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 .
Claims 1-9 and 11-34 are pending.
Status of the Application
Applicant’s response and amendment filed 12 June 2026 are acknowledged and entered.
Applicant has made no claim amendments but has added Claim 34.
Claims 1-9 and 11-33 remain rejected under 35 U.S.C. 103 as being unpatentable for reasons of record set forth in the Office Action of 29 December 2026 and reiterated below.
Claims 1-9 and 11-34 are examined.
Arguments applicable to newly applied rejections to amended or newly presented claims are addressed below. Arguments that are no longer relevant are not addressed.
Rejections not reiterated here are withdrawn.
Claim Objections
Applicant is advised that should Claim 12 be found allowable, Claim 14 will be objected to under 37 CFR 1.75 as being a substantial duplicate thereof. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m).
Claims 12 and 14 have the same scope because they both recite providing the components besides for the crRNA (i.e., the liposome containing the tracrRNA portion of the guideRNA, the liposome containing the Cas enzyme/nucleic acid sequence encoding the Cas enzyme, and the target cell) to the well BEFORE providing to the well the liposome containing the crRNA portion of the guideRNA. Whether the guideRNA is added after those components or if those components are added before the guideRNA, the scope of the claims is the same.
NEW REJECTIONS
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 25-27 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.
Claims 25-27 recite the limitation "guide RNA" in L1. There is insufficient antecedent basis for this limitation in the claim because these claims depend from Claim 1 which recites only a crRNA portion of a guideRNA and a tracrRNA portion of a guideRNA, so it is unclear what to portion the “guideRNA” recited in Claims 25-27 refer.
In the interest of compact prosecution, the claims are interpreted as meaning that the total concentration of crRNA + tracrRNA must be within the ranges specified in the claims.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claim 24 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 24 fails to incorporate all the limitations of the claim from which it depends, namely Claim 1, because Claim 1 explicitly recites that step (a) requires three separate liposomes but Claim 24 recites that the crRNA portion of a guideRNA and the tracrRNA portion of a guideRNA tare provided within a liposome. Therefore Claim 24 fails to include Claim 1’s limitation requiring three separate liposomes.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
REJECTIONS REITERATED FROM OFFICE ACTION DATED 20 AUGUST 2025
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1-2, 5, 8, 11-17, 19, 23-24, and 31-34 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application Publication No. US 2020/0071693 (published 05 March 2020, “App693”, of record), Khan (et al. 2019. Multiplexed CRISPR/Cas9 gene knockout with simple crRNA:tracrRNA co‑transfection. Cell Biosci. 9:41, “Khan”, of record) as evidenced by Wikipedia (“Lipofectamine”. Archived on 12 June 2020. Accessed on 13 August 2025, “Wikipedia”, of record), Zhen (and Li. 2020. Liposomal delivery of CRISPR/Cas9. Cancer Gene Ther. 27:515, “Zhen”, of record), Cong (et al. 2013. Multiplex Genome Engineering Using CRISPR/Cas Systems. Science 339:819-823, “Cong”, of record), US Patent Application Publication No. US 2023/0173106 (published on 08 June 2023 but effectively filed on 28 November 2018 and issued as US Patent No. 12168062, “App106”, of record), Scott (et al. 2019. Improved Cas9 activity by specific modifications of the tracrRNA. Sci. Report 9:16104; “Scott”, of record), and Filippova (et al. 2019. Guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems. Biochimie 167:49-60, “Filippova”, of record). This rejection is maintained and updated in response to the addition of Claim 34. The rejection has been edited for clarification and to fix some typographical errors. All references are of record.
Although App106 was published on 08 June 2023, the content relied upon for this rejection was effectively filed on 28 November 2018, which is nearly two years before the earliest provisional document of the instant application.
The App106 content discussed in this rejection are entitled to the priority date of 28 November 2018 and is considered prior art under 35 U.S.C. 102(a)(2).
If the issue date of the U.S. patent or publication date of the U.S. patent application publication or WIPO published application is not before the effective filing date of the claimed invention, it may be applicable as prior art under AIA 35 U.S.C. 102(a)(2) if it was "effectively filed" before the effective filing date of the claimed invention in question with respect to the subject matter relied upon to reject the claim. MPEP § 2152.01 discusses the "effective filing date" of a claimed invention. AIA 35 U.S.C. 102(d) sets forth the criteria to determine when subject matter described in a U.S. patent document was "effectively filed" for purposes of AIA 35 U.S.C. 102(a)(2).
2154.01(a) WIPO Published Applications [R-11.2013]
[Editor Note: This MPEP section is only applicable to applications subject to examination under the first inventor to file (FITF) provisions of the AIA as set forth in 35 U.S.C. 100 (note). See MPEP § 2159 et seq. to determine whether an application is subject to examination under the FITF provisions, and MPEP § 2131-MPEP § 2138 for examination of applications subject to pre-AIA 35 U.S.C. 102.]
The WIPO publication of a PCT international application that designates the United States is an application for patent deemed published under 35 U.S.C. 122(b) for purposes of AIA 35 U.S.C. 102(a)(2) under 35 U.S.C. 374. Thus, under the AIA , WIPO publications of PCT applications that designate the United States are treated as U.S. patent application publications for prior art purposes, regardless of the international filing date, whether they are published in English, or whether the PCT international application enters the national stage in the United States. Accordingly, a U.S. patent, a U.S. patent application publication, or a WIPO published application that names another inventor and was effectively filed before the effective filing date of the claimed invention, is prior art under AIA 35 U.S.C. 102(a)(2). This differs from the treatment of a WIPO published application under pre-AIA 35 U.S.C. 102(e), where a WIPO published application is treated as a U.S. patent application publication only if the PCT application was filed on or after November 29, 2000, designated the United States, and is published under PCT Article 21(2) in the English language. See MPEP § 2136.03, subsection II.
§MPEP 2154.01
See also §MPEP 2152.01.
App693 is drawn to a high-throughput (HTP) genomic engineering platform for improving fungal cells. App693 teaches:
(¶19) a HTP method of genomic engineering to evolve filamentous fungal strains to acquire a desired phenotype, comprising [the first step of]: perturbing the genomes of an initial plurality of filamentous fungal strains having the same strain background, to thereby create an initial HTP genetic design filamentous fungal strain library comprising individual strains with unique genetic variations.
App693 teaches (¶305) their strategies may be used with different kinds of cells, not just fungal cells. App693 teaches that one of its purposes is to:
(¶226) analyz[e] the genome-wide combinatorial effect of mutations across multiple disparate genomic regions, including expressed and non-expressed genetic elements, and uses gathered information (e.g., experimental results) to predict mutation combinations expected to produce strain enhancements.
App693 teaches their methods (¶21) can be used to make a subsequent plurality of fungi strains that each comprise a unique combination of genetic variations. App693 teaches (¶22-24) making combinatorial genetic changes and (¶25) perturbing the fungus’s genome in various ways including targeted sequence deletions. App693 teaches (¶28-32) introducing multiple genetic perturbations/variations into a fungal strain. App693 teaches (¶646-647) generating fungal mutant libraries:
In one embodiment, the methods and systems provided herein generate a plurality of protoplasts such that each protoplast from the plurality of protoplasts is transformed with a single first construct from a plurality of first constructs and a single second construct from a plurality of second constructs. Further to this embodiment, a first polynucleotide in each first construct from the plurality of first constructs comprises a different mutation and/or genetic control or regulatory element while a second polynucleotide in each second construct from the plurality of second constructs is identical. The method further comprises … in order to generate a library of filamentous fungal cells such that each filamentous fungal cell in the library comprises a first polynucleotide with a different mutation and/or genetic control or regulatory element…
App693 teaches that CRISPR systems may be used to produce the mutants. App693 teaches (¶54-56; ¶192) transforming cells with a ribonucleoprotein complex (RNP) using Type II, Type V, or Type VI CRISPR systems and teaches that such system comprises an endonuclease and a gRNA that can comprise a crRNA or may be annealed to a tracrRNA, or that the gRNA is a single gRNA molecule (sgRNA) comprising a tracrRNA and a crRNA. A single guide RNA (sgRNA) is a limitation of Claim 24. A Cas enzyme that is part of a type II CRISPR system is a limitation of Claim 31. App693 teaches that (¶191-192) the gRNA can be comprised of two separate molecules: a targeting segment and a scaffold segment (¶195) whose structure determines how the scaffold interacts with the nuclease and can affect binding kinetics to a target. App693 teaches (¶191) the scaffold can comprise a tracrRNA:
In some cases, binding kinetics of a guide nucleic acid to a nucleic acid guided nuclease is determined in part by secondary structures within the scaffold sequence. In some cases, binding kinetics of a guide nucleic acid to a nucleic acid guided nuclease is determined in part by nucleic acid sequence with the scaffold sequence.
Separate crRNA and tracrRNA are limitations of Claim 1. App693 teaches (¶355) that a protoplast (i.e., a single fungal cell) can be transformed with 2 or more RNP-complexes such that each RNP-complex comprises a nucleic acid guided endonuclease complexed with a guide nucleic acid and that each gRNA in each RNP can target a different target gene or can target the same target gene. App693’s Example 13 teaches (starts at ¶724) knocking out multiple genes, so knocking out multiple genes in a single cell was clearly contemplated by App693. Knocking out at least two different target genes in a target cell is a limitation of Claim 2. App693 teaches that once combined, (Fig. 47, ¶693) the crRNA and tracrRNA anneal together and then form a complex with Cas9. App693 teaches Example 9 (¶693-694) wherein the crRNA and tracrRNA were annealed and then mixed with Cas9 to make RNPs which were then added to protoplasts. A mixture comprising the tracrRNA and the Cas enzyme being provided to the well is a limitation of Claim 15.
The passages discussed in the preceding paragraph (i.e., ¶355, ¶646-647, together with the descriptions of CRISPR systems and gRNA) teach the App693 system encompasses transforming a plurality of protoplasts (i.e., “cells”) with a plurality of mutagenizing constructs (i.e., RNP systems comprising gRNAs that can target different genes) and that the crRNA, tracrRNA, and Cas enzyme colocalize and mutate a target gene or genes, a limitation of Claim 1.
App693 teaches (¶180-181) different Cas enzymes may be used and the Cas enzyme(s) may comprise mutations to produce a Cas protein with one or more altered characteristics compared to the parental Cas protein.
App693 teaches (¶618-619) tools and strategies for placing a single cell in a well. A single cell in a well is a limitation of Claim 23. App693’s Example 12 (starts at ¶709) teaches that protoplasts used in the experiments were derived from mycelia, so all the cells were the same cell type. That is a limitation of Claim 8. App693 teaches (¶217) the system can be used with protoplasts or other cell types and that (¶481) the system can be utilized with any host cell to engineer said host cell to have any desired phenotypic trait.
App693 teaches (¶488-489) using a reference strain whose role is simply to serve as an added normalization factor for making comparisons within or between plates. App693 teaches (¶86) FIG. 30 depicts steps to rapidly isolate genomic DNA and prepare amplicons that contain identifying sequences that associate specific amplicons with the well that they came from which contains the organism that was isolated following genetic alteration (emphasis added). Those two passages indicate that the way each cell is mutated is known. That means that the location of each well within a plate is identified or “addressed” with whatever was used to transform the cell(s) inside of that well. That means that the well location within each plate and the location of each genetic perturber (i.e., endonuclease + “gRNA” or “crRNA”) used in that well is known. Those are limitations of Claim 1. App693 doesn’t use the term “addressable” but it does make clear that each well is associated with a specific transformant, which requires that the wells are identifiable and the transformer (i.e., the genetic perturber, namely endonuclease + “gRNA” or “crRNA”) was known. Therefore App693 teaches limitations of Claim 33.
App693 teaches (¶550) using 96-well and 384-well plates as well as (¶51) 1536 well plates. 1536 well plates is a limitation of Claim 5. App693 further teaches (¶550):
as will be appreciated by those in the art, any number of different plates or configurations may be used. In addition, any or all of the steps outlined herein may be automated… [the system can be] capable of evaluating about 1,000 or more transformants per day, and particularly to those methods capable of evaluating 5,000 or more transformants per day, and most particularly to methods capable of evaluating 10,000 or more transformants per day.
App693 teaches (¶606) transformation of the protoplasts generated using the methods described herein can be facilitated through the use of any transformation reagent known in the art including Lipofectamine®, which is a transfection reagent that encapsulates a payload within a lipid (see discussion of Zhen, below, specifically Zhen §Lipoplexes, entire §).
App693 does not each administering each component in its own liposome.
The work of Khan teaches using liposome-encapsulated crRNA:tracrRNA in a HTP screen. Khan, directed to multiplexed CRISPR/Cas9 gene KO with simple crRNA:tracrRNA co-transfection, teaches a method of cotransfecting cells with a crRNA:tracrRNA combination. Khan teaches (§Methods-RNA transfection and cell viability assay) “stamp[ing]” into wells a crRNA-tracrRNA mixture. Khan teaches (same §) they combined a crRNA:tracrRNA mixture with Lipofectamine® and then delivered cells to the wells. The art of Wikipedia teaches that Lipofectamine® forms liposomes that encapsulate a substance: (¶1) Lipofectamine contains lipid subunits that can form liposomes in an aqueous environment, which entrap the transfection payload. Khan teaches (§Discussion ¶3) because the size of crRNA and tracrRNA is comparable to siRNA, they can be transfected into cells at high efficiency with conventional cationic lipids. Khan teaches (§Discussion ¶4) their platform in easily scalable because sequence-specific crRNAs can be chemically synthesized in an arrayed format and the tracrRNA can be synthesized in bulk, [so] this platform can be easily scaled to create genome-wide, arrayed CRISPR libraries that are analogous to arrayed siRNA libraries for high-content screens.
Khan teaches administering a crRNA:tracrRNA duplex within a liposome. Khan indicates it is routine and conventional to administer components of a CRISPR-Cas system wherein the components are delivered via liposome in a high-throughput editing system.
Khan does not teach administering each component in its own liposome.
Zhen teaches using liposomes for delivery of CRISPR/Cas9 components. Zhen teaches (§Challenges of CRISPR/Cas9 delivery ¶1-2) the CRISPR/Cas9 system can be used to develop disease models, facilitate genetic engineering, allow for more thorough epigenetic studies, knock out various genes of interest, and that CRISPR/Cas9 cocktails can simultaneously knockout multiple genes. Zhen teaches (same § ¶3) that CRISPR cannot readily enter cells because of its negative charge, so it requires a carrier. Zhen teaches (§Liposomes for CRISPR/Cas9 delivery ¶2) that liposomes’ surface properties affect their delivery. Zhen teaches that (§Lipoplexes ¶2) lipoplexes are easy to synthesize and good for in vitro use:
PNG
media_image1.png
310
557
media_image1.png
Greyscale
The cationic liposome formulations taught by Zhen are lipofection agents which is a limitation of instant Claim 32. Zhen teaches that (§Monovalent cationic liposomes ¶2) lipoplexes formed from monovalent cationic liposomes usually have a high gene knockout efficiency in vitro because they bind to cell membrane through nonspecific ionic interaction…Second, they have efficient endosomal escape by the ion-pair mechanism. Fig. 1 of Zhen shows that lipoplexes contain CRISPR/Cas9:
PNG
media_image2.png
254
458
media_image2.png
Greyscale
Although Zhen teaches some forms of conjugation that may be employed, for example PEGylation (§PEGylation on liposomal CRISPR/Cas9 delivery) and targeting ligands (§Surface targeting ligands on CRISPR/Cas9 delivery), the teachings of Zhen make it clear that conjugation has benefits in vivo but is not ideal or necessary and even interferes with system efficiency for in vitro applications. For example, Zhen teaches that (§PEGylation on liposomal CRISPR/Cas9 delivery) PEGylation increases circulation time in the blood stream but also reduces cellular uptake and endosomal escape and (§Conclusions and perspectives ¶5) in vitro studies show that current cleavable PEGylated liposomes are still less efficient than unPEGylated ones. Therefore, an artisan would have had no motivation to use any form of conjugation in the HTP mutagenesis platform of App693.
App693 teaches the crRNA and tracrRNA may be encoded on two separate molecules and teaches using Lipofectamine®, Khan teaches HTP screens using liposome-encapsulated elements (which encapsulate a combination crRNA:tracrRNA), and Zhen teaches benefits of administering biologics encapsulated within a liposome, but those references do not teach that each element of the RNP-complex (i.e., crRNA + tracrRNA + Cas enzyme or nucleic acid encoding Cas enzyme) is added to each well of a HTP system in its own separate liposome.
However, the prior art of Cong and App106 makes it clear that it was possible to separately administer those three elements: crRNA, tracrRNA, Cas enzyme or nucleic acid encoding Cas enzyme. The prior art of Scott and Filippova makes it clear that there was motivation to do so.
Cong teaches (§Main text ¶3) to test whether heterologous expression of the CRISPR system (SpCas9, SpRNase III, tracrRNA, and pre-crRNA) can achieve targeted cleavage of mammalian chromosomes, we transfected 293FT cells with different combinations of CRISPR/Cas components. Cong teaches (same §, ¶) they used the SURVEYOR assay to detect target cleavage to determine which combinations work. Cong shows (Fig. 1B) the constructs they used to express each element and (Fig. 1D) the results wherein each component was administered. Note that Fig. 1 calls the crRNA DR-EMX1(1)-DR or Dr-Spacer-DR. Cong teaches (§SUPPLEMENTARY MATERIALS AND METHODS-Suveryor assay and sequencing analysis for genome modification) cells were transfected with plasmid DNA for the SURVEYOR assay and (same §, §§ Cell culture and transfection) cells were seeded into 24-well plates and transfected using Lipofectamine 2000®.
Cong’s teachings indicate that it was known in the art to administer each element—Cas enzyme, crRNA, and tracrRNA—separately with each element encoded on its own plasmid and encapsulated in its own liposome. Cong’s liposomes comprise each element encoded on a plasmid.
Cong’s liposomes do not comprise each of the crRNA and tracrRNA in its own liposome.
However, App106, drawn to guideRNA that targets a specific mutation in a specific gene, teaches (¶7-11) a CRISPR system for knocking out a dominant mutation. App106 teaches (¶30-31) their invention encompasses a CRISPR nuclease (including Cas9), an RNA molecule, and a tracrRNA molecule. Reading App106, it is clear that what they refer to as “an RNA molecule” is what the art typically calls “a crRNA molecule” (i.e., App106 teaches [¶100] the crRNA base-pairs to a target DNA; see also ¶30-31 and claims 43 and 49). App106 teaches (¶31) embodiments of its invention include CRISPR complexes utilizing a separate tracrRNA molecule and separate RNA molecule comprising a guide sequence portion. Therefore, App106 clearly teaches using separate crRNA and tracrRNA. App106 further teaches (¶53-54) embodiments wherein :
(¶53) the CRISPR nuclease and the RNA molecule or RNA molecules are delivered to the subject and/or cells substantially at the same time or at different times… (¶54) the tracrRNA is delivered to the subject and/or cells substantially at the same time or at different times as the CRISPR nuclease and RNA molecule or RNA molecules [emphasis added].
PNG
media_image3.png
208
546
media_image3.png
Greyscale
Therefore App106 teaches each element of their system can be delivered separately: the CRISPR nuclease and the RNA molecule (passages above explained that the RNA molecule is App106’s terminology for what is claimed as crRNA), as well as the tracrRNA. App106 teaches (¶53-54) each of those components can be delivered to cells at different times.
App106 further clarifies that (¶75) the nuclease, crRNA, and tracrRNA can be delivered as separate components:
there is provided a kit for inactivating a mutant GUCY2D allele in a cell, comprising an RNA molecule comprising a guide sequence portion having 17-20 nucleotides in the sequence of 17-20 contiguous nucleotides set forth in any one of SEQ ID NOs: 1-3010, a CRISPR nuclease, and/or a tracrRNA molecule; and instructions for delivering the RNA molecule; CRISPR nuclease, and/or the tracrRNA to the cell.
App106’s use of and/or indicates that the components of the kit can be separate: an RNA molecule (i.e., a crRNA), a CRISPR nuclease, and a tracrRNA molecule as well as instructions for delivering the RNA molecule; CRISPR nuclease, and/or the tracrRNA to the cell. That indicates that each component of the kit can be delivered separately. Furthermore, App106 teaches that (¶95) the components can be delivered separately but are active at the same time:
Embodiments referred to above refer to a CRISPR nuclease, RNA molecule(s), and tracrRNA being effective in a subject or cells at the same time. The CRISPR, RNA molecule(s), and tracrRNA can be delivered substantially at the same time or can be delivered at different times but have effect at the same time. For example, this includes delivering the CRISPR nuclease to the subject or cells before the RNA molecule and/or tracrRNA is substantially extant in the subject or cells [emphasis added.]
App106 further teaches (¶126) the components of their system can be delivered in liposomes: Methods of non-viral delivery of nucleic acids and/or proteins include… lipofection… liposomes… or lipid:nucleic acid conjugates. Note that lipid:nucleic acid conjugates is an alternative to lipofection and liposomes; that indicates that liposomes are not conjugated to their contents.
Therefore, it is clear that App106 teaches each component—a crRNA, a tracrRNA, and a nuclease—may be delivered to a cell separately and within its own liposome, and that the components are effective at the same time. The reference also teaches the components can be delivered at the same time. That indicates that App106 teaches that both separate delivery and contemporaneous delivery are modes of delivery for the three components.
App693, Khan, Zhen, Cong, and App106 do not teach why an artisan would have been motivated to administer each crRNA and tracrRNA separately.
However, Scott teaches that tracrRNA modifications affect Cas9 activity and, therefore, genetic modification efficiency. Scott is drawn to improving Cas9 activity by making specific modifications to the tracrRNA. Scott teaches (§Abstract) a dual-guide RNA (dgRNA) with a modified tracrRNA can improve reporter knockdown and indel formation at several targets within the long terminal repeat (LTR) of HIV. Scott teaches (§Abstract) tracrRNA sequence can affect Cas9 activity. Scott teaches (§Sequence-modified tracrRNA can improve Cas9 RNP activity of alternative crRNAs) different tracrRNAs (i.e., tracrRNAs having different modifications or different sequences) affect different targets to varying extents …which suggests some target specific effects between tracrRNA-6 and tracrRNA-19 (i.e., two different tracrRNAs). Scott teaches (same §) their data demonstrate that the modified tracrRNAs can significantly improve Cas9 RNP activity with other crRNAs compared to an unmodified tracrRNA. Scott teaches (§Discussion ¶5) research studies have focused on chemically-modified dgRNAs for in vivo applications as a result of the technical and financial constraints synthesizing longer sgRNAs9, highlighting the need for approaches that improve dgRNA activity. Scott’s teachings indicate that different tracrRNAs have target specific effects and that it’s cheaper to use dual gRNAs (i.e., those that comprise both a crRNA and a tracrRNA).
Additionally, Filippova, drawn to guide RNA modification as a way to improve CRISPR/Cas9-based genome-editing systems, teaches (Fig. 2c; ) guidelines for modifying the backbone of both crRNA and tracrRNA. Filippova teaches regions of multiple nt on crRNA and tracrRNA that are preferable positions for 2’-OMe modification. Filippova teaches (§4. Backbone modifications ¶2) ribose modifications can affect crRNA–tracrRNA interactions, as well as targeting specificity and cleavage activity. Filippova teaches (Table 1) positions in each of a crRNA and tracrRNA wherein a certain modification has been linked to a certain improvement to CRISPR mutagenesis.
Altogether, App693 teaches a HTP system for altering cells with a CRISPR system, Khan teaches using liposomes to administer CRISPR components including in a HTP (i.e., “high-content”) system, Zhen teaches ease of using liposomes to deliver CRISPR components, Cong teaches separately administering liposomes wherein each contains a plasmid encoding each component (i.e., a liposome containing a plasmid encoding a crRNA, a liposome containing a plasmid encoding a tracrRNA, and a liposome containing a plasmid encoding a Cas enzyme) to induce mutagenesis in a cell, App106 teaches a CRISPR system wherein each component is administered separately in its own liposome but all components are effective in a cell at the same time, and Scott and Filippova teach that tracrRNAs comprising different sequence or tracrRNAs and crRNAs comprising different modifications can affect efficacy of CRISPR-induced mutagenesis.
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the HTP methods for altering cells including fungal cells of App693 with the teachings of Khan, Zhen, Cong, App106, Scott (including the fact that different tracrRNAs have target specific effects), and Filippova for the benefit of testing which tracrRNAs work best to knock out different targets and using differently-modified tracrRNAs to optimize knockout. One would have been motivated to do so with a reasonable expectation of success because Khan teaches (§Abstract) multiplex CRISPR knockouts and delivering components within liposomes, Scott teaches that tracrRNAs can have target-specific effects and that dual gRNAs are cheaper than sgRNAs, because Filippova teaches tracrRNAs comprising different modifications can affect efficacy of CRISPR-induced mutagenesis, because App693 teaches that the crRNA and tracrRNA may be two separate molecules and that (¶195) the sequence of the scaffold (which comprises a tracrRNA) determines how the scaffold interacts with the nuclease and can affect binding kinetics to a target, and because App106 teaches administering the three components separately but that they are effective in a cell at the same time.
It stands to reason that an artisan seeking to knock out multiple targets in a HTP mutagenesis would have wanted to simultaneously evaluate the effect of differently modified tracrRNAs (or tracrRNAs with different sequences and therefore different structures) on knockout efficiency of different genes and in different cell types. Since differently-modified tracrRNAs have target-specific effects (as taught by Scott and Filippova), an artisan would have wanted to know which tracrRNAs or tracrRNA modifications are most efficacious for each gene target and each cell type. Testing combinations of each crRNA (i.e., the spacer that targets a particular gene) with different tracrRNAs in a HTP format across various cell types would be most efficient if the crRNA and the tracrRNA were administered separately because separate administration would not require an extra annealing step and would facilitate making combinations and permutations of various crRNAs and various tracrRNAs. Testing those combinations would not be possible if sgRNAs were used.
One would have been motivated to do so with a reasonable expectation of success because Khan and App106 teach administering the components of a CRISPR gene editing system in liposomes, and App106 teaches administering each component in its own liposome and teaches that the components are effective within a cell at the same time. Success would have been a reasonable expectation because Cong teaches separate administration wherein each component is encoded on a plasmid which indicates a CRISPR editing system comprising delivery of three separate components is effective, and it would have been obvious to administer the actual components—rather than a plasmid encoding each one—including because App106 teaches doing so. One would have been motivated to use liposomes because Khan, Cong, Zhen, and App106 teach using them and for the benefits of easily producing a delivery vehicle that improves cellular uptake and is suited for in vitro use as well as the benefits of allowing easy administration of various combinations and permutations of crRNA and tracrRNA. One would have been motivated to do so with a reasonable expectation of success because Zhen teaches that lipoplexes are easy to synthesize using just two ingredients (cationic liposomes such as Lipofectamine® and CRISPR/Cas9). Using liposomes containing the CRISPR components would have made it easier to carry out the HTP mutagenesis of App693 including testing different tracrRNAs to identify which tracrRNA is best for each target. Placing each component in its own liposome would have made it easy to “mix and match” crRNA and tracrRNA combinations in a HTP system.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to use different Cas proteins because App693 teaches using mutated Cas protein[s] with one or more altered characteristics compared to the parental Cas protein. An artisan would have been motivated to administer separate Cas proteins in the HTP mutagenesis for the benefit of testing how the different Cas proteins affect knock out efficacy. A desire to test the efficacy of various crRNA + tracrRNA + Cas protein combinations would have motivated an artisan to keep the three components (Cas protein, crRNA, tracrRNA) separate and administer them separately or together in various combinations or permutations to carry out the experiments discussed above. Administration of each component in its own liposome would have been obvious for the benefits of improved cellular uptake.
Modifying the teachings of App693, Khan, Zhen, Cong, and App106 with the different tracrRNAs of Scott and Filippova would have produced a HTP mutagenesis wherein the Cas enzyme, crRNA, and tracrRNA are administered separately, each within its own liposome. It would have produced a method of genetically altering a plurality of target cells comprising combining the cells with a crRNA, a tracrRNA, and a Cas enzyme so the components come together and form a complex in a cell and are effective at the same time, wherein each cell is mutated, and wherein the location of each unique spacer sequence within each well is known, and the target cell within each well has a different target gene mutated (limitations of Claim 1) or wherein at least two crRNAs, each directed to different target genes, are used to knockout at least two or more different target genes in each target cell (limitations of Claim 2). Altogether, modifying the method of genetically altering a plurality of target cells and the method comprising knocking out at least two or more different target genes in each cell of App693 with the teachings of Khan, Zhen, Cong, App106, Scott, and Filippova would have produced the limitations of Claims 1-2, 5, 8, 15, 23-24, and 31-33.
The teaching of App106 that allows (¶95) administering the components either separately or together allows for any component to be added in any order. Therefore the limitations of Claims 11-14, 16-17, and 19 would have been obvious in view of App693, Khan, Zhen, Cong, App106, Scott, and Filippova.
Regarding Claim 34: the claim requires that the three separate liposomes are combined into a mixture before they are combined with the target cell. App106 teaches (¶53) the CRISPR nuclease and the RNA molecule or RNA molecules are delivered to the subject and/or cells substantially at the same time or at different times. And ¶54 teaches the tracrRNA is delivered to the subject and/or cells substantially at the same time or at different times as the CRISPR nuclease and RNA molecule or RNA molecules. That indicates that App106 permits administering each of the different components (the tracrRNA, the crRNA, the nuclease) to the cell in any combination or permutation. Therefore it would have been obvious to combine the three components into a mixture to ensure that they would be delivered to the cells at the exact same time. Therefore the limitations of Claim 34 would have been obvious in view of App693, Khan, Zhen, Cong, App106, Scott, and Filippova.
Claims 1, 3-6, 9, and 23 are rejected under 35 U.S.C. 103 as being unpatentable over App693, Khan, Zhen, Cong, App106, Scott, and Filippova as applied to Claims 1, 5, and 23 (and 2, 8, 11-17, 19, 24, and 31-34) above, and further in view of International Patent Application WO2017075294 (“WO294”, of record). This rejection is maintained. All references are of record.
The teachings of App693, Khan, Zhen, Cong, App106, Scott, and Filippova as applied to Claim 1 have been described in the 103 rejection above.
App693, Khan, Zhen, Cong, App106, Scott, and Filippova teach a method of genetically altering a plurality of target cells comprising (a) combining within each of a plurality of wells of a first well plate three liposomes, each containing within the liposome and without being conjugated thereto: a crRNA that includes a unique spacer complementary to a target gene, a tracrRNA, a Cas enzyme or a nucleic acid sequence encoding the Cas enzyme, as well as a target cell; and(b) incubating the plurality of target cells within the plurality of wells wherein the crRNA and the tracrRNA form a guide RNA and wherein the Cas enzyme and the guideRNA form a colocalization complex with the target gene of the target cell, and the Cas enzyme cuts and mutates the target gene, wherein the unique sequence of the crRNA in each well is complementary to a different target gene and wherein the location within the first well plate of each unique spacer sequence within each well is known, and the target cell within each well has a different target gene mutated.
App693, Khan, Zhen, Cong, App106, Scott, and Filippova do not explicitly teach the method wherein the plurality of wells includes 500 or more well (Claim 3), wherein the plurality of wells includes 1000 or more wells (Claim 4), or wherein the plurality of wells includes 3000 or more wells (Claim 6).
However, WO294 teaches tools and methods for systematic analysis of genetic interactions such as higher order interactions, as well as tools and methods for combinatorial probing of cellular circuits for uses such as dissecting cellular circuitry, delineating molecular pathways, and/or identifying relevant targets for therapeutics development. WO294 reports that such a system is necessary because interactions among components of mammalian cells can be multi-way, so studying them requires performing high order combinations of perturbations; the CRISPR/Cas9 system is named as one way to perturb cells.
Regarding numbers of wells, WO294 teaches (¶400) preferred embodiments of the invention are multi-well assay plates that use industry' standard multi-well plate formats for the number, size, shape and configuration of the plate and wells. Examples of standard formats include 96-, 384-, 1536- and 9600-well plates.
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the high-throughput genome alteration method of App693, Khan, Zhen, Cong, App106, Scott, and Filippova with the standard multiwell formats of WO924 for the benefit of altering a great many cells at one time. One would have been motivated to do so with a reasonable expectation of success because the methods set forth in WO924 at ¶24 can be used with CRISPR to perform many assays in a single experiment: …combining droplet based single cell transcriptomics with CRISPR-Cas based perturbations…allow[s] researchers to perform thousands of assays in a single pooled experiment and because App693 teaches (¶550) any number of different plates or configurations may be used. Modifying the method of App693, Khan, Zhen, Cong, App106, Scott, and Filippova with the teachings of WO924 would have produced the limitations of Claims 3-6.
Regarding instant Claims 9 and 23, App693, Khan, Zhen, Cong, App106, Scott, and Filippova do not explicitly teach the method wherein each target cell within the plurality of target cells is a different cell type (Claim 9).
However, WO294 teaches (¶51) perturbing cells, including a single cell, to investigate genetic interactions:
The method can include…perturbing and isolating a single cell with at least one labeling ligand specific for binding at one or more target RNA transcripts, or isolating a single cell with at least one labeling ligand specific for binding at one or more target RNA transcripts and perturbing the cell…
WO294 teaches (¶5) molecular profiling at the single cell level.
WO294 teaches (¶265) the discrete nature of cells allows for libraries to be prepared in mass with a plurality of cellular variants all present in a single starting media and then that media is broken up into individual droplet capsules that contain at most one cell. Perturbing only a single isolated cell would have required only one cell being present in a well which is a limitation of Claim 23. As described above, App693 teaches tools and methods of placing a single cell in a well.
WO924 teaches that their assay can be used to study different cell types. ¶22 teaches:
Applicants developed Perturb-seq, which combines single cell RNA-seq and CRISPR/Cas9 based perturbations…to perform many, tens of thousands in certain embodiments…assays in a single pooled experiment...Applicants develop a computational framework…to identify the regulatory effects of individual perturbations and their combinations at different levels of resolution: from effects on each individual gene to functional signatures to proportional changes in cell types.
¶334 teaches:
cells may be treated with drugs, small molecules, pathogens, hormones, cytokines, proteins, nucleic acids, virus particles, or grown in different cellular environments. Cells may be isolated from a diseased tissue. The cells from the diseased tissue may be compared to cells from non-diseased tissue.
An artisan knows that tissue by definition includes different cell types. Then ¶213 teaches that the invention allows a researcher to introduce biomolecules into almost any cell type and assess the outcome:
[the invention is] a generalized platform for introducing a diverse range of biomolecules into living cells in high-throughput could transform how complex cellular processes are probed and analyzed… This modality enables one to assess the phenotypic consequences of introducing a broad range of biological effectors (DNAs, RNAs, peptides, proteins, and small molecules) into almost any cell type.
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the high-throughput genome alteration method of App693, Khan, Zhen, Cong, App106, Scott, and Filippova to study different cell types or individual cells as taught by WO924 for the benefit of testing different hypotheses (e.g., “protein X coded by gene X has function 1 in cell type A, but has function 2 in cell type B” or “protein X coded by gene X has function 1 when cell type A is a single cell but has function 3 when there is a colony of cells type A”). One would have been motivated to do so with a reasonable expectation of success because it stands to reason that if individual cells were able to be isolated and targeted to identify effects on individual genes or functional signatures, then individual cells of different cell types could also have been isolated and targeted. One would also have been motivated because WO924 teaches (¶271) the cells or beads being encapsulated are generally variants on the same type of cell or bead. In one example, the cells may comprise cancer cells of a tissue biopsy, and each cell type is encapsulated to be screened for genomic data or against different drug therapies, and because WO924 teaches explicitly that their methods can be used with CRISPR-Cas based perturbations: (¶30)
The perturbing or perturbation(s) may comprise(s) genetic perturbing…single-order perturbations…combinatorial perturbations…gene knock-down, gene knock-out, gene activation, gene insertion, or regulatory element deletion…genome-wide perturbation…performing CRISPR-Cas-based perturbation…pooled single or combinatorial CRISPR-Cas-based perturbation with a genome-wide library of sgRNAs…[or] may be of a selected group of targets based on similar pathways or network of targets.
Modifying the method of App693, Khan, Zhen, Cong, App106, Scott, and Filippova with the teachings of WO924 would have produced the limitations of Claims 9 and 23.
Claims 1, 7, and 20-22 are rejected under 35 U.S.C. 103 as being unpatentable over App693, Khan, Zhen, Cong, App106, Scott, and Filippova as applied to Claim 1 (and 2, 5, 8, 11-17, 19, 23-24, and 31-34) above, and further in view of US Patent Application Publication No. US20080095673 (“App673”, of record) and evidenced by Yale University’s Stat course document Experimental Design Randomization and Replication (“Yale”, of record). This rejection is maintained. All references are of record.
The teachings of App693, Khan, Zhen, Cong, App106, Scott, and Filippova as applied to Claim 1 have been described above.
App693, Khan, Zhen, Cong, App106, Scott, and Filippova teach a method of genetically altering a plurality of target cells comprising (a) combining within each of a plurality of wells of a first well plate three liposomes each containing within the liposome and without being conjugated thereto: a crRNA that includes a unique spacer complementary to a target gene, a tracrRNA, a Cas enzyme or a nucleic acid sequence encoding the Cas enzyme, as well as a target cell; and(b) incubating the plurality of target cells within the plurality of wells wherein the crRNA and the tracrRNA form a guide RNA and wherein the Cas enzyme and the guideRNA form a colocalization complex with the target gene of the target cell, and the Cas enzyme cuts and mutates the target gene, wherein the unique sequence of the crRNA in each well is complementary to a different target gene and wherein the location within the first well plate of each unique spacer sequence within each well is known, and the target cell within each well has a different target gene mutated.
App693, Khan, Zhen, Cong, App106, Scott, and Filippova do not teach the method wherein the first well plate includes edge wells and wherein the edge wells are vacant (Claim 7). Nor do they teach the method of claim 1 wherein steps (a) and (b) are repeated for a second well plate, and wherein each unique spacer sequence provided to the wells of the first well plate is provided at a location within the second well plate that is different from the first well plate (Claim 20); claim 1 wherein steps (a) and (b) are repeated for a plurality of well plates, and wherein each unique spacer sequence provided to the wells of the first well plate is provided at a different location within each of the plurality of well plates and the first well plate (Claim 21); or claim 21 wherein the plurality of well plates is greater than 1000 well plates (Claim 22).
However, App673 relates to an improved microplate that is characterized by modifications that produce fewer artificially induced inaccuracies in peripheral wells, especially in comer wells. App673 discusses that peripheral wells in standard microwell plates can lead to experimental inaccuracies.
Regarding leaving edge wells vacant and repeating the steps of instant Claim 1 in more well plates, App673 teaches (¶20)
“Peripheral artifacts”, “lateral artifacts”, “quadrilateral artifacts”, or “edge artifacts” is defined as artificially induced difference(s) of experimental results specifically stemming from “peripheral wells” or “lateral wells” other than internal experimental wells. These artifacts are usually owing to disparities of thermal receptance, light exposure, and/or liquid evaporation between “peripheral wells” and “internal wells”.
App673 teaches in Figure 4B that sham wells (figure reference 19, ¶[64] 19. Sham wells) are located at the periphery of the plate:
PNG
media_image4.png
510
764
media_image4.png
Greyscale
and clarifies at ¶19 that “Sham wells” is defined as the wells from which any final experimental results obtained are predicted to be useless, no matter whether the said sham wells are used to host an assay, or they are just left blank without an assay. These passages indicate that it is advantageous to leave edge wells vacant.
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the high-throughput genome alteration method of App693, Khan, Zhen, Cong, App106, Scott, and Filippova with App673’s teaching regarding the deficiencies of edge wells for the benefit of preventing inaccurate experimental results. One would have been motivated to do so with a reasonable expectation of success because App673 teaches at ¶ [0016] It is inevitable that the above-mentioned limitations have caused some inaccurate experimental results… in the conventional microplates. Doing so would have produced the limitations of instant Claim 7.
Regarding instant Claims 20-22, a person of ordinary skill in the art would have recognized that since experimental artifacts are likely to arise based on well location, it would be an obvious solution to run a second replicate of an experiment with well locations altered from the first replicate, or to run many or a great many replicates with locations altered.
Evidence that altering locations in the manner disclosed by instant Claims 20-22 is standard in the art is provided by Yale §Randomized Block Design:
If an experimenter is aware of specific differences among groups of subjects or objects within an experimental group, he or she may prefer a randomized block design to a completely randomized design. In a block design, experimental subjects are first divided into homogeneous blocks before they are randomly assigned to a treatment group. If, for instance, an experimenter had reason to believe that age [i.e., in the instance case, “well location”] might be a significant factor in the effect of a given medication, he might choose to first divide the experimental subjects into age groups, such as under 30 years old, 30-60 years old, and over 60 years old. Then, within each age level, individuals would be assigned to treatment groups using a completely randomized design. In a block design, both control and randomization are considered.
Further, §Replication teaches:
Although randomization helps to insure that treatment groups are as similar as possible, the results of a single experiment, applied to a small number of objects or subjects, should not be accepted without question…To improve the significance of an experimental result, replication, the repetition of an experiment on a large group of subjects, [i.e., in the instance case, “a second well plate”, “a plurality of well plates”, or “wherein the plurality of well plates is greater than 1000 well plates”] is required…Replication reduces variability in experimental results, increasing their significance and the confidence level with which a researcher can draw conclusions about an experimental factor.
Modifying the methods of App693, Khan, Zhen, Cong, App106, Scott, and Filippova with the teachings of App673 before the effective filing date of the instant invention for the benefits of obtaining more robust data would have produced the limitations of Claims 20-22.
Claims 1-2 and 11-19 are rejected under 35 U.S.C. 103 as being unpatentable App693, Khan, Zhen, Cong, App106, Scott, and Filippova as applied to Claims 1-2 and 15 (and 5, 8, 11-14, 16-17, 19, 23-24, and 31-34) above, and further in view of Hultquist (of record), Jacobi 2017 (of record), and Gonzalez 2014 (of record). This rejection is maintained. All references are of record.
The teachings of App693, Khan, Zhen, Cong, App106, Scott, and Filippova as applied to Claim 1 have been described above.
App693, Khan, Zhen, Cong, App106, Scott, and Filippova teach a method of genetically altering a plurality of target cells comprising (a) combining within each of a plurality of wells of a first well plate three liposomes each containing within the liposome and without being conjugated thereto: a crRNA that includes a unique spacer complementary to a target gene, a tracrRNA, a Cas enzyme or a nucleic acid sequence encoding the Cas enzyme, as well as a target cell; and(b) incubating the plurality of target cells within the plurality of wells wherein the crRNA and the tracrRNA form a guide RNA and wherein the Cas enzyme and the guideRNA form a colocalization complex with the target gene of the target cell, and the Cas enzyme cuts and mutates the target gene, wherein the unique sequence of the crRNA in each well is complementary to a different target gene and wherein the location within the first well plate of each unique spacer sequence within each well is known, and the target cell within each well has a different target gene mutated.
App693, Khan, Zhen, Cong, App106, Scott, and Filippova do not teach the method wherein the target cell is provided to the well before the liposome containing the crRNA, the liposome containing the tracrRNA, and the liposome containing the Cas enzyme/nucleic acid sequence encoding the Cas enzyme are provided to the well (Claim 11); wherein the liposome containing the tracrRNA, the liposome containing the Cas enzyme/a nucleic acid sequence encoding the Cas enzyme, and the target cell are provided to the well before the liposome containing the crRNA (Claim 12); wherein the liposome containing the crRNA is provided to the well before the liposome containing the tracrRNA, the liposome containing the Cas enzyme/a nucleic acid sequence encoding the Cas enzyme, and the target cell are provided to the well (Claim 13); wherein the liposome containing the crRNA is provided to the well after the liposome containing the tracrRNA, the liposome containing the Cas enzyme/a nucleic acid sequence encoding the Cas enzyme, and the target cell are provided to the well (Claim 14); wherein the liposome containing the tracrRNA, the liposome containing the Cas enzyme/nucleic acid sequence encoding the Cas enzyme, and the target cell are provided to the well as a mixture (Claim 16); wherein the liposome containing the Cas enzyme or a nucleic acid sequence encoding the Cas enzyme, and the target cell are provided to the well as a mixture (Claim 17); wherein the cell is transfected with the nucleic acid encoding the Cas enzyme before the target cell is provided to the well (Claim 18); or wherein (1) the liposome containing the crRNA, (2) a mixture of the liposome containing the tracrRNA and the liposome containing the Cas enzyme/a nucleic acid sequence encoding the Cas enzyme, and (3) the target cell are provided separately to the well (Claim 19).
However, the references Hultquist, Jacobi, Gonzalez, and Cong teach adding the different CRISPR components in different permutations. Hultquist teaches a high-throughput platform for efficient, multiplex editing of host factors that control infection of T cells; in the platform, arrayed electroporation of CRISPR/Cas9 ribonucleoproteins (RNPs) enables production of cells with ablated “candidate” factors; the system is used to identify gene modifications that provide viral resistance. Jacobi is drawn to CRISPR tools for efficient genome editing and protocols for delivery into mammalian cells. Jacobi adds ctRNP to the plate before target cells. Gonzalez is drawn to a CRISPR platform for rapid, multiplexable genome editing in cells; Gonzalez transfects target cells with Cas9-encoding nucleic acids before proceeding with guideRNA steps. Cong is drawn to multiplex genome engineering using CRISPR; Cong varies the combinations of experimental components. All four references alter the order of adding ingredients in order to optimize their experimental success.
Hultquist teaches on P. 1447, Left Column, final ¶:
a screen of 45 genes described in the published literature that either directly or indirectly affect the function of HIV integrase. These included 21 genes…as well as 24 genes... Three crRNAs were designed per gene alongside multiple non-targeting controls and several previously analyzed crRNAs, including those targeting CXCR4 and LEDGF as positive controls and CDK9 as a toxicity control. In total, 146 crRNAs were designed and synthesized in 96-well plate arrayed format. These crRNAs were incubated with tracrRNA and Cas9 protein to form Cas9 RNPs, which were subsequently electroporated into activated primary T cells from two donors.
Regarding Claim 2, Hultquist teaches that the system can be used to produce double knockouts (i.e., KOs in different genes) on P. 1444, §Cas9 RNP Multiplexing Allows for the Generation of Double-Knockout Cells:
A potential strength of using in vitro synthesized Cas9 RNPs is the ease with which double knockouts could be generated…we attempted to knock out both CXCR4 and CD4 by co-electroporation of their respective Cas9 RNPs. For CD4, we designed three crRNAs that yielded different efficiencies…Immunostaining for both CXCR4 and CD4 demonstrated specific depletion of each cell surface marker only when the Cas9 RNP targeting that gene was included and furthermore demonstrated the clear accumulation of a double-negative population…we calculated the predicted percentage of double-knockout cells when delivering the Cas9 RNPs at a 1:1 ratio, assuming editing at each locus was independent of the other (Figure 4C). We found that this nearly perfectly reflected the observed percentage of double-knockout cells when staining for CD4 and CXCR4 (Figure 4C).
Hultquist further teaches in ¶2 under §Experimental Procedures Cas9 RNP-Mediated Editing of Primary Human T cells: Cas9 RNPs were prepared fresh for each experiment. crRNA and tracrRNA were first mixed 1:1 and incubated 30 min at 37°C to generate 40 µM crRNA: tracrRNA duplexes. An equal volume of 40 µM Cas9-NLS was slowly added to the crRNA:tracrRNA and incubated for 15 min at 37°C to generate 20 µM Cas9 RNPs. . . 3 µL of 20 µMCas9 RNP mix was added directly to these cells and the entire volume transferred to the 96-well reaction cuvette. Therefore Hultquist teaches providing the tracrRNA and the Cas enzyme as a mixture (limitations of Claim 15); providing the tracrRNA, the Cas enzyme, and target cell as a mixture (Claim 16); and providing the Cas enzyme and the target cell as a mixture (Claim 17).
Jacobi teaches on p. 18, Left Column, Step 8: Aliquot 50 µL of ctRNP transfection solution followed by 100 µL cell suspension to 3 wells in a 96-well tissue culture plate to create biological triplicates (reverse transfection format). The ctRNPs of Jacobi comprise (§2.1.1. Lipofection of ctRNP complexes for NHEJ into HEK293 cells Step 3) crRNA, tracrRNA, and Cas9 protein. Therefore Jacobi teaches that the CRISPR components are provided to the well before the cell.
Gonzalez teaches on p. 216 Right Column, ¶2: We reasoned that one could develop a more efficient and versatile genome-editing platform by first generating hPSCs that express Cas9, the invariable component of the CRISPR/Cas system and on p. 223 under gRNA or gRNA + SSDNA Transfection: iCas9 hPSCs were treated with doxycycline (2 mg/ml) for 1 or 2 days before and during transfection. For transfection, cells were dissociated using … and transfected in suspension with gRNAs or… Therefore Gonzalez teaches the limitation of instant Claim 18 that the cell is transfected with the nucleic acid encoding the Cas enzyme before the target cell is provided to the well.
Cong teaches on p. 820, Left Column, final ¶ that to test whether heterologous expression of the CRISPR system (SpCas9, SpRNase III, tracrRNA, and pre-crRNA) can achieve targeted cleavage of mammalian chromosomes, we transfected 293FT cells with different combinations of CRISPR/Cas components.
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the method of App693, Khan, Zhen, Cong, App106, Scott, and Filippova with different sequences of adding ingredients for the benefit of testing out which sequence is most effective for any given combination of the particular crRNAs, tracrRNAs, Cas enzyme(s), and cell type(s). One would have been motivated to do so with a reasonable expectation of success because the references of App693, Khan (described above), Hultquist, Jacobi, Gonzalez, and Cong demonstrate that such permutations of the sequence of adding ingredients were commonly used in the art at the time of filing. Furthermore it is known in the art, is taught by App106—and the instant claims demonstrate–that the structures of crRNA, tracrRNA, and Cas enzyme will—due to their structures—form a complex provided they are all in the same place at the same time. Changing the order of adding ingredients to the wells would not have changed that those three components would have come together and cut the target(s). Modifying the method of App693, Khan, Zhen, Cong, App106, Scott, and Filippova with the order of adding ingredients of App693, Hultquist, Jacobi, Gonzalez, and/or Cong would have produced the limitations of Claims 15-18. It is noted that instant Claims 11-14 and 19 merely change the order of adding ingredients. Therefore, an artisan undertaking routine optimization would have arrived at those permutations based on the teachings of App693, Khan, Zhen, Cong, App106, Scott, and Filippova, Hultquist, Jacobi, Gonzalez, and Cong. Therefore the limitations of Claims 11-19 would have been obvious in view of the references.
The Court has stated that generally such differences in the sequence of adding ingredients amount to mere optimization and will not support patentability unless there is evidence indicating the claimed feature is critical. Ex parte Rubin, 128 USPQ 440 (Bd. App. 1959) (Prior art reference disclosing a process of making a laminated sheet wherein a base sheet is first coated with a metallic film and thereafter impregnated with a thermosetting material was held to render prima facie obvious claims directed to a process of making a laminated sheet by reversing the order of the prior art process steps.). See also In re Burhans, 154 F.2d 690, 69 USPQ 330 (CCPA 1946) (selection of any order of performing process steps is prima facie obvious in the absence of new or unexpected results); In re Gibson, 39 F.2d 975, 5 USPQ 230 (CCPA 1930) (Selection of any order of mixing ingredients is prima facie obvious.
Applicant must provide either a showing that the particular sequences of adding ingredients recited within the claims is critical; and/or a showing that the prior art reference teaches away from the claimed sequences. In the instant case, the specification as filed provides no evidence that the particular sequence of ingredient addition is critical because 1) the various claims alter the order, indicating that no specific order is critical; and 2) the above cited references teach explicitly and/or implicitly that the claimed permutations were well known at the time of filing.
Claims 1 and 25-29 are rejected under 35 U.S.C. 103 as being unpatentable over App693, Khan, Zhen, Cong, App106, Scott, and Filippova as applied to Claim 1 (and 2, 5, 8, 11-17, 19, 23-24, and 31-34) above, and further in view of Jacobi, Gonzalez, Weinert 2018 (of record), and International Patent Application Publication No. WO 2016/028887 A1 (of record, “App887”). This rejection is maintained. All references are of record.
The teachings of App693, Scott, and Zhen as applied to Claim 1 have been described above.
App693, Khan, Zhen, Cong, App106, Scott, and Filippova teach a method of genetically altering a plurality of target cells comprising (a) combining within each of a plurality of wells of a first well plate three liposomes each containing within the liposome and without being conjugated thereto: a crRNA that includes a unique spacer complementary to a target gene, a tracrRNA, a Cas enzyme or a nucleic acid sequence encoding the Cas enzyme, as well as a target cell; and(b) incubating the plurality of target cells within the plurality of wells wherein the crRNA and the tracrRNA form a guide RNA and wherein the Cas enzyme and the guideRNA form a colocalization complex with the target gene of the target cell, and the Cas enzyme cuts and mutates the target gene, wherein the unique sequence of the crRNA in each well is complementary to a different target gene and wherein the location within the first well plate of each unique spacer sequence within each well is known, and the target cell within each well has a different target gene mutated.
App693, Khan, Zhen, Cong, App106, Scott, and Filippova do not teach the method wherein the well concentration of guide RNA is between 10 nM and 1 µM (Claim 25); wherein the well concentration of guide RNA is between 25 pmol and 125 pmol (Claim 26); wherein the well concentration of guide RNA is between 75 pmol and 125 pmol (Claim 27); wherein the well concentration of Cas enzyme is between 1 nM and 150 nM (Claim 28); or wherein the well concentration of Cas enzyme is between 60 nM and 80 nM (Claim 29).
However, Jacobi teaches on p. 18, Left Column, Step 8: The final concentration of the ctRNP complex in the transfection is 10 nM…per well (i.e., a limitation of instant Claim 28). As discussed above, Jacobi’s ctRNP complex includes crRNA, tracrRNA, and Cas enzyme. Jacobi teaches on p. 19 Right Column, Step 13 Therefore, the final concentrations during electroporation are 1.5 µM Alt-RTM S.p. Cas9 3NLS, 1.8 µM guide RNA complex... Therefore Jacobi teaches the guideRNA concentration close to that recited by instant Claim 25. Additionally, Khan teaches (§Methods-RNA transfection and cell viability assay ¶2) the concentration of crRNA:tracrRNA duplex optimal for a cell is 25 nM. That reads on the claimed limitation of a well concentration of guide RNA between 10 nM and 1 µM (Claim 25).
Gonzalez teaches (§gRNA or gRNA + ssDNA Transfection) final concentrations of guideRNA was 10 nM which is very close to the range recited in Claim 25 if not within that range. (The Spec. does not specify whether the bounds of the range are included.)
Jacobi and Gonzalez do not teach the limitations of the method wherein the well concentration of guide RNA is between 25 pmol and 125 pmol (Claim 26); wherein the well concentration of guide RNA is between 75 pmol and 125 pmol (Claim 27); or wherein the well concentration of Cas enzyme is between 60nM and 80nM (Claim 29).
However, Weinert and App887 teach the limitations that App693, Khan, Zhen, Cong, App106, Scott, and Filippova do not teach. Weinert is drawn to the relationship between CRISPR/Cas9 gene editing of primary cells/tissues and immune responses, as well as an optimized method of transfecting cells with guide RNA that recites specific concentrations. App887 is drawn to methods for selectively analyzing a mixed population of nucleic acids (i.e., pooled samples), and selectively enriching regions of interest; these methods recite specific concentrations. App887 teaches that its methods may be used with the CRISPR-Cas system.
Weinert teaches (p. 12 §Culture and transfection of primary HSPCs) HSPCs from mobilized peripheral blood (Allcells) were thawed and cultured…for 48 h before nucleofection with dCas9 or Cas9 RNP (75 pmol of dCas9, 75 pmol of gRNA) (i.e., limitations of instant Claims 26-27).
App887 teaches (¶120) that The Cas9 reaction mixture included…32 nM Cas9 enzyme (i.e., close to the limitations of instant Claim 29).
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the high-throughput genome alteration method of App693, Khan, Zhen, Cong, App106, Scott, and Filippova with the quantities taught by Jacobi and/or Khan and/or Gonzalez and/or Weinert and/or App887 as well as other similar quantities arrived at through routine experimentation for the benefit of optimizing the high-throughput screen. One would have been motivated to do so with a reasonable expectation of success because the references show that the instantly claimed quantities or quantities close to them are within the ranges commonly used in the art. Modifying method of App693, Khan, Zhen, Cong, App106, Scott, and Filippova with the teachings of Jacobi and/or Khan and/or Gonzalez and/or Weinert and/or App887 would have produced the limitations of Claims 25-29.
The Court has stated that generally such differences amount to mere optimization and will not support patentability unless there is evidence indicating the claimed feature is critical. “[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). (Claimed process which was performed at a temperature between 40°C and 80°C and an acid concentration between 25% and 70% was held to be prima facie obvious over a reference process which differed from the claims only in that the reference process was performed at a temperature of 100°C and an acid concentration of 10%.); see also Peterson, 315 F.3d at 1330, 65 USPQ2d at 1382 (“The normal desire of scientists or artisans to improve upon what is already generally known provides the motivation to determine where in a disclosed set of percentage ranges is the optimum combination of percentages.”); In re Hoeschele, 406 F.2d 1403, 160 USPQ 809 (CCPA 1969) (Claimed elastomeric polyurethanes which fell within the broad scope of the references were held to be unpatentable thereover because, among other reasons, there was no evidence of the criticality of the claimed ranges of molecular weight or molar proportions.).
For more recent cases applying this principle, see Merck & Co. Inc. v. Biocraft Laboratories Inc., 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert. denied, 493 U.S. 975 (1989); In re Kulling, 897 F.2d 1147, 14 USPQ2d 1056 (Fed. Cir. 1990); and In re Geisler, 116 F.3d 1465, 43 USPQ2d 1362 (Fed. Cir. 1997). In KSR International Co. v. Teleflex Inc., 550 U.S. 398 (2007), the Supreme Court held that "obvious to try" was a valid rationale for an obviousness finding, for example, when there is a "design need" or "market demand" and there are a "finite number" of solutions. 550 U.S. at 421. MPEP 2144 sets forth Applicant' s burden for rebuttal of a prima facie case of obviousness based upon routine optimization.
Applicant must provide either a showing that the particular amount or range recited within the claims is critical; and/or a showing that the prior art reference teaches away from the claimed amount. In the instant case, the specification as filed provides no evidence that the particular amount or range recited within the claims is critical because the amount of a specific ingredient in a composition is clearly a result-effective parameter that a person of ordinary skill in the art would have routinely optimized, as demonstrated by the different ranges in the references cited above. Optimization of parameters is a routine practice that would have been obvious for a person of ordinary skill in the art to employ.
Claims 1 and 30 are rejected under 35 U.S.C. 103 as being unpatentable over App693, Khan, Zhen, Cong, App106, Scott, and Filippova as applied to claim 1 (and 2, 5, 8, 11-17, 19, 23-24, and 31-34) above, and further in view of US Patent Application Publication No. US 2019/0249219 (“App219”, of record). This rejection is maintained. All references are of record.
The teachings of App693, Khan, Zhen, Cong, App106, Scott, and Filippova as applied to Claim 1 have been described above.
App693, Khan, Zhen, Cong, App106, Scott, and Filippova teach a method of genetically altering a plurality of target cells comprising (a) combining within each of a plurality of wells of a first well plate three liposomes each containing within the liposome and without being conjugated thereto: a crRNA that includes a unique spacer complementary to a target gene, a tracrRNA, a Cas enzyme or a nucleic acid sequence encoding the Cas enzyme, as well as a target cell; and(b) incubating the plurality of target cells within the plurality of wells wherein the crRNA and the tracrRNA form a guide RNA and wherein the Cas enzyme and the guideRNA form a colocalization complex with the target gene of the target cell, and the Cas enzyme cuts and mutates the target gene, wherein the unique sequence of the crRNA in each well is complementary to a different target gene and wherein the location within the first well plate of each unique spacer sequence within each well is known, and the target cell within each well has a different target gene mutated.
App693, Khan, Zhen, Cong, App106, Scott, and Filippova do not teach the method wherein the liposome containing a crRNA, the liposome containing a tracrRNA, the liposome containing a Cas enzyme/a nucleic acid sequence encoding the Cas enzyme, and the target cell are provided to the well by droplet transfer from a source container to the well using sound waves.
However, App219 teaches an automated method for obtaining a discrete microorganism colony or cells from a solution comprising multiple microorganism or cells. The method includes using acoustic liquid transfer to transfer a very small amount of material.
Regarding droplet transfer using sound waves, App219 teaches in §Abstract this method includes a step where acoustic liquid transfer is employed and (¶357)
The term “acoustic liquid transfer”… is based on the principle to use a pulse of ultrasound to move a low volume of a fluid (such as e.g. pl, nl or µl) without any physical contact. This technology focuses acoustic energy into a fluid sample in order to eject droplets as small as a nanoliter or even a picoliter. “Acoustic liquid transfer” can be used to transfer samples without damage.
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to combine the high-throughput genome alteration method of App693, Khan, Zhen, Cong, App106, Scott, and Filippova with the acoustic liquid transfer of App219 for the benefit of transferring a precise and very small amount of a liquid without damage. One would have been motivated to do so with a reasonable expectation of success because App219 teaches at (¶5) transfection comprises the transfer of nucleic acids such as e.g. RNA or DNA into eukaryotic cells; (¶365) that transfection can be carried out…by mixing a cationic lipid with the material to produce liposomes, which fuse with the cell membrane and deposit their cargo inside. Typical transfection protocols are known to the skilled person; (¶16) there is the need for high-throughput methods for the analysis of liquid samples as regards…cells potentially comprised therein; and (¶100) in high throughput cloning approaches, several transformation reactions (e.g. 6, 12, 24 or 96) are ideally handled in parallel, and only one culture plate or a multi well plate is ideally used to save time and resources. Modifying the method of App693, Khan, Zhen, Cong, App106, Scott, and Filippova with the acoustic transfer method of App219 would have produce the limitations of Claim 30.
Response to Arguments
Applicant’s arguments, see pp. 13-25, filed 12 June 2026, have been fully considered but they are not persuasive.
Objection
Claims 12 and 14 have the same scope because they both recite providing the components besides for the crRNA (i.e., the liposome containing the tracrRNA portion of the guideRNA, the liposome containing the Cas enzyme/nucleic acid sequence encoding the Cas enzyme, and the target cell) to the well BEFORE providing to the well the liposome containing the crRNA portion of the guideRNA. Whether the guideRNA is added after those components or if those components are added before the guideRNA, the scope of the claims is the same.
112(b)
Claims 25-27 recite the limitation "guide RNA" in L1. There is insufficient antecedent basis for this limitation in the claim because these claims depend from Claim 1 which recites only a crRNA portion of a guideRNA and a tracrRNA portion of a guideRNA, so it is unclear what to portion the “guideRNA” recited in Claims 25-27 refer.
112(d)
Claim 24 fails to incorporate all the limitations of the claim from which it depends, namely Claim 1, because Claim 1 explicitly recites that step (a) requires three separate liposomes but Claim 24 recites that the crRNA portion of a guideRNA and the tracrRNA portion of a guideRNA tare provided within a liposome. Therefore Claim 24 fails to include Claim 1’s limitation requiring three separate liposomes
103
All of Applicant’s arguments are directed to the App693, Khan, Zhen, Cong, App106, Scott, and Filippova references.
Briefly, the combination of cited prior art would have made obvious the claimed invention. The prior art shows:
App693, Khan: high-throughput transformation assays were known,
Zhen, Khan, Cong, App106: liposomes containing various components were known delivery vehicles for both proteins and nucleic acids,
Cong and App106: Using liposomes to separately deliver components of a Cas9 + crRNA + tracrRNA system was known, and it was known that doing so would result in the components being effective in the cell at the same time,
App106: delivery of CRIPSR nuclease, crRNA, and tracrRNA at different times or at the same time, and
Scott, Filippova: Separate modifications to each of the crRNA and tracrRNA were known to affect activity, efficiency, and specificity of a Cas9 system.
Applicant argues that the claimed invention requires… three separate liposomes administered to a cell and asserts that none of the references teaches the administration of the three separate components each in its own liposome.
In general, those arguments are not persuasive because the rejection is an obviousness rejection and it is based on synthesis of what is known in the art and by one of ordinary skill. The examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, the combination of references teaches that separately providing each element—a nuclease, a crRNA, and a tracrRNA—to a cell or cells was known in the art. HTP mutagenesis was known. Delivery via liposomes was known. The rejection explains why an artisan would have been motivated to administer each of the three components separately at different times or at the same time.
The bulk of Applicant’s remarks (pp. 19-22) argue against the App106 reference. Those arguments first assert that App106 fails to teach elements of the claimed invention because (¶75) is discussing a kit and does not teach separate delivery of the three components. Applicant asserts that and/or simply means that the kit for inactivating a mutant allele in a cell includes an RNA molecule, a CRISPR nuclease, with the tracrRNA being optional.
However, the plain language of ¶75 indicates that it says the kit can comprise instructions for delivering any combination or permutation of the three components in the kit. ¶75 states the kit includes instructions for delivering the RNA molecule; CRISPR nuclease, and/or the tracrRNA to the cell. That indicates that instructions can be for delivering each component—and/or—to the cell. Since the instructions can be for delivering each component to the cell, it follows that the components can be delivered separately.
Note also that Robbins (2018. “And/Or” and the Proper Use of Legal Language. Maryland Law Review 77[2]:2, “Robbins”) addresses (PDF pp. 26-27) this very subject. Robbins teaches that while “A, B and/or C” can be interpreted ambiguously—does that punctuation/phrasing provide a choice among A, B, C, or any combination thereof, equal to “A and/or B and/or C”? Or does the placement of and/or between B and C suggest that and/or is intended to provide a choice between only B and C, with A remaining a constant?—the workable solution is to:
always use a serial comma such that the structure becomes “A, B, and/or C.” With this construction, it becomes more clear that and/or should apply to all of the terms. The inclusion of this comma is key to ensuring that a phrase using and/or with more than two propositions is interpreted correctly. [emphasis added.]
App106 includes that serial comma and the meaning of their teachings is clear: and/or should apply to all of the terms within the list.
Further to the point about App106 teaching separate delivery, the rejection cites App106’s specific teachings that the components are delivered to cells at different times: App106 clearly teaches (¶30-31, ¶53-54, ¶95) separate RNA molecule (a.k.a. crRNA), nuclease, and tracrRNA, as well as (¶53-54, ¶95) separate or contemporaneous delivery.
App106 (¶30) teaches: alternatively, CRISPR nucleases may form a CRISPR complex between the CRISPR nuclease, an RNA molecule, and a tracrRNA molecule. How would that complex form between three components if there weren’t three separate components? App106 teaches (¶31) embodiments of its invention include CRISPR complexes utilizing a separate tracrRNA molecule and separate RNA molecule comprising a guide sequence portion. Therefore, App106 teaches using separate crRNA and tracrRNA.
Regarding Applicant’s arguments about App106’s claims, that is not relevant because those are merely some embodiments of the App106 disclosure.
Regarding Applicant’s arguments about ¶54, those aren’t persuasive because the pair of ¶53 and ¶54 clearly indicate the CRISPR nuclease and RNA molecule are delivered at the same time or different times and the tracrRNA can also be delivered at the same time or different times as the CRISPR nuclease and the RNA molecule.
PNG
media_image3.png
208
546
media_image3.png
Greyscale
¶53 teaches the CRISPR nuclease and the RNA molecule can be delivered at different times and ¶54 teaches the tracrRNA can be delivered at different times from the CRISPR nuclease or the RNA molecule, so altogether that means each component—the RNA molecule a.k.a. crRNA, the nuclease, and the tracrRNA—can be delivered at separate times.
Applicant’s arguments about ¶95 (on Remarks p. 20) are similarly unpersuasive. The rejection discusses that App106 teaches that (¶95) the components can be delivered separately but they are active at the same time; original text of ¶95 follows:
Embodiments referred to above refer to a CRISPR nuclease, RNA molecule(s), and tracrRNA being effective in a subject or cells at the same time. The CRISPR [nuclease], RNA molecule(s), and tracrRNA can be delivered substantially at the same time or can be delivered at different times but have effect at the same time. For example, this includes delivering the CRISPR nuclease to the subject or cells before the RNA molecule and/or tracrRNA is substantially extant in the subject or cells [emphasis added.]
PNG
media_image5.png
225
542
media_image5.png
Greyscale
That clearly teaches that those components can be delivered at substantially the same time or at different times. It then provides one single example—“for example”—of delivering the CRISPR nuclease to the cells:
before the RNA molecule or
before the tracrRNA or
before the RNA molecule and the tracrRNA.
But before providing the example, ¶95 teaches any of those components can be delivered at substantially the same time or at different times and provides one example. One exemplary statement doesn’t exclude separately delivering each component. Note again the use of Robbins’s serial comma: the CRISPR, RNA molecule(s), and tracrRNA which means the CRISPR nuclease can be delivered at substantially the same time or at different times, the RNA molecule(s) can be delivered at substantially the same time or at different times, and the tracrRNA can be delivered at substantially the same time or at different times. (App106 consistently and intentionally includes serial commas in its lists.) Each component can be delivered at substantially the same time or at different times which indicates that any or all components can be delivered at substantially the same time or at different times.
It stands to reason that in order for the components discussed in ¶95 to be delivered at different times (or to make one component present in the cell before the other[s]), the components have to be delivered separately. Those teachings clearly and unequivocally indicate that App106 teaches that (¶95) each component can be delivered at different times but have effect at the same time [emphasis added].
Specifically in their arguments regarding ¶95 Applicant states that (p. 21) throughout App106, it teaches that the RNA molecule and the tracrRNA molecule are delivered together. Certainly, App106 teaches embodiments where that is the case. However, App106 also discloses other embodiments wherein the components are delivered separately, as discussed (¶53-54, ¶95). Applicant’s arguments assert that App106’s ¶95 should be either ignored or interpreted against its plain meaning because other passages teach additional embodiments. However, it is improper to ignore one embodiment within a reference simply because the reference provides additional embodiments. Notably Applicant’s remarks have not addressed ¶30-31 or the combination of ¶53-54.
Altogether, Applicant’s arguments against App106 are not at all persuasive.
Applicant’s other arguments are similarly unpersuasive. All of these arguments amount to piecemeal analysis of the references. In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986).
It is reiterated that the rejection is a 103 rejection that concludes that the claimed invention would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention. Obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In this case, the prior art shows:
App693, Khan: high-throughput transformation assays were known,
Zhen, Khan, Cong, App106: liposomes containing various components were known delivery vehicles for both proteins and nucleic acids,
Cong and App106: Using liposomes to separately deliver components of a Cas9 + crRNA + tracrRNA system was known, and it was known that doing so would result in the components being effective in the cell at the same time,
App106: delivery of CRIPSR nuclease, crRNA, and tracrRNA at different times or at the same time, and
Scott, Filippova: Separate modifications to each of the crRNA and tracrRNA were known to affect activity, efficiency, and specificity of a Cas9 system.
It is, therefore, irrelevant that none of the references teaches exactly what is claimed. The rejection explains that what is claimed would have been obvious in view of what was known in the art. In addition, and contrary to Applicant’s argument that the rejection doesn’t provide a motivation to administer three separate components each in its own liposome, the rejection clearly states:
An artisan seeking to knock out multiple targets in a HTP mutagenesis would have wanted to simultaneously evaluate the effect of differently modified tracrRNAs (or tracrRNAs with different sequences and therefore different structures) on knockout efficiency of different genes and in different cell types. Since differently-modified tracrRNAs have target-specific effects (as taught by Scott and Filippova), an artisan would have wanted to know which tracrRNAs or tracrRNA modifications are most efficacious for each gene target and each cell type. Testing combinations of each crRNA (i.e., the spacer that targets a particular gene) with different tracrRNAs in a HTP format across various cell types would be most efficient if the crRNA and the tracrRNA were administered separately because separate administration would not require an extra annealing step and would facilitate making combinations and permutations of various crRNAs and various tracrRNAs. Testing those combinations would not be possible if sgRNAs were used.
An artisan would have been motivated to administer separate Cas proteins in the HTP mutagenesis for the benefit of testing how the different Cas proteins affect knock out efficacy. A desire to test the efficacy of various crRNA + tracrRNA + Cas protein combinations would have motivated an artisan to keep the three components (Cas protein, crRNA, tracrRNA) separate and administer them separately or together in various combinations or permutations to carry out the experiments discussed above. Administration of each component in its own liposome would have been obvious for the benefits of improved cellular uptake.
Applicant’s arguments in (a) are not persuasive because the combination of App693, Khan, Zhen, Cong, App106, Scott, and Filippova would have made it obvious to provide three separate liposomes each containing one of a crRNA, a tracrRNA, or a Cas enzyme or a nucleic acid sequence encoding the Cas enzyme, as explained above. Furthermore Zhen teaches liposomes improve cellular uptake and are suitable for in vitro use and explains why an artisan wouldn’t want to conjugate a liposome to its contents for in vitro uses. Facilitating entry to the cell is a feature inherent to liposomes.
Furthermore, in response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., facilitating entry of each component to a cell) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Applicant’s arguments in (b) are not persuasive because the combination of App693, Khan, Zhen, Cong, App106, Scott, and Filippova would have made it obvious to provide three separate liposomes each containing one of a crRNA, a tracrRNA, or a Cas enzyme or a nucleic acid sequence encoding the Cas enzyme, as explained above. Furthermore, Khan was applied to show it is possible to administer components of a CRISPR/Cas system in liposomes in a HTP system. Applicant argues that Khan teaches away from the claimed invention because of a passage on p. 2. That passage discusses that some kinds of multiplex gene co-targeting is cumbersome. Khan also teaches using complexed crRNA:tracrRNA in Cas9-expressing cells.
Just because Khan found System A cumbersome and that System B works better for their own purposes does not mean that System A is always cumbersome or that System B is always better for every purpose. The 103 rejection explains that (App693) different Cas enzymes have different efficacies, that (Scott) different tracrRNAs have target-specific effects, and that (Scott and Filippova) combinations of modifications on crRNA and tracrRNA affect efficacy. For an artisan who is testing combinations of each crRNA (i.e., the spacer that targets a particular gene) with different tracrRNAs in a HTP format across various cell types, it would be most efficient if the crRNA and the tracrRNA were administered separately because separate administration would facilitate making combinations and permutations of various crRNAs and various tracrRNAs. Testing those combinations would not be possible if Khan’s sgRNAs were used.
Similarly for the Cas enzyme or nucleic acid encoding it: An artisan would have been motivated to administer separate Cas proteins in the HTP mutagenesis for the benefit of testing how the different Cas proteins affect knock out efficacy. A desire to test the efficacy of various crRNA + tracrRNA + Cas protein combinations would have motivated an artisan to keep the three components (Cas protein, crRNA, tracrRNA) separate and administer them separately or together in various combinations or permutations. Administration of each component in its own liposome would have been obvious for the benefits of improved cellular uptake.
Since the structure of a HTP screen is merely a design choice that an artisan chooses depending on what they want to study, the arguments in (b) are not persuasive.
Applicant’s arguments in (c) are not persuasive. The arguments relating to App106 have been addressed above. The arguments against Zhen and Cong are not persuasive because the combination of App693, Khan, Zhen, Cong, App106, Scott, and Filippova would have made it obvious to provide three separate liposomes each containing one of a crRNA, a tracrRNA, or a Cas enzyme or a nucleic acid sequence encoding the Cas enzyme, as explained above. Applicant’s arguments that neither Zhen nor Cong teaches separate delivery of the components have been addressed above: this is an obviousness rejection and it is the combination of App693, Khan, Zhen, Cong, App106, Scott, and Filippova that would have made it obvious to provide three separate liposomes each containing one of a crRNA, a tracrRNA, or a Cas enzyme or a nucleic acid sequence encoding the Cas enzyme.
Furthermore, Applicant’s arguments concede that Cong teaches delivery of each of the three components encoded on its own plasmid, each plasmid within its own liposome, but argues that Cong teaches cotransfection but not separate delivery. However, cotransfection (i.e., simultaneous delivery of various nucleic acid components) is in fact no different from combining within each of a plurality of wells three separate liposomes and a target cell and incubating them all together. Only Claim 19 requires separately providing the liposomes to the well. The 103 rejection explains that differences in the sequence of adding ingredients amount to mere optimization and will not support patentability unless there is evidence indicating the claimed feature is critical.
Regarding the arguments against Scott and Filippova, those are not persuasive because the combination of App693, Khan, Zhen, Cong, App106, Scott, and Filippova would have made it obvious to provide three separate liposomes each containing one of a crRNA, a tracrRNA, or a Cas enzyme or a nucleic acid sequence encoding the Cas enzyme, as explained above. Similar to the arguments against Khan, Applicant argues that Scott’s method requires a step of annealing a crRNA to a tracrRNA, which then forms a Cas9 RNP complex.
Just because Scott found one system useful for their work doesn’t mean that system is universally better for every purpose. Applicant argues that Scott’s mix and match happens prior to being encapsulated in separate liposomes but provides no evidence or explanation of why, aside from their assertion that Scott did it that way, multiple component encapsulation would necessarily be the case. It has already been explained above that App106 teaches separate delivery, including at different times. It has already been explained above that delivery to a HTP system of separate liposomes would allow an artisan to avoid a time consuming annealing step. App106 indicates that such a time consuming step is unnecessary because each component can be delivered separately.
Furthermore, Scott wasn’t using a HTP system. Many people of ordinary skill in the art would recognize that when administering 3 or more components to numerous wells, it is far simpler to individually deliver each component to its target well(s) rather than produce a plethora of mixtures comprising various combinations and permutations of those components and subsequently adding each of the plethora of mixtures to its target well(s). Many people of ordinary skill in the art would recognize that producing intermediate mixtures of various combinations and permutations is an unnecessary and confusing step. Many people of ordinary skill in the art would recognize that simply labeling the mixture vials would be time consuming, inefficient, resource intensive, and cause their hand to cramp up. No person of ordinary skill would voluntarily choose that kind of unnecessary pain, especially since the vials of individual components already exist. Why would an artisan intending mix and match for the above-stated purposes undertake such an endeavor? Applicant has not explained why, aside from that Scott did it. But again, Scott wasn’t running HTP experiments. A person of ordinary skill would readily recognize that there is no reason to do it Scott’s way if App106’s way is easier, cheaper, and faster.
ATTORNEY ARGUMENTS CANNOT TAKE THE PLACE OF EVIDENCE
The arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965). Examples of attorney statements which are not evidence and which must be supported by an appropriate affidavit or declaration include statements regarding unexpected results, commercial success, solution of a long-felt need, inoperability of the prior art, invention before the date of the reference, and allegations that the author(s) of the prior art derived the disclosed subject matter from the inventor or at least one joint inventor.
See MPEP § 2145 generally for case law pertinent to the consideration of applicant’s rebuttal arguments.
MPEP §716.01(c)
"A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton." KSR, 550 U.S. at 421, 82 USPQ2d at 1397. "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle." Id. at 420, 82 USPQ2d at 1397. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ." Id. at 418, 82 USPQ2d at 1396.
In addition to the factors above, Office personnel may rely on their own technical expertise to describe the knowledge and skills of a person of ordinary skill in the art. The Federal Circuit has stated that examiners and administrative patent judges on the Board are "persons of scientific competence in the fields in which they work" and that their findings are "informed by their scientific knowledge, as to the meaning of prior art references to persons of ordinary skill in the art." In re Berg, 320 F.3d 1310, 1315, 65 USPQ2d 2003, 2007 (Fed. Cir. 2003). In addition, examiners "are assumed to have some expertise in interpreting the references and to be familiar from their work with the level of skill in the art ." PowerOasis, Inc. v. T-Mobile USA, Inc., 522 F.3d 1299, 86 USPQ2d 1385 (Fed. Cir. 2008) (quoting Am. Hoist & Derrick Co. v. Sowa & Sons, 725 F.2d 1350, 1360, 220 USPQ 763, 770 (Fed. Cir. 1984). See MPEP § 2141.03 for a discussion of the level of ordinary skill.
MPEP §2141(II)(c)
Regarding Applicant’s arguments against Filippova, those are not persuasive because the combination of App693, Khan, Zhen, Cong, App106, Scott, and Filippova would have made it obvious to provide three separate liposomes each containing one of a crRNA, a tracrRNA, or a Cas enzyme or a nucleic acid sequence encoding the Cas enzyme, as explained above.
Applicant’s arguments on pp. 23-25 simply reiterate their previous arguments which aren’t found persuasive for the reasons discussed above.
It is reiterated that the obviousness rejection synthesizes what would have been obvious to a person of ordinary skill in the art.
Many of Applicant’s arguments pertain to their assertion that the individual references teach only cotransfection or components being combined before being encapsulated in liposomes. However, it has been explained that an artisan, synthesizing teachings from the prior art, would have been motivated to do things differently from exactly what is taught in the art because they would have had different aims. For example, Khan (cited to teach that it is routine and conventional to encapsulate components of a CRISPR system within liposomes) teaches (§Abstract) they were targeting only three genes for knockout and only two cell types. In contrast, App693 teaches (¶238, ¶295) altering 10 genes or even more—any finite number of genes. App693 teaches (¶550) using their system to produce 10,000 or more transformants. That indicates App693 were using their system to modify a huge number of different target genes. If an artisan were simultaneously testing how combinations of differently-modified tracrRNA + different Cas enzymes affect mutation efficacy in different cell types, they would not have wanted to create additional solutions. A system wherein each component is administered separately within its own liposome would have been easier to apply to a HTP system transforming 10,000 or more transformants a day when aiming to test multiple variables at once.
Using Khan’s or Scott’s methods in the App693 system would have been cumbersome. But both of those references teach why and how an artisan would have wanted to improve App693’s HTP system.
Altogether, the 103 obviousness rejection is maintained.
The following arguments from previous Office Actions are still relevant
The remarks did not provide/discuss any mechanism by which the components would be delivered at different times but also be delivered together. Applicant asserts (p. 15) that App106 doesn’t teach separate delivery but does not explain how ¶54 and ¶95 fail to teach separate delivery. App106 literally says (¶95) the CRISPR, RNA molecule(s), and tracrRNA… can be delivered at different times.
The rejection discusses that App106 teaches (¶126) the components of their system can be delivered in liposomes: Methods of non-viral delivery of nucleic acids and/or proteins include… lipofection… liposomes… or lipid:nucleic acid conjugates.
Together those references teach the separate delivery and delivery in liposomes that Applicant alleges the references don’t teach.
Regarding Applicant’s arguments that (pp. 12-14) App693, Zhan, Zhen, and Cong fail to teach separate delivery with each element in its own liposome, those are not found persuasive because as pointed out, App106 unequivocally teaches separate delivery—the CRISPR, RNA molecule(s), and tracrRNA can be delivered substantially at the same time or can be delivered at different times but have effect at the same time [emphasis added]—and delivery of components in liposomes.
Regarding Applicant’s arguments that (pp. 14-15) App106 ¶75 doesn’t teach separate delivery, those arguments are not persuasive because of App106’s ¶54 and ¶95 which teach separate delivery. Notably, Applicant’s arguments didn’t address those portions of the rejection. Applicant then asserts that one of the claims (App106’s claim 43) does not teach separate delivery but that is not persuasive in view of the teachings at ¶54 and ¶95 which disclose the CRISPR, RNA molecule(s), and tracrRNA can be delivered substantially at the same time or can be delivered at different times but have effect at the same time [emphasis added]. Just because App106’s claim 43 doesn’t recite separate delivery doesn’t mean that ¶54 and ¶95 don’t exist or are no longer relevant. It is reiterated that the rejection is a 103 obviousness rejection which relies on synthesis of information from each reference. The reference as a whole teaches exactly what Applicant asserts no reference teaches: administration of the three separate components each in its own liposome.
Applicant asserts (p. 15) that none of the cited prior art references teaches three separate liposomes each containing one of a crRNA, a tracrRNA, or a Cas enzyme/nucleic acid encoding a Cas enzyme within the liposome and without being conjugated thereto. The inaccuracy of that assessment has been addressed above.
Finally, (pp. 16-17) Applicant argues against Scott and Filippova because each of those references doesn’t teach the exact same invention as what is recited in the claims. But Scott and Filippova were applied to show why an artisan would have been motivated to apply a multitude of different crRNAs and tracrRNAs to a cell: Scott teaches different tracrRNA can have target-specific effects and Filippova teaches (Fig. 2c; ) guidelines for modifying the backbone of both crRNA and tracrRNA and that (§4. Backbone modifications ¶2) ribose modifications can affect crRNA–tracrRNA interactions, as well as targeting specificity and cleavage activity. Applicant concedes that Scott has provided the motivation to mix and match crRNA and tracrRNA in a HTP system (p. 16 even if Scott might have provided the motivation to “mix and match” crRNA and tracrRNA combinations) but argues that the mix and match happens prior to being encapsulated in liposomes. That argument is not found persuasive because the rejection addressed that:
If differently-modified tracrRNAs have target-specific effects (as taught by Scott and Filippova), an artisan would have wanted to know which tracrRNAs or tracrRNA modifications are best for each target. Testing combinations of each crRNA (i.e., the spacer that targets a particular gene) with different tracrRNAs in a HTP format across various cell types would be most efficient if the crRNA and the tracrRNA were administered separately because separate administration would not require an extra annealing step and would facilitate making combinations of crRNAs and tracrRNAs. Testing those combinations would not be possible if sgRNAs were used. [emphasis added.]
Furthermore, the rejection was applied over the synthesis of information from the references. And, there is absolutely nothing in Scott and Filippova’s teachings regarding different efficacies of different tracrRNAs that requires tracrRNA be delivered only by Scott’s methods.
Applicant also argues that (pp. 16-17) Filippova is completely silent on liposome delivery and thus cannot be relied on as a source of motivation of the presently claimed method that requires three separate liposomes… That argument is not found persuasive because other references teach delivery via liposome and liposome delivery of biologics was routine and customary.
It is reiterated that App106 teaches separate delivery of tracrRNA and crRNA, including that the components are delivered to cells at different times:
PNG
media_image6.png
102
541
media_image6.png
Greyscale
And, App106 teaches that (¶95) the components can be delivered separately but are active at the same time:
PNG
media_image5.png
225
542
media_image5.png
Greyscale
In response to applicant's arguments against the references individually, one cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). The rejection explains (pp. 13-16) how an artisan would have synthesized the teachings of the different references:
They would have wanted to test which tracrRNAs work best to knock out different targets and would have used differently-modified tracrRNAs to optimize knockout. Khan teaches (§Abstract) multiplex CRISPR knockouts and delivering components within liposomes; Scott teaches tracrRNAs can have target-specific effects and dual gRNAs are cheaper than sgRNAs; Filippova teaches crRNAs and tracrRNAs comprising different modifications can affect efficacy of CRISPR-induced mutagenesis; App693 teaches the crRNA and tracrRNA may be two separate molecules and (¶195) the sequence of the scaffold (which comprises a tracrRNA) determines how the scaffold interacts with the nuclease and can affect binding kinetics to a target; App106 teaches administering components separately but that they are effective in a cell at the same time. An artisan would have wanted to simultaneously evaluate the effect of differently modified tracrRNAs on knockout efficiency of different genes and in different cell types. Testing combinations of each crRNA (i.e., the spacer that targets a particular gene) with different tracrRNAs in a HTP format across various cell types would be most efficient if the crRNA and the tracrRNA were administered separately because separate administration would not require an extra annealing step and would facilitate making combinations of crRNAs and tracrRNAs. Testing those combinations (i.e., crRNA/tracrRNA/cell type) would not be possible if sgRNAs were used.
App106 teaches administering each component in its own liposome at different times. Since Cong teaches separate administration wherein each component is encoded on a plasmid (which indicates the system is effective) separately administering the actual components—rather than a plasmid encoding each one—would have been obvious. It would have been obvious to use liposomes because Khan, Cong, and App106 teach using them and because Zhen teaches that lipoplexes are easy to synthesize and good for in vitro applications. Using liposomes containing the CRISPR components would have made it easier and cheaper to carry out the HTP mutagenesis of App693 including testing different tracrRNAs. Placing each component in its own liposome would have made it easy to “mix and match” crRNA and tracrRNA combinations in a HTP system.
A person of ordinary skill is a person of ordinary creativity.
"A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton." KSR, 550 U.S. at 421, 82 USPQ2d at 1397. "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle." Id. at 420, 82 USPQ2d at 1397. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ." Id. at 418, 82 USPQ2d at 1396.
In addition to the factors above, Office personnel may rely on their own technical expertise to describe the knowledge and skills of a person of ordinary skill in the art. The Federal Circuit has stated that examiners and administrative patent judges on the Board are "persons of scientific competence in the fields in which they work" and that their findings are "informed by their scientific knowledge, as to the meaning of prior art references to persons of ordinary skill in the art." In re Berg, 320 F.3d 1310, 1315, 65 USPQ2d 2003, 2007 (Fed. Cir. 2003). In addition, examiners "are assumed to have some expertise in interpreting the references and to be familiar from their work with the level of skill in the art ." PowerOasis, Inc. v. T-Mobile USA, Inc., 522 F.3d 1299, 86 USPQ2d 1385 (Fed. Cir. 2008) (quoting Am. Hoist & Derrick Co. v. Sowa & Sons, 725 F.2d 1350, 1360, 220 USPQ 763, 770 (Fed. Cir. 1984). See MPEP § 2141.03 for a discussion of the level of ordinary skill.
MPEP §2141(II)(c)
A person of ordinary skill would have understood that liposomes could be used to deliver Scott/Filippova’s modified tracrRNA and a person of ordinary skill would have understood that administering each component in its own liposome is efficient for a HTP system testing the effects of combinations of various crRNAs, various tracrRNAs, and Cas enzyme(s) on mutagenesis.
An artisan would have been motivated to administer the components separately because of the ability to simultaneously test the effects of crRNA, tracrRNA, and Cas enzyme on various targets in a HTP system. An artisan would have readily taken the teachings of App106, Scott, and Filippova and applied them to App693’s system of making combinatorial genetic changes for the benefit of testing different tracrRNAs. Altogether, the claimed invention would have been obvious in view of the cited prior art.
Conclusion
Claims 1-9 and 11-34 are rejected.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. International Publication Number WO 2020/180975 (published 10 September 2020, “WO975”). WO975 is directed to high-throughput gene editing systems that utilize CRISPR.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to RUTHIE S ARIETI whose telephone number is (571)272-1293. The examiner can normally be reached M-Th 8:30AM-4PM, alternate Fridays 8:30AM-4PM (ET).
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, Ram R Shukla can be reached at (571)272-0735. 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.
RUTHIE S ARIETI
Examiner
Art Unit 1635
/RUTH SOPHIA ARIETI/Examiner, Art Unit 1635
/NANCY J LEITH/Primary Examiner, Art Unit 1636
Prosecution Insights