DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Application Status and Withdrawn Rejections
Applicant’s amendment filed July 23, 2026, amending claims 9 and 12, and canceling claims 8 and 13-17 is acknowledged. Claims 1-6 and 9-12 are pending and under examination.
Terminal Disclaimers
The terminal disclaimers filed on April 2, 2024 disclaiming the terminal portion of any patent granted on this application which would extend beyond the expiration date of US Patents 10131870, 12065638, 10336982 and 10760045 have been approved.
Withdrawn Rejections
The cancelation of claim 8 renders the §112(d) rejection moot. The amendment to claim 12 overcomes the §112(b) rejection. The cancellation of claims 13-17 renders the nonstatutory double patenting (NSDP) rejections over US Patent 12447202 and copending US Application 19/202734 moot. The terminal disclaimers filed over the US patents listed above overcome the remaining NSDP rejections.
Applicant's arguments and evidence proffered in the 1.132 Declaration filed July 23, 2026 indicates that Zhang's method does not produce stable transformants of Spirulina/Arthrospira cells, which die soon after transformation. The §103 rejections over Zhang in view of Klanchui are withdrawn.
Applicant' s amendments and arguments have been thoroughly reviewed, but are not persuasive to place the claims in condition for allowance for the reasons that follow.
Claim Rejections - 35 USC § 103 – Kawata in view of Klanchui
Claims 1, 3, 6, 9-12 are rejected under 35 U.S.C. 103 as being unpatentable over Kawata (Kawata et al., Marine Biotechnology (2004), 6: 355-363) in view of Klanchui (Klanchui et al., BMC Systems Biology (2012), 6:71, pages 1-15). This is a maintained rejection.
Regarding claims 1 and 3, Kawata teaches transforming Spirulina platensis with PCR products of a Tn5 transposon comprising a cat gene (i.e., a vector comprising an exogenous nucleotide sequence) (page 257, ¶2; Fig 1). Kawata teaches detecting the cat gene in the genomic DNA of transformed Spirulina (i.e., exogenous nucleotide sequence is stably integrated) (page 257, ¶4; Fig 3). Kawata teaches growing the transformed cells under selection after 60 days of incubation (Fig 2, legend) and culturing for at least 12 months (Table 1). Kawata teaches that the transposon is integrated into the Spirulina chromosome (page 362, ¶2). Kawata characterizes the Tn5-mediated integration as “stable” (page 362; ¶3). Kawata teaches collecting transformed cells after 2 months and 12 months, freezing and pulverizing the cells (i.e., collecting a biomass) (page 257, ¶5). Kawata also teaches that Spirulina is a commercially important cyanobacteria and is a source of proteins, minerals, vitamin B12 and fatty acids.
Kawata does not teach drying the biomass and is silent on the double time of Spirulina platensis C1.
Klanchui teaches that Spirulina platensis is a source for commercially produced nutraceutical compounds, including use a human food supplements and animal feed, and has been consumed as a protein source for many years (page 71, ¶1). Klanchui teaches measuring biomass production under various growth conditions (page 11, ¶3, Table 4). Klanchui teaches determining the dry cell weight (i.e., collecting the biomass and drying the biomass) (page 11, ¶3, Table 4). Klanchui teaches the generation time for Spirulina platensis is approximately 40 hours (Table 4).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have collected the cells transformed by Tn5-mediated gene integration taught in Kawata as dried biomass. It would have amounted to the simple combination of method steps by known means to yield predictable results. Regarding the generation number, based on Klanchui’s growth rate analysis, Kawata’s cells grown for 2 months and 12 months would have undergone approximately 36 generations and 219 generations, respectively. Therefore, the exogenous cat gene was stably integrated into the Spirulina genome for at least five generations before the cells were collected. The skilled artisan would have predicted that Kawata’s cells could be collected and dried because Klanchui’s cells that are dried are the same species and strain. The skilled artisan would have been motivated to have done so to prepare the cells for inclusion in supplements or to measure metabolite output of the cells as taught by Klanchui.
Regarding claim 6, Kawata teaches the Tn5-cat constructs were provided as a PCR product (i.e., a linear vector) (page 357, ¶2).
Regarding claims 9, Kawata teaches electroporating the cells with the Tn5 transposon/transposase construct (i.e., artificial competence by electroporation) (page 357, ¶2).
Regarding claims 10-11, Kawata teaches washing the cells in HEPES buffer (i.e., a pH balancer) before contacting the cells with the Tn5-cat transposon (page 357, ¶2).
Regarding claim 12, Kawata teaches the HEPES buffer is pH 7.5 (page 357, ¶2).
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Kawata (Kawata et al., Marine Biotechnology (2004), 6: 355-363) and Klanchui (Klanchui et al., BMC Systems Biology (2012), 6:71, pages 1-15) as applied to claims 1, 3, 6, 9-12 above, and further in view of Toyomizu (Toyomizu et al., Journal of Applied Phycology (2001), 13: 209-214; cited in IDS). This is a maintained rejection.
The teachings of Kawata and Klanchui are recited above and applied as for claims 1, 3, 6, 9-12. As noted above, Kawata and Klanchui teach the strains Spirulina Platensis strain C1.
Kawata and Klanchui do not teach introducing targeted mutations into Arthrospira platensis strain NIES-39.
Toyomizu teaches that the NIES-39 strain of Spirulina platensis (i.e., Arthrospira platensis) may be genetically transformed by introducing DNA via electroporation in a HEPES buffer (¶ bridging pages 210-211).
It would have been obvious to one skilled in the art before the effective filing date of the claimed invention to have substituted the C1 strain used in the method of Kawata with the NIES-39 strain of the Spirulina alga Arthrospiria platensis as taught by Toyomizu. It would have amounted to the simple substation of one known algal strain for another of the same species by known means to yield predictable results. The skilled artisan would have expected that the strains could be substituted for each other because both are amenable to genetic transformation by electroporation in a HEPES buffer. Because the prior art recognizes the equivalence of the C1 and NIES-39 strains for the purpose of genetic transformation by electroporation an express suggestion to substitute one equivalent component or process for another is not necessary to render such substitution obvious. MPEP 2144.06.11.
Response to Arguments
Applicant argues that the Tn5 transformation protocol of Kawata is not enabling. Applicant provides evidence in the form of quote from two articles, Fang et al., published in 2013 and Jeamton published in 2017. Applicant quotes one passage from each article that suggests, but does not demonstrate, that Kawata’s method did not result in stable integration (Remarks, pages 7-9). This argument has been fully considered but is not persuasive.
First, the citation from Fang does not appear to be specifically regarding the Kawata reference. It is not clear if Fang is critical of Kawata for the “low efficiency” or “instability”. There is no requirement in the claims for a specific efficiency and Kawata demonstrates at least 5 generations of stable integration.
Second, the Jeamton quotation does not make sense when viewed in reference to Kawata. Kawata never attempts to isolate a clone. Thus, it is not clear what reference out of the four listed in the cited paragraph, the “single stable transformant clone” is referring to. There is no requirement in the claims for a single clone to be isolated. Kawata makes clear that the transgene was detected 12 months in the Spirulina population after transformation was performed and the cells were selected.
Finally, at least two additional references refer to Kawata as succeeding in producing stable Spirulina transformants. Dehghani recites, “There are only a few reports about the stable transformation of Spirulina by using a transposon-mediated plasmid (Jeamton et al. 2017; Kawata et al. 2004). Indeed, a natural Tn5 transposon, transposase, and cation liposome complex were used to transform the Spirulina genome by chloramphenicol acetyltransferase gene via electroporation. This gene was methylated to protect from the Spirulina restriction enzymes (Kawata et al. 2004).” (Dehghani et al., Appl. Microbiol Biotechnol (2018), 102: 9267-9278). A review by Durdakova and colleagues in 2022, described the Kawata method as a “stable recombinant DNA methodology” (Durdakova et al., Critical Reviews in Food Science and Nutrition (2024), 64: 3091-3102).
Therefore, without additional evidence that Kawata’s method did not actually result in stable integration, the preponderance of the evidence including the results in Kawata and two references referencing the method as “stable integration”, it was more likely than not that Kawata’s methylated Tn5 transposon method is enabled for integrating a generic exogenous nucleotide sequence stably in the Spirulina genome for at least five generations.
Allowable Subject Matter
Claims 2 and 5 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Claims 2 and 5 require the cells to be contacted with an osmotic stabilizer at some point during the transformation process. The closest prior art is Kawata, whose teachings are recited above. Kawata’s Tn5 DNA integration method does not require an osmotic stabilizer because the Spirulina cells are never protoplasted. As such there is no need for osmotic stabilization and it would not have been obvious to include an osmotic stabilizer such as sucrose or glycerol in Kawata’s method.
Conclusion
Claims 2 and 5 are objected to. Claims 1, 3-4, 6 and 9-12 are rejected.
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/CATHERINE KONOPKA/Primary Examiner, Art Unit 1635
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