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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 January 6, 2026 has been entered.
Application Status and Withdrawn Rejections
Applicant’s amendment filed January 6, 2026 amending claims 1, 18 and 23-26, and adding new claims 36 and 37 is acknowledged. Claims 1, 6, 10-15, 18-28, 30 and 32-37 are pending and under examination.
The claims amendments overcome the objections and §112(b) rejections of record.
Applicant’s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Claim Objections
Claim 18 is objected to because of the following informalities:
Claim 18 recites “wherein the plurality of nucleic acids comprising at least one…” which is grammatically incorrect. “comprising” should be changed to “comprises”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(a) – New Matter
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.
Claim 37 is 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. This is a NEW MATTER rejection.
MPEP 2163.II.A.3.(b) states, “when filing an amendment an applicant should show support in the original disclosure for new or amended claims” and “[i]f the originally filed disclosure does not provide support for each claim limitation, or if an element which applicant describes as essential or critical is not claimed, a new or amended claim must be rejected under 35 U.S.C. 112a, as lacking adequate written description". According to MPEP § 2163.I.B, "While there is no in haec verba requirement, newly added claim limitations must be supported in the specification through express, implicit, or inherent disclosure" and "The fundamental factual inquiry is whether the specification conveys with reasonable clarity to those skilled in the art that, as of the filing date sought, applicant was in possession of the invention as now claimed. See, e.g., Vas-Cath, Inc., 935 F.2d at 1563-64, 19 USPQ2d at 1117".
In the instantly rejected claims, the new limitation of “wherein said pharmaceutical composition is a therapeutic agent effective in reducing or ameliorating at least one pathological symptom when administered to a patient” appears to represent new matter. No specific basis for this limitation was identified in the specification, nor did a review of the specification by the examiner find any basis for the limitation. The Specification recites various “methods” (pages 31-38) and “applications” (pages 38-42), including generic “therapeutic” applications ([00102]) and to correct genetic mutations associated with a disease by making genomic modifications to cells ex vivo ([00105]). The specification also provides that pharmaceutical compositions can include a drug as a therapeutic agent ([00107]), but not that the Cas9 protein is itself a therapeutic agent. The Specification does not recite administering pharmaceutical compositions to patients or ameliorating pathological symptoms. Since no basis has been identified, the claims are rejected as incorporating new matter.
Response to Arguments – New Matter
Applicant argues that support for new claim 37 can be found in the specification as published in [0125]). This argument has been fully considered but is not persuasive. Paragraph [0125] is US 20230058352 A1 is equivalent to paragraph [00107] in the filed Specification. As indicated in the rejection above, paragraph [00107] on discloses that pharmaceutical compositions can include a drug such as a therapeutic agent. It does not disclose that Cas9/gRNA is the drug that is the therapeutic agent. Additionally, a thorough review of the Specification found no disclosure of administering the SpCas9 variants to patients or for the purpose of reducing/ameliorating pathological symptoms.
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.
Claims 1, 10-15, 18-23, 25-28, 30 and 32-35 are rejected under 35 U.S.C. 103 as being unpatentable over Cereseto (US 20200149020 A1, priority to at least February 14, 2018, of record), in view of Vakulskas (US 20180100148 A1, published April 12, 2018, of record) and Joung (US 20170058271 A1, published March 2, 2017, of record). Claims 22 and 25 is evidenced by DiCarlo (DiCarlo et al., Nucleic Acids Research (2013), 41: 4336-4343, of record).
Regarding claim 1, Cereseto teaches previous SpCas9 variants with high efficiency and reduced off-target activity include a K855A substitution ([0005]). Cereseto teaches making triple substitutions in SpCas9 (Fig 5). Cereseto also teaches mutagenesis methods to identify SpCas9 variants with higher cleavage fidelity (Abstract). Cereseto teaches preferred substitutions in SpCas9 to produce variants with higher on-target cleavage and/or lower off-target cleavage include R661L and R691L ([0018]). Cereseto also teaches substitutions at residues R661 and R691 in combination with other residue substitutions (Table 1). Cereseto teaches previously developed SpCas9 variants with high efficiency and specificity including the K855A substitution ([0005]). Cereseto teaches the K855A mutant was developed by design-based rational using the solved SpCas9 crystal structure ([0005]).
Cereseto does not teach the K855Q substitution.
Vakulskas teaches a mutant screen to identify SpCas9 mutants that have reduced off-target cleavage while maintaining on-target activity ([0063]). Vakulskas teaches developing a rational approach by making alanine substitutions of charged amino acids in close proximity to the Cas9 nucleic acid binding pockets base on published crystal structures ([0065]). Vakulskas then teaches a method of systematically testing every single amino acid substitution at a residue of interest, including leucine and glutamine ([0080]-[0082]). Vakulskas teaches the SpCas9 K691L variant exhibits much lower off-target cleavage efficiency than wildtype SpCas9 (FIG. 13). Vakulskas teaches that previously discovered high fidelity SpCas9 variants (i.e., SpCas9 variants with low off-target cleavage) include one or more alanine substitutions at R661, R780, K810, K848, K855, K1003 and R1060 ([0041]). Vakulskas teaches engineering SpCas9 variants with three amino acid substitutions in various combinations and testing the variants for on- and off-target cleavage efficiencies (FIG. 11; [0070]-[0071]; [0074]).
Joung teaches SpCas9 proteins comprising substitutions at K855 ([0010]), including the K855A substitution alone and in combination with other amino acid substitutions including with R661A ([0011], [0150]). Joung teaches an SpCas9 comprising R661A and K855A has higher fidelity than the SpCas9 with the wildtype lysine residue at position K855 (FIG 14). Joung teaches residues R780, K810, K1003 and R1060 appear to make contacts with the non-target DNA strand ([0144]). Joung also teaches alanine substitutions in SpCas9 at residues R780, K810, K1003 and R1060 can also increase the fidelity of SpCas9 (FIG. 13-14).
Regarding claim 1, it would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have used Vakulskas’s method of trying every amino acid at an SpCas9 residue known to be involved in target DNA interactions to create the K855Q substitution and combine it with the R691L and R661L substitutions taught in Cereseto. It would have amounted to using known methods of protein engineered and screening of SpCas9 variants for desired functions. The skilled artisan would have predicted that an R691L/R661L/K855Q triple SpCas9 substitution variant could be produced because from each of Vakulskas, Cereseto and Joung, it is evident that site directed mutagenesis of SpCas9 is a well-understood methodology in the art. The skilled artisan would have been motivated to make the SpCas9 R691L/R661L/K855Q variant because Vakulskas teaches that Q and A substitutions in residues making contact with the target DNA has similar on-target and off-target cleavage efficiencies, and to test the cleavage fidelity of a R691L/R661L/K855Q variant in the pursuit of a high fidelity SpCas9 variant.
Regarding claims 10-12 and 33, Cereseto teaches inclusion of two NLSs at the N-terminus and the C-terminus for expression of the SpCas9 variants in mammalian cells ([0106]). Vakulskas teaches fusion proteins of the SpCas9 variants to a heterologous domain, including a nuclear localizations signal ([0043]).
Regarding claims 13-14, Cereseto teaches SpCas9 having D10A (i.e., a mutation in the RuvC domain) and H840A (i.e., a mutation in the HNH domain) substitutions to decrease nuclease activity and then fused with a VP16 domain to create a transcriptional activator ([0010], [0043}).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have further created D10A and H840A mutations in the R691L/R661L/K855Q variant. It would have amounted to making known mutations in SpCas9 by known means to yield predictable results. The skilled artisan would have predicted that an R691L/R661L/K855Q/D10A/H840A variant could be created because Cereseto teaches making the D10A and H840A in other SpCas9 variants with reduced off-target activity. The skilled artisan would have been motivated to do so in order to use the obvious R691L/R661L/K855Q variant as a transcriptional activator.
Regarding claims 15, 18, 20, 23, 26-28 and 30, Cereseto teaches combining the SpCas9 variants with a gRNA targeting to an on-target cleavage site ([0089]). Ceresto teaches encoding the SpCas9 variants and guide RNAs in a plasmid (i.e., at least one vector that is DNA) for transformation into yeast (i.e., a single cell eukaryotic organism) ([0105]) and mammalian cells ([0106]). Cereseto teaches delivering both the SpCas9 variants and the guide RNA to yeast cells or mammalian cells by introducing a plasmid encoding the SpCas9 and guide RNA (i.e., comprising at least one nucleic acid, that is DNA, each encoding SpCas9 and guide RNA) ([0105]-[0106]).
Regarding claims 19 and 32, Ceresto teaches that the SpCas9 and guide RNA can be delivered as a ribonucleoprotein (i.e., one nucleic acid is RNA) ([0052]). Vakulskas teaches delivering the SpCas9 variant-encoding plasmid with guide RNAs (i.e., one nucleic acid is RNA) to HEK293 cells (i.e., a human, in vitro cultured cell) ([0070], [0082]). Vakulskas teaches the SpCas9 variants delivered to cells as an RNP complexed with a guide RNA ([0073], [0084]).
Regarding claims 21-22 and 25, Cereseto teaches the plasmids encoding the SpCas9 variants were codon optimized for expression in the mammalian cells ([0106]). Ceresto teaches delivering the nucleic acids encoding the SpCas9 variants and guide RNAs to 293T.17 cells (i.e., human, in vitro cultured cells) ([0116]). Cereseto teaches for expression in mammalian cells, the SpCas9 variant was driven by expression from a CBh promoter (i.e., a eukaryotic promoter sequence) and the gRNA expression cassette was driven by the U6 promoter (i.e., a eukaryotic promoter sequence). Cereseto teaches for expression in yeast, the SpCas9 expression plasmid from DiCarlo et al., was used ([0105]), which DiCarlo teaches uses an SpCas9 expression cassette with a codon-optimized coding sequence for SpCas9 (page 4338, ¶2) and a GAL1 promoter and SNR52 promoter (i.e., eukaryotic promoter sequences) for expression of SpCas9 and guide RNAs, respectively (Figure 1).
Regarding claim 34, the Specification teaches that pharmaceutical acceptable excipients encompassed inactive ingredients that impart cohesive, disintegrating, lubricant, solvent or surfactant functions ([00107]). Vakulskas teaches delivering SpCas9 variants as an RNP complexed with guide RNAs using RNAiMax (i.e., a pharmaceutically acceptable excipient) ([0074], [0084]).
Regarding claim 35, Cereseto teaches using the SpCas9 variants to induce genome mutations in yeast and mammalian cells (Figs 5-6). Vakulskas teaches using the SpCas9 variants to cleave (i.e., modify) a chromosomal sequence in HEK293 cells (i.e., eukaryotic cells) by delivering either plasmid ([0082]) or RNPs ([0084]) to the cells.
Claim 24 is rejected under 35 U.S.C. 103 as being unpatentable over Cereseto (US 20200149020 A1, priority to at least February 14, 2018, of record), Vakulskas (US 20180100148 A1, published April 12, 2018, of record) and Joung (US 20170058271 A1, published March 2, 2017, of record) as applied to claims 1, 10-15, 18-23, 25-28, 30 and 32-35 above, and further in view of Jinek (Jinek et al., Science (2012), 337: 816-821 and Supplemental Material, of record), evidenced by Agilent (pET System Vectors and Hosts Instruction Manual, Revision C0, of record).
The teachings of Cereseto, Vakulskas and Joung are recited above and applied as for claims 1, 10-15, 18-23, 25-28, 30 and 32-35. Vakulskas also teaches that guide RNAs can be produced via in vitro transcription ([0005]).
Cereseto, Vakulskas and Joung do not teach the promoter used to express SpCas9 variants from bacteria or a promoter for guide RNA in vitro transcription.
Jinek teaches bacterial expression of SpCas9 from pET-based vectors (Methods, page 2, ¶1). Agilent teaches that pET-3 and pET-11 vectors are for bacterial expression and comprise a T7 promoter (i.e., a phage promoter) for expression of the transgene (Figures 1-2). Jinek teaches that the guide RNAs were in vitro transcribed from PCR-generated DNA templates carrying a T7 promoter sequence (Methods, page 1, ¶4). Jinek teaches using the bacterial expressed and purified SpCas9 complexed with the in vitro transcribed guide RNAs for in vitro cleavage assays (Fig. 5).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have cloned the coding sequence for the SpCas9 R691L/R661L/K855Q variant rendered obvious above into nucleic acids to be driven by the T7 promoter. It would have amounted to using known promoter for the production of SpCas9 proteins and guide RNAs by known means to yield predictable results. The skilled artisan would have expected that the coding sequences for the obvious SpCas9 R691L/R661L/K855Q variant could be cloned behind the T7 promoter because Jinek demonstrates cloning the wildtype SpCas9 sequence and Vakulskas teaches that in vitro transcription of guide RNAs is known in the art. The skilled artisan would have been motivated to so for testing the in vitro cleavage activity of the high-fidelity mutants.
Claim 37 is rejected under 35 U.S.C. 103 as being unpatentable over Cereseto (US 20200149020 A1, priority to at least February 14, 2018, of record), Vakulskas (US 20180100148 A1, published April 12, 2018, of record) and Joung (US 20170058271 A1, published March 2, 2017, of record) as applied to claims 1, 10-15, 18-23, 25-28, 30 and 32-35 above, and further in view of Doudna (US 20140068797 A1, published March 6, 2014).
The teachings of Cereseto, Vakulskas and Joung are recited above and applied as for claims 1, 10-15, 18-23, 25-28, 30 and 32-35. Vakulskas also teaches that guide RNAs can be produced via in vitro transcription ([0005]). Vakulskas teaches Cas9 can be adapted to function as a therapeutic agent ([0005]).
Cereseto, Vakulskas and Joung do not teach administering Cas9 and guide RNA systems to a patient to relieve pathological symptoms.
Doudna teaches Cas9 polypeptides with DNA-targeting RNAs (i.e., guide RNAs) and methods for using them for site-specific modification of target DNA (Abstract). Doudna teaches DNA-targeting RNAs and the site-directed modifying polypeptides (i.e., Cas9 proteins and their guide RNAs) can be administered to cells in a targeting way “to treat a disease or as an antiviral, antipathogenic or anticancer therapeutic” ([0260], [0309]). Doudna teaches pharmaceutical preparations to use Cas9/guide RNAs for therapeutic purposes ([0310]-[0315]).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have prepared the SpCas9 R691L/R661L/K855Q variant rendered obvious for claim 1 in a pharmaceutical composition that is capable of treating symptoms of a disease. It would have amounted to using the obvious proteins in known therapeutic applications for Cas9/gRNA systems. The skilled artisan would have been motivated to use the obvious SpCas9 variant in a pharmaceutical composition capable of therapeutic results because both Doudna and Vakulskas teaches such an application of SpCas9 and variants. It would have been entirely predictable that the SpCas9 R691L/R661L/K855Q variant would be capable of ameliorating pathological symptoms since the obvious protein is predicted to have on-target DNA activity as indicated for claim 1 above, and the on-target activity could target DNA viruses in vivo for a predicted antiviral activity described by Doudna.
Response to arguments - §103
Applicant reviews the teachings of Cereseto, Vakulskas and Joung (Remarks, page 9, ¶4-6). Applicant argues that the claimed combinations of SpCas9 substitutions would have not been obvious at least because Joung teaches a glutamine (Q) substitution at N863 reduced Cas9 nuclease function (page 9, ¶6-7). This argument has been fully considered but it not persuasive. First, an N[Wingdings font/0xE0]Q substitution is not equivalent to a K[Wingdings font/0xE0]Q substitution. An N[Wingdings font/0xE0]Q substitution maintains polarity and elongates the side chain by a methyl group; whereas the claimed K[Wingdings font/0xE0]Q substitution shortens the side chain by a methyl group and reduces the overall positive charge of the protein. The N863 residue is well-known to be involved in catalytic activity of HNH domains; an N863A renders the Cas9 a nickase (See Nishimasu et al., Cell (2014), 156: 935-949; of record, page 939, ¶5). Thus, the K855 and N863 residues are known to have separate functions in Cas9/gRNA DNA binding and catalytic activity. Therefore, the Q substitution at N863 would not dissuade the skilled artisan from making the Q substitution at the K855 residue.
Allowable Subject Matter
Claims 6 and 36 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Claims 6 and 36 require three specific amino acid substitutions in SpCas9: R661L and K855Q, and either K562L or K562Q. The combination of K562L or K562Q specifically with R661L and K855Q would not have been obvious in view of Cereseto, Vakulskas and Joung, which are the closest prior art and whose teachings are recited above. Cereseto teaches SpCas9 variants with substitutions at K562 ([0012]), but specifically teaches the K562E mutation in combination with a K526N substitution. Although, Cereseto teaches that the identified amino acids can be modified with leucine (L) or glutamine (Q) ([0022]), it would not have been obvious to combine a K562L or K562Q substitution with the obvious K855Q mutation because Cereseto doesn’t teach mutating both those sites together. Additionally, Vakulskas’s method is based on systematic substitution of amino acids that are known from structural studies to be in close proximity to target DNA. There is no evidence from available crystal structure that K562 makes contacts with the target DNA. See e.g., Nishimasu et al., Cell (2014), 156: 935-949, of record, and Guo et al., Cell Research (2019), 29: 183-192, of record. Therefore, there is no motivation to specifically try the K562L or K562Q substitution or combine it with the obvious SpCas9 R661L/K855Q combination.
Conclusion
Claims 6, 18 and 36 are objected to. Claims 1, 10-15, 18-28, 30, 32-35 and 37 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CATHERINE KONOPKA whose telephone number is (571)272-0330. The examiner can normally be reached Mon - Fri 7- 4.
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/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635