DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group I and SEQ ID NO: 25 in the reply filed on 6/18/2026 is acknowledged.
Applicant is reminded that upon the cancelation of claims to a non-elected invention, the inventorship must be corrected in compliance with 37 CFR 1.48(a) if one or more of the currently named inventors is no longer an inventor of at least one claim remaining in the application. A request to correct inventorship under 37 CFR 1.48(a) must be accompanied by an application data sheet in accordance with 37 CFR 1.76 that identifies each inventor by his or her legal name and by the processing fee required under 37 CFR 1.17(i).
The restriction is made FINAL.
Status of Claims
Claims 1, 3-7 & 22-35 are under examination on the merits.
Specification
The disclosure is objected to because of the following informalities: Table 2 (page 37) provides examples of inducible promoters for expression. However, the table includes SEQ ID NO: 21, which is an amino acid sequence.
Appropriate correction is required.
Claim Objections
Claim 30 is objected to because of the following informalities:
Claim 30 (lines 2-3): benzenoids, flavonoids, phenylpropanoids, and terpenoids do not need capitalization.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
Indefiniteness
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 28 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 28 is drawn to the modified tobacco plant of claim 1, “wherein the polypeptide comprising SEQ ID NO: 25”. It is unclear how to interpret this limitation, whether the polypeptide must comprise SEQ ID NO: 25 or whether the polypeptide, having SEQ ID NO: 25, should have an additional limitation. Because the meaning and scope of the claim is unclear and could have more than one interpretation, the claim is indefinite.
Written Description
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1, 3-7 & 22-35 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claims 1, 3-7 & 22-35 are drawn to a modified tobacco plant comprising a non-natural mutation in an endogenous nucleic acid sequence encoding an amino acid sequence at least 95% identical to SEQ ID NO: 25, a method of preparing a tobacco product from such a plant, or a product of such a plant. The claims therefore all require a modified tobacco plant comprising an endogenous nucleic acid sequence encoding an amino acid sequence at least 95% identical to SEQ ID NO: 25 and producing at least one leaf comprising a reduced amount of at least one metabolite.
Applicant defines a genetic modification as a change in the genetic makeup of a plant, encompassing but not limited to mutagenesis, genome editing, and transformation (paragraph [0070]). Applicant defines "modified" as a plant comprising a genetic alteration introduced for purposes and beyond natural polymorphisms (paragraph [0071]). Applicant defines mutations as not natural polymorphisms, and "non-natural" as a non-spontaneous mutation generated via human intervention that does not correspond to a spontaneous mutation generated without human intervention (paragraph [0073]).
Thus, the claims broadly require a tobacco plant comprising a non-spontaneous mutation generated via human intervention in an endogenous nucleic acid sequence encoding an amino acid sequence at least 95% identical to SEQ ID NO: 25 and producing a leaf comprising a reduced amount of at least one metabolite.
Amino acid sequences with at least 95% identity to the 424 amino acid-long SEQ ID NO: 25 encompasses sequences with 21 amino acid substitutions relative to SEQ ID NO: 25.
The specification describes twenty coding sequences (SEQ ID NOs: 1-20) encoding twenty amino acid sequences (SEQ ID NOs: 21-40) and twenty corresponding genomic sequences (SEQ ID NOs: 41-60) for trichome density and flavor associated genes. Of the protein sequences provided, the most similar to SEQ ID NO: 25 is instant SEQ ID NO: 24, with just 88.3% match. See alignment below. Therefore, the specification does not describe plants comprising nucleic acids encoding amino acids over the full scope of the claims. Applicant describes instant SEQ ID NO: 25 as a cembratrienol synthase 3 (table 11), but the other sequence described as a cembratrienol synthase 3, SEQ ID NO: 40, is even less similar and has only 51.6% match to SEQ ID NO: 25. See second alignment below.
US-19-009-709-24
Sequence 24, US/19009709
Publication No. US20260117236A1
GENERAL INFORMATION
APPLICANT: Altria Client Services LLC (en)
TITLE OF INVENTION: METHODS AND COMPOSITIONS FOR MODULATING TRICHOME DENSITY AND FLAVOR MOLECULE SECRETION (en)
FILE REFERENCE: P35070
CURRENT APPLICATION NUMBER: US/19/009,709
CURRENT FILING DATE: 2025-01-03
NUMBER OF SEQ ID NOS: 90
SEQ ID NO 24
LENGTH: 598
TYPE: PRT
FEATURE:
NAME/KEY: source
LOCATION: 1..598
QUALIFIERS: mol_type = protein
organism = synthetic construct
Query Match 88.3%; Score 1945.5; Length 598;
Best Local Similarity 77.3%;
Matches 384; Conservative 13; Mismatches 27; Indels 73; Gaps 1;
Qy 1 MLVETPDNSTQKLVLIDTIQRLGLAYHFNDEIENSIQNIFYLSQNSEEDDEHNLYVVALR 60
|||||||||||||||||||||||||||||||||||||||| ||||||:|||||||| |||
Db 102 MLVETPDNSTQKLVLIDTIQRLGLAYHFNDEIENSIQNIFNLSQNSEDDDEHNLYVAALR 161
Qy 61 FRLARQQGNYMSSDVFKQFTNHDGKFKENHTNDVRGLLSLYEAAHMSVHDEEILEESLIF 120
|||||||| |||||||||||||||||||||||||:||||||||||| |||||||||:|||
Db 162 FRLARQQGYYMSSDVFKQFTNHDGKFKENHTNDVQGLLSLYEAAHMRVHDEEILEEALIF 221
Qy 121 TTTHLESMIPNLSNSLKVQVTEALSQPIRKTIPRVGARKYIYIYENIEAHNDLLLKFAKL 180
|||||||:||||||||||||||||| |||| ||||||||||:||||| |||||||||||
Db 222 TTTHLESVIPNLSNSLKVQVTEALSHPIRKAIPRVGARKYIHIYENIGTHNDLLLKFAKL 281
Qy 181 DFNMLQKLHRKELNELT------------------------------------------- 197
|||||||||||||||||
Db 282 DFNMLQKLHRKELNELTSWWKDLDRANKFPYAKDRLVEAYFWTVGIYFEPQYSRSRSLVT 341
Qy 198 ------------------------------RWDIGAMDSLPTYLRPIYQGLLDVFNEMEE 227
||| |||| |||||||||||||||||||||
Db 342 KVVKMNSIIDDTYDAYATFDELVLFTDAIQRWDEGAMDLLPTYLRPIYQGLLDVFNEMEE 401
Qy 228 VLAKEGKADRIYYAKKEMKKLVATYFKEAEWLNANYIPKCEEYMKNGLVSCTGMMYGTIS 287
||||||||| ||||||||||: |||||||||||||||||||||||||| || ||| ||
Db 402 VLAKEGKADHIYYAKKEMKKVAEVYFKEAEWLNANYIPKCEEYMKNGLVSSTGPMYGIIS 461
Qy 288 LVVMEEFITKETFEWLANEPLILRASSTICRLMDDIADHEVEQQRGHVASFVECYMKEYG 347
|||||| |||| |||| ||||||||:|||||||||:||||||||||||||||||||||||
Db 462 LVVMEEIITKEAFEWLTNEPLILRAASTICRLMDDMADHEVEQQRGHVASFVECYMKEYG 521
Qy 348 VSKQEAYVEMWKNITNAWKCINKELLRPTAVPMFVLERTLNYTRLVDTFFKDDDGYTNPK 407
|||||||||| | |||||| ||||||||||||||:|||:||::|| ||| ||||||||||
Db 522 VSKQEAYVEMRKKITNAWKDINKELLRPTAVPMFILERSLNFSRLADTFLKDDDGYTNPK 581
Qy 408 SKVKDLITLLFVESIDI 424
||||||| |||||:||
Db 582 SKVKDLIASLFVESVDI 598
US-19-009-709-40
Sequence 40, US/19009709
Publication No. US20260117236A1
GENERAL INFORMATION
APPLICANT: Altria Client Services LLC (en)
TITLE OF INVENTION: METHODS AND COMPOSITIONS FOR MODULATING TRICHOME DENSITY AND FLAVOR MOLECULE SECRETION (en)
FILE REFERENCE: P35070
CURRENT APPLICATION NUMBER: US/19/009,709
CURRENT FILING DATE: 2025-01-03
NUMBER OF SEQ ID NOS: 90
SEQ ID NO 40
LENGTH: 333
TYPE: PRT
FEATURE:
NAME/KEY: source
LOCATION: 1..333
QUALIFIERS: mol_type = protein
organism = synthetic construct
Query Match 51.6%; Score 1138; Length 333;
Best Local Similarity 66.7%;
Matches 236; Conservative 22; Mismatches 38; Indels 58; Gaps 5;
Qy 1 MLVETPDNSTQKLVLIDTIQRLGLAYHFNDEIENSIQNIFYLSQNSEEDDEHNLYVVALR 60
||||| ||||||||||||||||||||||| |||||||||||||||||::||||||| |||
Db 1 MLVETLDNSTQKLVLIDTIQRLGLAYHFNYEIENSIQNIFYLSQNSEDNDEHNLYVAALR 60
Qy 61 FRLARQQGNYMSSDVFKQFTNHDGKFKENHTNDVRGLLSLYEAAHMSVHDEEILEESLIF 120
||||||||||||||||||||||| ||||||||||:|||||||||| |||||||||:|||
Db 61 FRLARQQGNYMSSDVFKQFTNHDRKFKENHTNDVQGLLSLYEAAHTRVHDEEILEEALIF 120
Qy 121 TTTHLESMIPNLSNSLKVQVTEALSQPIRKTIPRVGARKYIYIYENIEAHNDLLLKFAKL 180
| |||||:||||||:||: : | | || | | : : ||| :
Db 121 TPTHLESVIPNLSNALKL-------------VERFGLRKQISICKG-QISRSLLLDGGNI 166
Qy 181 -------DFNMLQKLHRKE-------------------LNELTRWDIGAMDSLPTYLRPI 214
:|: |: : : : ||| |||||||||||||
Db 167 FEPQYGRSRSMITKVVKMNSIIDDTYDAYATFDELVLFTDAIQRWDEGAMDSLPTYLRPI 226
Qy 215 YQGLLDVFNEMEEVLAKEGKADRIYYAKKEMKKLVATYFKEAEWLNANYIPKCEEYMKNG 274
|||||||||||||||||| |:|||||||||||||||||||||
Db 227 YQGLLDVFNEMEEVLAKE------------------AYYKEAEWLNANYIPKCEEYMKNG 268
Qy 275 LVSCTGMMYGTISLVVMEEFITKETFEWLANEPLILRASSTICRLMDDIADHEV 328
:|: || ||| |||||||| |||| |||||||||||||:|||||||||:|||||
Db 269 VVTSTGTMYGIISLVVMEEIITKEAFEWLANEPLILRAASTICRLMDDMADHEV 322
The art likewise does not describe nucleic acid sequences encoding polypeptides at least 95% identical to SEQ ID NO: 25 over the full scope claimed. UniProtKB entry A0A1S3Z3X1_TOBAC (available 4/12/2017) describes a germacrene C synthase- or sesquiterpene synthase 12-like protein with 97.9% identity to instant SEQ ID NO: 25, but this sequence has no mismatches and a single deletion relative to instant SEQ ID NO: 25. See first alignment below. UniProt entry A0A1S3ZM31_TOBAC (available 4/12/2017), on the other hand, describes a germacrene C synthase-like protein with no indels and 28 mismatches. This sequence has only 90.2% identity to instant SEQ ID NO: 25 (see second alignment below). Plants comprising amino acid sequences within the range of 95% identity of SEQ ID NO: 25 are not described over the scope claimed, and in particular, plants comprising an endogenous nucleic acid sequence encoding such amino acid sequences are not described.
A0A1S3Z3X1_TOBAC
ID A0A1S3Z3X1_TOBAC Unreviewed; 597 AA.
AC A0A1S3Z3X1;
DT 12-APR-2017, integrated into UniProtKB/TrEMBL.
DT 12-APR-2017, sequence version 1.
DT 28-JAN-2026, entry version 39.
DE SubName: Full=Germacrene C synthase-like {ECO:0000313|RefSeq:XP_016458872.1};
DE SubName: Full=Sesquiterpene synthase 12-like {ECO:0000313|RefSeq:XP_016458872.1};
GN Name=LOC107782504 {ECO:0000313|RefSeq:XP_016458872.1};
OS Nicotiana tabacum (Common tobacco).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC asterids; lamiids; Solanales; Solanaceae; Nicotianoideae; Nicotianeae;
OC Nicotiana.
OX NCBI_TaxID=4097 {ECO:0000313|Proteomes:UP000790787, ECO:0000313|RefSeq:XP_016458872.1};
RN [1] {ECO:0000313|Proteomes:UP000790787}
RP NUCLEOTIDE SEQUENCE [LARGE SCALE GENOMIC DNA].
RX PubMed=24807620; DOI=10.1038/ncomms4833;
RA Sierro N., Battey J.N., Ouadi S., Bakaher N., Bovet L., Willig A.,
RA Goepfert S., Peitsch M.C., Ivanov N.V.;
RT "The tobacco genome sequence and its comparison with those of tomato and
RT potato.";
RL Nat. Commun. 5:3833-3833(2014).
RN [2] {ECO:0000313|RefSeq:XP_016458872.1}
RP IDENTIFICATION.
RC TISSUE=Leaf {ECO:0000313|RefSeq:XP_016458872.1};
RG RefSeq;
RL Submitted (AUG-2025) to UniProtKB.
CC -!- COFACTOR:
CC Name=Mg(2+); Xref=ChEBI:CHEBI:18420;
CC Evidence={ECO:0000256|ARBA:ARBA00001946};
CC -!- PATHWAY: Secondary metabolite biosynthesis; terpenoid biosynthesis.
CC {ECO:0000256|ARBA:ARBA00004721}.
CC -!- SUBUNIT: Monomer. {ECO:0000256|ARBA:ARBA00011245}.
CC ---------------------------------------------------------------------------
CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms
CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
CC ---------------------------------------------------------------------------
DR RefSeq; XP_016458872.1; XM_016603386.1.
DR RefSeq; XP_016458872.1; XM_016603386.2.
DR AlphaFoldDB; A0A1S3Z3X1; -.
DR SMR; A0A1S3Z3X1; -.
DR STRING; 4097.A0A1S3Z3X1; -.
DR PaxDb; 4097-A0A1S3Z3X1; -.
DR GeneID; 107782504; -.
DR KEGG; nta:107782504; -.
DR OMA; RDNLEYP; -.
DR OrthoDB; 1877784at2759; -.
DR UniPathway; UPA00213; -.
DR Proteomes; UP000790787; Chromosome 14.
DR GO; GO:0000287; F:magnesium ion binding; IEA:InterPro.
DR GO; GO:0010333; F:terpene synthase activity; IBA:GO_Central.
DR GO; GO:0016102; P:diterpenoid biosynthetic process; IEA:InterPro.
DR GO; GO:0046246; P:terpene biosynthetic process; IBA:GO_Central.
DR CDD; cd00684; Terpene_cyclase_plant_C1; 1.
DR FunFam; 1.10.600.10:FF:000007; Isoprene synthase, chloroplastic; 1.
DR FunFam; 1.50.10.130:FF:000001; Isoprene synthase, chloroplastic; 1.
DR Gene3D; 1.10.600.10; Farnesyl Diphosphate Synthase; 1.
DR Gene3D; 1.50.10.130; Terpene synthase, N-terminal domain; 1.
DR InterPro; IPR008949; Isoprenoid_synthase_dom_sf.
DR InterPro; IPR034741; Terpene_cyclase-like_1_C.
DR InterPro; IPR044814; Terpene_cyclase_plant_C1.
DR InterPro; IPR001906; Terpene_synth_N.
DR InterPro; IPR036965; Terpene_synth_N_sf.
DR InterPro; IPR050148; Terpene_synthase-like.
DR InterPro; IPR005630; Terpene_synthase_metal-bd.
DR InterPro; IPR008930; Terpenoid_cyclase/PrenylTrfase.
DR PANTHER; PTHR31225; OS04G0344100 PROTEIN-RELATED; 1.
DR PANTHER; PTHR31225:SF225; SESQUITERPENE SYNTHASE 12; 1.
DR Pfam; PF01397; Terpene_synth; 1.
DR Pfam; PF03936; Terpene_synth_C; 1.
DR SFLD; SFLDS00005; Isoprenoid_Synthase_Type_I; 1.
DR SFLD; SFLDG01019; Terpene_Cyclase_Like_1_C_Termi; 1.
DR SUPFAM; SSF48239; Terpenoid cyclases/Protein prenyltransferases; 1.
DR SUPFAM; SSF48576; Terpenoid synthases; 1.
PE 4: Predicted;
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723};
KW Reference proteome {ECO:0000313|Proteomes:UP000790787}.
FT DOMAIN 68..244
FT /note="Terpene synthase N-terminal"
FT /evidence="ECO:0000259|Pfam:PF01397"
FT DOMAIN 301..539
FT /note="Terpene synthase metal-binding"
FT /evidence="ECO:0000259|Pfam:PF03936"
SQ SEQUENCE 597 AA; 70075 MW; 0019F5537CB73712 CRC64;
Query Match 97.9%; Score 2157.5; Length 597;
Best Local Similarity 85.3%;
Matches 424; Conservative 0; Mismatches 0; Indels 73; Gaps 1;
Qy 1 MLVETPDNSTQKLVLIDTIQRLGLAYHFNDEIENSIQNIFYLSQNSEEDDEHNLYVVALR 60
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 101 MLVETPDNSTQKLVLIDTIQRLGLAYHFNDEIENSIQNIFYLSQNSEEDDEHNLYVVALR 160
Qy 61 FRLARQQGNYMSSDVFKQFTNHDGKFKENHTNDVRGLLSLYEAAHMSVHDEEILEESLIF 120
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 161 FRLARQQGNYMSSDVFKQFTNHDGKFKENHTNDVRGLLSLYEAAHMSVHDEEILEESLIF 220
Qy 121 TTTHLESMIPNLSNSLKVQVTEALSQPIRKTIPRVGARKYIYIYENIEAHNDLLLKFAKL 180
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 221 TTTHLESMIPNLSNSLKVQVTEALSQPIRKTIPRVGARKYIYIYENIEAHNDLLLKFAKL 280
Qy 181 DFNMLQKLHRKELNELT------------------------------------------- 197
|||||||||||||||||
Db 281 DFNMLQKLHRKELNELTSWWKDLDFANKFPYAKDRLVEAYFWMVGIYFEPQYSRSRRMIT 340
Qy 198 ------------------------------RWDIGAMDSLPTYLRPIYQGLLDVFNEMEE 227
||||||||||||||||||||||||||||||
Db 341 KVVKTNSIIDDTYDAYATFDELVLFTDAIQRWDIGAMDSLPTYLRPIYQGLLDVFNEMEE 400
Qy 228 VLAKEGKADRIYYAKKEMKKLVATYFKEAEWLNANYIPKCEEYMKNGLVSCTGMMYGTIS 287
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 401 VLAKEGKADRIYYAKKEMKKLVATYFKEAEWLNANYIPKCEEYMKNGLVSCTGMMYGTIS 460
Qy 288 LVVMEEFITKETFEWLANEPLILRASSTICRLMDDIADHEVEQQRGHVASFVECYMKEYG 347
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 461 LVVMEEFITKETFEWLANEPLILRASSTICRLMDDIADHEVEQQRGHVASFVECYMKEYG 520
Qy 348 VSKQEAYVEMWKNITNAWKCINKELLRPTAVPMFVLERTLNYTRLVDTFFKDDDGYTNPK 407
||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Db 521 VSKQEAYVEMWKNITNAWKCINKELLRPTAVPMFVLERTLNYTRLVDTFFKDDDGYTNPK 580
Qy 408 SKVKDLITLLFVESIDI 424
|||||||||||||||||
Db 581 SKVKDLITLLFVESIDI 597
A0A1S3ZM31_TOBAC
ID A0A1S3ZM31_TOBAC Unreviewed; 525 AA.
AC A0A1S3ZM31;
DT 12-APR-2017, integrated into UniProtKB/TrEMBL.
DT 12-APR-2017, sequence version 1.
DT 28-JAN-2026, entry version 36.
DE SubName: Full=Germacrene C synthase-like isoform X2 {ECO:0000313|RefSeq:XP_016465323.1};
GN Name=LOC107788173 {ECO:0000313|RefSeq:XP_016465323.1};
GN Synonyms=cyclase {ECO:0000313|RefSeq:XP_016465323.1};
OS Nicotiana tabacum (Common tobacco).
OC Eukaryota; Viridiplantae; Streptophyta; Embryophyta; Tracheophyta;
OC Spermatophyta; Magnoliopsida; eudicotyledons; Gunneridae; Pentapetalae;
OC asterids; lamiids; Solanales; Solanaceae; Nicotianoideae; Nicotianeae;
OC Nicotiana.
OX NCBI_TaxID=4097 {ECO:0000313|RefSeq:XP_016465323.1};
RN [1] {ECO:0000313|RefSeq:XP_016465323.1}
RP IDENTIFICATION.
RG RefSeq;
RL Submitted (AUG-2025) to UniProtKB.
CC ---------------------------------------------------------------------------
CC Copyrighted by the UniProt Consortium, see https://www.uniprot.org/terms
CC Distributed under the Creative Commons Attribution (CC BY 4.0) License
CC ---------------------------------------------------------------------------
DR RefSeq; XP_016465323.1; XM_016609837.1.
DR AlphaFoldDB; A0A1S3ZM31; -.
DR OrthoDB; 1877784at2759; -.
DR UniPathway; UPA00213; -.
DR GO; GO:0000287; F:magnesium ion binding; IEA:InterPro.
DR GO; GO:0010333; F:terpene synthase activity; IEA:InterPro.
DR GO; GO:0016102; P:diterpenoid biosynthetic process; IEA:InterPro.
DR CDD; cd00684; Terpene_cyclase_plant_C1; 1.
DR FunFam; 1.50.10.130:FF:000001; Isoprene synthase, chloroplastic; 1.
DR Gene3D; 1.10.600.10; Farnesyl Diphosphate Synthase; 1.
DR Gene3D; 1.50.10.130; Terpene synthase, N-terminal domain; 1.
DR InterPro; IPR008949; Isoprenoid_synthase_dom_sf.
DR InterPro; IPR044814; Terpene_cyclase_plant_C1.
DR InterPro; IPR001906; Terpene_synth_N.
DR InterPro; IPR036965; Terpene_synth_N_sf.
DR InterPro; IPR050148; Terpene_synthase-like.
DR InterPro; IPR005630; Terpene_synthase_metal-bd.
DR InterPro; IPR008930; Terpenoid_cyclase/PrenylTrfase.
DR PANTHER; PTHR31225; OS04G0344100 PROTEIN-RELATED; 1.
DR PANTHER; PTHR31225:SF225; SESQUITERPENE SYNTHASE 12; 1.
DR Pfam; PF01397; Terpene_synth; 1.
DR Pfam; PF03936; Terpene_synth_C; 1.
DR SUPFAM; SSF48239; Terpenoid cyclases/Protein prenyltransferases; 1.
DR SUPFAM; SSF48576; Terpenoid synthases; 1.
PE 4: Predicted;
KW Metal-binding {ECO:0000256|ARBA:ARBA00022723}.
FT DOMAIN 69..245
FT /note="Terpene synthase N-terminal"
FT /evidence="ECO:0000259|Pfam:PF01397"
FT DOMAIN 296..468
FT /note="Terpene synthase metal-binding"
FT /evidence="ECO:0000259|Pfam:PF03936"
SQ SEQUENCE 525 AA; 61027 MW; DFD296F39680A01F CRC64;
Query Match 90.2%; Score 1988; Length 525;
Best Local Similarity 90.3%;
Matches 383; Conservative 13; Mismatches 28; Indels 0; Gaps 0;
Qy 1 MLVETPDNSTQKLVLIDTIQRLGLAYHFNDEIENSIQNIFYLSQNSEEDDEHNLYVVALR 60
|||||||||||||||||||||||||||||||||||||||| ||||||:|||||||| |||
Db 102 MLVETPDNSTQKLVLIDTIQRLGLAYHFNDEIENSIQNIFNLSQNSEDDDEHNLYVAALR 161
Qy 61 FRLARQQGNYMSSDVFKQFTNHDGKFKENHTNDVRGLLSLYEAAHMSVHDEEILEESLIF 120
|||||||| |||||||||||||||||||||||||:||||||||||| |||||||||:|||
Db 162 FRLARQQGYYMSSDVFKQFTNHDGKFKENHTNDVQGLLSLYEAAHMRVHDEEILEEALIF 221
Qy 121 TTTHLESMIPNLSNSLKVQVTEALSQPIRKTIPRVGARKYIYIYENIEAHNDLLLKFAKL 180
|||||||:||||||||||||||||| |||| ||||||||||:||||| |||||||||||
Db 222 TTTHLESVIPNLSNSLKVQVTEALSHPIRKAIPRVGARKYIHIYENIGTHNDLLLKFAKL 281
Qy 181 DFNMLQKLHRKELNELTRWDIGAMDSLPTYLRPIYQGLLDVFNEMEEVLAKEGKADRIYY 240
|||||||||||||||||||| |||| |||||||||||||||||||||||||||||| |||
Db 282 DFNMLQKLHRKELNELTRWDEGAMDLLPTYLRPIYQGLLDVFNEMEEVLAKEGKADHIYY 341
Qy 241 AKKEMKKLVATYFKEAEWLNANYIPKCEEYMKNGLVSCTGMMYGTISLVVMEEFITKETF 300
|||||||: |||||||||||||||||||||||||| || ||| |||||||| |||| |
Db 342 AKKEMKKVAEVYFKEAEWLNANYIPKCEEYMKNGLVSSTGPMYGIISLVVMEEIITKEAF 401
Qy 301 EWLANEPLILRASSTICRLMDDIADHEVEQQRGHVASFVECYMKEYGVSKQEAYVEMWKN 360
||| ||||||||:|||||||||:||||||||||||||||||||||||||||| |||| |
Db 402 EWLTNEPLILRAASTICRLMDDMADHEVEQQRGHVASFVECYMKEYGVSKQETYVEMRKK 461
Qy 361 ITNAWKCINKELLRPTAVPMFVLERTLNYTRLVDTFFKDDDGYTNPKSKVKDLITLLFVE 420
|||||| ||||||||||||||:|||:||::|| ||| ||||||||||||||||| ||||
Db 462 ITNAWKDINKELLRPTAVPMFILERSLNFSRLADTFLKDDDGYTNPKSKVKDLIASLFVE 521
Qy 421 SIDI 424
|:||
Db 522 SVDI 525
Furthermore, a mutation generated via human intervention reads on a product-by-process claim. "[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985) (citations omitted). The claims do not require, and the specification does not describe, any particular characteristics of the encompassed mutation which would distinguish a “non-natural” mutation from one arising by natural means. Any mutation in an encompassed nucleic acid sequence would read on the limitation.
With respect to “non-natural mutation”, examples of inducing a mutation described by the specification include ethyl methanesulfonate application, irradiation, or use of a nuclease, base editor, prime editor, or transposon (paragraph [0077-0083]). Applicant describes that mutations comprise insertions, deletions, and substitutions (paragraph [0085]). The specification describes generating mutations in the genes of table 10 by editing the genes with CRISPR protein (paragraph [0699-00700]).
The specification describes that a tobacco plant can be any plant from the Nicotiana genus (paragraph [00174]), which encompasses many species. Even within Nicotiana tabacum, the claims encompass many varieties (paragraphs [00182-00196] & tables 3-9).
The specification describes that genome modification can provide a lower level of total alkaloid or nicotine by targeting NIC1, NIC2, QPT, PMT, ADC, AO or ODC (paragraph [00214]) or comprising a mutation in an ERF gene of a Nic locus (paragraphs [00220-00221]) or a modification in a locus encoding aspartate oxidase, agmatine deiminase, arginase, diamine oxidase, arginine decarboxylase, methylputrescine oxidase, NADH dehydrogenase, ornithine decarboxylase, phosphoribosylanthranilate isomerase, putrescine N-methyltransferase, quinolate phosphoribosyl transferase, and S-adenosyl-methionine synthetase, A622, NBB1, BBL, MYC2, ethylene response factor nicotine uptake permease, and MATE transporter (paragraph [00222]).
The specification describes screening known and suspected secretor and non-secretor tobacco lines (paragraph [00679], table 10). RNA sequencing was performed on the tobacco to uncover 2775 or 2501 differentially expressed genes (paragraphs [00683-00685]). The top 20 genes were selected based on highest change in expression (table 11). The specification describes transforming tobacco leaf discs with an ALCS1 expression vector comprising recombinant DNA constructs (paragraphs [00689-00692]). The specification describes transgenic tobacco plants overexpressing candidate genes demonstrated increased and decreased amounts of metabolites (paragraph [0246], table 14).
However, beyond listing SEQ ID NO: 25 as cembratrienol synthase 3 (table 11), Applicant has not described the function of SEQ ID NO: 25 nor any structural features required for its activity. Applicant has not described which metabolite(s) may be reduced in a plant comprising the non-natural mutation. Therefore, Applicant has not described specific mutations to SEQ ID NO: 25 that would lead to reduced amount of a metabolite in a leaf over the full scope of the claims.
Given the breadth of the genus claimed, the lack of structure provided, the lack of examples in the specification, and the lack of description in the prior art, one of ordinary skill in the art would not recognize that Applicant was in possession of tobacco plants comprising endogenous nucleic acid sequences encoding amino acid sequences at least 95% identical to SEQ ID NO: 25 over the full scope of the claims. In addition, one of ordinary skill in the art would not readily recognize which nucleic acid sequences comprising mutations would result in a reduced amount of at least one metabolite in a leaf.
Applicant has not, in fact, described plants comprising mutations in endogenous nucleic acids that encode an amino acid sequence of 95% over the full scope of the claims, and the specification fails to provide an adequate written description of the claimed invention. Therefore, given the lack of written description in the specification with regard to the structural and functional characteristics of the claimed compositions, Applicant does not appear to have been in possession of the claimed genus at the time this application was filed.
Scope of Enablement
Claims 1, 3-7 & 22-35 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for a plant comprising a mutation in a nucleic acid sequence encoding an amino acid sequence of SEQ ID NO: 25, does not reasonably provide enablement for plants comprising any mutation in any nucleic acid sequence encoding an amino acid sequence of at least 95% identity to SEQ ID NO: 25 and comprising a reduced amount of at least one metabolite . The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Claims 1, 3-7 & 22-35 broadly require a modified tobacco plant comprising a non-natural mutation in an endogenous nucleic acid sequence encoding an amino acid sequence at least 95% identical to SEQ ID NO: 25 and producing a leaf comprising a reduced amount of at least one metabolite.
Amino acid sequences with at least 95% identity to the 424 amino acid-long SEQ ID NO: 25 encompasses sequences with 21 amino acid substitutions relative to SEQ ID NO: 25.
The specification teaches twenty coding sequences (SEQ ID NOs: 1-20) encoding twenty amino acid sequences (SEQ ID NOs: 21-40) and twenty corresponding genomic sequences (SEQ ID NOs: 41-60) for trichome density and flavor associated genes. Of the protein sequences provided, the most similar to SEQ ID NO: 25 is instant SEQ ID NO: 24, with just 88.3% match. See alignment above. The next most similar sequence is SEQ ID NO: 40, with only 51.6% match to SEQ ID NO: 25. Alignment provided above. Therefore, the specification does not teach plants comprising nucleic acids encoding amino acids over the full scope of the claims.
The specification teaches that SEQ ID NO: 25 is a cembratrienol synthase 3, as is SEQ ID NO: 40 (table 11). The specification teaches metabolites that are higher in secretor vs non-secretor plants (table 15) and that the gene encoding SEQ ID NO: 25, labeled g58563, is expressed more in secretor than non-secretor lines (table 12). However, the expression of SEQ ID NO: 25 is not linked to any particular metabolites. The specification does not teach the structural features of SEQ ID NO: 25 that might be responsible for the production of a metabolite in a leaf. The specification does not teach working examples wherein the endogenous gene encoding SEQ ID NO: 25 is mutated leading to the reduction of a metabolite.
Cembratrienol synthase (CBTS) 3 is known in the art. Dluge et al (2018). BMC Genomics. 19: 484 (published 6/20/2018, hereafter Dluge) teaches that CBTS cyclizes geranylgeranyl diphosphate into α- and β-CBT-diols (page 2, left column, paragraph 4). Dluge teaches that isoforms of CBTS, alternative splicing variants, exist (page 4, left column, paragraph 2; figure 2).
Isoforms of a protein may comprise variation in amino acid sequence but do not provide examples of amino acid substitutions. The art does not teach nucleic acid sequences encoding polypeptides at least 95% identical to SEQ ID NO: 25 over the full scope claimed, and the most similar sequences to instant SEQ ID NO: 25 known in the art are not annotated as CBTS3.
UniProtKB entry A0A1S3Z3X1_TOBAC (available 4/12/2017) teaches a protein annotated as a germacrene C synthase- or sesquiterpene synthase 12-like protein with 97.9% identity to instant SEQ ID NO: 25, but this sequence has no mismatches and a single deletion relative to instant SEQ ID NO: 25. UniProtKB entry A0A1S3ZM31_TOBAC (available 4/12/2017), on the other hand, teaches a germacrene C synthase-like protein with no indels and 28 mismatches. This sequence has only 90.2% identity to instant SEQ ID NO: 25 (see both alignments above). Examples of plants comprising amino acid sequences within the range of 95% identity of SEQ ID NO: 25 are not known over the scope claimed, and in particular, plants comprising an endogenous nucleic acid sequence encoding such amino acid sequences are not known in the art. Therefore, the art does not teach amino acids over the full scope of the claims and which, if any, would be functional to produce a metabolite when a mutation is not present.
Because synthases involved in biosynthesis of metabolites are highly variable and because Applicant has not described the necessary features of amino acids with 95% identity to SEQ ID NO: 25, such that a mutation to the encoding sequence would lead to the reduction in a metabolite, one of ordinary skill in the art would require undue trial and error experimentation to discover which amino acid sequences over the scope of the claims are found endogenously in plants and which have a biological activity such that a mutation in the encoding gene would lead to a reduced amount of a metabolite.
Thus, the instantly claimed invention is not enabled over the full scope of any tobacco plant comprising any mutation in any nucleic acid sequence encoding an amino acid sequence at least 95% identical to SEQ ID NO: 25.
Conclusion
No claims are allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure: Ennajdaoui et al (2010). Plant Mol Biol. 73:673–685 (published 5/21/2010, hereafter Ennajdaoui) and GenBank reference sequence ADI87447.1 (available 6/23/2010).
Ennajdaoui teaches that secretory glandular trichomes produce large amounts of cembratrien-diols and are found in Nicotiana tabacum varieties such as Burley or Virginia (page 674, left column, paragraph 2). Ennajdaoui teaches that cembratrienol synthases (CBTS) are believed to catalyze the first step in the production of diterpenoid biosynthesis, leading to cembratrien-ols (page 674, right column, paragraph 2; figure 1). Ennajdaoui teaches that CBTS proteins are most closely related to the tomato sesquiterpene synthases (page 677, left column, paragraph 2).
Ennajdaoui suggests that tobacco lines devoid of endogenous diterpenoids could help identify novel terpenoids and also could increase the pool of geranylgeranyl diphosphate in the plants (page 675, left column, paragraph 2). Ennajdaoui teaches gene silencing of the CBTS genes in Nicotiana sylvestris using an intron hairpin construct transformed into the plant via Agrobacterium-mediated transformation (page 677, right column, paragraph 2-page 678, right column, paragraph 1). CBTS silenced lines had higher transformation efficiency and frequency when retransformed with a second T-DNA (page 678, right column, paragraph 1) and had reduced levels of cembranoids measured in leaf exudate (figure 5).
GenBank reference sequence ADI87447.1 provides evidence that the N. sylvestris cembratrienol synthase 2b sequence taught by Ennajdaoui comprises a sequence with just 87% identity to instant SEQ ID NO: 25, with a gap relative to instant SEQ ID NO: 25. See BLAST alignment statistics and MUSCLE alignment below.
Max Score
Total Score
Query Cover
E value
Per. Ident
Acc. Len
NsCBTS2b
428
810
100%
8e-150
87.01%
598
25 ------------------------------------------------------------
NsCBTS2b MSQSISPLMFSHFAKFQSNIWRCNTSQLRVIHSSYASFGGRRKERVRRMNRAMDLSSSSR
25 -----------------------------------------MLVETPDNSTQKLVLIDTI
NsCBTS2b HLADFPSTIWGDHFLSYNSEITEITTQEKNEHEMLKEIVRKMLVETPDNSTQKLVLIDTI
*******************
25 QRLGLAYHFNDEIENSIQNIFYLSQNSEEDDEHNLYVVALRFRLARQQGNYMSSDVFKQF
NsCBTS2b QRLGLAYHFNDEIENSIQNIFNLSQNSEDDDEHNLYVAALRFRLARQQGYYMSSDVFKQF
********************* ******:********.*********** **********
25 TNHDGKFKENHTNDVRGLLSLYEAAHMSVHDEEILEESLIFTTTHLESMIPNLSNSLKVQ
NsCBTS2b TNHDGKFKENHTNDVQGLLSLYEAAHMRVHDEEILEEALIFTTTHLESVIPNLSNSLKVQ
***************.*********** *********:**********:***********
25 VTEALSQPIRKTIPRVGARKYIYIYENIEAHNDLLLKFAKLDFNMLQKLHRKELNELT--
NsCBTS2b VTEALSHPIRKAIPRVGARKYIHIYENIGTHNDLLLKFAKLDFNMLQKLHRKELNELTSW
******:****:**********:***** :****************************
25 ------------------------------------------------------------
NsCBTS2b WKDLDRANKFPYAKDRLVEAYFWTVGIYFEPQYSRSRSLVTKVVKMNSIIDDTYDAYATF
25 -----------RWDIGAMDSLPTYLRPIYQGLLDVFNEMEEVLAKEGKADRIYYAKKEMK
NsCBTS2b DELVLFTDAIQRWDEDAMDLLPTYLRPIYQGLLDVFNEMEEVLAKEGKADHIYYAKKEMK
*** .*** ******************************.*********
25 KLVATYFKEAEWLNANYIPKCEEYMKNGLVSCTGMMYGTISLVVMEEFITKETFEWLANE
NsCBTS2b KVAAVYFKEAEWLNANYIPKCEEYMKNGLVSSTGPMYGIISLVVMEEIITKEAFEWLANE
*:.*.**************************.** *** ********:****:*******
25 PLILRASSTICRLMDDIADHEVEQQRGHVASFVECYMKEYGVSKQEAYVEMWKNITNAWK
NsCBTS2b PLILRAASTICRLMDDMADHEVEQQRGHVASFVECYMKEYGVSKQEAYVEMRKKITNAWK
******:*********:**********************************.*:******
25 CINKELLRPTAVPMFVLERTLNYTRLVDTFFKDDDGYTNPKSKVKDLITLLFVESIDI
NsCBTS2b DINKELLRPTAVPMFILERSLNFSRLADTFLKDDDGYTNPKSKVKDLIASLFVESVDI
**************:***:**::**.***:*****************: *****:**
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Victoria L DeLeo whose telephone number is (703)756-5998. The examiner can normally be reached M-F 8:00am-4pm EDT.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Bratislav Stankovic can be reached at (571) 270-0305. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/VICTORIA L DELEO/Examiner, Art Unit 1662
/Anne Kubelik/Primary Examiner, Art Unit 1663
Prosecution Insights