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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 03/31/2026 has been entered.
Claim Status
Claims 1, 4-6, 9 and 55-59 are pending and currently being examined.
All previous objections and rejections not set forth below have been withdrawn in view of applicant’s amendments to the claims.
Claim Rejections - 35 USC § 112(b)
Claims 1, 4-6, 9 and 55-59 are 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.
Amended claim 1 recites, “…selecting for transient expression of the site-specific DNA modifying agent, wherein embryogenic cells having stable integration of the one or more polynucleotides of the second vector are selected against by expression of the anti-regeneration gene”.
The claim does not recite more than one options to express the site-specific DNA modifying agent (the transgene) so that a skilled artisan can specifically select for transient expression. Moreover, the claim, as written, implies that there is a possibility to get stable integration of the one or more polynucleotides including the site-specific DNA modifying agent while expressing the polynucleotide sequences present in the second vector transiently.
Claim 1 specifies that both site-specific DNA modifying agent and the anti-regeneration gene are cloned in the same (second) vector. Fate of both the genes in terms of its integration into the host genome is expected to be the same. It is known in the art that genes that are transiently expressed are not stably integrated into the host genome (Eppendorf Lab Academy, Stable vs transient expression: Which to use and when?, 30 August 2024; p. 2. Para 2, line 4-6). If the second vector is transiently expressed, then the anti-regeneration gene would not be stably integrated into the host genome, and, thus, cannot be used for selection “against by expression of the anti-regeneration gene”.
The only active step (that a skilled artisan can take) recited herein is “selecting for transient expression of the site-specific DNA modifying agent” which implies that the second vector is transiently expressed.
It is not clear to the Examiner if “embryogenic cells having stable integration of the one or more polynucleotides of the second vector” is recited as an intended possibility and/or actively being selected against. The last paragraph of claim 1 is very confusing and do not seem to add much value to the claim.
It is suggested to delete the last paragraph in claim 1.
Claim Rejections - 35 USC § 112(a)
Enablement
Claims 1, 4-6, 9 and 55-59 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 enablement requirement. The claims contain subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
Claim 1 recites, “…. wherein the site-specific DNA modifying agent creates a double-strand break at or near the DNA target site and repairs the double-strand break via homology-directed repair…”.
The Applicant does not that a “site-specific DNA modifying agent”, i.e, CRISPR-Cas endonuclease as described by the Applicant (spec, p.6, para 0017, line 1-3), can actually repair “the double-strand break via homology-directed repair”. The Applicant does not provide any example or guidance/evidence that the site-specific DNA modifying agent (viz., Cas endonuclease) can repair any double-strand break via homology-directed repair, as currently recited in parent claim 1.
It is known in the art that Double-Strand Break (DSB) repair is essential to all forms of life. Depending on the forms of repair template and CRISPR system used, homology-mediated gene insertion and replacement (i.e., homology dependent repair after Cas endonuclease makes the double-stranded break in the target DNA), are carried out via specific DNA repair pathways such as homology-directed repair (HDR), synthesis-dependent strand annealing (SDSA), microhomology-mediated end joining (MMEJ), and homology-mediated end joining (HMEJ) pathways (Lau et al., CRISPR-based strategies for targeted transgene knock-in and gene correction, 2020, Faculty Reviews, 9:20; p. 3, left column, para 2, line 9-15). All the pathways are naturally present in a plant cell to repair double stranded breaks in DNA. However, none of these pathways actually needs a Cas endonuclease.
Moreover, the Applicant also teaches that endogenous cellular DNA repair mechanisms are activated to repair the (double stranded) breaks in DNA (spec, p. 10, para 0037, line 1-2) (as opposed to a site-specific DNA modifying agent repairing the double-stranded break, as recited in claim1).
Undue trial and error experimentations would be needed to repair a DNA break using site-specific DNA modifying agent(s).
Based on breadth of the claims, lack of any working example, lack of guidance in the instant description or in prior art, the specification at the time of the application filed would not have taught one skilled in the art how to make and use the full scope of the claimed invention without performing undue experiments.
Scope of Enablement
Claims 1, 4-6, 9 and 55-59 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 method that comprises- i) at least one gRNA and ii) a DNA modifying agent comprising a Cas endonuclease and/or a (nucleotide) base editor; does not reasonably provide enablement for a method that does not include a gRNA, and/or include any DNA modifying agent other than a Cas endonuclease or a (nucleotide) base editor. 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/or use the invention commensurate in scope with these claims.
Claims 1 and 56-57 recites “a site-specific DNA modifying agent”. The Applicant does not define the term “DNA modifying agent”, although the instant description mentions various embodiments of DNA modifying agents including a CRISPR-Cas polypeptide (endonuclease) and a base editor (spec, p. 6, para 0017). Both of the DNA modifying agents, as described, require at least one guide RNA to become site specific in its action (Tang et al. , Class 2 CRISPR/Cas: an expanding biotechnology toolbox for and beyond genome editing, 2018, Cell Biosci., 8:59; p. 2, right column, para 2, line 5-6; p. 8, right column, para 3, line 7-15).
However, claims 1 and 56-57 comprises all DNA modifying agents. DNA modifying agents are not restricted to proteins (enzymes) alone but would include RNA molecules as well. All the following agents can modify DNA structure and/or function: DNA exonuclease, endonuclease like restriction enzymes (both creates double stranded breaks), ligase, polymerase, helicase (some of its members can create double stranded breaks), transferase, methyltransferases, demethylases, kinase, phosphatases, topoisomerase and gyrase (creates double stranded breaks), recombinase, and non-coding RNAs (like lncRNA or piRNA) that guide epigenetic modifications and/or reverse transcriptase function that use RNA templates to alter DNA structure.
The Applicant does not describe any example of using any DNA modifying agent other than CIRSPR-Cas polypeptide. Current status of the art does describe all the foresaid DNA modifying agents, but none would act in a site-specific manner to modify a (one) specific site in the host genome, as done by CRISPR-Cas endonuclease (in presence of a gRNA).
Based on breadth of the claims, lack of any working example, lack of guidance in the instant description or in prior art, the specification at the time of the application filed would not have taught one skilled in the art how to make and use the full scope of the claimed invention without performing undue experiments.
Claim Rejections - 35 USC § 103
Claims 1, 4-6, 9 and 55-59 are rejected under 35 U.S.C. 103 as being unpatentable over Gordon-Kamm et al. (Using Morphogenic Genes to Improve Recovery and Regeneration of Transgenic Plants, 2019, Plants, 8:38), in view of He et al. (Technological breakthroughs in generating transgene-free and genetically stable CRISPR-edited plants, 2020, aBIOTECH, 1:88–96; published on 3 December 2019), Schaeffer et al. (CRISPR/Cas9-mediated genome editing and gene replacement in plants: Transitioning from lab to field, 2015, Plant Science, 240:130–142), and Liu et al. (Comparison of three Agrobacterium-mediated co-transformation methods for generating marker-free transgenic Brassica napus plants, 2020, Plant Methods 16:81).
In regard to the rejections under 35 U.S.C. 103, claim 1 is interpreted as the repair of the double-strand break is done by the endogenous cellular DNA repair mechanisms and the claim comprises at least one gRNA in any of the two vectors.
Claim 1 is drawn to a method of modifying a target site in the genome of a plant cell, the method comprising providing to the plant cell two distinct Rhizobiales bacteria comprising a first bacteria and a second bacteria, i) wherein the first bacteria comprises a first vector to edit a target site in the plant cell; ii) the second bacteria comprises a second vector encoding a DNA modifying agent, at least one morphogenic factor, and an anti-regeneration gene; and wherein the ratio of the first vector and the second vector or the ratio of the first bacteria and the second bacteria is about 1:1 to about 10:1; and obtaining embryogenic cells being selected against by expression of the anti-regeneration gene.
Gordon-Kamm et al. describes transforming a variety of important crop plants (abstract, line 13-14) including Arabidopsis, coffee, cocoa, soybean (G. max), Brassica and tobacco all of which are dicots and monocots including maize and sorghum (as recited in claim 5) with a T-DNA vector (reads on to “first vector”) containing the trait expression cassette (for the sequence/gene of interest in the plant genome) and a separate T-DNA vector (which reads on to “second vector”) containing the morphogenic factor WUSCHEL (WUS) expression cassette which is sufficient to enhance recovery of transformants without integration of the morphogenic gene BABY BOOM or WUS (p. 6, last para, line 6), in the genome (page 3, Fig. 1D). Gordon-Kamm et al. describes ectopic overexpression of genes involved in morphogenesis, i.e. morphogenic factors, improves transformation efficiencies, and facilitates transformation of numerous recalcitrant crops (page 1, abstract). It also describes various means including use of inducible and tissue-specific promoters (page 1, abstract) along with specific benefits of ectopic transient expression of morphogenic gene(s) to avoid the deleterious pleiotropic phenotypes observed in plants when such genes are constitutively expressed (page 10, para 4), which has been demonstrated in a variety of important crops (page 1, abstract). Gordon-Kamm et al. also describes use of Agrobacterium mediated transformation and co-transfection techniques used to obtain transgenic plants by mixing two different strains of Agrobacterium (as recited in claim 9), which belongs to Rhizobiales family (as recited in claim 1), wherein the first strain containing a morphogenic gene, WUS (page 3, Fig. 1D) or BABY BOOM (BBM) (page 12, para 4) (as recited in claim 6), expression cassette while the other strain contains a gene of interest or trait. The Rhizobiales bacteria (Agrobacterium) comprising two different and separate T-DNA vectors encoding different gene(s) and/or polynucleotide sequences are interpreted as two distinct Rhizobiales bacteria. Two separate T-DNA vectors are to be cloned in two different Agrobacterium which reads on to “first” and “second” bacteria.
However, although Gordon-Kamm et al. mentions CRISPR/Cas9-mediated genome editing (page 13, last para; page 14, para 2), it does not explicitly describe using the DNA modifying agent Cas9. It also does not describe any ratio of the amounts of the first vector and the second vector OR the ratio of the first bacteria and the second bacteria.
He et al. describes that “editing a gene in-vivo by CRISPR only requires three components - a programmable nuclease such as Cas9 (reads on to “DNA modifying agent”), a guide RNA (gRNA) and a protospacer adjacent motif (PAM) (present in the target site(s) in the host plant genome) in close proximity (reads on to “at or near”, as recited in claim 1) to the target DNA. The Cas9 and gRNA complex binds to the target DNA, which is complementary to part of the gRNA molecule. Subsequently, Cas9 generates a double-stranded break within the target sequence, providing opportunities for editing the target sequence through DNA-repair pathways” (p. 88, bridging paragraph between left and right column). He et al. describes self-elimination of transgene(s) using the Transgene Killer CRISPR system (TKC technology) (p. 93, left column, para 2). He et al. teaches use of the lethal BARNASE gene, which effectively kill plant cells when expressed inside cells (p. 93, right column, para 2, line 2-3), under the control of REG2 promoter which is active during the early embryo development stage in plants (e.g. rice) (p. 93, right column, para 2, line 11-13). Thus, the BARNESE gene reads on to “anti-regeneration” gene as it reads on to “factors that impede normal regeneration and normal plant fertility can be used to provide the anti-regeneration property” (spec, p. 47, para 1, last 2 lines) and also “transient expression of the anti-regeneration gene does not kill the cell, but stable integration inhibits regeneration, or is lethal” (spec, p. 8, para 0027, line 7-8). The TKC system automatically eliminates those plants that contain the transgene construct comprising the BARNASE gene is being expressed, but enables the genome edited plants to undergo targeted gene editing before the expression of the BARNASE gene starts and the transgenic DNA fragments comprising the gene are removed (p. 93, right column, para 3, first 4 lines). As the expression of the BARNESE gene is under the control of REG2 promoter, the transgenic and/or genome edited cells will not be affected before embryogenesis, but would be affected once embryogenesis starts leading to development of the embryogenic cells (as recited in claim 1). He et al. teaches gene editing by agrobacterium-mediated transient expression of transgenes (p. 91, right column, para 2), as recited in claim 1.
Schaeffer et al. describes various endonucleases including Zinc finger nuclease (p. 131, left column, para 3, line 4; p. 133, right column, para 3, line 7), meganuclease (p. 139, left column, para 1, line 7) and Cas9 endonuclease (Title; abstract; p. 131, right column, para 3, line 2-3), as recited in claim 56. Schaeffer et al. teaches that Cas9 endonuclease introduces double-stranded breaks at or around defined positions in the host genome in plants (page 131, right column, para 2) guided or aided by a polynucleotide sequence encoding a guide RNA (sgRNA or gRNA), as recited in claims 57-58. It also describes homologous recombination mediated genome editing (reads on to “homology-directed repair”, as recited in claim 4) if the heterologous or exogenous DNA contains arms with sequence homology to the regions flanking the genomic or DNA target site (page 132, Fig. 2), as recited in claim 55. Site specific genome editing or gene knock-in has been described using CRISPR/Cas9 and shown it to be heritable in Arabidopsis (page 132, Fig. 2; page 136, left column, para 2).
Liu et al. describes a two Agrobacterium strains harboring two plasmids, and each contains an independent T-DNA: the two strains/two plasmids method or the mixed-strain system (page 2, right column, first para). Liu et al. also describes various ratios ranging from 1:8 to 8:1 of the two T-DNA vectors from two different Agrobacterium strains and the 1:1 ratio perform the best by producing the highest co-transformation frequencies in T0 transformants of the mixed-strain system (page 9, Fig. 3), as recited in claim 1.
Before the effective filing date, it would have been obvious to a person with ordinary skill in the art to modify the method taught by Gordon-Kamm et al. by expressing two different vectors. One of the vectors comprise a heterologous polynucleotide comprising a gene of interest (as described by Gordon-Kamm et al.) to be inserted into a specific site in the host genome wherein the heterologous polynucleotide is flanked by polynucleotides comprising homology to a nucleotide sequence at the target site in the plant genome. The other (second) vector comprises a polynucleotide encoding a Cas endonuclease (Cas9), as described by He et al. and Schaeffer et al., to edit one or more specific sequence(s) including endogenous gene(s) in the host genome while expressing a morphogenic factor (e.g. WUS or BBM), as taught by Gordon-Kamm et al., using suitable gRNA(s). Stable integration of the anti-regeneration BARNASE transgene (present in the second vector) would render the cell(s) non-viable and, thus, reduce regeneration frequency, as described by He et al. The site of the homologous recombination based gene-editing would be decided by the gRNA(s), which can be cloned in any of the two T-DNA vectors or in both (as described in the Claim interpretation section) (as recited in claims 58-59). The heterologous polynucleotide (present in the first vector) would act as a template for homologous recombination as described by Schaeffer et al. Stable integration any other foreign polynucleotide sequence including nucleotide sequences encoding Cas endonuclease (e.g., Cas9), gRNA (if present in the same vector), morphogenic factor(s), or the anti-regeneration factor (BARNESE gene) would be avoided while improving transformation efficiency, as described by Gordon-Kamm et al. and He et al. It is prudent to mention here that the heterologous polynucleotide cloned in the first vector acts in trans in conjunction with Cas9 and the gRNA and, thus, does not need to be cloned in the same vector, and that need to be achieved by transient expression (as described by Schaeffer et al.) to avoid integrating any foreign DNA present in the second vector encoding Cas9, gRNA (if present), the morphogenic agent and the anti-regeneration gene.
The ratio of the two Agrobacterium strains containing two different T-DNA vectors can be calibrated to increase transformation efficiency of genome edited plants according to the method described by Liu et al. (1). The ratio of the two Agrobacterium strains containing two different T-DNA vectors is an experimental design choice of an ordinarily skilled artisan without affecting the outcome in terms of modifying the target DNA site in the host genome.
Before the effective filing date of the invention, one ordinarily skilled artisan would have been motivated to transform a plant using two different T-DNA vectors using agrobacterium mediated transformation with a realistic goal to edit a specific target site in any commercially important plant genome and achieve higher transformation and regeneration efficient by providing a morphogenic factor (WUS or BABY BOOM protein) without integrating any foreign nucleotide sequence present in the second T-DNA vector comprising the Cas endonuclease (Cas9), the morphogenic factor, and the anti-regeneration (BARNESE) gene.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 1, 4-6, 9 and 55-59 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 5 and 13 of copending Application No. 17/760,273 (reference application, hereafter referred as ‘273) in view of He et al. and Liu et al.
Reference claim 1 is drawn to a method for obtaining a plant with a modified genomic target site in the genome of a somatic cell in a plant leaf tissue by using a Cas endonuclease to introduce a double stranded break at the target site, a guide RNA comprising a sequence sharing (reads on to “flanking”) homology with a genomic target site, and at least two morphogenic polypeptides to stimulate modification of the genomic target site, inserting the donor DNA into the target site via homology-dependent repair of the double stranded break (as recited in instant claim 4), and obtaining an embryogenic cell followed by regenerating a plant with the modified genomic target site.
Reference claims 5 and 13 recite, “… Wuschel (WUS) polypeptide” and “Babyboom (BBM) polypeptide”. WUS and BBM are described by the Applicant as morphogenic factors and recited in in instant claim 6.
However, reference claim 1 does not describe any anti-regeneration gene. It also does not describe using two distinct Rhizobiales bacteria comprising a first vector and a second vector in two distinct bacteria. Is also does not teach any ratio of the first bacteria and the second bacteria, or the first vector and the second vector.
He et al. describes self-elimination of transgene(s) using the Transgene Killer CRISPR system (TKC technology) (p. 93, left column, para 2). He et al. teaches the use of the anti-regeneration BARNASE gene, which effectively kill plant cells (lethal) when expressed inside cells (p. 93, right column, para 2, line 2-3), under the control of REG2 promoter which is active during the early embryo development stage in plants (e.g. rice) (p. 93, right column, para 2, line 11-13), as described above. Thus, cells successfully undergone embryogenesis to make embryogenic cells will not be having any expression of the lethal BARNASE gene. He et al. also describes gene editing by agrobacterium-mediated transient expression of transgenes (p. 91, right column, para 2).
Liu et al. describes a two Agrobacterium strains (as recited in claim 9) harboring two plasmids, and each contains an independent T-DNA: the two strains/two plasmids method or the mixed-strain system (page 2, right column, first para). Liu et al. also describes various ratios ranging from 1:8 to 8:1 of the two T-DNA vectors from two different Agrobacterium strains and the 1:1 ratio perform the best by producing the highest co-transformation frequencies in T0 transformants of the mixed-strain system (page 9, Fig. 3), as recited in claim 1.
Before the effective filing date, it would have been obvious to a person with ordinary skill in the art to modify the method taught by claim 1 of ‘273 by transiently expressing the site-specific DNA modifying agent (i.e., Cas9) while expressing the anti-regeneration gene BARNESE under the control of REG2 promoter (as described by He et al.). The heterologous polynucleotide encoding the gene/polynucleotide of interest and all the other factors (viz. gRNA, site-specific DNA modifying enzyme, morphogenic factor, and the anti-regeneration gene) needed to achieve the genome modification at the target site in the host genome act in trans. Thus, it is an experimental design choice of the ordinarily skilled artisan to clone the polynucleotides encoding these factors in separate (T-DNA) vectors, which is then cloned in two (or more) separate agrobacteria. One of the vectors (interpreted as “first vector” cloned in “first bacteria”) comprises a heterologous polynucleotide (comprising a gene of interest), wherein the heterologous polynucleotide is flanked by polynucleotides comprising homology to a nucleotide sequence at the target site in the plant genome. The other vector (interpreted as “second vector” cloned in “first bacteria”) comprises a polynucleotide encoding a DNA-modifying agent (a Cas endonuclease like Cas9), as described by He et al. and Schaeffer et al., to edit one or more specific sequence(s) including endogenous gene(s) in the host genome while expressing a morphogenic factor (e.g. WUS or BBM), as recited by reference claims 1 and 5. Stable integration of the anti-regeneration BARNASE transgene (present in the second vector) would render the cell(s) non-viable and, thus, reduce regeneration frequency, as described by He et al. The site of the homologous recombination based gene-editing would be decided by the gRNA(s) (which can be cloned in any of the two vectors) while the heterologous polynucleotide (present in the first vector) would act as a template for homologous recombination as described by Schaeffer et al. Stable integration of the T-DNA region within the right and left border of the Ti Plasmid comprising nucleotide sequences encoding Cas9, the morphogenic factor, and the anti-regeneration factor (BARNESE gene) would be avoided while improving gene editing efficiency.
The ratio of the two Agrobacterium strains containing two different T-DNA vectors can be calibrated to increase transformation efficiency of genome edited plants according to the method described by Liu et al. (1) and an experimental design choice of an ordinarily skilled artisan without affecting the outcome in terms of modifying the target DNA site in the host genome.
Before the effective filing date of the invention, one ordinarily skilled artisan would have been motivated to transform a plant using two different T-DNA vectors using agrobacterium mediated transformation with a realistic goal to edit a specific sequence/gene in any commercially important plant genome and achieve higher transformation and regeneration efficiency by providing a morphogenic factor (WUS protein) in trans without integrating any foreign nucleotide sequence present in the second T-DNA vector comprising the Cas endonuclease (Cas9), the morphogenic factor (WUS), and the anti-regeneration (BARNESE) gene.
Regarding claim 5, reference claim 7-8 recite monocot and maize, respectively.
Regarding claim 6, reference claims 5 and 13 recite a Babyboom polypeptide.
Regarding claim 9, using agrobacterium is a routine and standard process, as also described by He et al. (p. 89. right column, para 2, line 5-6) and Liu et al. (page 2, right column, first para) to introduce one of more heterologous polynucleotide sequence(s) into a plant cell.
Regarding claim 55, reference claim 1 of ‘273 recites “… inserting the donor DNA into the genomic target site via homology-directed repair of the double-strand break…” (line 17-18).
Regarding claims 56-59, reference claim 1 recites Cas endonuclease (line 10 and 14) (as recited in instant claim 56) and guide RNA (line 11 and 14) (as recited in claims 57-59). It is known in the art that gRNA(s) act in trans and can be cloned any either of the two vectors or in both the vectors, as discussed above.
This is a provisional nonstatutory double patenting rejection.
Claims 1, 4, 6, 9 and 55-59 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 15-16, 20 and 24 of copending Application No. 17/053,663 (reference application, hereafter referred as ‘273) in view of He et al. and Liu et al.
Reference claim 15 recites: “A method for modifying a genomic target site of a plant cell, the method comprising providing to the plant cell:
(a) a first double-strand-break-inducing agent,
(b) a polynucleotide sequence, further comprising:
(i) a heterologous polynucleotide;
(ii) a set of two homology regions flanking the heterologous polynucleotide, wherein one homology region comprises a sequence sharing sufficient homology with the polynucleotide sequence upstream of the genomic target site and the other homology region comprises a sequence sharing sufficient homology with the polynucleotide sequence downstream of the genomic target site; and,
(iii) a second target site sequence that is recognized and cleaved by a second double-strand-break-inducing agent, wherein the second target site sequence is next to one of the homology regions of (b)(ii) but is not next to the heterologous polynucleotide of (b)(i);
wherein the second double-strand-break-inducing agent cleaves the second target site sequence to create a double-strand-break in the second polynucleotide sequence;
wherein the first double-strand-break-inducing agent creates a double-strand break at the genomic target site, and wherein the polynucleotide sequence of (b) promotes homologous recombination repair of the double-strand break at the first target site sequence.”
Reference claim 16 and 24 recites “double strand breaking agent” (which reads on to “DNA modifying agent”) and “a morphogenic factor”, respectively.
However, reference claims 15-16 and 24 do not describe any anti-regeneration gene. It also does not describe using two distinct Rhizobiales bacteria comprising a first vector and a second vector in two distinct bacteria. Is also does not teach any ratio of the first bacteria and the second bacteria, or the first vector and the second vector.
He et al. describes self-elimination of transgene(s) using the Transgene Killer CRISPR system (TKC technology) (p. 93, left column, para 2). He et al. teaches the use of the anti-regeneration BARNASE gene, which effectively kill plant cells (lethal) when expressed inside cells (p. 93, right column, para 2, line 2-3), under the control of REG2 promoter which is active during the early embryo development stage in plants (e.g. rice) (p. 93, right column, para 2, line 11-13), as described above. Thus, cells successfully undergone embryogenesis to make embryogenic cells will not be having any expression of the lethal BARNASE gene. He et al. also describes gene editing by agrobacterium-mediated transient expression of transgenes (p. 91, right column, para 2).
Liu et al. describes a two Agrobacterium strains harboring two plasmids, and each contains an independent T-DNA: the two strains/two plasmids method or the mixed-strain system (page 2, right column, first para). Liu et al. also describes various ratios ranging from 1:8 to 8:1 of the two T-DNA vectors from two different Agrobacterium strains and the 1:1 ratio perform the best by producing the highest co-transformation frequencies in T0 transformants of the mixed-strain system (page 9, Fig. 3), as recited in instant claim 1.
Before the effective filing date, it would have been obvious to a person with ordinary skill in the art to modify the method taught by claim 15-16 and 24 of ‘663 by expressing the site-specific DNA modifying agent (i.e., Cas9) while expressing the anti-regeneration gene BARNESE under the control of REG2 promoter (as described by He et al.). The heterologous polynucleotide encoding the gene/polynucleotide of interest and all the other factors (viz. gRNA, site-specific DNA modifying enzyme, morphogenic factor, and the anti-regeneration gene) needed to achieve the genome modification at the target site in the host genome act in trans. Thus, it is an experimental design choice of the ordinarily skilled artisan to clone the polynucleotides encoding these factors in separate (T-DNA) vectors, which is then cloned in two (or more) separate agrobacteria. One of the vectors (interpreted as “first vector” cloned in “first bacteria”) comprises a heterologous polynucleotide (comprising a gene of interest), wherein the heterologous polynucleotide is flanked by polynucleotides comprising homology to a nucleotide sequence at the target site in the plant genome. The other vector (interpreted as “second vector” cloned in “first bacteria”) comprises a polynucleotide encoding a DNA-modifying agent (a Cas endonuclease like Cas9), as recited by reference claim 15, to edit one or more specific sequence(s) including endogenous gene(s) in the host genome while expressing a morphogenic factor (e.g. WUS or BBM), as taught by reference claim 24. Stable integration of the anti-regeneration BARNASE transgene (present in the second vector) would render the cell(s) non-viable and, thus, reduce regeneration frequency, as described by He et al. The site of the homologous recombination based gene-editing would be decided by the gRNA(s) (which can be cloned in any of the two vectors) while the heterologous polynucleotide (present in the first vector) would act as a template for homologous recombination as described by Schaeffer et al. Stable integration of the T-DNA region within the right and left border of the Ti Plasmid comprising nucleotide sequences encoding Cas9, the morphogenic factor, and the anti-regeneration factor (BARNESE gene) would be avoided while improving gene editing efficiency.
The ratio of the two Agrobacterium strains containing two different T-DNA vectors can be calibrated to increase transformation efficiency of genome edited plants according to the method described by Liu et al. (1) and an experimental design choice of an ordinarily skilled artisan without affecting the outcome in terms of modifying the target DNA site in the host genome.
Before the effective filing date of the invention, it would have been obvious to an ordinarily skilled artisan and the artisan would have been motivated to transform a plant using two different T-DNA vectors using agrobacterium mediated transformation with a realistic goal to edit a specific sequence/gene in any commercially important plant genome and achieve higher transformation and regeneration efficiency by providing a morphogenic factor (WUS protein) in trans without integrating any foreign nucleotide sequence present in the second T-DNA vector comprising the Cas endonuclease (Cas9), the morphogenic factor (WUS), and the anti-regeneration (BARNESE) gene.
Regarding claim 6, reference claim 1 recites, “WUS morphogenic factor” (line 22).
Regarding claim 9, using agrobacterium is a routine and standard process, as also described by He et al. (p. 89. right column, para 2, line 5-6) and Liu et al. (page 2, right column, first para) to introduce one of more heterologous polynucleotide sequence(s) into a plant cell.
Regarding claim 55, reference claim 15 b(ii) reads on to instant claim 55.
Regarding claims 56-59, reference claim 20 recites, “… wherein the method further comprises providing a first guide RNA to the first polynucleotide, wherein the first guide RNA selectively hybridizes with a polynucleotide sequence at or near the first target site, and wherein the first guide RNA and the Cas endonuclease form a complex that nicks or cleaves the first target site.” It is known in the art that gRNA(s) act in trans and can be cloned any either of the two vectors or in both the vectors, as discussed above.
This is a provisional nonstatutory double patenting rejection.
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.
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Jay Chatterjee
Patent Examiner
Art Unit 1662
/Jay Chatterjee/Examiner, Art Unit 1662
/BRATISLAV STANKOVIC/Supervisory Patent Examiner, Art Units 1661 & 1662
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