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 1-20 are pending.
Claims 1-20 are examined on the merits.
Claim Rejections - 35 USC § 112
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.
Scope of Enablement
Claims 1-20 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specifications, while being enabling for certain embodiments of an engineered genetic incompatibility (EGI) system in Arabidopsis thaliana employ CRISPR-based programmable transcription activators to activate the endogenous WUSCHEL (UUS) gene through a specifically engineered promoter containing a limited number of mutations; does not reasonably provide enablement for the full scope of the claimed invention. 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.
An “analysis of whether a particular claim is supported by the disclosure in an application requires a determination of whether that disclosure, when filed, contained sufficient information regarding the subject matter of the claims as to enable one skilled in the pertinent art to make and use the claimed invention.” MPEP 2164.01. “A conclusion of lack of enablement means that. . . the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention [i.e. commensurate scope] without undue experimentation.” In re Wright, 999 F.2d 1557,1562, 27 USPQ2d 1510, 1513 (Fed. Cir. 1993); MPEP 2164.01.
In In re Wands, 858 F.2d 731,8 USPQ2d 1400 (Fed. Cir. 1988), several factors implicated in determination of whether a disclosure satisfies the enablement requirement and whether any necessary experimentation is “undue” are identified. These factors include, but are not limited to:
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. In re Wands, 858 F.2d 731,737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988). No single factor is independently determinative of enablement; rather “[i]t is improper to conclude that a disclosure is not enabling based on an analysis of only one of the above factors while ignoring one or more of the others.” MPEP 2164.01. Likewise, all factors may not be relevant to the enablement analysis of any individual claim.
Under the broadest reasonable interpretation consistent with the specification, claim 1 is directed to a transgenic plant cell comprising a functionally coordinated biocontainment system. The system requires a coding region whose overexpression alters cell growth or impairs cell division, an upstream transcription regulatory region comprising a mutation, and a polynucleotide encoding a programmable transcription activator that does not initiate overexpression from the mutated regularity region but, after sexual reproduction, activates the corresponding nonmutated regulatory region and thereby overexpresses the coding region.
Claim 1 is not limited to a particular plant species, cell type, coding region, regulatory region, mutation, programmable transcription activator, activation domain, guide sequence, degree of overexpression, issue, developmental stage, or phenotype. The claim therefore encompasses a broad functional genus extending across multiple technical dimensions, including the identity and dosage sensitivity of the coding region, the location and architecture of the regulatory target site, the type and position of the protective mutation, the binding specificity and activation strength of the programmable transcription activator, the plant species and cellular context, the inheritance and expression of the system after sexual reproduction, and the resulting growth or cell-division phenotype. These variables must operate together in a coordinated manner to satisfy the claim.
The specification provides a proof-of-concept embodiment involving Arabidopsis thaliana, the endogenous WUSHEL gene, WUS-sgRNA1, one-base A or T insertions within the WUS-sgRNA1 binding site, and a dCas9-based MoonTag programmable transcription activator. The disclosure further describes crossing the engineered plants with wild-type Arabidopsis and observing increased WUSCHEL expression and variable developmental phenotypes in at least some hybrid progeny.
The disclosed experiments, however, demonstrate substantial context dependence. Four sgRNAs directed to sites within an approximately 250-nuclearotide region upstream of the WUSCHEL transcriptional start site were tested, but WUS-sgRNA1 produced activation more than three orders of magnitude greater than the other guide RNAs. Different transformed EGI lines produced different levels of programmable-transcription-activator expression and different levels of WUSCHEL activation. The crosses also produced variable frequencies and severities of phenotype, including smaller seedling, cotyledon browning, ectopic embryos, yellowing, delayed germination, slower growth, and defects in flowers and siliques. Some seedlings died, while others survived.
The specification does not provide a generally applicable rule by which a skilled artisan could predictably select a coding region throughout the claimed scope whose overexpression would produce the recited effect in a desired plant cell. Although numerous classes of coding regions are listed, including developmental, cytoskeletal, ER-Golgi vesicle, mRNA-processing, electron-transport, nuclear-trafficking, chromosome-segregation, spindle-pole-duplication, and oxidative-stress polypeptides, the disclosure does not establish that members through those classes share a common structural or functional property making them suitable targets for the claimed system.
The specification likewise does not provide a predictive rule for selecting an effective regulatory target site. The application’s own results show that target sites within the same promoter are not functionally equivalent. Nor does the disclosure provide a general method for selecting a mutation that both prevents activation by the programmable transcription activator and preserves sufficient native expression and function of the associated coding region. Only two one-nucleotide insertions at one WUS target site were demonstrated.
The disclosure further does not establish a generally applicable correlation among programmable-transcription-activator architecture, activation-domain identity, guide sequence, target-site location, chromatin environment, plant species, target-gene expression level, and resulting phenotype. A skilled artisan could not determine from the disclosure alone whether a proposed combination would provide insufficient activation, inappropriate activation in the engineered parent, loss of ordinary target-gene expression, failure to activate the inherited nonmutated allele, or an effect too weak to satisfy the claim.
Accordingly, to practice the full scope of claim 1, a skilled artisan would need to identify candidate expression-sensitive genes, experimentally test their overexpression, identify and screen regulatory target sites, design and compare multiple guides or binding domains, generate and test candidate mutations, verify preservation of native gene function, construct and introduce the programmable transcription activator, screen independent transformation events, identify lines having suitable expression levels, conduct sexual crosses, genotype the progeny, quantify target-gene activation, and evaluate growth, development, fertility, and variability over multiple lines and generations. Although individual procedures may have been know, the disclosure does not teach which combinations will work, and successful embodiments would have to be discovered through iterative screening.
The breadth of the claims is extensive, and the claim is multidimensional because numerous independent biological and molecular variables must function together. The nature of the invention involves coordinated regulation of endogenous gene expression, mutation-dependent sequence discrimination, plant transformation, inheritance, sexual reproduction, and phenotypic outcome. The application’s own results demonstrate unpredictability in guide performance, transformation-line behavior, activation level, and phenotype. The specification provides detailed guidance of the particular Arabidopsis/WUSCHEL embodiment but does not provide predicative rules or representative working examples sufficient to enable the remaining genus. The quantity of experimentation required is therefore not limited to routine verification but would involve substantial iterative screening to discover operative combinations.
Claims 2-16 do not cure these deficiencies. These claims narrow individual features, such as the cell type, Cas9-based activator, MoonTag or VP64 activation domain, silent mutation, coding-region class, WUSCHEL or LEC1, lethality, comparative growth phenotype, whole plant or crop category, but they do not narrow the claims to the particular operative embodiment demonstrated int eh specification. Claims 9-11 further increases the required coordination by reciting a second biocontainment system, yet the specification does not provide a working example of a plant cell containing two independently operative system or sufficient guidance for selecting two compatible target genes, two regulatory target sites, two protective mutations, and two non-cross-reactive programmable transcription activators. Claims 15 and 16 extend the system to whole plants and broad crop categories, although the complete system was demonstrated only in Arabidopsis thaliana.
Claim 17 is directed to a method of limiting hybridization by providing a genetically modified plant comprising the biocontainment system of the preceding claims, wherein crossing the genetically modified plant with a genetically dissimilar variant results in progeny exhibiting phenotype distance from the genetically modified plant. Thus, claim 17 incorporates the broad and multidimensional biocontainment system discussed above the respect to claim 1 and further extends the claimed scope to hybridization between genetically modified plants and genetically dissimilar variants across diverse plant species and genetic backgrounds.
As discussed above with respect to claim 1, the specification provides only a proof-of-concept embodiment using a WUSCHEL-based engineered genetic incompatibility system in Arabidopsis thaliana. Although the application demonstrate crosses between particular Arabidopsis EGI lines and wild-type Arabidopsis, the disclosure does not provide sufficient guidance to enable a person of ordinary skill in the art to predictably implement the claimed method across the full scope of genetically modified plants, genetically dissimilar variants, coding regions, transcriptional regulatory regions, mutations, programmable transcription activators, and resulting hybrid phenotypes encompassed by the claim. Rather, a skilled artisan would first need to identify an operative biocontainment system and then determine whether the desired post-cross phenotypes is achieved in each plant species and genetic background, requiring substantial iterative screening and experimentation.
Claims 18-29 do not overcome these deficiencies. Limiting the genetically dissimilar variant to a wild-type plant, to a differently genetically modified plant, or requiring the progeny phenotype to comprise lethality merely narrows individual aspects of the method but does not provide sufficient guidance to enable the full scope of the claimed genus.
Accordingly, while the specification enables certain proof-of-concept embodiments involving a WUSCHEL-based engineered genetic incompatibility stem in Arabidopsis thaliana, it does not enable the full scope of the claimed invention. The speciation does not provide sufficient representative embodiments, productive guidance, or generally applicable design principles that would allow a person of ordinary skill int the art to identify and coordinate the claimed coding regions, transcription regulatory regions, mutations, programmable transcription activators, and plant systems throughout the full claimed scope without undue experimentation.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claims 1-6 and 8-20 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Maselko (Maciej Maselko et. al., US20180327762A1, Application of 2016-11-10, Publication of 2018-11-15).
Claim 1 recites a transgenic plant cell, the cell comprising a biocontainment system that comprises: a coding region whose overexpression alters growth of the cell or impairs cell division; a transcription regulatory region operably linked upstream of the coding region, and comprising a mutation; and a polynucleotide that encodes a programmable transcription activator engineered to bind to the transcription regulatory region in the absence of the mutation after sexual reproduction, thereby overexpressing the coding region in the absence of the mutation, but does not initiate overexpression of the coding region when the transcription regulatory region comprises the mutation.
Maselko discloses a cell comprising a biocontainment system having a coding region whose overexpression alters growth of the cell, a transcription regulatory region operably linked upstream of the coding region and comprising a mutation, and a polynucleotide encoding a programmable transcription activator engineered to bind to the transcription regulatory region in the absence of the mutation, thereby overexpressing the coding region when the mutation is absent but not initiating overexpression when the regulatory region comprises the mutation (Abstract; pa0003-0004, 0026-0027, 0039 and 0042-0047; claim 1).
Maselko further discloses that, upon sexual reproduction with a wild-type organism, the programmable transcription activator binds the nonmutated regulatory sequence contributed by the wild-type parent and causes overexpression of the associated coding region (pa0014-0015, 0027, 0035-0039, and 0042).
Although Maselko’s experimental example uses Saccharomyces cerevisiae, Maselko expressly includes the disclosed biocontainment system may be introduced into cells of a multicellular organism, including a plant, and identifies tobacco, corn, soybean, rice, poplar, rubber tree, and creeping bentgrass as exemplary plant hosts. Thus, Maselko expressly discloses a transgenic plant cell comprising the claimed biocontainment system.
Claim 2 recites the transgenic plant cell of claim 1, wherein the cell is a germ cell.
Maselko discloses that the cell comprising the biocontainment system may be a germ cell of a multicellular organism and expressly identifies plants as suitable multicellular organisms (pa0005, 0040-0042; claim 3).
Claim 3 recites the transgenic plant cell of claim 1, wherein the programmable transcription activator comprises dCas9 fused to an activation domain. Claim 4 recites the transgenic plant cell of claim 3, wherein the activation domain comprises MoonTag or VP64.
Maselko discloses that the programmable transcription activator comprises dCas9 fused to an activation domain, including dCas9-VP64 (pa0006, 0026, and 0046; claim 4). Maselko discloses VP64 as the activation domain (pa0006, 0026, 0031-0032 and 0046).
Claim 5 recites the transgenic plant cell of claim 1, wherein the mutation comprises a silent mutation.
Maselko discloses that the transcription regulatory region comprises a silent mutation. Maselko defines a silent mutation as one that does not significantly alter the organism’s phenotype outside the biocontainment system and demonstrates a single-nucleotide ACT1-promoter deletion that did not substantially change native expression but prevented targeted transcriptional activation (pa0003, 0032, 0042, and 0045; claim 1).
Claim 6 recites the transgenic plant cell of claim 1, wherein the coding region encodes a developmental polypeptide, a cytoskeletal polypeptide, an ER-Golgi vesicle polypeptide, an mRNA processing polypeptide, an electron transport polypeptide, a nuclear trafficking polypeptide, a chromosome segregation polypeptide, a spindle pole duplication polypeptide, or an oxidative stress polypeptide.
Maselko discloses coding regions encoding cytoskeletal, ER-Golgi vesicle, mRNA-processing, electron-transport, nuclear-trafficking, chromosome-segregation, spindle-pole-duplication, and oxidative-stress polypeptides (pa0007, 0030, 0033, and 0048; claim 5).
Claim 8 recites the transgenic plant cell of claim 1, wherein overexpression of the coding region is lethal to the cell.
Maselko discloses that overexpression of the coding region may be lethal to the cell. Maselko further demonstrates that cells resulting form an incompatible mating undergo limited divisions, swelling, and lysis (pa0008, 0027, 0033, 0038-0039, 0044, and 0047; claim 6).
Claim 9 recites the transgenic plant cell of claim 1, further comprising a second biocontainment system comprising: a second coding region whose overexpression decreases growth of the cell or impairs cell division; a second transcription regulatory region operably linked upstream of the second coding region, and comprising a second mutation; a polynucleotide that encodes a second programmable transcription activator engineered to bind to the second transcription regulatory region in the absence of the second mutation, thereby overexpressing the second coding region in the absence of the second mutation, but does not initiate overexpression of the second coding region when the second transcription regulatory region comprises the second mutation.
Maselko discloses a cell comprising a second biocontainment system having a second coding region whose overexpression decreases growth, a second upstream regulatory region comprising a second silent mutation, and a second programmable transcription activator direct to the corresponding nonmutated regulatory region. Maselko further discloses that multiple orthogonal circuits may be introduced into the same organismal background (pa0009, 0027, and 0049; claim 7).
Claim 10 recites the transgenic plant cell of claim 9, wherein the second mutation comprises a silent mutation.
Maselko’s second biocontainment system expressly includes a second silent mutation (pa0049; claim 7).
Claim 11 recites the transgenic plant cell of claim 9, wherein the second coding region encodes a developmental polypeptide, a cytoskeletal polypeptide, an ER-Golgi vesicle polypeptide, an mRNA processing polypeptide, an electron transport polypeptide, a nuclear trafficking polypeptide, a chromosome segregation polypeptide, a spindle pole duplication polypeptide, or an oxidative stress polypeptide.
Maselko discloses that the coding regions used in the biocontainment systems may encode the same cytoskeletal, ER-Golgi vesicle, mRNA-processing, electron-transport, nuclear-trafficking, chromosome-segregation, spindle-pole-duplication, and oxidative-stress polypeptide classes recited in the claim (pa0030, 0033, 0048-0049; claim 8).
Claim 12 recites the transgenic plant cell of claim 1, wherein the altered cell growth comprises a decrease in growth rate of a cell heterozygous for the biocontainment system compared to a suitable control.
Maselko discloses that a cell or organism heterozygous for the biocontainment system may exhibit a decreased growth rate relative to a suitable control, while and organism homozygous for the system grows normally (pa0004, 0042-0044; claim 9).
Claim 13 recites the transgenic plant cell of claim 1, wherein the altered cell growth comprises an increase in growth rate of a cell heterozygous for the biocontainment system, the increase in growth rate decreasing fitness of the cell compared to a suitable control.
Maselko discloses that a heterozygous cell may exhibit increased growth that nevertheless decrease fitness relative to a suitable control, including growth resulting in deformity or death (pa0043-0044; claim 10).
Claim 14 recites transgenic plant cell of claim 12, wherein the suitable control comprises a wild-type cell or a cell homozygous for the biocontainment system.
Maselko discloses comparison of the altered growth of the heterozygous cell with a wild-type cell or with a cell homozygous for the biocontainment system (claims 11, 12, 17 and 18).
Claim 15 recites a plant comprising the transgenic plant cell of claim 1.
Maselko discloses a plant comprising cells containing the disclosed biocontainment system (pa0022-0025, and pa0040-0042).
Claim 16 recites the plant of claim 15, wherein the plant is an oil-producing plant, a vegetable, a fruit, or a grain.
Maselko discloses soybean as an exemplary plant host, thereby disclosing an oil-producing plant, and identifies corn and rice, thereby disclosing grain plants (pa0040).
Claim 17 recites a method of limiting hybridization of a genetically-modified plant with a genetically dissimilar variant, the method comprising: providing a plant genetically modified to comprise the biocontainment system of any preceding claim, wherein a cross between the genetically-modified plant and the genetically dissimilar variant plant results in progeny that exhibit a phenotype that is distinct from the genetically-modified plant.
Maselko discloses a method of limiting hybridization of a genetically modified organism with a genetically dissimilar variant by providing an organism genetically modified to include the disclosed biocontainment syst, wherein crossing the genetically modified organism with the genetically dissimilar variant produced progeny exhibiting a phenotype distinct from the genetically modified organism (pa0010, 0022, 0027, 0039, and 0042-0044; claim 13).
Maselko further discloses application of the biocontainment system to plants and explains that the system may reduce unwanted genetic recombination and gene flow between genetically modified crop varieties and other plant variants (pa0022-0025, and 0040).
Thus, Maselko discloses the claimed method of limiting hybridization between a genetically modified plant and a genetically dissimilar plant variant.
Claim 18 recites the method of claim 17 wherein the genetically dissimilar variant comprises a wild-type plant.
Maselko discloses that the genetically dissimilar variant may be a wild-type organism (pa0011, 0027; and claim 14).
Claim 19 recites the method of claim 17 wherein the genetically dissimilar variant comprises a different genetic modification compared to the genetically-modified plant having the biocontainment system.
Maselko discloses that the genetically dissimilar variant may comprise a different genetic modification from the genetically modified organism containing the biocontainment system (pa0011, 0012; and claim 15).
Claim 20 recites the method of claim 17 wherein the phenotype exhibited by the progeny comprises lethality.
Maselko discloses that the phenotype exhibited by the progeny may comprise lethality or nonviability (pa0012, 0027; and claim 16).
Accordingly, Maselko discloses each and every limitation of claims 1-6 and 8-20, as required by the claims, and therefore anticipated the claims 1-6 and 8-20.
Claim Rejections - 35 USC § 103
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.
Claim 7 is rejected under 35 U.S.C. §103 as being unpatentable over Maselko (US20180327762A1) as apply to claim 6, and in view of Zinselmeier (Matthew H Zinselmeier et. al., Plant Biotechnology Journal (3/19/2024), 22(11): 3202-3204, pp1-29).
Claim 6 as the teachings of Maselko are discussed above.
Claim 7 is interpreted as dependent of claim 6.
Claim 7 recites the transgenic plant cell of claim 6, wherein the coding region is the WUSCHEL gene or the LECI gene.
For the same reason set forth above with respect to claim 16, Zinselmeier teaches programmable transcriptional activation of the endogenous plant WUSCHEL (WUS) gene in plant cells, demonstrating that WUS is a suitable target coding region for activation by programmable transcription activator (Abstract, p2 “Building a library of putative plant-derived transcriptional activation domains”).
Therefore, it would have been obvious to select WUSCHEL as the coding region in the transgenic plant cell of Maselko.
Accordingly, claim 7 would have been obvious over Maselko and Zinselmeier.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to YANXIN SHEN whose telephone number is (571)272-7538. The examiner can normally be reached Monday-Friday.
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/YANXIN SHEN/ Examiner, Art Unit 1663
/WEIHUA FAN/Primary Examiner, Art Unit 1663
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