DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Response to Amendment/Status of Claims
Claims 1 and 2 were amended. Claims 5-7 were cancelled. Claims 1-4 and 8-20 are under examination.
Withdrawn Objections
Applicant’s arguments and amendments, see page 6, filed 06/15/2026, with respect to objection to the drawings have been fully considered and are persuasive due to amendments correctly labeling the drawings. The objection to the drawings has been withdrawn.
Maintained Objection
Specification
The use of the term “TALENs” (See page 1 of instant specification), which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Response to Arguments
Applicant's arguments filed 06/15/2026 have been fully considered but they are not persuasive.
Applicant argues on page 6 of response that Applicant does not believe that TALEN is a tradename and is merely an acronym for Transcription Activator-Like Effector Nucleases and that the specification includes both the full name and acronym together.
This is not found persuasive, as TALEN is a Trademark and has the US Serial Number 79107519 and US Registration Number 729507. Therefore, the specification should include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Maintained Rejections
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1,8,10,11,13-17 and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Yamagishi et al. (Applied Sciences, Vol. 9, No. 5, 7 March 2019, page 965) in view of Guillaume-Gentil et al. #1 (Trends in Biotechnology, Vol. 32, No 7, July 2014, pages 381-387) and Guillaume-Gentil et al. #2 (Small, Vol. 9, No. 11, 10 June 2013), all cited on an IDS.
Claim Interpretation: Regarding the instant amendments regarding “a single cell”, each nanoneedle in the array of Yamagishi would still perform direct injection of a genome editing composition into a nucleus of a single-cell, and therefore still reads on the claims as amended.
Regarding claim 1, Yamagishi et al. teach the necessity to develop efficient direct Cas9-sgRNA delivery tools that ensure safe genome editing technology, and that the delivery of macromolecules into living cells via the insertion of nano-scale acicular materials such as carbon nanofiber arrays, silicon nanowires, diamond nanoneedle arrays and porous silicon nanoneedle arrays has been reported as a new delivery platform, however successful delivery of RNPs using acicular materials has not been reported (page 2, paragraphs 1 and 2). Yamagishi et al. teach developing cell manipulation techniques such as the AFM cantilever type nanoneedle (AFM-NN) and silicon nanoneedle arrays (NNAs) as able to efficiently deliver naked DNA or zinc-finger proteins into cells, and therefore studied the efficiency of genome editing via RNP delivery using NNA (page 2, third paragraph). Yamagishi et al teach the RNP complexes were constructed by mixing Cas9 and sgRNA and Cas9 buffer, which were then adsorbed on the surface of NNA containing tens of thousands of nanoneedles (Section 2.4, page 3). Yamagishi et al. teach delivery of the RNPs comprising the Cas9 and sgRNA into various cells using the NNA, and evaluating knockout of the target gene (Sections 2.6, 2.7, pages 4-5). Yamagishi et al. teach the conclusion that the NNA system may be used for direct delivery of Cas9-sgRNA RNPs into living cells (page 2, first paragraph, Conclusion page 9). Yamagishi et al. teach evaluating the nucleus insertion efficiency of the nanoneedle in HEK 293 cells wherein the NNA adsorbed with the Cas9-mEmGFP containing the sgRNA was inserted into the cells, images were obtained and when one or more fluorescent spots derived from Cas9-mEmGFPR adsorbed needles were observed in the nucleus, that cell was defined as a nucleus inserted cell (Section 2.5, page 4).
Regarding claims 8 and 13, Yamagishi et al. teach the sgRNA that forms the RNP complexes, contains green fluorescent protein (GFP), and the GFP was used to evaluate the cleavage activity of RNP (Sections 2.3 and 2.4, pages 3-4). Yamagishi et al. teach evaluating the nucleus insertion efficiency of the nanoneedle in HEK 293 cells wherein the NNA adsorbed with the Cas9-mEmGFP containing the sgRNA was inserted into the cells, images were obtained and when one or more fluorescent spots derived from Cas9-mEmGFPR adsorbed needles were observed in the nucleus, that cell was defined as a nucleus inserted cell (Section 2.5, page 4).
Regarding claims 10 and 11, Yamagishi et al. teach the Cas9 protein was prepared and stored in HEPES-based buffer containing HEPES, NaCl, MgCl2, glycerol and had a pH of 7.4 (page 3, first paragraph).
Regarding claim 20, Yamagishi et al. teach genetically engineered cells obtained by the direct delivery method of the RNP containing Cas9 and sgRNA using the NNA, as it teaches immune-stained FP106SC2 cells after delivery of RNP with the NNA (Figure 3D page 8), shown below.
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Yamagishi et al. do not teach that the direct injection method is performed with a microelectromechanical systems injection chip comprising a cantilever, the cantilever comprising a microchannel being in fluid communication with a nanosyringe, wherein the direct injection comprises providing a fluid communication between the microchannel and the nucleus of the cell by insertion of the nanosyringe into the nucleus of the cell and injecting the genome editing composition via the microchannel through the nanosyringe into the nucleus of the cell.
Before the effective filing date, Guillaume-Gentil et al. #1 taught that atomic force microscopy (AFM) has been used as an imaging tool traditionally, but recent developments have extended the variety of cell-manipulation protocols, and that fluidic force microscopy (FluidFM) combines AFM with microfluidics via microchanneled cantilevers with nano-sized apertures, wherein hollow cantilevers are connected to a pressure controller, allowing operation in liquid as force-controlled nanopipettes under optical control (Abstract, page 381). Guillaume-Gentil et al. #1 taught FluidFM combines a conventional atomic force microscope mounted on top of an optical microscope with microchanneled cantilevers that are connected to a pressure controller for operation in liquid, allowing for isolation of single cells and allows for well-controlled perturbation experiments (Intro, page 381). See Figure 3A below for the structure of the FluidFM, which teaches the recited structure of the system of instant claims 1 and 16.
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Guillaume-Gentil et al. #1 taught the ability of the FluidFM system applying a pressure allows for negative pressure experiments involving suction for application such as cell adhesion or positive pressure experiments resulted in controlled dispensing for applications such as cell injection (page 383, right column). Guillaume-Gentil et al. #1 taught using FluidFM to dispense selected molecules in solution to a single targeted cell, whereby the microfluidic probe was filled with a membrane-permeable fluorescent dye and brought into contact with the cell membrane via force-controlled positioning (page 385, left column). Guillaume-Gentil et al. #1 taught using FluidFM is a versatile tool to pick-and-place single living cells for patterning and isolation (page 385, right column).
Guillaume-Gentil et al. #2 taught that preliminary results on cytoplasmic injection revealed the great potential of the fluidic force microscope (FluidFM) which combines small size probes, the nanoscale resolution of AFM and a pressure controlled fluid delivery through an integrated microchannel, and therefore developed and demonstrated for the first time, the ability of the FluidFM for straightforward and standardized injection into cell nuclei (Page 1904, and Fig. 1A).
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Guillaume-Gentil et al. #2 taught force and pressure controlled intranuclear injection using FluidFM and shows fluorescent images of HeLa cells following intranuclear injection of Lucifer yellow CH (LY) at different volumes, including 300fL, with no noticeable morphological changes visible, 900 fL with the formation of small vesicles at the cell periphery observed, and 1000 fL with the cell massively inflated (page 1905, Figure 2).
Guillaume-Gentil et al. #2 also taught delivery of DNA into HeLA cell nuclei and that plasmid DNAs encoding the fluorescent GFP were co-injected with a fluorescent marker and cells were monitored for viability and protein expression, and effective protein expression in addition to well preserved nuclear and cytoplasmic membranes and multiple cell divisions following days after injection demonstrate cell integrity and function (pages 1905-1906).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to substitute the direct injection method of the gene editing composition into the cell nucleus using the NNAs of Yamagishi et al. with the injection method using the FluidFM system of Guillaume-Gentil et al. #1 and Guillaume-Gentil et al. #2, to arrive at the instant claims with a reasonable expectation of success. There would be a reasonable expectation of success, as this would amount to simple substitution of the NNA system used in the method of Yamagishi et al. for inserting Cas9 and sgRNA into the nucleus of a cell, with the FluidFM system of Guillaume-Gentil et al. #1 and Guillaume-Gentil et al. #2 which is also taught as being used for cell injection and fluid injection into cell nuclei. One of ordinary skill in the art would have been motivated to do so because Guillaume-Gentil et al. #1 taught FluidFM combines a conventional atomic force microscope mounted on top of an optical microscope with microchanneled cantilevers that are connected to a pressure controller for operation in liquid, allowing for isolation of single cells and allows for negative pressure experiments involving suction for application such as cell adhesion or positive pressure experiments resulted in controlled dispensing for applications such as cell injection and can be used to dispense selected molecules in solution to a single targeted cell, and is a versatile tool to pick-and-place single living cells for patterning and isolation. Additionally, Guillaume-Gentil et al. #2 taught the ability of the FluidFM for straightforward and standardized injection into cell nuclei and taught delivery of DNA into HeLA cell nuclei and that plasmid DNAs encoding the fluorescent GFP were co-injected with a fluorescent marker and cells were monitored for viability and protein expression, and effective protein expression in addition to well preserved nuclear and cytoplasmic membranes and multiple cell divisions following days after injection demonstrate cell integrity and function.
Accordingly, the limitations of claims 1,8,10,11,13,16 and 17 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Regarding claims 14 and 15, while the speed of the nanosyringe in claim 14 and the pressure applied to the microchannel of the cantilever in claim 15 is not disclosed by Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2, and are silent as to these limitations regarding the specific speed and pressure used, since the desire is to get the genome editing composition into the nucleus, one skilled in the art would manipulate the conditions of injection including the speed and pressure applied, in order to achieve the desired injection. In addition, it is generally noted that "[W]here 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." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Given that applicant did not point out the criticality of the speed and pressure of the invention, it is concluded that the normal desire of scientists or artisans to improve upon what is already generally known would provide the motivation to determine where in a disclosed set of ranges is the optimum speed and pressure. NOTE: MPEP 2144.05.
Accordingly, the limitations of claims 14 and 15 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Regarding claim 20, Note MPEP 2113: “[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). The MPEP also indicates that “the structure implied by the process steps should be considered when assessing the patentability of product-by-process claims over the prior art, especially where the product can only be defined by the process steps by which the product is made, or where the manufacturing process steps would be expected to impart distinctive structural characteristics to the final product. See, e.g., In re Garnero, 412 F.2d 276, 279, 162 USPQ 221, 223 (CCPA 1979). “In determining validity of a product-by-process claim, the focus is on the product and not the process of making it.” Amgen Inc. v. F. Hoffman-La Roche Ltd., 580 F.3d 1340, 1369 (Fed.Cir.2009). The process of making is only relevant “if the process by which a product is made imparts ‘structural and functional differences’ distinguishing the claimed product from the prior art” Greenliant Systems, Inc. v. XicorLLC, 692 F.3d 1261, 1268 (Fed. Cir. 2012). In the instant case, Yamagishi et al. teach genetically engineered cells obtained by the direct delivery method of the RNP containing Cas9 and sgRNA using the NNA, as it teaches immune-stained FP106SC2 cells after delivery of RNP with the NNA (Figure 3D page 8), and the product of claim 20 does not appear to be different than the product of Yamagishi et al., Guillaume-Gentil et al. #1 and Guillaume-Gentil et al. #2.
Accordingly, the limitations of claim 20 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Response to Arguments
Applicant's arguments filed 06/15/2026 have been fully considered but they are not persuasive.
Applicant argues on pages 6-7 that Yamagishi teaches away from the claimed subject matter and does not perform injection in any conventional sense, and uses solid, non-hollow nanoneedles arranged in a massive array of tens of thousands and the Cas9-sgRNA RNPs are adsorbed on the outer surface of the needles, the needles then puncture cells in bulk and the RNPs desorb or diffuse into the punctured cells, and cites Conclusion and Section 2.4 of Yamagishi. Applicant argues that the adsorption in Yamagishi is distinct from the claimed subject matter which recites fluidic loading of composition inside a microchannel, active pressure-driven injection through a nanosyringe lumen into the nucleus, and that a person of ordinary skill would not view these as interchangeable “direct delivery methods”. Replacing a solid adsorption need with a hollow pressure-driven nanosyringe is not a substitution of one known element for another performing the same function, it is a wholesale change in the physical mechanism of delivery. Applicant also argues that the central feature of Yamagishi is performing bulk, population delivery via tens of thousands of needles simultaneously and is a non-single-cell method in contrast with amended claim 1 requiring injection into the nucleus of a single cell.
This is not found persuasive because Yamagishi was cited for the motivation of needing to develop efficient direct Cas9-sgRNA delivery tools that ensure safe genome editing technology, and also Yamagishi did teach “We have been developing cell manipulation techniques such as AFM cantilever type nanoneedle and the silicon NNA” on page 2 which was cited in the office action. Therefore, Yamagishi suggests using an AFM type cantilever needle for cell manipulation. Regarding Applicants argument about Yamagishi performing bulk, population delivery via tens of thousands of needles simultaneously and is a non-single-cell method in contrast with claim 1 as amended, this is not found persuasive, because each nanoneedle in the array of Yamagishi would still perform direct injection of a genome editing composition into a nucleus of a single-cell, and therefore still reads on the claims as amended.
Applicant argues on page 7 that the Office’s selection of Yamagishi as the primary reference while acknowledging that it lacks every structural element of the claimed system suggests the combination was constructed with the claimed invention as a roadmap, as the only feature Yamagishi supplies is the CRISPR-Cas use, and the structure and functional aspects of the claimed injection method must come from the Guillaume-Gentil references and is clearly improper hindsight reconstruction.
This is not found persuasive. In response to applicant’s argument that the examiner’s conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant’s disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971). See also MPEP 2145 X.A., “there is no requirement that an "express, written motivation to combine must appear in prior art references before a finding of obviousness." Ruiz v. A.B. Chance Co., 357 F.3d 1270, 1276, 69 USPQ2d 1686, 1690 (Fed. Cir. 2004). See KSR, 550 U.S. at 402, 82 USPQ2d at 1389 ("The diversity of inventive pursuits and of modern technology counsels against confining the obviousness analysis by a formalistic conception of the words teaching, suggestion, and motivation, or by overemphasizing the importance of published articles and the explicit content of issued patents."). The Examiner chose Yamagishi as the primary reference to provide motivation for needing to develop efficient direct Cas9-sgRNA delivery tools that ensure safe genome editing technology.
Applicant argues on page 8 that Yamagishi’s approach is a high-throughput, population-level technique while the Guillaume-Gentil references teach controlled operations to an individually optically-selected target cell. These approaches solve different problems, as Yamagishi is trying to reduce off-target effects in a bulk transfection-like workflow; Guillaume-Gentil 2013 demonstrates proof-of-concept single-cell fluidic manipulation originally with dyes and plasmid DNAs, not CRISPR RNPs. Applicant argues a person of ordinary skill starting from Yamagishi’s bulk NNA platform would have no reason to abandon its crucial defining feature (massive parallelism) in favor of one-cell-at-a time FluidFM injection which would destroy the throughput advantage Yamagishi explicitly touts. Applicant argues, a skilled artisan starting from the Guillaume-Gentil references would have had no indication that FluidFM could or should be used for CRISPR-Cas RNP delivery as the references do not mention CRISPR, Cas9 or gRNA, and instead injection a small-molecule dye and GFP-encoding plasmid DNA. Applicant argues on page 8 that Yamagishi does more than fail to suggest microfluidic nanosyringe injection, it identifies the problem (off-target effects from prolonged Cas9 exposure) and solves it by a non-fluidic adsorption based mechanism and directs the skilled artisan to further optimize the NNA system not abandon it. Applicant argues a reference that explicitly identifies its own delivery platform as the path forward for CRISPR RNA delivery teaches away from abandoning that platform for an entirely different approach, and the skilled artisan is taught switching from large parallel injection to a single injection approach would decrease efficiency which is a reason why those of skill would not consider modifying Yamagishi with the Guillaume-Gentil references.
This is not found persuasive, See MPEP 2145 X.A., “there is no requirement that an "express, written motivation to combine must appear in prior art references before a finding of obviousness." Ruiz v. A.B. Chance Co., 357 F.3d 1270, 1276, 69 USPQ2d 1686, 1690 (Fed. Cir. 2004). Guillaume-Gentil 2014 taught controlled dispensing for applications such as cell injection (page 383, right column) and taught using FluidFM to dispense selected molecules in solution to a single targeted cell as well as using FluidFM is a versatile tool to pick-and-place single living cells for patterning and isolation (page 385, right column), and Guillaume-Gentil 2013 taught the ability of the FluidFM for straightforward and standardized injection into cell nuclei. These uses and motivations cited in the rejection from the Guillaume-Gentil references provide motivation for why one of ordinary skill in the art would have been motivated to use the FluidFM device of Guillaume-Gentil 2013 and Guillaume-Gentil 2014 (for controlled dispensing for application such as cell injection, to dispense selected molecules in solution to a single targeted cell, to pick-and-place single living cells for patterning and isolation, and injection into cell nuclei, in order to directly inject the gene editing composition of Yamagishi into single cells). In addition, as Guillaume-Gentil et al. #2 taught delivery of DNA into HeLA cell nuclei and that plasmid DNAs encoding the fluorescent GFP which are large molecules, there would be a reasonable expectation of success that a Cas protein and gRNA composition would be capable of being delivered into cell nuclei, without evidence to the contrary. The United States Court of Appeals for the Federal Circuit in Intel Corp. v. PACT XPP Schweiz AG, 61 F.4th 1373, 2023 USPQ2d 297 (Fed. Cir. 2023) held that establishing a motivation to combine prior art for KSR analysis based on the “known-technique” rationale does not require showing that the “known-technique” is an improvement. The Court held that it only requires showing that the “known-technique” is a “suitable option”. The Examiner maintains that the FluidFM system of Guillaume-Gentil 2013 and Guillaume-Gentil 2014 that can directly inject fluid containing molecules into the nucleus of single cells would be a suitable option to directly inject the genome editing composition of Yamagishi and there would be a reasonable expectation of success.
Applicant argues on pages 9-10 that Cas9-sgRNA RNPs are well known to aggregate and precipitate in solution, especially at the concentrations needed for genome editing, and agglomeration is illustrated in Camperi et al. Applicant copies Figure 1 from that reference, as well as citing the problem of precipitation, and argues the skilled artisan would have known the clogging is a problem to be avoided with nanoneedles and knows that CRISPR Cas have strong tendency to agglomerate, and contemplating the substitution proposed by the Examiner would have had serious doubts that RNP-containing solutions could flow reliably through the sub-micrometer aperture of the FluidFM nanosyringe without clogging. In addition, a person reviewing the art and finding Yamagishi would teach the skilled person to delivery RNPs not by nanosyringe but by the nanoneedle array allowing the components to diffuse into punctured cells and avoiding clogging. Applicant argues that Yamagishi avoids this problem by design because the RNPs are adsorbed to the exterior of the solid needles rather than flowing through a lumen, and aggregation does not impede delivery and an artisan would recognize this is an affirmative technical reason Yamagishi chose surface adsorption over microfluidic injection for CRISPR components- even though Guillaume-Gentil’s FluidFM platform was known and available at the time Yamagishi was written, and chose a completely different mechanism to deliver CRISPR RNPs which demonstrates that the skilled artisan would not have had a reasonable expectation of success combining these disclosures. Applicant also argues the instant specification at paragraph 0013 emphasizes that direct intranuclear injection via the claimed microchannel/nanosyringe enables accurate stoichiometric control of the Cas protein and gRNA delivered as well as direct delivery past the nuclear envelope without reliance on NLS sequences or endosomal escape, and Yamagishi’s mechanism is incapable of controlled stoichiometry, and estimates each needle delivers uncontrolled ~0.1 amol depending on adsorption and desorption kinetics that vary cell-to-cell. The claimed methods therefore achieves a qualitatively different, superior results that a POSA would not have predicted from the cited combination and would not have been possible with the Yamagishi approach.
This is not found persuasive. As stated above, the FluidFM system is a suitable option, and does not have to be the best option. Regarding Applicant’s arguments pertaining to Camperi et al., it does not appear that Camperi et al. discuss precipitation or use that term at all. The section on page 9 of Applicants response regarding steps 14 and 15 and mentioning precipitation and precipitated material was not found in Camperi et al. as claimed by Applicant. While Camperi et al. discuss sgRNA aggregates, Camperi et al. found that the data supported that the different forms and the quantity of the aggregates are sgRNA sequence-dependent (page 1436), and that to reduce the heterogeneity and aggregates, all sgRNAs were denatured by heating to 70 Degrees Celsius and allowed to cool to room temperature, and that the heating of the sgRNA eliminated all forms of aggregates, and the sgRNA monomers are stable and do not go back into aggregate form (page 1437, left column). Therefore, an ordinary artisan reading Camperi et al. would know that one way to avoid or eliminate sgRNA aggregates is to heat the sgRNA, which the instant claims do not exclude.
In addition, Guillaume-Gentil (“FluidFM Applications in Single-Cell Biology”, Open-Space Microfluidics: Concepts, Implementations and Applications, Published 2018) taught a critical issue during cell manipulation consists of biofouling, that is, the non specific adsorption of biomacromolecules to the probe surface, and that upon specific contact with cells, fouling can damage cells and the excessive adsorption of biological material within the microchannel or at the probe aperture can lead to clogging or to the loss of material flown through the probe. Protein and cell adhesion to the cantilever surface thus limits the practical use of the microchanneled probes. In order to ensure nondestructive cell handling and allow for serial manipulations, the FluidFM probes are therefore functionalized with an antifouling coating prior to experimentation. The hydrophilic copolymer poly(l-lysine)-graft-poly(ethylene glycol) (PLL-g-PEG) has been most extensively applied to confer antifouling properties to the FluidFM probes (Section 15.2, page 326). Therefore, Guillaume-Gentil acknowledges and addresses the problem of clogging in its FluidFM system, and that the way to prevent fouling or clogging is with an antifouling coating on the FluidFM probes. Therefore, one of ordinary skill in the art would know that clogging of the FluidFM probes can be overcome with an antifouling coating on the FluidFM probes, which the instant claims do not exclude.
It is also noted that the instant specification does not mention clogging, precipitation, or agglomeration as a problem at all and therefore does not seem to be an issue that Applicant was trying to provide a solution to with the instant invention. Regarding Applicant’s argument about the instant specification and controlled stoichiometry and that Yamagishi’s mechanism is incapable of controlled stoichiometry, and that the claimed methods therefore achieves a qualitatively different, superior results that a POSA would not have predicted from the cited combination and would not have been possible with the Yamagishi approach, this is not found persuasive, because the rejection provided by the Examiner does not use the mechanism or device in Yamagishi but uses the FluidFM system of Guillaume-Gentil 2014 and Guillaume-Gentil 2013 to directly inject the gene editing composition of Yamagishi to the cell nucleus, and as the structure of the FluidFM system of Guillaume-Gentil 2014 and Guillaume-Gentil 2013 is the same as the instant claims, the Examiner argues using the FluidFM system of Guillaume-Gentil 2014 and Guillaume-Gentil 2013 would also be able to provide controlled stoichiometry with a reasonable expectation of success in the absence of unexpected results.
No separate or additional arguments were made regarding the other references and 103 rejections of claims 2-4 as obvious over Yamagishi, Guillaume-Gentil 2014, Guillaume-Gentil 2013, English translation of Yi et al. and Yeh et al., claims 9 and 12 as obvious over Yamagishi, Guillaume-Gentil 2014, Guillaume-Gentil 2013 and Zhu, and claims 18 and 19 as obvious over Yamagishi, Guillaume-Gentil 2014, Guillaume-Gentil 2013 and Hess. The Examiner has responded to Applicant’s arguments above regarding Yamagishi, Guillaume-Gentil 2014 and Guillaume-Gentil 2013 and as the rejection has been maintained, the 103 rejections of the dependent claims is likewise maintained and they follow below.
Claims 2-4 are rejected under 35 U.S.C. 103 as being unpatentable over Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2 as applied to claims 1,8,10,11,13-17 and 20 above, and further in view of the English translation of Yi et al. (CN108130314, Published 08 June 2018) and Yeh et al. (Cells, 2020, 9, 1482).
The teachings of Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2 as applicable to claims 1,8,10,11,13-17 and 20 have been described above.
Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2 do not teach expanding the single genetically engineered cell to a generate monoclonal cell culture.
Before the effective filing date, Yi et al. taught a monoclonal cell culture method in the process of constructing a stably transfected cell line using a gene editing technology (Technical field, page 1 of translation). Yi et al. taught aiming at some problems in the existing process of constructing stable expression cell lines using gene editing technology, the purpose of the present application is to provide a more convenient and efficient monoclonal cell culture technology so that genetic modification can be screened uniformly (page 2 of translation). Yi et al. taught a monoclonal cell culture method based on CRISPR/Cas9 gene editing technology which includes the first step of editing the genome using CRISPR/Cas9 technology in the cell to be edited, and flow-sorting the cells to obtain the target cell (page 2 of translation). The obtained cells were then diluted to ensure that each well in the well plate is a single cell during subsequent plating (page 2 of translation). Yi et al. taught after the cultured cells in step 7 are cultured for 12-16 days, they are inoculated and plated in 24-well plates and subsequently 6-well plates, followed by Western blot and sequencing (page 3 of translation). Yi et al. taught their method of constructing monoclonal cell populations greatly improves the survival of single cells and rapidly selects genetically modified uniform genetic backgrounds with the same stability (page 3 of translation).
Additionally, Yeh et al. taught single-cell cloning is a critical step in generating monoclonal cell lines which are widely used as in vitro models and for producing proteins with high reproducibility for research, and that in monoclonal cell line generation, the development time can be shortened by validating the monoclonality of the cloned cells (Abstract). Yeh et al. taught monoclonal cells are groups of cells originating from a single parental cell and the have cognate genomic DNA sequences and express similar phenotypes (Intro, page 1). Yeh et al. taught to construct monoclonal cell lines, the genomic DNA of cells must be modified by transfecting foreign DNA into the cells, and that transfected cells usually have highly diverse gene complements and highly expressing cells are rare in the heterogenous cell populations and the transfected cells also have different cell viabilities. Therefore, screening and selecting for large numbers of monoclonal cells with a desired phenotype and high reproductive capacity are necessary (Intro, page 1). Yeh et al. taught using a disposable microfluidic chip device in which single cells can be isolated and grown into monoclonal colonies, which can subsequently be transferred from the device to the wells of conventional plates for further expansion and can be used in general laboratories (page 2, third paragraph).
Yeh et al. taught using actin-GFP-transfected non-small cell lung cancer A549 cells as a model to demonstrate using the SCC device for monoclonal cell generation, and after 9 days of culture, the transfected cell colonies formed on the SCC device were transferred to a 96-well culture plate, and subsequently some of the colonies could be expanded into larger populations and transferred to 48-well plates at different time points, depending on their proliferation rates, and that using the SCC device, three single-cell derived colonies (clones 3-5) of the A549 cells could be obtained (page 11 section 3.5 and Figure 5(b)). Yeh et al. taught that the 5 colonies expressed higher, more distinct GFP signals indicating monoclonality of the colonies, and clones 3 and 4 had high proliferation and high protein expression (pages 11-12).
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Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to have modified the method of Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2, with the teachings of Yi et al. and Yeh et al. to arrive at the instant claims with a reasonable expectation of success, as this would have amounted to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to modify the method of Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2, to provide a step of expanding the single genetically engineered cell to generate a first monoclonal cell culture because Yi et al. taught providing a more convenient and efficient monoclonal cell culture technology so that genetic modification can be screened uniformly and that their method of constructing monoclonal cell populations greatly improves the survival of single cells and rapidly selects genetically modified uniform genetic backgrounds with the same stability (page 3 of translation), and because Yeh et al. taught single-cell cloning is a critical step in generating monoclonal cell lines which are widely used as in vitro models and for producing proteins with high reproducibility for research, and that in monoclonal cell line generation, the development time can be shortened by validating the monoclonality of the cloned cells. Regarding the limitations of claims 3 and 4 of dividing the first monoclonal cell culture into a first and second sub-group, wherein the cells of the first sub-group are analyzed by sequencing and the cells of the second sub-group are expanded to further generate a second monoclonal cell culture, and wherein the cells of the first monoclonal cell culture are additionally divided into a third-sub-group which are then expanded to further produce a third monoclonal cell culture, while the prior art does not explicitly teach these additional steps as recited, Yi et al. taught after the cultured cells in step 7 are cultured for 12-16 days, they are inoculated and plated in 24-well plates and subsequently 6-well plates, followed by Western blot and sequencing (page 3 of translation), and their method of constructing monoclonal cell populations greatly improves the survival of single cells and rapidly selects genetically modified uniform genetic backgrounds with the same stability (page 3 of translation), and Yeh et al. taught obtaining multiple clones in different steps at different time points using A549 cells as a model to demonstrate using the SCC device for monoclonal cell generation, and the transfected cell colonies formed on the SCC device were transferred to a 96-well culture plate, and subsequently some of the colonies could be expanded into larger populations and transferred to 48-well plates at different time points, depending on their proliferation rates, and that using the SCC device, three single-cell derived colonies (clones 3-5) of the A549 cells could be obtained (page 11 section 3.5 and Figure 5(b)), in which the 5 colonies expressed higher, more distinct GFP signals indicating monoclonality of the colonies, and clones 3 and 4 had high proliferation and high protein expression (pages 11-12).
Accordingly, the limitations of claims 2-4 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claims 9 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2 as applied to claims 1,8,10,11,13-17 and 20 above, and further in view of Zhu (Embryo Project Encyclopedia, Published 27 Dec 2017).
The teachings of Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2 as applicable to claims 1,8,10,11,13-17 and 20 have been described above.
Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2 do not teach wherein the genome editing composition comprises a plurality of Cas proteins and plurality of gRNA molecules each direct to a genomic location to be edited or wherein the genome editing composition further comprises a double stranded or single stranded DNA for homologous recombination.
Before the effective filing date, Zhu taught that George Church and his colleagues at Harvard University published an article in 2013 detailing their use of RNA-guided Cas9 to modify the DNA of human cells (page 1, 1st paragraph). Zhu taught when the Cas9 protein cuts out a sequence of DNA, researchers rely on cell repair mechanisms to incorporate a new sequence into the genome, and one of the methods is homologous recombination which requires a donor sequence provided by the scientist that matches the guide RNA and contains the desired sequence for gene editing, then the guide RNA targets the site and Cas9 cuts it. The donor sequence that matches the guide RNA will then insert itself into the site to repair the DNA (page 3, 2nd paragraph). Zhu taught that experiments were performed to repair a non-functioning GFP protein using RNA-guided Cas9 technology, in which two guide RNAs which had slight variations in their sequences were used but both were targeted to the GFP gene region. Zhu taught the donor sequences had the correct sequence for GFP for insertion into the genome of the human cell (page 4, third paragraph). Zhu taught that it is possible to use multiple guide RNAs at once in the system, and that multiplexing in gene editing is the ability to edit different genes at the same time, and that two new guide RNAs that targeted different sites in the human genome were desired and cells were treated with two and three different guide RNAs at once and report that Cas9 could edit the three different gene sites at once (page 5).
Zhu taught testing using homologous recombination repair, Church and his team detail the same protocol with the addition of including donor sequences to repair the DNA. Church and his colleagues point out that they designed a double-stranded DNA donor sequence for a specific segment of DNA, and treating human cell lines with Cas 9, guide RNA, and the donor sequence which resulted in successful homologous recombination confirmed with DNA sequencing (page 6).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to have modified the method of Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2, with the teachings of Zhu to arrive at the instant claims with a reasonable expectation of success. There would be a reasonable expectation of success because Zhu also pertains to gene editing using gRNA and Cas9 technology in cells, and would amount to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to modify the genome editing composition used in the method of Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2 to provide a plurality of Cas proteins and plurality of gRNA molecules each directed to a genomic location to be edited because Zhu taught using multiple guide RNAs at once in the system, and that multiplexing in gene editing is the ability to edit different genes at the same time, and that two new guide RNAs that targeted different sites in the human genome were desired and cells were treated with two and three different guide RNAs at once and report that Cas9 could edit the three different gene sites at once. It would have been obvious that multiple Cas proteins be provided to cut out each sequence of DNA at the different target sites. One of ordinary skill in the art would have been motivated to modify the genome editing composition used in the method of Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2 to provide a double stranded DNA for homologous recombination because Zhu taught when the Cas9 protein cuts out a sequence of DNA, researchers rely on cell repair mechanisms to incorporate a new sequence into the genome, and one of the methods is homologous recombination which requires a donor sequence provided by the scientist that matches the guide RNA and contains the desired sequence for gene editing, then the guide RNA targets the site and Cas9 cuts it. The donor sequence that matches the guide RNA will then insert itself into the site to repair the DNA and taught using a double-stranded DNA donor sequence for a specific segment of DNA, treating human cell lines with Cas 9, guide RNA, and the donor sequence which resulted in successful homologous recombination confirmed with DNA sequencing.
Accordingly, the limitations of claims 9 and 12 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claims 18 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2 as applied to claims 1,8,10,11,13-17 and 20 above, and further in view of Hess et al. (US 20190309288, Published 20 Oct 2019).
The teachings of Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2 as applicable to claims 1,8,10,11,13-17 and 20 have been described above.
Yamagishi et al., Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2 do not teach wherein the at least one gRNA molecule comprises a tracrRNA annealed with a crRNA. While Yamagishi et al. teach using an sgRNA, Yamagishi et al. does not explicitly recite that the sgRNA consists of a single RNA molecule comprising the sequence of a crRNA and a tracrRNA.
Before the effective filing date, Hess et al. taught that Cas9/gRNA complexes have found use in genome editing (paragraph 0095). Hess et al. taught that some Cas9/RNA complexes comprise two RNA molecules: (1) a CRISPR RNA (crRNA), possessing a nucleotide sequence complementary to the target nucleotide sequence and (2) a trans-activating crRNA (tracrRNA), and that in this mode, Cas9 functions as an RNA-guided nuclease that uses both the crRNA and tracrRNA to recognize and cleave the target sequence (paragraph 0096). Hess et al. taught recently, a single chimeric guide RNA mimicking the structures of the annealed crRNA/tracrRNA has become more widely used than crRNA/tracrRNA because the gRNA approach provides a simplified system with only two components, and thus sequence-specific binding to a nucleic acid can be guided by a natural dual RNA complex comprising a crRNA, a tracrRNA and Cas9, or a chimeric single-guide RNA (e.g., a sgRNA and Cas9) (paragraph 0096).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to have modified the sgRNA molecule used in the method of Yamagishi et al. and using the FluidFM system of Guillaume-Gentil et al. #1, and Guillaume-Gentil et al. #2, to comprising at least one gRNA molecule comprising a tracrRNA annealed with a crRNA, or provide an sgRNA consisting of a single RNA molecules comprising the sequence of a crRNA and tracrRNA based on the teachings of Hess et al. with a reasonable expectation of success. There would be a reasonable expectation of success because Hess et al. also pertains to gene editing using Cas9 and gRNA and is therefore relevant art. One of ordinary skill in the art would have been motivated to provide the gRNA used in the gene editing method of Yamagishi et al. as a gRNA comprising a tracrRNA annealed with a crRNA or a gRNA being a sgRNA consisting of a single RNA molecule comprising the sequence of the crRNA and a tracrRNA because Hess et al. taught that some Cas9/RNA complexes comprise two RNA molecules: (1) a CRISPR RNA (crRNA), possessing a nucleotide sequence complementary to the target nucleotide sequence and (2) a trans-activating crRNA (tracrRNA), and that in this mode, Cas9 functions as an RNA-guided nuclease that uses both the crRNA and tracrRNA to recognize and cleave the target sequence (paragraph 0096) and taught a single chimeric guide RNA mimicking the structures of the annealed crRNA/tracrRNA has become more widely used than crRNA/tracrRNA because the gRNA approach provides a simplified system with only two components, and thus sequence-specific binding to a nucleic acid can be guided by a natural dual RNA complex comprising a crRNA, a tracrRNA and Cas9, or a chimeric single-guide RNA (e.g., a sgRNA and Cas9) (paragraph 0096).
Accordingly, the limitations of claims 18 and 19 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Conclusion
Claims 1-4 and 8-20 are rejected.
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/STEPHANIE L SULLIVAN/Examiner, Art Unit 1635
/ABIGAIL VANHORN/Primary Examiner, Art Unit 1636
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