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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 09/29/2025 has been entered.
Applicant’s Supplemental Claim Amendment filed on 12/05/2025 has been entered.
Claims 7, and 11-19 are cancelled. Claims 23-29 are new.
Claims 1-6, 8-10, and 20-29 are pending and under examination.
Priority
The most recent filing receipt filed on May 9, 2024 is controlling.
Applicant's claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. 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) or under 35 U.S.C. 120, 121, 365(c), or 386(c) 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).
The disclosure of the priority documents fail to provide adequate support or enablement in the manner provided by 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph for one or more claims of this application as follows.
The US Provisional 61/717,324 does not disclose the invention of base claims 1 and 25. The '324 Provisional does not disclose a Cas/guide RNA complex formed in vitro before being introduced into the eukaryotic cell. Further, US Provisional 61/803,599 does not disclose the invention of base claims 1 and 25. The '599 Provisional is three pages and refers to guide RNA but does not refer to a chimeric or dual guide RNA. The '599 Provisional does not refer to a Cas/guide RNA complex formed in vitro before being introduced into the eukaryotic cell.
Thus, present claims 1-3, 5-6, 8-10, and 21-29 receive an effective filing date to US Provisional 61/837,481 (06/20/2013).
Regarding instant claims 4 and 20, the US Provisional 61/837,481 does not disclose the Cas9 protein is a mutant form (D10A Cas9). Thus, present claims 4 and 20 receive an effective filing date to PCT/KR2013/009488 filed on October 23, 2013.
Information Disclosure Statement
The IDS filed on 12/05/2025 has been considered by the examiner.
Response to Amendment
Any/all objections and rejections made in the previous office action and not repeated in this office action are withdrawn.
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.
Currently amended claims 1-6, 8-10, and 20-29 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. This is a new grounds of rejection.
Independent claims 1 and 25 are drawn to a composition comprising a Cas9 protein, a guide RNA (sgRNA, claim 25), and an electroporation buffer.
However, the claim 1 recites the following limitations which appear to require active method steps: “wherein the Cas9 protein and the guide RNA are added to the electroporation buffer”, “where the Cas9 protein and the guide RNA form an in vitro Cas9/guide RNA complex in the electroporation buffer”, and “wherein the in vitro Cas9/guide RNA complex serves to induce the modification of the target human endogenous nucleic acid sequence in the nucleus of the human cell”.
Further, the claim 25 recites the following limitations which appear to require active method steps: “wherein the sgRNA is added to the electroporation buffer in a molar excess of the Cas9 protein”, “where the Cas9 protein and the sgRNA form an in vitro Cas9/sgRNA complex in the electroporation buffer” and “wherein the in vitro Cas9/sgRNA complex serves to induce the modification of the target human endogenous nucleic acid sequence in the nucleus of the human cell”.
It is unclear if the claims are drawn to a composition comprising a Cas9 protein, a guide RNA (sgRNA, claim 25), and an electroporation buffer or rather intend to require a Cas9/guide RNA complex. The fact pattern for the instant claims is that it is known that the Cas9 protein and guide RNA in electroporation buffer would form a complex essentially immediately. The scope of the claims is unclear as to whether the active method steps of “adding”, “forming a complex”, and “serving to induce sequence modification in the nucleus of the human cell” are intended to be required of the claims.
For purpose of applying prior art claims must be given their broadest reasonable interpretation consistent with the specification. Present claims are construed as a product composition that do not require these active method steps.
Claims 2-6, 8-10, and 20-24, and 26-29 are indefinite for the same reasoning as they depend from base claims 1 or 25 and are not remedial.
Claim Rejections - 35 USC § 103 – new grounds
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.
Claims 1-6, 8-10, and 20-29 are rejected under 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 1/28/2013; of record), in view of WO2005/123962 to Cui et al (of record) in view of US Patent 10,731,181 to Chen et al (with priority to US Provisional 61/794,422 filed on 03/15/2013), in view of Gaj et al & Supplemental (Nature Methods 2012, 9: 805-807), in view of Hwang et al (Nature Biotechnology 2013, 31: 227-229 and Supplemental Material, published January 29, 2013), in view of Addgene (pMLM3613, https://www.addgene.org/browse/seguence/59208/ [retrieved June 2, 2013]), and Raemdonck et al (Biochemistry 2006, 45: 10614-10623), in view of Hayashi et al (Developmental Dynamics 2010, 239: 2034-2040). Doudna et al citations in the body of the rejection refer to the ‘640 provisional. Chen et al citations refer to the ‘422 provisional. This is a new grounds of rejection.
Claim interpretation: The presently amended claims are drawn to a product and are construed to not require the active method steps which are recited in the claims.
Also, note that in base claims 1 and 25, the phrase “for inducing a modification of a target human endogenous nucleic acid sequence in a nucleus of a human cell” is construed as an intended use phrase.
Further, regarding the product-by-process limitations in the claims, MPEP 2113 informs that product-by-process claims are not limited to the manipulations of the recited steps, only the structure implied by the steps, stating that: "[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985) (citations omitted). Claim was directed to a novolac color developer. The process of making the developer was allowed. The difference between the inventive process and the prior art was the addition of metal oxide and carboxylic acid as separate ingredients instead of adding the more expensive pre-reacted metal carboxylate. The product-by-process claim was rejected because the end product, in both the prior art and the allowed process, ends up containing metal carboxylate. The fact that the metal carboxylate is not directly added, but is instead produced in-situ does not change the end product.). Furthermore, "[b]ecause validity is determined based on the requirements of patentability, a patent is invalid if a product made by the process recited in a product-by-process claim is anticipated by or obvious from prior art products, even if those prior art products are made by different processes." Amgen Inc. v. F. Hoffman-La Roche Ltd., 580 F.3d 1340, 1370 n 14, 92 USPQ2d 1289, 1312, n 14 (Fed. Cir. 2009). See also Purdue Pharma v. Epic Pharma, 811 F.3d 1345, 117 USPQ2d 1733 (Fed. Cir. 2016). However, in the context of an infringement analysis, a product-by-process claim is only infringed by a product made by the process recited in the claim. Id. at 1370 ("a product in the prior art made by a different process can anticipate a product-by-process claim, but an accused product made by a different process cannot infringe a product-by-process claim").
Regarding claims 1, 2, and 24-25, Doudna et al discloses a composition comprising:
a Cas9 protein to which an NLS is linked (see Reference claims 5, 14-17, 19, 39, 42, 44, 48, 58-59, & 60, 73; para 00408 & 00522);
a single guide RNA (sgRNA) comprising the crRNA fused to the tracrRNA, where the crRNA comprises
a portion to be hybridized with a portion of the tracrRNA, and
a portion complementary to at least a portion of the target human endogenous sequence in the nucleus of the human cell (see Reference claims 5, 70; para 00374); and
an electroporation buffer which is free of DNA comprising such target human endogenous nucleic acid sequence and which is suitable for electroporation of an in vitro Cas9/guide RNA complex into a human cell. (See para 00108;00206; 00232; 00260; Reference claims 44, 59, 63, 110, 112). For example, in para 00107-108, Doudna disclose that for mammalian cells, suitable method of genetic manipulation include electroporation.
Further, in Reference claim 110, Doudna et al recite a kit comprising the DNA-targeting RNA of ref claim 1 and a buffer for introducing into cells the DNA-targeting RNA. Further, in Reference claim 112, Doudna et al recite a kit comprising a site-directed modifying polypeptide of ref claim 44, a buffer for introducing into cells the polypeptide. Also, Reference claim 60 recites a composition comprising
a DNA-targeting RNA (gRNA) and
the site-directed modifying polypeptide (Cas9).
Reference claim 63 recites a composition comprising the site-directed polypeptide of Ref claim 60 and a buffer for stabilizing nucleic acids and proteins. Also, Reference claim 58 recites a composition comprising the DNA-targeting RNA of Ref claim 44 (gRNA) and a buffer for stabilizing nucleic acids. Reference claim 59 recites a composition comprising the site-directed polypeptide of Ref claim 44 and a buffer for stabilizing nucleic acids and proteins. Further, the ‘640 Specification recites that electroporation is a suitable method for introducing the guide RNA and mRNA encoding the Cas9 into cells…(see para 00260).
Regarding the limitation of an NLS, 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.) Doudna et al disclose that the purpose of the NLS is to target nucleic acid sequence in the nucleus of the cell.
Further, Doudna et al disclose the guide RNA is a single guide RNA (sgRNA) 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 Fig3B, para 0031 reciting “RNA chimera A”, para 00374).
Regarding the limitation of an electroporation buffer free of target DNA, Doudna et al discloses a buffer for introducing into cells the DNA-targeting RNA, and a buffer for introducing into cells the Cas9 polypeptide. Doudna et al does not explicitly recite an electroporation buffer but contemplate electroporation as a method to introduce sgRNA and mRNA into eukaryotic cells. For example, each of claims 110 and 112 depend from Ref claim 44 which recites a combination of the guide RNA and the Cas9 polypeptide. In Reference claim 110, Doudna et al recite a kit comprising the DNA-targeting RNA of ref claim 1 and a buffer for introducing into cells the DNA-targeting RNA. In Reference claim 112, Doudna et al recite a kit comprising a site-directed modifying polypeptide of ref claim 44, a buffer for introducing into cells the polypeptide. Further, regarding the limitation of an electroporation buffer free of target DNA, note that it is prima facie obvious that an electroporation buffer for introducing a Cas9 and sgRNA into a human cell for targeting an endogenous nucleic acid sequence in the nucleus of the human cell would be free of DNA comprising such target human endogenous nucleic acid sequence because the target is already present in the nucleus of the cell and thus would not be electroporated into the cell. 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 00273; 00274, see especially para 00408 & 00522). 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 electroporation buffer in the specification the cleavage buffer of Doudna is construed to meet the limitation of a elctroporation 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 KCI, 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.
Regarding claim 8, Doudna et al discloses that the guide RNA is in vitro transcribed RNA. (See para 0260-0261). Doudna et al disclose that the guide RNA is transcribed in vitro. (See ‘640 Prov Working Example 2, para 0413). Doudna et al disclose in vitro synthesized RNA which may be introduced into a cell by microinjection, electroporation, transfection. (See para 0260-0261).
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”.
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).
Regarding claim 3, 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 claims 4 and 20, Doudna et al discloses that the Cas9 protein is a mutant form (D10A Cas9) where a catalytic aspartate residue is modified to alanine. See ‘640 reference claim 14 which recites a variant Cas9 having a D10A mutation.
Regarding claim 5, Doudna et al discloses that the Cas9 protein may comprise a hemagglutinin (HA) tag. (See para 0409).
Regarding claim 6, Doudna et al discloses that the Cas9 protein may comprise a 6xHis-tag. (See para 00204, line 8; para 00222, last sentence).
Regarding claim 8, Doudna et al discloses that the guide RNA is in vitro transcribed RNA. (See para 0260-0261). Doudna et al disclose that the guide RNA is transcribed in vitro. (See ‘640 Prov Working Example 2, para 0413). Doudna et al disclose in vitro synthesized RNA which may be introduced into a cell by microinjection, electroporation, transfection. (See para 0260-0261).
Regarding claim 9, Doudna et al disclose that the guide RNA is synthetic or in vitro-transcribed tracrRNA and crRNA. “Synthetic” is construed to meet the limitation of chemically synthesized RNA. (See para 00366, line 1).
Regarding claim 10, 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; FIG13E, 15, 16A-C; para 00398, 00416).
Regarding claim 24, 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 00325, 00384).
Also, regarding new claims 21-22, Doudna et al disclose human cancer cells and suggest targets in cancer genes. (See para 00239; 00287; 00289; see para 00417 targeting human CLTA locus which is a cancer-related gene.) Also, Chen et al explicitly suggest cancer cells and cancer gene targets. (see below).
However, while Doudna et al disclose a composition for inducing targeted modification of an endogenous target nucleic acid sequence in a human cell nucleus, the system comprising a Cas9 protein linked to an NLS; an 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 for introducing the guide RNA and Cas9 protein into mammalian cells to target endogenous DNA, which would be free of DNA comprising endogenous target nucleic acid sequence, and further suggest electroporation as a method to introduce nucleic acids into mammalian cells, they do not explicitly disclose the Cas9 protein and sgRNA in a composition with electroporation buffer. The Doudna teach forming the Cas9/sgRNA complex in vitro and in the human cell and directing such complex to the cell nucleus using an NLS attached to the Cas9. They do not explicitly disclose the method of electroporation of a Cas9/sgRNA complex formed in vitro, into the human cells. However, note that present claims are not drawn to a method but rather to a product composition.
Cui et al discloses that purified RNPs can be delivered into cultured eukaryotic cells by electroporation. (See para 0022; 0057; 0064; 0094 FIG8 & legend). Cui et al disclose that an NLS is linked to the group II intron-encoded protein of their RNPs to localize RNPs to the nucleus in eukaryotic cells. (See para 0052). Success chromosomal targeting in tissue culture cells by electroporation was shown. (See para 0023; 0057; 0064; 0094 FIG9 & legend). Cui et al disclose that for electroporation of an RNP into eukaryotic cells in culture, ten microgram of RNP was mixed with 400 ul of cells and incubated on ice for less than 2 min. Electroporations for introducing the RNPs into eukaryotic cells were carried out at 230v and various capactitance values. (See para 0094).
Chen et al suggest introducing Cas9/sgRNA complexes into eukaryotic cells by means of transfection. Regarding claims 1 and 25, Chen et al disclose Cas9 fused to an nuclear localization sequence called an NLS (para 0010 cites Cas9 protein; para 0023 cites NLS). The purpose of the NLS is to introduce the Cas9 complex into the nucleus of the eukaryotic cell. Regarding currently amended claims 1 and 25, 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). In para 0075-0076 lists suitable type of eukaryotic cells including human cells. 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”. (See para 0080-0081). 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”.
Regarding claim 3, 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).
Regarding claims 21-22, Chen et al disclose that suitable cells include the human cervical carcinoma cells (HELA) and human U2-OS osteosarcoma cells, and human A549 cells, A-431 cells, and human K562 cells. Regarding claim 22, Chen et al disclose that the target site in the host cells can be in the coding region of a gene or in a control region between genes. (See para 0046).
Chen et al disclose that the ratio of protein to gRNA in an in vitro complex will generally be approximately stoichiometric “such that they can form an RNA-protein complex”. See para 0067. 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”. Chen et al recite in para 105: “Transfection methods are well known in the art (see, e.g., “Current Protocols in Molecular Biology” Ausubel et al., John Wiley & Sons, New York, 2003 or “Molecular Cloning: A Laboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 3.sup.rd edition, 2001).
However, regarding claims 23, and 25-29 Doudna et al and Chen et al do not disclose the limitations of a molar excess of gRNA to Cas9 protein.
Hwang teaches methods of introducing Cas9 and sgRNA into Zebrafish embryos (i.e., eukaryotic 21. cells) into induce targeted genetic modifications in vivo (Abstract). Hwang teaches the Cas9 was fused to an NLS (Supplemental methods, 1). Hwang teaches introducing Cas9-encoding mRNA and sgRNA into the zebrafish embryos (page 227, 13; Supp Table 3). Hwang teaches varying the ratio of sgRNA and the Cas9-encoding mRNA to optimize the target site modification efficiency (Supp Table 3). Hwang teaches mass ratios of sgRNA:Cas9-mRNA (Supp Table 3). Hwang teaches the length of the sgRNA is 100 nt (Supp Table 1). Hwang teaches the Cas9-encoding mRNA was produced from pMLM3613, which is available from Addgene. Addgene teaches the mRNA-encoding Cas9-NLS-HA including the polyadenylation signal is approximately 4442 nt. Hwang teaches a mass ratio of sgRNA : Cas9 mRNA of 5 : 100 (Supp Table 3, row 1), which, using a nucleic acid mass calculator, is equivalent to a 2.2 molar ratio. Hwang teaches a mass ratio of sgRNA : Cas9 mRNA of 12.5 : 100 (Supp Table 3, row 2), which is equivalent to a 5.5 molar ratio. Hwang teaches a mass ratio of sgRNA : Cas9 mRNA of 36.7 : 100 (Supp Table 3, row 2), which is equivalent to a 16.3 molar ratio. Hwang teaches the above ratios resulted in between 21-33% average indel mutation frequency (Supp Table 3).
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, 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).
Raemdonck teaches a substantial level of single stranded RNA (ssRNA) degradation in cellular 22. extracts and in intact cells (Abstract). Raemdonck teaches ssRNA is almost completely degraded after 60 minutes of incubation in cytoplasmic and nuclear fractions, indicating that both the cytoplasm and the nucleus has substantial ssRNAse activity (1 spanning pages 10619-10620; Fig 7A). Raemdonck teaches that ssRNA is also degraded when injected into the cytoplasm of intact cells (page 10620, 14; Fig 9B,D). Raemdonck teaches that when ssRNA is injected into the cytoplasm, a fraction of the ssRNA is rapidly degraded, whereas another fraction of ssRNA will reach the nucleus and experience a slower rate of degradation (page 10620, 14; Fig 9B,D).
It would have been obvious to one skilled in the art before the effective filing date to have combined the SpCas9 and sgRNA taught in Doudna and delivered it to cells in the instantly claimed molar ratio range. Although Doudna and Chen do not explicitly disclose that the sgRNA present in the claimed ratios with SpCas9, they disclose that the ratio of Cas9-fusion proteins to gRNA will generally be approximately stoichiometric "such that they can form an RNA-protein complex". 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. Therefore, the claimed ratio of the sgRNA being present in >2-fold to 20-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. The skilled artisan would have been motivated to optimize the molar ratio of sgRNA : Cas9 between ~2- 20 because Hwang teaches these ratios within the claimed range for a different delivery modality, all of which resulted in above 20% mutation frequency. The skilled artisan would have also been motivated to increase the in vitro sgRNA : Cas9 molar ratio taught in Doudna when using Cas9/sgRNA for in vivo genetic modification because Raemdonck teaches ssRNA is rapidly degraded in the cytoplasm before reaching the nucleus. 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.
It would have been obvious to one skilled in the art before the effective filing date to have combined the Cas9 and sgRNA taught in Doudna and delivered it to cells in the instantly claimed molar ratio range. Although Doudna and Chen do not explicitly disclose that the sgRNA present in the claimed ratios with Cas9, they disclose that the ratio of Cas9-fusion proteins to gRNA will generally be approximately stoichiometric "such that they can form an RNA-protein complex". 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. Therefore, the claimed ratio of the sgRNA being present in >2-fold to 20-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. The skilled artisan would have been motivated to optimize the molar ratio of sgRNA : Cas9 between ~2- 20 because Hwang teaches these ratios within the claimed range for a different delivery modality, all of which resulted in above 20% mutation frequency. The skilled artisan would have also been motivated to increase the in vitro sgRNA : Cas9 molar ratio taught in Doudna when using Cas9/sgRNA for in vivo genetic modification because Raemdonck teaches ssRNA is rapidly degraded in the cytoplasm before reaching the nucleus. 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, regarding the limitations of excess gRNA over Cas9 protein, it is noted above that Doudna teaches methods of introducing nucleic acids into cells include electroporation and Chen also teaches methods of introducing RNA- targeted endonucleases and their guide RNAs include electroporation. Rationale to optimize an excess amount of sgRNA in buffer with Cas9 protein would be to reduce the possible degradation of the sgRNA both in the in vitro buffer and within the cells. Note that it is known in the art (see Doudna and Chen) that the functional Cas9/sgRNA complex is essentially in a 1:1 ratio. However, it is also known that the composition of Cas9 and sgRNA in an electroporation buffer would change as the complex forms in the buffer. The composition intended to be electroporated would thus contain the complex and the excess gRNA. Doudna et al contemplates a composition comprising the site-directed polypeptide of Ref claim 60 and a buffer for stabilizing nucleic acids and proteins.
Hayashi teaches introducing mRNA (i.e., ssRNA) into mouse cells via electroporation (page 2035, 34. col 1, 12). Hayashi teaches the mRNA is nearly completely degraded within 12 hours of electroporation (Fig. 1F). It would have been obvious to one skilled in the art before the effective filing date of the 35. claimed invention to have introduced the obvious Cas9/sgRNA combination in the claimed sgRNA and Cas9 ratios into the eukaryotic cells via electroporation. It would have amounted to substituting one known means of introducing proteins and ssRNA into cells for another known means by known methods to yield predictable results. The skilled artisan would have been motivated to use the increased sgRNA : Cas9 ratio rendered obvious above in electroporation methods because Hayashi teaches that ssRNA is not stable in cells. The skilled artisan would have predicted that by increasing the molar ratio of sgRNA : Cas9, sufficient numbers of Cas9/sgRNA complexes taught in Doudna and Chen would still form even upon the ssRNA degradation using the ssRNA electroporation method disclosed in Hayashi.
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 in a composition a Cas9 protein comprising an NLS, an sgRNA for targeting a human gene, and an electroporation buffer which did not contain a target DNA, for the intended use of introducing such complex into human cells to edit an endogenous target gene in the cell nucleus to arrive at the presently claimed invention.
Response to Arguments
The applicants’ arguments filed on 12/05/2025 have been fully considered but are unpersuasive and are addressed as they relate to this new grounds of rejection. The applicants argue that claim 1 is presently amended to:
"composition for inducing a modification of a target human endogenous nucleic acid sequence in a nucleus of a human cell, the composition comprising a Cas9 protein a guide RNA and an electroporation buffer that is free of DNA comprising the target human endogenous nucleic acid sequence; wherein the Cas9 protein and the guide RNA are added to the electroporation buffer; wherein the Cas9 protein and the guide RNA form an in vitro Cas9/guide RNA complex in the electroporation buffer" and "wherein the electroporation buffer is suitable for electroporation of the in vitro Cas9/guide RNA complex into the human cell.”
Further, the applicants argue that presently amended “independent claim 1 and newly added base claim 25 are patentably distinct over Doudna, Jinek, and Chen, whether considered separately or in any combination thereof: and that the patentability of the claims is further supported by secondary considerations, including industry recognition and unexpected results.
First, the applicants argue that a POSA would not have viewed a combination of Doudna, Jinek, and Chen as disclosing or suggesting a composition comprising an in vitro Cas9/guide RNA complex that forms in an electroporation buffer that is free of target human endogenous DNA, where the electroporation buffer is suitable for electroporation of the in vitro Cas9/guide complex into a human cell.
However, this argument is unpersuasive because the present rejection is over Doudna, Cui, Chen, Hwang et al, Addgene and Raemdonck et al and Hayashi et al. Doudna et al explicitly recites in their reference claims a composition of a Cas9 protein in a buffer suitable for introducing into mammalian cells including human cells and a composition of an sgRNA in a buffer suitable for introducing into mammalian cells. Also, Chen explicitly suggests introducing a Cas9 protein/sgRNA complex into mammalian cells including human cells. Each of Doudna and Chen suggest electroporation/nucleofection as a means to introduce Cas9 and sgRNA into cells. Further, the applicants argument that they are claiming a Cas9/guide RNA complex formed in the electroporation buffer is unpersuasive because features must not be read into the claims. The presently amended claims recite a composition comprising a Cas9 protein, a guide RNA, and an electroporation buffer. They do not recite a Cas9/gRNA complex. However, each of Douda and Chen recite a Cas9/gRNA complex.
Further, the argument that Doudna does not explicitly disclose an electroporation buffer, the Cui et al reference explicitly disclose successful electroporation of a functional RNP complex into human cells.
Also, the applicants argument that the cited references do not suggest an electroporation buffer that is free of target human endogenous DNA is unpersuasive. Doudna explicitly recites a buffer for introducing Cas9 and sgRNA into cells where the sgRNA is designed to target human endogenous DNA. They do not disclose adding target DNA to such composition for the logical rationale that the intended use is to target (cleave) endogenous DNA in the nucleus (genome) and not to add such target DNA to the cells. Further, the applicants’ argument that Doudna Example 1 (para 0366) recites an example of a Cas9/sgRNA complex in an in vitro cleavage buffer but with a target DNA is unpersuasive. 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, the applicants argue that Chen is directed to fusion proteins with expanded CRISPR DNA-binding word length. However, this argument is unpersuasive because the present claims require an NLS attached to the Cas9 protein which is itself a type of fusion protein. Further, present claims recite open claim language “comprising” and thus do not exclude a Cas9 fusion.
Further, the applicants argument that a POSA would not have been motivated or had a reasonable expectation of success in practicing the claimed composition, specifically an electroporation buffer suitable for electroporation of the in vitro Cas9/guide RNA complex into the human cell, stating that electroporation would result in unfolding and destabilization of the Cas9 protein, citing Bekard et al and Rodrigues et al. However, the Cui et al reference explicitly disclose successful electroporation of a functional RNP complex into human cells.
Lastly, the applicants’ arguments regarding industry recognition and unexpected results is unpersuasive because the argument is not commensurate with the scope of the present claims because the evidence provided by the applicant shows a narrower scope than is presently recited in the claims. For example, the argument refers to the Amaxa 4D-Nucleofector but the claims recite a generic electroporation buffer rather that a specific nucleofection buffer.
Also, the showing in the ‘481 Provisional does not support the finding that a molar increase in sgRNA over Cas9 protein in an electroporation buffer results in the improvement of an intended use of inducing a modification of a target human endogenous nucleic acid in a nucleus of a human cell. For example, regarding unexpected results, in the paragraph bridging pages 34-35, the ‘481 Provisional discloses a single example of a Cas9 protein/sgRNA complex in an electroporation buffer for introducing the complex directly into K562 cells by electroporation, specifically by nucleofection which is a type of electroporation. 1x106 K562 cells were transfected with 22.5-225 ug of Cas9 protein mixed with 100ug of in vitro transcribed sgRNA (or crRNA 40 ug and tracrRNA 80ug) using a the SF Cell Line 4D-Nucleofector X Kit, Program FF-120 (Lonza) “according to the manufacturer’s protocol”. Note that the sgRNA was 103 bp in length. This resulted in induced targeted mutation at the CR5 locus. Results are shown in Fig10 and disclose that targeted mutation at the CCR5 locus ranges from 4.8% to 38% in a sgRNA or Cas9 protein dose-dependent manner (9.4% for dual gRNA), stating that this result was “on par with the frequency obtained with Cas9 plasmid transfection (45%). Figure 10 is shown just below. Note that Fig10 shows that the plasmid transfection showed 42% frequency compared to the Cas9protein/dual gRNA. Further, Fig10 shows that constant 100ug sgRNA with increasing Cas9 protein showed increasing frequency as Cas9 protein increased from 22.5ug, 75ug, to 225ug. Thus the % frequency increased as the sgRNA to Cas9 ratio decreased. Further, Fig10 shows that increasing the amount of sgRNA from 22.5ug, to 75ug, to 225 ug appears to improve % frequency from 4.8% to 18% to 38%.
PNG
media_image1.png
379
451
media_image1.png
Greyscale
Further, the argument of unexpected results regarding the results shown in Kim 2014 and Lin et al are not commensurate with the scope of the present claims. For example, Kim 2014 and Lin et al recites K562 cells or HEK292T cells, specific target sequences, specific gRNA constructs, and buffers not recited in the present claims. Further, a POSA reviewing the instant Fig10 would understand that increasing the amount of Cas9 while keeping the amount of sgRNA actually improved the indel(%). See Fig10 just above.
Further, the applicants argument that introducing a Cas9/guide RNA complex may be less toxic to cells than introducing plasmids encoding such is unpersuasive because the phenomenon of plasmids being toxic to cells was known in the prior art (see Gaj above) and was cited as a rationale for forming a Cas9/guide complex rather than using plasmids encoding such. The fact that the inventor has recognized another advantage which would flow naturally from following the suggestion of the prior art cannot be the basis for patentability when the differences would otherwise be obvious. See Ex parte Obiaya, 227 USPQ 58, 60 (Bd. Pat. App. & Inter. 1985).
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Response to Arguments
The applicants’ arguments filed on 12/05/2025 have been fully considered. However, the applicants response filed on 12/05/2025 does not address the NSDP rejections. The applicants response filed on 09/29/2025 has also been fully considered but these Remarks do not address the presently amended claims. The applicants state in the Remarks filed on 09/29/2025 that they acknowledge NSDP rejections over US Applications: 18/314050, 18/313,946, 18/467,952, 18/147156, 17/440350, and US Patent 11,999952 with the statement that they will consider filing a Terminal Disclaimer as appropriate upon final resolution of the claims. Thus, the NSDP rejection are maintained and updated. The applicants state in the Remarks filed on 09/29/2025 that regarding the NSDP rejections over US Patent 10,767174, US Patent 11,123409, and US Patent 11,572574, that using these patents as a reference patent against the instant claims “is antithetical to the principles of ODP” because “this is the first application (and thus will be the first patent) from Applicant directed to the instantly claimed subject matter (see Remarks page 7).
However, this argument is unpersuasive for reasons provided in the body of the rejections below which show that the patented claims in combination with cited prior art references anticipate or render obvious the present claims.
Claims 1-3, 8-10, 24-26, and 28-29 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-8 of U.S. Patent No. 12,473,559. Although the claims at issue are not identical, they are not patentably distinct from each other because the patented claims anticipate or render obvious the instant claims.
Regarding instant claims 1, 24-26, and 28-29, patented claim 1 recites a method of inducing a modification of a target endogenous nucleic acid in a nucleus of a human cell, the method comprising a Cas9 protein comprising an NLS; an sgRNA comprising a crRNA and a tracrRNA Cas9/RNA in a buffer where the sgRNA is disposed in the buffer in at least a two-fold molar excess over the Cas9 protein in the buffer, and a electroporation of the Cas9/RNA into the cell.
Regarding instant claim 2, patented claim 1 recites a single-chain guide RNA (sgRNA).
Regarding instant claim 3, patented claim 4 recites the Cas9 protein has the NLS in proximity to its C-terminus or N-terminus.
Regarding instant claims 8-9, patented claim 1 recites that the guide RNA is in vitro transcribed RNA or chemically synthesized RNA.
Regarding instant claims 10, patented claim 3 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'.
Claims 1-3, 8, 21-22, and 24 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 15 of U.S. Patent No. US 11,572,574 in view of US2014/0068797 to Doudna et al (with priority to US Provisional 61/757,640 filed on filed on 1/28/2013), in view of Chen et al (above). All Doudna et al citations in the body of the rejection refer to the ‘640 Provisional). Although the claims at issue are not identical, they are not patentably distinct from each other because the patented claims render obvious the instant claims.
Regarding instant claims 1 and 24, patented claim 6 recites a method comprising a composition comprising a Cas9 protein and a guide RNA comprising a crRNA and a tracrRNA. Although the claim does not recite an electroporation buffer in the composition it is considered an electroporation buffer would have been prima facie obvious in view of the limitation in claim 6 that the composition is introduced into a eukaryotic cell using electroporation. Further, patented claim 4 recites the gRNA and Cas9 protein are in a form of a ribonucleoprotein.
Regarding instant claim 2, patented claim 2 recites single guide RNA.
Regarding instant claims 21-22, patented claims 1, 7, and 11 recited the target is the human wmN1 enhancer region of the PLP1 gene and while PLP1 itself is not generally classified as a classic oncogene, the wmN1 enhancer is directly linked to the regulation of PLP1 expression, which has been associated with cancer development and progression and thus is being interpreted to meet the limitation of an endogenous human oncogene target.
Regarding instant claim 8, patented claims do not recite the guide RNA is transcribed in vitro. Doudna et al discloses that the guide RNA is transcribed in vitro. ((See ‘640 Prov para 00260, lines 1-4). It would have been obvious to use the in vitro transcribed gRNA method of Doudna et al because their reference is in the same field of making Cas9/gRNA complexes in vitro for delivery to eukaryotic cells.
Further, regarding instant claims, the patented claims do not recite an NLS is attached to the Cas9.
Each of Doudna et al and Chen et al disclose an NLS attached to the Cas9 for the targeting to genes in the eukaryotic cell nucleus. It would have been prima facie obvious to add an NLS to the Cas9 as disclosed in Doudna et al, and Chen et al, because the patented claims explicitly recite a method of targeting a nuclear gene in a eukaryotic cell.
For example, Chen et al suggest introducing Cas9/sgRNA complex into eukaryotic cells by means of transfection. 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”. (See para 0080-0081). 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”. Chen et al disclose that suitable cells include the human cervical carcinoma cells (HELA) and human U2-OS osteosarcoma cells, and human A549 cells, A-431 cells, and human K562 cells. Regarding instant claims 1 and 3, Chen et al disclose Cas9 fused to an nuclear localization sequence called 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 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). In para 0075-0076 lists suitable type of eukaryotic cells including human cells. In para 117, Chen et al recite: “A variety of eukaryotic cells and embryos are suitable for use in the method. For example, the cell can be a human cell…”. In para 121, Chen et al recite:
(121) Fusion proteins comprising a CRISPR/Cas-like protein or a fragment thereof and an effector domain are detailed above in section (II). In general, the fusion proteins disclosed herein further comprise at least one nuclear localization signal. Nucleic acids encoding fusion proteins are described above in section (III). In some embodiments, the fusion protein can be introduced into the cell or embryo as an isolated protein (which can further comprise a cell-penetrating domain). Furthermore, the isolated fusion protein can be part of a protein-RNA complex comprising the guide RNA.
Chen et al recite in para 105: “Transfection methods are well known in the art (see, e.g., “Current Protocols in Molecular Biology” Ausubel et al., John Wiley & Sons, New York, 2003 or “Molecular Cloning: A Laboratory Manual” Sambrook & Russell, Cold Spring Harbor Press, Cold Spring Harbor, N.Y., 3.sup.rd edition, 2001). “
One or ordinary skill in the art would have been motivated to combine the elements of the patented claims with the method suggest by Chen et al to transfect the Cas9/sgRNA complex into a human cell as a complex because Chen et al expressly suggest transfecting a Cas9/sgRNA complex into eukaryotic cells and attaching an NLS to the Cas9 to achieve transport to the cell nucleus to edit genomic DNA, including human cells. It would have been obvious to do such because patented claims and Chen et al are in the same field of Cas9/sgRNA gene editing in human cells.
Claims 1-3, 8-10, and 24 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 58-60, 62-65, 67, and 69-72 of US Application 18/314,050 as evidenced by US2014/0068797 to Doudna et al (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). Although the claims at issue are not identical, they are not patentably distinct from each other because the combination of copending claims render obvious the instant claims.
Regarding instant claims 1, and 24, copending claim 63 depends from base claim 58 and recites a method that comprises a composition for inducing a modification of a target endogenous nucleic acid sequence in a eukaryotic 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). As evidenced by Doudna et al and as defined in the instant specification, it is well-established in the art that the crRNA of a CRISPR-guide RNA system comprises: a portion to be hybridized with a portion of the tracrRNA, and a portion complementary to a target DNA of the endogenous genes. Further, base claim 58 recites that the Cas/guide RNA complex is formed in vitro before being introduced into the eukaryotic cell. Further, copending claims 59-60 and 72 recite the complex transfected into the eukaryotic cell is performed using electroporation.
Regarding instant claim 2, copending claim 58 recites that the guide RNA is a single-chain guide RNA (sgRNA) comprising the crRNA fused to the tracrRNA.
Regarding instant claim 3, copending claim 63 recites that the Cas9 protein has the NLS in proximity to its C-terminal.
Regarding instant claims 8-9, copending base claim 58 recites that the guide RNA is in vitro transcribed RNA or synthetic RNA which meets the limitation of chemically synthesized RNA.
Regarding instant claim 10, copending claim 62 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'.
In view of the high skill level in the art it is considered that one of ordinary skill in the art would have been motivated to combine the elements of the copending claims for the rationale of making an efficient CRISPR gene-editing system for use in eukaryotic cells. It would have been prima facie obvious to do such because the claims cited all depend from base claim 58 and are the preferred embodiments.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Currently amended claims 1-3, 5, 8-10, and 23-29 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 58, 61, 63-64, 67-68, and 70-73 of US Application 18/313,946 in view of US Patent 10,731,181 to Chen et al (with priority to US Provisional 61/794,422 filed on 03/15/2013) Although the claims at issue are not identical, they are not patentably distinct from each other because the copending claims anticipate the present claim(s).
Regarding instant claims 1-2 and 25, copending base claim 58 recites a system for inducing a modification of a target endogenous gene in a eukaryotic 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 single guide RNA having a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). Further, base claim 58 recites that the Cas/guide RNA complex is formed in vitro before being introduced into the eukaryotic cell. Also, copending claim 70 recites a human cell.
Regarding instant claim 3, copending claim 61 recites that the Cas9 protein has the NLS in proximity to its C-terminal.
Regarding claim 5, copending claim 64 recites that the Cas9 protein comprises a HA-tag.
Regarding instant claim 8, copending claim 67 recites that the guide RNA is in vitro transcribed RNA.
Regarding instant claim 9, copending claim 68 recites that the guide RNA is in chemically synthesized RNA.
Regarding instant claim 10 copending base claim 58 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'.
Regarding instant claims 23, and 25-29, copending claims 58, recite at least a two-fold molar excess of sgRNA over the Cas9 protein. Copending claims 71-73 recite molar ratios of sgRNA:Cas9 protein of 2.1 : 1 to 6.2:1; 4:9 to 1.3:1; and 8:15 to 20:15.
However, copending claims differ from instant claims because while they recite a transfection buffer they do not specify the transfection buffer is an electroporation buffer.
Chen et al suggest introducing Cas9/sgRNA complexes into eukaryotic cells by means of transfection. Chen et al disclose Cas9 fused to an nuclear localization sequence called an NLS (para 0010 cites Cas9 protein; para 0023 cites NLS). The purpose of the NLS is to introduce the Cas9 complex into the nucleus of the eukaryotic cell. Chen et al disclose that methods of introducing into human cells include transfection, nucleofection, and electroporation and state that “Transfection methods are well known in the art” (see para 0066). In para 0075-0076 lists suitable type of eukaryotic cells including human cells. 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”. (See para 0080-0081). 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”.
It would have been obvious for one of ordinary skill in the art to use electroporation because Chen et al disclose this was routinely used in the art as a method to introduce nucleic acids into human cells.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Currently amended claims 1-3, 5, 8-10, and 23-29 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 15-27 and 29-33 of US Application 19/002,832.
Although the claims at issue are not identical, they are not patentably distinct from each other because the copending claims anticipate the present claim(s).
Regarding instant claims 1-2, 8-10, and 23-29, copending claim 27 recites a method comprising a composition for inducing a modification of a target endogenous gene in a eukaryotic 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 single guide RNA having a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). Copending claims 26-27 recite an sgRNA. Further, claim 27 recites that the Cas/guide RNA complex is formed in vitro before being introduced into the eukaryotic cell. Copending claim 27 recites and the Cas9 has an NLS. Also, copending claims 23 and 31 recite a human cell. Copending claims 16 and 28 recite electroporation which would inherently include an electroporation buffer.
Regarding instant claim 3, copending claim 18 recites that the Cas9 protein has the NLS in proximity to its C-terminal.
Regarding claim 5, copending claim 64 recites that the Cas9 protein comprises a HA-tag.
Regarding instant claim 8, copending claim 67 recites that the guide RNA is in vitro transcribed RNA.
Regarding instant claim 9, copending claim 68 recites that the guide RNA is in chemically synthesized RNA.
Regarding instant claim 10 copending base claim 17 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'.
Regarding instant claims 23, and 25-29, copending claims 58, recite at least a two-fold molar excess of sgRNA over the Cas9 protein. Copending claims 71-73 recite molar ratios of sgRNA:Cas9 protein of 2.1 : 1 to 6.2:1; 4:9 to 1.3:1; and 8:15 to 20:15.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Currently amended claims 1-3, 10, and 23-29 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 11-19, and 21-24 of US Application 18/932,745.
Although the claims at issue are not identical, they are not patentably distinct from each other because the copending claims anticipate or render obvious the present claims.
Regarding instant claims 1-2, 10, and 23-29, copending claim 11 recites a method comprising a composition for inducing a modification of a target endogenous gene in a eukaryotic 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 single guide RNA having a CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA). Copending claim 11 recites an sgRNA. Further, copending claim 11 recites that the Cas/guide RNA complex is formed in vitro before being introduced into the eukaryotic cell. Copending claim 16 recites a human cell. Copending claim 11 recites and the Cas9 has an NLS. Copending claim 11 recites that the sgRNA and Cas9 protein are present in a molar ratio ranging from 29:14 to 29:1.4. Copending claim 19 recites electroporation which would inherently include an electroporation buffer.
Regarding instant claim 3, copending claim 22 recites that the Cas9 protein has the NLS in proximity to its C-terminal.
Regarding instant claim 10 copending base claim 21 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'.
This is a provisional nonstatutory double patenting rejection because the patentably indistinct claims have not in fact been patented.
Conclusion
No claim is allowed.
Protocol for Electroporation (downloaded February 2026).
Avoiding Ribonuclease Contamination (downloaded February 2026).
Li et al (Virus Research 37 1995 pages 153-161).
Liang et al 2025.
Shen et al (Cell Research 2013 Vol 23: pages 720-723, published online April 2, 2013; of record). Shen et al describe co-injecting Cas9 mRNA and gRNAs targeting an endogenous EGFP incorporated into the mouse genome (knock-in) into one-cell mouse embryos. Shen et al showed that pre-annealing the gRNA before injection to facilitate its correct folding generated stronger cleavage bands. Shen et al added a linker (32 amino acids) between the NLS and Cas9 which resulted in enhanced cleavage activity in the nucleus of human 293T cells.
WO2014/093661 to Zhang (with priority to US Provisional 61/835,931 filed on June 17, 2013; IDS reference). The ‘931 provisional discloses delivery methods for Cas9 and gRNAs and/or nucleic acids encoding such.
WO2014/093712 to Zhang (with priority to US Provisional 61/836,127 filed on June 17, 2013; IDS reference). The ‘127 provisional discloses delivery methods for Cas9 and gRNAs.
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