intalce,
oral fixation, and
taste. The following section describes tobacco products that can be used with
the tobacco-
use cessation programs described herein.
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Tobacco products for use in nicotine reduction and/or tobacco-use cessation.
programs
Wild type tobacco varies significantly in the amount of TSNAs and nicotine
depending on the variety and the manner it is grown, harvested, and cured. For
example, a
typical Burley tobacco leaf can have about 30,000 parts per million (ppm)
nicotine and
8,000 parts per billion (ppb) TSNA; a typical Flue-Cured Burley leaf can have
about
20,000 ppm nicotine and 300 ppb TSNA; and a typical Oriental cured leaf can
have about
10,000 ppm nicotine and 100 ppb TSNA. A tobacco plant or portion thereof
having a
reduced amount of nicotine and/~r TSNA, for use with aspects of the invention,
can have
no detectable nicotine and/or TSNA, or may contain some detectable amounts of
one or
more TSNA and/or nicotine so long as the amount of nicotine and/or TSNA is
less than that
found in a control plant of the same variety.
That is, a Burley tobacco leaf embodiment of the invention having a reduced
amount of nicotine can have between about 0 and about 30,000 ppm nicotine and
about 0
and about 8,000 ppb TSNA desirably, between about 0 and ab~ut 20,000 ppm
nicotine and
about 0 and about 6,000 ppb TSNA more desirably, between about 0 and about
10,000 ppm
nicotine and about 0 and about 5,000 ppb TSNA preferably, between about 0 and
about
5,000 ppm nicotine and about 0 and about 4,000 ppb TSNA more preferably,
between
about 0 and about 2,500 ppm nicotine and about 0 and about 2,000 ppb TSNA even
more
preferably, and most preferably between about 0 and about 1,000 ppm nicotine
and about 0
and about 1,000 ppb TSNA. Embodiments of Burley leaf prepared by the methods
described herein can also have between about 0 and about 1000 ppm nicotine and
about 0
and about 500 ppb TSNA and some embodiments of Burley leaf prepared by the
methods
described herein have virtually no detectable amount of nicotine or TSNA.
Similarly, a Flue-cured tobacco leaf for use with the disclosed methods can
have a
reduced amount of nicotine which is between about 0 and about 20,000 ppm
nicotine and
about 0 and about 300 ppb TSNA desirably between about 0 and about 15,000 ppm
nicotine and about 0 and about 250 ppb TSNA more desirably between about 0 and
about
10,000 ppm nicotine and about 0 and about 200 ppb TSNA preferably between
about 0 and
about 5,000 ppm nicotine and about 0 and about 150 ppb TSNA more preferably
between
about 0 and about 2,500 ppm nicotine and about 0 and about 100 ppb TSNA and
most
preferably between about 0 and about 1,000 ppm nicotine and about 0 and about
50 ppb
TSNA. Embodiments of flue-cured tobacco prepared by the methods described
herein can
also have between about 0 and about 500 ppm nicotine and about 0 and about 25
ppb
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TSNA and some embodiments of flue-cured tobacco prepared by the methods
described
herein have virtually no detectable amount of nicotine or TSNA.
Further, an Oriental cured tobacco for use with the embodied methods can have
a
reduced amotmt of nicotine having between about 0 and about 10,000 ppm
nicotine and
about 0 and about 100 ppb TSNA desirably between about 0 and about 7,000 ppm
nicotine
and about 0 and about 75 ppb TSNA more desirably between about 0 and about
5,000 ppm
nicotine and about 0 and about 50 ppb TSNA preferably between about 0 and
about 3,000
ppm nicotine and about 0 and about 25 ppb TSNA more preferably between about 0
and
about 1,500 ppm nicotine and about 0 and about 10 ppb TSNA and most preferably
between about 0 and about 500 ppm nicotine and essentially no TSNA.
Embodiments of
Oriental cured tobacco prepared by the methods described herein can also have
between
about 0 and about 250 ppm nicotine and essentially no TSNA and some
embodiments of
Oriental cured tobacco prepared by the methods described herein have virtually
no
detectable amount of nicotine or TSNA.
As discussed above, TSNAs and nicotine contribute significantly to the
carcinogenic potential and addictive properties of tobacco and tobacco
products. Thus,
tobacco and tobacco products that have a reduced amount of TSNA and nicotine ~
have
tremendous utility. It was found that the reduction of nicotine in tobacco was
directly
related to the reduction of TSNAs. Unexpectedly, the methods described herein
not only
produce tobacco with a reduced addictive potential but, concomitantly, produce
a tobacco
that has a lower carcinogenic potential.
It should be emphasized that the phrase "a reduced amount" is intended to
refer to
an amount of nicotine and or TSNA in a treated or transgenic tobacco plant,
tobacco, or a
tobacco product that is less than what would be found in a tobacco plant,
tobacco, or a
tobacco product from the same variety of tobacco processed in the same manner,
which has
not been treated or was not made transgenic for reduced nicotine and/or TSNA.
Thus, in
some contexts, wild-type tobacco of the same variety that has been processed
in the same
manner is used as a control by which to measure whether a reduction in
nicotine and/or
TSNA has been obtained.
In some contexts, the phrase reduced amount of nicotine and/or TSNAs refers to
the
tobacco plants, tobacco and tobacco products of the invention that have less
nicotine and/or
TSNAs by weight than the same variety of tobacco grown, processed, and cured
in the
same way. For example, wild type tobacco can contain approximately 1-4% dry
weight
nicotine and approximately 0.2% - 0.~% dry weight TSNAs depending on the
variety, and
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the manner in which it was grown, harvested and cured. A typical cigarette has
11 mg of
nicotine and 8~,g of TSNAs. Thus, the tobacco plants, tobacco and tobacco
products of the
invention can have, in dry weight for example, less than 0.01%, 0.015%, 0.02%,
0.025%,
0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%, 0.07%, 0.075%,
0.08%,
0.085%, 0.09%, 0.095%, 0.1%, 0.15%, 0.175%, 0.2%, 0.225%, 0.25%, 0.275%, 0.3%,
0.325%, 0.35%, 0.375%, 0.4%, 0.425%, 0.45%, 0.475%, 0.5%, 0.55%, 0.6%, 0.65%,
0.7%,
0.75%, 0.8%, 0.85%, 0.9%, 0.95%, and 1.0% nicotine and less than 0.01%,
0.015%,
0.02%, 0.025%, 0.03%, 0.035%, 0.04%, 0.045%, 0.05%, 0.055%, 0.06%, 0.065%,
0.07%,
0.075%, and 0.08% TSNA.
Alternatively, a tobacco product, e.g., a cigarette, can have, for example,
less than
O.Olmg, O.OSmg, O.lmg, O.lSmg, 0.2mg, 0.25mg, 0.3mg, 0.35mg, 0.4mg, 0.45mg,
O.Smg,
O.SSmg, 0.6mg, 0.65mg, 0.7mg, 0.75mg, 0.8mg, 0.85mg, 0.9mg, 0.95mg, l.Omg,
l.lmg,
1.15mg, l.2mg, 1.25mg, l.3mg, 1.35mg, l.4mg, 1.45mg, l.5mg, l.SSmg, l.6mg,
1.65mg,
l.7mg, 1.75mg, l.8mg, 1.85mg, l.9mg, 1.95mg, 2.Omg, 2.lmg, 2.15mg, 2.2mg,
2.25mg,
2.3mg, 2.35mg, 2.4mg, 2.45mg, 2.Smg, 2.SSmg, 2.6mg, 2.65mg, 2.7mg, 2.75mg,
2.8mg,
2.85mg, 2.9mg, 2.95mg, 3.Omg, 3.lmg, 3.15mg, 3.2mg, 3.25mg, 3.3mg, 3.35mg,
3.4mg,
3.45mg, 3.Smg, 3.SSmg, 3.6mg, 3.65mg, 3.7mg, 3.75mg, 3.8mg, 3.85mg, 3.9mg,
3.95mg,
4.Omg,. 4.lmg, 4.15mg, 4.2mg, 4.25mg, 4.3mg, 4.35mg, 4.4mg, 4.45mg, 4.4mg,
4.45mg,
4.Smg, 4.SSmg, 4.6mg, 4.65mg, 4.7mg, 4.75rng, 4.8mg, 4.85mg, 4.9mg, 4.95mg,
S.Omg,
S.Smg, 5.7mg, 6.Omg, 6.Smg, 6.7mg, 7.Omg, 7.Smg, 7.7mg, B.Omg, 8.Smg, 8.7mg,
9.Omg,
9.Smg, 9.7mg, lO.Omg, 10.5mg, 10.7mg, and ll.Omg nicotine and less than 0.1
micrograms, 0.15 micrograms, 0.2 micrograms, 0.25 micrograms, 0.3 micrograms,
0.35
micrograms, 0.4 micrograms, 0.45 micrograms, 0.5 micrograms, 0.55 micrograms,
0.6
micrograms, 0.65 micrograms, 0.7 micrograms, 0.75 micrograms, 0.8 micrograms,
0.85
micrograms, 0.9 micrograms, 0.95 micrograms, 1.0 micrograms, 1.1 micrograms,
1.15
micrograms, 1.2 micrograms, 1.25 micrograms, 1.3 micrograms, 1.35 micrograms,
1.4
micrograms, 1.45 micrograms, 1.5 micrograms, 1.55 micrograms, 1.6 micrograms,
1.65
micrograms, 1.7 micrograms, 1.75 micrograms, 1.8 micrograms, 1.85 micrograms,
1.9
micrograms, 1.95 micrograms, 2.0 micrograms, 2.1 micrograms, 2.15 micrograms,
2.2
micrograms TSNA.
Several methods for reducing endogenous levels of nicotine and TSNAs in a
plant
have been discovered. These approaches can be used to create the tobacco
products
described above. Tobacco plants having a reduced amount of nicotine and/or
TSNAs that
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retain good smoking characteristics and taste, as manufactured by the methods
described in
the following section, can be used in the embodied tobacco-use cessation
programs.
Appz~oaches to snake tobacco pz~oducts having reduced nicotine azzdlor TSNA
levels
Nicotine is produced in tobacco plants by the condensation of nicotinic acid
and 4-
methylaminobutanal. Two regulatory loci (Nicl and Nic2) act as co-dominant
regulators of
nicotine production. These two loci are unlinked and the gene action is semi-
dominant and
primarily additive (Legg et al. (1969) J. Hez-ed., 60, 213-217).
Genetic and enzyme analyses have been used to investigate the Nicl and Nic2
genes. Collins et al. ((1974) Gz~op Sci., 14, 77-80) prepared doubled haploid
tobacco
breeding lines of these four alkaloid genotypes. The genotype of standard
cultivars is
NicllNicl N. ic2/Nic2 and that of low nicotine lines is nicllnicl nic~lzzic2.
NicllNicl
nic2/nic2 is a high intermediate and nicllrzicl Nic2/Nic2 is a low
intermediate (Legg and
Collins (1971) Can. J. Genet. Cytol. 13, 287-291). These lines are similar in
days-to-
flower, number of leaves, leaf size, and plant height. Enzyme analyses of
roots of single
and double Nic mutants show that the activities of two enzymes, quinolate
phosphoribosyl
transferase (QPTase) and putrescine methyl transferase (PMTase), are directly
proportional
to levels of nicotine biosynthesis (Saunders and Bush (1979) Plant Plzysiol
64:236). Both
Nicl and Nic2 affect PMTase and QPTase activities in roots, and thus, regulate
nicotine
synthesis (Leete (1983) In: Alkaloids: Chemical and Biological Pez~spectives,
S.W.
Pelletier, ed. John Wiley & Sons, pp. 85-152).
Enzyme analyses of roots of single and double Nic mutants show that the
activities
of QPTase and PMTase are directly proportional to levels of nicotine
biosynthesis. An
obligatory step in nicotine biosynthesis is the formation of nicotinic acid
from quinolinic
acid, which step is catalyzed by QPTase. QPTase appears to be a rate-limiting
enzyme in
the pathway supplying nicotinic acid for nicotine synthesis in tobacco (See,
eg., Feth et al.,
Plazzta, 168, pp. 402-07 (1986) and Wagner et al., Physiol. Plant., 68, pp.
667-72 (1986)).
A comparison of enzyme activity in tobacco tissues (root and callus) with
different
capacities for nicotine synthesis shows that QPTase activity is strictly
correlated with
nicotine content (Wagner and Wagner, Planta 165:532 (1985)). In fact, Saunders
and Bush
(Plant Plzysiol 64:236 (1979) showed that the level of QPTase in the roots of
low nicotine
mutants is proportional to the levels of nicotine in the leaves.
to
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Hibi et al. ((1994) Plant Cell, 6, 723-735) isolated the cDNA encoding PMTase,
PMT, and showed that PMT transcript levels are regulated by Nicl and Nic2. The
QPTase
cDNA and genomic clones (NtQPTl ) have also been isolated and the transcript
levels of
NtQPTl are also regulated by Nicl and Nic2. Thus, it appears that the Nic
genes regulate
nicotine content by regulating the transcript levels of genes encoding the two
rate-limiting
enzymes, PMTase and QPTase. Further, Nicl and Nick have been shown to be
positive
regulators of NtQPTl transcription and that promoter sequences upstream of the
transcription initiation site contain the cis-acting sequences necessary for
Nic gene product
activation of NtQPTl transcription. Because expression of QPTase and PMTase
are
coordinately-regulated by the Nic gene products, it likely that the Nic gene
products also
directly regulate transcription of the PMT gene.
One approach for reducing nicotine involves reducing the amount of a required
enzyme (e.g., QPTase and PMTase) in the biosynthetic pathway leading to its
production.
Where the affected enzyme naturally occurs in a rate-limiting amount (relative
to the other
enzymes required in the pathway), any reduction in that enzyme's abundance
will decrease
the production of the end product. If the amount of the enzyme is not normally
rate-
limiting, its presence in a cell must be reduced to rate-limiting levels in
order to diminish
the pathway's output. Conversely, if the naturally-occmTing amount of enzyme
is rate
limiting, then any increase in the enzyme's activity will result in an
increase in the
biosynthetic pathway's end product.
The modification of nicotine levels in tobacco plants by antisense regulation
of
PMTase expression is proposed in US Patents 5,369,023 and 5,260,205 to
Nalcatani and
Malik. PCT application WO 94/28142 to Wahad and Malilc describes DNA encoding
PMT
and the use of sense and antisense PMT constructs. Additionally, PCT
Application
W098/56923 to Conkling et al. describes DNA encoding a plant QPTase enzyme,
constructs comprising such DNA, and methods of altering QPTase expression to
increase
or decrease nicotine production in tobacco plants. Still further, U.S. patent
application
serial number 09/941,042 to Conlcling describes the use of DNA encoding
regulatory
sequences for the QPTase enzyme and methods of using these sequences as
molecular
decoys to sequester transcription factors at sites distant to the endogenous
promoter for the
QPTase gene, thereby decreasing nicotine production in tobacco plants. The
following
section describes in greater detail the antisense approach to making tobacco
products
having a reduced nicotine and/or TSNA level.
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Antisense teclaraology can be used to cf°eate tobacco pnoducts having a
reduced
level of nicotine aradlor TSNA
Antisense technology may be used to create tobacco plants with reduced
nicotine
levels. The preferred enzyme for antisense regulation of nicotine levels is
the TobRD2
gene (see Conkling et al., Plant Phys. 93, 1203 (1990)) encoding a Nicotiana
tabacufn
QPTase (see Example 1) (SEQ. ID. No. 1). In addition to the description of the
technology provided herein, general aspects of the technology are described in
PCT/IJS98/11893.
Regulation of gene expression in plant cell genomes can be achieved by
integration
of heterologous DNA under the transcriptional control of a promoter which is
functional in
the host, and in which the transcribed strand of heterologous DNA is
complementary to the
strand of DNA that is transcribed from the endogenous gene to be regulated.
The
introduced DNA, referred to as antisense DNA, provides an RNA sequence which
is
complementary to naturally produced (endogenous) mRNAs and which inhibits
expression
of the endogenous mRNA. Although the mechanism of antisense is not completely
understood, it is lcnown that antisense constructs can be used to regulate
gene expression.
A preferred approach for reducing QPTase levels through molecular modification
is
provided in Example 2 and Example 3.
In some methods of the invention, the antisense product may be complementary
to
coding or non-coding (or both) portions of naturally occurnng target RNA. The
antisense
construct may be introduced into the plant cells in any suitable mmner, and
may be
integrated into the plant genome for inducible or constitutive transcription
of the antisense
sequence. Tobacco plants are then regenerated from successfully transformed
cells using
conventional techniques. It is most preferred that the antisense sequence
utilized be
complementary to the endogenous sequence, however, minor variations in the
exogenous
and endogenous sequences may be tolerated. It is preferred that the antisense
DNA
sequence be of sufficient sequence similarity that it is capable of binding to
the endogenous
sequence in the cell to be regulated, under stringent conditions as described
below.
Although the preferred enzyme for antisense regulation is QPTase, other
enzymes
that are suitable for antisense regulation include, for example, putrescine N-
methyltransferase, N-methylputrescine oxidase, ornithine decarboxylase, S-
adenosylmethionine synthetase, NADH dehydrogenase, phosphoribosylanthranilate
isomerase, and any other enzyme linked to nicotine biosynthesis.
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WO 2005/000352 PCT/US2004/016958
As an example of the use of antisense technology, tobacco having a reduced
amount
of nicotine and TSNA is generated from a tobacco plant that is created by
exposing at least
one tobacco cell of a selected tobacco variety (preferably Burley 21) to an
exogenous DNA
construct having, in the 5' to 3' direction, a promoter operable in a plant
cell and DNA
containing a portion of a DNA sequence that encodes an enzyme in the nicotine
synthesis
pathway or a complement thereof (e.g., SEQ. ID. No. 1). The DNA is operably
associated
with said promoter and the tobacco cell is transformed with the DNA construct.
The
transformed cells are selected using either negative selection or positive
selection
techniques and at least one tobacco plant is regenerated from transformed
cells. The
regenerated tobacco plant or portion thereof is preferably analyzed to
determine the amount
of nicotine and/or TSNA present and these values can be compared to the amount
of
nicotine and/or TSNA present in a control tobacco plant or portion, preferably
of the same
variety.
The DNA constructs having a portion of a DNA sequence that encodes an enzyme
in the nicotine synthesis pathway may have the entire coding sequence of the
enzyme a
complement of this sequence, or any portion thereof. A portion of a DNA
sequence that
encodes an enzyme in the nicotine synthesis pathway or the complement thereof
may have
at least 25, 27, 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500, 750, 1000,
1500, 2000, 2500,
or 5000 bases, or the entire coding sequence of the enzyme or complement
thereof (e.g.,
SEQ. ID. No. 1). Accordingly, these DNA constructs have the ability to perturb
the
production of endogenous enzyme in the nicotine biosynthesis pathway through
either an
antisense or cosuppression mechanism. It is contemplated that both antisense,
RNAi, and
cosuppression constructs are effective at reducing the levels of nicotine
and/or nitrosamines
in tobacco plants.
Nucleic acid sequences employed in the constructs described herein include
those
with sequence similarity to the gene encoding QPTase, and encoding a protein
having
quinolate phosphoribosyl transferase activity, including, for example, allelic
variations in
QPTase proteins. Thus, DNA sequences that hybridize to DNA of the QPTase-
encoding
gene and code for expression of QPTase, particularly plant QPTase enzymes, may
also be
employed in carrying out the present invention. Multiple forms of tobacco QPT
enzyme
may exist. Multiple forms of an enzyme may be due to post-translational
modification of a
single gene product, or to multiple forms of the NtQPTl gene.
As used herein, the term 'gene' can refer to a DNA sequence that incorporates
(1)
upstream (5') regulatory signals including the promoter, (2) a coding region
specifying the
13
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WO 2005/000352 PCT/US2004/016958
product, protein or RNA of the gene, (3) downstream regions including
transcription
termination and polyadenylation signals and (4) associated sequences required
for efficient
and specific expression. In some contexts, a gene can include only (2), above,
or some
combination of items (1), (3), and (4) with (2). The DNA sequence of the
present invention
may comprise or consist essentially of the sequence encoding the QPTase
enzyme, or
equivalent nucleotide sequences representing alleles or polymorphic variants
of these
genes, or coding regions thereof. Use of the phrase "substantial sequence
similarity" in the
present specification and claims means that DNA, RNA or amino acid sequences
which
have slight and non-consequential sequence variations from the actual
sequences disclosed
and claimed herein are considered to be equivalent to the sequences of the
present
invention. hi this regard, "slight and non-consequential sequence variations"
mean that
"similar" sequences (i.e., the sequences that have substantial sequence
similarity with the
DNA, RNA, or proteins disclosed and claimed herein) will be functionally
equivalent to the
sequences disclosed and claimed in the present invention. Functionally
equivalent
sequences will function in substantially the same manner to produce
substantially the same
compositions as the nucleic acid and amino acid compositions disclosed and
claimed
herein.
By one approach, a novel cDNA sequence encoding a plant QPTase may be used.
As QPTase activity is strictly correlated with nicotine content, construction
of transgenic
tobacco plants in which QPTase levels are lowered in the plant roots (compared
to levels in
wild-type plants) result in plants having reduced levels of nicotine in the
leaves.
Embodiments of the invention provide methods and nucleic acid constructs for
producing
such transgeiuc plants, as well as the transgenic plants themselves. Such
methods include
the expression of antisense NtQPTl RNA, which lowers the amount of QPTase in
tobacco
roots.
Aspects of the present invention also concern sense and antisense recombinant
DNA molecules encoding QPTase or QPTase antisense RNA molecules, and vectors
comprising those recombinant DNA molecules, as well as transgenic plant cells
and plants
transformed with those DNA molecules and vectors. Transgenic tobacco cells and
the
plants described herein are characterized in that they have a reduced amount
of nicotine
and/or TSNA as compared to unmodified or control tobacco cells and plants.
Promoters to be linked to the antisense constructs of the present invention
may be
constitutively active promoters. Numerous constitutively active promoters
which are
operable in plants are available. A preferred example is the Cauliflower
Mosaic Virus
14
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WO 2005/000352 PCT/US2004/016958
(CaMV) 35S promoter which is expressed constitutively in most plant tissues. W
the
alternative, the promoter may be a root-specific promoter or root cortex
specific promoter,
as explained in greater detail below.
Antisense sequences have been expressed in transgenic tobacco plants utilizing
the
Cauliflower Mosaic Virus (CaMV) 35S promoter. See, e.g., Cornelissen et al.,
"Both RNA
Level and Translation Efficiency are Reduced by Anti-Sense RNA in Transgenic
Tobacco", Nucleic Acids Res. 17, pp. 833-43 (1989); Rezaian et al., "Anti-
Sense RNAs of
Cucumber Mosaic Virus in Transgenic Plants Assessed for Control of the Virus",
Plaht
Mol. Biol. 11, pp. 463-71 (1988); Rodermel et al., "Nuclear-Organelle
Interactions:
Nuclear Antisense Gene Inhibits Ribulose Bisphosphate Carboxylase Enzyme
Levels in
Transformed Tobacco Plants", Cell 55, pp. 673-81 (1988); Smith et al.,
"Antisense RNA
Inhibition of Polygalacturonase Gene Expression in Transgenic Tomatoes",
Nature 334,
pp. 724-26 (1988); Vaxi der Krol et al., "An Anti-Sense Chalcone Synthase Gene
in
Transgenic Plants Inhibits Flower Pigmentation", Nature 333, pp. 866-69
(1988).
Use of the CaMV 35S promoter for expression of antisense QPTase genes in the
transformed tobacco cells and plants of this invention is preferred. Use of
the CaMV
promoter for expression of other recombinant genes in tobacco roots has been
well
described (Lam et al., "Site-Specific Mutations Alter In Vitro Factor Binding
and Change
Promoter Expression Pattern in Transgenic Plants", Proc. Nat. Acad Sci. USA
86, pp. 7890-
94 (1989); Poulsen et al. "Dissection of 5' Upstream Sequences for Selective
Expression of
the Nicotiana plumbaginifolia rbcS-8B Gene", Mol. Gen. Genet. 214, pp. 16-23
(1988).
Other promoters which are active only in root tissues (root specific
promoters) are
also particularly suited to the methods of the present invention. See, e.g.,
US Patent No.
5,459,252 to Conlcling et al.; Yamamoto et al., Plaf2t Cell, 3:371 (1991). The
TobRD2 root-
cortex specific promoter may also be utilized. See, eg., US Patent application
SN
08/508,786, now allowed, to Conkling et al; PCT WO 9705261.
Some of the nucleic acids described herein may also be used in methods of
sense
co-suppression or RNAi-mediated suppression of nicotine production. Sense DNAs
employed in these methods are preferably of a length sufficient to, when
expressed in a
plant cell, suppress the native expression of the plant QPTase protein as
described herein in
that plant cell. Such sense DNAs may be essentially an entire genomic or
complementary
DNA encoding the QPTase enzyme, or a fragment thereof, with such fragments
typically
being at least 15, 25, 27, 30, 35, 40, 45, 50, 60, 75, 100, 150, 250, 500,
750, nucleotides in
CA 02527648 2005-11-29
WO 2005/000352 PCT/US2004/016958
length. Methods of ascertaining the length of sense DNA that results in
suppression of the
expression of a native gene in a cell are available to those skilled in the
art.
In an alternate embodiment, Nicotiaha plant cells are transformed with a DNA
construct containing a DNA segment encoding an enzymatic RNA molecule termed a
"ribozyme", which enzymatic RNA molecule is directed against and cleaves the
mRNA
transcript of DNA encoding plant QPTase as described herein. Production of
such an
enzymatic RNA molecule in a plant cell and disruption of QPTase protein
production
reduces QPTase activity in plant cells in essentially the same manner as
production of an
antisense RNA molecule: that is, by disrupting translation of mRNA in the cell
which
produces the enzyme. The section below describes yet another method to
decrease levels
of specific enzymes involved in nicotine biosynthesis, using decoy nucleic
acid fragments.
Molecular decoy tecla~cology to lomer nicotine andloY TSNA levels
The use of nucleic acid-based decoy fragments to reduce gene expression is
referred
to as "molecular decoys". In a preferred example, the "decoy fragment"
corresponds to
promoter sequences upstream of the QPTase, to reduce QPTase expression.
In some embodiments, an isolated nucleic acid, or a fragment thereof
consisting of
at least 20-450 consecutive nucleotides desirably, at least 30-400 consecutive
nucleotides
preferably, 50-350 consecutive nucleotides more preferably, and 100-300 or 200-
400
consecutive nucleotides most preferably, that is or contains at least one cis-
acting
regulatory element, which exists upstream of the plant QPTase andlor
putrescine methyl
transferase PMTase coding sequences (e.g., SEQ. ID. No. 1). Another example is
the Nic
gene product responsive element obtained from the sequence disclosed in U.S.
Patent No.
5,459,252. In some embodiments, the Nic gene product responsive element
resides
between -1000 and -600 or -700 by of the NtQPTl promoter. Accordingly, some
embodiments involve a 300-400 nucleotide long fragment of the NtQPTl promoter
that
corresponds to the sequence of the NtQPTl promoter between -1000 and -600 or -
700, as
disclosed in U.S. Patent No. 5,459,252.
Thus, in several embodiments, the embodied nucleic acids have a structure that
promotes an interaction with one or more transcription factors (e.g., Nicl and
Nic2), which
are involved in initiating transcription of QPTase and/or PMTase. Accordingly,
said
nucleic acids are said to be or contain at least one transcription factor
(e.g., Nicl and Nic2)
binding sequences, which are also referred to as "cis-acting regulatory
elements." By
introducing multiple copies of these cis-acting regulatory elements (e.g.,
sequences that
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WO 2005/000352 PCT/US2004/016958
interact with Nicl and/or Nic2) into a plant cell, the ability of the
transcription factor to
initiate transcription of the targeted gene (e.g., QPTase and/or PMTase genes)
can be
reduced or squelched.
By one approach, tobacco plants are transformed with an excess number of DNA
sequences (cis-acting elements) from the promoters of genes encoding, but not
limited to,
QPTase and PMTase that are regulated in nicotine biosynthesis. These cis-
acting elements
are preferably integrated into the plant genome so as to allow for transfer to
successive
generations. Preferred approaches are provided in Example 4 and Example 5.
Typically,
the Nicl and Nic2 DNA-binding proteins that interact with these cis-acting DNA
sequences
are expressed at relatively low levels in the cell, thus the excess of
transgenic cis-acting
elements will compete with the endogenous elements associated with the genes
encoding,
but not limited to, QPTase and PMTase for available Nicl and Nic2 Accordingly,
these cis-
acting DNA sequences (and those of other cis-acting elements) are referred to
herein as
"decoys" or "molecular decoys". The competition decreases occupancy of traps-
acting
DNA-binding proteins on their cognate cis-acting elements, thereby down-
regulating the
synthesis of nicotine biosynthesis enzymes.
Embodiments of the present invention also provide DNA molecules of cis-acting
elements of QPTase or PMTase, and vectors comprising those DNA molecules, as
well as
transgenic plant cells and plants transformed with those DNA molecules and
vectors.
Transgenic tobacco cells and plants of this invention are characterized by
lower nicotine
content than untransformed control tobacco cells and plants.
Any of a variety of cis-acting elements can be used in carrying out the
molecular
decoy methods, depending upon the particular application. Examples of cis-
acting
elements (and corresponding transcription factors) that may be used, alone or
in
combination with one another, which may be used in embodiments of the present
invention
include, but are not limited to, AS-1 and ASF-1 (see U.S. Patents 4,990,607
and
5,223,419), the AATT repeat element and PABF (see U.S. Patents 5,834,236 and
6,191,258), a wounding-responsive cis-acting element from potato (Siebert et
al., Plant
Cell 1:961-8 (1989)), an embryo-specific cis-acting element from bean (Bustos
et al, Plah.t
Cell 1:839-853 (1989)), a root-specific cis-acting element from the tobacco
RB7 promoter
(US patent 5,459,252 and Yamamoto et al., Plant Cell 3:371-382 (1991)), a
positive
poly(dA-dT) regulatory element and binding protein and negative CCCAA repeat
element
and binding protein (Wang et al., Mol. Cell Biol. 12:3399-3406 (1992)), a root-
tip
regulatory element from the tobacco phytochrome A1 promoter of tobacco (Adam
et al.,
1~
CA 02527648 2005-11-29
WO 2005/000352 PCT/US2004/016958
Plant Mol Biol 29:983-993 (1995)), an anaerobiosis-responsive element from the
maize
glyceraldehyde-3-phosphate dehydrogenase 4 gene (Geffers et al., Plant Mol
Biol 43:11-21
(2000)), and a seed-specific regulatory region from an Arabidopsis oleosin
gene (see US
patent 5,792,922).
The status of the art is such that large databases list identified cis-acting
regulatory
regions (e.g., Plant Cis-acting Regulatory elements, "PLACE", with about 1,340
entries,
and Plant Cis-acting Regulatory Elements "PlantCARE", which lists about 159
plant
promoters. The listed cis-acting regulatory elements in these databases and
the cis-acting
regulatory elements that are provided in Raumbauts et al., Nucleic acids
Research 27:295-
296 (1999), and Higo et al., Nucleic acids Reseaf-ch 27:297-300 (1999) can be
used with
embodiments of the invention. The section below describes general methods for
transformation of tobacco plants with modified sequences to create tobacco
plants with low
nicotine andlor TSNA levels.
T~arasgefzic Plant Cells and Plants
DNA sequences provided herein can be transformed into a variety of host cells.
A
variety of suitable host cells, having desirable growth and handling
properties, are readily
available in the art. As used herein, a "native DNA sequence" or "natural DNA
sequence"
means a DNA sequence which can be isolated from non-transgenic cells or
tissue. Native
DNA sequences are those which have not been artificially altered, such as by
site-directed
mutagenesis. Once native DNA sequences are identified, DNA molecules having
native
DNA sequences may be chemically synthesized or produced using recombinant DNA
procedures as are known in the art. A native plant DNA sequence typically can
be isolated
from non-transgenic plant cells or tissue.
DNA constructs, or "transcription cassettes," of the present invention may
include,
5' to 3' in the direction of transcription, a promoter as discussed herein, a
DNA sequence as
discussed herein operatively associated with the promoter, and, optionally, a
termination
sequence including stop signal for RNA polymerase and a polyadenylation signal
for
polyadenylase. The term "operatively associated," as used herein, refers to
DNA
sequences on a single DNA molecule which are associated so that the function
of one is
affected by the other. Thus, a promoter is operatively associated with a DNA
when it is
capable of affecting the transcription of that DNA (i.e., the DNA is under the
transcriptional control of the promoter). The promoter is said to be
"upstream" from the
DNA, which is in turn said to be "downstream" from the promoter.
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WO 2005/000352 PCT/US2004/016958
In embodiments of the invention wherein a termination signal is used, any
suitable
termination signal may be employed in carrying out the present invention,
examples thereof
including, but not limited to, the nopaline synthase (nos) terminator, the
octopine synthase
(ocs) terminator, the CaMV terminator, or native termination signals derived
from the same
gene as the transcriptional initiation region or derived from a different
gene. See, e.g.,
Rezian et al. (1988) supra, and Rodermel et al. (1988), supra. Alternatively,
if nicotine
levels are decreased by molecular decoy technology rather than by antisense or
other
methods, the molecular decoy fragments, with or without additional sequences,
may be
provided to the plant cell by any means. For example, the molecular decoy
fragment may
have an accompanying gene encoding a selectable marker, other suitable genes,
or may be
present as part of a plasmid vector. The molecular decoy fragment may consist
of a single
or double stranded DNA or RNA molecule. The molecular decoy may be integrated
into
the genome or may exist freely in the cell.
The transcription cassette may be provided in a DNA construct that also has at
least
one replication system. For convenience, it is common to have a replication
system
functional in Esche~ichia coli, such as ColEl, pSC101, pACYC184, or the lilce.
In this
manner, at each stage after each manipulation, the resulting construct may be
cloned,
sequenced, and the correctness of the manipulation determined. In addition, or
in place of
the E. coli replication system, a broad host range replication system may be
employed, such
as the replication systems of the P-1 incompatibility plasmids, e.g., pRK290.
In addition to
the replication system, there will frequently be at least one marlcer present,
which may be
useful in one or more hosts, or different markers for individual hosts. That
is, one marker
may be employed for selection in a prokaryotic host, while another marker may
be
employed for selection in a eukaryotic host, particularly the plant host. The
marlcers may
be protection against a biocide, such as antibiotics, toxins, heavy metals, or
the like; may
provide complementation, by imparting prototrophy to an auxotrophic host; or
may provide
a visible phenotype through the production of a novel compound in the plant.
The various fragments comprising the various constructs, transcription
cassettes,
marlcers, and the like may be introduced consecutively by restriction enzyme
cleavage of an
appropriate replication system, and insertion of the particular construct or
fragment into the
available site. After ligation and cloning the DNA construct may be isolated
for further
manipulation. All of these techniques are amply exemplified in the literature
as
exemplified by J. Sambroolc et al., Molecular Cloning, A Laboratory Manual (2d
Ed.
1989)(Cold Spring Harbor Laboratory).
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WO 2005/000352 PCT/US2004/016958
Vectors which may be used to transform plant tissue with nucleic acid
constructs of
the present invention include both Ag~obacterium vectors and ballistic
vectors, as well as
vectors suitable for DNA.-mediated transformation. The term 'promoter' refers
to a region
of a DNA sequence that incorporates the necessary signals for the efficient
expression of a
coding sequence. This may include sequences to which an RNA polymerase binds
but is
not limited to such sequences and may include regions to which other
regulatory proteins
bind together with regions involved in the control of protein translation and
may include
coding sequences.
The QPTase recombinant DNA molecules and vectors used to produce the
transformed tobacco cells and plants of this invention may further comprise a
dominant
selectable marker gene. Suitable dominant selectable markers for use in
tobacco include,
inter alia, antibiotic resistance genes encoding neomycin phosphotransferase
(NPTII), and
hygromycin phosphotransferase (HPT). Other well-known selectable markers that
are
suitable for use in tobacco include a mutant dihydrofolate reductase gene that
encodes
methotrexate-resistant dihydrofolate reductase. DNA vectors containing
suitable antibiotic
resistance genes, amd the corresponding antibiotics, are commercially
available.
Transformed tobacco cells are selected out of the surrounding population of
non-
transformed cells by placing the mixed population of cells into a culture
medium
containing an appropriate concentration of the antibiotic (or other compound
normally
toxic to tobacco cells) against which the chosen dominant selectable marker
gene product
confers resistance. Thus, only those tobacco cells that have been transformed
will survive
and multiply. Additionally, the positive selection techniques described by
Jefferson (e.g.,
WO 00055333; WO 09913085; U.S. Pat. Nos. 5599670; 5432081; and 5268463) can be
used.
Methods of malting recombinant plants of the present invention, in general,
involve
first providing a plant cell capable of regeneration (the plant cell typically
residing in a
tissue capable of regeneration). The plant cell is then transformed with a DNA
construct
comprising a transcription cassette of the present invention (as described
herein) and a
recombinant plant is regenerated from the transformed plant cell. As explained
below, the
transforming step is carried out by techniques as are known in the art,
including but not
limited to bombarding the plant cell with microparticles, carrying the
transcription cassette,
infecting the cell with an Agrobacte~ium tumefaciens containing a Ti plasmid
carrying the
transcription cassette or any other technique suitable for the production of a
transgenic
plant.
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WO 2005/000352 PCT/US2004/016958
Numerous Agf~obacterium vector systems useful in carrying out the present
invention are known. For example, U.S. Patent No. 4,459,355 discloses a method
for
transforming susceptible plants, including dicots, with an Agrobacter~iunz
strain containing
the Ti plasmid. The transformation of woody plants with an AgYObacte~ium
vector is
disclosed in U.S. Patent No. 4,795,855. Further, U.S. Patent No. 4,940,838 to
Schilperoort
et al. discloses a binary Ag~obacterium vector (i.e., one in which the
Agrobacte~ium
contains one plasmid having the vir region of a Ti plasmid but no T region,
and a second
plasmid having a T region but no vir region) useful in carrying out the
present invention.
Microparticles carrying a DNA construct of the present invention, which
microparticle is suitable for the ballistic transformation of a plant cell,
are also useful for
making transformed plants of the present invention. The microparticle is
propelled into a
plant cell to produce a transformed plant cell, and a plant is regenerated
from the
transformed plant cell. Any suitable ballistic cell transformation methodology
and
apparatus can be used in practicing the present invention. Exemplary apparatus
and
procedures are disclosed in Sanford and Wolf, U.S. Patent No. 4,945,050, and
in Christou
et al., U.S. Patent No. 5,015,580. When using ballistic transformation
procedures, the
transcription cassette may be incorporated into a plasmid capable of
replicating in or
integrating into the cell to be transformed. Examples of microparticles
suitable for use in
such systems include 1 to 5 ~m gold spheres. The DNA construct may be
deposited on the
microparticle by any suitable technique, such as by precipitation.
Plant species may be transformed with the DNA construct of the present
invention
by the DNA-mediated transformation of plant cell protoplasts; and subsequent
regeneration
of the plant from the transformed protoplasts in accordance with procedures
well known in
the art. Fusion of tobacco protoplasts with DNA-containing liposomes or via
electroporation is lcnown in the art. (Shillito et al., "Direct Gene Transfer
to Protoplasts of
Dicotyledonous and Monocotyledonous Plants by a Number of Methods, Including
Electroporation", Methods Enzymol. 153, pp. 313-36 (1987)).
As used herein, transformation refers to the introduction of exogenous DNA
into
cells, so as to produce transgenic cells stably transformed with the exogenous
DNA.
Transformed cells are induced to regenerate intact tobacco plants through
application of
tobacco cell and tissue culture techniques that are well known in the art. The
method of
plant regeneration is chosen so as to be compatible with the method of
transformation. The
stable presence and the orientation of the QPTase sequence in transgenic
tobacco plants can
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WO 2005/000352 PCT/US2004/016958
be verified by Mendelian inheritance of the QPTase sequence, as revealed by
standard
methods of DNA analysis applied to progeny resulting from controlled crosses.
After
regeneration of transgenic tobacco plants from transformed cells, the
introduced DNA
sequence is readily transferred to other tobacco varieties through
conventional plant
breeding practices and without undue experimentation.
Any plant tissue capable of subsequent clonal propagation, whether by
organogenesis or embryogenesis, may be transformed with a vector of the
present
invention. The term "organogenesis," as used herein, means a process by which
shoots and
roots are developed sequentially from meristematic centers; the term
"embryogenesis," as
used herein, means a process by which shoots and roots develop together in a
concerted
fashion (not sequentially), whether from somatic cells or gametes. The
particular tissue
chosen will vary depending on the clonal propagation systems available for,
and best suited
to, the particular species being transformed. Exemplary tissue targets include
leaf disks,
pollen, embryos, cotyledons, hypocotyls, callus tissue, existing meristematic
tissue (e.g.,
apical meristems, axillary buds, and root meristems), and induced meristem
tissue (e.g.,
cotyledon meristem and hypocotyl meristem).
Plants of the present invention may take a variety of forms. The plants may be
chimeras of transformed cells and non-transformed cells; the plants may be
clonal
transformants (e.g., all cells transformed to contain the transcription
cassette); the plants
may comprise grafts of transformed and untransformed tissues (e.g., a
transformed root
stock grafted to an untransformed scion in citrus species). The transformed
plants may be
propagated by a variety of means, such as by clonal propagation or classical
breeding
techniques. For example, first generation (or TI) transformed plants may be
selfed to give
homozygous second generation (or T2) transformed plants, and the T2 plants
further
propagated through classical breeding techniques. A dominant selectable marker
(such as
nptII) can be associated with the transcription cassette to assist in
breeding.
As used herein, a crop comprises a plurality of plants of the present
invention, and
of the same genus, planted together in an agricultural field. Thus, the
present invention
provides a method of producing a crop of plants having lowered QPTase or
PMTase
activity and thus having decreased nicotine and/or TSNA levels, as compared to
a similar
crop of non-transformed plants of the same species and variety.
Levels of nicotine in the transgenic tobacco plants of the present invention
can be
detected by standard nicotine assays. Transformed plants in which the level of
QPTase or
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PMTase is reduced compared to untransformed control plants will accordingly
have a
reduced nicotine level compared to the control.
The modified tobacco plants described herein are suitable for conventional
growing
and harvesting techniques (e.g. topping or no topping, bagging the flowers or
not bagging
the flowers, cultivation in manure rich soil or without manure). The harvested
tobacco
leaves and stems are suitable for conventional methods of processing such as
curing and
blending. The modified tobacco is suitable for use in any traditional tobacco
product
including, but not limited to, pipe, cigar and cigarette tobacco, and chewing
tobacco in any
form including leaf tobacco, shredded tobacco, or cut tobacco. The section
below describes
typical curing methods which may be used to prepare the tobacco once it is
harvested.
Curing
The curing process, which typically lasts about 1 week, brings out the flavor
and
aroma of tobacco. Several methods for curing tobacco may be used, and indeed
many
methods have been previously disclosed. For example, U.S. Pat. Nos. 4,499,911
to
Johnson; 5,685,710 to Martinez Sagrera; 3,905,123 to Fowler; 3,840,025 to
Fowler; and
4,192,323 to Horne describe aspects of the tobacco curing process which may be
used for
some embodiments of the present invention. Conventionally, "sticks" that are
loaded with
tobacco are placed into bulls containers and placed into closed buildings
having a heat
source known as a curing barn. A flue is often used to control the smoke (thus
earning the
term "flue-cured"). The method of curing will depend, in some cases, on the
type of
tobacco-use cessation product desired, (i.e., snuff, cigarettes, or pipe
tobacco may
preferably utilize different curing methods) and preferred methods may vary
from region to
region and in different countries. In some approaches, the stems and midveins
of the leaf
are removed from the leaves prior to curing to yield a high quality, low
nitrosamine tobacco
product.
"Flue curing" is a popular method for curing tobacco in Virginia, North
Carolina,
and the Coastal Plains regions of the United States. This method is used
mainly in the
manufacture of cigarettes. Flue curing requires a closed building equipped
with a system
of ventilation and a source of heat. The heating can be direct or indirect
(e.g., radiant heat).
When heat and humidity are controlled, leaf color changes, moisture is
quiclcly removed,
and the leaf and stems dry. Careful monitoring of the heating and humidity can
reduce the
accumulation of nitrosamines.
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Another curing method is termed "air curing". In this method, an open
framework
is prepared in which sticks of leaves (or whole plants) are hung so as to be
protected from
both wind and sun. Leaf color changes from green to yellow, as leaves and
stems dry
slowly.
"Fire curing" employs an enclosed barn similar to that used for flue curing.
The
tobacco is hung over low temperature fire so that the leaves cure in a smolce-
laden
atmosphere. This process uses lower temperatures, so the process may take up
to a month,
in contrast to flue curing, which takes about 6 to 8 days.
A fiu~ther curing method, termed "sun curing" is the drying of uncovered
sticks or
strings of tobacco leaves in the sun. The best known sun-cured tobaccos are
the so-called
oriental tobaccos of Turkey, Greece, Yugoslavia, and nearby countries.
The curing process, and most particularly the flue-curing process, is
generally
divided into the following four stages:
A) Firing Up: During this step, the tobacco leaves turn bright lemon-orange in
color. This
is achieved by a gradual increase in temperature.
B) Leaf Yellowing: In this step any moisture is removed. This creates the
"yellowing" of
the tobacco. It also prepares the tobacco for drying in the next step.
C) Leaf Dr 'n~,n~: Leaf drying, an important step in the curing process,
requires much time
for the tobacco to dry properly. Additionally, air flow is increased in this
step to facilitate
the drying process.
D) Stem Dryiy The drying process continues, as the stem of the tobacco leaf
becomes
dried.
The cured tobacco may then be blended with other tobaccos or other materials
to
create the product to be used for the tobacco-use cessation method. The
section below
describes typical methods of blending and preparing the tobacco product.
Tobacco blefading
It may be desirable to blend tobacco of varying nicotine levels to create the
cessation product having the desired level of nicotine. This blending process
is typically
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performed after the curing process, and may be performed by conventional
methods.
Preferred tobacco blending approaches are provided in Examples 6 and 7. In
some
embodiments, blending of the transgenic tobacco is conducted to prepare the
tobacco so
that it will contain specific amounts of nicotine and/or TSNA in specific
products.
Preferably, the blending is conducted so that tobacco products of varying
amounts of
nicotine and/or TSNAs are made in specific products.
A mixture that contains different types of tobacco is desirably substantially
homogeneous throughout in order to avoid undesirable fluctuations in taste or
nicotine
levels. Typically, tobacco to be blended may have a moisture content between
30 and 75%.
As an example, the tobacco is first cut or shredded to a suitable size, then
mixed in a
mixing device, such as a rotating drum or a blending box. One such known
mixing device
is a tumbling apparatus that typically comprises a rotating housing enclosing
mixing
paddles which are attached to and, therefore, rotate with the housing to stir
the tobacco
components together in a tumbling action as the drum turns.
After the desired tobaccos are thoroughly mixed, the resulting tobacco blend
is
removed from the mixing apparatus and bulked to provide a continuous,
generally uniform
quantity of the tobacco blend. The tobacco is then allowed to remain
relatively undisturbed
(termed the "bulking step") for the required period of time before subsequent
operations are
performed. The bullring step typically takes 30 minutes or less, and may be
carried out on
a conveyor belt. The conveyor belt allows the blended tobacco to remain in
bulls form in
an undisturbed condition while it is continuously moving the tobacco blend
through the
process from the mixing stage to the expansion stage.
The tobacco blend is typically expanded by the application of steam. The
tobacco
mixture is typically subjected to at least 0.25 pounds of saturated steam at
atmospheric
conditions per pound of blended tobacco for at least 10 seconds to provide an
increase in
moisture of at least 2 weight percent to the tobacco blend. After the tobacco
blend has been
expanded, it is dried. A typical drying apparatus uses heated air or
superheated steam to
dry the tobacco as the tobacco is conveyed by the heated air or steam stream
through a
drying chamber or series of drying chambers. Generally, the wet bulb
temperature of the
drying air may be from about 150 degrees F. to about 211 degrees F. The
tobacco blend is
typically dried to a moisture content of from about 60 percent to about 5
percent. The
dried, expanded tobacco blend is then in a suitable mode to be processed into
the tobacco-
use cessation product as described below.
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Nicotine t~eduction andlo~ tobacco-use cessation progf~ams methods
It is also contemplated that the low nicotine and/or TSNA tobacco described
herein
can be processed and blended with conventional tobacco so as to create a wide-
range of
tobacco products with varying amounts of nicotine and/or nitrosamines. These
blended
tobacco products can be used in nicotine reduction and/or tobacco-use
cessation programs
so as to move a consumer from a high nicotine and TSNA product to a low
nicotine and
TSNA product.
In some embodiments of the invention, a stepwise nic~tine reduction and/or
tobacco-use cessation program can be established using the low nicotine, low
TSNA
products described above. As an example, the program participant initially
determines his
or her current level of nicotine intake. The program participant then begins
the program at
step 1, with a tobacco product having a reduced amount of nicotine, as
compared to the
tobacco product that was used prior to beginning the program. After a period
of time, the
program participant proceeds to step 2, using a tobacco product with less
nicotine than the
products used in step 1. The program participant, after another period of
time, reaches step
3, wherein the program participant begins using a tobacco product with less
nicotine than
the products in step 2, and so on. Ultimately, the program participant uses a
tobacco
product having an amount of nicotine that is less than that which is
sufficient to become
addictive or to maintain an addiction. Thus, the nicotine reduction and/or
tobacco-use
cessation program limits the exposure of a program participant to nicotine
and,
concomitantly, the harmful effect of nicotine yet retains the secondary
factors of addiction,
including but not limited to, smoke intake, oral fixation, and taste.
For example, a smoker can begin the program smoking blended cigarettes having
Smg of nicotine and 1.S~,g of nitrosamine, gradually move to smoking
cigarettes with 3mg
of nicotine and l~.g of nitrosamine, followed by cigarettes having lmg
nicotine and O.S~.g
nitrosamine, followed by cigarettes having O.Smg nicotine and 0.25~,g
nitrosamine,
followed by cigarettes having less than 0.lmg nicotine and less than 0.1~,g
TSNA until the
consumer decides to smolce only the cigarettes having virtually no nicotine
and
nitrosamines or quitting smoking altogether. Preferably, a three-step program
is followed
whereby at step 1, cigarettes containing 0.6mg nicotine and less than 2~,g/g
TSNA are
used; at step 2, cigarettes containing 0.3mg nicotine and less than 1 p,g/g
TSNA are used;
and at step 3, cigarettes containing less than O.lmg nicotine and less than
0.7~g/g TSNA
are used. More preferably, a three-step program is followed whereby at step 1,
cigarettes
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WO 2005/000352 PCT/US2004/016958
containing 0.6mg nicotine and less than 2~g/g TSNA are used; at step 2,
cigarettes
containing 0.3mg nicotine and less than 1 ~.g/g TSNA are used; and at step 3,
cigarettes
containing less than O.OSmg nicotine and less than 0.7~g/g TSNA are used.
Accordingly,
the blended cigarettes described herein provide the basis for an approach to
reduce the
carcinogenic potential in a human in a step-wise fashion.
The methods described herein facilitate tobacco-use cessation by allowing the
individual to retain the secondary factors of addiction such as smoke intalce,
oral fixation,
and taste, while gradually reducing the addictive nicotine levels consumed.
Eventually,
complete cessation is made possible because the presence of addiction for
nicotine is
gradually decreased while the individual is allowed to maintain dependence on
the
secondary factors, above.
Embodiments, for example, include stepwise blends of tobacco products, which
are
prepared with a variety of amounts of nicotine. These stepwise blends are made
to have
reduced levels of TSNAs and varying amounts of nicotine. As an example,
cigarettes may
contain, for example, 5 mg, 4, 3, 2, 1, 0.5, 0.1, or 0 mg of nicotine per
cigarette. More
preferably, blended cigarettes contain less than 0.01%, 0.02%, 0.03%, 0.04%,
0.05%,
0.06%, 0.07%, 0.08%, 0.09%~ 0.1%, 0.2%, 0.3%, 0.4%, 0.5%, and 0.6% nicotine.
In another aspect of the invention, the cigarettes of vaxying levels of
nicotine axe
packaged to clearly indicate the level of nicotine present, and marketed as a
smoking
cessation program. A preferred approach to produce a product for nicotine
reduction
and/or tobacco-use cessation program is provided in Example 8. Individuals may
wish to
step up the program by skipping gradation levels of nicotine per cigarette or
staying at
certain steps until ready to proceed to the next level. Significantly, aspects
of the invention
allow a consumer to individually select the amount of nicotine that is
ingested by selection
of a particular tobacco product described herein. Furthermore, because the
secondary
factors of addiction are maintained, dependence on nicotine can be reduced
rapidly.
The nicotine reduction andlor tobacco-use cessation program limits the
exposure of
a program participant to nicotine while retaining the secondary factors of
addiction. These
secondary factors include but are not limited to, smoke intake, oral fixation,
and taste.
Because the secondary factors are still present, the program participant may
be more likely
to be successful in the nicotine reduction and/or tobacco-use cessation
program than in
programs that rely on supplying the program participant with nicotine but
remove the
above-mentioned secondary factors. Ultimately, the program participant uses a
tobacco
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WO 2005/000352 PCT/US2004/016958
product having an amount of nicotine that is less than that which is
sufficient to become
addictive.
In another aspect of the invention, individuals would choose to obtain only
cigarettes with less than 0.05 mg nicotine per cigarette. Some individuals,
such as
individuals needing to stop nicotine intake immediately (for example,
individuals with
medical conditions or individuals using drugs that interact with nicotine) may
find this
method useful. For some individuals, the mere presence of a cigarette in the
mouth can be
enough to ease withdrawal from nicotine addiction. Gradually, the addictive
properties of
smoking can decrease since there is no ucotine in the cigarettes. These
individuals are
then able to quit smol~ing entirely.
hi another aspect of the invention, paclcs of cigarettes containing the
gradations of
nicotine levels are provided as a "smoking cessation kit." An individual who
wishes to quit
smoking can buy the entire kit of cigarettes at the beginning of the program.
Thus any
temptation that may occur while buying cigarettes at the cigarette counter is
avoided. Thus,
the success of this method may be more likely for some individuals. A
preferred example
of such a lit is provided in Example 9.
The examples which follow are set forth to illustrate the present invention,
and are
not to be construed as limiting thereof.
EXAMPLE 1
Isolation and Seauencin~
TobRD2 cDNA (Conkling et. al., Plant Phys. 93, 1203 (1990)) encodes QPTase,
which is predicted to be a cytosolic protein. Comparisons of the NtQPTl amino
acid
sequence with the GenBank database revealed limited sequence similarity to
certain
bacterial and other proteins; quinolate phosphoribosyl transferase (QPTase)
activity has
been demonstrated for the S. typhifnuYium, E. coli. and N. tabacum genes. The
NtQPTI
encoded QPTase has similarity to the deduced peptide fragment encoded by an
As°abidopsis
EST (expression sequence tag) sequence (Genbank Accession number F20096),
which may
represent part of an Arabidopsis QPTase gene.
EXAMPLE 2
Transformation of Tobacco Plants
DNA of the QPTase gene, in antisense orientation, is operably linked to a
plant
promoter (CaIVIV 35S or TobRD2 root-cortex specific promoter) to produce two
different
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WO 2005/000352 PCT/US2004/016958
DNA cassettes: CaMV35S promoter/antisense QPTase-encoding gene and TobRD2
promoter/antisense QPTase-encoding gene.
A wild-type tobacco line and a low-nicotine tobacco line are selected for
transformation, e.g., wild-type Burley 21 tobacco (Nicl+/Nic2+) and homozygous
Nicl-
lNic2- Burley 21. A plurality of tobacco plant cells from each line are
transformed using
each of the DNA cassettes. Transformation is conducted using an
Ag>~obacte>"iunz vector,
e.g., an Agrobacteriuzn-binary vector carrying Ti-border sequences and the
nptll gene
(confernng resistance to kanamycin and under the control of the nos promoter
(nptll)).
Transformed cells axe selected and regenerated into transgenic tobacco plants
called
R°. The R° plants are grown to maturity and tested for levels of
nicotine; a subset of the
transformed tobacco plants exhibit significantly lower levels of nicotine
compared to non-
transformed control plants.
R° plants are then selfed and the segregation of the transgene is
analyzed in next
generation, the Rl progeny. Rl progeny are grown to maturity and selfed;
segregation of
the transgene among R2 progeny indicate which Rl plants are homozygous for the
transgene.
EXAMPLE 3
Tobacco having reduced nicotine levels
Tobacco of the variety Burley 21 LA was transformed with the binary
Agrobacteriunz vector pYTY32 to produce a low nicotine tobacco variety, Vector
21-41.
The binary vector pYTY32 carried the 2.0 lcb NtQPTl root-cortex-specific
promoter
driving antisense expression of the NtQPTl cDNA and the nopaline synthase
(nos) 3'
termination sequences from Agz~obactef°ium tumefacierzs T-DNA. The
selectable marker
for this construct was neomycin phosphotransferase (nptll) from E. coli Tn5
which confers
resistance to kanamycin, and the expression nptll was directed by the zaos
promoter from
Agr~obactez°ium tumefaciens T-DNA. Transformed cells, tissues, and
seedlings were
selected by their ability to grow on Murashige-Skoog (MS) medium containing
300 ~,g/ml
kanamycin. Burley 21 LA is a variety of Burley 21 with substantially reduced
levels of
nicotine as compared with Burley 21 (i.e., Burley 21 LA has 8% the nicotine
levels of
Burley 21, see Legg et al., Can J Genet Cytol, 13:287-91 (1971); Legg et al.,
J Hered,
60:213-17 (1969)).
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One-hundred independent pYTY32 transformants of Burley 21 LA (To) were
allowed to self. Progeny of the selfed plants (T1) were germinated on medium
containing
kanamycin and the segregation of kanamycin resistance scored. T1 progeny
segregating
3:1 resulted from transformation at a single locus and were subjected to
further analysis.
Nicotine levels of T1 progeny segregating 3:1 were measured qualitatively
using a
micro-assay technique. Approximately 200 mg fresh tobacco leaves were
collected and
ground in 1 ml extraction solution (Extraction solution: 1 ml Acetic acid in
100 ml HZO).
Homogenate was centrifuged for 5 min at 14,000 x g and supenlatant removed to
a clean
tube, to which the following reagents were added: 100 ~L NH40AC (5 g/100 ml
HZO + 50
~,L Brij 35); 500 ~.L Cyanogen Bromide (Sigma C-6388, 0.5 g/100 ml H20 + 50
~.L Brij
35); 400 ~L Aniline (0.3 ml buffered Aniline in 100 ml NH40AC + 50 ~.L Brij
35). A
nicotine standard stock solution of 10 mg/ml in extraction solution was
prepared and
diluted to create a standard series for calibration. Absorbance at 460 mn was
read and
nicotine content of test samples were determined using the standard
calibration curve.
Tl progeny that had less than 10% of the nicotine levels of the Burley 21 LA
parent
were allowed to self to produce TZ progeny. Homozygous TZ progeny were
identified by
germinating seeds on medium containing kanamycin and selecting clones in which
100%
of the progeny were resistant to lcanamycin (i.e., segregated 4:0;
heterozygous progeny
would segregate 3:1). Nicotine levels in homozygous and heterozygous T2
progeny were
qualitatively determined using the micro-assay and again showed levels less
than 10% of
the Burley 21 LA parent. Leaf samples of homozygous TZ progeny were sent to
the
Southern Research and Testing Laboratory in Wilson, NC for quantitative
analysis of
nicotine levels using Gas Chromatography/Flame Ionization Detection (GC/FID).
Homozygous T2 progeny of transformant #41 gave the lowest nicotine levels (~70
ppm),
and this transformant was designated as "Vector 21-41."
Vector 21-41 plants were allowed to self cross, producing T3 progeny. T3
progeny
were grown and nicotine levels assayed qualitatively and quantitatively. T3
progeny were
allowed to self cross, producing T4 progeny. Samples of the bulked seeds of
the T4
progeny were grown and nicotine levels tested.
In general, Vector 21-41 is similar to Burley 21 LA in all assessed
characteristics,
with the exception of alkaloid content and total reducing sugars (e.g.,
nicotine and nor-
nicotine). Vector 21-41 may be distinguished from the parent Burley 21 LA by
its
substantially reduced content of nicotine, nor-nicotine and total alkaloids.
As shown
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WO 2005/000352 PCT/US2004/016958
below, total alkaloid concentrations in Vector 21-41 are significantly reduced
to
approximately relative to the levels in the parent Burley 21 LA, and nicotine
and nor-
nicotine concentrations show dramatic reductions in Vector 21-41 as compared
with Burley
21 LA. Vector 21-41 also has significantly higher levels of reducing sugars as
compared
with Burley 21 LA.
Field trials of Vector 21-41 T4 progeny were performed at the Central Crops
Research Station (Clayton, NC) and compared to the Burley 21 LA parent. The
design was
three treatments (Vector 21-41, a Burley 21 LA transformed line carrying only
the NtQPTl
promoter [Promoter-Control], and untransformed Burley 21 LA [Wild-type]), 15
replicates,
plants per replicate. The following agronomic traits were measured and
compared: days
from transplant to flowering; height at flowering; leaf number at flowering;
yield; percent
nicotine; percent nor-nicotine; percent total nitrogen; and percent reducing
sugars.
Vector 21-41 was also grown on approximately 5000 acres by greater than 600
farmers in five states (Pennsylvania, Mississippi, Louisiana, Iowa, and
Illinois). The US
Department of Agriculture, Agriculture Marketing Service (USDA-AMS) quantified
nicotine levels (expressed as percent nicotine per dry weight) using the FTC
method of
2,701 samples taken from these farms. Nicotine levels ranged from 0.01% to
0.57%. The
average percent nicotine level for all these samples was 0.09%, with the
median of 0.07%.
Burley tobacco cultivars typically have nicotine levels between 2% and 4% dry
weight
(Tso, T.C., 1972, Physiology and Biochemistfy of Tobacco Plants. Dowden,
Hutchinson,
and Ross, Inc. Stroudsbury).
EXAMPLE 4
Regulation of NtQPTl Gene Expression Using Molecular Decoys
Nucleotide sequence located between -1000 and -600 or -700 by of the NtQPTl
promoter is inserted in tandem arrays into a plant-Agj°obacterium
shuttle vector and
subsequently transformed into tobacco via methods known to one skilled in the
art. Plants
stably transformed with said vector are assessed for the level of expression
of NtQPTl and
for nicotine andlor TSNA content. These experiments demonstrate that tobacco
transformed with molecular decoys that interact with Nic gene products exhibit
a reduced
level of expression of NtQPTI.
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EXAMPLE 5
Tobacco Having Reduced Nicotine andlor TSNA Levels Generated Using
Molecular Decoys
Multiple copies of an approximately 300 or 400 nucleotide long fragment of the
NtQPTl promoter (e.g., including nucleotide sequence located between -1000 and
-600 or -
700 by of the NtQPTl promoter), are affixed to microparticles (e.g., by
precipitation) that are
suitable for the ballistic transformation of a plant cell (e.g., 1 to 5 ~,m
gold spheres). The
microparticles are propelled into tobacco plant cells (e.g., Burley 21 LA)
using any suitable
ballistic cell transformation methodology, so as to produce transformed plant
cells. Plants
are then regenerated from the transformed plant cells. Burley 21 LA is a
variety of Burley
21 with substantially reduced levels of nicotine as compared with Burley 21
(i. e., Burley 21
LA has 8% the nicotine levels of Burley 21, see Legg et al., Can J Genet
Cytol, 13:287-91
(1971); Legg et al., JHered, 60:213-17 (1969))
Transformed cells, tissues, and seedlings are grown on Murashige-Skoog (MS)
medium (with or without the selection compound, e.g., antibiotic, depending on
whether a
selectable marker was used. One-hundred independent transformants of Burley 21
LA (To)
are allowed to self. Progeny of the selfed plants (Tl)~ are germinated.
Nicotine levels of Tl
progeny are measured qualitatively using a micro-assay technique.
Approximately 200 mg
fresh tobacco leaves are collected and ground in 1 ml extraction solution.
(Extraction
solution: 1 ml Acetic acid in 100 ml H20) Homogenate is centrifuged for 5 min
at 14,000
x g and supernatant removed to a clean tube, to which the following reagents
are added:
100 ~L NH40AC (5 81100 ml H2O + 50 ~.L Brij 35); 500 ~,L Cyanogen Bromide
(Sigma
C-6388, 0.5 8/100 ml H20 + 50 ~L Brij 35); 400 p,L Aniline (0.3 ml buffered
Aniline in
100 ml NH40AC + 50 ~,L Brij 35). A nicotine standard stock solution of 10
mg/ml in
extraction solution is prepared and diluted to create a standard series for
calibration.
Absorbance at 460 nm is read and nicotine content of test samples are
determined using the
standard calibration curve.
TI progeny that have less than 10% of the nicotine levels of the Burley 21 LA
parent are allowed to self to produce Ta progeny. Homozygous TZ progeny are
then
identified. Nicotine levels in homozygous and heterozygous TZ progeny are also
qualitatively determined using the micro-assay. Leaf samples of homozygous TZ
progeny
can also be sent to the Southern Research and Testing Laboratory in Wilson, NC
for
quantitative analysis of nicotine levels using Gas ChromatographylFlame
Ionization
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WO 2005/000352 PCT/US2004/016958
Detection (GC/Fm). Homozygous T2 progeny will have nicotine levels that are
substantially reduced as compared to the untransformed tobacco (e.g., ~70
ppm). Because
the nicotine levels in such plants are substantially reduced, the TSNA levels
in these plants
is concomitantly reduced.
These experiments demonstrate that tobacco transformed with molecular decoys
that
interact with Nic gene products exhibit a reduced amount of nicotine and/or
TSNA. Plants
with multiple tandem insertions of the molecular decoy that have reduced
NtQPTI expression
and reduced nicotiile/TSNA levels are used to generate commercially valuable
tobacco
products.
EXAMPLE 6
Low Nicotine and Nitrosamine blended Tobacco
The following example describes several ways to create tobacco products having
specific amounts of nicotine and/or TSNAs through blending. Some blending
approaches
begin with tobacco prepared from varieties that have extremely low amounts of
nicotine
and/or TSNAs., By blending prepared tobacco from a low nicotine/TSNA variety
(e.g.,
undetectable levels of nicotine and/or TSNAs ) with a conventional tobacco
(e.g., Burley,
which has 30,000 parts per million (ppm) nicotine and 8,000 parts per billion
(ppb) TSNA;
Flue-Cured, which has 20,000 ppm nicotine and 300 ppb TSNA; and Oriental,
which has
10,000 ppm nicotine and 100 ppb TSNA), tobacco products having virtually any
desired
amount of nicotine and/or TSNAs can be manufactured. Other approaches blend
only low
nicotine/TSNA tobaccos (e.g., genetically modified Burley, genetically
modified Virginia
flue, and genetically modified Oriental tobaccos that contain reduced amounts
of nicotine
and/or TSNAs). Tobacco products having various amounts of nicotine and/or
TSNAs can
be incorporated into tobacco-use cessation kits and programs to help tobacco
users reduce
or eliminate their dependence on nicotine and reduce the carcinogenic
potential.
By one approach, a step 1 tobacco product is comprised of approximately 25%
low
nicotine/TSNA tobacco and 75% conventional tobacco; a step 2 tobacco product
can be
comprised of approximately 50% low nicotine/TSNA tobacco and 50% conventional
tobacco; a step 3 tobacco product can be comprised of approximately 75% low
nicotine/TSNA tobacco and 25% conventional tobacco; and a step 4 tobacco
product can
be comprised of approximately 100% low nicotine/TSNA tobacco and 0%
conventional
tobacco. A tobacco-use cessation kit can comprise an amount of tobacco product
from
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each of the aforementioned blends to satisfy a consumer for a single month
program. That
is, if the consumer is a one pack per day smoker, for example, a single month
kit would
provide 7 packs from each step, a total of 28 packs of cigarettes. Each
tobacco-use
cessation kit would include a set of instructions that specifically guide the
consumer
through the step-by-step process. Of course, tobacco products having specific
amounts of
nicotine and/or TSNAs would be made available in conveniently sized amounts
(e.g., boxes
of cigars, packs of cigarettes, tins of snuff, and pouches or twists of chew)
so that
consumers could select the amount of nicotine and/or TSNA they individually
desire.
There are many ways to obtain various low nicotine/low TSNA tobacco blends
using the
teachings described herein and the following is intended merely to guide one
of skill in the
art to one possible approach.
To obtain a step 1 tobacco product, which is a 25% low nicotine/TSNA blend,
prepared tobacco from an approximately 0 ppm nicotine/TSNA tobacco can be
mixed with
conventional Burley, Flue-cured, or Oriental in a 25%/75% ratio respectively
to obtain a
Burly tobacco product having 22,500 ppm nicotine and 6,000 ppb TSNA, a Flue-
cured
product having 15,000 ppm nicotine and 225 ppb TSNA, and an Oriental product
having
7,500 ppm nicotine and 75 ppb TSNA. Similarly, to obtain a step 2 product,
which is 50%
low nicotine/TSNA blend, prepared tobacco from an approximately 0 ppm
nicotine/TSNA
tobacco can be mixed with conventional Burley, Flue-cured, or Oriental in a
50%/50%
ratio respectively to obtain a Burly tobacco product having 15,000 ppm
nicotine and 4,000
ppb TSNA, a Flue-cured product having 10,000 ppm nicotine and 150 ppb TSNA,
and an
Oriental product having 5000 ppm nicotine and 50 ppb TSNA. Further, a step 3
product,
which is a 75%/25% low nicotine/TSNA blend, prepared tobacco from an
approximately 0
ppm nicotine/TSNA tobacco can be mixed with conventional Burley, Flue-cured,
or
Oriental in a 75%/25% ratio respectively to obtain a Burly tobacco product
having 7,500
ppm nicotine and 2,000 ppb TSNA, a Flue-cured product having 5,000 ppm
nicotine and
75 ppb TSNA, and an Oriental product having 2,500 ppm nicotine and 25 ppb
TSNA.
It should be appreciated that tobacco products are often a blend of many
different
types of tobaccos, which were grown in many different parts of the world under
various
growing conditions. As a result, the amount of nicotine and TSNAs will differ
from crop to
crop. Nevertheless, by using conventional techniques one can easily determine
an average
amount of nicotine and TSNA per crop used to create a desired blend. By
adjusting the
amount of each type of tobacco that malces up the blend one of skill can
balance the amount
of nicotine and/or TSNA with other considerations such as appearance, flavor,
and
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WO 2005/000352 PCT/US2004/016958
smokability. In this manner, a variety of types of tobacco products having
varying level of
nicotine and/or nitrosamine, as well as, appearance, flavor and smolcability
can be created.
EXAMPLE 7
Low Nicotine and Nitrosamine blended Tobacco
By a preferred method, conventional Virginia flue tobacco was blended with
genetically modified Burley (i.e., Burley containing a significantly reduced
amount of
nicotine and nitrosamine) to yield a blended tobacco that was incorporated
into three levels
of reduced nicotine cigarettes: a step 1 cigarette containing 0.6mg nicotine,
a step 2
cigarette containing 0.3mg nicotine, and a step 3 cigarette containing less
than O.OSmg
nicotine. The amount of total TSNA was found to range between approximately
0.17~,g/g -
0.6~,g/g.
In some cigarettes, approximately, 28% of the blend was Virginia flue tobacco,
approximately 29% of the blend was genetically modified (i.e., reduced
nicotine Burley),
approximately 14% of the blend was Oriental, approximately 17% of the blend
was
expanded flue-cured stem, and approximately 12% was standard commercial
reconstituted
tobacco. The amount of total TSNAs in cigarettes containing this blend was
approximately
1.5 ~.g/g.
EXAMPLE 8
Nicotine Reduction and/or Smoking Cessation Program Containing Low
Nicotine And Nitrosamine Levels
The following example describes a nicotine reduction and/or smoking cessation
program utilizing the low nicotine, low TSNA tobacco products of the present
invention.
The modified tobacco containing very low levels of TSNAs and essentially no
nicotine is
mixed with tobacco having a known amount of nicotine to create specific,
stepwise levels
of nicotine per cigarette. As an example, Virginia flue tobacco was blended
with
genetically modified Burley (i.e., Burley containing a significantly reduced
amount of
nicotine and nitrosamine) to yield a blended tobacco that was incorporated
into three levels
of reduced nicotine cigarettes: a step 1 cigarette containing 0.6mg nicotine,
a step 2
cigarette containing 0.3mg nicotine, and a step 3 cigarette containing less
than O.OSmg
nicotine. The stepwise packs of cigarettes axe clearly marlced as to their
nicotine content,
and the step in the stepwise nicotine reduction program is also clearly marked
on the
CA 02527648 2005-11-29
WO 2005/000352 PCT/US2004/016958
package. Each week, the user purchases packs containing cigarettes having the
next lower
level of nicotine, but limits himself to no more cigarettes per day than
consumed
previously. The user may define his/her own rate of nicotine reduction and/or
smoking
cessation according to individual needs by choosing a) the number of
cigarettes smoked per
day b) the starting nicotine levels c) the change in nicotine level per
cigarette each week,
and d) the final level of nicotine consumed per day. To keep better traclc of
the program,
the individual keeps a daily record of total nicotine intake, as well as the
number of
cigarettes consumed per day. Eventually, the individual will be consuming
tobacco
products with essentially no nicotine. Since the nicotine-free tobacco
products of the final
step are non-addictive, it should then be much easier to quit the use of the
tobacco products
altogether.
EXAMPLE 9
Nicotine Reduction and/or Smoking Cessation Kit Coritainin~ Packs Of
Cigarettes With Low TSNA Levels And Stepwise Reductions In Nicotine Levels
Various nicotine reduction and/or smoking cessation kits are prepared, geared
to
heavy, medium, or light smokers. The kits provide all of the materials needed
to quit
smoking in either a two-week period (fast), a one-month period (medium) or in
a two-
month period (slow), depending on the kit. Each kit contains a set number of
packs of
cigarettes modified according the present invention, containing either step 1
cigarettes
containing 0.6rng nicotine, step 2 cigarettes containing 0.3mg nicotine, and
step 3 cigarettes
containing less than O.OSmg nicotine. For example, 1 pack a day smokers would
receive 7
packs of cigarettes, each pack containing the above amounts of nicotine per
each cigarette.
Several weelcs worth of additional cigarettes containing less than 0.05 mg
nicotine/cigarette
would also be provided in the lcit, to familiarize the consumer with smoking
no nicotine
cigarettes. The kit would also contain a diary for keeping track of daily
nicotine intake,
motivational literature to keep the individual interested in continuing the
cessation
program, health information on the benefits of smoking cessation, and web site
addresses to
find additional axiti-smol~ing information, such as chat groups, meetings,
newsletters, recent
publications, and other pertinent links.
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Although the invention has been described with reference to embodiments and
examples, it should be understood that various modifications can be made
without
departing from the spirit of the invention. Accordingly, the invention is
limited only by the
following claims.
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SEQUENCE LISTING
<110> Vector Tobacco, Ltd.
Conkling, Mark
<120> METHOD OF REDUCING THE HARMFUL EFFECTS
OF ORALLY OR TRANSDERMALLY DELIVERED NICOTINE
<130> VTOB.138VPC
' <150> 60/475,945
<151> 2003-06-04
<160> 1
<170> FastSEQ for Windows Version 4.0
<210> 1
<211> 1399
<212> DNA
<213> Nicotiana tabacum
<400> 1
caaaaactat tttccacaaa attcatttca caaccccccc aaaaaaaaac catgtttaga 60
gctattcctt tcactgctac agtgcatcct tatgcaatta cagctccaag gttggtggtg 120
aaaatgtcag caatagccac caagaataca agagtggagt cattagaggt gaaaccacca 180.
gcacacccaa cttatgattt aaaggaagtt atgaaacttg cactctctga agatgctggg 240
aatttaggag atgtgacttg taaggcgaca attcctcttg atatggaatc cgatgctcat 300
tttctagcaa aggaagacgg gatcatagca ggaattgcac ttgctgagat gatattcgcg 360
gaagttgatc cttcattaaa ggtggagtgg tatgtaaatg atggcgataa agttcataaa 420
ggcttgaaat ttggcaaagt acaaggaaac gcttacaaca ttgttatagc tgagagggtt 480
gttctcaatt ttatgcaaag aatgagtgga atagctacac taactaagga aatggcagat 540
gctgcacacc ctgcttacat cttggagact aggaaaactg ctcctggatt acgtttggtg 600
gataaatggg 'cggtattgat cggtgggggg aagaatcaca gaatgggctt atttgata'tg 660
gtaatgataa aagacaatca catatctgct gctggaggtg tcggcaaagc tctaaaatct 720
gtggatcagt atttggagca aaataaactt caaatagggg ttgaggttga aaccaggaca 780
attgaagaag tacgtgaggt tctagactat gcatctcaaa caaagacttc gttgactagg 840
ataatgctgg acaatatggt tgttccatta tctaacggag atattgatgt atccatgctt 900
aaggaggctg tagaattgat caatgggagg tttgatacgg aggcttcagg aaatgttacc 960
cttgaaacag tacacaagat tggacaaact ggtgttacct acatttctag tggtgccctg 1020
acgcattccg tgaaagcact tgacatttcc ctgaagatcg atacagagct cgcccttgaa 1080
gttggaaggc gtacaaaacg agcatgagcg ccattacttc tgctataggg ttggagtaaa 1140
agcagctgaa tagctgaaag gtgcaaataa gaatcatttt actagttgtc aaacaaaaga 1200
tccttcactg tgtaatcaaa caaaaagatg taaattgctg gaatatctca gatggctctt 1260
ttccaacctt attgcttgag ttggtaattt cattatagct~ttgttttcat gtttcatgga 1320
atttgttaca atgaaaatac ttgatttata agtttggtgt atgtaaaatt ctgtgttact 1380
tcaaatattt tgagatgtt 1399
1/1
