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Application Status and Withdrawn Rejections
Applicant’s amendments filed March 31, 2026, amending claims 26-27, 30, 32, 38-39 and 44-45, canceling claims 1-2, 4-5, 8-9 and 19-22, and adding new claims 109-117. Note that there are two claims numbered “111”. The second misnumbered claim 111 reciting “wherein the napDNAbp domain comprises an amino acid sequence that has at least 85% sequence identity to SEQ ID NO: 226 or 235” is objected to and has been renumbered claim 118. Claims 26-28, 30, 32, 38-40, 44-45 and 109-118 are pending and under examination.
Applicant’s amendments removing the options for A3G and “variants thereof” of other rABOBEC1 variates overcome the §102 rejection over Koblan, the §102 and §103 rejections over Gao in view of others, and the nonstatutory double patenting (NSDP) rejections of record.
Any other 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 – Koblan in view of Komor and Kim
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.
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.
Claims 26-28, 32, 38-40, 44-45, 109, 112 are rejected under 35 U.S.C. 103 as being unpatentable over Koblan (Koblan et al., Nature Biotechnology (2018), 36: 843-846 and Supplemental Materials; of record) in view of Komor (Komor et al., Science Advances (2017), 3: eaao4774) and Kim (Kim et al., Nature Biotechnology (2017), 35: 371-376 and Supplemental Material). This is a new rejection necessitated by amendment.
Regarding claims 26-28 and 112, Koblan teaches the "BE4max" fusion protein comprising (a) a rat APOBEC1 cytidine deaminase, (b) Cas9n D10A nickase, (c) two NLSs, and (d) two UGI domains (Fig 1a; Supp Sequence 2). Koblan teaches BE4-max is based on optimization of the 4th generation cytidine base editor BE4 taught in Komor (page 843, ¶1).
Koblan does not teach a base editor comprising a YE1, YE2, YEE or EE deaminase domain.
Komor teaches BE4 is based on the 3rd generation BE3 cytidine base editor with the addition of a second UGI domain (Fig 3).
Kim teaches BE-based base editors comprising a deaminase domain comprising the rAPOBEC1 with W90Y+R126E substitutions (i.e., YE1), with W90Y+R132E substitutions (i.e., YE2), with R126E+R132E substitutions (i.e., EE), and with W90Y+R126E+ R132E substitutions (i.e., YEE) (Fig 3; page 375, ¶1-5). Kim teaches the base editors with the YE1, YE2, EE and YEE deaminase domains altered the editing window of the base editors (Figs 2-3). Kim teaches that altering the base editing window is important for targeting only the desired C[Wingdings font/0xE0]T change (page 372, ¶5). Kim teaches using the YE1, YE2, EE or YEE deaminases increases preferential targeting from 31% to 59% over BE3 with rAPOBEC1 (Fig 3).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have substituted the rAPOBEC1 deaminase domain in Koblan’s BE4max base editor with the rAPOBEC1 variants YE1, YE2, YEE or EE taught in Kim. It would have amounted to the simple substitution of one rAPOBEC1 version for another by known means to yield predictable results. The skilled artisan would have a reasonable expectation that the YE1, YE2, YEE or EE domains could be used in Koblan’s BE4max because they are based on the same overall BE3 structure: rAPOBEC1 fused to dCas9 fused to at least one NLS. One would have been motivated to make the substitution to narrow the editing window of BE4max for the purpose of increasing preferential targeting as taught by Kim.
Regarding claims 32 and 109, Koblan teaches the amino acid sequence of BE4max (Supp Sequence 2). Koblan teaches the Cas9(D10A) nickase comprise the amino acid sequence of SEQ ID NO 215 (Supp Sequence 2).
Regarding claim 38, Koblan teaches the amino acid sequence of BE4max (Supp Sequence 2). Koblan teaches the NLS sequence in BE4max comprises SEQ ID NO 285 at the N-terminus and SEQ ID NO 286 at the C-terminus.
Regarding claim 39, Koblan teaches the amino acid sequence of BE4max (Supp Sequence 2). Koblan teaches order of the domains in BE4Max is NH2-NLS-APOBEC1-linker-Cas9n-linker-UGl-linker- UGl-linker-NLS (Supp Sequence 2).
Regarding claim 40, Koblan teaches the amino acid sequence of BE4max (Supp Sequence 2). Koblan teaches the linker between second UGI and the second NLS is SGGS (Supp Sequence 2).
Regarding claims 44-45, Koblan teaches the amino acid sequence of BE4max and the sequence of rAPOBEC1 that is present in BE4max (Supp Sequence 2), which is 100% identical to SEQ ID NO 257. SEQ ID NOs 257 and SEQ ID NO 258 differ by a single amino acid R[Wingdings font/0xE0]E at position 145. Based on the amino acid sequence of rAPOBEC1 provided by Koblan (Supp Sequences 2), the R[Wingdings font/0xE0]E at position 145 is equivalent to the R126E YE1 substitution taught in Kim. Thus, the base editor comprising the YE1 deaminase domain rendered obvious above for claim 26 comprised SEQ ID NO 258.
Claim Rejections - 35 USC § 103 – Koblan in view of Komor, Kim, Eid and Jinek (dSpCas9)
Claim 30 is rejected under 35 U.S.C. 103 as being unpatentable over Koblan (Koblan et al., Nature Biotechnology (2018), 36: 843-846 and Supplemental Materials; of record), Komor (Komor et al., Science Advances (2017), 3: eaao4774) and Kim (Kim et al., Nature Biotechnology (2017), 35: 371-376 and Supplemental Material) as applied to claims 26-28, 32, 38-40, 44-45, 109 and 112 above, and further in view of Eid (Eid et al., Biochemical Journal (2018), 475: 1955-1964; of record) and Jinek (Jinek et al., Science (2012), 337: 816-821; of record). This is a new rejection necessitated by amendment.
Regarding claim 30, the Specification teaches that SEQ ID NO 213 is wildtype, catalytically active SpCas9 (page 83). SEQ ID NO 214 differs from SEQ ID NO 213 by including a D10A and a H840A substitution. The Specification teaches that the D10A and H840A substitutions inactivates cleavage of the non-protospacer strand and protospacer strand, respectively (page 85). Therefore, the napDNAbp that comprises SEQ ID NO 214 is catalytically inactive SpCas9, also known as dSpCas9 or dCas9.
The teachings of Koblan, Komor and Kim are recited above and applied for 26-28, 32, 38-40, 44-45, 109 and 112. As noted above, each of Koblan, Komor and Kim teach base editors with SpCas9 D10A nickase.
Koblan, Komor and Kim do not teach a base editor comprising catalytically inactive dSpCas9,
Eid reviews the state of the art of CRISPR base editors (Abstract). Eid teaches that catalytically inactive Cas9 variants have been fused to deaminases to tether the deaminases for gene editing purposes (page 1956, ¶1). Eid teaches dCas9 was fused to APOBEC deaminases, which successfully converted C[Wingdings font/0xE0] in a catalytic window (page 1956, ¶5).
Jinek teaches ma king D10A and H840A substitutions to render SpCas9 catalytically inactive (Fig 2A).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have substituted dCas9 for the Cas9 nickase in the BE4-YE1 base editor rendered obvious above. It would have amounted to the simple substitution of one known Cas9-based DNA-binding domain for another by known means to yield predictable results. The skilled artisan would have predicted that the dSpCas9 with the D10A and H840A substitutions taught in Jinek could be used in the BE4-YE1 base editor because Eid teaches that dCas9 has been fused to APOBEC-based base editors previously for successful base editing in cells. One would have been motivated to do so to reduce nicking damage to off-targets sites of genomic DNA.
Claim Rejections - 35 USC § 103 – Koblan in view of Komor, Kim, Liu and Nishimasu (SpCas9-NG)
Claims 110, 113-114 and 118 are rejected under 35 U.S.C. 103 as being unpatentable over Koblan (Koblan et al., Nature Biotechnology (2018), 36: 843-846 and Supplemental Materials; of record), Komor (Komor et al., Science Advances (2017), 3: eaao4774) and Kim (Kim et al., Nature Biotechnology (2017), 35: 371-376 and Supplemental Material) as applied to claims 26-28, 32, 38-40, 44-45, 109 and 112 above, and further in view of Liu (Liu et al., "Highly precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions”. bioRxiv (2019): 658351). Claim 111 is evidenced by Nishimasu (Nishimasu et al., Science (2018), 361: 1259-1262). This is a new rejection necessitated by amendment.
The teachings of Koblan, Komor and Kim are recited above and applied for 26-28, 32, 38-40, 44-45, 109 and 112. As noted above, each of Koblan, Komor and Kim teach base editors with SpCas9 D10A nickase.
Koblan, Komor and Kim do not teach a base editor comprising an SpCas9-NG.
Regarding claims 110, 113 and 114, Liu teaches base editors comprising APOBEC3G and BE4-based editors using an SpCas9-NG variant (Abstract; page 4, ¶3). Liu teaches the SpCas9-NG variant has a relaxed PAM requirement compared to SpCas9, which affords expanded genome-targeting (Fig 2a; page 3, ¶4).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have substituted SpCas9-NG variant for the SpCas9 protein in the BE4-YE1 base editor rendered obvious above. It would have amounted to the simple substitution of one known Cas9-based DNA-binding domain for another by known means to yield predictable results. The skilled artisan would have predicted that the SpCas9-NG could be used in the BE4-YE1 base editor because Liu teaches that the SpCas9-NG variant in the highly similar APOBEC1-based BE4 editor. One would have been motivated to do so to expand the genome targeting availability of the obvious BE4-YE1 base editor.
Regarding claim 118, SEQ ID NO 235 differs from SEQ ID NO 214 (i.e., SpCas9(D10A)) by seven amino acid substitutions, L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R. Liu teaches that the SpCas9-NG variant was first developed by Nishimasu (page 4, ¶3). Nishimasu teaches that SpCas9-NG variant differs from the wild type SpCas9 by the following substitutions: L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R (Fig 2; ¶ spanning pages 1259 and 1261). Therefore, the BE4-YE1-SpCas9-NG rendered obvious for claims 110, 113 and 114 comprises SEQ ID NO 235.
Claim Rejections - 35 USC § 103 – Koblan in view of Komor, Kim and Huang (CP1028-Cas9)
Claims 111, 115-116 and 118 are rejected under 35 U.S.C. 103 as being unpatentable over Koblan (Koblan et al., Nature Biotechnology (2018), 36: 843-846 and Supplemental Materials; of record), Komor (Komor et al., Science Advances (2017), 3: eaao4774) and Kim (Kim et al., Nature Biotechnology (2017), 35: 371-376 and Supplemental Material) as applied to claims 26-28, 32, 38-40, 44-45, 109 and 112 above, and further in view of Huang (Huang et al., Nature Biotechnology (2019), 37: 626-631; published May 20, 2019). This is a new rejection necessitated by amendment.
The teachings of Koblan, Komor and Kim are recited above and applied for 26-28, 32, 38-40, 44-45, 109 and 112. As noted above, each of Koblan, Komor and Kim teach base editors with SpCas9 D10A nickase.
Koblan, Komor and Kim do not teach a base editor comprising CP1028 Cas9.
Regarding claims 111, 115 and 116, Huang teaches CP1028 Cas9 is a circular permutated version of SpCas9 split after amino acid 1028 and the N- and C-terminus fused together (Fig 2b). Huang teaches a BE4max cytidine base editor varied to comprise CP1028 Cas9 instead of SpCas9 (page 626, ¶6; Fig 2). Huang teaches CP1028-BE4max were capable of base editing at all five tested sites without substantial indel formation and had a broadened window for base editing (¶ spanning pages 629-630). Huang also teaches that CP1028-BE4max exhibited greatly reduced, undesired C-G and C-A edits, giving rise to greater product purity (page 360, ¶3). Huang teaches that the features of CP1028-BE4max enable efforts to perform base editing at currently inaccessible target nucleotides and substantially improve product purity (page 630, ¶7).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have substituted CP1028-Cas9 variant for the SpCas9 protein in the BE4-YE1 base editor rendered obvious above. It would have amounted to the simple substitution of one known Cas9-based DNA-binding domain for another by known means to yield predictable results. The skilled artisan would have predicted that the CP1028-Cas9 could be used in the BE4-YE1 base editor because Huang teaches that the CP1028-Cas9 variant in the highly similar APOBEC1-based BE4max editor. One would have been motivated to do so to perform base editing at currently inaccessible target nucleotides for the obvious BE4-YE1 base editor.
Regarding claim 118, SEQ ID NO 226 differs from SEQ ID NO 214 (i.e., SpCas9(D10A)) by the fusion of the N- and C-termini and split after amino acid position 1028. Therefore, the BE4-YE1-CP1028Cas9 rendered obvious for claims 111, 115 and 116 comprises SEQ ID NO 226.
Response to Arguments – §103
Applicant argues that the claim amendments overcome the § 103 rejections of record because Gao does not teach the deaminases recited in claim 26 (page 11). This argument has been fully considered and is persuasive – the previous §103 rejections are withdrawn. However, most of the claims are still obvious over prior art teachings for the reasons recited in the §103 rejections above.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 26-28, 32, 38-40, 44-45, 109-116 and 118 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-21 of U.S. Patent No. 11319532 in view of Koblan (Koblan et al., Nature Biotechnology (2018), 36: 843-846 and Supplemental Materials) and Kim (Kim et al., Nature Biotechnology (2017), 35: 371-376 and Supplemental Material). Claims 110, 113-114 and 118 are rejected further in view of Liu (Liu et al., "Highly precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions”. bioRxiv (2019): 658351) and Nishimasu (Nishimasu et al., Science (2018), 361: 1259-1262). Claims 111, 115-116 and 118 are rejected further in view of Huang (Huang et al., Nature Biotechnology (2019), 37: 626-631; published May 20, 2019). This is a new rejection necessitated by amendment.
Patented claim 1 recites “A fusion protein comprising: (i) a nucleic acid programmable DNA binding protein (napDNAbp) domain; (ii) a cytidine deaminase domain. Patented claims 2 and 6 recite further comprising (iv) a first and a second uracil glycosylase inhibitor (UGI) domain. Patented claims 2-3 recite wherein the napDNAbp domain is a Cas9 domain, including a nuclease active Cas9 domain, a Cas9 nickase (nCas9) domain, or a nuclease inactive Cas9 (dCas9) domain. Patented claim 8 recites wherein the deaminase domain is from the APOBEC family. Patented claim 21, recites wherein the cytidine deaminase domain comprises an amino acid sequence that is at least 90% identical to the amino acid sequence of SEQ ID NO: 349, which according to the patented Specification is derived from rAPOBEC1. Patented claim 11 recites wherein the fusion protein comprises the structure: NH2-[Gam protein domain]-[cytidine deaminase domain]-[napDNAbp domain]-[first UGI domain]-[second UGI domain]-COOH, and wherein each instance of “]-[” comprises an optional linker.
The patented claims do not recite the fusion protein comprises one or two NLSs at the N and C terminus. The patented claims do not recite a YE1 deaminase domain. The patented claims do not recite SpCas9-NG or CP1028-Cas9.
Koblan teaches the "BE4max" fusion protein comprising (a) a rat APOBEC1 cytidine deaminase, (b) Cas9n D10A nickase, (c) two NLSs, and (d) two UGI domains (Fig 1a; Supp Sequence 2). Koblan teaches BE4-max is based on optimization of the 4th generation cytidine base editor BE4 taught in Komor (page 843, ¶1).
Kim teaches BE-based base editors comprising a deaminase domain comprising the rAPOBEC1 with W90Y+R126E substitutions (i.e., YE1), with W90Y+R132E substitutions (i.e., YE2), with R126E+R132E substitutions (i.e., EE), and with W90Y+R126E+ R132E substitutions (i.e., YEE) (Fig 3; page 375, ¶1-5). Kim teaches the base editors with the YE1, YE2, EE and YEE deaminase domains altered the editing window of the base editors (Figs 2-3). Kim teaches that altering the base editing window is important for targeting only the desired C[Wingdings font/0xE0]T change (page 372, ¶5). Kim teaches using the YE1, YE2, EE or YEE deaminases increases preferential targeting from 31% to 59% over BE3 with rAPOBEC1 (Fig 3).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have included the two NLSs taught in Koblan at the N- and C-termini of the patented fusion protein and included the rAPOBEC1 variants YE1, YE2, YEE or EE taught in Kim. It would have amounted to the simple combination of known protein domains used in base editors by known means to yield predictable results. The skilled artisan would have predicted that the NLSs could be added to the patented fusion protein because both fusion proteins are based on the BE4 architecture using variants of the rAPOBEC1 deaminase domain. Additionally, there would have been reasonable expectation that the YE1, YE2, YEE or EE domains could be used in Koblan’s BE4max because they are based on the same overall base editor structure: rAPOBEC1 fused to dCas9 fused to at least one NLS. One would have been motivated to do so because Koblan teaches the specific NLS sequences provide superior editing efficiencies in mammalian cells and to narrow the editing window of BE4max for the purpose of increasing preferential targeting as taught by Kim.
Regarding claims 110, 113-114 and 118, Liu teaches base editors comprising APOBEC3G and BE4-based editors using an SpCas9-NG variant (Abstract; page 4, ¶3). Liu teaches the SpCas9-NG variant has a relaxed PAM requirement compared to SpCas9, which affords expanded genome-targeting (Fig 2a; page 3, ¶4). Liu teaches that the SpCas9-NG variant was first developed by Nishimasu (page 4, ¶3). Nishimasu teaches that SpCas9-NG variant differs from the wild type SpCas9 by the following substitutions: L1111R, D1135V, G1218R, E1219F, A1322R, R1335V, T1337R (Fig 2; ¶ spanning pages 1259 and 1261). Therefore, the BE4-YE1-SpCas9-NG rendered obvious for claims 110, 113 and 114 comprises SEQ ID NO 235.
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have additionally substituted SpCas9-NG variant for the generic SpCas9 proteins in the variant of the patented base editor rendered obvious above. It would have amounted to the simple substitution of one known Cas9-based DNA-binding domain for another by known means to yield predictable results. The skilled artisan would have predicted that the SpCas9-NG could be used in the patented base editor because Liu teaches that the SpCas9-NG variant in the highly similar APOBEC1-based BE4 editor. One would have been motivated to do so to expand the genome targeting availability of the obvious variant of the patented base editor.
Regarding claims 111, 115, 116 and 118, Huang teaches CP1028 Cas9 is a circular permutated version of SpCas9 split after amino acid 1028 and the N- and C-terminus fused together (Fig 2b). Huang teaches a BE4max cytidine base editor varied to comprise CP1028 Cas9 instead of SpCas9 (page 626, ¶6; Fig 2). Huang teaches CP1028-BE4max were capable of base editing at all five tested sites without substantial indel formation and had a broadened window for base editing (¶ spanning pages 629-630). Huang also teaches that CP1028-BE4max exhibited greatly reduced, undesired C-G and C-A edits, giving rise to greater product purity (page 360, ¶3). Huang teaches that the features of CP1028-BE4max enable efforts to perform base editing at currently inaccessible target nucleotides and substantially improve product purity (page 630, ¶7).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have substituted CP1028-Cas9 variant for the SpCas9 protein in the obvious variant of the patented BE4 base editor rendered obvious above. It would have amounted to the simple substitution of one known Cas9-based DNA-binding domain for another by known means to yield predictable results. The skilled artisan would have predicted that the CP1028-Cas9 could be used in the obvious variant because Huang teaches that the CP1028-Cas9 variant in the highly similar APOBEC1-based BE4max editor. One would have been motivated to do so to perform base editing at currently inaccessible target nucleotides for the obvious variant of the patented base editor.
Claims 26-28, 32, 38-40, 44-45, 109-116 and 118 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-21 of U.S. Patent No. 11913044 in view of Koblan (Koblan et al., Nature Biotechnology (2018), 36: 843-846 and Supplemental Materials) and Kim (Kim et al., Nature Biotechnology (2017), 35: 371-376 and Supplemental Material). Claims 110, 113-114 and 118 are rejected further in view of Liu (Liu et al., "Highly precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions”. bioRxiv (2019): 658351) and Nishimasu (Nishimasu et al., Science (2018), 361: 1259-1262). Claims 111, 115-116 and 118 are rejected further in view of Huang (Huang et al., Nature Biotechnology (2019), 37: 626-631; published May 20, 2019). This is a new rejection necessitated by amendment.
Patented claim 1 recites A cytidine deaminase protein variant comprising an amino acid sequence that is at least 95% identical to the amino acid sequence of SEQ ID NO: 15 [rAPOBEC1], wherein the amino acid sequence of the cytidine deaminase protein variant includes at least three mutations at a position selected from the group consisting of R33, G45, N57, N65, Y75, T101, F113, A123, S149, A165, H166, T204, F205, and W224 of SEQ ID NO: 15 [rAPOBEC1]. Patented claim 13 recites A fusion protein comprising: (i) a RNA-programmable nuclease (i.e., an napDNAbp); (ii) the cytidine deaminase protein variant of claim 1; and (iii) a uracil glycosylase inhibitor (UGI) domain. Patented claims 14-15 recite wherein the RNA-programmable nuclease is a Cas9 domain comprising an amino acid sequence that is at least 85% identical to the amino acid sequence provided in SEQ ID NO: 20 or 21 and is a Cas9 nickase domain (i.e., is SEQ ID NO 215).
The patented claims do not recite the fusion protein comprises one or two NLSs at the N and C terminus. The patented claims do not recite a YE1 deaminase domain. The patented claims do not recite SpCas9-NG or CP1028-Cas9.
The teachings of Koblan and Kim are recited above in paragraphs 41-43 and incorporated here. The obviousness of having included the two NLSs taught in Koblan at the N- and C-termini of the patented fusion protein and included the rAPOBEC1 variants YE1, YE2, YEE or EE taught in Kim is recited above in paragraph 44.
Regarding claims 110, 113-114 and 118, the teachings of Liu and Nishimasu are recited above in paragraph 45 and incorporated here. The obviousness of having additionally substituted the SpCas9-NG variant for the generic SpCas9 proteins in the obvious variant of the patented base editor is recited above in paragraph 46.
Regarding claims 111, 115, 116 and 118, the teachings of Huang are recited above in paragraph 47. The obviousness of having substituted CP1028-Cas9 variant for the SpCas9 protein in the obvious variant of the patented base editor is recited above in paragraph 48.
Claims 26-28, 32, 38-40, 44-45, 109-116 and 118 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-34 of U.S. Patent No. 12344869 in view of Koblan (Koblan et al., Nature Biotechnology (2018), 36: 843-846 and Supplemental Materials). Claims 110, 113-114 and 118 are rejected further in view of Liu (Liu et al., "Highly precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions”. bioRxiv (2019): 658351) and Nishimasu (Nishimasu et al., Science (2018), 361: 1259-1262). Claims 111, 115-116 and 118 are rejected further in view of Huang (Huang et al., Nature Biotechnology (2019), 37: 626-631; published May 20, 2019). This is a new rejection necessitated by amendment.
Patented claim 1 recites A complex comprising: (i) a Cas9 protein; (ii) a cytidine deaminase; (iii) a uracil glycosylase inhibitor (UGI) protein, wherein the complex deaminates a cytidine in a target nucleic acid sequence. Patented claim 2 recites wherein the Cas9 protein is a Cas9 nickase. Patented claim 3 recites wherein the nCas9 comprises an amino acid sequence having at least 85% sequence identity to the amino acid sequence provided in SEQ ID NO: 10, and wherein the amino acid sequence provided in SEQ ID NO: 10 further comprises a D10A mutation (i.e., the Cas9n comprises SEQ ID NO 215 of the examined application). Patented claim 16 recites wherein the deaminase is a rat APOBEC1 (rAPOBEC1) deaminase comprising one or more mutations selected from the group consisting of W90Y, R126E, and R132E of SEQ ID NO: 284 (i.e., a YE1, YE2, EE, or YEE deaminase).
The patented claims do not recite a second UGI or one or more NLSs. The patented claims do not recite the elements are in a fusion protein. The patented claims do not recite SpCas9-NG or CP1028-Cas9.
The teachings of Koblan are recited above in paragraphs 41-43 and incorporated here. The obviousness of having included the two NLSs taught in Koblan at the N- and C-termini of a patented base editor comprising a napDNAbp and a APOBEC1-based base editor is recited above in paragraph 44. Additionally, one would have been motivated to add a second UGI because Koblan teaches that two UGIs in the base editor provide nearly 80% editing efficiency.
Regarding claims 110, 113-114 and 118, the teachings of Liu and Nishimasu are recited above in paragraph 45 and incorporated here. The obviousness of having additionally substituted the SpCas9-NG variant for the generic SpCas9 proteins in the obvious variant of the patented base editor is recited above in paragraph 46.
Regarding claims 111, 115, 116 and 118, the teachings of Huang are recited above in paragraph 47. The obviousness of having substituted CP1028-Cas9 variant for the SpCas9 protein in the obvious variant of the patented base editor is recited above in paragraph 48.
Claims 26-28, 32, 38-40, 44-45, 109-116 and 118 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-34 of U.S. Patent No. 12281338 in view of Koblan (Koblan et al., Nature Biotechnology (2018), 36: 843-846 and Supplemental Materials) and Kim (Kim et al., Nature Biotechnology (2017), 35: 371-376 and Supplemental Material). Claims 110, 113-114 and 118 are rejected further in view of Liu (Liu et al., "Highly precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions”. bioRxiv (2019): 658351) and Nishimasu (Nishimasu et al., Science (2018), 361: 1259-1262). Claims 111, 115-116 and 118 are rejected further in view of Huang (Huang et al., Nature Biotechnology (2019), 37: 626-631; published May 20, 2019). This is a new rejection necessitated by amendment.
Patented claim 1 recites a fusion protein comprising an amino acid sequence that is at least 85% identical to SEQ ID NO: 81. The patented Specification teaches that SEQ ID NO 81 is also called “GeoBE4” whose structure is illustrated in Fig 2. Patented figure 2 indicated that SEQ ID NO 81 comprises (a) an APOBEC1 deaminase domain-a linker- XTEN-GeoCas9n (a Cas9 napDNAbp)-UGI-UGI. Therefore, patented claim 1 encompasses a base editor comprising elements (a), (b) and (d). The patented SEQ ID NO 81 comprises a SGGSGGSGGS.
The patented claims do not recite the fusion protein comprises one or two NLSs at the N and C terminus. The patented claims do not recite a YE1 deaminase domain. The patented claims do not recite SpCas9-NG or CP1028-Cas9.
The teachings of Koblan and Kim are recited above in paragraphs 41-43 and incorporated here. The obviousness of having included the two NLSs taught in Koblan at the N- and C-termini of the patented fusion protein and included the rAPOBEC1 variants YE1, YE2, YEE or EE taught in Kim is recited above in paragraph 44.
Regarding claims 110, 113-114 and 118, the teachings of Liu and Nishimasu are recited above in paragraph 45 and incorporated here. The obviousness of having additionally substituted the SpCas9-NG variant for a GeoCas9 protein in the obvious variant of the patented base editor is recited above in paragraph 46.
Regarding claims 111, 115, 116 and 118, the teachings of Huang are recited above in paragraph 47. The obviousness of having substituted CP1028-Cas9 variant for a GeoCas9 protein in the obvious variant of the patented base editor is recited above in paragraph 48.
Claims 26-28, 32, 38-40, 44-45, 109-116 and 118 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-2, 9, 21-22, 24, 35, 41, and 52-54 of copending Application No. 17593020 in view of Koblan (Koblan et al., Nature Biotechnology (2018), 36: 843-846 and Supplemental Materials) and Kim (Kim et al., Nature Biotechnology (2017), 35: 371-376 and Supplemental Material). Claims 110, 113-114 and 118 are rejected further in view of Liu (Liu et al., "Highly precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions”. bioRxiv (2019): 658351) and Nishimasu (Nishimasu et al., Science (2018), 361: 1259-1262). Claims 111, 115-116 and 118 are rejected further in view of Huang (Huang et al., Nature Biotechnology (2019), 37: 626-631; published May 20, 2019). This is a new rejection necessitated by amendment.
Copending claim 1 recites fusion proteins compris[ing] (i) a nuclease inactive Cas9 (dCas9) domain and (ii) a deaminase domain, and a guide RNA (gRNA) bound to the dCas9 domain, wherein each of the fusion proteins of the plurality comprises an amino acid sequence that is at least 90% identical to any one of SEQ ID NOs: 3,6-8, or 10-15. The copending Specification teaches that SEQ ID NO 11 comprises cytidine base editor 4 ([00131]). Copending SEQ ID NO 11 has 100% identity to SEQ ID NO 257 except for amino acids 1-20 and the last ~ 20 amino acids. Copending SEQ ID NO 11 comprises PKKKRK at its C-terminus (i.e., a NLS sequence). Thus, the copending fusion protein comprises (a) an rAPOBEC1 deaminase, (b) a dCas9 napDNAbp, (c) at least one NLS and (d) two UGI domains.
The copending claims do not recite the fusion protein comprises one or two NLSs at the N and C terminus. The copending claims do not recite a YE1 deaminase domain. The copending claims do not recite SpCas9-NG or CP1028-Cas9.
The teachings of Koblan and Kim are recited above in paragraphs 41-43 and incorporated here. The obviousness of having included the two NLSs taught in Koblan at the N- and C-termini of the patented fusion protein and included the rAPOBEC1 variants YE1, YE2, YEE or EE taught in Kim is recited above in paragraph 44.
Regarding claims 110, 113-114 and 118, the teachings of Liu and Nishimasu are recited above in paragraph 45 and incorporated here. The obviousness of having additionally substituted the SpCas9-NG variant for the SpCas9 protein in the obvious variant of the copending base editor is recited above in paragraph 46.
Regarding claims 111, 115, 116 and 118, the teachings of Huang are recited above in paragraph 47. The obviousness of having substituted CP1028-Cas9 variant for the SpCas9 protein in the obvious variant of the copending base editor is recited above in paragraph 48.
Claims 26-28, 32, 38-40, 44-45, 109-116 and 118 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 303-325 of copending Application No. 19/202976 in view of Koblan (Koblan et al., Nature Biotechnology (2018), 36: 843-846 and Supplemental Materials). Claims 110, 113-114 and 118 are rejected further in view of Liu (Liu et al., "Highly precise base editing with CC context-specificity using engineered human APOBEC3G-nCas9 fusions”. bioRxiv (2019): 658351) and Nishimasu (Nishimasu et al., Science (2018), 361: 1259-1262). Claims 111, 115-116 and 118 are rejected further in view of Huang (Huang et al., Nature Biotechnology (2019), 37: 626-631; published May 20, 2019). This is a new rejection necessitated by amendment.
Copending claim 303 A nucleotide sequence encoding recites a fusion protein comprising (b) a Cas9 domain, (a) a cytidine deaminase domain, and (d) a uracil glycosylase inhibitor (UGI) domain. Copending claim 305 recites wherein the Cas9 domain is a Cas9 nickase (nCas9) domain that cuts a nucleotide target strand of a nucleotide duplex, wherein the nucleotide target strand is the strand that binds to a gRNA. Copending claim 306 recites wherein the Cas9 domain is a Cas9 nickase (nCas9) domain that comprises a D10A mutation in the amino acid sequence provided in SEQ ID NO: 10 (i.e., comprises SEQ ID NO 215). Copending claim 309 recites wherein the APOBEC family deaminase is selected from the group consisting of APOBEC1 deaminase. Copending claim 311 recites wherein the cytidine deaminase domain is a rat APOBECI (rAPOBEC1) deaminase comprising one or more mutations selected from the group consisting of W90Y, R126E, and R132E of SEQ ID NO: 284, or one or more corresponding mutations in another APOBEC deaminase (i.e. YE1). Copending claim 322 recites wherein the fusion protein comprises the structure: NH2-[cytidine deaminase domain]-[Cas9 domain]-[UGI domain]-[NLS]-COOH, and wherein each instance of "-" comprises an optional linker. Copending claim 323 recites wherein the UGI domain and the NLS are linked via a linker comprising the amino acid sequence: SGGS.
The copending claims do not recite a second UGI or a second NLS with the recited sequence at the N-terminus. The copending claims do not recite SpCas9-NG or CP1028-Cas9.
The teachings of Koblan are recited above in paragraphs 41-43 and incorporated here. The obviousness of having included the two NLSs taught in Koblan at the N- and C-termini of the patented fusion protein that included the rAPOBEC1 variants YE1, YE2, YEE or EE is recited above in paragraph 44.
Regarding claims 110, 113-114 and 118, the teachings of Liu and Nishimasu are recited above in paragraph 45 and incorporated here. The obviousness of having additionally substituted the SpCas9-NG variant for the SpCas9 protein in the obvious variant of the copending base editor is recited above in paragraph 46.
Regarding claims 111, 115, 116 and 118, the teachings of Huang are recited above in paragraph 47. The obviousness of having substituted CP1028-Cas9 variant for the SpCas9 protein in the obvious variant of the copending base editor is recited above in paragraph 48.
Response to Arguments – NSDP
Applicant argues that the claim amendments overcome the NSDP rejections of record (page 12). This argument has been fully considered and is persuasive – the previous NSDP rejections are withdrawn. However, most of the claims are still not patently distinct based on prior art teachings.
Allowable Subject Matter
Claim 117 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.
Claim 117 requires the deaminase domain in the base editor to be AALN. The Specification indicates that “AALN” is rAPOBEC1 with the R33A + K34A + H122L + D124N substitutions, and has SEQ ID NO 254 ([0034]). The rAPOBEC1 R33A+K34A double substitution is known in the art and first disclosed by Grunewald (Grunewald et al., Nature (May 16, 2019), 569: 433-437). Grunewald developed the R33A+K34A variant base editor by screening previously known APOBEC1 variants that had reduced RNA deaminase activity (page 435, ¶2). Likewise, the rAPOBEC1 H112L+D124N double substitution was known in the art and first disclosed by Thuronyi (Thuronyi et al., Nature Biotechnology (July 22, 2019), 37:1070-1079). Thuronyi used continuous evolution to identify the H112L and D124N substitutions to find variants with higher on-target efficiencies. However, there is no teaching or suggestion in the prior art of combining the two sets of substitutions or suggestion to combine the features (i.e., lower RNA targeting and broader target range) into a single deaminase domain in a base editor. Therefore, a base editor comprising the AALN deaminase is free of the prior art.
It is noted that the Specification teaches that the AALN deaminase was disclosed in WO 2019023680. Although the WIPO publication disclosed the H122L and D124N substitutions, a thorough search of the publication found no disclosure of R33A, K34A or any sequence that appeared to comprise the R33A and K34A substitutions.
Conclusion
Claim 117 is objected to. Claims 26-28, 30, 32, 38-40, 44-45 and 109-116 and 118 are rejected.
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 CATHERINE KONOPKA whose telephone number is (571)272-0330. The examiner can normally be reached Mon - Fri 7- 4.
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 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.
/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635
Prosecution Insights