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 .
Specification
The disclosure is objected to because of the following informalities: The disclosure is not fully legible (see Table 2-1, for example).
Appropriate correction is required.
Sequence Compliance
This application contains sequence disclosures that are encompassed by the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.821(a)(1) and (a)(2). However, this application fails to comply with the requirements of 37 CFR 1.821 through 1.825 because there are sequences in Tables 1, 2-1, 2-2, 3-1, 3-2, 4-1, 4-2, and 5, as well as Figure 1D of the drawings that do not contain a SEQ ID NO.
A complete response to this office action must correct the defects cited above regarding compliance with the sequence rules and a response to the action on the merits which follows.
The aforementioned instance of failure to comply is not intended as an exhaustive list of all such potential failures to comply in the instant application. Applicants are encouraged to thoroughly review the application to ensure that the entire application is in full compliance with all sequence rules. This requirement will not be held in abeyance.
Drawings
The drawings filed on 6/23/23 are objected to because 1) the drawings are not fully legible and 2) Figure 1D, for example, contains sequences that are encompassed by the definitions for nucleotide and/or amino acid sequences set forth in 37 CFR 1.821(a)(1) and (a)(2), but each sequence does not contain a SEQ ID NO., as explained in the “Sequence Compliance” section above. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-14 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.
The claims are directed to “allowing” a “sequence-specific nucleic acid cleaving molecule capable of sequence-specifically cleaving target sequences at two locations on genomic DNA” to act on genomic DNA. The method does not require introduction of any specific compound, but rather “allowing” a genus of compounds to “act” on genomic DNA. The specification only describes introduction of guide RNAs, a single species of “allowing” and “sequence-specific nucleic acid cleaving molecule capable of sequence-specifically cleaving target sequences at two locations on genomic DNA”.
The claim language is not definite because the metes and bounds of “allowing” and “acting” cannot be ascertained. It is not clear what step is required to allow the compound to act. For purposes of the instant examination, the claims are interpreted as requiring introduction or delivery of the molecule.
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-14 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The claims are directed to “allowing” any “sequence-specific nucleic acid cleaving molecule capable of sequence-specifically cleaving target sequences at two locations on genomic DNA”. The method does not require introduction of any specific compound, but rather “allowing” a genus of compounds to “act” on genomic DNA. The specification only describes introduction of guide RNAs, a single species of “allowing” and “sequence-specific nucleic acid cleaving molecule capable of sequence-specifically cleaving target sequences at two locations on genomic DNA”. The single species is not representative of the entire claimed genus.
Without further description of the structure required for the sequence-specific nucleic acid cleaving molecule to have the required function of being capable of sequence -specifically cleaving target sequences at two locations on genomic DNA, one would not be able to recognize which molecules are necessarily included or excluded from the recited genus and one would not be able to readily recognize that applicant was in possession of the entire genus at the time of filing.
Importantly, the instant claims are directed to allowing a single molecule to cleave two locations in the genomic DNA. The specification does not adequately describe the structure required for this single molecule to function as claimed.
The instant claims are directed to allowing any sequence-specific nucleic acid cleaving molecule capable of sequence-specifically cleaving target sequences at two locations on genomic DNA to act on genomic DNA, although the specification only discloses introduction of gRNAs.
The MPEP states that for a generic claim, the genus can be adequately described if the disclosure presents a sufficient number of representative species that encompass the genus. See MPEP § 2163. If the genus has a substantial variance, the disclosure must describe a sufficient variety of species to reflect the variation within that genus. See MPEP § 2163. Although the MPEP does not define what constitute a sufficient number of representative species, the courts have indicated what do not constitute a representative number of species to adequately describe a broad genus. In Gostelli, the courts determined that the disclosure of two chemical compounds within a subgenus did not describe that subgenus. In re Gostelli, 872, F.2d at 1012, 10 USPQ2d at 1618. Additionally, in Carnegie Mellon University v. Hoffman-La Roche Inc., Nos. 07-1266, -1267 (Fed. Cir. Sept. 8, 2008), the Federal Circuit affirmed that a claim to a genus described in functional terms was not supported by the specification’s disclosure of species that were not representative of the entire genus. Furthermore, for a broad generic claim, the specification must provide adequate written description to identify the genus of the claim. In Regents of the University of California v. Eli Lilly & Co. the court stated:
"A written description of an invention involving a chemical genus, like a description of a chemical species, 'requires a precise definition, such as by structure, formula, [or] chemical name,' of the claimed subject matter sufficient to distinguish it from other materials." Fiers, 984 F.2d at 1171, 25 USPQ2d 1601; In re Smythe, 480 F.2d 1376, 1383, 178 USPQ 279, 284985 (CCPA 1973) ("In other cases, particularly but not necessarily, chemical cases, where there is unpredictability in performance of certain species or subcombinations other than those specifically enumerated, one skilled in the art may be found not to have been placed in possession of a genus ...") Regents of the University of California v. Eli Lilly & Co., 43 USPQ2d 1398.
The claims are rejected under the written description requirement for failing to disclose adequate species to represent the claimed genus, the genus being “allowing” any “sequence-specific nucleic acid cleaving molecule capable of sequence-specifically cleaving target sequences at two locations on genomic DNA” to act in any manner on genomic DNA.
The Guidelines for Examination of Patent Applications under the 35 USC § 112, first paragraph, “Written Description” Requirement”, published at Federal Register, Vol. 66, No. 4, pp. 1099-1111 outline the method of analysis of claims to determine whether adequate written description is present. The first step is to determine what the claim as a whole covers, i.e., discussion of the full scope of the claim. Second, the application should be fully reviewed to understand how applicant provides support for the claimed invention including each element and/or step, i.e., compare the scope of the claim with the scope of the description. Third, determine whether the applicant was in possession of the claimed invention as a whole at the time of filing.
To achieve the desired function, it appears that the structure is required to be a gRNA with specific structural relationship to the target. For example, Matson et al. (PLOS ONE, 2019, 14(12): e0226107, 1-16) teach: This study highlights a strategy for minimizing off-target events when employing CRISPR/ Cas9 for potential use in the field of xenotransplantation for GTKO. Moreover, analysis of potential OTC may be critical to improving efficiency while maintaining the integrity of cells intended for use in functional screening of mutated genes in cells or the production of GE animals. OTM occurring in cells used to create GE animals could lead to the development of Optimization of sgRNA length to improve CRISPR/Cas9 specificity for animals with unknown gene modifications with the potential to affect both the donor animal and recipients of xenografts. No OTM were found in any of the GTKO cell lines or the pig we screened leading us to believe that if OTM occur in critical genes those cells may not develop and survive in culture. Findings presented here underscore the importance of evaluating and screening guide efficiency and specificity to produce GTKO GE cells, as well as the importance of PAM site selection and its role in optimizing both efficiency and specificity of the CRISPR/ Cas9 gene editing system (pages 13 and 14).
Thus, having analyzed the claims with regard to the Written Description guidelines, it is clear that the specification does not disclose a representative number of species for agents within the instant enormous genus that function as claimed. Thus, one skilled in the art would be led to conclude that Applicant was not in possession of the claimed invention at the time the application was filed.
Claims 1-14 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 an in vitro method comprising introducing gRNAs via the construct of the instant specification with resultant DNA region deletions, does not reasonably provide enablement for allowing by any mode any sequence-specific nucleic acid cleaving molecule with resultant DNA region deletions. 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.
Factors to be considered in a determination of lack of enablement 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)
The claims are directed to “allowing” any “sequence-specific nucleic acid cleaving molecule capable of sequence-specifically cleaving target sequences at two locations on genomic DNA”. The method does not require introduction of any specific compound, but rather “allowing” a genus of compounds to “act” on genomic DNA. The specification only discloses introduction of guide RNAs, a single species of “allowing” and “sequence-specific nucleic acid cleaving molecule capable of sequence-specifically cleaving target sequences at two locations on genomic DNA”. The single species is not commensurate in scope or enabling for the claimed genus.
The specification demonstrates transfection of cells in vitro with a specific construct comprising gRNAs that are complementary to a specific target, which is not enabling for a method of “allowing” any “sequence-specific nucleic acid cleaving molecule capable of sequence-specifically cleaving target sequences at two locations on genomic DNA” to act on genomic DNA and result in DNA region deletions.
The specification does not draw an adequate nexus between delivery of any possible “sequence-specific nucleic acid cleaving molecule capable of sequence-specifically cleaving target sequences at two locations on genomic DNA” and the recited outcome.
The scope of the claims in view of the specification as filed together do not reconcile the unpredictability in the art to enable one of skill in the art to make and/or use the claimed invention, namely a broad method of introducing multiple DNA region deletions with a genus of compounds that have not been adequately described or demonstrated.
MPEP 2164.01
Any 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.
Also, MPEP 2164.01(a)
A conclusion of lack of enablement means that, based on the evidence regarding each of the above factors, 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 without undue experimentation. In re Wright, 999 F.2d 1557,1562, 27 USPQ2d 1510, 1513 (Fed. Cir. 1993).
Given the teachings of the specification as discussed above, one skilled in the art could not predict a priori whether “allowing” any “sequence-specific nucleic acid cleaving molecule capable of sequence-specifically cleaving target sequences at two locations on genomic DNA” by the broadly disclosed methodologies of the instantly claimed invention, would result in successful DNA region deletions. To practice the claimed invention, one of skill in the art would have to de novo determine; the stability of the molecule, the structure of the molecule, delivery of the molecule to the cell, and entry of the molecule into the cell and the effective action therein. Without further guidance, one of skill in the art would have to practice a substantial amount of trial and error experimentation, an amount considered undue and not routine, to practice the instantly claimed invention.
A conclusion of lack of enablement means that, based on the evidence regarding each of the above factors, 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 without undue experimentation (see MPEP 2164.01(a)).
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1-14 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zheng et al. (BioTechniques. 2014, vol. 57, pp. 115-124), in view of Paulsen et al. (Nat Biomed Eng., 2017, 1(11): 878–888), Cai et al. (Int. J. Mol. Sci. 2018, 19, 3835, 1-13), and Kmiec et al. (WO 2018/132390 A1).
Zheng et al. teach: The prokaryotic type II CRISPR/Cas9 system has been adapted to perform targeted genome editing in cells and model organisms. Here, we describe targeted gene deletion and replacement in human cells via the CRISPR/Cas9 system using two guide RNAs. The system effectively generated targeted deletions of varied length, regardless of the transcriptional status of the target gene. It is notable that targeted gene deletions generated via CRISPR/Cas9 and two guide RNAs resulted in the formation of correct junctions at high efficiency. Moreover, in the presence of a homology repair donor, the CRISPR/Cas9 system could guide precise gene replacement. Our results illustrate that the CRISPR/Cas9 system can be used to precisely and effectively generate targeted deletions or gene replacement in human cells, which will facilitate characterization of functional domains in protein-coding genes as well as noncoding regulatory sequences in animal genomes (abstract).
Zheng et al. therefore teaches that a targeted DNA region in human cells was deleted with a CRISPR/Cas9 system that uses two single-guide RNA (sgRNA) molecules, and teaches that utilizing said method can enable the analysis of functional domains in protein-coding genes as well as noncoding regulatory sequences (abstract; figure 1, etc.).
Therefore, utilization of a CRISPR/Cas9 system with two sgRNAs when deleting a targeted DNA region was already known. Instant claim 1 differs in that there is a step of determining the influence of the DNA region deletion on cell proliferation and survival.
Although Zheng et al. do not explicitly state this step, Zheng et al. do teach that the technique of deleting a targeted region of DNA will enable the analysis of functional domains in protein-coding genes as well as noncoding regulatory sequences, and therefore a person skilled in the art could easily investigate cell growth or cell survival as effects of deleting the DNA region. When performing functional domain analysis, it would have been obvious to determine if the deletion has an influence on cell proliferation or survival, and the magnitude of the effect as a matter of design choice. Additionally, it is routine in the gene deletion field to perform such methods for the purpose of effecting cell proliferation and survival.
Additionally, selecting a size of region to be deleted is routine and obvious in view of Zheng et al. that teaches the steps necessary to perform targeted gene deletion (Figure 1).
Moreover, during that process a person skilled in the art could, as appropriate, investigate whether or not the deleted DNA region contains a gene that regulates cell survival or growth by comparison with a cell population serving as a control, and could choose the region to be deleted as the region between two cleavage sites, such as a marker gene for negative selection, depending on the intended purpose. Performing such determination by estimating a ratio or counting is considered a matter of design choice. Furthermore, the abovementioned configuration cannot be considered to exhibit a particularly prominent advantageous effect that could not be envisioned by a person skilled in the art.
Zheng et al. teach that in the presence of a homologous repair donor, the CRISPR/Cas9 system may be used to generate precise and defined modifications and insertions at a targeted locus through the HDR process (page 115). Therefore, it would have been obvious to manipulate the system to perform a deletion or insertion as desired.
Additionally, Paulsen et al. teach that ectopic expression of RAD52 and dn53BP1 improves homology-directed repair during CRISPR–Cas9 genome editing (title). Paulsen et al. teaches: Gene disruption by clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR associated protein 9 (Cas9) is highly efficient and relies on the error-prone non-homologous end joining pathway. Conversely, precise gene editing requires homology-directed repair (HDR), which occurs at a lower frequency than non-homologous end-joining in mammalian cells. Here, by testing whether manipulation of DNA repair factors improves HDR efficacy, we show that transient ectopic co-expression of RAD52 and a dominant-negative form of tumor protein p53- binding protein 1 (dn53BP1) synergize to enable efficient HDR using a single-stranded oligonucleotide DNA donor template at multiple loci in human cells, including patient-derived induced pluripotent stem cells. Co-expression of RAD52 and dn53BP1 improves multiplexed HDR-mediated editing, whereas expression of RAD52 alone enhances HDR with Cas9 nickase. Our data show that the frequency of non-homologous end-joining-mediated double-strand break repair in the presence of these two factors is not suppressed and suggest that dn53BP1 competitively antagonizes 53BP1 to augment HDR in combination with RAD52. Importantly, coexpression of RAD52 and dn53BP1 does not alter Cas9 off-target activity. These findings support the use of RAD52 and dn53BP1 co-expression to overcome bottlenecks that limit HDR in precision genome editing (Abstract).
Paulsen et al. teaches incorporation of a control sequence (page 10).
Therefore, Paulsen et al. offers motivation with a reasonable expectation of success in ectopically introducing at least one gene that controls cell survival and/or proliferation to the genomic DNA having the DNA region deletion, the gene being operably linked to a control sequence.
It would have been obvious for the gRNAs to be at two different locations thereby causing a DNA region deletion in two locations as a matter of design choice, as it was known that multiple gRNAs at different locations can be utilized simultaneously (see Figures 2 and 3 of Zheng et al.).
Further evidence of this is taught by Cai et al. Cai et al. teaches: we employed CRISPR/Cas9 technology to specifically induce targeted deletions of DNA fragments in GmFT2a (Glyma16g26660) and GmFT5a (Glyma16g04830) in soybean using a dual-sgRNA/Cas9 design. We achieved a deletion frequency of 15.6% for target fragments ranging from 599 to 1618 bp in GmFT2a. We also achieved deletion frequencies of 12.1% for target fragments exceeding 4.5 kb in GmFT2a and 15.8% for target fragments ranging from 1069 to 1161 bp in GmFT5a. In addition, we demonstrated that these CRISPR/Cas9-induced large fragment deletions can be inherited. The T2 ‘transgene-free’ homozygous ft2a mutants with a 1618 bp deletion exhibited the late-flowering phenotype. In this study, we developed an efficient system for deleting large fragments in soybean using CRISPR/Cas9; this system could benefit future research on gene function and improve agriculture via chromosome engineering or customized genetic breeding in soybean (abstract).
Cai et al. teach: To obtain targeted fragment deletions in GmFT2a and GmFT5a, three combinatory dual-sgRNA/Cas9 vectors—GmFT2a-D1/D2, GmFT2a-D1/D3, and GmFT5a-D1/D2—were used to delete the DNA fragments between each pair of sgRNAs (Figure 1D). Therefore, it was known to design combinatory dual-sgRNA/Cas9 vectors for gene editing. It would have been obvious to employ combinatory dual-sgRNA/Cas9 vectors to delete multiple sites simultaneously with a reasonable expectation of success.
Kmiec et al. is additional evidence that it was known to perform multiple deletions simultaneously, as well as insertions in genomic DNA via utilizing gRNAs. FIG. 27C displays a DNA sequence in which three NotI fragment insertions accompanied by multiple deletions surrounding the inserted NotI fragment; a single base pair deletion upstream from the first inserted fragment and a seven base pairs deletion downstream from the third inserted fragment. Approximately 8% of the plasmid DNA had no alteration in the DNA sequence as seen in FIG. 27D.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Amy R Hudson whose telephone number is (571)272-0755. The examiner can normally be reached M-F 8:00am-6:00pm.
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/AMY ROSE HUDSON/Primary Examiner, Art Unit 1636