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 .
Claim Status
The amended claim set filed on 18 September 2025 is acknowledged. Claims 17-23 and 26-49 are currently pending. Of those, claims 17, 19, and 21 are amended. Claims 26-49 are new, and claims 1-16 and 24-25 are cancelled. Claims 17-23 and 26-49 will be examined on the merits herein.
Election/Restrictions
Applicant’s election without traverse of Group III (claims 17-23) in the reply filed on 18 September 2025 is acknowledged.
Priority
The instant application is a 371 of application PCT/AU2021/051121 (filed 24 September 2021) and claims priority to Australian Provisional Application No. 2020903460 (filed 25 September 2020). Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55. However, it appears that the submitted foreign priority document filed on 24 March 2023 may be incomplete and only contains support for the subject matter of claims 17-23 (see para. 7-17). WO 2022/061417 A1 (PCT/AU2021/051121) contains support for the subject matter of claims 26-49 in at least para. 9-13, 15-18, and 29. Therefore, for the purposes of searching the prior art, the effective filing date of instant claims 17-23 is 25 September 2020 and claims 26-49 is 24 September 2021.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 24 March 2023 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Specification
The disclosure is objected to because of the following informalities: in at least para. 10, 30, 65-68, and 127, bacterial genera and/or species (e.g., Synechocystis, Synechococcus, etc.) should be italicize. Appropriate correction is required.
The use of the term “GenBank” (see at least para. 62 and 64), which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
Claims 19-20, 29-31, and 47-49 are objected to because of the following informalities:
In claim 19, line 8, “a the third landing zone” should read “and a third landing zone”;
In claim 20, line 1, “comprising” should be followed by a colon; and
In claims 29-31 and 47-49, bacterial genera and species names (e.g., Synechocystis sp., Synechococcus sp., Synechococcus elongatus, Anabaena variabilis, and Leptolyngbya sp.) should be italicized.
The attempt to incorporate subject matter into this application by reference to GenBank® identifiers in claims 29-31 is ineffective because the root words "incorporate" and/or "reference" have been omitted, see 37 CFR 1.57(c)(1).
Also, the incorporation of essential material in the specification by reference to an unpublished U.S. application, foreign application or patent, or to a publication is improper. Applicant is required to amend the disclosure to include the material incorporated by reference, if the material is relied upon to overcome any objection, rejection, or other requirement imposed by the Office. The amendment must be accompanied by a statement executed by the applicant, or a practitioner representing the applicant, stating that the material being inserted is the material previously incorporated by reference and that the amendment contains no new matter. 37 CFR 1.57(g).
The incorporation by reference will not be effective until correction is made to comply with 37 CFR 1.57(c), (d), or (e). If the incorporated material is relied upon to meet any outstanding objection, rejection, or other requirement imposed by the Office, the correction must be made within any time period set by the Office for responding to the objection, rejection, or other requirement for the incorporation to be effective. Compliance will not be held in abeyance with respect to responding to the objection, rejection, or other requirement for the incorporation to be effective. In no case may the correction be made later than the close of prosecution as defined in 37 CFR 1.114(b), or abandonment of the application, whichever occurs earlier.
Any correction inserting material by amendment that was previously incorporated by reference must be accompanied by a statement that the material being inserted is the material incorporated by reference and the amendment contains no new matter. 37 CFR 1.57(g).
Appropriate correction is required.
Claim Interpretation
The instant claims recite elements that are referred to by number; for example, claim 19 recites the elements: a first nucleic acid of interest, a first nucleic acid insert, a first landing zone, a fifth landing zone, a second selectable marker, and a third landing zone. The numbering of such elements in the instant claims is interpreted as a naming convention specifying distinct elements, and does not necessarily indicate a particular number of elements are present in a given structure. For example, claim 19 recites a first nucleic acid insert comprising a fifth landing zone, but the presence of the “fifth” landing zone does not necessarily indicate that five landing zones be present; only that the fifth landing zone is distinct from the other landing zones recited.
Claims 17, 19, and 21 are drawn to separate methods for generating a recombinant Cyanobacterium cell and do not depend one another. Thus, the numbered claim elements are interpreted as having the same structure only when recited in the same group of method claims (i.e., claim 17 and its dependents, claim 19 and its dependents, and claim 21 and its dependents). For example, a “first landing zone” recited in claim 19 or one of its dependents is not necessarily the same “first landing zone” recited in claim 21 or one of its dependents because they are drawn to separate methods.
Additionally, claims 17, 19, and 21 each recite a step of contacting a Cyanobacterium cell with a nucleic acid insert “under conditions that allow recombination of the nucleic acid insert with a nucleic acid cassette….” This limitation does not actually require the presence of a nucleic acid cassette or construct in the cell or cell genome, only that recombination of the nucleic acid insert with the nucleic acid cassette/construct be possible at some later possible step.
Claim Rejections - 35 USC § 112(b)
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.
Claim 17-23 and 26-49 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 17 (upon which claims 18, 23, 26, 29, 32, 35, 38, 41, 44, and 47 depend), in lines 6-7, the claim recites, “the nucleic acid insert comprises the nucleic acid of interest and a second selectable marker flanked by two landing zones”. As written, it is unclear if only the second selectable marker is flanked by two landing zones or if the nucleic acid of interest and the second selectable marker are located between the two landing zones. In the interest of compact prosecution, the scope of this limitation is assumed to encompass both interpretations.
Also, claim 17 recites the limitation “the landing zones in the cell’s genome” in line 8. It is unclear if there is antecedent basis for this limitation. MPEP 2173.05(e) states: “Inherent components of elements recited have antecedent basis in the recitation of the elements themselves.” However, cyanobacteria cell genomes do not inherently have landing zones, so the recitation of the “landing zones in the cell’s genome” cannot claim antecedent basis to the earlier recitation of “the genome of the Cyanobacterium cell.”
Regarding claim 19 (upon which claims 20, 27, 30, 33, 36, 39, 42, 45, and 48 depend), lines 8-9 recite the limitation "the first and/or third landing zones". There is insufficient antecedent basis for this limitation in the claim. Step (a) of claim 19 recites two alternative wherein clauses, and the limitation “the first and/or third landing zones” in the second wherein clause cannot claim antecedent basis to the earlier recitation of “a first landing zone” and “a third landing zone” because the first wherein clause is not required by the second wherein clause.
Claim 19, lines 9-10 recite, “the first and/or third landing zones in the cell, respectively.” There is insufficient antecedent basis for this limitation in the claim. MPEP 2173.05(e) states: “Inherent components of elements recited have antecedent basis in the recitation of the elements themselves.” However, cyanobacteria cells do not inherently have landing zones, so the recitation of the “landing zones in the cell” cannot claim antecedent basis to the earlier recitation of “a Cyanobacterium cell.”
Claim 20 recites the limitation "the nucleic acid construct" in line 4. There is insufficient antecedent basis for this limitation in claim 20 or claim 19, upon which claim 20 depends.
Claim 20 recites the limitation "the fourth landing zone" in line 7. There is insufficient antecedent basis for this limitation in claim 20 or claim 19, upon which claim 20 depends.
Claim 20 recites the limitation “the fourth… landing [zone]” in lines 7-8. There is insufficient antecedent basis for this limitation in the claim. Step (c) of claim 20 recites two alternative wherein clauses, and the limitation “the fourth… landing [zone]” in the second wherein clause cannot claim antecedent basis to the earlier recitation of a “fourth landing zone” because the first wherein clause is not required by the second wherein clause.
Claim 20 recites the limitation “the fourth… landing [zone] in the cell” in lines 8-9. There is insufficient antecedent basis for this limitation in the claim. MPEP 2173.05(e) states: “Inherent components of elements recited have antecedent basis in the recitation of the elements themselves.” However, cyanobacteria cells do not inherently have landing zones, so the recitation of the “landing zones in the cell” cannot claim antecedent basis to the earlier recitation of “a Cyanobacterium cell.”
Claim 21 (upon which claims 22, 28, 31, 34, 37, 40, 43, 46, and 49 depend) recites the limitation "the first nucleic acid insert" in lines 3-4. There is insufficient antecedent basis for this limitation in the claim.
Claim 21 recites the limitation "the nucleic acid construct" in line 5. There is insufficient antecedent basis for this limitation in the claim.
Claim 21 recites the limitation "the second and/or fourth landing zones" in line 8. There is insufficient antecedent basis for this limitation in the claim. Step (a) of claim 21 recites two alternative wherein clauses, and the limitation “the second and/or fourth landing zones” in the second wherein clause cannot claim antecedent basis to the earlier recitation of “a second landing zone” and “a fourth landing zone” because the first wherein clause is not required by the second wherein clause.
Claim 21, lines 9-10 recite, “the second and/or fourth landing zones in the cell, respectively.” There is insufficient antecedent basis for this limitation in the claim. MPEP 2173.05(e) states: “Inherent components of elements recited have antecedent basis in the recitation of the elements themselves.” However, cyanobacteria cells do not inherently have landing zones, so the recitation of the “landing zones in the cell” cannot claim antecedent basis to the earlier recitation of “a Cyanobacterium cell.”
Claim 22 recites the limitation "the first landing zone" in line 5. There is insufficient antecedent basis for this limitation in claim 22 or claim 21, upon which claim 22 depends.
Claim 22 recites the limitation “the first… landing [zone]” in lines 7-8. There is insufficient antecedent basis for this limitation in the claim. Step (c) of claim 22 recites two alternative wherein clauses, and the limitation “the first… landing [zone]” in the second wherein clause cannot claim antecedent basis to the earlier recitation of “the first landing zone” because the first wherein clause is not required by the second wherein clause.
Claim 22 recites the limitation "the first… landing [zone] in the cell" in 8-9. There is insufficient antecedent basis for this limitation in the claim. MPEP 2173.05(e) states: “Inherent components of elements recited have antecedent basis in the recitation of the elements themselves.” However, cyanobacteria cells do not inherently have landing zones, so the recitation of the “landing zones in the cell” cannot claim antecedent basis to the earlier recitation of “a Cyanobacterium cell.”
Claim 23 recites the limitation "the one or more of the nucleic acid of interest, first nucleic acid of interest, or second nucleic acid of interest." There is insufficient antecedent basis for this limitation in claim 23 or claim 17 (upon which claim 23 depends), which only recites “a nucleic acid of interest.”
Regarding claims 26-28, each recites, “wherein the nucleic acid cassette is integrated into the genome of the Cyanobacterium cell….” It is not clear if this limitation is an active step in the methods set forth in claims 17, 19, and 21, or if this limitation limits the structure of the Cyanobacterium cell used in the methods of claims 17, 19, and 21. If claims 26-28 limit the structure of the Cyanobacterium cell, to which step(s) of the methods of claims 17, 19, and 21 does the limitation apply? In the interest of compact prosecution, the limitation of claims 26-28 has been interpreted to limit the structure of the Cyanobacterial cell prior to contact with a nucleic acid insert (for claim 17) or first nucleic acid insert (for claims 19 and 21) when searching the art.
Regarding claims 26-28, the term “substantially homologous” is a relative term which renders the claim indefinite. The term “substantially homologous” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. While one of ordinary skill in the art would be able to determine if two sequences are homologous using tools and teachings known in the art, one would not be able to determine the scope of neutral site sequences claimed because it is not clear what constitutes a substantially homologous sequence. In the interest of compact prosecution, and for the purposes of searching the prior art, a neutral site sequence is “substantially homologous” if it allows for chromosomal integration of DNA via homologous recombination.
Regarding claims 26-28, the term “non-essential” is a relative term which renders the claim indefinite. The term “non-essential” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. Para. 61 of the instant specification states, “neutral sites can be homologous to any region of the microbial genome is not required for viability and can therefore be tailored using methods known in the art…. Non-essential regions are known in the art.” However, there is not a clear definition of a “non-essential region” in the art, and genes essential for viability vary based on the media and conditions with which bacteria are grown. In the interest of compact prosecution, a region taught as being “non-essential” or not required/necessary for viability will be interpreted as meeting the limitation.
Claims 29-31 contain the trademark/trade name GenBank®. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to describe accession numbers identifying several non-essential regions of bacterial genomes and, accordingly, the identification/description is indefinite because the regions recited in the claims cannot be identified without the GenBank® accession numbers.
Also, the incorporation of essential material in the specification by reference to an unpublished U.S. application, foreign application or patent, or to a publication is improper. 37 CFR 1.57(d) states "Essential material" is material that is necessary to (2) describe the claimed invention in terms that particularly point out and distinctly claim the invention as required by 35 U.S.C. 112(b). Without the sequences cited by the GenBank® accession numbers, the non-essential regions cannot be identified by one of ordinary skill in the art. Therefore, the claim is indefinite.
Regarding claims 32-34, the term “about” is a relative term which renders the claim indefinite. The term “about” is not defined by the claim, the definition in the specification (para. 35) is not sufficient to provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. While one of ordinary skill in the art would be able to determine the length of a nucleic acid sequence, one would not be able to determine the full scope of sequence lengths encompassed by the term “about 500bp,” etc.
Regarding claims 35-37, the term “approximately” is a relative term which renders the claim indefinite. The term “approximately” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. While one of ordinary skill in the art would be able to determine the GC content of a nucleic acid using the tools and teachings known in the art, one would not be able to determine the scope of GC content encompassed by the phrase “approximately 50%.”
Regarding claims 35-37, the phrase “the landing zones comprises” (in lines 1 and 5 of each claim) is grammatically incorrect, and should read “landing zones comprise” or “landing zone comprises.” It is unclear which phrase is intended and thus, it is unclear whether the claim requires each landing zone to comprise a core sequence consisting of a randomly generated nucleic acid sequence with a GC content of approximately 50% and lacking a bacterial promoter sequence, least one transcriptional terminator, and at least one translational insulator, and whether each landing zone comprises a transcriptional terminator and a translational insulator at either end of the core sequence, or if each pair/group of landing zones are collectively required to have all of these requires elements. Additionally, the antecedent basis for “the landing zones” is unclear as claims 17, 19, and 21 (upon which claims 35-37 depend, respectively) each recite one or more landing zones within a nucleic acid insert and two landing zones in the cell or cell genome. In the interest of compact prosecution, these claims have been interpreted to refer to each landing zone, as is supported by para. 48 and 68-69 and FIG. 11 of the instant specification.
Claim 41 recites “the selectable marker gene.” However, claim 41 depends upon both claims 17-18, which recite a “second selectable marker gene,” and claim 26, which recites a “first selectable marker gene.” Thus, it is unclear which selectable marker gene claim 41 refers to, or if the claim is intended to limit both the first and second selectable marker genes, meaning that both selectable marker genes confer resistance to the same antibiotic. In the interest of compact prosecution, “the selectable marker gene” has been interpreted to mean “one or both of the first and second selectable marker genes.”
Claim 42 recites “the selectable marker gene.” However, claim 42 depends upon claim 19, which recites a “second selectable marker gene,” claim 20, which recites a “further selectable marker gene”, and claim 27, which recites a “first selectable marker gene.” Thus, it is unclear which of the three selectable marker genes claim 42 refers to, or if the claim is intended to limit all three selectable marker genes, meaning that all three selectable marker genes confer resistance to the same antibiotic. In the interest of compact prosecution, “the selectable marker gene” has been interpreted to mean “one or more of the first, second, and further selectable marker genes.”
Claim 43 recites “the selectable marker gene.” However, claim 43 depends upon claim 21, which recites a “second selectable marker gene,” claim 22, which recites a “further selectable marker gene”, and claim 28, which recites a “first selectable marker gene.” Thus, it is unclear which of the three selectable marker genes claim 43 refers to, or if the claim is intended to limit all three selectable marker genes, meaning that all three selectable marker genes confer resistance to the same antibiotic. In the interest of compact prosecution, “the selectable marker gene” has been interpreted to mean “one or more of the first, second, and further selectable marker genes.”
Clarification is requested.
Claim Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 17-23, 26, and 29-49 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
The focus of the enablement inquiry is whether everything within the scope of the claim(s) is/are enabled, at the time of filing, without requiring undue experimentation to make or use the invention. The factors to be considered in determining whether a disclosure would require undue experimentation include: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the inventor; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. In re Wands, 8 USPQ2d, 1400 (CAFC 1988) and MPEP 2164.01.
The breadth of the claims: With respect to claim breadth, the standard under 35 U.S.C. §112(a) or 35 U.S.C. §112, first paragraph, entails the determination of what the claims recite and what the claims mean as a whole. The claims are drawn to methods for generating a recombinant Cyanobacterium cell.
Claim 17 (upon which claims 18, 23, 26, 29, 32, 35, 38, 41, 44, and 47 depend), recites the step, “contacting a Cyanobacterium cell with a nucleic acid insert under conditions that allow recombination of the nucleic acid insert with a nucleic acid cassette in the genome of the Cyanobacterium cell; wherein the nucleic acid insert comprises the nucleic acid of interest and a second selectable marker flanked by two landing zones, each comprising a sequence at least 90% identical to the landing zones in the cell’s genome.” The broadest reasonable interpretation of the claim is that the nucleic acid cassette is not required to be part of the genome, only that recombination of the nucleic acid insert and the nucleic acid cassette in the genome be possible in a possible later step. Additionally, the broadest reasonable interpretation is that the two landing zones may be any two landing zones.
Claim 19 (upon which claims 20, 27, 30, 33, 36, 39, 42, 45, and 48 depend), recites a step of “contacting a Cyanobacterium cell with a first nucleic acid insert under conditions that allow recombination of the first nucleic acid insert with a nucleic acid cassette in the genome of the cell… wherein the first and/or third landing zones have at least 90% sequence identity to the first and/or third landing zones in the cell, respectively….” As with claim 17, the nucleic acid cassette is not required to be part of the genome.
Claim 21 (upon which claims 22, 28, 31, 34, 37, 40, 43, 46, and 49 depend), recites a step of “contacting a Cyanobacterium cell with the first nucleic acid insert under conditions that allow recombination of the first nucleic acid insert with the nucleic acid construct in the genome of the cell… wherein the second and/or fourth landing zones have at least 90% sequence identity to the second and/or fourth landing zones in the cell, respectively….” As with claims 17 and 19, the nucleic acid cassette is not required to be part of the genome.
Claim 26, which is dependent upon claim 17, recites, “a heterologous nucleic acid comprising at least two landing zones….” The broadest reasonable interpretation of this limitation is that the nucleic acid cassette may comprise any two landing zones; thus, the two landing zones recited in claim 17 and the two landing zones recited in claim 26 are not necessarily the same.
The state of the prior art and the level of predictability in the art: George et al. (WO 2017/201311 A2; cited in IDS) teaches insertion of exogenous donor DNA into a host cell using “landing pads” inserted into the genome that contain sequences homologous to library sequences attached to upstream and downstream regions of nucleic acids of interest (Abstract and para. 13). Targeted landing pads are inserted into the genome via homologous recombination between neutral loci in the genome and homologous endogenous genomic sequences on either end of the landing pad (George et al., para. 89, 190, and 192). Homologous recombination occurs between the homologous sequences of the exogenous DNA insert and the landing pads in the genome (George et al., Abstract). George does not teach any examples of integrating exogenous nucleic acids into a genome that does not comprise at least one landing pad or integrating into the genome a landing pad that is not flanked by endogenous genomic sequences.
Based on the prior art, one of ordinary skill in the art would not be able to predict that a recombinant Cyanobacterial cell could be generated under conditions that allow for recombination of a nucleic acid insert with a nucleic acid cassette without the presence of the nucleic acid insert in the cell genome, because the recited nucleic acid insert does not contain sequences that are homologous with any part of the genome that are necessary for homologous recombination to occur.
The amount of direction provided by the inventor and the existence of working examples: The instant specification teaches that homologous recombination occurs with the alignment of homologous nucleic acid sequences (para. 57). The instant specification teaches an example in which WT Synechococcus sp. PCC 7002 is transformed with a first plasmid comprising a spectinomycin resistance gene between two landing zones, which is flanked by portions of a neutral site in the cell’s genome (para. 126). Transformed cells (i.e., those containing the two landing zones) were then transformed with a different plasmid comprising a different selection marker flanked by the same two landing zones inserted by the first plasmid (para. 127 and 129). The specification does not teach any examples in which a nucleic acid insert comprising two or more landing zones was integrated into a Cyanobacterial cell genome without the presence of a nucleic acid cassette containing landing zones homologous to those in the nucleic acid insert. The instant specification also does not teach any examples of a nucleic acid cassette comprising two landing zones able to homologously recombine with a nucleic acid insert comprising two landing zones that do not match the landing zones in the cassette. The specification also does not explain how homologous recombination could occur between a nucleic acid insert and a Cyanobacterium cell genome that do not share any homologous sequences. Therefore, what is enabled by the instant specification and working examples is narrow in comparison to the scope of the claims, and the specification does not provide enough information with which one may overcome the known unpredictability in the art.
The quantity of experimentation needed to make or use the invention based on the content of the disclosure: The standard of an enabling disclosure is not the ability to make and test if the invention works but one of the ability to make and use with a reasonable expectation of success. A patent is granted for a completed invention, not the general suggestion of an idea (MPEP 2164.03 and Chiron Corp. v. Genentech Inc., 363 F.3d 1247, 1254, 70 USPQ2d 1321, 1325-26 (Fed. Cir. 2004)). The instant specification is not enabling for the claimed invention because one cannot follow the guidance presented therein, or within the art at the time of filing, and perform the claimed method without first making a substantial inventive contribution.
The instant specification and the teachings of the art prior to the effective filing date of the claimed invention both teach that, in order for an exogenous nucleic acid to be inserted into a bacterial genome, there must be sequences in both the exogenous nucleic acid insert and the genome that are homologous with each other. Therefore, one of ordinary skill in the art could not generate a recombinant Cyanobacterium using the methods of claims 17, 19, 21, and 26 because there is no teaching in the art or the specification that would allow one to induce conditions that would allow for recombination of a nucleic acid insert into a genome that does not share any homologous sequences with that insert.
Therefore, claims 17-23, 26, and 29-49 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, for failing to meet the enablement requirement.
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 17-23 and 47-49 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Cheah et al. (2012, Biotechnol. Progress, herein “Cheah”).
Regarding claims 17-19 and 21, Cheah teaches a method for generating a recombinant Cyanobacterium cell comprising contacting a Synechocystis cell with a mazF/aphII cassette (i.e., a nucleic acid insert) comprising the nrsR-nrsS cluster (i.e., nucleic acid of interest or first nucleic acid of interest) and, aphII, a kanamycin resistance gene (i.e., selectable marker gene) (pg. 26, right col., para. 5). The nrsR-nrsS cluster and aphII gene are flanked by an upstream homologous region and a downstream homologous region (i.e., landing zones) that were cloned from the slr1609 gene or slr1993 and slr1994 genes (i.e., having at least 90% identity to the landing zones in the genome) (pg. 24, right col., para. 2 and Table 1). Cheah also teaches culturing the recombinant Synechocystis cell on kanamycin plates, thereby selecting for cells comprising the mazF/aphII cassette (pg. 26, left col., para. 3).
Regarding claims 20 and 22, Cheah teaches transforming the Synechocystis comprising the mazF/aphII cassette with either plasmid pUC19m 1609 HR or pUC19m PHB HR, each comprising an upstream homologous region and a downstream homologous region (i.e., landing zones) clones from either the slr1609 gene or slr1993 and slr1994 genes (i.e., having at least 90% identity to the landing zones in the genome) (pg. 26, left col., para. 4). This step was followed by culturing the cell on media containing nickel or kanamycin, thereby selecting for cells that were transformed by either pUC19m 1609 HR or pUC19m PHB HR, removing the nrsR-nrsS cluster and/or the aphII gene (pg. 26, left col., para. 4).
Regarding claim 23, Cheah teaches that the nrsR-nrsS cluster is operably linked to the inducible promoter PnrsB (pg. 26, right col., para. 5).
Regarding claims 47-49, Cheah teaches that the Cyanobacterium is Synechocystis sp. PCC 6803 (pg. 23, left col., para. 1).
Claim Rejections - 35 USC § 103
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.
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.
Claims 17-23, 26-43, and 47-49 are rejected under 35 U.S.C. 103 as being unpatentable over George (WO 2017/201311) in view of Vogel et al. (2017, J. Biol. Eng.; cited in IDS; herein “Vogel”), Brophy et al. (2014, Nat. Methods; herein “Brophy”), and Li et al. (2013, “Synechococcus sp. PCC 7002, complete genome”; herein “Li”).
Regarding claims 17-19 and 21, George teaches methods of generating recombinant bacterial cells comprising contacting the cell containing exogenous landing pads, which comprise library sequences for homologous recombination, with an exogenous donor nucleic acid (i.e., nucleic acid insert), which may comprise, in a 5’ to 3’ orientation, an upstream library sequence (i.e., landing zone), a first nucleic acid of interest (such as a selectable marker, see para. 119), a linker (i.e., a landing zone), another nucleic acid of interest (such as a selectable marker, see para. 119), and a downstream library sequence (i.e., landing zone), in which the upstream library sequence, linker, and downstream library sequence (i.e., landing zones) are capable of homologously recombining with the upstream landing pad homology sequence, linker, and downstream landing pad homology sequence in the cell genome (i.e., landing zones in the cell genome) (para. 12-13 and 70). George teaches that the upstream and downstream landing pad homology sequences (i.e., landing zones) may comprise the same sequence as the upstream and downstream library sequences (i.e., landing zones) of the exogenous donor nucleic acid, respectively (para. 92). George also teaches a step of growing the transformed cell on selective media, thereby selecting for cells in which the exogenous nucleic acid (comprising the first nucleic acid of interest and selectable marker) has been integrated into the cells genome (para. 119).
Regarding claims 20 and 22, George teaches that a plurality of component polynucleotides (i.e., polynucleotides comprising at least an upstream or downstream library sequence, nucleic acid of interest, and a linker) homologously recombine to form assembled component polynucleotides (i.e., nucleic acid inserts having different nucleic acids of interest and landing zones), which can then homologously recombine with exogenous landing pads containing nucleic acid inserts integrated in to the cell genome (para. 8).
Regarding claim 23, George teaches that a nucleic acid of interest may comprise a promotor (para. 118) and teaches the inducible promoter, pGAL1 (para. 251).
Regarding claims 26-28, George teaches methods of targeted integration of nucleic acids, in which a host cell comprises one or more exogenous landing pads integrated in the host cell’s genome, which comprises endogenous genomic sequences (i.e., neutral sites) adjacent to landing pad homology sequences (i.e., landing zones) in a landing pad allow for targeted insertion of exogenous donor nucleic acid (para. 190). George teaches that the landing pads may be integrated at “neutral loci” such as intergenic regions of the host cell genome with no known function or a non-coding region (para. 89), i.e., the endogenous genomic sequences are “substantially homologous to a part of a non-essential region of the bacterial genome. In order for these endogenous genomic sequences (i.e., neutral sites) to facilitate insertion of the exogenous landing pad, they must be flanking the exogenous landing pad (see para. 24 and FIG. 1A). The method comprises contacting the host cell comprising one or more exogenous landing pads integrated in the host cell’s genome with one or more exogenous donor nucleic acids (i.e., heterologous nucleic acid), comprising, one or more first component polynucleotides (para. 190(a)(ii), comprising, in a 5’ to 3’ direction, an upstream library sequence (i.e., landing zone), a first nucleic acid of interest (such as a selectable maker), a first linker (i.e., landing zone, see para. 70), and one or more last component polynucleotide (para. 192(a)(iii)), comprising, in a 5’ to 3’ direction, a last linker (i.e., landing zone), a second nucleic acid of interest (such as a selectable marker), and a downstream library sequence (i.e., landing zone), wherein the one or more first component polynucleotides and one or more last component polynucleotides (i.e., an exogenous donor nucleic acid that may comprise, in a 5’ to 3’ direction, a first landing zone, a second landing zone, a selectable marker, a third landing zone, and a fourth landing zone) are homologously recombined with the upstream and downstream landing pad homology sequences of the exogenous landing pad within the cell’s genome, producing a cell comprising one or more exogenous donor nucleic acids (i.e., heterologous nucleic acid) between two endogenous genomic sequences (i.e., neutral site portions), i.e., a nucleic acid cassette.
Regarding claims 35-37, George teaches that the upstream and downstream library sequences (i.e., landing zones) within the landing pad may be comprise a randomly generated synthetic nucleic acid sequence and may comprise about 50% GC content (para. 98). George also teaches that the exogenous landing pad may comprise an insulator sequence, such as a transcriptional terminator (para. 73-74). George teaches that insulators are inserted into an exogenous landing pad and insulate the landing pad from unintended “read-through” (para. 74). George does not teach that the upstream or downstream library sequences or upstream or downstream landing pad homology sequences in the genome comprise a bacterial promotor sequence.
Regarding claims 41-43, George teaches that the selectable marker may confer resistance to antibiotics such as bleomycin, kanamycin, neomycin, and streptomycin (para. 120).
However, George does not teach that the recombinant cell is a Cyanobacterial cell as in claims 17-23 and 26-49, the non-essential regions recited in claims 29-31, the length of the neutral site sequence portions, as in claims 32-34, landing zones comprising both at least one transcriptional terminator and at least one translational insulator at either end of the core sequence, as in claims 35-37, and neutral site portions selected from a portion of any one of SEQ ID NOs: 8-17, as in claims 38-40.
Regarding claims 17-22 and 26-49, Vogel teaches a method of producing recombinant Synechococcus sp. PCC 7002 (pg. 2, left col., para. 7), comprising contacting a Synechococcus sp. PCC 7002 cell with a nucleic acid insert comprising a kanamycin resistance gene flanked by two portions of a neutral integration site sequence followed by culturing the cell in the presence of kanamycin to confirm insertion of the kanamycin resistance gene (pg. 2, right col., para. 2 and 4).
Regarding claims 29-31, Vogel teaches that the neutral site sequences are homologous to upstream and downstream neutral integration sites A0159 of Synechococcus sp. PCC 7002 or A2842 of Synechococcus sp. PCC 7002 (pg. 2, right col., para. 4, and Fig. 1).
Regarding claims 32-34, Vogel teaches that the length of each neutral site sequence portion may be 400 or 800 bp (pg. 3, left col., para. 1).
Regarding claims 35-37, Brophy teaches that the use of insulators improves reliability of constructs that tune and control gene expression (referred to as “genetic circuits”), which vary based on their application (pg. 511, right col., para. 4). Brophy also teaches that insulator sequences standardize the DNA sequences flanking promotors, thereby attenuating the activity of upstream promotors (pg. 513-514, para. bridging pages) and strong, tandem (i.e., one after the other) transcription terminators can be placed on either side of a gene to ensure isolated expression of individual operons (pg. 514, left col., para. 3).
Regarding claims 38-40, Li teaches nucleotide sequences identical to instant SEQ ID NO: 12 (see Figure 1 below for alignment) and SEQ ID NO: 13 (see Figure 2 below for alignment), which correspond to upstream and downstream portions of the A0159 locus (pg. 2-3).
Therefore, it would have been prima facie obvious, before the effective filing date of the claimed invention, to a person of ordinary skill in the art, to modify the method of generating recombinant cells taught by George by using Synechococcus sp. PCC 7002 taught by Vogel as the host cell (thereby arriving at the inventions of claims 17-23, 26-29, and 47-49), and further modify the method taught by George by using 400 or 800 bp portions of the non-essential region A0159 taught by Vogel and Li as the endogenous genomic sequences (thereby arriving at the inventions of claims 29-34 and 38-43). The person of ordinary skill in the art would have been motivated to make the modification because genomic integration of “landing pads” comprising randomly generated homology sequences allows for the targeted insertion of exogenous nucleic acids without adding custom homologous regions to every nucleic acid of interest; one need only add homologous sequences to the initial landing pad(s), making the process of generating recombinant strains more cost-effective and efficient (George, para. 5-6). Therefore, the combination is also desirable (see MPEP 2144(II)). The person of ordinary skill in the art would have had a reasonable expectation of success because George teaches methods of inserting exogenous nucleic acids using landing pads that utilize homologous recombination in the host cell, and Vogel teaches that heterologous genes can be integrated into the chromosome of Synechococcus sp. PCC 7002 via homologous recombination between neutral sites on a vector and homologous neutral sites in the chromosome. Because the methods taught by George are generic to the type of cells and endogenous genomic sequences, one would expect that the exogenous landing pads of George may be inserted into Synechococcus sp. PCC 7002 using portions of the A0159 region taught by Vogel and Li, thereby allowing subsequent insertions of exogenous nucleic acids into the landing pads. Therefore, the combination leads to expected results because each element performs the same function as is does individually.
Regarding claims 35-37, it would have been prima facie obvious, before the effective filing date of the claimed invention, to a person of ordinary skill in the art, to duplicate the “insulators” which flank the exogenous landing pad (i.e., only one insulator adjacent to each landing zone) and rearrange the insulators and homologous library/landing pad sequences as taught by George in order to obtain a single landing zone that is flanked by different types of insulators, such as transcription terminators and another insulator that prevents unintended “read-through” as is suggested by Brophy. The person of ordinary skill in the art would have been motivated to make the modification because both George and Brophy teach that “read-through” from upstream promotors can be a problem for engineering of certain genetic elements, and that transcription terminators and other insulators protect target genes from those effects. Thus, adding additional insulators to the homology sequences taught by George, such as the structures in which insulators and/or terminators flank a gene of interest taught by Brophy, would insulate the sequence between the homology sequences, not just the landing pad as a whole. Therefore, the combination is also desirable (see MPEP 2144(II)). The person of ordinary skill in the art would have had a reasonable expectation of success because the function of the library or landing pad homology sequence would not be affected by the addition of additional insulators (i.e., the homology sequence still functions as a site for homologous recombination) flanking the homology sequence. See MPEP 2144.04(VI)(B) and (C): “Although the reference did not disclose a plurality of ribs, the court held that mere duplication of parts has no patentable significance unless a new and unexpected result is produced.” (In re Harza, 274 F.2d 669, 124 USPQ 378 (CCPA 1960)) and “Claims to a hydraulic power press which read on the prior art except with regard to the position of the starting switch were held unpatentable because shifting the position of the starting switch would not have modified the operation of the device.” (In re Japikse, 181 F.2d 1019, 86 USPQ 70 (CCPA 1950) Therefore, the combination leads to expected results because each element performs the same function as is does individually.
Additionally, KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007), discloses that combining prior art elements according to known methods to yield predictable results, is obvious unless its application is beyond that person's skill. KSR International Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741 (2007) also discloses that the combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results. In the instant case, all elements (i.e., Cyanobacterium cell, landing zones, nucleic acid of interest, selectable marker, neutral site portions, non-essential regions, insulator sequences, etc.) were known in the art. In addition, combining these elements yields a method wherein each element merely performs the same function as it does separately; thus, the results of the combination would be recognized as predictable to one of ordinary skill in the art. Therefore, the claimed invention is prima facie obvious in view of the teachings of the prior art, absent any convincing evidence to the contrary.
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Figure 1: Alignment of instant SEQ ID NO: 12 (Qy) with Synechococcus sp. PCC 7002 (Db).
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Figure 2: Alignment of instant SEQ ID NO: 13 (Qy) and Synechococcus sp. PCC 7002 (Db).
Conclusion
No claim is allowed.
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/BAILEY M MORGAN/Examiner, Art Unit 1645
/VANESSA L. FORD/Supervisory Patent Examiner, Art Unit 1674