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 .
Claim Status
Claims 1-3, 5-7 and 13-27 are pending.
Claims 4 and 8-12 are cancelled by the Applicant.
Claims 24-27 are newly added.
Claims 1-3, 5-7 and 13-27 are being examined.
All previous objections and rejections not set forth below have been withdrawn in view of applicant’s amendments to the claims.
The claim amendments by the Applicant by adding new issues, which was not present in the claims before, necessitated new grounds of rejections and new prior art reference(s), as discussed below.
Acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy of the foreign priority application GB application No. 2109586.4 filed on 02 Jul 2021 was filed on December 28, 2023.
Claim Rejections - 35 USC § 112(b), § 102 and Double Patenting
Response to Applicants’ arguments: Amendments made to the claims and/or the arguments filed in Applicant’s response submitted on 01/07/2026 overcame the rejections of record.
Claim Rejections - 35 USC § 103
Claims 1-3, 5-7, 13-20 and 22-27 are rejected under 35 U.S.C. 103 as being unpatentable over Malabarba et al. (New Strategies to Overcome Present CRISPR/Cas9 Limitations in Apple and Pear: Efficient Dechimerization and Base Editing, 2021, Int. J. Mol. Sci., 22:319; first published on 30 Dec. 2020) in view of Ntui et al. (Robust CRISPR/Cas9 mediated genome editing tool for banana and plantain (Musa spp.), 2020, Curent Plant Biology, 21:100128) and Tripathi et al. (Efficient regeneration and transformation of plantain cv. “Gonja manjaya” (Musa spp. AAB) using embryogenic cell suspensions, 2012, In Vitro Cell. Dev. Biol., Plant, 48:216-224) and
Claims 1 and 24 are drawn to a method of generating at least one embryo or a plant from an embryogenic cell suspension comprising at least one mutation in at least one endogenous acetolactate synthase (ALS) gene providing resistance to an ALS inhibitor.
Malabarba et al. describes a method using the CRISPR-Cas9 based base editing technique using a Cas9 nickase and cytosine base editor (CBE), which introduces at least one desired C- to- T mutation, to successfully edit target gene(s) that include at least one endogenous acetolactate synthase (ALS) gene resulting in ALS inhibitor (chlorsulfuron), resistant plants (abstract; page 3, para 1, line 7-8). Malabarba et al. also teaches successful use of CRISPR/Cas based gene editing in banana (page 2, para 1, line 6-10). Malabarba et al. describes a nCas9 (nickase) fused to a cystine deaminase base editor and gRNAs to edit the endogenous ALS genes (page 6, para 1, line 1-16), as recited in claim 3. It is known in the art that the gene editing function of sequence-specific Cas9 endonuclease comprises homology-directed repair (HDR) in conjunction with a donor template, which is often an exogenous DNA encoding the gRNA sequence1 comprising the desired mutation for the target gene (i.e. ALS gene, in this case), as recited in claims 5-6. Malabarba et al. teaches Agrobacterium-mediated transformation (page 6, para 1, line 2-3), as recited in claim 7.
Malabarba et al. describes the nCas9, cytidine deaminase, and gRNAs (reads on to “agents”) operable to introduce the at least one desired (C- to T-) mutation in the target ALS sequence encoded by the vector, pDen-Cas9_PmCDA1_UGI developed using the Gateway vector cloning system (Fig. 5) (reads on to “nucleic acid construct”) which also encodes a selectable marker (NPTII gene), as recited in claim 15. The method (base editing using CRISPR-Cas technique) described by Malabarba et al. does not generate any genome-edited plant comprising heterologous DNA, as recited in claim 20.
Malabarba et al. describes that herbicides from the sulfonylurea class comprising chlorsulfuron are ALS inhibitors (page 3, para 1, line 7-8), as recited in claims 18 and 27. Malabarba et al. describes the transgenic plants (with mutant ALS genes) grew for two months on a medium deprived of ALS inhibitor (chlorsulfuron), to ensure a sufficient development and thereafter the most vigorous lines were transferred to a ALS inhibitor chlorsulfuron selection medium (page 14, para 2, line 1-3). It is a standard practice and well known in the art that any explant, be it an embryo or any other cell, needs to be treated in presence of a selection agent and undergo the selection process (i.e., selection in presence of an ALS inhibitor like chlorsulfuron, in this case) to generate one or more transgenic cell(s)/embryo(s) containing the gene (i.e., ALS mutant gene, in this case) conferring resistance to the ALS inhibitor like chlorsulfuron.
It is known in the art that culturing a mixed population of plant cells that contains transgenic or genome edited herbicide-resistant cells in the presence of the specific herbicide acts as a strong selection pressure resulting in at least 90% (nearing 100%) of the surviving population possessing the mutated (ALS) resistance gene, as recited in claim 19.
However, Malabarba et al. does not describe a banana embryogenic cell or a protoplast derived from an embryogenic cell suspension and generating at least one banana embryo or plant.
Tripathi et al. describes creating embryogenic cell suspension using friable embryogenic calli with many translucent proembryos (which ultimately become embryos before regenerating) that were transferred to liquid medium and homogenous cell suspensions were established (page 216, left column, para 1, line 9-11).
Ntui et al. describes a robust CRISPR/Cas9 mediated genome editing tool for banana, Musa spp. (title and abstract). It describes regenerating transgenic (genome edited) banana plants (page 5, left column, para 5, line 4-7) after transforming embryogenic cells in an embryogenic cell suspension (page 3, left column, para 7, line 1-2; page 5, left column, para 5, line 1-2), as recited in claims 1-2 and 8. Ntui et al. describes regenerating (reads on to “growing”) several banana plants from at least one cell (page 5, left column, para 5, line 4-6), as recited in claims 22-23. Ntui et al. teaches achieving at least 90% (100%) mutation efficiency (which also reads on to “cells in the population comprise mutated ALS”) in at least one banana cultivar (page 8, right column, para 2, line 9-10), as recited in claim 19.
It would have been obvious to an ordinarily skilled artisan to modify the method of editing the ALS gene using CRISPR-Cas technique and select herbicide resistant banana plants using chlorsulfuron, as described by Malabarba et al., in embryogenic cells in commercially important banana varieties, as described by Ntui et al.
The ordinarily skilled artisan would have been motivated to edit the endogenous ALS gene in banana with a realistic objective to develop commercially important banana plants that are resistant to herbicides from the sulfonylurea class comprising chlorsulfuron, which is also a commercially valuable trait.
Regarding claims 13-14 and 25, Physically separating the mutated (transgenic or genome edited) cells from non-mutated ones, as recited in claim 13, during subculturing in selection media containing the selection agent, is a standard practice while using (embryogenic) cell suspension culture comprising small clumps of friable callus for transformation (as described by Tripathi et al.) Genotyping the mutated cells and/or the genome edited plants to confirm the presence of the desired mutation, as recited in claims 14 and 25, is also a routine and standard practice in the art, as also described by Ntui et al. (page 3, right column, para 3).
Regarding claims 16-17 and 26, Malabarba et al. teaches the ALS sequences from apple (Gene ID: MD06G1028700 and Uniprot Accession No. A0A498JQ03) and pear (Gene ID NO. PCP000109 and Uniprot Accession No. A0A5N5HW78) (page 13, para 2, line 1-2; Fig. 3A). It describes designing the guide RNA (sgRNA) around the coding sequence for the polypeptide “SIPVVAITGQVPRRMIGTDAFQETPIVE” (page 7, Fig. 3A). Identifying homologues, in any plant including banana, based on sequence identity is a routine and standard practice in the art. It would have been obvious to an ordinarily skilled artisan to identify different alleles of the ALS gene (ALS1 and/or ALS2) in one or more commercially important banana species, as recited in claim 16, using the banana genome hub, as described by Ntui et al. (page 2, right column, para 3, line 1-4).
Malabarba et al. describes mutating the ALS genes in apple and peach so that P185 and P192 are mutated to leucine (P185L) (page 8, para 1, line 4-5) and/or serine (page 6, para 1, line 11-14) resulting in a mutant apple plant resistant to ALS inhibitor chlorsulfuron. The proline residue at 187 in ALS1 and at 181 in ALS2 in banana are aligned to the apple ALS polypeptide at position P185. That proline residue, as shown in the red box above, is conserved in all the ALS sequences and would be functional equivalents. It would have been obvious to an ordinarily skilled artisan to substitute P187 in ALS1 and/or P181 in ALS2 in banana with leucine and/or serine (as recited in claims 17 and 26) with a realistic objective to get a mutant banana plant that is resistant to ALS inhibitor chlorsulfuron.
All the ALS sequences from apple, pear, and banana (MsALS1 and MsALS2 consisting of instant SEQ ID NOs: 9-10) are aligned in respect to “SIPVVAITGQVPRRMIGTDAFQETPIV E”, as shown below.
CLUSTAL 2.1 multiple sequence alignment
AppleALS MAAVAPPSSSSFSKPSLFSSSAKSHHPISRFNLPFSPLPQKPSPLRHLRISSSLSPKTDT
PearALS MAAIA PPSSYSFSKPSPFSSSAKSHHPISRFNPPFSPLPQKPSPLRHLRIFSSLSPKTDT
Malabarba3A ------------------------------------------------------------
MsALS1 --MASSAAAAVAAAGAVITPSKSSHPPFSAAYRLPFPLPKPLHSLSTRHPHVRPISASAD
MsALS2 --------MASSTPGAAIA PSNPSLWPFSAASRLLVPFPKPLLSLSPSHRNVRPISASAD
AppleALS TITTTTS----NPLDLEAPISRFAPDEPRKGADVLVEALERQGVTDVFAYPGGASMEIHQ
PearALS TITTATTTTTTNPLHPEVPISRFAPDEPRKGADVLVEALERQGVTDVFAYPGGASMEIHQ
Malabarba3A ------------------------------------------------------------
MsALS1 RRQLPSAAAPTADATTAPLLRNFAPDEPRKGADILVEALEREGVTDLFAYPGGASMEIHQ
MsALS2 RHQTPSAAASTAATTAVPLLRNFAPDEPRKGSDILVEALEREGVTDLFAYPGGASMEIHQ
AppleALS ALTRSSSIRNVLPRHEQGGVFAAEGYARASGRPGVCIATSGPGATNLVSGLADALMDSIP 176
PearALS ALTRSSSIRNVLPRHEQGGVFAAEGYARASGRPGVCIATSGPGATNLVSGLADALMDSIP 180
Malabarba3A ---------------------------------------------------------SIP 3
MsALS1 ALTRSPSITNHLLRHEQGEVFAASGYARSTGRPGVCIATSGPGATNLVSGLADALLDSVP 178
MsALS2 ALTRSPSITNHLLRHEQGEIFAASGYARSTGRPGVCIATSGPGATNLVSGFADALLDSVP 172
*:*
[AltContent: rect]AppleALS VVAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVEDIPRVVREAFFLATSGRPG
PearALS VVAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLDVEDIPRVVREAFFLATSGRPG
Malabarba3A VVAITGQVPRRMIGTDAFQETPIVE-----------------------------------
MsALS1 LVAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLNVDDIPRIIKEAFFVATTGRPG
MsALS2 LVAITGQVPRRMIGTDAFQETPIVEVTRSITKHNYLVLNVDDIPRIIKEAFFLASTGRPG
:************************
AppleALS PVLIDIPKDVQQQLVVPNWDQPIKLPGYMSRLPKSPNEGHLEQIVRLVSESKKPVLYVGG
PearALS PVLIDIPKDVQQQLVVPNWDQPIKLPGYMSRLPKSPNEGHLEQIVRLVSESKKPVLYVGG
Malabarba3A ------------------------------------------------------------
MsALS1 PVLVDIPKDIQQQLAIPVWDPPLRLPGYISRLPRLPARPLLDQILRLVSESRRPVLYVGG
MsALS2 PVLVDIPKDIQQQLAIPVWDPPLRLPGYISRLPKPPSRCLLDQIIRLVSESHRPVLYVGG
AppleALS GCLNSSEELRRFVELTGIPVASTLMGLGAYPCNDEELSLQMLGMHGTVYANYAVDKSDLL
PearALS GCLNSSEELRRFVELTGIPVASTLMGLGAYPCNDEELSLQMLGMHGTVYANYAVDKSDLL
Malabarba3A ------------------------------------------------------------
MsALS1 GCLNSSEELHRFADLTGIPIASTLMGLGAYP-TDAELSLKMLGMHGTVYANYAIDKADLL
MsALS2 GCLNSSEELRRFADLTGIPIASTLMGLGVYP-TDAELSLKMLGMHGTVYANYSVDKADLL
AppleALS LAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADVKPALEGLNRILE
PearALS LAFGVRFDDRVTGKLEAFASRAKIVHIDIDSAEIGKNKQPHVSVCADVKSALEGLNRILE
Malabarba3A ------------------------------------------------------------
MsALS1 LAFGVRFDDRVTGKIEAFASRAKIVHIDIDPAEIGKNKQPHVSICADVKLALQGMNALME
MsALS2 LALGVRFDDRVTGKLEAFASRAKIVHIDIDPAEIGKNKQPHVSLCADVRLALQGMNALME
AppleALS QKESKVKLDFSAWRAELKEEKVKFPLGFKTFGDSISPQNAIQVLDELTDGNAIISTGVGQ
PearALS QKESKVKLDFSAWRAELKEEKVKFPLGFKTFGDSISPQNAIQVLDELTDGNAIISTGVGQ
Malabarba3A ------------------------------------------------------------
MsALS1 ETEIYRKFDFSTWREELDKQKKIYPLNYKTFGDQIPPQYAIQVLDELTNGEAIITTGVGQ
MsALS2 ESGIHQKFDFSTWRKELDQLKKAYPLSYKTFGDLIPPQYAIQVLDELTNGEAIISTGVGQ
AppleALS HQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPDSVVVDIDGDGSFLMNVQE
PearALS HQMWAAQFYKYKRPRQWLTSGGLGAMGFGLPAAIGAAVANPDSVVIDIDGDGSFLMNVQE
Malabarba3A ------------------------------------------------------------
MsALS1 HQMWAAQYYSYKRARQWLTSAGLGAMGFGLPAAAGAAVGNPGVTVVDIDGDGSFQMNAQE
MsALS2 HQMWAAQYYSYKRARQWLTSGGLGAMGFGLPAAAGAAVGNPGVTVVDIDGDGSFQMNAQE
AppleALS LATISVEKLPVKIMLLNNQHLGMVVQWEDRFYKANRAHTYLGNPANESEIFPNMLKFADA
PearALS LATISVEKLPVKIMLLNNQHLGMVVQWEDRFYKANRAHTYLGNPANESEIFPNMLKFADA
Malabarba3A ------------------------------------------------------------
MsALS1 LAMIRIENLPVKIMVLNNQHLGMVVQWEDRFYHRNRAHTYLGNPANETEVFPDFLKISEG
MsALS2 LAMIRIENLPVKMMVLNNQHLGMVVQWEDRFYRSNRGHTYLGNPANESEIFPDFLKITEA
AppleALS CGIPAARVTKKEDLKAAIQKMLDTPGPYLLDVIVPHQEHVLPMIPSGGAFKDVITEGDGR
PearALS CGIPAARVTKKEDLKAAIQKMLDTPGPYLLDVIVPHQEHVLPMIPSGGAFKDVITEGDGR
Malabarba3A ------------------------------------------------------------
MsALS1 YGIPAARVTKKSEVREAIRKMLETSGPYLLDVIVPHEEHVLPMIPSGGAFKDMILDGDGR
MsALS2 YGIPAARVTKKSEVREAIRKMLKTPGPYLLDVIVPHEEHVLPMIPSGGAFKDMILDGDGR
AppleALS SSY-
PearALS SSY-
Malabarba3A ----
MsALS1 TPY-
MsALS2 TPPH
Claims 21 is rejected under 35 U.S.C. 103 as being unpatentable over Malabarba et al. in view of Ntui et al. and Tripathi et al. as applied to claims 1-3, 5-7, 13-20 and 22-27 above, and further in view of Hu et al. (CRISPR/Cas9-mediated genome editing of MaACO1 (aminocyclopropane-1-carboxylate oxidase 1) promotes the shelf life of banana fruit, 2021, Plant Biotech. J., 19:654–656; published on 28th January 2021).
Malabarba et al. in view of Ntui et al. and Tripathi et al. describes a method of generating at least one banana embryo and/or a banana plant comprising at least one desired mutation in at least one of the endogenous acetolactate synthase (ALS) gene(s) so as to provide resistance to an ALS inhibitor, wherein the banana cell is an embryogenic cell or a protoplast derived from an embryogenic cell suspension, as discussed above.
However, Malabarba et al. in view of Ntui et al. and Tripathi et al. does not describe gene stacking by mutating ACO, ACS or PPO gene(s) in the banana plant already having the mutated ALS gene(s).
Hu et al describes commercially important trait of increased shelf-life of banana by suppressing the production of ethylene by editing the ACO gene using CRISPR-Cas system in banana (MaACO1) (title; page 656, left column, para 4, line 2-4). Gene stacking is a well-known standard technique in the art to introduce multiple desirable genes and/or mutations in a single plant to introduce valuable traits into crop varieties.
It would have been obvious to an ordinarily skilled artisan to edit the endogenous ACO gene(s) in banana, as described by Hu et al., in the same plant having the mutated ALS gene(s), as described by Malabarba et al. in view of Ntui et al. and Tripathi et al. The artisan would have been motivated to do it with a realistic objective to extend the shelf-life of the already herbicide resistant banana plants comprising ALS mutation(s).
Response to Applicant’s arguments
The argument set forth in the Applicant’s response on 01/07/2026 has been fully considered but is not found persuasive.
Regarding rejection under 35 U.S.C. 103: the Applicant argues that “none of Malabarba, Ntui, or Tripathi, either alone in combination, teach or suggest all the features of the claimed method nor provide some suggestion or motivation to a person of skill in the art to arrive at the claimed invention with a reasonable expectation of success” (page 12, para 3, line 1-4). The argument continues, “starting from the objectives of Malabarba (reducing chimerism and validating the base editor), there is also no reason that a person having ordinary skill in the art would consider developing the claimed lab-based selection method from that disclosure” (page 13, para 3, line 1-3); and “other than showing transformation of banana embryonic tissue, Ntui does not disclose any of the other features of the amended independent claims: editing of ALS; co-editing of ALS and a target gene; and selection of ALS-inhibitor resistance in embryonic tissue” (page 13, para 5, line 1-3) while “Tripathi does not mention gene-editing, or any of the other claimed features” (page 13, last para, last line).
The Examiner disagrees. Malabarba et al. teaches editing ALS genes to develop commercially important trait of herbicide resistance in apple and peach plants using CRISPR-Cas technique while Ntui et al., in conjunction with Tripathi et al., described CRISPR-Cas9 based technique comprising culturing banana embryogenic cells to regenerate fully matured banana plant. There is no evidence to indicate that that the method used in apple and peach would not work in banana in terms of developing specific herbicide resistance in banana by mutating ALS gene(s). There is no reason to believe that the genome edited herbicide resistant banana embryogenic cells would not respond to the method described by Ntui et al. and Tripathi et al. It is a standard practice and well known in the art that any explant, be it an embryo or any other cell, needs to be treated in presence of a selection agent and undergo the selection process (i.e., selection in presence of an ALS inhibitor like chlorsulfuron, in this case) to generate one or more transgenic cell(s)/embryo(s) containing the gene (i.e., ALS mutant gene, in this case) conferring resistance to the ALS inhibitor like chlorsulfuron.
Applicant is reminded that the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981).
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Communication
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Jay Chatterjee
Patent Examiner
Art Unit 1662
/Jay Chatterjee/ Examiner, Art Unit 1662
1Chen et al. (CRISPR/Cas Genome Editing and Precision Plant Breeding in Agriculture, 2019, Annu. Rev. Plant Biol. 2019. 70:667–97) provides the evidence that CRISPR/Cas9 based gene editing function of sequence-specific Cas9 endonuclease comprises homology-directed repair (HDR) in conjunction of a donor template (Fig.2b; page 673, para 2.2.2, line 1-8).