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 .
Applicant’s amendment filed on 02/13/2026 has been entered.
Claims 2, 6-7, 9-10, 12, 19-21, 26-28, 41-43, 45-46 and 50-54 are pending in the present application.
Applicant previously elected the following species: (a) a plasmid; (b) a pUC origin; (c) an antibiotic resistance gene; and (d) the insert encodes a mRNA.
The species of replication origin derived from ColE1 plasmid had been rejoined and examined together previously with the elected replication origin derived from a pUC plasmid.
Claim 52 was withdrawn previously from further consideration because it is drawn to a non-elected species.
Accordingly, amended claims 2, 6-7, 9-10, 12, 19-21, 26-28, 41-43, 45-46, 50-51 and 53-54 are examined on the merits herein with the above elected species.
Response to Amendment
1. The rejection under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, for New Matter was withdrawn upon further considerations and in light of Applicant’s cited written supports in the Amendment filed on 02/13/2026 (last full paragraph at page 7).
2. The rejection under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, was withdrawn in light of the cancellation of claims 47-48.
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.
Claims 6-7, 9-10, 19, 26-28, 41-42, 45 and 50-51 are rejected under 35 U.S.C. 103 as being unpatentable over Zibert et al (J. Virol. 64:2467-2473, 1990) in view of Carnes et al (US 7,943,377) and Romero Ramos et al (US 2018/0135060); and evidenced by Shyadehi et al (J. Biol. Chem. 271:12445-12450, 1996) and Forss et al (Nucleic Acids Research 12:6587-6601, 1984) for the same reasons set forth in the Non-Final Office Action dated 08/13/2025 (pages 7-14). The same rejection is restated below.
The instant claims are directed to a method for fermentative production of a covalently closed recombinant DNA molecule comprising the steps of: (a) providing an E. coli bacterium comprising the covalently closed circular recombinant DNA molecule comprising: - a bacterial origin of replication, and - an insert, preferably the insert encodes a mRNA, comprising: (i) a RNA polymerase promoter sequence; (ii) an open reading frame (ORF) encoding a polypeptide; and (iii) a homopolymeric region, wherein the homopolymeric region comprises at least one poly(A) sequence of about 20 to about 400 adenosine nucleotides and is located at a distance of at least 2200 bp from the bacterial origin of replication, wherein the at least one poly(A) sequence is oriented so that the direction of transcription of the insert is the same as the direction of the origin of replication and the ORF is positioned between the RNA polymerase promoter sequence and the homopolymeric region, wherein the covalently closed circular recombinant DNA molecule further comprises a kanamycin resistance gene as a selection marker; and (b) fermenting the E. coli bacterium of step (a), wherein the yield of the covalently closed circular recombinant DNA molecule is increased compared to the yield of an otherwise identical covalently closed circular recombinant DNA molecule in which the homopolymeric region is: (i) located at a distance of less than 500 bp from the origin of replication in the direction of replication; and (ii) oriented so that the direction of transcription of the homopolymeric region is opposite to the direction of replication of the origin of replication, wherein the fermentative production is performed using a fed-batch process.
Zibert et al already disclosed construction of YEp51-derived pFMDV-YEP-polyC plasmids containing a full-length cDNA of foot-and-mouth disease virus flanked by a poly(C) of an average length of 50 to 150 nucleotides at the 5’ end and a homopolymeric adenosyl tract of approximately 90 nucleotides at the 3’ end, operably linked to the SP6 promoter in tandem with the GAL10 promoter (RNA polymerase promoters) in the yeast vector YEp51, along with a ColE1 origin (a high copy number origin), an ampicillin resistance gene for replication and selection in E. coli, the autonomous replication portion of the 2 um circle, and the LEU2 marker for growth in Saccharomyces cerevisiae (see at least Abstract; sections titled “Construction of plasmid pFMDV-L” on page 2467; “Construction of plasmids pFMDV-YEP and pFMDV-YEP-polyC” on page 2468; particularly page 2468, left column, last paragraph continues to first paragraph on right column; page 2469, right column, second and third paragraphs; and Figures 1-3). FIG. 1 depicts nucleotide sequence of the viral 5’ region in plasmid pFMDV-YEP-poly C, while top of FIG. 2 depicts cDNA of the viral genome as shown below.
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The poly(A) sequence in the YEp51-derived pFMDV-YEP-polyC is located at a distance at least 500 bp (e.g., at least 1000 bp, at least 2000 bp) from the ColE1 origin of replication situated between the ampicillin resistance gene and the LEU2 marker gene; and both the SP6 promoter in tandem with the GAL10 promoter along with the ColE1 origin of replication are in the same direction as evidenced at least by Figure 1 showing the YEp51 vector map in the Shyadehi reference (J. Biol. Chem. 271:12445-12450, 1996) below. Additionally, the 7.8 kb full-length cDNA of foot-and-mouth disease virus comprises ORFs encoding viral polypeptides as evidenced by the teachings of Forss et al (Nucleic Acids Research 12:6587-6601, 1984; see at least the Abstract), and it is positioned between the tandem promoters and the polyA sequence. Moreover, since the FMDV RNA genome is a single positive strand RNA that acts directly as a messenger RNA as evidenced by the teachings of Forss et al (page 6587, first full paragraph of the “Introduction” section), the 7.8 kb full-length cDNA of FMDV encodes a mRNA.
Zilbert et al taught that the pFMDV-YEP-polyC plasmids were used to transform yeasts or E. Coli cells (page 2468, right column, last sentence of first paragraph; section titled “Infectious cDNA clone in E. coli” on page 2470). Zibert et al stated clearly “The use of yeast cells to propagate infectious FMDV cDNA is limited by the fact that there is no simple protocol to purify the 2um replicons, which represent less than 1% of the total cellular DNA. For future manipulations of the cDNA to study gene functions or construct attenuated strains, it was desirable to have larger amounts of purified cDNA available. Therefore, we made another attempt to establish an infectious cDNA clone in bacteria and transformed E. coli cells with the same batch of polycytidylated pFMDV-YEP DNA as has been used to transfect yeast cells. Plasmid DNA was prepared from 20 individual ampicillin-selected clones and transcribed into RNA. One of these RNAs led to the production of infectious viruses after transfection of BHK-21 cells……It was possible to expand this clone (pFMDV-YEP-polyC) under standard conditions, and the isolated plasmid DNA could also be used to transform other E. Coli cells without complications. Transfection of BHK-21 cells with RNA transcribed from these second-generation plasmids did not always lead to the production of viruses. We initially interpreted this fact as a certain instability of the infectious clone. More careful analysis revealed, however, this result was due to the low relative infectivity of the in vitro-
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derived RNA of this clone” (page 2470, left column, first two complete paragraphs). Zibert et al also determined the differences between the infectious and non-infectious cDNA clones, and analysis of the poly(C) region by DNA sequencing revealed that the infectious cDNA clone contained probably more than 30 cytidyl residues, whereas the homopolymer in non-infectious clones ranged from 17 to 25 nucleotides (section titled “Correlation of poly(C) length and infectivity”). Zibert et al also stated “The lowest number of nucleotides in the poly(C) tract necessary for infectivity of plasmid-derived RNA was found to be 32 in our experiments. This number of C residues seems to be crucial in two respects. On the one hand, it seems to be at the upper limit to be tolerated in a high copy-number plasmid in E. coli. We do not know why we were unable to establish a correspondingly long C tract in the smaller vector pSP64. Possibly the large size of the yeast vector construct (17 kb) or the resulting reduced plasmid copy number per cell led to the maintenance of this sequence” (page 2472, first full paragraph).
Zibert et al did not disclose explicitly at least a method for fermentative production of the covalently closed circular recombinant pFMDV-YEP-polyC plasmid comprising a kanamycin resistance gene instead of an ampicillin resistance gene as a selection marker via a fed-batch process to obtain large amounts of plasmids for the production of infectious transcribed RNAs.
Before the effective filing date of the present application (8/10/2015), Carnes et al already taught improvements in plasmid DNA production technology to ensure economic feasibility of future DNA vaccines and DNA therapeutics, including fed-batch fermentation strategies combining novel growth and induction phase temperature shifts (see at least Abstract; Disclosure of the Invention; Brief Summary of the Invention; issued claims 1-7). Specifically, Carnes et al disclosed a method of fed-batch fermentation in which plasmid-containing E. Coli cells are grown at a reduced temperature (e.g., around 300C) during the fed-batch phase, during which growth rate is restricted by a carbon limiting exponential feeding strategy (e.g., preferred growth rate ranges are u=0.05 to 0.3 per hour, preferably u=0.12 per hour via control nutrient feed), followed by a temperature up-shift (e.g., to approximately 36-450C) and continued growth at elevated temperature in order to accumulate plasmid, whereby the temperature shift and restricted growth rate improves yield and purity of plasmids, and wherein the plasmid contains a ColE1-derived replication origin, a pMB1 replication origin containing the pUC G to A mutation, or a pUC origin containing plasmid (col. 5, first full paragraph; col. 9, lines 53-63). Carnes et al also defined specifically the term “pUC origin” as pBR322-derived origin, with G to A transition that increases copy number at elevated temperature (col. 7, lines 21-22).
Additionally, Romero Ramos et al also taught preparation of the Escherichia Coli T7 expression pMRKA plasmid vector that allows an enhanced production, in industrial scale, of recombinant proteins, by modifying plasmids containing the gene sequence of the T7 promoter of E. coli (e.g., pAE vector) and which modification comprises the substitution of the ampicillin-resistance gene by the kanamycin-resistance gene (Abstract; Summary of the Invention; particularly paragraphs [0051]-[0052], [0134]-[0138]; and Figure 1). Romero Ramos et al stated “The need to modify the pAE plasmid was due to the following reasons: (a) as the ampicillin resistant gene encodes a beta-lactamase which is located in periplasmic space of E. coli in high density culture, this enzyme has the possibility to migrate of the culture means and, consequently, to degrade the antibiotic, decreasing the selective pressure of the means and providing the buildup of cells without plasmid; and (b) in the production of expression vectors in industrial scale it is not recommended to use antibiotic similar to the ones used in medical and veterinary fields, as it is the case of ampicillin. As kanamycin does not have medical application, the ampicillin resistant gene in pAE vector was replaced with kanamycin resistance gene” (paragraph [0052]). Romero Ramos et al also disclosed that T7 promoter of T7 bacteriophage is a strong promoter, and the expression systems based on T7 are broadly used for super-expression of in large scale of recombinant proteins in prokaryotes and eukaryotes (paragraph [0003]).
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to modify the teachings of Zibert et al by also using the fed-batch fermentation method of Carnes et al for fermentative production of the covalently closed circular recombinant pFMDV-YEP-polyC plasmid modified by the substitution of the ampicillin-resistance gene by the kanamycin-resistance gene as a selection marker to obtain large amounts of plasmids for at least future manipulations of the cDNA to study gene functions or construct attenuated strains, and optionally further modified by the substitution of the SP6 promoter by the T7 promoter, or optionally further modified by the substitution of the origin of replication from ColE1 plasmid by the origin of replication from pUC plasmid with G to A transition that increases copy number at elevated temperature, in light of the teachings of Carnes et al and Romero Ramos et al as presented above.
An ordinary skilled artisan would have been motivated to carry out the above modifications because Carnes et al already disclosed successfully a method of fed-batch fermentation in which plasmid-containing E. Coli cells are grown at a reduced temperature (e.g., around 300C) during the fed-batch phase, during which growth rate is restricted (e.g., preferred growth rate ranges are u=0.05 to 0.3 per hour, preferably u=0.12 per hour via control nutrient feed), followed by a temperature up-shift (e.g., to approximately 36-450C) and continued growth at elevated temperature in order to accumulate plasmid, whereby the temperature shift and restricted growth rate improves yield and purity of plasmids, and wherein the plasmid contains a ColE1-derived replication origin, a pMB1 replication origin containing the pUC G to A mutation, or a pUC origin containing plasmid. Particularly, Carnes et al taught that pUC origin as pBR322-derived origin, with G to A transition that increases copy number at elevated temperature. Moreover, Romero Ramos et al taught advantages offered by substitution of the ampicillin-resistance gene by the kanamycin-resistance gene for an Escherichia Coli T7 expression plasmid vector, and that T7 promoter of T7 bacteriophage is a strong promoter, and the expression systems based on T7 are broadly used for super-expression of in large scale of recombinant proteins in prokaryotes and eukaryotes.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Zibert et al, Carnes et al and Romero Ramos et al as set forth above; coupled with a high level of skill of an ordinary skilled artisan in the relevant art.
The modified method for fermentative production of a covalently closed recombinant DNA molecule resulting from the combined teachings of Zibert et al, Carnes et al and Romero Ramos et al is indistinguishable from the presently claimed method regarding to the materials and method steps used. Accordingly, it would also possess the same properties recited in the “wherein functional clause” regarding to the yield of the covalently circular recombinant DNA molecule. Moreover, it is noted that the “wherein functional clause” regarding to the yield of the covalently circular recombinant DNA molecule does not add any further structural details for the covalently closed circular recombinant DNA molecule and/or any further step/condition for the fermentation method of claim 26.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Zibert et al (J. Virol. 64:2467-2473, 1990) in view of Carnes et al (US 7,943,377) and Romero Ramos et al (US 2018/0135060) as applied to claims 6-7, 9-10, 19, 26-28, 41-42, 45 and 50-51 above, and further in view of Williams (US 2010/0184158).
The combined teachings of Zibert et al, Carnes et al and Romero Ramos et al were presented above. However, none of the cited references teaches specifically the use of a ccc recombinant DNA molecule (e.g., modified pFMDV-YEP-polyC plasmid) comprising a primosome assembly site in the heavy strand (PAS-BH).
Before the effective filing date of the present application (8/10/2015), Williams already taught vector modifications that improve production yield of ccc recombinant DNA molecules such as plasmids, including a plasmid with a pUC origin (see at least Abstract; Summary of the Invention; particularly paragraphs [0011]-[0012], [0015], [0060]-[0064], [0067], [0070] and Fig. 2). Williams taught explicitly that by adding one or more components selected from the group consisting of an SV40 enhancer, PAS-BH region, and PAS into a plasmid vector to improve plasmid yield in shake flask and/or fermentation culture. Williams stated explicitly “It has been surprisingly found that this PAS-BH site improves plasmid yield in subsequent shake flask and/or fermentation culture” (paragraph [0067]).
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to further modify the combined teachings of Zibert et al, Carnes et al, Romero Ramos et al by also further incorporating at least the PAS-BH region and/or an SV40 enhancer into the modified pFMDV-YEP-polyC plasmid vector to improve plasmid yield in a fermentation culture, in light of the teachings of Williams as presented above.
An ordinary skilled artisan would have been motivated to further carry out the above modifications because Williams taught explicitly that by adding one or more components selected from the group consisting of an SV40 enhancer, PAS-BH region, and PAS into a plasmid vector, including a plasmid vector with a pUC origin, to improve plasmid yield in shake flask and/or fermentation culture.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Zibert et al, Carnes et al, Romero Ramos et al and Williams as set forth above; coupled with a high level of skill of an ordinary skilled artisan in the relevant art.
The modified method resulting from the combined teachings of Zibert et al, Carnes et al, Romero Ramos et al and Williams is indistinguishable and encompassed by the presently claimed invention.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Response to Argument
Applicant’s arguments related to the above 103 rejections in the Amendment filed on 02/13/2026 (pages 8-10) have been fully considered, but they are respectfully not found persuasive for the reason discussed below. It is noted that Applicant presented the same arguments as those presented in the Amendment filed on 07/10/2025 (pages 11-13).
Applicant argued basically that Zibert does not provide any motivation to increase the production of pFMDV-YEP-polyC plasmid beyond the levels already achieved, and the difficulties encountered by Zibert were not related to the availability of plasmid material, but rather the low infectivity of the RNA derived from the plasmid. Additionally, Applicant argued that Zibert was not concerned with the yield of the plasmid DNA, and there is no mention of the importance of optimizing the distance between the homopolymeric poly(A) sequence and the origin of replication, nor is there any discussion regarding the orientations of prokaryotic elements of the plasmid. Once again, Applicant argued that since Carnes teaches that the orientations of prokaryotic elements in the plasmid had no effect and that the inducible process is not specific to a particular plasmid, an ordinary skill in the art would have expected all plasmids to work equally well regardless of the orientation of prokaryotic elements. Accordingly, Applicant argued that combining Zibert and Carnes teachings would not have led a skilled artisan to the claimed invention, as it was not obvious to modify the plasmid characteristics based on the combination of the two prior arts. Applicant further argued that numerous widely used plasmids, such as pNIC-CH and pcDNA3.1, exhibit opposite directionality between transcription and replication; and consequently there was no reason to believe altering the directionality of these elements would reasonably result in an increase in yield. Applicant also argued that the Williams reference does not cure the deficiencies/defects of the combination Zibert, Carnes, Romero Ramos, Shyadehi and Forss as discussed above.
First, Zibert stated clearly “The use of yeast cells to propagate infectious FMDV cDNA is limited by the fact that there is no simple protocol to purify the 2um replicons, which represent less than 1% of the total cellular DNA. For future manipulations of the cDNA to study gene functions or construct attenuated strains, it was desirable to have larger amounts of purified cDNA available. Therefore, we made another attempt to establish an infectious cDNA clone in bacteria and transformed E. coli cells with the same batch of polycytidylated pFMDV-YEP DNA as has been used to transfect yeast cells. Plasmid DNA was prepared from 20 individual ampicillin-selected clones and transcribed into RNA. One of these RNAs led to the production of infectious viruses after transfection of BHK-21 cells……It was possible to expand this clone (pFMDV-YEP-polyC) under standard conditions, and the isolated plasmid DNA could also be used to transform other E. Coli cells without complications. Transfection of BHK-21 cells with RNA transcribed from these second-generation plasmids did not always lead to the production of viruses. We initially interpreted this fact as a certain instability of the infectious clone. More careful analysis revealed, however, this result was due to the low relative infectivity of the in vitro-derived RNA of this clone” (page 2470, left column, first two complete paragraphs). On the basis of the above paragraphs, it would have been obvious for an ordinary skilled in the art that Zibert desired to have a large amount of purified cDNA available for future manipulations of the cDNA to study gene functions or construct attenuated strains, which can be obtained via the fed-batch fermentation method of Carnes for fermentative production of the covalently closed circular recombinant pFMDV-YEP-polyC plasmid, regardless whether Zibert recognized the importance of optimizing the distance between the homopolymeric poly(A) sequence and the origin of replication, and/or the orientations of prokaryotic elements in the recombinant pFMDV-YEP-polyC plasmid.
Second, it is also irrelevant whether or not Carnes recognized the importance of optimizing the distance between the homopolymeric poly(A) sequence and the origin of replication, and/or the orientations of prokaryotic elements in any plasmid vector. As set forth in the above 103 rejection, an ordinary skilled in the art would have been motivated to modify the teachings of Zibert by also at least using the fed-batch fermentation method of Carnes to obtain large amounts of purified cDNA in the form of plasmids for at least future manipulation studies because Carnes already taught improvements in plasmid DNA production technology to ensure economic feasibility of future DNA vaccines and DNA therapeutics, including fed-batch fermentation strategies combining novel growth and induction phase temperature shifts.
Third, the pFMDV-YEP-polyC plasmid of Zibert contains all elements except for the kanamycin resistance gene and in the same directionality as those recited in the DNA molecule used in a fermentative method of the presently claimed invention. The above 103 rejections are directed specifically to the pFMDV-YEP-polyC plasmid of Zibert, and not related to any modifications for other widely used plasmids, such as pNIC-CH and pcDNA3.1. Please refer to the above 103 rejections for more details, particularly the motivation on why an ordinary skilled artisan would modify the teachings of Zibert by also using the fed-batch fermentation method of Carnes for fermentative production of the covalently closed circular recombinant pFMDV-YEP-polyC plasmid modified by the substitution of the ampicillin-resistance gene by the kanamycin-resistance gene as a selection marker to obtain large amounts of plasmids for at least future manipulations of the cDNA to study gene functions or construct attenuated strains. It is also important to note that the replacement/substitution of the ampicillin-resistance gene with the kanamycin-resistance gene should not affect the yield of a covalently closed circular (ccc) recombinant DNA molecule since similar yields were obtained for ccc recombinant DNA plasmids P1140-K2 containing kanamycin-resistance gene and P1140-AF2 containing RNA OUT selective marker (see Table 1 of the specification).
Fourth, since the modified method for fermentative production of a covalently closed recombinant DNA molecule resulting from the combined teachings of Zibert et al, Carnes et al and Romero Ramos et al is indistinguishable from the presently claimed method regarding to the materials and method steps used, it would also possess the same properties recited in the “wherein functional clause” regarding to the yield of the covalently circular recombinant DNA molecule. Moreover, it is noted that the “wherein functional clause” regarding to the yield of the covalently circular recombinant DNA molecule does not add any further structural details for the covalently closed circular recombinant DNA molecule and/or any further step/condition for the fermentation method of claim 26. Please, also note that where, as here, the claimed and prior art products are identical or substantially identical, or are produced by identical or substantially identical processes, the PTO can require an applicant to prove that the prior art products do not necessarily or inherently possess the characteristics of his claimed product. See In re Ludtke. Whether the rejection is based on "inherency" under 35 USC 102, or "prima facie obviousness" under 35 USC 103, jointly or alternatively, the burden of proof is the same, and its fairness is evidenced by the PTO's inability to manufacture products or to obtain and compare prior art products. In re Best, Bolton, and Shaw, 195 USPQ 430, 433 (CCPA 1977) citing In re Brown, 59 CCPA 1036, 459 F.2d 531, 173 USPQ 685 (1972).
Fifth, with respect to any robust and surprising yields for plasmids P1140-AF2 and P1140-K2 of the present application they must be commensurate with the scope of the claims. However, the claims under rejection are not necessarily limited to a method for fermentative production of plasmids P1140-AF2 (3617 bp-plasmid vector; Figure 2) and P1140-K2 (4385 bp-plasmid vector; Figure 4) in E. Coli bacteria. It is noted that the P1140-K2 plasmid has a single 64-adenosine-nucleotide poly(A) tail sequence that is followed by a single 31-cytidine-nucleotide poly(C) sequence, and both poly(A) tail sequence and poly(C) sequence are 3’ of the ORF (see Figure 2).
Sixth, the Williams reference was cited primarily to supplement the combined teachings of Zibert, Carnes and Romero Ramos for the additional limitation recited in dependent claim 12.
Claims 6-7, 9-10, 19, 26-28, 41-42, 45 and 50-51 are rejected under 35 U.S.C. 103 as being unpatentable over Bancel et al (WO 2014/152027; IDS) in view of Carnes et al (US 7,943,377), Romero Ramos et al (US 2018/0135060) and evidenced by the Notice of Opposition against EP Patent No. 4155409, on behalf of Margaret Dixon Limited (C127 in IDS filed on 10/29/2024, the first 5 paragraphs at page 9) for the same reasons set forth in the Non-Final Office Action dated 08/13/2025 (pages 19-24). The same rejection is restated below.
Bancel et al already disclosed at least a method for isolating and purifying the exemplary plasmid pJ344:91543-TC-GCSF that includes the coding sequence for GCSF (granulocyte colony stimulating factor) and 141 nucleotide sequence coding for Poly A tail, using an E. coli strain DH10B harboring the plasmid, with the plasmid map is shown in Figure 5 below (Abstract; particularly paragraphs [00409]-[00410]; Example 1 and Figure 5). The exemplary plasmid pJ344:91543-TC-GCSF also contains a T7-promoter used to express the coding sequence for GCSF (granulocyte colony stimulating factor) and 141 nucleotide sequence coding for Poly A tail (Insert 91543), a high copy pUC origin of replication, an Ampicillin resistance gene, and the transcription direction of the T7 promoter is the same as the direction of replication of the pUC origin of replication as shown in Figure 5.
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Bancel et al also disclosed the isolation and purification of another exemplary plasmid pJ204109475 that includes the coding sequence for Factor IX and 141 nucleotide sequence coding for PolyA tail, using an E. coli strain DH10B harboring the plasmid, with the plasmid pJ204109475 map is shown in Figure 11A below (Abstract; Summary of the Invention; particularly paragraphs [0040]-[0050], [00409]-[00410], [0419]; Example 6; Figure 6 and 11A-B).
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The exemplary plasmid pJ204:104975 contains a T7-promoter used to express the coding sequence for Factor IX and 141 nucleotide sequence coding for Poly A tail (Insert 109475), a high copy bacterial origin of replication, an Ampicillin resistance gene, and the transcription direction of the T7 promoter is the same as the direction of replication of the pUC origin of replication as shown in Figure 11A reproduced above. Alignment of the pJ204:109475 and pJ344:91543 sequences (Fig. 11B and Fig. 6, respectively) with the pUC origin sequence of SEQ ID NO: 6 of the present application (page 70, lines 16-20) revealed that pJ204:109475 plasmid also contains a pUC origin of replication which is separated from the poly(A) sequence by 2168 base pairs (in the alignment, the pUC origin ends at position 790 of plasmid pJ204:10975, and the poly(A) tract begins at position 2958 of the plasmid) as evidenced by the Notice of Opposition against EP Patent No. 4155409, on behalf of Margaret Dixon Limited (C127 in IDS filed on 10/29/2024, the first 5 paragraphs at page 9).
Bancel et al stated at least “PCR generated DNA templates have disadvantages that can be mitigated using linearized whole plasmid DNA templates. A PCR-free process has the following advantages: Scalability: Plasmid DNA template can be produced at microgram, milligram and grand gram scale in a cGMP compliant fashion. Large scale production of PCR generated templates is not commercially viable” (paragraphs [0023]-[0024]). Bancel et al also disclosed the plasmid DNA template comprises a gene of interest coding for a polypeptide of interest (e.g., GCSF, Factor IX, or one or more vaccines that improve immunity to an infectious agent), an RNA polymerase promoter (e.g., a T7 RNA polymerase, SP6 RNA polymerase) located 5’ to and operably linked to the gene of interest, a sequence coding for a polyA tail located 3’ to the gene of interest, and immediately downstream of the polyA tail coding sequence on the plasmid DNA template is a recognition site for a restriction endonuclease to linearize the plasmid (Abstract; Summary of the Invention; particularly paragraphs [0042]-[0045], [0052] and [0098]). Bancel et al also taught the region encoding the polypeptide of interest is at least greater than about 35, 50, 100, 500, 1,000, 2000, 2500, 3000, 4000, 5000 nucleotides or more in length, and the polyA tail is at least about 80 nucleotides or 160 nucleotides in length (paragraphs [0067], [0072], [0075]; and Figure 4).
Bancel et al did not disclose explicitly at least a method for fermentative production of the covalently closed circular recombinant DNA plasmid template comprising a kanamycin resistance gene instead of an ampicillin resistance gene as a selection marker to obtain large amounts of plasmids for the production of RNA transcripts.
Before the effective filing date of the present application (8/10/2015), Carnes et al already taught improvements in plasmid DNA production technology to ensure economic feasibility of future DNA vaccines and DNA therapeutics, including fed-batch fermentation strategies combining novel growth and induction phase temperature shifts (see at least Abstract; Disclosure of the Invention; Brief Summary of the Invention; issued claims 1-7). Specifically, Carnes et al disclosed a method of fed-batch fermentation in which plasmid-containing E. Coli cells are grown at a reduced temperature (e.g., around 300C) during the fed-batch phase, during which growth rate is restricted by a carbon limiting exponential feeding strategy(e.g., preferred growth rate ranges are u=0.05 to 0.3 per hour, preferably u=0.12 per hour via control nutrient feed), followed by a temperature up-shift (e.g., to approximately 36-450C) and continued growth at elevated temperature in order to accumulate plasmid, whereby the temperature shift and restricted growth rate improves yield and purity of plasmids, and wherein the plasmid contains a ColE1-derived replication origin, a pMB1 replication origin containing the pUC G to A mutation, or a pUC origin containing plasmid (col. 5, first full paragraph; col. 9, lines 53-63).
Additionally, Romero Ramos et al also taught preparation of the Escherichia Coli T7 expression pMRKA plasmid vector that allows an enhanced production, in industrial scale, of recombinant proteins, by modifying plasmids containing the gene sequence of the T7 promoter of E. coli (e.g., pAE vector) and which modification comprises the substitution of the ampicillin-resistance gene by the kanamycin-resistance gene (Abstract; Summary of the Invention; particularly paragraphs [0051]-[0052], [0134]-[0138]; and Figure 1). Romero Ramos et al stated “The need to modify the pAE plasmid was due to the following reasons: (a) as the ampicillin resistant gene encodes a beta-lactamase which is located in periplasmic space of E. coli in high density culture, this enzyme has the possibility to migrate of the culture means and, consequently, to degrade the antibiotic, decreasing the selective pressure of the means and providing the buildup of cells without plasmid; and (b) in the production of expression vectors in industrial scale it is not recommended to use antibiotic similar to the ones used in medical and veterinary fields, as it is the case of ampicillin. As kanamycin does not have medical application, the ampicillin resistant gene in pAE vector was replaced with kanamycin resistance gene” (paragraph [0052]). Romero Ramos et al also disclosed that T7 promoter of T7 bacteriophage is a strong promoter, and the expression systems based on T7 are broadly used for super-expression of in large scale of recombinant proteins in prokaryotes and eukaryotes (paragraph [0003]).
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to modify the teachings of Bancel et al by also at least using the fed-batch fermentation method of Carnes et al for fermentative production of the covalently closed circular recombinant DNA plasmid template comprising a gene of interest with 2500, 3000, 4000, 5000 nucleotides or more in length in the same configuration as those described in the exemplary plasmids pJ344:91543-TC-GCSF and pJ204:104975 that is modified by the substitution of the ampicillin-resistance gene by the kanamycin-resistance gene as a selection marker to obtain large amounts of plasmids for the production of RNA transcripts, in light of the teachings of Carnes et al and Romero Ramos et al as presented above.
An ordinary skilled artisan would have been motivated to carry out the above modifications because Carnes et al already disclosed successfully a method of fed-batch fermentation in which plasmid-containing E. Coli cells are grown at a reduced temperature (e.g., around 300C) during the fed-batch phase, during which growth rate is restricted (e.g., preferred growth rate ranges are u=0.05 to 0.3 per hour, preferably u=0.12 per hour via control nutrient feed), followed by a temperature up-shift (e.g., to approximately 36-450C) and continued growth at elevated temperature in order to accumulate plasmid, whereby the temperature shift and restricted growth rate improves yield and purity of plasmids, and wherein the plasmid contains a ColE1-derived replication origin, a pMB1 replication origin containing the pUC G to A mutation, or a pUC origin containing plasmid. Moreover, Romero Ramos et al taught advantages offered by substitution of the ampicillin-resistance gene by the kanamycin-resistance gene for an Escherichia Coli T7 expression plasmid vector.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Bancel et al, Carnes et al and Romero Ramos et al as set forth above; coupled with a high level of skill of an ordinary skilled artisan in the relevant art.
The modified method for fermentative production of a covalently closed recombinant DNA molecule resulting from the combined teachings of Bancel et al, Carnes et al and Romero Ramos et al is indistinguishable from the presently claimed method regarding to the materials and method steps used. Accordingly, it would also possess the same properties recited in the “wherein functional clause” regarding to the yield of the covalently circular recombinant DNA molecule. Moreover, it is noted that the “wherein functional clause” regarding to the yield of the covalently circular recombinant DNA molecule does not add any further structural details for the covalently closed circular recombinant DNA molecule and/or any further step/condition for the fermentation method of claim 26.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Bancel et al (WO 2014/152027; IDS) in view of Carnes et al (US 7,943,377) and Romero Ramos et al (US 2018/0135060) as applied to claims 6-7, 9-10, 19, 26-28, 41-42, 45 and 50-51 above, and further in view of Williams (US 2010/0184158).
The combined teachings of Bancel et al, Carnes et al and Romero Ramos et al were presented above. However, none of the cited references teach specifically a ccc recombinant DNA plasmid template comprising a primosome assembly site in the heavy strand (PAS-BH).
Before the effective filing date of the present application (8/10/2015), Williams already taught vector modifications that improve production yield of ccc recombinant DNA molecules such as plasmids, including a plasmid with a pUC origin (see at least Abstract; Summary of the Invention; particularly paragraphs [0011]-[0012], [0015], [0060]-[0064], [0067], [0070] and Fig. 2). Williams taught explicitly that by adding one or more components selected from the group consisting of an SV40 enhancer, PAS-BH region, and PAS into a plasmid vector to improve plasmid yield in shake flask and/or fermentation culture. Williams stated explicitly “It has been surprisingly found that this PAS-BH site improves plasmid yield in subsequent shake flask and/or fermentation culture” (paragraph [0067]).
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to further modify the combined teachings of Bancel et al, Carnes et al and Romero Ramos et al by also further incorporating at least the PAS-BH region and/or an SV40 enhancer into the modified pJ344:91543-TC-GCSF plasmid vector to improve plasmid yield in a fermentation culture, in light of the teachings of Williams as presented above.
An ordinary skilled artisan would have been motivated to further carry out the above modifications because Williams taught explicitly that by adding one or more components selected from the group consisting of an SV40 enhancer, PAS-BH region, and PAS into a plasmid vector, including a plasmid vector with a pUC origin, to improve plasmid yield in shake flask and/or fermentation culture.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Bancel et al, Carnes et al, Romero Ramos et al and Williams as set forth above; coupled with a high level of skill of an ordinary skilled artisan in the relevant art.
The modified method resulting from the combined teachings of Bancel et al, Carnes et al, Romero Ramos et al and Williams is indistinguishable and encompassed by the presently claimed invention.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Claims 2, 20-21, 43, 46 and 53-54 are rejected under 35 U.S.C. 103 as being unpatentable over Bancel et al (WO 2014/152027; IDS) in view of Carnes et al (US 7,943,377) and Romero Ramos et al (US 2018/0135060) as applied to claims 6-7, 9-10, 19, 26-28, 41-42, 45 and 50-51 above, and further in view of Thess et al (WO 2013/143698) and Priess et al (RNA 4:1321-1331, 1998).
The combined teachings of Bancel et al, Carnes et al and Romero Ramos et al were presented above. However, none of the cited references teach specifically that the insert further comprises at least one poly(C) sequence, preferably a sequence of about 15 to 200 cytidine nucleotides, wherein the ORF is positioned between the RNA polymerase promoter sequence and the at least one poly(C)sequence; or the insert comprises at least two homopolymeric regions of poly(A) sequence connected by a heterologous linker sequence and wherein each of said region of poly(A) sequence comprises 20 to 400 adenosine nucleotides in length.
Before the effective filing date of the present application (8/10/2015), Thess et al already disclosed an artificial nucleic acid molecule (e.g., mRNA and/or a DNA vector encoding the mRNA) comprising at least one open reading frame and at least one 3’-UTR for use in gene therapy and/or genetic vaccination applications, wherein the artificial nucleic acid molecule further comprises elements such as a poly(A) and a poly(C) sequence (see at least the Abstract; and page 45, last paragraph continues to first paragraph on page 46). Thess et al stated “[t]he inventive artificial nucleic acid molecule, particularly if the nucleic acid is in the form of an mRNA or codes for an mRNA, may be modified by a sequence of at least 10 cytidines, preferably at least 20 cytidines, more preferably at least 30 cytidines (so-called “poly(C) sequence”). Particularly, the inventive nucleic acid molecule may contain, especially if the nucleic acid is in the form of an (m)RNA or codes for an mRNA, a poly(C) sequence of typically about 10 to 200 cytidine nucleotides, preferably about 10 to 10 cytidine nucleotides, more preferably about 10 to 70 cytidine nucleotides or even more preferably about 20 to 50 or even 20 to 30 cytidine nucleotides. Thus, preferably the artificial nucleic acid molecule according to the present invention comprises, preferably in 5’-to-3’ direction, an ORF, at least one 3’UTR element as described above, a poly(A) sequence or a polyadenylation signal, and a poly(C) sequence” (page 45, last paragraph continues to first paragraph on page 46). Thess et al also taught that the poly(A) sequence may have a length of about 20 adenine nucleotides up to about 300 adenine nucleotides, more preferably from about 50 to about 100 adenine nucleotides, such as 60, 70, 80 or 100 adenine nucleotides (page 40, last paragraph); and the poly(A) sequence is located 3’ to at least one 3’UTR element via a direct connection or indirect connection such as via a linker of 1-50 or preferably 20 nucleotides (page 38, second paragraph).
Additionally, Priess et al already investigated the effect of different poly(A) tail length systematically by generating a series of capped IRE.CAT mRNAs with poly(A) tails ranging from 15 to 98 adenosines in length using plasmid DNAs encoded homopolymeric stretches of various lengths pIRE.CAT(A)15, pIRE.CAT(A)51, pIRE.CAT(A)98 and pIRE.CAT(A)150; and they found that CAT yields begin to increase significantly at and beyond 31 adenosines, and reach an early saturation at 51 adenosines under non-competitive conditions, whereas under competitive conditions CAT translation increases steadily between 15 and 98 adenosines (section titled “Effects of variations in poly(A) tail length on translation” on page 1326-1327; and Fig. 7). Moreover, Preiss et al reported generation of spontaneous deletion mutants during amplification of the plasmids pIRE.CAT(A)98 and pIRE.CAT(A)150 in bacteria; and found that the construct with the (A/T)150 proved too unstable for isolation in pure form but allowed the isolation of another spontaneous deletion clone pIRE.CAT(A)72 (section titled “Plasmid constructs” on page 1329).
Accordingly, it would have been obvious for an ordinary skilled artisan before the effective filing date of the present application to further modify the combined teachings of Bancel et al, Carnes et al and Romero Ramos et al by also further incorporating at least a poly(C) sequence of about 10 to 200 cytidine nucleotides, or 20 to 30 cytidine nucleotides; as well as using a poly(A) sequence having a length of about 20 adenine nucleotides up to about 300 adenine that is comprised of multiple of poly(A) sequence of about 30-70 adenosine nucleotides each that are indirectly connected via a heteropolymeric linker into the modified plasmid DNA template vector to improve plasmid yield in a fermentation culture, in light of the teachings of Thess et al and Priess et al as presented above.
An ordinary skilled artisan would have been motivated to further carry out the above modifications because Thess et al already disclosed an artificial nucleic acid molecule (e.g., mRNA and/or a DNA vector encoding the mRNA) comprising at least one open reading frame and at least one 3’-UTR for use in gene therapy and/or genetic vaccination applications, wherein the artificial nucleic acid molecule further comprises elements such as a poly(A) and a poly(C) sequence, and wherein the poly(C) sequence is about 10 to 200 cytidine nucleotides, or 20 to 30 cytidine nucleotides. Additionally, Thess et al also taught that the poly(A) sequence may have a length of about 20 adenine nucleotides up to about 300 adenine nucleotides and using a linker of 1-50 or preferably a 20 nucleotide-linker to indirectly connect the poly(A) sequence to the 3’ of at least one 3’UTR element; while Preiss et al reported generation of spontaneous deletion mutants during amplification of the plasmids pIRE.CAT(A)98 and pIRE.CAT(A)150 in bacteria; and found that the construct with the (A/T)150 proved too unstable for isolation in pure form but allowed the isolation of another spontaneous deletion clone pIRE.CAT(A)72. By segmenting a poly(A) sequence having a length of about 20 adenine nucleotides up to about 300 adenine nucleotides into multiple poly(A) sequences of about 30-70 adenosine nucleotides each that are indirectly connected via a heteropolymeric linker, spontaneous generation of deletion mutants during amplification of the plasmids could be avoided.
An ordinary skilled artisan would have a reasonable expectation of success in light of the teachings of Bancel et al, Carnes et al, Romero Ramos et al, Thess et al and Priess et al as set forth above; coupled with a high level of skill of an ordinary skilled artisan in the relevant art.
The modified method resulting from the combined teachings of Bancel et al, Carnes et al, Romero Ramos et al, Thess et al and Priess et al is indistinguishable and encompassed by the presently claimed invention.
Therefore, the claimed invention as a whole was prima facie obvious in the absence of evidence to the contrary.
Response to Argument
Applicant’s arguments related to the above 103 rejections in the Amendment filed on 02/13/2026 (pages 11-14) have been fully considered, but they are respectfully not found persuasive for the reason discussed below. It is noted that Applicant presented similar arguments as those presented in the Amendment filed on 07/10/2025 (pages 11-13).
A. Once again, Applicant argued that since Carnes teaches that the orientations of prokaryotic elements in the plasmid had no effect and that the inducible process is not specific to a particular plasmid, an ordinary skill in the art would have expected all plasmids to work equally well regardless of the orientation of prokaryotic elements. Applicant argued that at the time of filing the application, it was neither known nor reasonably expected that a plasmid with a specific orientation of replication and transcription, or a particular distance between the origin of replication and the poly(A) sequence, would be particularly suited for use in a fermentative production method. Additionally, Applicant argued that numerous widely used plasmids, such as pNIC-CH and pcDNA3.1, exhibit opposite directionality between transcription and replication. Consequently, there was no reason to believe altering the directionality of these elements would reasonably result in an increase in yield. None of Bancel, Carnes and Romero Ramos identified directionality as a point of optimization, and a skilled in the art would not have been motivated to specifically optimize the system with these two factors, directionality and distance, considered together. Applicant noted that the Action failed to acknowledge that 2168 bp is not at least 2200 bp, which is required by the claims. Applicant also argued that the Williams reference does not cure the aforementioned defects in the combination of Bancel, Carnes and Romero Ramos.
First, it irrelevant whether or not Carnes recognized the importance of optimizing the distance between the homopolymeric poly(A) sequence and the origin of replication, and/or the orientations of prokaryotic elements in any plasmid vector. As set forth in the above 103 rejection, an ordinary skilled in the art would have been motivated to modify the teachings of Bancel by also at least using the fed-batch fermentation method of Carnes for fermentative production of the covalently closed circular recombinant DNA plasmid template comprising a gene of interest with 2500, 3000, 4000, 5000 nucleotides or more in length in the same configuration as those described in the exemplary plasmids pJ344:91543-TC-GCSF and pJ204:104975 that is modified by the substitution of the ampicillin-resistance gene by the kanamycin-resistance gene as a selection marker to obtain large amounts of plasmids for the production of RNA transcripts because Carnes already taught improvements in plasmid DNA production technology to ensure economic feasibility of future DNA vaccines and DNA therapeutics, including fed-batch fermentation strategies combining novel growth and induction phase temperature shifts.
Second, the covalently closed circular recombinant pJ344:91543-TC-GCSF plasmid and/or pJ204109475 plasmid containing a gene of interest with 2500, 3000, 4000, 5000 nucleotides or more in length, instead of the GCSF gene and FIX gene, respectively, of Bancel contain all elements except for the kanamycin resistance gene and in the same directionality as those recited in the DNA molecule used in a fermentative method of the presently claimed invention. The above 103 rejections are directed specifically to the plasmid platform of the exemplary pJ344:91543-TC-GCSF plasmid and/or pJ204109475 plasmid in Bancel, and not related to any modifications for other widely used plasmids, such as pNIC-CH and pcDNA3.1. Please refer to the above 103 rejections for more details, particularly the motivation on why an ordinary skilled artisan would modify the teachings of Bancel by also using the fed-batch fermentation method of Carnes et al for fermentative production of the covalently closed circular recombinant DNA plasmid template comprising a gene of interest with 2500, 3000, 4000, 5000 nucleotides or more in length in the same configuration as those described in the exemplary plasmids pJ344:91543-TC-GCSF and pJ204:104975 that is modified by the substitution of the ampicillin-resistance gene by the kanamycin-resistance gene as a selection marker to obtain large amounts of plasmids for the production of RNA transcripts. It is also important to note that the replacement/substitution of the ampicillin-resistance gene with the kanamycin-resistance gene should not affect the yield of a covalently closed circular (ccc) recombinant DNA molecule since similar yields were obtained for ccc recombinant DNA plasmids P1140-K2 containing kanamycin-resistance gene and P1140-AF2 containing RNA OUT selective marker (see Table 1 of the specification).
Third, please note that the teachings of Bancel et al are not necessarily limited to the exemplary plasmid pJ204:104975 containing the coding sequence of Factor IX with a pUC origin of replication which is separated from the poly(A) sequence by 2168 base pairs. A covalently closed circular recombinant DNA plasmid template comprising a gene of interest with 3000, 4000, 5000 nucleotides or more in length in the same configuration as those described in the exemplary plasmids pJ344:91543-TC-GCSF (Fig. 5) and pJ204:104975 (Fig. 11A) would have a pUC origin of replication that is separated from the poly(A) sequence by at least 2200 bp as required by the instant claims.
Fourth, since the modified method for fermentative production of a covalently closed recombinant DNA molecule resulting from the combined teachings of Bancel et al, Carnes et al and Romero Ramos et al is indistinguishable from the presently claimed method regarding to the materials and method steps used, it would also possess the same properties recited in the “wherein functional clause” regarding to the yield of the covalently circular recombinant DNA molecule. Moreover, it is noted that the “wherein functional clause” regarding to the yield of the covalently circular recombinant DNA molecule does not add any further structural details for the covalently closed circular recombinant DNA molecule and/or any further step/condition for the fermentation method of claim 26. Please, also note that where, as here, the claimed and prior art products are identical or substantially identical, or are produced by identical or substantially identical processes, the PTO can require an applicant to prove that the prior art products do not necessarily or inherently possess the characteristics of his claimed product. See In re Ludtke. Whether the rejection is based on "inherency" under 35 USC 102, or "prima facie obviousness" under 35 USC 103, jointly or alternatively, the burden of proof is the same, and its fairness is evidenced by the PTO's inability to manufacture products or to obtain and compare prior art products. In re Best, Bolton, and Shaw, 195 USPQ 430, 433 (CCPA 1977) citing In re Brown, 59 CCPA 1036, 459 F.2d 531, 173 USPQ 685 (1972).
Fifth, with respect to any robust and surprising yields for plasmids P1140-AF2 and P1140-K2 they must be commensurate with the scope of the claims. However, the claims under rejection are not necessarily limited to a method for fermentative production of plasmids P1140-AF2 (3617 bp-plasmid vector; Figure 2) and P1140-K2 (4385 bp-plasmid vector; Figure 4) in E. Coli bacteria. It is noted that the P1140-K2 plasmid has a single 64-adenosine-nucleotide poly(A) tail sequence that is followed by a single 31-cytidine-nucleotide poly(C) sequence, and both poly(A) tail sequence and poly(C) sequence are 3’ of the ORF (see Figure 2).
Sixth, the Williams reference was cited primarily to supplement the combined teachings of Bancel, Carnes and Romero Ramos for the additional limitation recited in dependent claim 12.
B. With respect to the rejection based on Bancel, Carnes, Romero Ramos, Thes and Priess, Applicant argued that none of the cited prior art references teach a plasmid having two poly(A) sequence regions. Specifically, Applicant argued that Thess teaches a linker positioned between the insert and a single poly(A) sequence, while Priess reports the generation of spontaneous deletion mutants during plasmid amplification; and it is stated in the Office action that such mutants should be avoided, implying no inherent benefit from using a heteropolymeric linker between poly(A) sequences. Therefore, the combination of Thess and Priess does not suggest the claimed configuration of two poly(A) sequences separated by a linker, and a skilled artisan would not have been motivated to arrive at this invention by combining these two references with any other cited art.
First, since the above rejection was made under 35 U.S.C 103 none of the cited references have to teach every limitation of the instant claims. For example, neither Thess nor Priess alone has to teach a configuration of two poly(A) sequences connected by a heteropolymeric linker sequence.
Second, since Thess et al already taught that the poly(A) sequence may have a length of about 20 adenine nucleotides up to about 300 adenine nucleotides and using a linker of 1-50 or preferably a 20 nucleotide-linker to indirectly connect the poly(A) sequence to the 3’ of at least one 3’UTR element; and Preiss et al reported generation of spontaneous deletion mutants during amplification of the plasmids pIRE.CAT(A)98 and pIRE.CAT(A)150 in bacteria; and found that the construct with the (A/T)150 proved too unstable for isolation in pure form but allowed the isolation of another spontaneous deletion clone pIRE.CAT(A)72; it would have been obvious for an ordinary skilled in the art to recognize that by segmenting a poly(A) sequence having a length of about 20 adenine nucleotides up to about 300 adenine nucleotides into multiple poly(A) sequences of about 30-70 adenosine nucleotides each that are indirectly connected via a heteropolymeric linker, spontaneous generation of deletion mutants during amplification of the plasmids could be avoided.
Third, please also note that the standard under 35 U.S.C. 103 is a “reasonable” expectation of success.
Conclusions
No claim is allowed.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Quang Nguyen, Ph.D., at (571) 272-0776.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s SPE, James Douglas (Doug) Schultz, Ph.D., may be reached at (571) 272-0763.
To aid in correlating any papers for this application, all further correspondence regarding this application should be directed to Group Art Unit 1631; Central Fax No. (571) 273-8300.
Any inquiry of a general nature or relating to the status of this application or proceeding should be directed to (571) 272-0547.
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/QUANG NGUYEN/Primary Examiner, Art Unit 1631
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