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 .
Response to arguments under 35 USC § 112(d)
Applicant’s arguments, see page 1 in “Remarks” document, under “Claim rejections under 35USC 112d” section, filed 5/6/2026, with respect to claims 4, 10, 20-22, claims 5-9, 12-15, 23, and 25 and, claims 11, 16-19, and claim 25 have been fully considered and are persuasive. The applicant has corrected claims 4, 10, 20-22 so they are no longer multiple dependent claims that do not refer back in the alternative, and claims 5-9, 12-15, 23, and 25 and, claims 11, 16-19, and claim 25 are now in proper dependent form. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of 35 USC 102(a)(1) using Ishii. (see rejection necessitated by amendment).
Response to arguments under 35 USC § 112(b)
Applicant’s arguments, see page 1 in “Remarks” document, under “Claim rejections under 35USC 112b” section, filed 5/6/2026, with respect to claims 26-27 have been fully considered and are persuasive. Claim 26 has been amended and clearly refers back and further limits the “cell”. Claim 27 is now definite because the claims from which it depends, claim 1 has been amended to include multiple transcription units. The rejection of claims 26-27 has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of 35 USC 102(a)(1) using Ishii. (see rejection necessitated by amendment).
Response to arguments under 35 USC § 102(a)(1)
Applicant’s arguments filed 5/6/2026 have been fully considered and have been found persuasive, however a new ground of rejection has been issued under 102(a)(1) (see rejections necessitated by amendment below). Applicant argues that Ishii does not fully anticipate claim 1 because the reference does not disclose that the three promoters have high, medium, or low expression levels. The examiner notes that the previous action does not address those limitations due to the fact they were previously found in claim 4 which was not examined as an improper multiple dependent claim.
Applicant’s arguments, see page 1 in “Remarks” document, under “Claim rejections under 35USC 102” section, filed 5/6/2026, with respect to the rejections of claims 24,27 under 35 USC 102(a)(1) have been fully considered and are persuasive. They argue that Gossen does not disclose a multigene expression vector with three separate expression cassettes, wherein each expression cassette is controlled by its own promoter and that the three promoters have high, medium, or low expression levels. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of 102(a)(1) using Cox SR et al. (see rejection necessitated by amendment).
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 23 is 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.
Claim 23 recites the limitation "expression vector of claim 21, wherein the nucleic acid linkers" in the first line of the claim. There is insufficient antecedent basis for this limitation in the claim. There is no mention of “nucleic acid linkers” in any of the preceding claims.
Claim rejections - 35 USC § 102
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 1,6-7,10-11,16,20-22,27 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Ishii et al 2014, of record.
Ishii, et al. (2014) discloses, “We constructed a series of multi-copy and integration vector sets for concurrently expressing two or three genes in S. cerevisiae by embedding three classical promoters.” (abstract). Furthermore, “The DNA fragment containing PADH1-MCS1-TADH1, PTDH3-MCS2-TTDH3 and PPGK1-MCS3-TPGK1 was prepared by digesting pATP425 with XhoI/SacI and then inserted into the XhoI and SacI sites of pRS40x + 2 µm (x = 2, 3, 4 and 6) yeast multi-copy vectors, generating pATP422, pATP423temp, pATP424 and pATP426, respectively.” (Materials and Methods section, 6th paragraph in the “plasmid constructs subsection”) (also refer to figure 1b). Sequence analysis revealed that the construction outlined here resulted in PADH1 being truncated by 60bp on the 5’ end and 257 bp on the 3’ end. (See additional material named “ADH1 promoter sequence”), the PTDH3 promoter being truncated by 453bp on the 5’ end and 77bp on the 3’ end. (See additional material named “TDH3 promoter (aka GAP)”) and the PGK1 promoter being truncated by 371bp at the 5’ end and 1bp at the 3’ end. (See additional material named “PGK1 YCR012W”). Next, the researchers performed the following cloning workflow. “The DNA fragment encoding the TagBFP gene was prepared by digesting pBlue-B1 with AvrII and FseI and inserted into the MCS linked to the PADH1 promoter of pATP422 and pATP402, constructing pATP422-B1 and pATP402-B1. The DNA fragment encoding the mKate2 gene was prepared by digesting pBlue-R2 with SalI and NotI and inserted into MCS linked to the PTDH3 promoter of pATP422-B1 and pATP402-B1, producing pATP422-B1R2 and pATP402-B1R2. The DNA fragment encoding the TagGFP2 gene was prepared by digesting pBlue-G3 with XmaI and AscI and inserted into the MCS linked to the PPGK1 promoter of pATP422-B1R2 and pATP402-B1R2, creating pATP422-B1R2G3 and pATP402-B1R2G3.” (Materials and Methods section, 11th paragraph in the “plasmid constructs subsection”). This is to say that pATP422-B1R2G3 has BFP downstream of the PADH1 promoter, mKate2 downstream of PTDG3, and finally, GFP directly downstream of PPGK1. (Materials and Methods section, 8th paragraph in the “plasmid constructs subsection”). Therefore, Jun Ishii, et al. discloses the creation of a multigene expression plasmid with three separate expression cassettes wherein each expression cassette has a different synthetic promoter driving expression of a different gene. Importantly, in view of the applications definition of how to calculate “high, medium and low”, the three promoters can be classified as having a level of transcriptional activity equal to “high, medium, and low”. Specifically, “Although the relative fluorescence intensities were considerably different among three promoters, the order of fluorescence intensities was equivalent to the above described EGFP evaluations of the plasmids harboring only the single promoters (PTDH3 > PADH1 > PPGK1).” (paragraph 2 of “Expression of single fluorescent protein with pATP42x vector sets” in the results and discussion section). (See Figure 2A). Additionally, figure 2B discloses that the pADH1 and pPGK1 can be qualified has having the same level of transcriptional activity (see pATP402--ADE2 condition). These data indicate each promoter can have different levels of transcriptional activity under 1 condition (Figure 2A), but those same promoters can have equal levels of transcriptional activity under a different condition (Figure 2B pATP402--ADE2 condition). These disclosures anticipate claim 1, and claim 6-7.
Using the primers disclosed by Ishii et al and the full-length sequences of each wildtype promoter, the transcription factor regulatory elements (TFRE) occurring in the synthetic promoter were identified. (See additional materials). The following promoters and their TFREs are defined: 1) pTAG3 has 1 ETS site. 2) pADH1 has 12 ETS sites and 2 GC-boxes. 3) pPGK1 promoter has 4 ETS sites. This indicates that each promoter has at least one TFRE and therefore anticipates claim 10. It also indicates that each promoter contains either ETS or GC-box TFREs. Therefore, claim 16 is anticipated. Importantly, it is known that the number of TFREs controls transcriptional activity, as supported by von der Ahe D et al. Therefore, claim 11 is anticipated. Claims 20-22 are also anticipated by the Ishii disclosure, and those specific disclosures can be found in the rejection above. Briefly, they disclose the creations of each individual transcription unit wherein each unit is defined by a promoter, a gene, and a terminator. They disclose the construction of a three-color multigene expression plasmid wherein each expression cassette has one of the following fluorescent genes; TagBFP, mKate2 or TagGFP2. Importantly, each cassette is joined together (forms a single plasmid) by nucleic acid linkers. (see pATP422-B1R2G3 and pATP402-B1R2G3.”(see 11th paragraph in the “plasmid constructs subsection” under materials and methods and Figure 1b). They verify all three expression cassettes are functional. Specifically, “Yeast cells transformed with pATP422-B1R2G3 or pATP402-B1R2G3 displayed blue, red and green fluorescence (Fig. 4a and b)” (2nd paragraph of “Expression of three fluorescent proteins with pATP422 and pATP402 vectors” in results and discussion section). Therefore, they detail a method of regulating gene expression by introducing the multigene expression plasmid of claim 1 into a cell and incubating the cell to promote expression of all three genes, and therefore anticipate claim 27. Specifically, “All multi-copy plasmids pATP42x-G1, pATP42x-G2, pATP42x-G3 and pATP422-B1R2G3 (x = 2–6) transformed YPH499 without the restriction enzyme digestions…..All transformants were grown in the appropriate SD selectable media overnight and were then inoculated into10 mL of fresh SD media to give an initial optical density of 0.1 at 600 nm. Yeast cells were grown at 30 °C on a rotary shaker set at 150 r.p.m. for 24 h (or up to 96 h). Cultured cells were harvested for use in the following experiments.” 2nd and 3rd paragraph of “Yeast transformation and cultivation” in materials and methods section).
Claims 1, 5,24-26 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Cox SR et al.
Cox SR et al discloses a bacterial multigene expression plasmid containing three distinct expression cassettes wherein each cassette has a different promoter and a different gene See figure 1). They also disclose a bacterial cell contain the plasmid which is cultured under conditions that result in transgene expression. Specifically, “We used promoters PLtetO-1 (P1) to control cfp, PLlacO-1 (P2) to control yfp, and Plac/ara-1 (P3) to control rfp [34].” (2nd paragraph of “Synthetic DNA design” in methods section). Importantly, each promoter has a different expression level. (Figure 2). Additionally, each promoter can be categorized as either high, medium, or low as shown by the culture and induction of each promoter individually, and all three together. (Figure 2). Therefore, they disclose a bacterial expression vector containing the limitations of claim 1 and they also disclose the expression vector is in a bacterial cell. This anticipates claims 24-26. Comparison of lane D and Lane I in figure 2 indicates a repression of at least 20% which anticipated claim 5.
Claim rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claims 12-15 are rejected under 35 U.S.C. 103 as being unpatentable over Jun Ishii, et al. as applied to claim 1 above, and further in view of Huang J et al.
Jun Ishii, et al. discloses the creation of a yeast multigene expression plasmid with three separate expression cassettes wherein each expression cassette has a different synthetic promoter driving expression of a different gene.
Jun Ishii, et al. does not disclose the specific limitations regarding the number of TFREs in each of the low, medium, and high promoters.
Huang J et al. discloses the optimization of promoter transcriptional activity by altering the number of specific TFREs in a promoter. Specifically, “We developed a strategy to exponentially increase native promoter activity while retaining inherent regulation by inserting multi-copy response elements (REs) into non-adjacent locations. For the hepatocyte nuclear factor (HNF) 4α-dependent Hnf1a (MODY3) gene, HNF4α stimulation increased from 5- to 90-fold by inserting 3 additional HNF4α REs (H4REs). Constructing a promoter with two 4xH4REs 0.25 kb apart by duplicating the 4xH4RE fragment increased stimulation to >1000-fold.” (abstract)
Therefore, it would be obvious to a person of ordinary skill in the art, before the effective filing date, to vary the number the of particular TFREs in a promoter as taught by Huang J et al when designing and constructing the multigene expression plasmid of Jun Ishii et al. Huang J et al provides an explicit teaching that promoter activity is a function of TFRE number, and that modifying TFRE copy number changes promoter activity significantly. Therefore, one of ordinary skill in the art would be motivated to vary the number of TFREs in a particular promoter to achieve a desired level of expression (ie; low, medium high). Importantly, the creation of different variations is done using the routine practice of cloning, and, more importantly, the art teaches that optimization of individual TFRE copy number is done using the same range of claimed TFRE numbers (aka a range from 1-12 copies of an individual TRFE in a single promoter is clamed and used in optimization. See Figure 1). Therefore, claims 12-15 are obvious.
Claims 16-19 are rejected under 35 U.S.C. 103 as being unpatentable over Jun Ishii, et al. as applied to claim 1 above, and further in view of Brown et al.
Jun Ishii, et al. discloses the creation of a yeast multigene expression plasmid with three separate expression cassettes wherein each expression cassette has a different synthetic promoter driving expression of a different gene.
Jun Ishii, et al. does not disclose the specific limitations regarding the number and identity of TFREs in each of the low, medium, and high promoters.
Brown et al provides a teaching/suggestion to modify the identity and composition of different TFRE combinations in a promoter when trying to optimize transcriptional activity. Specifically, “Initially, 10 common viral promoter sequences known to be active in CHO cells were analyzed using bioinformatic sequence analysis programs to determine the identity and relative abundance of transcription factor regulatory elements (TFREs; or transcription factor binding sites) they contained. Based on this, 28 synthetic reporters were constructed that each harbored seven repeats of a discrete TFRE sequence upstream of a minimal CMV core promoter element and secreted alkaline phosphatase (SEAP) reporter gene. After evaluation of the relative activity of TFREs by transient expression in CHO-S cells, we constructed a first generation library of 96 synthetic promoters derived from random ligation of six active TFREs inserted into the same reporter construct backbone. Comparison of the sequence and relative activity of first generation promoters revealed that individual TFRE blocks were either relatively abundant in active promoters (NFκB, E-box), equally distributed across promoters of varying activity (C/EBPα, GC-box) or relatively abundant in low activity promoters (E4F1, CRE). These data were utilized to create a second generation of 44 synthetic promoters based on random ligation of a fixed ratio of 4 TFREs (NFκB 5: E-box 3: C/EBPα 1: GC-box 1). Comparison of the sequence and relative activity of second generation promoters revealed that the most active promoters contained relatively high numbers of both NFκB and E-box TFREs in approximately equal proportion, with a correspondingly low number of GC-box and C/EBPα blocks.” (abstract). Therefore, they teach modulating TFRE identity and amounts in promoters based on a defined starting pool of TFREs and a defined starting architecture (28 synthetic reporters were constructed that each harbored seven repeats of a discrete TFRE sequence upstream of a minimal CMV core promoter). This indicates that TFRE composition is an optimizable parameter and therefore renders claims 16-19 obvious.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Tracy Vivlemore/Supervisory Primary Examiner, Art Unit 1638
