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 .
Status of the Claims
Claim 3 is canceled.
Claims 1-2 and 4-21 are pending.
Claims 1-2 and 4-21 are examined herein.
Claims 1-2 and 4-21 are rejected.
Priority
Application No. 18/743,454 filed on 06/14/2024 is a continuation of PCT
Application No. PCT/EP2022/086257 and also claims foreign priority to Application No.
EP21215549.3 filed on 12/17/2021.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-2, 4-15, and 18-21 are rejected under 35 U.S.C. 103 as being unpatentable over Ron (WO-2020051283-A1) and Larriba (Larriba, E., Sánchez-García, A. B., Justamante, M. S., Martínez-Andújar, C., Albacete, A., & Pérez-Pérez, J. M. (2021). Dynamic hormone gradients regulate wound-induced de novo organ formation in tomato hypocotyl explants. International Journal of Molecular Sciences, 22(21), 11843).
Claim 1 is drawn to a method for producing a shoot of a plant, wherein the shoot comprises one or more cells having a genetic modification, and wherein the method comprises:
introducing in a cell of a plant a vector expressing at least one of a guide element and nuclease element of a site-directed nuclease;
removing the shoot apical meristem of the plant at a first location; and
regenerating a shoot at a second location in the plant by wounding the plant at the second location one or more days after removing the shoot apical meristem of the plant at the first location, wherein the shoot comprises one or more cells having a genetic modification.
Claim 2 is drawn to the method according to claim 1, wherein removal of the shoot apical meristem is by decapitation.
Claim 4 is drawn to the method according to claim 1, wherein regeneration of the shoot is by decapitation.
Claim 5 is drawn to the method according to claim 1, wherein the first location is above the cotyledons.
Claim 6 is drawn to the method according to claim 5, wherein the first location is in the epicotyl above the cotyledons and below the first true leaves.
Claim 7 is drawn to the method according to claim 1, wherein the second location is below the cotyledons.
Claim 8 is drawn to the method according to claim 7, wherein the second location is in the hypocotyl below the cotyledons.
Claim 9 is drawn to the method according to claim 1, wherein the site-directed nuclease is selected from the group consisting of a CRISPR-nuclease complex, an Argonaute, a Zinc finger nuclease, a TALEN and a meganuclease.
Claim 10 is drawn to the method according to claim 1, wherein the site-specific nuclease is a CRISPR-nuclease complex, wherein the plant expresses the nuclease element of the complex, and the vector expresses a gRNA.
Claim 11 is drawn to the method according to claim 1, wherein the period between the introducing (i) and the removing (ii) is 1 - 21 days.
Claim 12 is drawn to method according to claim 11, wherein the period is 1 - 7 days.
Claim 13 is drawn to the method according to claim 1, wherein the plant is a seedling.
Claim 14 is drawn to the method according to claim 1, wherein the introduced vector is a virus.
Claim 15 is drawn to the method according to claim 14, wherein the virus is selected from the group consisting of a Tobacco Rattle Virus (TRV), a Tobacco Mosaic Virus (TMV), a Potato virus X (PVX), a geminivirus, a Sonchus yellow net virus (SYNV).
Claim 18 is drawn to the method according to claim 1, further comprising (iv) selecting the shoot comprising one or more cells having a genetic modification and regenerating a plant thereof.
Claim 19 is drawn to the method according to claim 1, further comprising (iv) growing the plant to produce a gamete comprising the genetic modification.
Claim 20 is drawn to the method according to claim 19, further comprising (v) growing a seed from the gamete and optionally a progeny plant from the seed, wherein the seed and optional progeny plant comprises one or more cells having the genetic modification.
Claim 21 is drawn to a plant having a shoot comprising one or more cells having a genetic modification, wherein the plant is obtainable by the method according to claim 1.
Regarding claim 1, Ron teaches generating transgenic tomato plants expressing Cas9 and the negative selection marker Herpes Simplex Virus-I Thymidine Kinase (HSVtk) (¶0078-0079), then transiently expressing sgRNAs in the plant using a viral expression system (¶0080) (i.e. part (i) introducing in a cell of a plant a vector expressing at least one of a guide element and nuclease element of a site-directed nuclease). Ron teaches the Cas9 expressing mother plants were injected with the viral replicon vector for sgRNA expression, then the plants were decapitated at the epicotyl a week later, then regenerated shoots that developed were detached from the decapitation site and propagated (i.e. removing the shoot apical meristem of the plant at a first location and regenerating a shoot) (¶0093). Ron further discloses mutation level and type were analyzed by PCR and sequencing of amplicons flanking the target genomic sites, and regenerated stems were evaluated for mutation in the marker and POTATO LEAF (C) sequences and the expected change in leaf shape typical of a C knockout (i.e. wherein the shoot comprises one or more cells having a genetic modification) (¶0093). Ron teaches approximately 11-25% of regenerated shoots (up to 25% if additional Cas9 nuclease is injected into the Cas9-expressing mother plant at the time of injecting the viral replicon vector for sgRNA expression) had mutations in both targeted C and HSVtk genes (¶0121, Table 2).
Regarding claim 2, Ron teaches the seedlings were decapitated at the epicotyl (¶0093).
Regarding claim 4, Ron teaches shoots are regenerated from the wound site that is decapitation (¶0093).
Regarding claims 5-6, Ron teaches the seedlings were decapitated at the epicotyl (¶0093) (i.e. above the cotyledons and below first true leaves).
Regarding claim 9, Ron teaches the site-directed nuclease is Cas9 (i.e. a CRISPR-nuclease complex) (¶0080, 0082-0083, 0093).
Regarding claim 10, Ron teaches the plant expresses Cas9 and the sgRNAs are transiently expressed using a viral replicon vector (i.e. wherein the site-specific nuclease is a CRISPR-nuclease complex, wherein the plant expresses the nuclease element of the complex, and the vector expresses a gRNA) (¶0078-0080, 0087, 0093).
Regarding claims 11-12, Ron teaches the Cas9 expressing mother plants were injected with the viral replicon vector for sgRNA expression, then the plants were decapitated at the epicotyl a week later (i.e. the period between introducing and removing is a week or 7 days) (¶0093).
Regarding claim 13, Ron teaches the plant is a seedling (¶0093).
Regarding claim 14, Ron teaches the introduced vector is a viral replicon vector (i.e. wherein the introduced vector is a virus) (¶0093).
Regarding claim 15, Ron teaches the virus is Tobacco Rattle Virus (TRV) (¶0080, 0087).
Regarding claim 18, Ron teaches regenerated shoots that developed were detached from the decapitation site and propagated, mutation level and type were analyzed by PCR and sequencing of amplicons flanking the target genomic sites, and regenerated stems were evaluated for mutation in the marker and POTATO LEAF (C) sequences and the expected change in leaf shape typical of a C knockout (i.e. selecting the shoot comprising one or more cells having a genetic modification and regenerating a plant thereof) (¶0093).
Regarding claims 19-20, Ron teaches all shoots that were detached from the decapitation site were propagated to fruiting then analyzed for mutation (¶0093) and also teaches selected double mutants at HSVtk and C have been taken to the next generation to test for heritability (¶0126).
Regarding claim 21, Ron teaches the decapitated plant produced shoots that comprised the genetic mutations (¶0093).
However, Ron does not explicitly teach in a single embodiment:
regenerating a shoot at a second location in the plant by wounding the plant at the second location one or more days after removing the shoot apical meristem of the plant at the first location (remaining limitation of claim 1)
the method according to claim 1, wherein the second location is below the cotyledons (claim 7)
the method according to claim 7, wherein the second location is in the hypocotyl below the cotyledons (claim 8)
the method according to claim 1, further comprising (iv) growing the plant to produce a gamete comprising the genetic modification (claim 19)
the method according to claim 19, further comprising (v) growing a seed from the gamete and optionally a progeny plant from the seed, wherein the seed and optional progeny plant comprises one or more cells having the genetic modification (claim 20)
Regarding the remaining limitation of claim 1 and claims 7-8, in an alternative embodiment, Ron teaches wound generation/ shoot decapitation in the stem or hypocotyl or epicotyl, or runners, or internodes, seedlings or woody buds will generate a wound and reprogramming to produce axillary buds or callus and new shoots (¶0070). Ron teaches thus in some embodiments, the wound is formed at the location at which the nuclease and/or guide molecule(s) have been introduced to the plant (¶0070). In other analogous art, Larriba further teaches de novo shoot formation is observed in tomato from explants decapitated at the hypocotyl (below the cotyledons) (p. 2, ¶1, Fig. 1).
Regarding claims 19-20, in an alternative embodiment, Ron teaches generating transgenic tomato plants expressing Cas9 and the negative selection marker D-amino acid oxidase (DAAO) (¶0078-0079), then transiently expressing sgRNAs in the plant using a viral expression system to knockout the DAAO gene and also the ENTIRE gene for visual confirmation of gene-editing (¶0080). In this alternative embodiment, Ron also teaches a regenerated stems from the decapitation site were evaluated for mutation in the marker and ENTIRE sequences and the expected change in leaf shape typical of an ENTIRE knockout (¶0092). Ron teaches the plants were propagated to fruiting and heritability of the phenotype and genotype was determined, and further teaches plants without Cas9 segregated from these T1 and showed heritable E mutant phenotypes (¶0091 and 0117) (i.e. because the mutations were heritable into the progeny plants, the plant must have been grown to produce a gamete comprising the genetic modification, and the seed grown from the gamete and optionally a progeny plant from the seed, wherein the seed and optional progeny plant comprises one or more cells having the genetic modification).
It would be prima facie obvious to combine the teachings of Ron and Larriba for the purpose of regenerating more gene-edited shoots from the plant because Ron teaches i. decapitation at either location where the nuclease and/or guide molecule(s) have been introduced will result in shoot regeneration (¶0070), and ii. a low number of shoots regenerated from a single decapitation event comprise mutations at all targeted sites (Table 2). Therefore, the steps of decapitation at both locations were known in the prior art, their combination does not change the function of performing each step separately, and it would be prima facie obvious to combine the steps to produce more regenerated shoots with the targeted edits. One having ordinary skill in the art would have a reasonable expectation of regenerating the instantly claimed shoots by decapitating first at the epicotyl then at the hypocotyl success because Ron and Larriba teach shoots, including gene-edited shoots, can be regenerated from decapitation at both locations, and the combination of the steps does not present any special technical difficulties. Furthermore, one having ordinary skill in the art would have been motivated to combine the alternative embodiments of Ron for the purpose of generating progeny plants having the inherited mutation and the Cas9 T-DNA segregated out (¶0091 and 0117). One would have a reasonably expectation of success in generating gametes, seeds, and progeny plants having the mutation because in an alternative embodiment Ron shows the mutations are heritable.
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Ron and Larriba as applied to claims 1, 14, and 15 above, and further in view of Lindbo (Lindbo, J.A. (2007). TRBO: a high-efficiency tobacco mosaic virus RNA-based overexpression vector. Plant physiology, 145(4), 1232-1240).
Claim 16 is drawn to the method according to claim 15, wherein the virus is a tobacco mosaic virus RNA-based overexpression vector (TRBO).
Regarding claim 16, Ron and Larriba teach the limitations of claims 1, 14, and 15 as set forth in the previous obviousness rejection. The teachings of Ron and Larriba as they are applied to claims 1, 14, and 15 are set forth previously herein and are incorporated by reference.
However, Ron and Larriba do not explicitly teach wherein the virus is a tobacco mosaic virus RNA-based overexpression vector (TRBO).
In analogous art, Lindbo teaches about the construction of an improved
agroinfection-compatible TMV vector that lacks the TMV CP gene coding sequence,
called the TMV RNA-based overexpression (TRBO) vector (p. 1233, 4-5). Lindbo teaches that the TRBO vector will be a useful transient expression vector for production of recombinant proteins in plants (p. 1233, 4-5).
It would therefore have been obvious to a person of ordinary skill in the art to
modify the invention of as taught by Ron and Larriba to include the limitations of Lindbo to arrive at the instantly claimed method with a reasonable expectation of success because both methods are directed to Agroinfection of plants with a viral vector, and successful incorporation of the TRBO vector taught by Lindbo into the method taught by Ron could be achieved by one of ordinary skill in the art without encountering any special technical obstacles. One having ordinary skill in the art would have been motivated to do so because Lindbo teaches the TRBO vector has several significant improvements, such as (1) much higher agroinfection efficiency; (2) higher recombinant protein expression levels; and (3) inability to form virus particles during its infection/ replication cycle (p. 1233, 14-5).
Claim 17 is rejected under 35 U.S.C. 103 as being unpatentable over Ron and Larriba as applied to claim 1 above, and further in view of Mahas (Mahas, A., Ali, Z., Tashkandi, M., & Mahfouz, M.M. (2019). Virus-mediated genome editing in plants using the CRISPR/Cas9 system. In Plant Genome Editing with CRISPR Systems: Methods and Protocols (pp. 311-326). New York, NY: Springer New York).
Claim 17 is drawn to the method according to claim 1, wherein the introducing is
by contacting the cotyledon with an agrobacterium or virus particle carrying the vector.
Regarding claim 17, Ron and Larriba teach the limitations of claim 1 set forth in the previous obviousness rejection. The teachings of Ron and Larriba as they are applied to claim 1 set forth previously herein and are incorporated by reference.
However, Ron does not explicitly teach wherein the introducing is by contacting
the cotyledon with an agrobacterium or virus particle carrying the vector.
In analogous art, Mahas teaches about virus-mediated genome editing in plants
using CRISPR/Cas9 system (title). Specifically, Mahas teaches using the Tobacco
Rattle Virus to employ CRISPR/Cas9 genome editing, and teaches the initially infected
cells, such as leaves, presumably serve as a source for further replication of viral RNAs
and their systemic infection and spread into a variety of tissues and cells, such as
developing and meristematic tissues, including germline cells (p. 313, ¶2, Fig. 1).
It would therefore have been obvious to a person of ordinary skill in the art to
modify the invention of as taught by Ron and Larriba to include the limitations of Mahas to arrive at the instantly claimed method with a reasonable expectation of success because Mahas also teaches a method of Tobacco Rattle Virus (TRV)-mediated genome editing via agroinfection of plants, and Mahas teaches the utilization of TRV leads to further replication of viral RNAs and their systemic infection throughout the plant (p. 313, ¶2, Fig. 1). It would therefore be obvious to introduce the Agrobacterium comprising the recombinant TRV vectors into any tissue of the plant, including the cotyledons as instantly claimed, because Mahas teaches TRV is an effective vehicle for systemic delivery of CRISPR/Cas9 elements, and their systemic infection will spread into a variety of tissues and cells, such as developing and meristematic tissues, including germline cells plant (p. 313, ¶2-3, Fig. 1).
Response to Arguments
Applicant argues beginning on p. 5 of remarks dated 11/26/2025 the
following arguments:
The Office Action alleges at page 5 that "Ron discloses seedlings were decapitated at the first internode (i.e. removing the shoot apical meristem of the plant at a first location), the Cas9 expressing mother plants were injected with the viral replicon vector for sgRNA expression, the plants were decapitated at the epicotyl a week later, then regenerated shoots that developed were detached from the decapitation site and propagated (i.e. regenerating a shoot at a second location in the plant at the second location) (0093)."
However, Ron does not teach or suggest "decapitated at the first internode" and "regenerating a shoot at a second location" because the decapitation according to Ron at paragraph [0093] is performed at a single location in a single step. In particular, paragraph [0093] of Ron teaches that "we decapitated the seedlings at the first internode so that cells that were genome edited could form a callus, regenerate new meristems and stems. The Cas9 expressing mother plants were injected with the viral replicon vector and decapitated at the epicotyl a week later." (Emphasis added). In other words, Ron teaches decapitation in the first sentence of paragraph [0093], and the second sentence only further explains that the genome editing was performed by first injecting the Cas9 expressing mother plants with the viral replicon vector to obtain the genome edited cells referenced in the first sentence. Accordingly, the first two sentences of Ron refer to the same step of decapitation, and Ron only teaches or discloses a single step of decapitation at a single location. Thus, Ron does not teach, suggest, or disclose "removing the shoot apical meristem of the plant at a first location; and ... regenerating a shoot at a second location in the plant by wounding the plant at the second location one or more days after removing the shoot apical meristem of the plant at the first location," as recited by the present claims. For the above many reasons, Ron cannot anticipate the present claims. Reconsideration and withdrawal of the subject rejection is therefore respectfully requested.
Applicant further argues because claim 1 is not anticipated, the rejections under 35 USC 103 to the remaining claims that ultimately depend from claim 1 should also be withdrawn.
This argument has been fully considered and is found not persuasive for
the following reason(s): Upon further review and consideration, Applicant’s argument that the first two sentences of Ron refer to the same step of decapitation, and Ron only teaches or discloses in the referenced example a single step of decapitation at a single location. However, in view of the appropriate interpretation of the teachings of Ron and in view of Applicant’s amendments, the claims are now rejected under 35 USC 103.
Conclusion
No claims are allowed.
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JESSICA N. STOCKDALE
Examiner
Art Unit 1663
/JESSICA NICOLE STOCKDALE/Examiner, Art Unit 1663
/CHARLES LOGSDON/Primary Examiner, Art Unit 1662