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 .
Application Status
This action is written in response to applicant’s correspondence received 5 August 2025. Claims 1-4, 8-9, 16, 21-22, 24, 35, 41, 52-54, and 86-90 are currently pending. Claims 6-7, 15, 65, and 85 are canceled. Accordingly, claims 1-4, 8-9, 16, 21-22, 24, 35, 41, 52-54, and 86-90 are examined herein. The restriction requirement mailed 25 November 2024 is still deemed proper. Applicant's elected Group I, SEQ ID NO: 18, SEQ ID NO:6, and SEQ ID NO: 3 without traverse in the reply filed 25 February 2025. SEQ ID NOs: 4 and 7-15 were previously rejoined into examination in the Non-Final Office Action mailed 3 April 2025.
Any rejection or objection not reiterated herein has been overcome by amendment. Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Claim 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, 4, 8-9, 16, 21-22, 24, 35, and 86-90 is/are rejected under 35 U.S.C. 103 as being unpatentable over Thompson (ACS Chem (2017): 313-325) in view of Billon (Molecular cell 67.6 (2017): 1068-1079) and Liu (PG Pub No. US 2017/0121693 A1).
Regarding claims 1 and 16, Thompson is drawn to a review study concerned with multiplexed eukaryotic genome engineering (Abstract). Thompson teaches a method of multiplexed base editing via the use of CRISPR-Cas comprising contacting a target nucleic acid molecule with at least one fusion protein comprising a dCas9 domain, a base editor domain selected from a deaminase (pg. 318), and a gRNA bound to the dCas9 domain (pg. 317; see Figure 2). Thompson teaches that multiplex genome engineering entails the use of multiple, unique, gRNAs that bind to the fusion protein such that edits across distinct loci may occur (pg. 315). Thompson teaches that the utilization of 12 different gRNAs (i.e., unique guide RNAs that each individually require the use of at least one fusion protein) has been successfully achieved in order to edit a target genomic sequence (pg. 315). Thompson teaches that the fusion proteins can be utilized to edit eukaryotic genomes (Abstract) and be functionally expressed in mammalian cells (pg. 315).
Thompson does not teach or suggest that each of the fusion proteins comprise an amino acid sequence that is at least 90% identical to the claimed SEQ ID NO: 8 (Claims 1 and 16). Thompson does not teach or suggest that the unique gRNAs comprise at least 10 nucleotides (Claims 1 and 16). Thompson does not teach or suggest that at least 25 copies of the target sequence are present in the genomic DNA of a eukaryotic cell (Claims 1 and 16). Thompson does not teach or suggest that at least 50 copies of the target sequence are present in the genomic DNA of a eukaryotic cell (Claim 22).
However, one of ordinary skill in the art would have considered the teachings of Billon and Liu as both references in a common field pertaining to the use of Cas9 nucleases.
Billon is drawn to a study concerned with CRISPR-mediated gene disruption strategies that rely on Cas9-APOBEC1 fusion proteins (i.e., Cas9-deaminase fusion proteins) (Abstract). Billon teaches the use of Cas9 gRNAs that are 20 nucleotides in length (pg. e2). Billon teaches the use of a gRNA library comprising over 3.4 million single gRNAs to facilitate gene inactivation by induction of STOP codons in 97%-99% of genes in eight eukaryotic species (i.e., Billon teaches species of gRNA molecules that comprise at least 50 copies of a target sequence in genomic DNA) (Abstract). Billon teaches that a major advantage to inducing STOP codons at target genomic sites, as opposed to inducing double-strand break formation, the target cells exhibit a reduced cell death compared to conventional CRISPR-Cas9-based methods (pg. 1077).
Liu is drawn towards an invention concerned with Cas9 fusion proteins (Abstract). Liu teaches the use of an rAPOBEC1-XTEN-dCas9 fusion protein (i.e., a fusion protein comprising a dCas9, and a deaminase domain) comprising 99.6% sequence identity to the claimed SEQ ID NO: 8 that was capable of inducing C-to-T edits in human HEK293 cells ([0352]; see SEQ ID NO: 657 in attached sequence alignment). Liu teaches that the fusion protein can be directed to a target nucleic acid via the use of a guide RNA molecule ([0357]).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the fusion proteins of Thompson for the fusion protein of Liu and the unique guide RNAs of Thompson for the unique guide RNAs of Billon. A person of ordinary skill in the art would have been motivated to do so in order to reduce cell death of target cells comprising the target nucleic acid by inducing STOP codons are genomic locations of interest as opposed to cleavage in order to reduce cell death compared to conventional CRISPR methods. A person of ordinary skill in the art would have had a reasonable expectation of success because both Thompson and Liu teach the use of dCas9-deaminase fusion proteins that can be directed towards a target nucleic acid through the use of a guide RNA while Thompson and Billon both teach the use of guide RNAs that can direct Cas9 molecules to a target genomic location of interest.
Regarding claims 2 and 21, Thompson teaches that a large number of gRNAs are able to be packaged on a 150-kb HSV replicon (i.e., Thompson is interpreted as teaching the use of at least 20 unique guide RNAs) (pg. 320-321).
Regarding claim 4, Thompson teaches the use of a single dCas9-base editor fusion protein structure (i.e., Thompson teaches that the fusion proteins have the same structure) (pg. 317; see Figure 2).
Regarding claim 8, It is noted that the difference between the claimed SEQ ID NO: 8 and Liu’s SEQ ID NO: 657 is the presence of an SGGS motif present in Liu’s fusion protein ([0352]; see SEQ ID NO: 657 in attached sequence alignment).
Liu teaches that the SGGS motif is drawn towards an optional linker sequence that links the dCas9 to the adenosine deaminase ([0227]). Liu teaches that the optional linker sequence is not required by the fusion protein ([0026], [0305]).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the fusion protein rendered obvious by Thompson in view of Billon and Liu for a fusion protein that does not comprise an optional linker sequence and has 100% sequence identity to the claimed SEQ ID NO: 8, as described by Liu. A person of ordinary skill in the art would have had a reasonable expectation of success because Liu teaches that the optional linker sequence is not required for the fusion protein to function.
Regarding claim 9, Thompson teaches the use of an adenosine deaminase (pg. 318).
Regarding claims 24 and 86, Thompson teaches that repetitive elements in genomes such as Alu, L1 retrotransposons, SVA, and human endogenous retroviruses (HERVs) can be good potential targets for the fusion proteins (pg. 317-318).
Regarding claim 35, Billon teaches that the sgRNAs can be utilized to generate STOP codons (Abstract).
Regarding claim 87, Billon teaches the use of Cas9 sgRNAs (Abstract).
Regarding claim 88, Liu teaches that the target cell can be a human cel ([0352]).
Regarding claims 89-90, Liu teaches that the fusion proteins can be utilized both in vivo and in vitro ([0043]-[0044]). Liu teaches that utilizing the fusion proteins for targeted DNA editing in vitro allowed for the generation of mutant cells while utilizing the fusion proteins in vivo allowed for the editing of a subject cell’s genome ([0180], [0313]).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to utilize the method of base editing rendered obvious by Thompson in view of Billon and Liu in a cell that is in vivo or in vitro. A person of ordinary skill in the art would have been motivated to do so in order to generate mutant cells or edit a subject’s genome. A person of ordinary skill in the art would have had a reasonable expectation of success because Liu and Thompson in view of Billon and Liu teach the use of Cas9:deamianse fusion proteins that can be utilized to edit target cells of interest.
Claim(s) 41 and 52-54 are rejected under 35 U.S.C. 103 as being unpatentable over Thompson (ACS Chem (2017): 313-325) in view of Billon (Molecular cell 67.6 (2017): 1068-1079) and Liu (PG Pub No. US 2017/0121693 A1) as applied to claims 1, 2, 4, 8-9, 16, 21-22, 24, 35, and 86-90 above, and further in view of Niu (Science 357.6357 (2017): 1303-1307).
Regarding claims 41 and 52-54, Thompson in view of Billon and Liu renders obvious claims 1, 2, 4, 8-9, 16, 21-22, 24, 35, and 86-90 as described above.
Thompson in view of Billon and Liu does not teach or suggest that the step of contacting results in less than 30% cell death when the plurality of fusion proteins and the gRNAs are administered to a population of eukaryotic cells (Claim 41). Thompson in view of Billon and Liu does not teach or suggest does not teach or suggest the use of an anti-apoptotic molecule selected from pifithin-α (Claims 52-53). Thompson in view of Billon and Liu does not teach or suggest does not teach or suggest further contacting the eukaryotic cell with a growth factor (Claim 54).
However, one of ordinary skill in the art would have considered the teachings of Niu as both references are in a common field pertaining to the use of Cas9 proteins.
Niu is drawn to a study concerned with the inactivation of porcine endogenous retroviruses (i.e., PERVs) in a porcine primary cell line using CRISPR-Cas9 (Abstract). Niu teaches that simultaneous editing (i.e., multiplexed editing) of multiple PERV sites with Cas9 in a genome may trigger DNA damage-induced apoptosis (pg. 1). Niu teaches that administering pifithin-α alongside basic fibroblast growth factor rescued the porcine cells and increased the editing efficiency of the cell line (pg. 3). Niu teaches that cells that did not have pifithin-α and basic fibroblast growth factor administered alongside the Cas9 did not display any editing, or very low amount of editing, at different PERV sites while the administration of the pifithin-α and basic fibroblast growth factor allowed for greater than 80% editing efficiency across the PERV sites (i.e., cell apoptosis induced by the multiplexed editing was less than 20%) (pg. 1; see Figure S6).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method rendered obvious by Thompson in view of Billon and Liu such that the contacting further comprised the use of pifithin-α and basic fibroblast growth factor in order to ensure that less than 30% of the target eukaryotic cells died, as described by Niu. A person of ordinary skill in the art would have been motivated to do so in order to reduce cell death induced by Cas9 multiplexed editing and increase the Cas9’s targeting efficiency. A person of ordinary skill in the art would have had a reasonable expectation of success because both Niu and Thompson in view of Billon and Liu are drawn towards the use of Cas9 for multiplexed editing in a eukaryotic cell.
Claim(s) 1, 2, 4, 9, 16, 21-22, 24, 35, and 86-90 is/are rejected under 35 U.S.C. 103 as being unpatentable over Thompson (ACS Chem (2017): 313-325) in view of Billon (Molecular cell 67.6 (2017): 1068-1079) and “Liu 2” (PG Pub No. US 2020/0172931, filed 27 July 2018).
Regarding claims 1 and 16, Thompson is drawn to a review study concerned with multiplexed eukaryotic genome engineering (Abstract). Thompson teaches a method of multiplexed base editing via the use of CRISPR-Cas comprising contacting a target nucleic acid molecule with at least one fusion protein comprising a dCas9 domain, a base editor domain selected from a deaminase (pg. 318), and a gRNA bound to the dCas9 domain (pg. 317; see Figure 2). Thompson teaches that multiplex genome engineering entails the use of multiple, unique, gRNAs that bind to the fusion protein such that edits across distinct loci may occur (pg. 315). Thompson teaches that the utilization of 12 different gRNAs (i.e., unique guide RNAs that each individually require the use of at least one fusion protein) has been successfully achieved in order to edit a target genomic sequence (pg. 315). Thompson teaches that the fusion proteins can be utilized to edit eukaryotic genomes (Abstract) and be functionally expressed in mammalian cells (pg. 315).
Thompson does not teach or suggest that each of the fusion proteins comprise an amino acid sequence that is at least 90% identical to the claimed SEQ ID NO: 3 (Claims 1 and 16). Thompson does not teach or suggest that the unique gRNAs comprise at least 10 nucleotides (Claims 1 and 16). Thompson does not teach or suggest that at least 25 copies of the target sequence are present in the genomic DNA of a eukaryotic cell (Claims 1 and 16). Thompson does not teach or suggest that at least 50 copies of the target sequence are present in the genomic DNA of a eukaryotic cell (Claim 22).
However, one of ordinary skill in the art would have considered the teachings of Billon and Liu 2 as both references in a common field pertaining to the use of Cas9 nucleases.
Billon is drawn to a study concerned with CRISPR-mediated gene disruption strategies that rely on Cas9-APOBEC1 fusion proteins (i.e., Cas9-deaminase fusion proteins) (Abstract). Billon teaches the use of Cas9 gRNAs that are 20 nucleotides in length (pg. e2). Billon teaches the use of a gRNA library comprising over 3.4 million single gRNAs to facilitate gene inactivation by induction of STOP codons in 97%-99% of genes in eight eukaryotic species (i.e., Billon teaches species of gRNA molecules that comprise at least 50 copies of a target sequence in genomic DNA) (Abstract). Billon teaches that a major advantage to inducing STOP codons at target genomic sites, as opposed to inducing double-strand break formation, the target cells exhibit a reduced cell death compared to conventional CRISPR-Cas9-based methods (pg. 1077).
Liu 2 is drawn towards an invention concerned with evolved base editor fusion proteins (Abstract; [0013]). Liu 2 teaches the use of an APOBEC BE4Max fusion protein (i.e., a fusion protein comprising a dCas9 and a deaminase domain ([0013], [0020]) comprising 98% sequence identity to the claimed SEQ ID NO: 3 ([0374]; see SEQ ID NO: 15 in attached sequence alignment). Liu 2 teaches that the fusion protein can be directed to a target nucleic acid via the use of a guide RNA molecule ([0340]).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the fusion proteins of Thompson for the fusion protein of Liu 2 and the unique guide RNAs of Thompson for the unique guide RNAs of Billon. A person of ordinary skill in the art would have been motivated to do so in order to reduce cell death of target cells comprising the target nucleic acid by inducing STOP codons are genomic locations of interest as opposed to cleavage in order to reduce cell death compared to conventional CRISPR methods. A person of ordinary skill in the art would have had a reasonable expectation of success because both Thompson and Liu 2 teach the use of dCas9-deaminase fusion proteins that can be directed towards a target nucleic acid through the use of a guide RNA while Thompson and Billon both teach the use of guide RNAs that can direct Cas9 molecules to a target genomic location of interest.
Regarding claims 2 and 21, Thompson teaches that a large number of gRNAs are able to be packaged on a 150-kb HSV replicon (i.e., Thompson is interpreted as teaching the use of at least 20 unique guide RNAs) (pg. 320-321).
Regarding claim 4, Thompson teaches the use of a single dCas9-base editor fusion protein structure (i.e., Thompson teaches that the fusion proteins have the same structure) (pg. 317; see Figure 2).
Regarding claim 9, Thompson teaches the use of an adenosine deaminase (pg. 318).
Regarding claims 24 and 86, Thompson teaches that repetitive elements in genomes such as Alu, L1 retrotransposons, SVA, and human endogenous retroviruses (HERVs) can be good potential targets for the fusion proteins (pg. 317-318).
Regarding claim 35, Billon teaches that the sgRNAs can be utilized to generate STOP codons (Abstract).
Regarding claim 87, Billon teaches the use of Cas9 sgRNAs (Abstract).
Regarding claim 88, Liu 2 teaches that the target cell can be a human cel ([0596]).
Regarding claims 89-90, Liu 2 teaches that the fusion proteins can be utilized both in vivo and in vitro ([0056]). Liu teaches that utilizing the fusion proteins for targeted DNA editing in vitro allowed for the validation of gene editing activity ([0596]) while utilizing the fusion proteins in vivo allowed for the editing of a subject genome ([0594]).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to utilize the method of base editing rendered obvious by Thompson in view of Billon and Liu 2 in a cell that is in vivo or in vitro. A person of ordinary skill in the art would have been motivated to do so in order to validate editing activity or edit a subject’s genome. A person of ordinary skill in the art would have had a reasonable expectation of success because Liu 2 and Thompson in view of Billon and Liu teach the use of Cas9:deamianse fusion proteins that can be utilized to edit target cells of interest.
Claim(s) 41 and 52-54 are rejected under 35 U.S.C. 103 as being unpatentable over Thompson (ACS Chem (2017): 313-325) in view of Billon (Molecular cell 67.6 (2017): 1068-1079) and Liu 2 (PG Pub No. US 2020/0172931, filed 27 July 2018) as applied to claims 1, 2, 4, 9, 16, 21-22, 24, 35, and 86-90 above, and further in view of Niu (Science 357.6357 (2017): 1303-1307).
Regarding claims 41 and 52-54, Thompson in view of Billon and Liu 2 renders obvious claims 1, 2, 4, 9, 16, 21-22, 24, 35, and 86-90 as described above.
Thompson in view of Billon and Liu 2 does not teach or suggest that the step of contacting results in less than 30% cell death when the plurality of fusion proteins and the gRNAs are administered to a population of eukaryotic cells (Claim 41). Thompson in view of Billon and Liu 2 does not teach or suggest does not teach or suggest the use of an anti-apoptotic molecule selected from pifithin-α (Claims 52-53). Thompson in view of Billon and Liu 2 does not teach or suggest does not teach or suggest further contacting the eukaryotic cell with a growth factor (Claim 54).
However, one of ordinary skill in the art would have considered the teachings of Niu as both references are in a common field pertaining to the use of Cas9 proteins.
Niu is drawn to a study concerned with the inactivation of porcine endogenous retroviruses (i.e., PERVs) in a porcine primary cell line using CRISPR-Cas9 (Abstract). Niu teaches that simultaneous editing (i.e., multiplexed editing) of multiple PERV sites with Cas9 in a genome may trigger DNA damage-induced apoptosis (pg. 1). Niu teaches that administering pifithin-α alongside basic fibroblast growth factor rescued the porcine cells and increased the editing efficiency of the cell line (pg. 3). Niu teaches that cells that did not have pifithin-α and basic fibroblast growth factor administered alongside the Cas9 did not display any editing, or very low amount of editing, at different PERV sites while the administration of the pifithin-α and basic fibroblast growth factor allowed for greater than 80% editing efficiency across the PERV sites (i.e., cell apoptosis induced by the multiplexed editing was less than 20%) (pg. 1; see Figure S6).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method rendered obvious by Thompson in view of Billon and Liu 2 such that the contacting further comprised the use of pifithin-α and basic fibroblast growth factor in order to ensure that less than 30% of the target eukaryotic cells died, as described by Niu. A person of ordinary skill in the art would have been motivated to do so in order to reduce cell death induced by Cas9 multiplexed editing and increase the Cas9’s targeting efficiency. A person of ordinary skill in the art would have had a reasonable expectation of success because both Niu and Thompson in view of Billon and Liu 2 are drawn towards the use of Cas9 for multiplexed editing in a eukaryotic cell.
Claim(s) 1, 2, 4, 9, 16, 21-22, 24, 35, and 86-90 is/are rejected under 35 U.S.C. 103 as being unpatentable over Thompson (ACS Chem (2017): 313-325) in view of Billon (Molecular cell 67.6 (2017): 1068-1079) and “Liu 3” (PG Pub No. US 2018/0312828, published 1 November 2018, filed 23 March 2018).
Regarding claims 1 and 16, Thompson is drawn to a review study concerned with multiplexed eukaryotic genome engineering (Abstract). Thompson teaches a method of multiplexed base editing via the use of CRISPR-Cas comprising contacting a target nucleic acid molecule with at least one fusion protein comprising a dCas9 domain, a base editor domain selected from a deaminase (pg. 318), and a gRNA bound to the dCas9 domain (pg. 317; see Figure 2). Thompson teaches that multiplex genome engineering entails the use of multiple, unique, gRNAs that bind to the fusion protein such that edits across distinct loci may occur (pg. 315). Thompson teaches that the utilization of 12 different gRNAs (i.e., unique guide RNAs that each individually require the use of at least one fusion protein) has been successfully achieved in order to edit a target genomic sequence (pg. 315). Thompson teaches that the fusion proteins can be utilized to edit eukaryotic genomes (Abstract) and be functionally expressed in mammalian cells (pg. 315).
Thompson does not teach or suggest that each of the fusion proteins comprise an amino acid sequence that is at least 90% identical to the claimed SEQ ID NO: 3 (Claims 1 and 16). Thompson does not teach or suggest that the unique gRNAs comprise at least 10 nucleotides (Claims 1 and 16). Thompson does not teach or suggest that at least 25 copies of the target sequence are present in the genomic DNA of a eukaryotic cell (Claims 1 and 16). Thompson does not teach or suggest that at least 50 copies of the target sequence are present in the genomic DNA of a eukaryotic cell (Claim 22).
However, one of ordinary skill in the art would have considered the teachings of Billon and Liu 3 as both references in a common field pertaining to the use of Cas9 nucleases.
Billon is drawn to a study concerned with CRISPR-mediated gene disruption strategies that rely on Cas9-APOBEC1 fusion proteins (i.e., Cas9-deaminase fusion proteins) (Abstract). Billon teaches the use of Cas9 gRNAs that are 20 nucleotides in length (pg. e2). Billon teaches the use of a gRNA library comprising over 3.4 million single gRNAs to facilitate gene inactivation by induction of STOP codons in 97%-99% of genes in eight eukaryotic species (i.e., Billon teaches species of gRNA molecules that comprise at least 50 copies of a target sequence in genomic DNA) (Abstract). Billon teaches that a major advantage to inducing STOP codons at target genomic sites, as opposed to inducing double-strand break formation, the target cells exhibit a reduced cell death compared to conventional CRISPR-Cas9-based methods (pg. 1077).
Liu 3 is drawn towards an invention concerned with evolved base editor fusion proteins (Abstract; [0007]). Liu 3 teaches the use of an rABOPEC1-linker-dCas9-UGI-NLS fusion protein comprising 96.3% sequence identity to the claimed SEQ ID NO: 3 ([0849]; see SEQ ID NO: 167 in attached sequence alignment). Liu 3 teaches that the fusion protein can be directed to a target nucleic acid via the use of a guide RNA molecule ([0035]).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to substitute the fusion proteins of Thompson for the fusion protein of Liu 3 and the unique guide RNAs of Thompson for the unique guide RNAs of Billon. A person of ordinary skill in the art would have been motivated to do so in order to reduce cell death of target cells comprising the target nucleic acid by inducing STOP codons are genomic locations of interest as opposed to cleavage in order to reduce cell death compared to conventional CRISPR methods. A person of ordinary skill in the art would have had a reasonable expectation of success because both Thompson and Liu 3 teach the use of dCas9-deaminase fusion proteins that can be directed towards a target nucleic acid through the use of a guide RNA while Thompson and Billon both teach the use of guide RNAs that can direct Cas9 molecules to a target genomic location of interest.
Regarding claims 2 and 21, Thompson teaches that a large number of gRNAs are able to be packaged on a 150-kb HSV replicon (i.e., Thompson is interpreted as teaching the use of at least 20 unique guide RNAs) (pg. 320-321).
Regarding claim 4, Thompson teaches the use of a single dCas9-base editor fusion protein structure (i.e., Thompson teaches that the fusion proteins have the same structure) (pg. 317; see Figure 2).
Regarding claim 9, Thompson teaches the use of an adenosine deaminase (pg. 318).
Regarding claims 24 and 86, Thompson teaches that repetitive elements in genomes such as Alu, L1 retrotransposons, SVA, and human endogenous retroviruses (HERVs) can be good potential targets for the fusion proteins (pg. 317-318).
Regarding claim 35, Billon teaches that the sgRNAs can be utilized to generate STOP codons (Abstract).
Regarding claim 87, Billon teaches the use of Cas9 sgRNAs (Abstract).
Regarding claim 88, Liu 3 teaches that the target cell can be a human cel ([0402]).
Regarding claims 89-90, Liu 3 teaches that the fusion proteins can be utilized both in vivo and in vitro ([0056]). Liu 3 teaches that utilizing the fusion proteins for targeted DNA editing in vitro allowed for the generation of mutant cells ([0243]) while utilizing the fusion proteins in vivo allowed for the editing of a subject genome ([0400]).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to utilize the method of base editing rendered obvious by Thompson in view of Billon and Liu 3 in a cell that is in vivo or in vitro. A person of ordinary skill in the art would have been motivated to do so in order to generate mutant cells or edit a subject’s genome. A person of ordinary skill in the art would have had a reasonable expectation of success because Liu 3 and Thompson in view of Billon and Liu 3 teach the use of Cas9:deamianse fusion proteins that can be utilized to edit target cells of interest.
Claim(s) 41 and 52-54 are rejected under 35 U.S.C. 103 as being unpatentable over Thompson (ACS Chem (2017): 313-325) in view of Billon (Molecular cell 67.6 (2017): 1068-1079) and Liu 3 (PG Pub No. PG Pub No. US 2018/0312828, published 1 November 2018, filed 23 March 2018) as applied to claims 1, 2, 4, 9, 16, 21-22, 24, 35, and 86-90 above, and further in view of Niu (Science 357.6357 (2017): 1303-1307).
Regarding claims 41 and 52-54, Thompson in view of Billon and Liu 3 renders obvious claims 1, 2, 4, 9, 16, 21-22, 24, 35, and 86-90 as described above.
Thompson in view of Billon and Liu 3 does not teach or suggest that the step of contacting results in less than 30% cell death when the plurality of fusion proteins and the gRNAs are administered to a population of eukaryotic cells (Claim 41). Thompson in view of Billon and Liu 3 does not teach or suggest does not teach or suggest the use of an anti-apoptotic molecule selected from pifithin-α (Claims 52-53). Thompson in view of Billon and Liu 3 does not teach or suggest does not teach or suggest further contacting the eukaryotic cell with a growth factor (Claim 54).
However, one of ordinary skill in the art would have considered the teachings of Niu as both references are in a common field pertaining to the use of Cas9 proteins.
Niu is drawn to a study concerned with the inactivation of porcine endogenous retroviruses (i.e., PERVs) in a porcine primary cell line using CRISPR-Cas9 (Abstract). Niu teaches that simultaneous editing (i.e., multiplexed editing) of multiple PERV sites with Cas9 in a genome may trigger DNA damage-induced apoptosis (pg. 1). Niu teaches that administering pifithin-α alongside basic fibroblast growth factor rescued the porcine cells and increased the editing efficiency of the cell line (pg. 3). Niu teaches that cells that did not have pifithin-α and basic fibroblast growth factor administered alongside the Cas9 did not display any editing, or very low amount of editing, at different PERV sites while the administration of the pifithin-α and basic fibroblast growth factor allowed for greater than 80% editing efficiency across the PERV sites (i.e., cell apoptosis induced by the multiplexed editing was less than 20%) (pg. 1; see Figure S6).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the method rendered obvious by Thompson in view of Billon and Liu 3 such that the contacting further comprised the use of pifithin-α and basic fibroblast growth factor in order to ensure that less than 30% of the target eukaryotic cells died, as described by Niu. A person of ordinary skill in the art would have been motivated to do so in order to reduce cell death induced by Cas9 multiplexed editing and increase the Cas9’s targeting efficiency. A person of ordinary skill in the art would have had a reasonable expectation of success because both Niu and Thompson in view of Billon and Liu 3 are drawn towards the use of Cas9 for multiplexed editing in a eukaryotic cell.
Allowable Subject Matter and Closest Prior Art
Regarding claim 3, the claim requires the use of a fusion protein having 100% sequence identity to the claimed SEQ ID NO: 3.
The closest prior art is Liu 2 (PG Pub No. US 2020/0172931, filed 27 July 2018). Liu 2 is drawn towards an invention concerned with evolved base editor fusion proteins (Abstract; [0013]). Liu 2 teaches the use of an APOBEC BE4Max fusion protein (i.e., a fusion protein comprising a dCas9 and a deaminase domain ([0013], [0020]) comprising 98% sequence identity to the claimed SEQ ID NO: 3 ([0374]; see SEQ ID NO: 15 in attached sequence alignment). Liu 2 teaches that the fusion protein can be directed to a target nucleic acid via the use of a guide RNA molecule ([0340]).
However, neither Liu 2 nor the prior art teaches or suggests the use of a fusion protein comprising 100% identity to the claimed SEQ ID NO: 3 (see Claim 3).
Accordingly, claim 3 is objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Regarding the claimed SEQ ID NO: 6, the closest prior art is Liu (US Patent No. 11,306,324 B2, filed 13 October 2017). Liu is drawn towards an invention concerned with the use of split Cas9 proteins in methods of base editing (Abstract). Liu teaches the use of a nucleobase editor having 83.3% sequence identity to the claimed SEQ ID NO: 6 (Col 125-126; see SEQ ID NO: 406 in attached sequence alignment).
However, neither Liu nor the prior art teaches or suggests the use of a fusion protein comprising at least 90% identity to the claimed SEQ ID NO: 6 (see Claim 1).
Response to Arguments
Applicant’s amendments, filed 5 August 2025, have been full considered and are deemed persuasive. Accordingly, the previously pending rejections under 35 USC 103 have been withdrawn.
However, Applicant’s amendments have necessitated the newly filed rejections above.
Insofar as Applicant’s arguments pertain to the newly filed rejections of record, Applicant’s arguments are not found persuasive.
Applicant alleges that the incorporation of the claimed SEQ ID NO: 3 into the newly amended Claims 1 and 16 alongside the subject matter of claim 8 (i.e., SEQ ID NOs: 6-8 or 10-15) renders the claimed invention allowable.
This argument is not found persuasive because, as discussed above, Thompson in view of Billon and Liu renders obvious the use of a fusion protein comprising 90% identity to the claimed SEQ ID NO: 8 (see above 35 USC 103 rejection of record). Claim 1 is not limited to solely sequences having 100% identity to the claimed SEQ ID NOs: 3 or at least 90% identity to the claimed SEQ ID NO: 6. Accordingly, the claim is rejected under 35 USC 103 as discussed above.
Conclusion
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to KYLE T REGA whose telephone number is (571)272-2073. The examiner can normally be reached M-R 8:30-4:30, every other F 8:30-4:30 (EDT/EST).
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, Neil Hammell can be reached at 571-270-5919. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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/KYLE T REGA/Examiner, Art Unit 1636
/NEIL P HAMMELL/Supervisory Patent Examiner, Art Unit 1636