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 .
Applicant’s response filed 12/23/2025 has been received and considered entered. This is a response to amendments and arguments filed 12/23/2025.
Election/Restrictions
Claims 152-153 stand withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 07/01/2025.
Priority
This application discloses and claims only subject matter disclosed in prior Application No. 16816172, filed 03/11/2020, and names the inventor or at least one joint inventor named in the prior application. Accordingly, this application may constitute a continuation. This application has been filed as a divisional. However, claims 147-150 of this application are directed to the elected invention of the prior application of Application No. 16816172, as described in the restriction requirement mailed 08/05/2021 in the prior application. As such, while the instant application does disclose and claim subject matter disclosed in a prior application, it is not directed to an invention that is independent and distinct from that claimed in the prior/parent application 16816172 (see MPEP § 201.06). Given this, the instant application is considered a continuation of Application No. 16816172.
Claims Status
Claims 1-146 is/are cancelled. Claims 147-153 is/are currently pending with claims 152-153 withdrawn. Claims 147-151 is/are under examination.
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) 147-151 is/are rejected under 35 U.S.C. 103 as being unpatentable over Larson (2013), in view of Alting-Mees (1995) and Li (2008). This is a new grounds of rejection necessitated by amendment.
Regarding claim 147, Larson teaches a composition comprising: a first circular plasmid comprising an sgRNA sequence and an ampicillin resistance gene, and a second circular plasmid encoding a Cas9 protein (Fig. 1). The base-pairing region of the sgRNA can be replaced by restriction digest (linearizing the first plasmid), PCR (inserting the new sgRNA sequence), and ligation (Fig. 1b-c).
Regarding claim 148, Larson teaches that the composition is present in a bacterial cell (Fig. 1).
Regarding claim 149, Larson teaches a cell culture composition comprising a cell culture medium and the host cell (pages 2185-2186).
Regarding claim 150, Larson teaches that the medium comprises ampicillin (pages 2185-2188).
However, Larson does not teach that the first circular plasmid, when linearized, comprises a first homology region and a second homology region, wherein the first and second homology regions are capable of recombining via homologous recombination, thereby circularizing the plasmid.
Regarding claim 147, Li teaches a composition comprising: a site-specific nuclease (DpnI); and a linear nucleic acid, wherein the linear nucleic acid comprises two internal homology regions that are capable of homologously recombining with each other in a host cell, whereupon homologous recombination of the internal homology regions results in formation of a circular nucleic acid comprising a coding sequence for a selectable marker (selectable marker AmpR of plasmid pBluescriptII, see Alting-Mees for the plasmid map of pBluescriptII) (Fig. 1; the entirety of Li teaches this composition and methods of using this composition).
Regarding claim 148, Li teaches a cell comprising the composition (Fig. 1 and page 390, the linear nucleic acid and site-specific nuclease are introduced into the same host cell).
Regarding claim 149, Li teaches a cell culture composition comprising a cell culture medium and the host cell (page 390-391 teach transformed bacterial colonies, which constitutes a cell culture).
Regarding claim 151, Li teaches a linear nucleic acid comprising a first homology region and a second homology region, wherein the first and second homology regions are capable of recombining with each other via homologous recombination, whereupon homologous recombination of the first and second homology regions results in formation of a circular nucleic acid comprising a coding sequence for a selectable marker (Fig. 1; pages 390-391: the plasmid of Fig. 1 can be pThyA-FL, which comprises the thyA selectable marker gene).
Li teaches that the two-step process of PCR (to insert a new sequence of site-specific mutation, or to delete a sequence in a plasmid) followed by homologous recombination (to re-circularize a plasmid) is more efficient than other site-directed mutagenesis methods (page 389). Fig. 1 shows that any desired deletion, insertion, or mutation can be introduced with properly designed PCR primers, with “efficiency approaching 100% in the least hands-on time” (see Fig. 1 of Li and page 391). Larson uses inverse PCR to insert an sgRNA sequence with novel restriction digest sites, and uses digestion, PCR, and ligation to introduce new sgRNA sequences into the plasmid (see Fig. 1). Compared to the method of Li, the method of Larson has more hands-on steps, more opportunity for error, and less efficiency. It would have been obvious to an artisan at the time of filing that the method of Larson, for the replacement of one sgRNA sequence for a new sgRNA sequence, would be improved by using the inverse PCR-homologous recombination method of Li. This modification would improve efficiency, reduce costs (including the costs of the reagents and other materials required for digestion and ligation), and reduce human labor required, while not substantially changing the host cell species—both Larson and Li introduce their plasmids into E. coli, with Li further requiring a strain of E. coli expressing the enzyme DpnI to digest the template plasmid in vitro, and Larson requiring that isolated DpnI enzyme be used to digest the template vector (Larson page 2188).
However, Li does not teach that the cell culture composition further comprises a compound that selects for expression of the selectable marker.
Alting-Mees teaches that the plasmid used by Li comprises the ampicillin resistance gene.
Regarding claim 150, Alting-Mees teaches that ampicillin can be used to select for cells comprising the pBluescriptII plasmid (see abstract, page 180).
While Li does not teach that the cell culture comprises ampicillin in order to select for the selectable marker, and Larson merely teaches that ampicillin is a required material in a protocol that uses a vector encoding an ampicillin resistance gene (page 2185 and Fig. 1 of Larson), it would have been obvious to a person of ordinary skill in the art at the time of filing, given that the plasmid used comprises an ampicillin resistance gene, to add ampicillin to the cell culture medium in order to select for cells containing the plasmid. This selection would be obvious for such an artisan to perform, as this would eliminate any cells that were not transformed with the plasmid, and thus could not be further used.
Response to Arguments
Applicant's arguments filed 12/23/2025 have been fully considered but they are not persuasive.
Applicant argues that Li does not “teach or suggest a linear nucleic acid comprising two internal homology regions that are capable of homologous recombination, whereupon homologous recombination of the internal homology regions results in the formation of an extra-chromosomal circular nucleic acid comprising a coding sequence for a selectable marker and a guide RNA sequence” (page 6). Applicant further argues that “Li teaches using a linear nucleic acid to introduce mutations at a target site on another plasmid by homologous recombination, but does not teach the formation of an extra-chromosomal circular nucleic acid with a selectable marker and a guide RNA sequence after homologous recombination. The mutations introduced by the linear nucleic acid are incorporated into the plasmid, and the remaining linear nucleic acid is degraded by the DpnI restriction enzyme in the host cell” (page 6).
The instant specification does not provide a limiting definition of an “internal homology region”. On page 35, the specification states that “recombination regions can be positioned at or near the termini of the linear pre-recombination vector…, internal to the termini (e.g., each recombination region is flanked on both ends by additional sequences)”; based on this phrasing, an internal homology region is not limited to a recombination region flanked by additional sequences. Paragraph [00219] describes the only sequences in the drawings described as comprising internal overlap or internal homology regions (“three split constructs (with internal overlap for reconstitution by homologous recombination in vivo)…FIG. 24”). Fig. 24B shows three pairs of linear nucleic acids wherein the homology regions (described in the specification as “internal”) are positioned at the termini of the linear fragments, whereupon homologous recombination, these homology regions become internal to the resulting circular plasmid. As such, “internal homology regions” are interpreted as encompassing paired homology regions which become internal to a circular plasmid upon recombination, as in Fig. 24B.
Li, in Fig. 1, teaches the following method for targeted deletion of a fragment of a sequence of interest. The plasmid of Fig. 1 prior to “Step 1” comprises a backbone and a sequence of interest, wherein the sequence of interest comprises a 5’ fragment “a” in tan, adjacent fragment “b” in red, fragment for deletion in gray, and 3’ fragment in tan, wherein after completion of the method, the gray fragment is deleted and the sequence of interest consists of “a”-“b”-“3’ tan fragment”. PCR forward (F) and reverse (R) primers are designed, such that the reverse primer consists of sequence “b” and an immediately adjacent fragment of sequence “a” of the sequence of interest, and the forward primer consists of sequence “b” and a fragment of the 3’ conserved sequence comprising the nucleotides directly 3’ of the deleted sequence. Inverse PCR (Step 1) using these two primers linearizes the initial vector and through inverse PCR extension introduces a second red region 5’ of the 3’ fragment and homologous to the red region “b” (“HR”, for homologous regions). After Step 1, the reaction composition comprises two nucleic acid species: unmodified template plasmid and a linearized plasmid comprising two homologous regions. In Step 2, the post-Step 1 reaction DNA mixture, comprising the modified and linearized vector and any unmodified template vector remaining, is introduced into E. coli strain BUNDpnI. In the bacterial cell, the two red homology regions recombine to re-circularize the modified plasmid, forming an extrachromosomal circular plasmid. DpnI only digests methylated and hemimethylated DNA sequences, allowing DpnI to digest the “template [unmodified] DNA isolated from most E. coli strains, but not the PCR-amplified DNA” (Li pate 389). As a result, the modified and in vitro-circularized plasmid is not digested but the unmodified template plasmid is digested, leaving only the desired modified plasmid able to be used by the cell. Fig. 1B and 1C shows alternative PCR primer designs for the same methodology as shown in 1A, but allowing for insertion of any desired sequence (1B) or insertion of a single nucleotide mutation (1C).
The only nucleic acids used in this method are a template plasmid, a forward PCR primer, and a reverse PCR primer. The primers are used to introduce, through inverse PCR, a 3’ homology arm to pair with an existing sequence within the template plasmid.
Li teaches that this method can be used for any insertion, deletion, or point mutation desired. As described in the rejection above, it would have been obvious to an artisan that, given the short length of the base-pairing segment of an sgRNA, the method of Li could be used to insert or swap base-paring sgRNA sequences in a plasmid vector more efficiently than the method used by Larson.
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.
US 10041092 B1:
Claims 147-151 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-26 of U.S. Patent No. 10041092 B1. Although the claims at issue are not identical, they are not patentably distinct from each other because the claims of ~092 recite limitations which encompass all the limitations recited in the instant claims, with further limitations not recited in the instant claims.
Issued claims 12-13 recite that the nuclease is an RNA-guided nuclease; issued claims 14-15 recite that the extrachromosomal nucleic acid comprises a guide RNA (wherein the extrachromosomal nucleic acid is formed by the circularization of the linear nucleic acid).
US 11390888 B2:
Claims 147-151 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-4 of U.S. Patent No. 11390888 B2 in view of Larson (2013) and Li (2008). This is a new grounds of rejection.
The issued claims recite a composition comprising a sequence encoding a site-specific nuclease and a linear nucleic acid comprising the following: “two internal homology regions that upon introduction into a host cell are capable of homologously recombining with each other in the host cell, whereupon homologous recombination of the internal homology regions results in formation of a circular nucleic acid comprising a coding sequence for a selectable marker, and further wherein the linear nucleic acid is linear prior to introduction into the host cell.” In col. 18 lines 40-48, ~888 teaches that nucleases encompass “a homing endonuclease, zinc-finger nuclease, TAL-effector nuclease, or RNA-Guided DNA endonuclease (e.g., CRISPR/Cas9)” (emphasis added). RNA-guided endonucleases thus are clearly encompassed by the broad recitation of “site-specific nuclease” of ~888 claim 1.
~888 does not recite in the claims that the linear nucleic acid comprises a guide RNA sequence.
Regarding claim 1, Larson teaches two-plasmid compositions introduced into cells, wherein a first plasmid comprises a guide RNA sequence and a first selectable marker gene, and a second plasmid comprises a gene encoding an RNA-guided endonuclease and a second selectable marker gene, for use in a CRISPRi system (Fig. 1).
Regarding claim 1, Li teaches that linear nucleic acids such as that claimed by ~888, which are circularized in vitro through homologous recombination, can be created in a highly efficient method of introducing desired insertions, deletions, or mutations in a plasmid (Fig. 1; page 389).
Based on the teachings of Larson, it would have been obvious to an artisan that a composition comprising a Cas9 protein should also comprise a guide RNA, such that the Cas9 protein could be used to target a sequence in vitro. As the site-specific nuclease of ~888 encompasses Cas9, it would have been obvious to an artisan that a guide RNA sequence should also be comprised in the claimed composition. The teachings of Li provide a motivation for creating a linear nucleic acid such as that claimed by ~888; using inverse PCR to introduce an insertion into a plasmid and a 3’ homology region homologous to the plasmid sequence desired to be immediately 5’ of the insert in the final circularized plasmid creates such a linear nucleic acid, in a method which increases the efficiency of site-specific insertional mutagenesis (Fig. 1; pages 389-391). Thus, an artisan would recognize that the invention of ~888 encompasses a CRISPR system (with the claimed nuclease encompassing Cas9), requiring a guide RNA sequence (as per Larson), and would recognize that this guide RNA sequence could be comprised in the linear nucleic acid of ~888 claim 1, particularly adjacent to the homology regions, as, per Li, these homology regions are ideal sites for introduction of new sequences into a plasmid (ideal because of the efficiency of inverse PCR and re-circularization through homologous recombination).
Response to Arguments
Applicant's arguments filed 12/23/2025 have been fully considered but they are not persuasive.
Applicant argues that ~888 does not “recite an RNA-guided endonuclease” (Page 14). ~888 claim 1 recites “a site-specific nuclease”, broadly encompassing any nuclease. In col. 18 lines 40-48, ~888 teaches that nucleases encompass “a homing endonuclease, zinc-finger nuclease, TAL-effector nuclease, or RNA-Guided DNA endonuclease (e.g., CRISPR/Cas9)” (emphasis added). RNA-guided endonucleases thus are clearly encompassed by the broad recitation of “site-specific nuclease” of ~888 claim 1. The new required limitation that the linear nucleic acid comprise a guide RNA sequence is addressed in the new grounds of rejection above.
Applicant has noted that the instant application claims priority to US 9476065 B2 and the instant claims are limited to a non-elected invention restricted from ~065 and not rejoined. Examiner has withdrawn the nonstatutory double patenting rejection over ~065.
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.
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/AFRICA M MCLEOD/ Examiner, Art Unit 1635
/KIMBERLY CHONG/ Primary Examiner, Art Unit 1636