DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims 1-9,11-13,17,18,20,21,31,34,35,66 and 67 are under examination.
Priority
This application is a 371 of PCT/US2022/019012 filed 03/04/2022, which claims benefit of 63/157,498, filed 03/05/2021 and claims benefit of 63/157,497, filed 03/05/2021 as reflected by the most recent filing receipt.
Drawings
The drawings are objected to because FIG. 7H is not clear and legible. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
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 2 and 34 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 2, lines 3-4 recite “a third or subsequent oligonucleotide encoding a third or subsequent DNA element fragment (oligo3, oligo4,…oligoN)”. It is not clear if what is in parenthesis requires more than 3 oligonucleotides, while what is not in parenthesis requires a third oligonucleotide or subsequent oligonucleotides, and therefore, what is in parenthesis does not appear to be of the same scope as the limitation outside of the parenthesis, and therefore the use of parentheses raises the question as to which term is required by the claim.
Regarding claim 34, the phrase "such as" in line 2 renders the claim indefinite because it is unclear whether the limitations following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-3,8,34,66 and 67 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Church (US 20070004041, Published 4 Jan 2007), cited on an IDS.
Regarding claims 1 and 66, Church teaches a method for hierarchical assembly of very large, including genome sized, nucleic acid products (paragraph 0123) and the polynucleotide constructs assemble in a desired manner by integrating into the host cell genome by homologous recombination (paragraph 0018) and the polynucleotide constructs may be contained on an extrachromosomal plasmid (paragraph 0019). Church teaches the hierarchical assembly process is illustrated with reference to Figs. 2-4 (paragraph 0129).
Regarding step (a)(i), Church teaches for each pairwise combination, the donor and recipient strains are mixed and the donor cells transfer their DNA to the recipient cells by conjugation (paragraphs 0068, 0129). Church teaches that Fig. 4 illustrates an embodiment of a hierarchical assembly method, which involves two sets of starting polynucleotide constructs each having four types of components including selected genes (illustrated as 2 and 3 in Fig. 4), meganuclease sites (illustrated as 4 and 5 in Fig. 4), conjugative transfer sites (oriT sites, illustrated as 6 and 7 in Fig. 4), and origins of replication (illustrated as 8 and 9 in Fig. 4) and the polynucleotide constructs are introduced into cells, the cells are then mixed pairwise and in each case the cell containing the lower polynucleotide construct of the pair (donor) is transferred into the cell containing the upper polynucleotide construct of the pair (recipient), and the cell containing the upper polynucleotide construct contains a meganuclease that recognizes the meganuclease cleavage sites of the incoming (lower) polynucleotide construct, and regarding step (a)(ii), cleavage by the meganuclease stimulates homologous recombination between the upper and lower polynucleotide constructs (paragraph 0136).
Regarding the first donor plasmid and recipient oligonucleotide, Church teaches starting materials include a cell and polynucleotide constructs that together comprise the sequence of the modified genome (hatched; labeled A,B,C and D) in which polynucleotide construct A is shown as containing a first meganuclease site at the beginning of the construct and a second meganuclease site at the end of the construct (Fig 3A and paragraphs 0132, 0139), and Fig. 4 shows a donor cell comprising first and second meganuclease sites (illustrated as 4 and 5 in Fig. 4) a first recombination site b (equivalent to instant HR1) and a second recombination site yz (equivalent to instant HR 2.1 and 2.2) flanking a sequence of interest (equivalent to instant oligo 1), and the recipient oligonucleotide comprises a third homologous recombination region (HR3) homologous to HR1 and a fourth homologous recombination region (HR4) homologous to HR2.2 as Fig. 4 teaches in Round 1 a recipient comprising a first recombination site b (equivalent to instant HR3) and a second recombination site yz (equivalent to instant HR4) flanking a sequence of interest 3 (equivalent to instant oligo 1) thereby providing following the homologous recombination of HR1 with HR3 and HR2.2 with HR4, a first recombined recipient oligonucleotide comprising the first DNA element fragment.
Regarding step b, Church teaches assembly into larger polynucleotide constructs, or whole genome replacement is achieved by repeated rounds of conjugation and integration (Fig. 3C and D, paragraph 0133) and teaches contacting the recipient cell with a second donor cell under conditions to transfer the second donor plasmid from the second donor cell to the first recipient cell by conjugation (Fig. 4). Church teaches that the product produced by introduction of the polynucleotide constructs are a plurality of cells comprising a single integrated polynucleotide construct replacing the wild-type sequence at that location (Fig 3B, paragraph 0132).
Church teaches that Fig. 3 discloses a second donor plasmid comprising a first homologous region flanking AB (equivalent to instant HR5) and a second homologous region flanking AB (equivalent to instant HR6), and the final modified genome produced by further rounds of conjugation and homologous recombination in which recombination between homologous region flanking AB with homologous region flanking CD forms a DNA assembly (Fig. 3D, paragraph 0099). Figure 4 again shows the second donor plasmids (Round 2) as comprising a first and second endonuclease site (meganuclease sites illustrated as 4 and 5) and the recombination regions
Regarding claims 2 and 3, Church teaches the process is repeated using further rounds of pairwise mixing, conditional cleavage of the incoming polynucleotide construct at the meganuclease site, selection of the incoming selectable marker and facilitated loss of the unwanted portions of the incoming polynucleotide construct using a conditional original of replication and/or negative selectable marker (see e.g. Rounds 2-4 in Fig. 4) until the desired product is achieved (paragraph 0138). Figure 4 shows repeated iterations of third and subsequent donor cells for conjugation with the recipient cell and discloses a plurality of donor cells for each of the first and second rounds.
Figures 3 and 4 of Church are shown below:
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Regarding claim 8, Church teaches providing a plurality of polynucleotide constructs comprising a sequence encoding a first selectable marker and a portion of the plurality of polynucleotide constructs comprise a sequence encoding a second selectable marker (Fig. 3 and paragraphs 0012-0014,0024,0132), and the desired product may be selected using the appropriate selectable marker (see FIG. 3C, paragraph 0133).
Regarding claims 34 and 67, Church teaches the hierarchical assembly methods described herein require polynucleotide constructs for assembly of large product nucleic acids, such as, for example, a modified, partially synthetic, or fully synthetic genome. The sequences obtained from such sources may then be modified using standard molecular biology and/or recombinant DNA technology to produce polynucleotide constructs having desired modifications for reintroduction into, or construction of, a large product nucleic acid, including a modified, partially synthetic or fully synthetic genome (paragraph 0155). Therefore, Church teaches the method is used to combine genetic regions of genes.
Claim Rejections - 35 USC § 103
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.
Claims 9,11,17,18,20 and 21 are rejected under 35 U.S.C. 103 as being unpatentable over Church as applicable to claims 1-3,8,34,66 and 67 above.
Claim Interpretation: Regarding the homologous DNA repair genes and recombination-mediated genetic engineering genes of claims 17 and 18, the instant specification discloses that the donor cell or recipient cell of the methods described herein includes an oligonucleotide encoding one or more homologous DNA repair genes, such as RecA, and in embodiments the homologous DNA repair gene is RecA, and in embodiments the homologous DNA repair genes are the recombineering genes Red alpha, Red beta and Red gamma (paragraph 0120). The instant specification discloses that in embodiments, the recombineering genes are lambda red genes; the recombination-mediated genetic engineering genes are Red alpha, Red beta, and Red gamma (paragraph 0148). Therefore, art teaching RecA reads on a homologous DNA repair gene for claim 17, and art teaching Red alpha, Red beta or Red gamma reads on recombination-mediated genetic engineering genes for claim 18.
The teachings of Church as applied to claims 1-3,8,34,66 and 67 have been described above.
Church does not explicitly teach wherein the recipient oligonucleotide comprises a counter-selectable marker, or wherein the donor plasmid comprises a conditional replication origin, or wherein the donor plasmid or recipient oligonucleotide comprises an oligonucleotide encoding one or more homologous DNA repair genes or encoding one or more recombination-mediated genetic engineering genes, or wherein the assembled DNA element is from 100-500,000 nucleotides in length, or wherein the first, second or subsequence homologous recombination regions and their corresponding HR regions on the recipient oligonucleotide each comprise from about 20 to about 500 base pairs.
However, Church teaches in certain embodiments, negative or counter selectable markers could be placed on the polynucleotide constructs to facilitate loss of the unwanted portions of the donor polynucleotide construct that are transferred to the recipient strain. The negative selectable marker may be incorporated into the polynucleotide construct outside of the region that homologously recombines with the recipient genome such that portions of the polynucleotide construct not incorporated into the genome may be removed by negative selective pressure and/or cells which have incorporated undesired regions of the polynucleotide construct into the recipient genome may be removed using negative selective pressure (paragraph 0137), and that polynucleotide constructs may comprise both selectable and counter selectable markers (paragraph 0143).
Church teaches the upper cell supports replication of the upper polynucleotide construct but not does not support replication of the lower (incoming) polynucleotide construct. This may be achieved using different conditional origins of replication such as, for example, IncX R6K oriγ (dependent on pir protein) or IncPα ori V (dependent on the trfA protein) for the polynucleotide constructs contained in the donor and recipient cells (paragraph 0137). Conditional origins of replication are origins that require the presence or expression of a trans-acting factor in the host cell for replication. A variety of conditional origins of replication functional in prokaryotic hosts (e.g., E. coli) are known to the art. Exemplary conditional origins of replication that may be used in accordance with the hierarchical assembly methods and genome excision methods described herein include, for example, the R6Kγ origin (paragraph 0149).
Church teaches the frequency of homologous recombination in prokaryotes is significantly enhanced by the presence of recombinase activities and several purified proteins catalyze homologous pairing and/or strand exchange in vitro, including: E. coli recA protein, and recombinases, like the recA protein of E. coli are proteins which promote strand pairing and exchange. Church teaches the most studied recombinase to date has been the recA recombinase of E. coli, which is involved in homology search and strand exchange reactions (paragraph 0189). Church teaches RecA is required for induction of the SOS repair response, DNA repair, and efficient genetic recombination in E. coli, and that RecA can catalyze homologous pairing of a linear duplex DNA and a homologous single strand DNA in vitro. In contrast to site-specific recombinases, proteins like recA which are involved in general recombination recognize and promote pairing of DNA structures on the basis of shared homology, as has been shown by several in vitro experiments (paragraph 0189).
Church teaches the polynucleotide constructs assemble in a desired manner by integrating into the host cell genome by homologous recombination, site-specific recombination, or combinations thereof, and when assembly involves homologous recombination, the host cells may express a recombinase such as, for example, recE and recT from E. coli or the Redα and Redβ proteins from lambda (paragraph 0018). Church teaches each polynucleotide construct is separately introduced into a cell, for example, constructs then integrate into the host cell genome by homologous recombination, and that it may be desirable to utilize a host cell that is overexpressing a recombinase and/or comprises a recombinase under the control of an inducible/repressible promoter. Exemplary recombinase systems include, for example, RedE and RecT proteins (from E. coli) or Redα, Redβ and Gam proteins (from lambda) (paragraph 0132).
Church teaches the plurality of polynucleotide segments are each at least about 50 kilobases in length, 100 kilobases in length, or longer (paragraph 0047) and embodiments wherein at least one terminal sequence of each polynucleotide construct is homologous with the terminal sequences of another polynucleotide construct. Such homologous terminal regions may be at least about 20, 50, or more nucleotides in length (paragraph 0020).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to have modified the assembly method of Church to provide the recipient oligonucleotide with a counter-selectable marker with a reasonable expectation of success. There would be a reasonable expectation of success because Church teaches negative or counter selectable markers could be placed on the polynucleotide constructs. One of ordinary skill in the art would have been motivated to do so because Church teaches negative or counter selectable markers could be placed on the polynucleotide constructs to facilitate loss of the unwanted portions of the donor polynucleotide construct that are transferred to the recipient strain, and the negative selectable marker may be incorporated into the polynucleotide construct outside of the region that homologously recombines with the recipient genome such that portions of the polynucleotide construct not incorporated into the genome may be removed by negative selective pressure and/or cells which have incorporated undesired regions of the polynucleotide construct into the recipient genome may be removed using negative selective pressure (paragraph 0137).
Accordingly, the limitations of claim 9 would have been prima facie obvious to one of ordinary skill in the art.
It would have been obvious to one of ordinary skill in the art before the effective filing date, to have modified the assembly method of Church to provide the donor plasmid with a conditional replication origin with a reasonable expectation of success. There would be a reasonable expectation of success because Church teaches a variety of conditional origins of replication functional in prokaryotic hosts (e.g., E. coli) are known to the art. One of ordinary skill in the art would have been motivated to do so because Church teaches that the upper cell supports replication of the upper polynucleotide construct but not does not support replication of the lower (incoming) polynucleotide construct, and this may be achieved using different conditional origins of replication such as, for example, IncX R6K oriγ (dependent on pir protein) or IncPα ori V (dependent on the trfA protein) for the polynucleotide constructs contained in the donor and recipient cells (paragraph 0137) and conditional origins of replication are origins that require the presence or expression of a trans-acting factor in the host cell for replication (paragraph 0149).
Accordingly, the limitations of claim 11 would have been prima facie obvious to one of ordinary skill in the art.
It would have been obvious to one of ordinary skill in the art before the effective filing date, to have modified the assembly method of Church to provide the donor plasmid or recipient cell with an oligonucleotide encoding a homologous DNA repair gene, and an oligonucleotide encoding one or more recombination-mediated genetic engineering genes with a reasonable expectation of success based on the teachings of Church. One of ordinary skill in the art would have been motivated to provide the donor plasmid or recipient cell with an oligonucleotide encoding a homologous DNA repair gene because Church teaches the frequency of homologous recombination in prokaryotes is significantly enhanced by the presence of recombinase activities and that the E. coli recA protein catalyze homologous pairing and/or strand exchange in vitro and promote strand pairing and exchange, and the recA recombinase of E. coli is the most studied recombinase to date and RecA is required for induction of the SOS repair response, DNA repair, and efficient genetic recombination in E. coli (paragraph 0189).
One of ordinary skill in the art would have been motivated to provide the donor plasmid or recipient cell with an oligonucleotide encoding one or more recombination-mediated genetic engineering genes because Church teaches the polynucleotide constructs assemble in a desired manner by integrating into the host cell genome by homologous recombination and the host cells may express a recombinase such as the Redα and Redβ proteins from lambda (paragraph 0018), and that it may be desirable to utilize a host cell that is overexpressing a recombinase and/or comprises a recombinase including RedE and RecT proteins (from E. coli) or Redα, Redβ and Gam proteins (from lambda) (paragraph 0132).
Accordingly, the limitations of claims 17 and 18 would have been prima facie obvious to one of ordinary skill in the art.
It would have been obvious to one of ordinary skill in the art before the effective filing date, that the method of Church would provide an assembled DNA element that is from 100-500,000 nucleotides in length with a reasonable expectation of success. While the exact length of the assembled DNA element is not disclosed by Church, Church teaches the plurality of polynucleotide segments are each at least about 50 kilobases in length, 100 kilobases in length, or longer and it is generally noted that differences in a range of oligonucleotide length do not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such length is critical. "[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). Given that applicant did not point out the criticality of the length of the assembled DNA element of the invention, it is concluded that the normal desire of scientists or artisans to improve upon what is already generally known would provide the motivation to determine where in a disclosed set of ranges is the optimum length NOTE: MPEP 2144.05.
It would have been obvious to one of ordinary skill in the art before the effective filing date to have modified the assembly method of Church wherein the first, second or subsequent homologous recombination regions and their corresponding HR regions on the recipient oligonucleotide each comprise from about 20 to about 500 base pairs based on the teachings of Church. One of ordinary skill in the art would have been motivated to do so because Church teaches wherein at least one terminal sequence of each polynucleotide construct is homologous with the terminal sequences of another polynucleotide construct, and such homologous terminal regions may be at least about 20, 50, or more nucleotides in length (paragraph 0020).
Accordingly, the limitations of claims 20 and 21 would have been prima facie obvious to one of ordinary skill in the art.
Claims 4-7,31 and 35 are rejected under 35 U.S.C. 103 as being unpatentable over Church as applied to claims 1-3,8,34,66 and 67 above, and further in view of Harvard (US 20200392538, Published 17 Dec 2020), cited on an IDS.
The teachings of Church as applied to claims 1-3,8,34,66 and 67 have been described above.
Church does not teach an oligonucleotide encoding a guide RNA or a first endonuclease targeting the first, third and/or fourth endonuclease site present on the first donor plasmid, or an oligonucleotide encoding a guide RNA or a second endonuclease targeting the second, fifth and/or sixth endonuclease site present on the second donor plasmid.
Before the effective filing date, Harvard also teaches methods for hierarchical assembly of synthetic genomes using nuclease assisted homologous recombination which enable scarless and iterative replacement of wild-type DNA with large (at least 50 kb) synthetic DNA segments at desired genomic loci (paragraph 0004). Harvard recites a method of introducing into a parental cell a donor DNA segment flanked by first homology sequences, wherein the parental cell comprises a selectable marker gene and flanked by second homology sequences homologous to the first homology sequences, and an inducible recombineering system, introducing into the parental cell a RNA-guide nuclease or nucleic acid encoding the RNA-guide nuclease and at least one nucleic acid encoding at least one guide RNA targeting the selectable marker gene and inducing expression of the inducible recombineering system (claim 2), and recites the RNA-guided nuclease is Cas9 nuclease (claims 15-16) (an RNA-guided endonuclease). Harvard teaches that gRNA targets cutting of the selectable marker gene in the genome of the parent cell to initiate and enhances recombination (paragraph 0007).
Harvard teaches the parental cells are engineered to carry an inducible sequence-specific nuclease (e.g., genomically), thus, nuclease activity may be induced before, during or after induction of expression of the recombineering system (paragraph 0011) and recites the expression of the nucleic acid encoding the RNA-guided nuclease is inducible (claims 67-68). Harvard teaches a RNA-guided nuclease (e.g., Cas9) and guide RNA (gRNA) targeting the genomic loci for integration are introduced in the cells (e.g., by plasmid transformation), before, during or after induction of expression of the recombineering system and in other embodiments, the parental cells are engineered to carry a RNA-guided nuclease (e.g., genomically), thus, only the gRNA targeting the genomic loci for integration is introduced before, during or after induction of expression of the recombineering system (paragraph 0012).
It would have been obvious to one of ordinary skill in the art before the effective filing date, to modify the first donor plasmid used in the method of Church to comprise an oligonucleotide encoding a guide RNA or endonuclease targeting the first, third and/or fourth endonuclease sites, and to modify the second donor plasmid used in the method of Church to comprise an oligonucleotide encoding a guide RNA or second endonuclease target the second, fifth and/or sixth endonuclease site on the second donor plasmid, and wherein the first and/or second endonuclease is inducible, and wherein the endonuclease is an RNA-guided endonuclease (Cas9) based on the teachings of Harvard with a reasonable expectation of success. There would be a reasonable expectation of success as Harvard also pertains to methods for hierarchical assembly of synthetic genomes using nuclease assisted homologous recombination and would amount to combining prior art elements according to known methods to yield predictable results.
One of ordinary skill in the art would have been motivated to modify the first donor plasmid used in the method of Church to comprise an oligonucleotide encoding a guide RNA or endonuclease targeting the first, third and/or fourth endonuclease sites, and to modify the second donor plasmid used in the method of Church to comprise an oligonucleotide encoding a guide RNA or second endonuclease target the second, fifth and/or sixth endonuclease site on the second donor plasmid because Harvard recites a method of introducing into a parental cell a donor DNA segment flanked by first homology sequences, wherein the parental cell comprises a selectable marker gene and flanked by second homology sequences homologous to the first homology sequences, and an inducible recombineering system, introducing into the parental cell a RNA-guide nuclease or nucleic acid encoding the RNA-guide nuclease and at least one nucleic acid encoding at least one guide RNA targeting the selectable marker gene and inducing expression of the inducible recombineering system (claim 2), and recites the RNA-guided nuclease is Cas9 nuclease (claims 15-16) (an RNA-guided endonuclease), and teaches that gRNA targets cutting of the selectable marker gene in the genome of the parent cell to initiate and enhances recombination (paragraph 0007). One of ordinary skill in the art would be motivated to provide an inducible first and/or second endonuclease and to induce expression of the first and/or second endonuclease because Harvard recites the expression of the nucleic acid encoding the RNA-guided nuclease is inducible (claims 67-68) and teaches the parental cells are engineered to carry an inducible sequence-specific nuclease (e.g., genomically), thus, nuclease activity may be induced before, during or after induction of expression of the recombineering system (paragraph 0011). One of ordinary skill in the art would be motivated to provide an RNA-guided endonuclease as the first and/or second endonuclease because Harvard teaches a RNA-guided nuclease (e.g., Cas9) and guide RNA (gRNA) targeting the genomic loci for integration are introduced in the cells (e.g., by plasmid transformation), before, during or after induction of expression of the recombineering system and in other embodiments and the parental cells are engineered to carry a RNA-guided nuclease (e.g., genomically) (paragraph 0012).
Accordingly, the limitations of claims 4-7 would have been prima facie obvious to one of ordinary skill in the art.
Church does not teach the method using two or more recipient oligonucleotides having compatible homologous recombination regions to construct a DNA library.
Harvard teaches the methods of the present disclosure are used for multiplexed integration of large-size DNA libraries, for example recoded segment libraries.
It would have been obvious to one of ordinary skill in the art before the effective filing date, to modify the method of Church to use two or more recipient oligonucleotides to construct a DNA library based on the teachings of Harvard with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to do so based on the teachings of Harvard suggesting that the methods of Harvard can be used for multiplexed integration of large-size DNA libraries, including recoded segment libraries.
Accordingly, the limitations of claim 31 would have been prima facie obvious to one of ordinary skill in the art.
Church does not teach the first and second oligonucleotides comprising the first and second DNA element fragments are inserted into the first and second donor plasmids prior to steps (a) and (b).
Harvard teaches methods and compositions (e.g., cells, genetic constructs, and kits) for targeted scarless integration of large DNA segments (e.g., at least 50 kb) from a donor into a receiver (parental) strain genome, and each assembly cycle (integration of a single segment) of these methods for genome assembly can be iterated for integration of multiple donor DNA segments in a sequential or parallel manner. Harvard teaches donor DNA segments may be assembled on a plasmid from synthetic DNA segments and the donor DNA is then introduced into the parental cells (paragraph 0010). Therefore, Harvard teaches insertion of donor DNA segments into the plasmid prior to the donor DNA segments being introducing into the parental cells (donor cells).
It would have been obvious to one of ordinary skill in the art before the effective filing date, to modify the method of Church to include a step of inserting the first and second oligonucleotides comprising the first and second DNA element fragments into the first and second donor fragments prior to the steps of contacting the first donor cells comprising the donor plasmids with the recipient cells based on the teachings of Harvard with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to do so because Harvard teaches methods for genome assembly can be iterated for integration of multiple donor DNA segments in a sequential or parallel manner and that donor DNA segments may be assembled on a plasmid from synthetic DNA segments and the donor DNA is then introduced into the parental cells (paragraph 0010).
Accordingly, the limitations of claim 35 would have been prima facie obvious to one of ordinary skill in the art.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Church as applied to claims 1-3,8,34,66 and 67 above, and further in view of Szybalski et al. (EP2423314, Published 29 Feb 2012).
The teachings of Church as applied to claims 1-3,8,34,66 and 67 have been described above.
Church does not teach wherein the donor plasmid or recipient oligonucleotide comprises an inducible high-copy replication origin.
Before the effective filing date, Szybalski et al. taught the bacterial artificial chromosome (BAC) vectors were developed for the construction and faithful maintenance of genomic libraries in bacteria, and BAC vectors are based on the F plasmid, which maintains the vector at 1-2 copies per cell. Low copy number is an important feature for clone stability by limiting the amount of homologous sequence subject to recombination. However, the crucial disadvantage of low copy number is that it makes both preparing the vector for cloning and downstream analyses of clones, such as by sequencing and fingerprinting, more costly and laborious (paragraph 0003). Szybalski et al. taught medium- to high-copy number plasmids are often preferred over the low- or single-copy number plasmids for over-production of recombinant DNA and protein, because they typically lead to high yields of the target products (paragraph 0004). Szybalski et al. taught a vector may comprise an excisable fragment comprising an insertion site, a first and second origin of replication, and a pair of transcriptional terminators flanking the excisable fragment… the second origin of replication may be an inducible high-copy number origin of replication, such asoriV (paragraph 0007). Szybalski et al. taught the origin of replication may also be a high-copy origin of replication, which may not have a partitioning system. A high-copy origin of replication may segregate by mass action, which may cause empty cells to be minimized by the low probability in view of the number of vector copies. The high-copy origin of replication may maintain a high number of vectors in a host cell. The high-copy origin of replication may maintain 5-100 copies of the vector in a host cell. The high-copy origin of replication may also maintain more than 100 to more than 1000 copies of the vector in a host cell and a representative example of a high-copy origin of replication isoriV, and that a high-copy origin of replication may be used to increase production of the vector in a host cell.
It would have been obvious to one of ordinary skill in the art to have modified the donor plasmid used in the method of Church to comprise an inducible high-copy replication origin based on the teachings of Szybalski et al. with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to do so because Szybalski et al. taught the disadvantages of low copy number is that it makes both preparing the vector for cloning and downstream analyses of clones, such as by sequencing and fingerprinting, more costly and laborious, and that high-copy plasmids typically lead to high yields of the target products (paragraph 0004) and the high-copy origin of replication may maintain 5-100 copies or 100 to more than 1000 copies of the vector in a host cell and that a high-copy origin of replication may be used to increase production of the vector in a host cell.
Accordingly, the limitations of claim 12 would have been prima facie obvious to one of ordinary skill in the art.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Church as applied to claims 1-3,8,34,66 and 67 above, and further in view Jones et al. (PLOS One July 2013, Vol. 8. Issue 7, e69319).
Claim Interpretation: The instant specification discloses the donor plasmid or recipient plasmid comprises a replicon that can replicate plasmids at least 30 kilobases in length (paragraph 0270). In further embodiments, the replicon is from a P1-derived artificial chromosome or a bacterial artificial chromosome (paragraph 0269). Therefore, art teaching a replicon from a P1-derived artificial chromosome or bacterial artificial chromosome reads on claim 13.
The teachings of Church as applied to claims 1-3,8,34,66 and 67 have been described above.
Church does not teach wherein the donor plasmid or recipient oligonucleotide comprises a replicon that can replicate plasmids of lengths greater than 30 kilobases.
Before the effective filing date, Jones et al. taught a procedure for the conjugative transfer of phage P1-derived Artificial Chromosome (PAC) library clones containing large natural product gene clusters (greater than or equal to 70 kilobases) to Streptomyces coelicolor strains that have been engineered for improved heterologous production of natural products (Abstract). Jones et al. taught while heterologous expression has been used successfully with small biosynthetic gene clusters (less than or equal to 40 kb), standard techniques are not as straightforward with large biosynthetic gene clusters (greater than or equal to 70 kb), largely because of the amount of DNA that needs to be cloned and transferred into a suitable expression host (introduction, left column). Jones et al. taught an approach which transfers large natural product gene clusters using phage P1-derived Artificial Chromosomes (PACs) for stable integration of PAC DNA via conjugation which creates a platform for gene cluster analysis, and enables targeted systems and synthetic biology approaches that may not be possible in wild-type strains.
It would have been obvious to one of ordinary skill in the art to have modified the donor plasmid used in the method of Church to comprise a P1-derived Artificial Chromosome (PAC) in order to replicate and transfer large natural product gene clusters based on the teachings of Jones et al. with a reasonable expectation of success, as this would amount to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to do so because Jones et al. taught that standard techniques are not as straightforward with large biosynthetic gene clusters (greater than or equal to 70 kb), because of the amount of DNA that needs to be cloned and transferred into a suitable expression host, and therefore taught an approach which transfers large natural product gene clusters using phage P1-derived Artificial Chromosomes (PACs) for stable integration of PAC DNA via conjugation which creates a platform for gene cluster analysis, and enables targeted systems and synthetic biology approaches that may not be possible in wild-type strains.
Accordingly, the limitations of claim 35 would have been prima facie obvious to one of ordinary skill in the art.
Conclusion
Claims 1-9,11-13,17,18,20,21,31,34,35,66 and 67 are rejected.
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/STEPHANIE L SULLIVAN/Examiner, Art Unit 1635
/ABIGAIL VANHORN/Primary Examiner, Art Unit 1636
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