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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 July 30, 2025 has been entered.
Applicant's Amendment to the Claims filed on July 30, 2025 has been entered.
Claims 1-57, 59-60, 62, 65-66, 69 are canceled. Claims 70-73 are new.
Claims 58, 61, 63-64, 67-68, and 70-73 are pending.
Claims 64 and 68 are withdrawn.
Claims 58, 61, 63, 67, and 70-73 are under examination.
Information Disclosure Statement
The IDS filed on 07/30/2025 has been considered by the examiner. The IDS has checked the box for No IDS size fee required under 37 CFR 1.17(v).
Priority
This US18/313,946 filed on 05/08/2023 is a CON of 17/004,338 filed on 08/27/2020 which is a CON of 14/685,568 filed on 04/13/2015 (now US Patent 10,851,380) which is a CON of PCT/KR2013/009488 filed on 10/23/2013 which claims US priority benefit of US Provisional 61/837,481 filed on 06/20/2013. The Filing Receipt filed on September 05, 2024 is acknowledged.
Regarding newly added claims 71 and 73, applicant has not complied with one or more conditions for receiving the benefit of an earlier filing date under 35 U.S.C. 119(e) as follows: The later-filed application must be an application for a patent for an invention which is also disclosed in the prior application (the parent or original nonprovisional application or provisional application). The disclosure of the invention in the parent application and in the later-filed application must be sufficient to comply with the requirements of 35 U.S.C. 112(a) or the first paragraph of pre-AIA 35 U.S.C. 112, except for the best mode requirement. See Transco Products, Inc. v. Performance Contracting, Inc., 38 F.3d 551, 32 USPQ2d 1077 (Fed. Cir. 1994).
Specifically, new claims 71 and 73 recite limitations that are not found in the ‘481 Provisional. The Applicants’ Remarks, page 4, filed on July 30, the applicants state that support for new claims can be found in US Provisional 61/837,481, page 35 (Example 2), and FIG 10; and PCT/KR2013/009488, Fig 10(b). However, a review of ‘481 page 35 (Example 2), and FIG 10 does not appear to provide support for new claims 71 and 73. For new claim 71, support is not found for the limitation, wherein the at least two-fold molar excess comprises a molar ratio range of sgRNA:Cas9 of 2.1:1 to 6.2:1. Although the PCT/KR2013/009488 Fig 10 legend (para 89) states: “Fig. 10 shows Cas9 protein/sgRNA complex induced targeted mutation” and Fig 10(b) shows a result of 8.7 µM sgRNA:1.4 Cas9 protein which equals a 6.2 : 1 molar ratio, this result is not shown in the ‘481 Provisional. Further, for new claim 73 support is not found for the limitation, wherein the sgRNA and the Cas9 protein in the transfection buffer are present at a weight ratio range of 8:15 to 20:15. The weight ratio of 8:15 is not found. Specifically regarding the new limitations of weight ratios in claim 73: Example 2 of the ‘481 Provisional (pages 34-35) discloses that Cas9 protein/sgRNA complex was introduced directly into K562 cells by nucleofection: 1x106 K562 cells were transfected with 22.5-225 µg of Cas9 protein mixed with 100 µg of in vitro transcribed sgRNA. However, FIG10 of ‘481 (shown below) shows only four specific weight ratios of sgRNA:Cas9 protein and does not show the weight ratio of 8:15 of new claim 73.
The applicants are invited to show where support for new claims 71 and 73 is found in the ‘481 Provisional.
Response to Amendment
Any rejection(s) made in the previous office action and not repeated in this office action are withdrawn in light of the Applicants’ Amendment to the Claims filed on July 30, 2025.
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.
Currently amended claims 58, 61, 63, 67, and 70-73 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.
Independent claim 58 recites a system which is being construed as a product. Such product comprises a Cas9 protein linked to an NLS; an sgRNA; and a transfection buffer which is free of DNA comprising the endogenous target nucleic acid sequence. Claim 58 is presently amended to recite the limitation: “…wherein the sgRNA is present in at least a two-fold molar excess over the Cas9 in the transfection buffer; wherein the sgRNA and the Cas9 protein form an extracellular Cas9/sgRNA complex in the transfection buffer prior to being introduced into the eukaryotic cell…”. It is unclear whether the product claim intends to require the active method step of “wherein the sgRNA and the Cas9 protein form an extracellular Cas9/sgRNA complex in the transfection buffer prior to being introduced into the eukaryotic cell. The fact pattern in the instant case is that the claims include a limitation regarding the molar ratio of the sgRNA and Cas9 protein in the transfection buffer. However, as the sgRNA forms a complex with the Cas9 protein in the transfection buffer this molar ratio changes. Thus one of ordinary skill in the art would not be able to determine the metes and bounds of the claim as presently written. For purpose of examination, the limitation “wherein the sgRNA and the Cas9 protein form an extracellular Cas9/sgRNA complex in the transfection buffer prior to being introduced into the eukaryotic cell…”. is not construed to require an active method step. Note that the final wherein clause in claim 58 refers to the extracellular Cas9/sgRNA complex which is formed in the transfection buffer. However, the active method step for forming such complex is not construed as required of the claimed product.
Dependent claims 61, 63, 67, and 70-73 are indefinite for the same reasoning as they depend from claim 58 and are not remedial.
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.
Further regarding 35 USC 103(a) rejections, the Supreme Court in KSR International Co. v. Teleflex Inc., 550 U.S. 398, 127 S. Ct. 1727, 82 USPQ2d 1385, 1395-97 (2007) (KSR) identified a number of rationales to support a conclusion of obviousness. The key to supporting any rejection under 35 U.S.C. 103 is the clear articulation of the reason(s) why the claimed invention would have been obvious. The Supreme Court in KSR noted that the analysis supporting a rejection under 35 U.S.C. 103 should be made explicit. Exemplary rationales that may support a conclusion of obviousness include:
(A) Combining prior art elements according to known methods to yield predictable results;
(B) Simple substitution of one known element for another to obtain predictable results;
(C) Use of known technique to improve similar devices (methods, or products) in the same way;
(D) Applying a known technique to a known device (method, or product) ready for improvement to yield predictable results;
(E} "Obvious to try" - choosing from a finite number of identified, predictable solutions, with a reasonable expectation of success;
(F) Known work in one field of endeavor may prompt variations of it for use in either the same field or a different one based on design incentives or other market forces if the variations are predictable to one of ordinary skill in the art;
(G) Some teaching, suggestion, or motivation in the prior art that would have led one of ordinary skill to modify the prior art reference or to combine prior art reference teachings to arrive at the claimed invention.
Note that the list of rationales provided is not intended to be an all-inclusive list.
Currently amended claims 58, 61, 63, 67, 70, and 71-73 are rejected 35 U.S.C. 103 as being unpatentable over US 2014/0068797 to Doudna et al (with priority to US Provisional 61/757,640 filed on filed on 1/28/2013), in view of WO2013/176772 to Jinek et al (EP 2800811 B1 filed on March 15, 2013, published 11/12/2014), in view of US Patent 10,731,181 to Chen et al (with priority to US Provisional 61/794422 filed on 03/15/2013), in view of Gaj et al (Nature Methods (2012), 9: 805-807 and Supplemental Material). Doudna et al citations refer to the ‘640 Provisional. Chen et al citations refer to the ‘422 Provisional. This is a new grounds of rejection.
Claim interpretation: The claimed system is construed as being drawn to a product rather than to a process. Claims are drawn to a product. The present claims are not drawn to a complex but rather to a system (which reads on a kit) comprising a Cas9 linked to an NLS, an sgRNA, and a transfection buffer which is free of endogenous target DNA. The instant specification does not provide a limiting definition for the term “transfection buffer”. In page 37 of the instant specification it discloses that an injection buffer was 0.25 mM EDTA, 10 mM Tris, pH 7.4. Thus, the term transfection buffer is construed to include a simple microinjection buffer such as 0.25 mM EDTA, 10 mM Tris, pH 7.4 as well as other buffers known in the art to be used in methods of introducing proteins and nucleic acids into eukaryotic cells.
Regarding presently amended base claim 58, Doudna et al disclose a system for inducing targeted disruption of endogenous genes in a eukaryotic cell, the system comprising a Cas9/RNA complex comprising a site directed polypeptide that is a Cas9 protein and DNA-targeting RNA that is single guide RNA (aka sgRNA or chimeric guide RNA). Doudna et al recites:
Generally, a subject method involves contacting a target DNA with a complex (a “targeting complex”), which complex comprises a DNA-targeting RNA and a site directed modifying polypeptide. [para 00233].
(See also para 0027300274, see especially para 00408 & 00522). In para 00325, Doudna et al disclose a kit comprising a complex that comprises a site-directed modifying polypeptide and a DNA-targeting RNA. Figure 31 shows Cas9 complexed with tracrRNA:crRNA (see para 00384). Doudna et al disclose that their Cas9 and sgRNA are capable of performing the intended use limitations in the claim. Doudna et al disclose a working example of a method for inducing targeted disruption of an endogenous human clathrin light chain gene in a eukaryotic cell nuclease, the method comprising introducing a Cas9 nuclease and gRNA specific for target DNA in a human HEK298T cell nucleus, wherein a Cas9-gRNA complex forms in the cell, wherein the Cas9/RNA complex functions as an endonuclease that is capable of inducing targeted disruption of the target DNA in the human cell nucleus. (See Working Example 2: para 0408-0423; also para 0134, 0168, 0238). See para 0064 which states that FIG 36A-D “demonstrate that co-expression of Cas9 and guide RNA in human cells generates double-strand DNA breaks at the target locus”. Further, in para 00234, Doudna et al recite that the target DNA includes “chromosomal DNA in cells, in vivo”. Further, Doudna et al disclose methods of introducing nucleic acid encoding Cas9 into a host cell include transfection, direct microinjection, and protoplast fusion. (See para 00232). Figures 16A-C shows the Cas9-tracrRNA:crRNA complex. Doudna et al discloses that the Cas9 protein is from Streptococcus pyogenes. (See para 0030-31, 0037, Figs 2-3).
Further, regarding the in vitro nature of the presently claimed system, Doudna et al disclose a kit (which reads on a system) comprising a Cas9 polypeptide, an sgRNA, and a transfection buffer that is for introducing into cells the Cas9 polypeptide and the sgRNA, that is free of DNA comprising the endogenous target nucleic acid sequence. (See para 23-25 and reference claims). The structure of the Cas9 linked to an NLS, the sgRNA, and the buffer of Doudna are not different from the Cas9, sgRNA, and transfection buffer of the present claims. (See especially references claims of the‘640 Provisional.) For example, regarding the buffer, Doudna et al disclose a kit comprising a reagent selected from the group consisting of: “a buffer for introducing into cells the site-directed modifying polypeptide”. (See ‘640 Prov claim 112). Further, Claim 44 recites a composition comprising a DNA-targeting RNA and a site-directed modifying polypeptide. Claim 48 depends from claim 44 and recites that the site-directed modifying polypeptide is Cas9. Claim 58 depends from claim 44 and recites a composition comprising the DNA-targeting RNA of Claim 44 and a buffer for stabilizing nucleic acids. Claim 59 depends from claim 59 and recites a composition comprising site-directed modifying polypeptide of Claim 44 and a buffer for stabilizing nucleic acids and proteins. The reference claims of Doudna describe a system comprising a single guide RNA corresponding to a Cas9 polypeptide (see claim 5), site-directed modifying polypeptide which is a Cas9, and a buffer for introducing into cells the site-directed modifying polypeptide, or for stabilizing nucleic acids and proteins. (see reference claims 1-121).
Further, Doudna et al explicitly teaches a working in vitro Example (para 00368) comprising the combination of the recombinant Cas9 protein and the guide RNA with a cleavage buffer before the target DNA has been added to the buffer. Absent a limiting definition for a transfection buffer in the specification the cleavage buffer of Doudna is construed to meet the limitation of a transfection buffer without endogenous target DNA. See Working Example: para 00368. Cas9 (25 nM) was pre-incubated 15 min at 37°C in cleavage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 10 mM MgCl₂, 0.5 mM DTT, 0.1 mM EDTA) with duplexed tracrRNA:crRNAsp2 (25 nM, 1:1) or both RNAs (25 nM) not preannealed and the reaction was started by adding protospacer 2 plasmid DNA (5 nM). Thus, the working example of para 00368 discloses the recombinant Cas 9 protein in a buffer with the sgRNA before the target DNA has been added to the buffer.
Further, regarding claim 58, Doudna et al discloses that the Cas9 protein is linked to one or more tagging sequences, wherein the one or more tagging sequence comprises a nuclear localization signal (NLS) (See para 00242, 00256, especially para 00416).
Further, regarding claim 58, Doudna et al disclose a guide RNA having a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), wherein the crRNA comprises: a portion to be hybridized with a portion of the tracrRNA, and a portion complementary to a target DNA of the endogenous genes. (See para 0031 reciting “RNA chimera A”, 00374). Doudna et al disclose that the guide RNA is a single-chain guide RNA comprising the crRNA portion fused to the tracrRNA portion. (See para 0031 reciting “RNA chimera A”, 00374). In addition, Doudna et al disclose that the target DNA sequence comprises a first strand having a 20-base pair region complementary to the second portion of the crRNA and a second strand having a PAM consisting of 5’-NGG-3’ motif. (See para 044; FIG 16A-C; para 00416-417).
Further, regarding claim 58, Doudna et al disclose that the extracellular Cas9/RNA complex functions as an endonuclease that is capable of inducing targeted disruption of the target DNA in a eukaryotic cell. (See entire document, especially para 00408, 00416, 00646). Doudna et al disclose that the Cas9 protein may be complexed with the guide RNA to form a ribonucleoprotein which is capable of being introduced into the eukaryotic cell wherein the ribonucleoprotein functions as an endonuclease that is capable of inducing targeted disruption of the target DNA. (See para 00273, see especially para 00408 & 00522). Note that the claim is drawn to a product and thus is being construed to not require an active method step of introducing into a eukaryotic cell.
Regarding claim 61, Doudna et al disclose that the Cas9 protein has the NLS in proximity to its C-terminal. (See para 00222, 00243; page 120, line 1; para 00411, line 1; para 00416).
Regarding claim 63, Doudna et al discloses that the RNA-guided endonuclease (Cas9) may comprise a 6XHis-tag which meets the limitation of a further peptide tag. (See para 00222, 00239, 00243, 00364, 00382).
Regarding claim 67, Doudna et al disclose that the guide RNA is transcribed in vitro. (See ‘640 Prov para 00260, lines 1-4).
Regarding new claim 70, Doudna et al disclose a working example of a method for inducing targeted disruption of an endogenous human clathrin light chain gene in a eukaryotic cell nuclease, the method comprising introducing a Cas9 nuclease and gRNA specific for target DNA in a human HEK298T cell nucleus, wherein a Cas9-gRNA complex forms in the cell, wherein the Cas9/RNA complex functions as an endonuclease that is capable of inducing targeted disruption of the target DNA in the human cell nucleus. (See Working Example 2: para 0408-0423; also para 0134, 0168, 0238). See para 0064 which states that FIG 36A-D “demonstrate that co-expression of Cas9 and guide RNA in human cells generates double-strand DNA breaks at the target locus”.
The instant specification provides two methods of introducing the Cas9/sgRNA into eukaryotic cells, one by nucleofection, and the other by injection. Fig 4 of instant specification shows T7/E1 assay for cells transfected with the Cas9/RNA complex. Fig4(B) of ‘481 Provisional shows T7E1 assay in cells transfected with Cas9/RNA complex. FIG7 shows targeting results of rCas9 protein: Foxn1-sgRNA complex (a and b) by pronuclear or intracytoplasmic injection. Fig7(c) shows highest dose. Pages 34-35 shows Cas9 protein complexed with gRNA: Cas9 protein/sgRNA complex was introduced directly into K562 cells by nucleofection: K562 cells were transfected with 22.5-225 ng of Cas9 protein mixed with 100ug of in vitro transcribed sgRNA using the 4D-Nucleofector, SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza) according to the manufacturer's protocol. Results are shown in Fig10. On page 36, the specification recites that “[a]lternatively, we directly injected the RGEN in the form of recombinant Cas9 protein (0.3 to 30 ng/ul) complexed with the two-fold molar excess of Foxnl-specific sgRNA (0.14 to 14 ng/ul)…” However, this statement shown on page 36 which states: “[a]lternatively, we directly injected the RGEN in the form of recombinant Cas9 protein (0.3 to 30 ng/ul) complexed with the two-fold molar excess of Foxnl-specific sgRNA (0.14 to 14 ng/ul)…” has not actually been demonstrated in the instant specification. It was known in the art (e.g., as evidenced by Jinek et al) that sgRNA and Cas9 protein form a stable functional complex in a precise 1:1 molar ratio. When equal initial concentrations of sgRNA and Cas9 are mixed, they bind completely, assuming the binding reaction goes to completion. According to the reaction stoichiometry, for example, 1 mole of sgRNA reacts with 1 mole of Cas9 to produce 1 mole of the final sgRNA:Cas9 complex. (While present claims recite an single-guide RNA complexed with Cas9, Doudna et al also shows that if dual-guide RNAs are used, the molar ratio would start as two guide RNAs for each Cas9 protein to produce in one guide RNA/Cas9 complex. Thus, absent evidence to the contrary, the structure of the sgRNA/Cas9 would be complexed at about a one to one molar ratio. While the transfection buffer may contain a two-fold molar excess at the original disposal of reagents into the transfection buffer, this ratio would quickly change as the complex forms. Thus, a starting molar ratio of a two-fold molar excess would most likely result in about a one to one molar ratio at the time of transfection/injection/microinjection into the cell, such ratio being one part sgRNA to one part sgRNA/Cas9 protein complex.
Note that the limitations wherein the sgRNA and the Cas9 protein form an extracellular Cas9/sgRNA complex in the transfection buffer prior to being introduced into the eukaryotic cell; and wherein the extracellular Cas9/sgRNA complex is capable of functioning as an endonuclease that induces targeted modification of the endogenous target nucleic acid sequence upon introduction into the eukaryotic cell appear to require active method steps. For purpose of examination, the claims are construed to be drawn to a product and the limitation “form an extracellular Cas9/sgRNA complex” is not construed to require an active method step but is considered to be functional language. Doudna et al discloses that the structure of their Cas9 protein comprising an NLS, sgRNA, and stabilizing buffers meet the limitation of being capable of forming an extracellular Cas9/sgRNA complex.
However, Doudna et al differs from the presently amended base claim 58 because while Doudna et al disclose a system for inducing targeted modification of an endogenous target nucleic acid sequence in a eukaryotic cell, the system comprising a Cas9 protein linked to an NLS; a single guide RNA (called sgRNA) having a cRNA fused to a tracrRNA, where the crRNA has i) a first portion to be hybridized with a portion of the tracrRNA, and 2) a second portion complementary to the endogenous target nucleic acid sequence, and a buffer free of DNA comprising such target nucleic acid sequence, they do not explicitly disclose that the sgRNA is present in at least a two-fold molar excess over the Cas9 protein in the transfection buffer.
Jinek et al (EP 2 800 811 B1) discloses guide RNA and Cas9 polypeptide form a complex. (See para 0397). Further, Jinek et al disclose Cas9 fused to an NLS and a hemagglutinin (HA) tag. (See para 0405). Further, Jinek et al disclose using in vitro transcribed gRNAs. (See para 0454; 0479). Jinek et al disclose Cas9 protein synthesis in and purification from E.coli (See para 0482). In addition, Jinek et al disclose a complex of Cas9 (25nM) preincubated with sgRNA (25 nM) in buffer without endogenous target DNA. (See para 0484-0486).
Regarding the limitations of at least two-fold molar excess of sgRNA over the Cas9 protein, the Doudna and Jinek cited references do not explicitly disclose a two-fold molar excess in the transfection buffer. Doudna et al suggests a 1:1 ratio and in addition, Jinek et al disclose a complex of Cas9 (25nM) preincubated with gRNA (25 nM). (See para 0484-0486).
Further, regarding new claim 71, Doudna and Jinek do not disclose that the at least two-fold molar excess comprises molar ratios of sgRNA:Cas9 of 2.1 : 1 to 6.2 :1.
Further, regarding new claim 72, Doudna and Jinek do not disclose that the sgRNA and the Cas9 protein in the transfection buffer are present at a weight ratio of 4 : 9 to 1.3 : 1.
Further, regarding new claim 73, Doudna and Jinek do not disclose that the sgRNA and the Cas9 protein in the transfection buffer are present at a weight ratio of 8 : 15 to 20 : 15.
Chen et al suggest introducing the Cas9 protein and a guide RNA into a eukaryotic cell or embryo. (para 0041). For example, in para 0047, lines 11-13, Chen et al state: “In still other embodiments, the fusion protein can be introduced into the cell or embryo as an RNA-protein complex comprising the fusion protein and the guiding RNA”. In para 0080-0081, Chen et al disclose that the RNA-guided endonuclease is Cas9 and the RNA-guided endonuclease is “introduced into the cell as a RNA-protein complex comprising the endonuclease protein and the guiding RNA”. Chen et al disclose Cas9 fused to an NLS (para 0010 cites Cas9 protein; para 0023 cites NLS). Chen et al disclose this link can be at the N- or C-terminal (see para 0024). The purpose of the NLS is to introduce the Cas9 complex into the nucleus of the eukaryotic cell. Chen et al disclose additional peptide tags (para 0025). Chen et al disclose that methods of introducing into eukaryotic cells include transfection, nucleofection, and electroporation and state that “Transfection methods are well known in the art” (see para 0066). Further, introducing may be by microinjection (para 0066, last sentence). In para 0075-0076 lists suitable type of eukaryotic cells including human cells. Also, reference claims 28 & 37 recite a human cell.
In addition, although Chen et al does not explicitly disclose that the sgRNA is present in at least a two-fold molar excess over the Cas9 protein in the transfection buffer, they disclose that the ratio of protein to gRNA will generally be approximately stoichiometric “such that they can form an RNA-protein complex”. See para 0067. However, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05. Chen et al discloses the general conditions where the ratio of protein to gRNA will generally be approximately stoichiometric “such that they can form an RNA-protein complex”. Therefore, the claimed ratio of the sgRNA being present in at least a two-fold molar excess over the Cas9 protein is considered optimization through routine experimentation, and would be obvious to one of ordinary skill in the art. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
Further, the claimed ratios of new claims 71-73 in view of the copending limitation of at least a two-fold molar excess over the Cas9 protein is considered optimization through routine experimentation, and would be obvious to one of ordinary skill in the art. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
Gaj teaches direct delivery of genome-editing zinc-finger nuclease (ZFN) proteins for endogenous gene disruption (Abstract). Gaj teaches the ZFN comprised an NLS (Fig 1a). Gaj teaches purifying ZFNs and diluted into serum-free medium (i.e., cell-free buffer and a transfection mixture) (Methods, page 1, last ]). Gaj teaches treating eukaryotic cells with the medium/ZFN mixture (i.e., transfecting the cells) (Methods, page 2, 4/2). Gaj teaches the ZFN modified the host cell genome (Fig 1fc-f). Gaj teaches an advantage of delivering ZFN to cells as a protein instead of plasmid is reduced ZFN-induced modifications at off-target sites (page 807, 42). Gaj teaches this is likely due to the short half-lives of the transduced ZFN and limiting the duration of ZFN in cells (page 807, 4/2). Gaj also teaches that directly delivering the gene editing proteins, instead of expressing them from plasmids, avoids the risk of insertional mutagenesis (page 807, 75). Gaj teaches “that protein delivery and the benefits afforded therein will be extended to other designer nucleases” (page 807, 45).
The level of skill in the art was high before the effective filing date of the presently claimed invention.
It is considered that one of ordinary skill in the art would have been motivated to use the constructs for making the Cas9 endonuclease and chimeric (single) guide RNA having a crRNA complementary to a 20 base pair target DNA, where the target DNA contained the NGG PAM sequence disclosed in Doudna et al to be recognized by the S. pyogenes Cas9, in a eukaryotic cell, for the rationale of making a successful gene-editing complex for gene editing (disruption) in a eukaryotic cell. Further, one of ordinary skill in the art would have been motivated to transfect Cas9 protein and in vitro transcribed sgRNA into cells in place of using plasmids encoding such because Gaj also teaches that directly delivering the gene editing proteins, instead of expressing them from plasmids, avoids the risk of insertional mutagenesis (page 807, 75). Gaj teaches “that protein delivery and the benefits afforded therein will be extended to other designer nucleases” (page 807, 45).
Further, a system comprising a Cas9 protein, an sgRNA, and a transfection buffer are further rendered obvious over each of the references of Doudna et al, Jinek et al and Chen et al because each of these references explicitly disclose such system, suggesting forming a Cas9-sgRNA complex for transfecting into a eukaryotic cell. Further, it would have been obvious to combine the elements of Chen et al because Chen et al suggest the ratio of protein to gRNA will generally be approximately stoichiometric“ such that they can form an RNA-protein complex”. See para 0067. Therefore, the claimed ratio of the sgRNA being present in at least a two-fold molar excess over the Cas9 protein is considered optimization through routine experimentation, and would be obvious to one of ordinary skill in the art.
Absent evidence to the contrary, it is considered that one of ordinary skill in the art having the cited references before the effective filing date of the presently claimed invention would have had a reasonable expectation of success to combine a Cas9 protein, and an sgRNA in a transfection buffer that was free of endogenous target DNA, and in a molar ratio of at least a two-fold molar excess of the gRNA, to arrive at the presently claimed invention.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have an in vitro composition comprising a Cas9 protein linked to an NLS; an sgRNA; and a transfection buffer free of DNA comprising the endogenous target nucleic acid sequence. Further, it would have been obvious for one of ordinary skill in the art to add to optimize the disposal of the initial molar amounts of sgRNA and Cas9 nuclease in the transfection buffer because it is routine lab procedure to increase the amount of gRNA compared to Cas9 in such buffer. One of ordinary skill in the art would have been motivated to do such because the sgRNA may be unstable in the buffers as evidenced by Doudna et al. It was known in the art by Doudna et al and Jinek et al that the functional Cas9/sgRNA complex was essentially a 1:1 molar ratio. However, Chen et al suggests the starting amounts should be optimized to determine the best ratio for delivery to cells.
It would have amounted to using known Cas9/gRNA complexes in known ways to yield predictable results. The skilled artisan would have predicted that the Cas9/sgRNA complex could be introduced into cells because Doudna et al suggest such and Chen suggest such. Gaj demonstrates the protein delivery of a different designer nuclease. The skilled artisan would have been motivated to do so because Gaj teaches that direct delivery of a designer nuclease avoids the risks of plasmid-based delivery including off-target cleavage and insertional mutagenesis. Further, the skilled artisan would have specifically motivated to deliver the Cas9/sgRNA complex that had formed in the buffer because Doudna and Jinek teach the ribonucleoprotein is the active form for Cas9.
Response to arguments
The applicants’ response filed on July 30, 2025 has been fully considered but is unpersuasive. The applicants argue that the presently claimed invention is amended to recite: “wherein the sgRNA is present in at least a two-fold molar excess over the Cas9 protein in the transfection buffer” and “wherein the sgRNA and the Cas9 protein form an extracellular Cas9/sgRNA complex in the transfection buffer”. The applicants argue that the cited references fail to teach or suggest such features.
However, the applicants arguments are not persuasive because they are not commensurate with the scope of the present claims. The claims are not drawn to a Cas9/sgRNA complex or to a method of forming such complex. Instead, the claims are drawn to a product comprising a Cas9 protein, an sgRNA, and a transfection buffer. In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., Cas9/sgRNA complex or to a method of forming such complex) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Further, regarding the argument regarding lack of sufficient motivation or rationale to form the sgRNA/Cas9 complex before transfection into eukaryotic cells, the reference of Gaj provides evidence for such rationale and motivation. As described in the body of the rejection above, the Chen reference suggests forming the sgRNA/Cas9 complex before transfecting into the eukaryotic cells. Gaj teaches an advantage of delivering ZFN to cells as a protein instead of plasmid is reduced ZFN-induced modifications at off-target sites (page 807, 42). Gaj teaches this is likely due to the short half-lives of the transduced ZFN and limiting the duration of ZFN in cells (page 807, 4/2). Gaj also teaches that directly delivering the gene editing proteins, instead of expressing them from plasmids, avoids the risk of insertional mutagenesis (page 807, 75). Gaj teaches “that protein delivery and the benefits afforded therein will be extended to other designer nucleases” (page 807, 45).
Further, as discussed in the body of the rejection above, it was known in the art that a functional sgRNA/Cas9 complex is made of one sgRNA for each Cas9 protein. Chen suggests forming the sgRNA/Cas9 complex using amounts of sgRNA and Cas9 protein appropriate to form such complex. Note that the molar ratio of sgRNA to Cas9 protein changes in a transfection buffer as the sgRNA/Cas9 protein forms. The claims are interpreted consistent with the specification but without reading limitations into the claims. However, On page 36, the ‘481 priority document specification recites that “[a]lternatively, we directly injected the RGEN in the form of recombinant Cas9 protein (0.3 to 30 ng/ul) complexed with the two-fold molar excess of Foxnl-specific sgRNA (0.14 to 14 ng/ul)…”. However, nothing in the applicants’ disclose actually shows a Cas9 protein complexed with the two-fold molar excess of sgRNA. Further, the applicants argue against “routine experimentation” rationale to optimize the ratio of sgRNA and Cas9 complex in the transfection buffer submitting that the Office Action “never established that the claimed molar excess was recognized in the art to be a result-effective variable when forming an extracellular Cas9/sgRNA in a transfection buffer. Lastly, applicants argue that “unexpected results confirm the nonobviousness of the pending claims”. Specifically, the applicants argue that common sense is typically invoked to provide a known motivation to combine, not to supply a missing claim limitation. However, this argument is unpersuasive because the Gaj reference teaches a motivation to use direct delivery of genome-editing nuclease proteins for endogenous gene disruption (Abstract) in place of delivering plasmids expressing such. Gaj teaches the ZFN comprised an NLS (Fig 1a). Gaj teaches purifying ZFNs and diluted into serum-free medium (i.e., cell-free buffer and a transfection mixture) (Methods, page 1, last ]). Gaj teaches treating eukaryotic cells with the medium/ZFN mixture (i.e., transfecting the cells) (Methods, page 2, 4/2). Gaj teaches the ZFN modified the host cell genome (Fig 1fc-f). Gaj teaches an advantage of delivering ZFN to cells as a protein instead of plasmid is reduced ZFN-induced modifications at off-target sites (page 807, 42). Gaj teaches this is likely due to the short half-lives of the transduced ZFN and limiting the duration of ZFN in cells (page 807, 4/2). Gaj also teaches that directly delivering the gene editing proteins, instead of expressing them from plasmids, avoids the risk of insertional mutagenesis (page 807, 75). Gaj teaches “that protein delivery and the benefits afforded therein will be extended to other designer nucleases” (page 807, 45).
Further, the motivation for routine optimization of the molar ratio of the sgRNA and Cas9 protein in the transfection buffer is provided in Chen in stating the amounts would be to form the sgRNA/Cas9 complex.
Further, the applicants argue that it would not have been obvious to arrive at a system comprising a Cas9/sgRNA complex in a transfection buffer that is free of DNA comprising the endogenous target nucleic acid sequence, stating that the Office Action does not cite any examples of an “extracellular Cas9/sgRNA complex formed in a transfection buffer being introduced to a eukaryotic cell”.
However, the applicants’ argument against Doudna and Jinek is unpersuasive because the rejection is over Doudna, Jinek, Chen, and Gaj. The claims are drawn to a product and active method steps recited in the claims are not read into the claimed product.
Further, the applicants argue that evidence is lacking to use a rationale that the unstable nature of RNA in a transfection buffer would motivate or provide a rationale underpinning explaining why a POSA would use routine optimization. The applicants argue that while RNA may be fragile in intracellular environments, it is “far more stable in in vitro, extracellular environments..”
Further, the applicants argue that the claimed subject matter achieves unexpected results that confirm nonobviousness.
However, this argument is unpersuasive because the arguments are not commensurate with the scope of the present claims. The examples of presumed unexpected results show specific reaction conditions, Cas9 constructs, and sgRNA constructs and specific cell types which are not required of the present claims.
In addition, the applicants argue that Doudna fails to teach or suggest a system comprising a Cas9 protein, an sgRNA, and a transfection buffer that is free of DNA comprising the endogenous target nucleic acid sequence” (Point II of page 7 of Remarks).
This argument is unpersuasive because Doudna et al disclose a kit comprising a reagent selected from the group consisting of: “a buffer for introducing into cells the site-directed modifying polypeptide”. (See ‘640 Prov claim 112).
Further, the applicants argument that the cleavage buffer of Doudna does not meet the limitation of a transfection buffer and that Doudna does not disclose an embodiment where the cleavage buffer is free of DNA comprising the endogenous target nucleic acid is unpersuasive. Doudna et al explicitly disclose working Examples where the recombinant Cas9 protein is combined with the sgRNA before the addition of a target DNA.
Further, the applicants argue against the rationale that RNA is especially susceptible to degradation in a buffer with a recombinant protein is unpersuasive. Doudna et al discloses that RNA may be unstable and explicitly suggest addressing the problem of unstable sgRNA. For example, the Doudna et al reference claim 58 depends from claim 44 and recites a composition comprising the DNA-targeting RNA of Claim 44 and a buffer for stabilizing nucleic acids. Claim 59 depends from claim 59 and recites a composition comprising site-directed modifying polypeptide of Claim 44 and a buffer for stabilizing nucleic acids and proteins. Further, Doudna et al suggest methods to modify the sgRNA to improve stability:
In some embodiments, a subject nucleic acid (e.g., a DNA-targeting RNA) comprises one or more modifications, e.g., a base modification, a backbone modification, etc, to provide the nucleic acid with a new or enhanced feature (e.g., improved stability). As is known in the art, a nucleoside is a base-sugar combination. The base portion of the nucleoside is normally a heterocyclic base. The two most common classes of such heterocyclic bases are the purines and the pyrimidines. Nucleotides are nucleosides that further include a phosphate group covalently linked to the sugar portion of the nucleoside. [00174]
Doudna et al also disclose that Locked nucleic acids (LNAs) may be used to modify the RNA to form a bicyclic sugar moiety for “stability towards 3'- exonucleolytic degradation”. (See para 00184)
Further, the secondary reference of Chen et al disclose that methods of introducing into eukaryotic cells include transfection, nucleofection, and electroporation and state that “Transfection methods are well known in the art” (see para 0066). Further, introducing may be by microinjection (para 0066, last sentence). In para 0075-0076 lists suitable type of eukaryotic cells. Regarding the limitation that the transfection buffer is free of endogenous target DNA, it is prima facie obvious that a system for introducing a Cas9 protein and sgRNA into a eukaryotic cell using a known method of transfection or microinjection into a eukaryotic cells would include a transfection buffer that is free of the endogenous target DNA because such endogenous target DNA is present within the cell to be transfected. The reference claims of Doudna et al explicitly support a limitation where the Cas9 protein and/or sgRNA are present in a buffer
10ug sgRNA: 22.5ug Cas9 protein; (weight ratio is 4:9)
30ug sgRNA: 75ug Cas9 protein; (weight ratio is 2:5 aka 0.4:1 & 1:2.5)
100 ug sgRNA: 22.55ug Cas9 protein; (weight ratio is 4.4:1)
100ug sgRNA: 75ug Cas9 protein; (weight ratio is 4:3 aka 1.3:1), and
100ug sgRNA: 225ug Cas9 protein (weight ratio is 4:9).
Further, regarding the new limitations of molar ratios: Example 2 of the ‘481 Provisional (pages 34-35) recites the following:
Here, we used recombinant Cas9 protein complexed with in vitro transcribed guide RNA to induce targeted disruption of endogenous genes in human cells. Recombinant Cas9 protein fused with the hexa-histidine tag was expressed in and purified from E. coli using standard Ni ion affinity chromatography and gel filtration. Purified recombinant Cas9 protein was concentrated in storage buffer (20 mM HEPES pH 7.5, 150 mM KCl, 1 mM DTT, and 10% glycerol).
As shown in Fig. 10, Cas9 protein/sgRNA complex induced targeted mutation at the CCR5 locus at frequencies that ranged from 4.8 to 38% in an sgRNA or Cas9 protein dose-dependent manner, on par with the frequency obtained with Cas9 plasmid transfection (45%).
FIG 10 of the ‘481 Provisional is shown just below:
PNG
media_image1.png
765
636
media_image1.png
Greyscale
10ug : 22.5ug; (molar ratio is 2.9 µM:1.4 µM = 2.1 : 1)
30ug : 75ug; (molar ratio is 8.8 µM:4.5 µM = 2:1)
100 ug : 22.55ug; (molar ratio is 29 µM:1.4 µM = 20.7 : 1)
100ug : 75ug; (molar ratio is 29 µM:4.5 µM = 6.4 : 1), and
100ug : 225ug (molar ratio is 29 µM:14 µM = 2.1 : 1).
Thus, regarding the present claims, FIG 10 (1st panel) of the ‘481 Provisional shows 10 ug sgRNA with 22.5 ug Cas9 protein, 30 ug sgRNA with 75 ug Cas9 protein, and 100 ug sgRNA with 225 ug Cas9 protein. FIG 10 (2nd panel) shows 100 ug sgRNA with 22.5ug, 75ug, or 225 ug Cas9 protein.
PNG
media_image2.png
406
584
media_image2.png
Greyscale
PNG
media_image3.png
505
621
media_image3.png
Greyscale
Double Patenting
Response to arguments regarding NSDP
The applicants’ response filed on July 30, 2025 regarding NSDP rejections has been fully considered but is unpersuasive. The applicants argue that the provisional NSDP rejections were each made in view of the Chen reference. The applicants argue that the limitation regarding the sgRNA being present in at least a two-fold molar excess over the Cas9 protein is not recited in the co-pending Applications and that this limitation is not rendered obvious in view of the Chen reference. The applicants argue the Chen reference for the same reasoning applied above regarding the rejection made under 35 U.S.C. 103 for obviousness. The applicants arguments regarding the Chen references are unpersuasive for the same reasoning applied above regarding the rejection made under 35 U.S.C. 103 for obviousness. Several new grounds of rejection are included below in light of present claim amendments and claim amendments of reference applications/patents.
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.
Currently amended claims 58, 61, 63, 67 and 70-73 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 11-19, 21-24 of co-pending Application No. 18/932,745 (reference application). This is a new grounds of rejection.
Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are rendered obvious over the combination of co-pending claims.
Regarding instant claims 58 and 67, copending claim 11 recites a method comprising a CRISPR/Cas9 complex comprising a Cas9 protein having an NLS and an in vitro single guide RNA having a crRNA and a tracrRNA portion where the complex is capable of cleaving an endogenous target DNA in a eukaryotic cell, where the Cas9/RNA complex is formed in vitro before transfer into cell. Co-pending claim 21 recites that the target DNA sequence has a PAM consisting of 5’-NGG-3’. Further, co-pending claims do not specify that the target DNA has a 20-base pair region complementary to the crRNA. However, present claims are drawn to a product complex in a transfection buffer and do not require this element of the target DNA. Regarding amended claim 58, copending claim 11 recites that the guide RNA is an in vitro single-chain guide RNA (sgRNA) comprising the crRNA portion fused to the tracrRNA portion. Further, copending claim 11 recites a two-fold molar excess over the Cas9 protein, specifically a molar ratio of gRNA to Cas9 ranging from 29:14 to 29:1.4 .
Regarding instant claim 61, copending claim 22 recites an NLS disposed at a C-terminus of the Cas9 protein.
Regarding instant claim 63, the NLS of copending claim 11 is construed to meet the limitation of a peptide tag.
Regarding instant claim 70, copending claim 16 recites a human cell.
Further, regarding new claims 71-73, copending claim 11 recites a molar ratio of gRNA to Cas9 ranging from 29:14 to 29:1.4 which is considered to render obvious the molar ratios and weight ratios of the instant claims 71-73, specifically: sgRNA:Cas9 of a molar ratio range being 2.1:1 to 6.2:1; a weight ratio range of 4:9 to 1.3:1, and a weight ratio range of 8:15 to 20:15. Therefore, the claimed ratios of instant claims 71-73 in view of the copending limitation is considered optimization through routine experimentation, and would be obvious to one of ordinary skill in the art. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
The level of skill in the art was high before the effective filing date of the presently claimed invention.
One of ordinary skill in the art would have been motivated to combine the elements of the copending claims for the rationale of performing successful gene editing in eukaryotic cells. It would have been obvious to combine the elements of copending claims because these are preferred embodiments in the claims.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Currently amended claims 58, 61, 63, 67 and 70-73 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 15-27, 29-33 of co-pending Application No. 19/002,832 (reference application). This is a new grounds of rejection.
Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are rendered obvious over the combination of co-pending claims.
Regarding instant claims 58 and 67, copending claims 27 and 31 recite a method comprising a CRISPR/Cas9 complex comprising a Cas9 protein and an in vitro single guide RNA having a crRNA and a tracrRNA portion where the complex is capable of cleaving an endogenous target DNA in a eukaryotic cell, where the Cas9/RNA complex is formed in vitro before transfer into cell. Copending claims 27 and 31 recite a Cas9 protein having an NLS. Co-pending claim 17 recites that the target DNA sequence has a PAM consisting of 5’-NGG-3’. Further, co-pending claims do not specify that the target DNA has a 20-base pair region complementary to the crRNA. However, present claims are drawn to a product complex in a transfection buffer and do not require this element of the target DNA. Regarding amended claim 58, copending claim 26 recites that the guide RNA is an in vitro single-chain guide RNA (sgRNA) comprising the crRNA portion fused to the tracrRNA portion. Further, regarding instant claim 58, copending claim 27 recites a two-fold molar excess over the Cas9 protein.
Regarding instant claim 61, copending claim 18 recites an NLS disposed at a C-terminus of the Cas9 protein.
Regarding instant claim 63, the NLS of copending claim 11 is construed to meet the limitation of a peptide tag.
Regarding instant claim 70, copending claims 23 and 31 recite a human cell.
Further, regarding new claims 71-73, copending claims 21, 29-30 and 31-32 specify a molar ratio of gRNA to Cas9 ranging from 29:1.4 to 29:14, 2:1 to 29:4.5 and a weight ratio of 8:15 to 20:15 which is considered to render obvious the molar ratios and weight ratios of the instant claims 71-73, specifically: sgRNA:Cas9 of a molar ratio range being 2.1:1 to 6.2:1; a weight ratio range of 4:9 to 1.3:1, and a weight ratio range of 8:15 to 20:15. Therefore, the claimed ratios of instant claims 71-73 in view of the copending limitation is considered optimization through routine experimentation, and would be obvious to one of ordinary skill in the art. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
The level of skill in the art was high before the effective filing date of the presently claimed invention.
One of ordinary skill in the art would have been motivated to combine the elements of the copending claims for the rationale of performing successful gene editing in eukaryotic cells. It would have been obvious to combine the elements of copending claims because these are preferred embodiments in the claims.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Currently amended claims 58, 61, 63, 67, and 71-73 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 58, 61-65, 67, and 69-72, and 75-77 of co-pending Application No. 17/004,355 (reference application).
Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are rendered obvious over the combination of co-pending claims. New grounds of rejection.
Regarding instant claims 58 and 67, copending claim 77 depends from copending claim 58 and recites a method comprising a CRISPR/Cas9 complex comprising a Cas9 having an NLS and an in vitro guide RNA having a crRNA and a tracrRNA portion where the complex is capable of cleaving an endogenous target DNA in a eukaryotic plant cell, where the Cas9/RNA complex is formed before transfer into the plant cell. Copending claim 75 recites that the gRNA is an sgRNA. Co-pending claim 71 recites that the target DNA sequence has a PAM that consists of 5’-NGG-3’. Further, co-pending claim 70 recites that the target DNA is genomic DNA of eukaryotic plant cell and co-pending claim 69 recites that the crRNA is 20 nucleotides in length. Regarding amended claim 58, copending claim 58 recites that the guide RNA is a single-chain guide RNA (sgRNA) comprising the crRNA portion fused to the tracrRNA portion. Further, copending claim 77 recites a two-fold molar excess over the Cas9 protein in the in vitro environment when complexing the Cas9/RNA complex.
Regarding instant claim 61, copending claim 76 recites an NLS disposed at a C-terminus of the Cas9 protein.
Regarding instant claim 63, the NLS of copending claim 58 is construed to meet the limitation of a peptide tag.
However, copending claims do not recite the limitation of a transfection buffer without endogenous target DNA. It is prima facie obvious in view of copending claim 67 that the Cas/guide RNA complex formed in vitro before being introduced into the eukaryotic cell would be in a transfection buffer without the endogenous target DNA because the complex needs to be in a buffer to exist as a functional complex without the endogenous target DNA because it is formed in vitro before being introduced into the cell.
Further, regarding new claims 71-73, copending claim 77 recites at least two-fold molar excess of gRNA to Cas9 which is considered to render obvious the molar ratios and weight ratios of the instant claims 71-73, specifically: sgRNA:Cas9 of a molar ratio range being 2.1:1 to 6.2:1; a weight ratio range of 4:9 to 1.3:1, and a weight ratio range of 8:15 to 20:15. Note that the difference is 2.1 : 1 (instant claim 71) versus 2 : 1 (copending claim 77). Therefore, the claimed ratios of instant claims 71-73 in view of the copending limitation of at least a two-fold molar excess over the Cas9 protein is considered optimization through routine experimentation, and would be obvious to one of ordinary skill in the art. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
The level of skill in the art was high before the effective filing date of the presently claimed invention. One of ordinary skill in the art would have been motivated to combine the elements of the copending claims for the rationale of making a functional Cas9/sgRNA complex to cleave target DNA in a eukaryotic cell. It would have been obvious to combine the elements of copending claims because these are preferred embodiments in the claims which depend from co-pending base claim 58.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Currently amended claims 58, 61, 63, and 67 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 58-60, 62-65, 67, and 69-72 of copending Application No. 18/314,050 (reference application), in view of US Patent 10,731,181 to Chen et al (with priority to US Provisional 61/794422 filed on 03/15/2013). Chen et al citations refer to their ‘181 Provisional.
Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are either anticipated or rendered obvious over the copending claims in view of Chen et al.
Regarding instant claims 58, 61, and 67, copending claim 63 recites a method using a CRISPR/Cas9 complex comprising a Cas9 having an NLS at the C-terminus and a gRNA having a crRNA fused to a tracrRNA portion where the complex is capable of modification of a target endogenous nucleic acid in a nucleus of eukaryotic cell.
Regarding amended claim 58, copending claim 58 recites that the guide RNA is a single-chain guide RNA (sgRNA) comprising the crRNA portion fused to the tracrRNA portion.
Further, copending claim 59 meets the limitation that the Cas9-gRNA complex is in a form capable of being introduced into the eukaryotic cell, specifically by transfection. Copending claim 62 recites that the target DNA sequence has a PAM that consists of 5’-NGG-3’. Co-pending claim 64 recites that the crRNA is 20 nucleotides in length. Co-pending claim 67 recites the assembly of the Cas9 and gRNA in vitro.
Regarding instant claim 63, the NLS of copending claim 63 is construed to meet the limitation of a peptide tag.
Regarding instant claim 67, copending claim 67 recites that the gRNA is transcribed in vitro.
However, copending claims do not recite the new limitation of a transfection buffer without endogenous target DNA or the limitation of molar amounts. It is prima facie obvious in view of copending claim 67 that the Cas/guide RNA complex formed in vitro before being introduced into the eukaryotic cell would be in a transfection buffer without the endogenous target DNA because the complex needs to be in a buffer to exist as a functional complex without the endogenous target DNA because it is formed in vitro before being introduced into the cell.
Regarding the limitation of molar amounts, Chen et al suggest introducing the Cas9 protein and a guide RNA into a eukaryotic cell or embryo by methods including transfection and microinjection. (para 0041). For example, in para 0047, lines 11-13, Chen et al state: “In still other embodiments, the fusion protein can be introduced into the cell or embryo as an RNA-protein complex comprising the fusion protein and the guiding RNA”. In para 0080-0081, Chen et al disclose that the RNA-guided endonuclease is Cas9 and the RNA-guided endonuclease is “introduced into the cell as a RNA-protein complex comprising the endonuclease protein and the guiding RNA”. Chen et al disclose Cas9 fused to an NLS (para 0010 cites Cas9 protein; para 0023 cites NLS). Chen et al disclose this link can be at the N- or C-terminal (see para 0024). Chen et al disclose additional peptide tags (para 0025). Chen et al disclose that methods of introducing into eukaryotic cells include transfection, nucleofection, and electroporation and state that “Transfection methods are well known in the art” (see para 0066). Further, introducing may be by microinjection (para 0066, last sentence). In para 0075-0076 lists suitable type of eukaryotic cells. Regarding the limitation that the transfection buffer is free of endogenous target DNA, it is prima facie obvious that a system for introducing a Cas9 protein and sgRNA into a eukaryotic cell using a known method of transfection or microinjection into a eukaryotic cells would include a transfection buffer that is free of the endogenous target DNA because such endogenous target DNA is present within the cell to be transfected. In addition, although Chen et al does not explicitly disclose that the sgRNA is present in at least a two-fold molar excess over the Cas9 protein in the transfection buffer, they disclose that the ratio of protein to gRNA will generally be approximately stoichiometric “such that they can form an RNA-protein complex”. See para 0067. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05. Chen et al discloses the general conditions where the ratio of protein to gRNA will generally be approximately stoichiometric “such that they can form an RNA-protein complex”. Therefore, the claimed ratio of the sgRNA being present in at least a two-fold molar excess over the Cas9 protein is considered optimization through routine experimentation, and would be obvious to one of ordinary skill in the art. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
The level of skill in the art was high before the effective filing date of the presently claimed invention.
In view of the high level of skill in the art before the effective filing date of the presently claimed invention it is considered that one of ordinary skill in the art would have been motivated to combine the elements of the copending claims in view of Chen et al for the rationale of making a functional Cas9/sgRNA complex to cleave target DNA in a eukaryotic cell.
It would have been obvious to combine the elements of copending claims because these are preferred embodiments in the claims which depend from co-pending base claim 58. Further, Chen et al suggest the ratio of protein to gRNA will generally be approximately stoichiometric “such that they can form an RNA-protein complex”. See para 0067. Therefore, the claimed ratio of the sgRNA being present in at least a two-fold molar excess over the Cas9 protein is considered optimization through routine experimentation, and would be obvious to one of ordinary skill in the art.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Currently amended claims 58, 61, 63, 67, and 70-73 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-6, 8-10, 20-25 of co-pending Application No. 18/467,967 (reference application), in view of Doudna et al (US 2014/0068797, with priority to US Provisional 61/757,640 filed on filed on 1/28/2013. All Doudna et al citations in the body of the rejection refer to the ‘640 Provisional.) This is a new grounds of rejection.
Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are rendered obvious over the combination of copending claims.
Regarding instant claims 58 and 70, co-pending claim recites a composition for inducing targeted disruption of endogenous genes in a human cell, the composition comprising a Cas9/guide RNA complex, wherein the Cas9/RNA complex comprises: a Cas9 protein to which a nuclear localization signal (NLS) is linked; and a guide RNA having a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA), wherein the crRNA comprises: i) a portion to be hybridized with a portion of the tracrRNA, and ii) a portion complementary to a target DNA of the endogenous genes, wherein the Cas/guide RNA complex is formed in vitro before being introduced into the eukaryotic cell. Further, copending claim 10 recites that the target DNA comprises a first strand having a region complementary to the crRNA and a second strand having a trinucleotide protospacer adjacent motif (PAM), wherein the PAM is the trinucleotide of 5'-NGG-3'.
Also, regarding amended claim 58, copending claim 2 recites that the guide RNA is a single-chain guide RNA (sgRNA) comprising the crRNA fused to the tracrRNA. Also, copending claim 1 recites a transfection buffer without endogenous target DNA.
Regarding instant claim 61, copending claim 3 recites that the Cas9 protein has the NLS in proximity to its C-terminal.
Regarding instant claim 63, copending claims 5 and 6 recite an HA-tag and His-tag, respectively which meet the limitation of a peptide tag.
Regarding instant claim 67, copending claim 8 recites that the guide RNA is in vitro transcribed RNA.
Regarding instant claims 71-73, copending claims 23 and 25 recite that the guide RNA and the Cas9 protein are added to the buffer in a molar ratio of gRNA:Cas9 protein ranging from 29:14 to 29:1.4. It is considered that this range of molar ratios renders obvious the molar ratios and weight ratios of the instant claims 71-73, specifically: sgRNA:Cas9 of a molar ratio range being 2.1:1 to 6.2:1; a weight ratio range of 4:9 to 1.3:1, and a weight ratio range of 8:15 to 20:15. Therefore, the claimed ratios of instant claims 71-73 in view of the copending limitation of a molar ratio of gRNA:Cas9 protein ranging from 29:14 to 29:1.4 is considered optimization through routine experimentation, and would be obvious to one of ordinary skill in the art. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
The level of skill in the art was high before the effective filing date of the presently claimed invention.
One of ordinary skill in the art would have been motivated to combine the elements of the copending claims for the rationale of making a functional Cas9/sgRNA complex to cleave target DNA in a eukaryotic cell. It would have been obvious to combine the elements of copending claims because these are preferred embodiments in the claims which depend from co-pending base claim 1.
In addition, regarding base claim 58, copending claims do not recite that the DNA target comprises a 20-base pair region complementary to the second portion of the crRNA. However, present claims are drawn to a product of a Cas9/sgRNA complex in transfection buffer and this limitation of the target DNA is not required of the presently claimed product. Doudna et al disclose functional single guide RNA (sgRNA)/Cas 9 protein complexes where the target DNA has a 20-base pair region complementary to the second portion of the crRNA. For example, Doudna et al disclose a guide RNA having a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), wherein the crRNA comprises: a portion to be hybridized with a portion of the tracrRNA, and a portion complementary to a target DNA of the endogenous genes. (See para 0031 reciting “RNA chimera A”, 00374). Doudna et al disclose that the guide RNA is a single-chain guide RNA comprising the crRNA portion fused to the tracrRNA portion. (See para 0031 reciting “RNA chimera A”, 00374). In addition, Doudna et al disclose that the target DNA sequence comprises a first strand having a 20-base pair region complementary to the second portion of the crRNA and a second strand having a PAM consisting of 5’-NGG-3’ motif. (See para 044; FIG 16A-C; para 00416-417).
One of ordinary skill in the art would have been motivated to combine the elements of the copending claims in view of Doudna et al for the rationale of making a functional Cas9/sgRNA complex to cleave target DNA in a eukaryotic cell.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Currently amended claims 58, 61, 63, 67, and 70-73 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-8 of US Patent 12/473,559 (reference application).
Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are rendered obvious over the combination of patented claims.
Regarding instant claims 58, 67, and 70, patented claim 1 recites a method of inducing a modification of a target endogenous nucleic acid sequence in a nucleus of a human cell, comprising: preparing a Cas9 protein, wherein the Cas9 protein comprises a nuclear localization signal (NLS); preparing a single-guide RNA (sgRNA), wherein the sgRNA comprises a crRNA and a tracrRNA, wherein the sgRNA is transcribed in vitro or synthesized chemically, and wherein the target endogenous nucleic acid sequence includes a portion complementary to the crRNA of the sgRNA; providing a buffer in an in vitro environment; disposing the Cas9 protein into the buffer; disposing the sgRNA into the buffer, wherein the sgRNA is disposed in at least a two-fold molar excess over the Cas9 protein in the buffer, allowing the Cas9 protein and the sgRNA to complex in the in vitro environment to form a Cas9/sgRNA complex; transfecting the Cas9/sgRNA complex into the human cell by electroporation, whereby the Cas9/sgRNA complex induces the modification of the target endogenous nucleic acid sequence in the nucleus of the human cell. Also, regarding instant claim 58, patented claim 3 recites that the target endogenous nucleic acid comprises a trinucleotide protospacer adjacent motif (PAM) recognized by the Cas9 protein, wherein the PAM consists of trinucleotide 5′-NGG-3′. Regarding instant claim 58, patented claim 5 recites that the crRNA is 20 nt in length.
Regarding instant claim 61, patented claim 4 recites that the NLS is at a C-terminus of the Cas9 protein.
Regarding instant claim 63, the NLS of copending claim 1 is construed to meet the limitation of a peptide tag.
The level of skill in the art was high before the effective filing date of the presently claimed invention.
Regarding new claims 71-73, copending claims 10 and 12 recites at least two-fold molar excess of gRNA to Cas9 which is considered to render obvious the molar ratios and weight ratios of the instant claims 71-73, specifically: sgRNA:Cas9 of a molar ratio range being 2.1:1 to 6.2:1; a weight ratio range of 4:9 to 1.3:1, and a weight ratio range of 8:15 to 20:15. Note that the difference is 2.1 : 1 (instant claim 71) versus 2 : 1 (copending claim 10). Therefore, the claimed ratios of instant claims 71-73 in view of the copending limitation of at least a two-fold molar excess over the Cas9 protein is considered optimization through routine experimentation, and would be obvious to one of ordinary skill in the art. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Currently amended claims 58, 61, 63, and 70-73 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-16 of co-pending Application No. 19/027,181 (reference application), in view of Doudna et al (US 2014/0068797, with priority to US Provisional 61/757,640 filed on filed on 1/28/2013. All Doudna et al citations in the body of the rejection refer to the ‘640 Provisional.) This is a new grounds of rejection.
Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are rendered obvious over the combination of co-pending claims in view of Doudna et al.
Regarding instant claims 58, and 70, copending claim 1 (see Examiner’s Amendment mailed on 11/20/2025, recites a method comprising a CRISPR/Cas9 complex comprising a Cas9 protein and a guide RNA having a crRNA and a tracrRNA portion where the complex is capable of cleaving an endogenous target DNA in a human cell, where the Cas9/RNA complex is formed before transfer into the human cell. Copending claim 11 recites that the gRNA is an sgRNA. Co-pending claim 8 recites that the target DNA sequence has a PAM. Further, co-pending claim 8 recites that the target DNA is genomic DNA and that the crRNA is 20 nucleotides in length. Regarding amended claim 58, copending claims 7 and 11 recites that the guide RNA is an in vitro-transcribed single-chain guide RNA (sgRNA) comprising the crRNA portion fused to the tracrRNA portion. Further, copending claims 10 and 12 recites a two-fold molar excess over the Cas9 protein in the in vitro environment when complexing the Cas9/RNA complex.
Regarding instant claim 61, copending claim 76 recites an NLS disposed at a C-terminus of the Cas9 protein.
Regarding instant claim 63, the NLS of copending claim 58 is construed to meet the limitation of a peptide tag.
Further, regarding new claims 71-73, copending claims 10 and 12 recites at least two-fold molar excess of gRNA to Cas9 which is considered to render obvious the molar ratios and weight ratios of the instant claims 71-73, specifically: sgRNA:Cas9 of a molar ratio range being 2.1:1 to 6.2:1; a weight ratio range of 4:9 to 1.3:1, and a weight ratio range of 8:15 to 20:15. Note that the difference is 2.1 : 1 (instant claim 71) versus 2 : 1 (copending claim 10). Therefore, the claimed ratios of instant claims 71-73 in view of the copending limitation of at least a two-fold molar excess over the Cas9 protein is considered optimization through routine experimentation, and would be obvious to one of ordinary skill in the art. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
However, regarding instant claims 58 and 67, copending claims do not recite that the guide RNA is an in vitro-transcribed and copending claims do not recite that the Cas9 protein comprising an NLS. Also, copending claims do not recite that the PAM sequence consists of 5’-NGG-3’.
The level of skill in the art was high before the effective filing date of the presently claimed invention.
Doudna et al disclose functional single guide RNA (sgRNA)/Cas 9 protein complex, the Cas9 protein containing an NLS sequence complexed where the target DNA has a 20-base pair region complementary to the second portion of the crRNA. For example, Doudna et al disclose a guide RNA having a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), wherein the crRNA comprises: a portion to be hybridized with a portion of the tracrRNA, and a portion complementary to a target DNA of the endogenous genes. (See para 0031 reciting “RNA chimera A”, 00374). Doudna et al disclose that the guide RNA is an in vitro-transcribed single-chain guide RNA comprising the crRNA portion fused to the tracrRNA portion. (See para 0031 reciting “RNA chimera A”, 00374). In addition, Doudna et al disclose that the target DNA sequence comprises a first strand having a 20-base pair region complementary to the second portion of the crRNA and a second strand having a PAM consisting of 5’-NGG-3’ motif. (See para 044; FIG 16A-C; para 00416-417).
One of ordinary skill in the art would have been motivated to combine the elements of the copending claims in view of Doudna et al for the rationale of making a functional Cas9/sgRNA complex to cleave target DNA in a eukaryotic cell. The NLS was used before the effective filing date to target the Cas9/sgRNA complex to the nucleus and the conventional PAM sequence for Cas9 consisted of a 5’-NGG-3’ motif.
It would have been obvious to combine the elements of copending claims because these are preferred embodiments in the claims and Doudna et al disclosed functional Cas9/sgRNA complexes successfully editing genomic DNA in human cells.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Currently amended claims 58, 61, 63, 67, 70 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-11 of co-pending Application No. 19/023,934 (reference application), in view of Doudna et al (US 2014/0068797, with priority to US Provisional 61/757,640 filed on filed on 1/28/2013), in view of Chen (above), in view of Gaj et al (above). All Doudna et al citations in the body of the rejection refer to the ‘640 Provisional.) This is a new grounds of rejection.
Although the claims at issue are not identical, they are not patentably distinct from each other because the instant claims are rendered obvious over the combination of co-pending claims in view of Doudna et al, Chen, and Gaj et al.
Regarding instant claim 58, copending claim 1 recites a method comprising a CRISPR/Cas9 complex comprising a Cas9 protein and a guide RNA having a crRNA and a tracrRNA portion where the complex is capable of cleaving an endogenous target DNA in a eukaryotic cell, where the Cas9/RNA complex is formed before transfer into the eukaryotic cell. Copending claim 1 recites that the gRNA is an sgRNA. Regarding amended claim 58, copending claim 1 recites that the guide RNA is a single-chain guide RNA (sgRNA) comprising the crRNA portion fused to the tracrRNA portion.
Regarding instant claim 63, the NLS of copending claim 1 is construed to meet the limitation of a peptide tag.
Regarding instant claim 70, copending claim 10 recites a human cell.
However, regarding instant claims 58, 61, and 67, copending claims do not recite that the PAM sequence consists of 5’-NGG-3’. Also, copending claims do not recite that the guide RNA is an in vitro-transcribed and copending claims do not recite that the NLS disposed at a C-terminus of the Cas9 protein.
The level of skill in the art was high before the effective filing date of the presently claimed invention.
Doudna et al disclose functional single guide RNA (sgRNA)/Cas 9 protein complex, the Cas9 protein containing an NLS sequence disposed at a C-terminus of the Cas9 protein. Doudna et al disclose the target DNA has a 20-base pair region complementary to the second portion of the crRNA. For example, Doudna et al disclose a guide RNA having a CRISPR RNA (crRNA) and a transactivating crRNA (tracrRNA), wherein the crRNA comprises: a portion to be hybridized with a portion of the tracrRNA, and a portion complementary to a target DNA of the endogenous genes. (See para 0031 reciting “RNA chimera A”, 00374). Doudna et al disclose that the guide RNA is an in vitro-transcribed single-chain guide RNA comprising the crRNA portion fused to the tracrRNA portion. (See para 0031 reciting “RNA chimera A”, 00374). In addition, Doudna et al disclose that the target DNA sequence comprises a first strand having a 20-base pair region complementary to the second portion of the crRNA and a second strand having a PAM consisting of 5’-NGG-3’ motif. (See para 044; FIG 16A-C; para 00416-417).
One of ordinary skill in the art would have been motivated to combine the elements of the copending claims in view of Doudna et al for the rationale of making a functional Cas9/sgRNA complex to cleave target DNA in a eukaryotic cell. The NLS was used before the effective filing date to target the Cas9/sgRNA complex to the nucleus and the conventional PAM sequence for Cas9 consisted of a 5’-NGG-3’ motif.
It would have been obvious to combine the elements of copending claims because these are preferred embodiments in the claims and Doudna et al disclosed functional Cas9/sgRNA complexes successfully editing genomic DNA in human cells.
Further, regarding claim 58, athough Doudna teaches delivering nucleic acids encoding Cas9 and guide RNA (gRNA) to cells for in vivo gene editing and teaches that the Cas9 protein can be delivered to cells, Doudna does not teach forming the Cas9/RNA complex before cell transfection and specifically copending claims do not recite a two-fold molar excess over the Cas9 protein in a transfection buffer.
Chen et al suggest introducing the Cas9 protein and a guide RNA into a eukaryotic cell or embryo. (para 0041). For example, in para 0047, lines 11-13, Chen et al state: “In still other embodiments, the fusion protein can be introduced into the cell or embryo as an RNA-protein complex comprising the fusion protein and the guiding RNA”. In para 0080-0081, Chen et al disclose that the RNA-guided endonuclease is Cas9 and the RNA-guided endonuclease is “introduced into the cell as a RNA-protein complex comprising the endonuclease protein and the guiding RNA”. Chen et al disclose Cas9 fused to an NLS (para 0010 cites Cas9 protein; para 0023 cites NLS). Chen et al disclose this link can be at the N- or C-terminal (see para 0024). Chen et al disclose additional peptide tags (para 0025). Chen et al disclose that methods of introducing into eukaryotic cells include transfection, nucleofection, and electroporation and state that “Transfection methods are well known in the art” (see para 0066). Further, introducing may be by microinjection (para 0066, last sentence). In para 0075-0076 lists suitable type of eukaryotic cells. Regarding the limitation that the transfection buffer is free of endogenous target DNA, it is prima facie obvious that a system for introducing a Cas9 protein and sgRNA into a eukaryotic cell using a known method of transfection or microinjection into a eukaryotic cells would include a transfection buffer that is free of the endogenous target DNA because such endogenous target DNA is present within the cell to be transfected. In addition, although Chen et al does not explicitly disclose that the sgRNA is present in at least a two-fold molar excess over the Cas9 protein in the transfection buffer, they disclose that the ratio of protein to gRNA will generally be approximately stoichiometric “such that they can form an RNA-protein complex”. See para 0067. However, where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. See MPEP § 2144.05. Chen et al discloses the general conditions where the ratio of protein to gRNA will generally be approximately stoichiometric “such that they can form an RNA-protein complex”. Therefore, the claimed ratio of the sgRNA being present in at least a two-fold molar excess over the Cas9 protein is considered optimization through routine experimentation, and would be obvious to one of ordinary skill in the art. Furthermore, a prima facie case of obviousness based on optimization may only be rebutted by evidence showing that the claimed range is critical, generally by proof that the claimed range achieves unexpected results relative to the prior art. See MPEP § 2144.05.
Further, one of ordinary skill in the art would have been motivated to transfect Cas9 protein and in vitro transcribed sgRNA into cells in place of using plasmids encoding such because use of plasmids in view of Gaj et al. Gaj teaches direct delivery of genome-editing zinc-finger nuclease (ZFN) proteins for endogenous gene disruption (Abstract). Gaj teaches the ZFN comprised an NLS (Fig 1a). Gaj teaches purifying ZFNs and diluted into serum-free medium (i.e., cell-free buffer and a transfection mixture) (Methods, page 1, last ]). Gaj teaches treating eukaryotic cells with the medium/ZFN mixture (i.e., transfecting the cells) (Methods, page 2, 4/2). Gaj teaches the ZFN modified the host cell genome (Fig 1fc-f). Gaj teaches an advantage of delivering ZFN to cells as a protein instead of plasmid is reduced ZFN-induced modifications at off-target sites (page 807, 42). Gaj teaches this is likely due to the short half-lives of the transduced ZFN and limiting the duration of ZFN in cells (page 807, 4/2). Gaj also teaches that directly delivering the gene editing proteins, instead of expressing them from plasmids, avoids the risk of insertional mutagenesis (page 807, 75). Gaj teaches “that protein delivery and the benefits afforded therein will be extended to other designer nucleases” (page 807, 45).
In view of the high skill level in the art of transfection of proteins directly into eukaryotic cells before the effective filing date of the presently claimed invention it would have been obvious to form the Cas9/sgRNA complex in vitro before transfecting into eukaryotic cells using standard known transfection lab procedures. It is considered that one of ordinary skill in the art having the cited references combined with the copending claims would have had a reasonable expectation of success to do such.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
No claim is allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CATHERINE S HIBBERT whose telephone number is (571)270-3053. The examiner can normally be reached M-F 8:00-5:00.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Melissa Fisher can be reached at 571-270-7430. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/CATHERINE S HIBBERT/Primary Examiner, Art Unit 1658