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 on 2/9/2023. Claims 21-40 are pending. All pending claims are currently under examination.
Claim Objections
Claim 32 is objected to because of the following informalities:
Claim 32 recites “comprises-growing,” which should be amended to recite “comprises growing”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 21-40 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Regarding claim 21, claim 21 recites “thereby producing cured cells” (final line of step “(d)”). Step (d) therefore establishes a meaning of the phrase “cured cells,” to mean cells cured of the editing vector in step (d). Steps (e) and (f) recite “curing the engine vector by growing the cured cells at a temperature that restricts replication of the first sensitive origin of replication,” (step (e), emphasis added) and “curing the curing vector by growing the cured cells at a temperature that restricts replication of the second temperature sensitive origin (step (f), emphasis added). Thus, the “cured cells” of step (d) are recited to further undergo growth in two steps: e and f. However, it is unclear from the claim if the cells produced in step (d) are necessarily required to undergo both steps e and f, since both steps e and f recite “the cured cells.” It is unclear if “cured cells” recited in step f are in reference to “the cured cells” rendered by step e (i.e., cells which are cured of both the editing vector and the engine vector) or “the cured” cells rendered in step d (i.e., cells that are cured of the editing vector). The phrase “cured cells” therefore lacks proper antecedent basis in step f, as it is unclear to which group of “cured cells” is being referenced. It is recommended that step e be amended to define the cured cells created in that step (e.g., “second cured cells”) and, if such an amendment is made, to continue the language into step f (e.g., “curing the curing vector by growing the second cured cells”).
Claims 22-33 depend from claim 21 and do not resolve this issue and are therefore also rejected.
Regarding claim 33, claim 33 recites “the selecting step” which lacks proper antecedent basis because no “selecting step” is recited either previously in claim 33 or the claim from which claim 33 depends (claim 1).
Regarding claim 34, claim 34 recites a similar 112(b) issue, where “cured cells” are defined in step c of claim 34 and recited in two separate steps (steps d and e). Thus, recitation of “cured cells” in step e lacks proper antecedent basis because it is unclear if the “cured cells” being referenced are the cells rendered in step d or if the “cured cells” are in reference to step c.
Claim 35 recites similar 112(b) issues, where “cured cell” is defined in step c, followed by additional curing steps in step c. Step d then recites curing the “cured cell;” recitation of “cured cell” in step d lacks proper antecedent basis as it is unclear to which step in the process is being referred as there are multiple curing steps in step c. Furthermore, step c recites curing the editing or engine vector to generate a “cured cell” (lines 1-3), followed by a curing step of curing the editing or engine vector by growing the ”transformed edited cells.” (line 3) It is unclear if step c is meant to generate two separate pools of cells, where first the editing or engine vector is cured (to make a “cured cell” which still comprises whichever component, the engine or editing vector, that was not cured) and separately curing the engine or editing vector of the “transformed edited cell.” If the method is meant to be sequential, it is recommended that the “cured cell” language be carried through each part of steps c and d, where the second curing in step c generates “a second cured cell” to which step d could reference.
Claims 36-40 depend from claim 35 and do not resolve this 112(b) issue and are rejected.
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.
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 21-26 and 29-40 are rejected under 35 U.S.C. 103 as being unpatentable over Belgrader (WO 2020/005383 A1, published 1/2/2020; see the entire reference) in view of Lauritsen (Lauritsen I . Microb Cell Fact. 2017 Aug 2;16(1):135).
Regarding claim 21, Belgrader teach methods that employ cells comprising editing vectors comprising editing cassettes comprising a guide RNA (gRNA) sequence covalently linked to donor DNA, where the gRNA and the donor nucleic acid are under the control of a single inducible promoter (e.g., paragraphs 61, 63, and 66). Belgrader teach the assembly of an editing cassette comprising the gRNA, donor and editing vector backbone (e.g., paragraph 66). Belgrader teach that the editing vectors comprise a selectable marker (e.g., paragraph 103). Belgrader teach the introduction of a library of editing vectors (i.e., the cells comprise “an editing vector”) by electroporation into cultured cells that comprise an “engine plasmid” comprising a sequence encoding a nuclease under the control of an inducible promoter and a selectable marker (e.g., paragraph 103). Belgrader teach that the nuclease is a RNA guided nuclease, such as Cas9, Cpf1 or MAD7 (e.g., paragraphs 7 and 69). Belgrader teach that the transformed cells are diluted and plated such that they are singulated onto selective medium that selects for both the engine and editing vectors, such as a medium containing both chloramphenicol and carbenicillin and therefore teaches that the selection markers are different, i.e., a first a second selectable marker gene (e.g., paragraph 104). Belgrader teaches inducing editing by inducing the promoters to produce edited cells (e.g., paragraph 9). Belgrader teach that it is desirable to cure the cells of one or more vectors used in the editing:
“In any recursive process, it is advantageous to "cure" the previous engine and editing vectors ( or single engine + editing vector in a single vector system). "Curing" is a process in which one or more vectors used in the prior round of editing is eliminated from the transformed cells. Curing can be accomplished by, e.g., cleaving the vector(s) using a curing plasmid thereby rendering the editing and/or engine vector (or single, combined vector) nonfunctional; diluting the vector(s) in the cell population via cell growth (that is, the more growth cycles the cells go through, the fewer daughter cells will retain the editing or engine vector(s)), or by, e.g., utilizing a heat-sensitive origin of replication on the editing or engine vector ( or combined engine + editing vector). The conditions for curing will depend on the mechanism used for curing; that is, in this example, how the curing plasmid cleaves the editing and/or engine plasmid,” (paragraph 219, emphasis added).
Belgrader therefore teaches the introduction of a curing plasmid/vector into cells in order to cure them, and furthermore teaches that it is advantageous to do so (paragraph 219).
Furthermore regarding the claim limitation that the engine vector comprises a temperature sensitive origin of replication, Belgrader teaches such a heat-sensitive/temperature sensitive origin of replication to be used for curing the engine vector (paragraph 219). Additionally, the embodiment taught by Belgrader in the curing process using a heat-sensitive origin of replication makes no mention of the presence of curing target sequence within an engine vector comprising temperature-sensitive origin of replication; thus, Belgrader teaches an engine plasmid comprising a temperature sensitive origin of replication which does not comprise a curing target sequence (i.e., (“a heat-sensitive origin of replication on the editing or engine vector,” paragraph 219 without mention of an additional curing target sequence). To further elaborate upon this point, the final sentence of paragraph 219 states that: “the curing plasmid cleaves the editing and/or engine plasmid.” Belgrader therefore directly teaches that the curing plasmid need not cleave the engine plasmid (“and/or”); thus the engine vectors comprising temperature sensitive origins or replication taught by Belgrader are taught in certain embodiments to lack curing target sequences (above).
With regards to the application of the engine and editing vectors, Belgrader specifically teaches within the same paragraph on curing vectors (i.e., paragraph 219) that:
“It should be apparent to one of ordinary skill in the art given the present disclosure that the process described may be recursive and multiplexed; that is, cells may go through the workflow described in relation to FIG. 10, then the resulting edited culture may go through another (or several or many) rounds of additional editing (e.g., recursive editing) with different editing vectors,” (paragraph 219).
Thus, Belgrader specifically teaches that the cells undergo iterative, recursive editing, and that such would be immediately apparent to one of ordinary skill in the art (paragraph 219). Thus, not only does Belgrader teach an engine vector comprising a temperature sensitive origin which does not comprise a curing target sequence, Belgrader also teaches a motivation for using an engine vector in an embodiment of their methods which lacks a curing target sequence, so that such an engine vector could be used in multiple, recursive rounds of editing and not cured during the curing process (paragraph 219). Belgrader therefore teaches an engine vector comprising a temperature sensitive origin and no curing target sequence both directly (“a heat-sensitive origin of replication on the editing or engine vector,” paragraph 219 without mention of an additional curing target sequence) and implicitly by teaching that such compositions are designed to go through multiple rounds of editing, where it would be apparent to a practitioner that the engine vector would not be designed to be cured by the curing vector in order to carry out the recursive (i.e., repetitive, multi-round) methods of Belgrader.
Belgrader, while teaching a curing vector and transforming such vectors into cells to cure them with a strong motivation to do so, does not teach the method where the curing vector comprises a third promoter driving transcription of an anti-curing target gRNA, a coding sequence for an RNA-guided nuclease compatible with the anti-curing target gRNA; a temperature-sensitive origin of replication, and a coding sequence for a third antibiotic resistance gene, which is different from the antibiotic resistance gene of the engine vectors. Belgrader does not teach curing the curing/engine vectors at a temperature that restricts the replication of the temperature sensitive origin of replication. Belgrader does not teach curing the editing vector by growing the edited cells in conditions to transcribe the anti-curing target gRNA.
Lauritsen teaches a broadly applicable CRISPR-Cas9-based curing platform that enables fast and efficient curing of all major plasmid replicons used in modern molecular biology laboratories (e.g., page 2/10, right column, 1st paragraph). Lauritsen teaches the design of CRISPR-Cas9 compatible guide RNA to the ColE1-like origins (e.g., paragraph bridging pages 2/10-3/10). Lauritsen teaches combining the gRNA and Cas9 encoding components, each under the control of an inducible promoter, into a single vector containing all parts necessary to form a fully functional curing system, including a kanamycin resistance gene (e.g., paragraph bridging pages 3/10-4/10; Fig. 2b, i.e., “a third selectable marker gene”). Lauritsen teaches that the curing vector comprises a temperature sensitive origin of replication (e.g., page 8/10, right column, 1st full paragraph; Fig. S3). Lauritsen teaches transforming cells containing the target plasmids for curing with the curing system vector, and selecting cells with the curing vector by the addition selection medium containing (e.g., page 6/10; page 810, right column, last paragraph; page 9/10, left column, 2nd paragraph; Fig. 2). Lauritsen teaches curing the curing vector by curing at elevated temperatures (e.g., paragraph bridging pages 5/10-6/10).
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 including a curing step of Belgrader to include the use of the curing vector including the temperature sensitive origin of replication and kanamycin resistance gene followed by selection with kanamycin to cure the editing vector with subsequent curing of the curing vector or the curing vector and engine vector by culture at elevated temperature as taught by Lauritsen, because Belgrader teach it is within the ordinary skill in the art to transform edited cells with a an engine vector with a temperature sensitive origin, an editing vector that is cleavable, and a curing vector that results in the cleavage of the editing vector and curing of the engine vector using temperature-sensitive ORIs, and Lauritsen teaches a curing vector for curing of vectors from cells, where the curing vector results in cleavage of the origin of replication, such as a ColE1 origin, to cure the cells of the vector, while the curing vector is cured by elevated temperature. One would have made such a motivation because Belgrader teaches curing by cleavage or by the use of a temperature-sensitive origin of replication according to the required mechanisms, and Lauritsen provide the appropriate mechanisms. One would have made such a substitution in order to achieve the predictable outcome of using a vector for curing that is known to have the required curing function according to the established protocol of Lauritsen and to cure vectors with a temperature sensitive origin by culturing at elevated temperature as taught by Lauritsen and furthermore directly taught and suggested by Belgrader. The combination is therefore the combination of known prior art elements with sufficient motivation to combine given the recursive nature of the methods taught by Belgrader, where such vectors as the engine vector would be advantageously cured using known methods such as temperature-sensitive origins of replication.
One would have been motivated to make such a modification in order to receive the expected benefit of using a broadly applicable CRISPR-Cas9-based curing platform that enables fast and efficient curing plasmid replicons used in modern molecular biology laboratories as taught by Lauritsen. One would have included the curing steps in order to allow for recursive editing as taught by Belgrader. As Belgrader teaches, the cells may undergo multiple rounds of editing before curing, and that vectors and how they are cured can be designed to accommodate the designs of a person of ordinary skill in the art (paragraph 219).
Regarding claim 22, Belgrader teaches that the first and second inducible promoter can be the same inducible promoter (paragraph 20).
Regarding claim 23, Belgrader teaches that the promoter can be a pL promoter (paragraph 20). Belgrader teaches that either the engine or editing vector comprises the cI857 gene under control of a constitutive promoter (paragraph 86, Figure 1C).
Regarding claim 24, Belgrader teaches that the first and second inducible promoters can be different (e.g., page 122 final sentence into top of page 123 and paragraph 87).
Regarding claims 25-26, Lauritsen teaches that the ColE1 origin is used in pUC18/19 plasmids (e.g., page 1/10, paragraph bridging columns). Thus, Lauritsen teaches that the curing target sequence and anti-curing target sequence (curing gRNA) can be a pUC origin.
Regarding claim 29, claim 29 recites that the anti-curing gRNA is under the control of either an inducible or a constitutive promoter. Thus, claim 29 encompasses any form of promoter capable of expression and broadly encompasses any promoter which expresses the anti-curing gRNA. Belgrader teaches that gRNA can be expressed from inducible promoters (paragraph 86). Furthermore, Lauritsen teaches that the curing gRNA is expressed, and therefore must be expressed from either an inducible or constitutive promoter (see Table S3, e.g., pMAZ-SK with rhamnose-inducible promoter and tracrRNA constitutively expressed).
Regarding claim 30, the combination of Belgrader and Lauritsen yields three different selectable marker genes (see rejection of claim 21).
Regarding claims 31-32, Belgrader teaches that after singulation, the cells are grown for 2-200 doublings (e.g., paragraphs 17, 61, and 97). Note also that the recited method does not require a specific ordering of steps per the present claim language.
Regarding claim 33, Belgrader teaches selecting cells based on the first and second selectable markers (e.g., paragraph 104, chloramphenicol and carbenicillin).
Regarding claim 34, the claim elements of (a), namely the editing and engine cassettes in two separate vectors with the recited elements are addressed in the rejection of claim 21, where the combination of Belgrader and Lauritsen render such claim elements obvious. Note that Belgrader also teaches that the editing and engine vectors can be separate vectors by teaching for instance that: “cleaving the vector(s) using a curing plasmid thereby rendering the editing and/or engine vector (or single, combined vector)” which implies that the editing and engine vectors can be separate vectors (paragraph 219). Belgrader teaches that the editing cassette can comprise a curing target sequence and that the engine vector can comprise a temperature sensitive origin of replication without a curing target sequence:
“cleaving the vector(s) using a curing plasmid thereby rendering the editing and/or engine vector (or single, combined vector) non-functional… or by, e.g., utilizing a heat-sensitive origin of replication on the editing or engine vector (or combined engine + editing vector). The conditions for curing will depend on the mechanism used for curing; that is, in this example, how the curing plasmid cleaves the editing and/or engine plasmid,” (paragraph 219)
The remainder of claim elements and their components (b-e) are addressed in the rejection of claim 21.
Regarding claim 35, as discussed above, each of the recited elements of claim 35 are rendered obvious by the combination of Belgrader and Lauritsen (see rejection of claim 21). For instance, the combination of Belgrader and Lauritsen render obvious a method of obtaining a cell with editing and engine vectors comprising editing and engine cassettes which respectively comprise guide RNA covalently linked with a donor template and RNA guided nuclease (see rejection of claim 21). Furthermore, Belgrader teaches the process of curing, where different approaches to curing can be adopted depending upon a practitioner’s design (paragraph 219). Furthermore, Belgrader teaches that either the editing vector or engine vector (or both) can be targeted by a curing/cutting vector (“The conditions for curing will depend on the mechanism used for curing; that is, in this example, how the curing plasmid cleaves the editing and/or engine plasmid,” which reasonable teaches that either vector can comprise a curing target sequence to be “cleaved”). Furthermore, Belgrader teaches that the temperature sensitive origin of replication can be on either vector: “utilizing a heat-sensitive origin of replication on the editing or engine vector,” (paragraph 219). Belgrader therefore taught and understood that the components being predictable and fungible between plasmid constructs could be used interchangeably on different plasmids for different effects as presently recited in claim 35 section “(a),” subsections “(A)” and “(B).” The additional curing steps and claim elements of sections b-d are addressed in the rejection of claim 21. In addition, note that Belgrader also teaches that:
“it should be recognized by one of ordinary skill in the art given the guidance of the present description that all elements of the nucleic acid-guided nuclease editing system may be contained on a single plasmid, or the elements shown on the engine and editing vectors of FIGs. 11A and 11B may reside on a different vector than shown. For example, the pBAD promoter and A Red recombineering system may be contained on the editing vector rather than the engine vector; likewise, the gene for the cl857 repressor may be contained on the editing vector rather than the engine vector,” (paragraph 279).
Thus, with regards to claims 34-35, it should be noted that Belgrader taught and understood that the components of their editing and engine vectors could be interchanged between vectors in order to suit the design of a practitioner. Claims 34-35 appear to be merely exchanging the positions of the elements, vectors, and compositions rendered obvious by the combination of Belgrader and Lauritsen, where such a combination of teachings is predictable given that the components are all the exact same as those rendered obvious by Belgrader and Lauritsen and used on the same vector designs for the same purposes.
Regarding claim 36, Belgrader teaches that the editing vector and not the engine vector is targeted by the curing vector, where such a teaching reasonably teaches that the editing vector comprises a curing target sequence for cleavage:
“Curing can be accomplished by, e.g., cleaving the vector(s) using a curing plasmid thereby rendering the editing and/or engine vector,” (paragraph 219).
Regarding claim 37, Belgrader teaches that the engine vector can comprise the first temperature sensitive origin of replication:
“utilizing a heat-sensitive origin of replication on the editing or engine vector,” (paragraph 219)
Regarding claim 38, Belgrader teaches that the nuclease can be Cpf1 or Cas9 (paragraphs 7 and 69).
Regarding claim 39, Belgrader teaches that the engine vector and not the editing vector can targeted for curing by cleavage by the curing vector, which reasonably teaches a curing target sequence:
“Curing can be accomplished by, e.g., cleaving the vector(s) using a curing plasmid thereby rendering the editing and/or engine vector,” (paragraph 219).
Regarding claim 40, Belgrader teaches that the editing vector can comprise a first temperature sensitive origin of replication:
“utilizing a heat-sensitive origin of replication on the editing or engine vector,” (paragraph 219).
Claims 27-28 are rejected under 35 U.S.C. 103 as being unpatentable over Belgrader (WO 2020/005383 A1, published 1/2/2020; see the entire reference) in view of Lauritsen (Lauritsen I . Microb Cell Fact. 2017 Aug 2;16(1):135) as applied to claims 21-26 and 29-40, above, and further in view of Haugan (Haugan K . J Bacteriol. 1992 Nov;174(21):7026-32).
The teachings of Belgrader and Lauritsen are described above and applied as before. Further, Lauritsen teach curing the curing vector by growing the cells at an elevated temperature (e.g., paragraph bridging pages 5/10-6/10). Lauritsen cites reference 33 for the culturing at an elevated temperature (paragraph bridging pages 5/10-6/10). Citation 33 is “Haugan.”
Belgrader and Lauritsen do not teach the method where the curing plasmid comprising the temperature sensitive origin is cured at a temperature of 42°C.
Haugan teach that the RK2 plasmid contains a temperature-sensitive origin of replication, which is sensitive at a temperature of 42°C (e.g. page 7027, paragraph bridging columns).
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 combined teachings of Belgrader and Lauritsen to include the temperature of 42°C taught by Haugan, because Lauritsen teach it is within the ordinary skill in the art to use an elevated temperature for the plasmid containing the RK2 origin, and Haugan teach the appropriate elevated temperature is specifically 42°C. Furthermore, Lauritsen directly references Haugan; a practitioner would therefore be reasonably guided to the teachings of Haugan as they apply to Lauritsen.
One would have been motivated to make such a modification in order to receive the expected benefit of using the specific temperature known to select against the RK2 origin as taught by Haugan.
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 USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The 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/process/file/efs/guidance/eTD-info-I.jsp.
Claims 21-40 are rejected on the ground of nonstatutory double patenting as being unpatentable over the claims of U.S. Patent No. 10,837,021 B1 (hereinafter the ‘021 patent) in view of Xie (US Patent Application Publication No. 2019/0264185 A1), and Pyne (Pyne e al. Appl Environ Microbiol. 2015 Aug;81(15):5103-14) and Bernate (US Patent Application Publication No. 2019/0100774 A1).
Although the claims at issue are not identical, they are not patentably distinct from each other because both sets of claims are directed to a method for curing cells during recursive nucleic acid-directed nuclease editing. Claim 1 of the ‘021 patent comprises the steps of “designing and synthesizing a first set of editing cassettes, wherein the first set of editing cassettes comprises one or more editing gRNA and donor DNA pairs wherein each editing gRNA and donor DNA pair is operably linked and under the control of a first inducible promoter; assembling the first set of editing cassettes into a vector backbone thereby forming a first set of editing vectors, wherein the vector backbone comprises a first selectable marker, and a curing target sequence; making cells of choice electrocompetent, wherein the cells of choice comprise an engine vector and the engine vector comprises a curing gRNA under the control of a second inducible promoter, a nuclease under the control of a third inducible promoter; and a second selectable marker; transforming the cells of choice with the first set of editing vectors to produce first transformed cells; selecting for the first transformed cells via the first and second selectable markers thereby selecting for first selected cells; inducing editing in the first selected cells by inducing the first and third inducible promoters thereby inducing transcription of the one or more editing gRNA and donor DNA pairs and the nuclease producing first edited cells; growing the first edited cells until the first edited cells reach a stationary phase of growth; curing the first set of editing vectors in the first edited cells by inducing the third and second inducible promoters thereby inducing transcription of the nuclease and curing gRNA which cuts the curing target sequence producing first cured cells; growing the first cured cells; rendering the first cured cells electrocompetent; and transforming the first cured cells with a second set of editing vectors to produce second transformed cells, wherein the second set of editing vectors comprises editing cassettes with one or more editing gRNA and donor DNA pairs operably linked and under the control of the first inducible promoter, a third selectable marker, and the curing target sequence.” Claim 11 of the ‘021 patent comprises the steps of “designing and synthesizing a first set of editing cassettes, wherein the first set of editing cassettes comprises one or more editing gRNA and donor DNA pairs wherein each editing gRNA and donor DNA pair is operably linked and under the control of a first inducible promoter; assembling the first set of editing cassettes into a vector backbone thereby forming a first set of editing vectors, wherein the vector backbone comprises a first selectable marker, a curing target sequence, and a curing gRNA under the control of a second inducible promoter; making cells of choice electrocompetent, wherein the cells of choice comprise an engine vector and the engine vector comprises a nuclease under the control of a third inducible promoter, and a second selectable marker; transforming the cells of choice with the first set of editing vectors to produce first transformed cells; selecting for the first transformed cells via the first and second selectable markers thereby selecting for first selected cells; inducing editing in the first selected cells by inducing the first and third inducible promoters thereby inducing transcription of the one or more editing gRNA and donor DNA pairs and nuclease producing first edited cells; growing the first edited cells until the first edited cells reach a stationary phase of growth; curing the first set of editing vectors in the first edited cells by inducing the third and second inducible promoters thereby inducing transcription of the nuclease and curing gRNA which cuts the curing target sequence producing first cured cells; growing the first cured cells; rendering the first cured cells electrocompetent; and transforming the first cured cells with a second set of editing vectors to produce second transformed cells, wherein the second set of editing vectors comprises editing cassettes with one or more editing gRNA and donor DNA pairs operably linked and under the control of the first inducible promoter, a third selectable marker, the curing target sequence, and the curing gRNA under the control of the second inducible promoter.” In independent claim 1 of the ‘021 patent, the curing gRNA is in the engine vector, which also encodes the nuclease. In independent claim 11 of the ‘021 patent, the curing gRNA is in the editing vector, which also encodes the gRNA and donor DNA pair for editing. The editing vector of claim 1 of the ‘021 patent is identical to the editing vector of the method of instant claim 21. The engine vector of claim 11 of the ‘021 patent is identical to the engine vector of instant claim 21, excepting that it does not comprise a temperature sensitive origin of replication. Instant claims 21 and 34-35 and their dependent claims differ in that the curing gRNA expression cassette is moved to a separate vector with a temperature sensitive origin of replication. The difference in scope of the claims is obvious as the duplication of parts would not alter the operation of the method. The claims of the ‘021 patent provide evidence that the curing gRNA expression cassette may be moved. A nuclease would be provided for editing whether it is present on one vector or two. Furthermore, Xie teach that it is within the skill of the art to conduct curing after editing and specifically teach curing by cultivation at 42°C (e.g., Example 1, especially paragraphs [0078]-[0079]). Furthermore, Xie teach it is within the skill of the art to use different inducible promoters and to use plasmids that contain a temperature sensitive pSC101 replication origin (e.g., paragraph [0050]). Thus, it would have been obvious to one of ordinary skill in the art to provide an additional vector encoding the nuclease with the curing gRNA after editing so that editing would be able to take place prior to curing and to use vectors with a temperature sensitive origin and different inducible promoters. Instant claims 21-40 are not patentably distinct from the claims of 021’, with additional teachings rendering the claims obvious with support from Xie, Pyne, and Bernate (below).
The claims of the ‘021 patent do not require three different antibiotic resistance genes. Pyne teach it is within the skill of the art to use three different antibiotic resistance genes in a three-plasmid configuration of gene editing (e.g., Fig. 2A). Thus, it would have been obvious to one to use three different antibiotic resistance genes in the method and to select based on one or more of the markers.
The claims of the ‘021 patent do not require the use of a singulating and doubling steps between transformation and selection. However, Bernate teach the use of an automated multi-module cell processing system for editing, where the modules direct transformation followed by singulation and doubling, induction of editing, and selection in that order (e.g., paragraphs [0072] and [0155]-[0158]). Bernate teach that once the cells are singulated they are allowed to grow through several to many doublings and establish colonies prior to induction (e.g., paragraph [0157]). Thus, it would have been obvious to one to include a singulating and doubling steps between transforming and selecting steps in an automated system as taught by Bernate to automate the process.
Regarding the additional claim limitations of the instant claims, 021’ claims the same limitations and/or renders the claims obvious in view of other teachings as follows: instant claim 22 reads on claim 2 of 021’, instant claim 23 reads on claim 3 of 021’, instant claim 24 reads on claim 1 of 021’, instant claims 25-26 read on claims 4-5 of 021’, instant claims 27-28 are rendered obvious in view of the teachings of Xie (above), instant claim 29 reads on claim 6 of 021’, instant claim 30 is rendered obvious in view of Pyne (above), instant claims 31-32 are rendered obvious by Bernate (above), instant claim 33 reads on claim 7 limitations of 021’, regarding claims 34-37 and 39-40, these claims simply recite obvious variations and arrangements of the components taught by 021’ and Xie, where each variation can simply be viewed as a combination of known prior art elements with predictable effects in light of the fact that 021’ teaches that different components of the compositions can be found on different vectors (claims 1 and 11), regarding claim 38, Cas9 is simply a well-known Cas enzyme and is therefore an obvious choice of RNA nuclease in the instant methods.
Conclusion
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/D.C.R./Examiner, Art Unit 1635
/KIMBERLY CHONG/Primary Examiner, Art Unit 1636