The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This office action is in response to Applicants’ amendments/remarks received November 12, 2025.
Rejections and/or objections not reiterated from previous office actions are hereby withdrawn.
Claim 12 is withdrawn. Claim 8 is canceled. Claims 1-7, 9-11 are under consideration.
Priority: This application is a 371 of PCT/CN2020/093500, filed May 29, 2020, which claims benefit of foreign application CN 201910460987, filed May 30, 2019. A copy of the foreign priority document has been received in the instant application on November 30, 2021 and is not in the English language.
Objections and Rejections
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 1-7, 9-11 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 written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a new matter rejection.
Claim 1 has been amended to recite a myriad of limitations and/or elements that do not appear to have explicit support in the originally filed specification. For instance, claim 1, step (3) has been amended to recite at least the limitations wherein, “in all in vitro cell-free protein synthesis systems in which the vector concentration ratios are varied”, total concentration of all expression vectors encoding the co-expressed target protein is held constant, and “only the relative concentration ratios of individual vectors are changed; a sufficient number of in vitro cell-free protein synthesis reactions are conducted under such varied vector concentration ratios to obtain, for each co-expressed target protein, a series of concentrations; and, by performing statistical regression fitting a relationship between (i) the series of concentrations and (ii) the percentage - relative to total vector concentration - of the concentration of vector carrying gene of corresponding co-expressed target protein that is added in each reaction, an equation of quantitative relationship is obtained”. The original specification does not appear to have express support for these limitations.
Claim 2 has also been amended to recite a myriad of limitations and/or elements that do not appear to have explicit support in the originally filed specification. For instance, claim 2, step 2, recites “stock solution” and “the stock solution is a liquid obtained by amplifying plasmid of the separated vector created in step (2). The original specification does not appear to have express support for these limitations. Claim 2, step 3, has also been amended to recite at least the limitations wherein, “in all in vitro cell-free protein synthesis systems in which the vector concentration ratios are varied”, total concentration of all expression vectors encoding the co-expressed target protein is held constant, and “only the relative concentration ratios of individual vectors are changed; a sufficient number of in vitro cell-free protein synthesis reactions are conducted under such varied vector concentration ratios to obtain, for each co-expressed target protein, a series of concentrations; and, by performing statistical regression fitting a relationship between (i) the series of concentrations and (ii) the percentage - relative to total vector concentration - of the concentration of vector carrying gene of corresponding co-expressed target protein that is added in each reaction, an equation of quantitative relationship is obtained”. The original specification does not appear to have express support for these limitations.
Claims 3-7, 9-11 are included in this rejection because they are dependent on the above claim(s).
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1-7, 9-11 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.
As previously noted, the claims are generally narrative, indefinite, and confusing in general. It is not clear what the actual method steps are and what is being achieved in the claimed method. The claims are replete with grammatical and idiomatic errors which make it difficult to determine and understand which limitations are active method steps and which limitations are merely results to be achieved from the claimed method. Applicants are requested to clarify and rewrite the claimed method so that is clear exactly what steps are being performed in the method. For instance, it is not clear whether the limitations and/or features recited in the “wherein” paragraphs in steps (1) to (5) in claims 1-2 are merely results to be achieved from the claimed method steps or actual steps that are performed as part of the claimed method. Further, even if the “wherein” clauses are interpreted as actual steps to be performed and/or carried out in the claimed method, as currently written, it is not clear what steps and features are being performed or carried out. The method steps and “wherein” clauses are all currently written in such a confusing fashion that it is difficult to ascertain exactly what the methods in claims 1-2 are trying to achieve. Claims 3-7, 9-11 are included in this rejection because they are dependent on the above claim(s) and fail to cure its defects.
A review of the instant specification indicates that the instant specification discloses that the invention is to quantitatively co-express multiple fluorescent proteins by adjusting the volume or concentration ratio of template DNA (patent application paragraph 0005). The methods recited in claims 1-2 appear to utilize different concentrations or ratios of vectors for adjusting the expression levels of target proteins, wherein the total concentration of the vectors remain the same.
As previously noted, in view of the indefiniteness of the instant claims noted above, the instant claims have been interpreted as a method for quantitatively co-expressing multiple proteins in vitro in a cell-free system comprising expressing multiple target proteins in the cell-free system by adjusting the levels of co-expressed target proteins based on the concentration ratios of vectors or template DNA encoding the multiple target proteins.
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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 1-5, 7, 9-11 are rejected under 35 U.S.C. 103 as being unpatentable over Stögbauer (2012 Experiment and quantitative modeling of cell-free gene expression dynamics Dissertation: 126 pages; previously cited) in view of Borkowski et al. (2018 Nature Communications 9:1457, 11 pages; previously cited) and Lentini et al. (2013 ACS Synth Biol 2: 482-489; previously cited).
In view of the indefiniteness of the instant claims noted above, the instant claims have been interpreted as a method for quantitatively co-expressing multiple proteins in vitro in a cell-free system comprising expressing multiple target proteins in the cell-free system by adjusting the levels of co-expressed target proteins based on the concentration ratios of vectors or template DNA encoding the multiple target proteins.
Stögbauer discloses cell-free systems denote mixtures of resources and enzymes that can be used to synthesize protein from a DNA template in vitro (at least p. 2). Stögbauer discloses establishing a model of cell-free gene expression kinetics based on differential equations for individual kinetics rates and to further account for different template DNA concentrations (at least p. 4). Stögbauer discloses a plasmid DNA encoding GFP for cell-free protein synthesis to establish a calibration standard, where GFP synthesis is measured fluorescently (at least p. 31, 35). Stögbauer discloses measuring the amount of synthesized GFP as a function of template DNA at different concentrations (at least p. 42-44). Stögbauer discloses developing a predictive model of cell-free transcription/translation for GFP synthesis dependent on DNA concentration (at least p. 49-52). Stögbauer discloses that the model predictively describes time courses mRNA and protein synthesis as a function of template concentration (at least p. 53). Stögbauer does not explicitly teach multiple plasmids encoding multiple proteins in the cell-free system.
Borkowski et al. disclose utilizing a cell-free lysate assay to predict in vivo burden of expressing multiple proteins (at least p. 1). Borkowski et al. disclose monitoring the concentrations of different plasmids encoding different proteins and expression (at least p. 2-5). For instance, Borkowski et al. disclose measuring in vitro expression in 10.5 µL cell lysate mix, with no competing plasmid, instead using increasing concentrations of a capacity monitor plasmid to determine the available capacity of the lysate (p. 2). Borkowski et al. disclose that the in vitro capacity reaches a plateau at 50 nM plasmid DNA and goes on to decrease at higher DNA concentrations (p. 2-3, also Fig. 1). Borkowski et al. disclose calculating the in vitro capacity per DNA concentration indicates that there is space capacity in the lysate assay below 30 nM total plasmid DNA, saturation at around 50 nM and decreased capacity per DNA above this, due to competition for expression within the pool of plasmids (p. 2-3, also Fig. 1). Borkowski et al. then set to determine the contributions of transcription and translation to resource competition in cell lysates (p. 3). Borkowski et al. disclose introducing two additional plasmids to compete with the capacity monitor plasmid; Borkowski et al. added different concentrations of each plasmid to the cell lysate mix along with 30 nM of the capacity monitor plasmid to measure the corresponding in vitro capacity via GFP production (p. 3). Borkowski et al. disclose that this demonstrates that the cell lysate assays when run with 20 nM of test plasmid plus 30 mM capacity monitor plasmid, expression is close to saturation (50 nM total DNA) and translational resources are the major limitation (p. 4). Borkowski et al. then disclose that using these determined conditions, the burden of a collection of plasmids expressing a protein of interest at different levels in the cell lysate assay was measured (p. 4-5). Therefore, Borkowski et al. fairly disclose monitoring the concentrations of different plasmids encoding different proteins, including the protein production levels, in an in vitro cell-free system. Borkowski et al. disclose predicting the burden of a multigene system comprising separate vectors or plasmids and measuring bioluminescence and/or expression (at least p. 6-7).
Lentini et al. disclose in vitro expression of 17 different fluorescent proteins in cell-free systems that give easily detectable fluorescence signals in real-time from in vitro transcription-translation reactions with a minimal system consisting of T7 RNA polymerase and E. coli translation machinery, i.e., the PUREsystem; the data were used to construct a ratiometric fluorescence assay to quantify the effect of genetic organization on in vitro expression levels (at least p. 482-483).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to combine the references and arrive at the claimed method for quantitatively co-expressing multiple proteins in vitro in a cell-free system comprising expressing multiple target proteins comprising a fluorescent protein in the cell-free system by adjusting the levels of co-expressed target proteins based on the concentration ratios of vectors or template DNA encoding the multiple target proteins, where the target proteins are expressed by different vectors and where it would be obvious that the different vectors added to the cell-free system at different concentrations result at a total concentration of the different vectors that is constant or remains the same prior to co-expression of the target proteins (instant claims 1-2, 4). The motivation to do so is given by the prior art. Stögbauer discloses a plasmid DNA encoding GFP for cell-free protein synthesis to establish a calibration standard, where GFP synthesis is measured fluorescently (at least p. 31, 35). Stögbauer discloses measuring the amount of synthesized GFP as a function of template DNA at different concentrations (at least p. 42-44). Stögbauer discloses developing a predictive model of cell-free transcription/translation for GFP synthesis dependent on DNA concentration (at least p. 49-52). Stögbauer discloses that the model predictively describes time courses mRNA and protein synthesis as a function of template concentration (at least p. 53). Borkowski et al. disclose monitoring the concentrations of different plasmids encoding different proteins and expression (at least p. 2-5). Borkowski et al. disclose predicting the burden of a multigene system comprising separate vectors or plasmids and measuring bioluminescence and/or expression (at least p. 6-7). Lentini et al. disclose in vitro expression of 17 different fluorescent proteins in cell-free systems that give easily detectable fluorescence signals in real-time. Therefore, one of ordinary skill would have reasonable motivation to incorporate the multiple plasmids encoding different proteins of Borkowski et al. where the different proteins comprise different fluorescent proteins as suggested in Lentini et al. into the model for developing a predictive model of cell-free transcription/translation for protein synthesis dependent on DNA template concentration disclosed in Stögbauer because there was interest in determining protein expression levels based on DNA template concentration in a cell-free system (instant claims 1-2, 4). One of ordinary skill would have a reasonable expectation of success because Stögbauer discloses establishing a model of cell-free gene expression kinetics based on differential equations for individual kinetics rates and to further account for different template DNA concentrations and fluorescent proteins in cell-free systems that give easily detectable fluorescence signals were known.
Regarding instant claim 3, Lentini et al. disclose at least the 17 different fluorescent proteins gave easily detectable signals that are unique in the in vitro cell-free system (at least p. 483, 485); therefore, it would be obvious that the luminescence value of each co-expressed protein does not interference with other expressed fluorescent proteins.
Regarding instant claim 5, Stögbauer discloses the cell-free system is one based on E. coli extract (at least p. 46) and Borkowski et al. and Lentini et al. also disclose cell-free systems based on E. coli extract (Borkowski et al. p. 2; Lentini et al. p. 482).
Regarding instant claim 7, Stögbauer discloses the fluorescent protein expressed in the in vitro cell-free system is measured as fluorescent units (at least p. 31-32). Therefore, it would be obvious that multiple co-expressed fluorescent proteins, such as those disclosed in Lentini et al., when expressed in an in-vitro cell-free system has a fluorescent unit.
Regarding instant claims 9-10, Stögbauer discloses the protein of interest is a fluorescent protein (GFP) (Stögbauer p. 31-53), Borkowski et al. also disclose the protein of interest is a fluorescent protein (p. 2-3, Fig. 1), and Lentini et al. disclose in vitro expression of 17 different fluorescent proteins in cell-free systems (p. 482-483). Therefore, it would be obvious to produce or co-express multiple proteins in the cell-free system where the multiple proteins are fluorescent proteins, including red fluorescent protein, yellow fluorescent protein, cyan fluorescent protein, blue fluorescent protein, etc.
Regarding instant claim 11, Stögbauer discloses that cell-free systems are developed to as a convenient means to synthesize proteins (p. 53-54). Therefore, it would be obvious that the protein synthesized in the cell-free system can be further isolated or purified for further studies or applications because purifying or isolating a synthesized protein is a routine and well-known activity done in the biotechnology field for maintaining and using the protein of interest.
Reply: Applicants’ amendments/remarks have been considered but they are not persuasive.
Applicants assert that the model of Stögbauer only involves the expression of a single protein (GFP). Applicants assert that it completely fails to account for the mutual influence of multiple DNA templates coexisting in the same system.
Applicants’ remarks are not persuasive.
In this instance, Stögbauer is cited as a 103 reference with at least Borkowski et al., where Borkowski et al. reasonably disclose measuring burden or concentrations of multiple plasmids and their capacity to express multiple proteins in a cell-free system.
As noted, in the 103 rejection, Stögbauer discloses developing a predictive model of cell-free transcription/translation for GFP synthesis dependent on DNA concentration in a cell-free system (at least p. 49-52). Stögbauer discloses that the model predictively describes time courses mRNA and protein synthesis as a function of template concentration (at least p. 53). Therefore, Stögbauer establishes a quantitative relationship and model that mRNA and protein synthesis is a function of DNA template concentration in a cell-free system.
While Stögbauer does not explicitly teach multiple plasmids encoding multiple proteins in the cell-free system, this deficiency is remedied by at least Borkowski et al., which disclose monitoring the concentrations of different plasmids encoding different proteins and the expression capacity of the multiple proteins in a cell-free system (at least p. 2-3, Fig. 1). Therefore, Borkowski et al., like Stögbauer, reasonably disclose the relationship and model that protein synthesis is a function of DNA template concentration. Therefore, it would be obvious to one of ordinary skill that the prior art Borkowski et al. disclose co-expressing multiple proteins in a cell-free system and disclose a relationship and/or model that expression of the multiple proteins is dependent on the concentrations of the corresponding plasmid encoding the protein and that expression of the multiple proteins is dependent on optimizing the concentrations of the multiple plasmids.
Applicants assert that Borkowski et al. is primarily focused on predicting in vivo expression burden and did not address the quantitative co-expression of multiple proteins in an in vitro cell-free system. Applicants assert that although Borkowski et al. monitored the expression of proteins encoded by the different plasmids, Borkowski et al. did not reveal any quantitative relationship between the concentrations of independent vectors and the concentrations of their expression products.
Applicants’ remarks are not persuasive. As also noted by Applicants, Borkowski et al. disclose monitoring the expression of multiple proteins encoded by the different plasmids (Borkowski et al. p. 2-3, Fig. 1, p. 9). Borkowski et al. disclose calculating and determining the expression capacity of the multiple plasmids expressing the multiple proteins, where expression capacity is measured as max GFP production rate (Fig. 1, p. 9), where the protein production rate is calculated from the protein produced (p. 9). Borkowski et al. disclose predicting the burden of a multigene system comprising separate vectors or plasmids and measuring bioluminescence and/or expression (at least p. 6-7). Therefore, it would be obvious to one of ordinary skill that Borkowski et al. reasonably disclose a quantitative relationship between the multiple proteins co-expressed and the multiple plasmids encoding the multiple proteins in the cell-free system and/or that the co-expression of the multiple proteins is a function of the multiple plasmids concentrations.
Additionally, Borkowski et al. is also cited with at least Stögbauer, where Stögbauer has already disclosed that protein expression or quantity is a function of DNA template concentration in a cell-free system (Stögbauer p. 43). Therefore, it would be obvious to one of ordinary skill in the art that the combination of the prior art discloses a system for co-expressing multiple proteins in a cell-free system and disclose a quantitative relationship and/or model that expression of the multiple proteins is dependent on the concentrations of the corresponding plasmid encoding the protein and that expression of the multiple proteins is dependent on optimizing the concentrations of the multiple plasmids.
Applicants assert that although Lentini et al. achieved the co-expression of fluorescent proteins, Lentini et al. focused on the impact of gene structure on expression levels. Applicants assert that Lentini et al. did not establish a mathematical model linking “vector concentration ratio to protein expression level.”
Applicants’ remarks are not persuasive. Lentini et al. is being relied on for the different types of multiple proteins that can be expressed in a cell-free system. The deficiency of Lentini et al. to not expressly teach a mathematical model linking “vector concentration ratio to protein expression level” is reasonably remedied by at least Stögbauer, where Stögbauer discloses that protein expression or quantity is a function of DNA template concentration in a cell-free system (Stögbauer p. 43) and establishes a quantitative relationship and model that mRNA and protein synthesis is a function of DNA template concentration in a cell-free system (see teachings of Stögbauer above), and Borkowski et al., where Borkowski et al. disclose that the quantity and production of multiple proteins in a cell-free system is dependent and/or a function of the multiple plasmids concentrations.
For at leas these reasons, the 103 rejection is maintained.
Claims 1-5, 6, 7, 9-11 are rejected under 35 U.S.C. 103 as being unpatentable over Stögbauer (Experiment and quantitative modeling of cell-free gene expression dynamics Dissertation 2012: 126 pages; previously cited) in view of Borkowski et al. (2018 Nature Communications 9:1457, 11 pages; previously cited), Lentini et al. (2013 ACS Synth Biol 2: 482-489; previously cited), and Gregorio et al. (2019 Methods Protoc 2(24): 34 pages; previously cited). The teachings of Stögbauer, Borkowski et al., and Lentini et al. over at least instant claims 1-5, 7, 9-11 are noted above.
Regarding instant claim 6, Stögbauer discloses other known cell-free systems including a eukaryotic extracted based on rabbit blood (p. 46-47). Gregorio et al. disclose high adoption platforms for cell-free protein synthesis are based on cell lines, including E. coli, S. cerevisiae (yeast), rabbit reticulocyte lysate, etc. (at least p. 3). Therefore, it would have been obvious to one of ordinary skill to incorporate the multiple plasmids encoding different proteins of Borkowski et al. where the different proteins comprise different fluorescent proteins as suggested in Lentini et al. into the model for developing a predictive model of cell-free transcription/translation for protein synthesis dependent on DNA template concentration disclosed in Stögbauer and where the cell-free system is based on a S. cerevisiae yeast lysate as suggested in Gregorio et al. because there was interest in determining protein expression levels based on DNA template concentration in a cell-free system. One of ordinary skill would have a reasonable expectation of success because Stögbauer discloses establishing a model of cell-free gene expression kinetics based on differential equations for individual kinetics rates and to further account for different template DNA concentrations and fluorescent proteins in cell-free systems that give easily detectable fluorescence signals were known.
Reply: Applicants’ amendments/remarks have been considered but they are not persuasive. The reasons for maintaining Stögbauer, Borkowski et al., and Lentini et al. are the same as noted above. In this instance, the teachings of the cited prior art noted above reasonably disclose quantitative modeling of cell-free gene expression dynamics and a system for co-expressing multiple proteins in a cell-free system and disclose a quantitative relationship and/or model that expression of the multiple proteins is dependent on the concentrations of the corresponding plasmid encoding the protein and that expression of the multiple proteins is dependent on optimizing the concentrations of the multiple plasmids, allowing for predicting the burden of a multigene system comprising separate vectors or plasmids and measuring bioluminescence and/or expression.
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.
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/Marsha Tsay/Primary Examiner, Art Unit 1656