DETAILED ACTION
This action is in reply to papers filed 11/24/2025. Claims 1, 6, 9-11, 15-16, 20-21, 23-25, and 30-31 are pending and examined herein.
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 .
Withdrawn Objection(s) and Rejection(s)
The cancellation of claims 2, 3, 8, 12, 14 and 18 renders any rejections thereof moot.
The objections to claims 10 and 25 regarding minor informalities are withdrawn in light of the amendments to the claims.
The rejection of claims 6 and 9 under 35 U.S.C. 112(b) is withdrawn in light of the amendments to the claims, which clarifies that ApoE is added to (claim 6) or not added to (claim 9) the culture medium during step (ii).
The rejection of claims 1, 6, 8-12, 15-16, and 23 under 35 U.S.C. 103 over Van Gaal (Journal of Controlled Release, 2011), in view of Fenton (Agnew. Chem., 2018; cited in IDS filed 08/24/2022) and Youn (Expert Opinion on Biological Therapy, 2015) is withdrawn in light of the amendment to claim 1.
The rejection of claims 1-3 under 35 U.S.C. 103 over Van Gaal (Journal of Controlled Release, 2011), in view of Fenton (Agnew. Chem., 2018; cited in IDS filed 08/24/2022) and Morozov (Molecular Biology, 2012) is withdrawn in light of the amendment to claim 1.
The rejection of claims 1, 11, 14, 18, 20, and 21 under 35 U.S.C. 103 over Van Gaal (Journal of Controlled Release, 2011), in view of Fenton (Agnew. Chem., 2018; cited in IDS filed 08/24/2022) and ThermoFisher (“Factors Influencing Transfection Efficiency,” retrieved from the Internet 11/21/2019 by Wayback Machine) is withdrawn in light of the amendment to claim 1.
The rejection of claims 1 and 24-25 under 35 U.S.C. 103 over Van Gaal (Journal of Controlled Release, 2011), in view of Fenton (Agnew. Chem., 2018; cited in IDS filed 08/24/2022) and Villeneuve (Environmental Toxicology and Chemistry, 2000) is withdrawn in light of the amendment to claim 1.
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 1, 6, 9-11, 15-16, and 23 are rejected under 35 U.S.C. 103 as being unpatentable over Van Gaal (Journal of Controlled Release, 2011, 154: 218-232), in view of Fenton (Agnew. Chem., 2018, 57(41): 13582-13586; cited in IDS filed 08/24/2022), Youn (Expert Opinion on Biological Therapy, 2015, 15(9): 1337-3148), ThermoFisher (“Factors Influencing Transfection Efficiency,” retrieved from the Internet 11/21/2019 by Wayback Machine), and Morozov (Molecular Biology, 2012), 46(3): 391-406).
Regarding claim 1: Van Gaal teaches an in vitro method for screening non-viral gene delivery candidates for transfection efficiency (reads on potency), wherein the method comprises reading out transgene expression levels relative to a reference formulation after transfection (Abstract, Section 7.3, Section 8). Van Gaal teaches that the plasmid used for transfection comprises a reporter gene driven by a viral promoter (Section 2.1). Van Gaal further teaches that the delivery vehicle used in the method may be a lipid nanoparticle (Sections 4.1, 7.2). Val Gaal teaches seeding HepG2 cells on a cell culture plate (Section 5.1; Fig 2 caption) (step (i)), then transfecting the cells with a test or reference sample of the composition (Sections 6.1-6.2) (steps (ii), (iii)). Val Gaal teaches that the readout of transgene expression may be a direct detection of the proteins, or an indirect method based on enzymatic conversion of an added substrate to a colored or fluorescent product (Section 7.1), and may be determined by flow cytometry or microscopic analysis (Section 7.1). Van Gaal further teaches that expression levels are calculated relative to control values from the reference sample (Section 7.3) (step (iv)).
Val Gaal does not teach 1) carrying out the method with mRNA encapsulated in a lipid nanoparticle (LNP), 2) seeding a population of Hep-G2 cells on a cell culture plate comprising wells coated with collagen or lysine, or 3) contacting the transfected cells with a first antibody specific for the polypeptide encoded by the mRNA and subsequently with a second, labeled antibody which is specific for the first antibody and detecting the second, labeled antibody.
Regarding difference 1: Val Gaal teaches carrying out the method disclosed therein with plasmid DNA. Val Gaal does not teaches carrying out the method with mRNA encapsulated in a lipid nanoparticle.
Fenton teaches transfecting HeLa cells with LNPs containing mRNAs (Abstract; p 13584, col 1, para 2).
Youn teaches that mRNA has major advantages to plasmid DNA for use in therapy (p 1345, col 1, para 2). Among other things, the use of mRNA reduces risk of insertion mutagenesis, eliminates the reliance on a promoter for modulating gene expression, allows for effective delivery of genetic cargo into non-dividing cells, and reduces vector-induced immunogenicity (p 1345, col 1, para 2).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Van Gaal to deliver mRNA instead of plasmid DNA in the LNPs, as taught by Fenton. One of ordinary skill in the art would have been motivated to make this modification because Youn teaches that mRNA has major advantages to plasmid DNA for use in transfection therapy. One of ordinary skill in the art would have had a reasonable expectation of reasonably making this modification because Fenton teaches that mRNAs can be encapsulated in LNPs, which can then be used to transfect cells in vitro.
Regarding difference 2: Val Gaal does not teach seeding a population of Hep-G2 cells on a cell culture plate comprising wells coated with collagen or lysine.
ThermoFisher teaches that some cell lines may need special coating materials, including collagen and poly-lysine, to attach to the culture plate and get the optimal transfection results (p 3, Media, para 3).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Van Gaal by using a cell culture plate wherein the wells are coated with collagen or lysine, as taught by ThermoFisher. One of ordinary skill in the art would have been motivated to make this modification because ThermoFisher teaches that collagen and lysing coating can help certain cell lines attach to the culture plate to get optimal transfection results. One of ordinary skill in the art would have had a reasonable expectation of reasonably making this modification because ThermoFisher teaches that plates comprising wells coated with collagen or lysine can be used as cell adhesion substrates for transfection experiments.
Regarding difference 3: Val Gaal does not teach contacting the transfected cells with a first antibody specific for the polypeptide encoded by the mRNA and subsequently with a second, labeled antibody which is specific for the first antibody and detecting the second, labeled antibody.
Morozov teaches transfecting HEK 239T cells with a vector encoding recombinant AFP (Abstract) using TransIT-LT1, a lipid-based transfection reagent (p 394, col 1, para 1), then performing immunofluorescence on the transfected cells (p 394, col 2, para 3). Morozov teaches treating the cells with an anti-AFP rabbit antibody (reads on first antibody), followed by Texas Red-labeled anti-rabbit IgG goat antibody (reads on second antibody), then imaging the cells using a fluorescent microscope (p 394, col 2, para 3).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Van Gaal by contacting the transfected cells with a first antibody specific for the polypeptide encoded by the mRNA and subsequently with a second, labeled antibody which is specific for the first antibody and detecting the second, labeled antibody, as taught by Morozov. One of ordinary skill in the art would have been motivated to make this modification to detect the amount of expression of the polypeptide encoded by the mRNA in the transfected cells. One of ordinary skill in the art would have had a reasonable expectation of reasonably making this modification because the method of Van Gaal comprises transfecting cells in vitro using an LNP, and Morozov teaches that immunofluorescence can be performed on cells transfected in vitro using an LNP.
Therefore, the method of claim 1 is rendered obvious over Van Gaal, in view of Fenton, Youn, ThermoFisher, and Mozorov.
Regarding claims 9-11, 15-16 and 23: Following the discussion of claim 1, the transfection method taught in Val Gaal does not comprise the addition of ApoE during transfection (claim 9). Van Gaal teaches that the lipid nanoparticle may be a cationic lipid (Sections 3.1, 3.3, 7.2) (claim 10). Val Gaal teaches that the transfection studies are performed in adherent monolayer cell culture systems (Section 5.1; claim 15), wherein the cells are seeded into 96-well plates (Fig 2 caption; claim 11) 24 hours prior to transfection (Section 6.1; claim 16) and grown to reach 50-95% confluency on the day of transfection (Section 6.2; claim 15).
Van Gaal teaches that transfection time varies, ranging from 1h to continuous exposure until the time of analysis, but that in general, expression increases steeply with increasing incubation times up to around 4 h (claim 23), after which expression levels may either reach a plateau, continue to increase less steeply, or decrease, depending on the type and dose of complexes and cell type used (Section 6.4). Val Gaal teaches that the transfection takes place at 37℃ with 5% CO2 (Supplementary Materials p 5, para 2; claim 23).
Regarding claim 6: Van Gaal does not teach adding ApoE to the transfection medium.
Fenton teaches transfecting HeLa cells with mRNA LNPs in the presence of additional ApoE than is endogenously present in media (p 13584, col 1, para 2).
It would have been prima facie obvious to a person of ordinary skill the art before the effective filing date of the claimed invention to have modified the method of Van Gaal by adding ApoE to the transfection medium, as taught in Fenton. One of ordinary skill in the art would have been motivated to make this modification because Fenton teaches that the presence of ApoE increased the total amount of protein expression by approximately 140% (p 13584, col 2, para 2). One of ordinary skill in the art would have had a reasonable expectation of successfully making this modification because Fenton teaches that ApoE can be added to media during transfection.
Claims 1, 20-21 and 31 are rejected under 35 U.S.C. 103 as being unpatentable over Van Gaal (Journal of Controlled Release, 2011, 154: 218-232), in view of Fenton (Agnew. Chem., 2018, 57(41): 13582-13586; cited in IDS filed 08/24/2022), Youn (Expert Opinion on Biological Therapy, 2015, 15(9): 1337-3148), ThermoFisher (“Factors Influencing Transfection Efficiency,” retrieved from the Internet 11/21/2019 by Wayback Machine), Morozov (Molecular Biology, 2012), 46(3): 391-406), and Riss (Assay and Drug Development Technologies, 2004, 2(1): 51-62).
Van Gaal, in view of Fenton, Youn, ThermoFisher, and Morozov, render obvious claim 1.
Regarding claims 20-21 and 31: Val Gaal teaches seeding HeLa CCL-2 cells at a density of 12,000 cells per 96-well (Supplementary Materials p 2, para 1).
Val Gaal does not teach a specific seeding density for HepG2 cells in a 96-well. Specifically, Van Gaal does not teach seeding each well of a 96-well plate at a density of 15,000 cells per well to 35,000 cells per well (claim 20), seeding each well of the culture plate with 20,000 cells per well to 30,000 cells per well (claim 21), or seeding each well of the culture plate with 30,000 cells per well (claim 31).
Riss teaches seeding HepG2 cells at a density of 25,000 cells per well in a 96-well plate to establish an in vitro system for determining the time- and dose-dependent toxic effects of tamoxifen on HepG2 cells using cell viability, cytotoxicity, and apoptosis assays (Abstract; Fig 1-5). Riss further teaches seeding HepG2 cells at a density of 1,000, 10,000, or 35,000 cells per well in a 96-well plate to determine whether cell density affects in vitro tamoxifen potency (p 57, col 2, para 2 – p 58, col 1, para 1; Fig 6). Riss teaches that cell density in the assay plates affects the outcome of a screening assay, and therefore, cell density should be tightly controlled across experiments (p 60, col 1, para 2). Riss further teaches that cell density is a parameter that is easily controlled using standard operating procedures (p 60, col 1, para 2).
It would have been prima facie obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Van Gaal, in view of Fenton, Youn, ThermoFisher, and Morozov, by using a fixed cell density, such as 25,000 or 35,000 HepG2 cells per well in a 96-well plate, as taught in Riss. One of ordinary skill in the art would have been motivated to make this modification because Riss teaches that cell density should be tightly controlled across experiments, and that HepG2 cells seeded at a density of 25,000 or 35,000 cells per well in a 96-well plate can be used in an in vitro assay. One of ordinary skill in the art would have had a reasonable expectation of successfully making this modification because Riss teaches that HepG2 cells seeded at a density of 25,000 or 35,000 cells per well in a 96-well plate can be used in an in vitro assay.
Regarding claims 20-21: In the case where the claimed ranges overlap or lie inside ranges disclosed by the prior art, a prima facie case of obviousness exists. See MPEP 2144.05(I).
Regarding claim 30: A prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are merely close. See MPEP 2144.05(I), In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955).
Claims 1, 6, and 30 are rejected under 35 U.S.C. 103 as being unpatentable over Van Gaal (Journal of Controlled Release, 2011, 154: 218-232), in view of Fenton (Agnew. Chem., 2018, 57(41): 13582-13586; cited in IDS filed 08/24/2022), Youn (Expert Opinion on Biological Therapy, 2015, 15(9): 1337-3148), ThermoFisher (“Factors Influencing Transfection Efficiency,” retrieved from the Internet 11/21/2019 by Wayback Machine), Morozov (Molecular Biology, 2012), 46(3): 391-406), and Dong (PNAS, 2014, 111(11): 3955-3960), as evidenced by ThermoFisher-2 (“Useful Numbers for Cell Culture,” retrieved from the Internet).
Van Gaal, in view of Fenton, Youn, ThermoFisher, and Morozov, render obvious claims 1 and 6.
Regarding claim 30: Van Gaal, in view of Fenton, Youn, ThermoFisher, and Morozov, does not teach the method of claim 6, wherein 4 μg/mL of ApoE is added. Specifically, Fenton, which teaches the addition of ApoE to culture medium, does not specify the concentration of ApoE used.
Dong, which is cited in Fenton (reference 6c), teaches lipopeptide nanoparticles for selective siRNA delivery (Abstract, Title). Dong teaches the use of 1 µg of ApoE per well in a 96-well plate (Supplementary Materials p 3, para 2). ThermoFisher-2 shows that each well of a 96-well plate holds approximately 0.1 to 0.2 mL of medium. Therefore, the concentration of ApoE taught in Dong is approximately 1 µg/0.1 mL to 1 µg/0.2 mL, or 10 µg/mL to 5 µg/ mL. In the case where the claimed ranges overlap or lie inside ranges disclosed by the prior art, a prima facie case of obviousness exists. See MPEP 2144.05(I).
Claims 1 and 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Van Gaal (Journal of Controlled Release, 2011, 154: 218-232), in view of Fenton (Agnew. Chem., 2018, 57(41): 13582-13586; cited in IDS filed 08/24/2022), Youn (Expert Opinion on Biological Therapy, 2015, 15(9): 1337-3148), ThermoFisher (“Factors Influencing Transfection Efficiency,” retrieved from the Internet 11/21/2019 by Wayback Machine), Morozov (Molecular Biology, 2012), 46(3): 391-406), and Villeneuve (Environmental Toxicology and Chemistry, 2000, 19(11): 2835-2843).
Van Gaal, in view of Fenton, Youn, ThermoFisher, and Morozov, render obvious claim 1.
Regarding claims 24 and 25: Van Gaal does not teach a method for generating a dose response curve for the test sample and the reference sample and determining the EC50 of the test sample and reference sample (claim 24), and calculating the relative potency as a percentage of the reference standard EC50 (claim 25).
Villeneuve teaches that relative potency estimates are widely used to characterize and compare the potency of a wide variety of samples analyzed using in vitro bioassays (Abstract). Villeneuve teaches that relative potency is generally calculated by first determining the dose-response curves for the sample and standard (claim 24), then calculating the EC50 of a standard divided by the EC50 of a sample (claim 25) (Abstract).
It would have been prima facie obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of Van Gaal by generating a dose response curve for the test sample and reference sample and determining the EC50 of each, as taught by Villeneuve. One of ordinary skill in the art would have been motivated to make this modification because Villeneuve teaches that relative potency estimates can be used to characterize and compare the potency of samples in in vitro assays. One of ordinary skill in the art would have had a reasonable expectation of reasonably making this modification because the method of Van Gaal is an in vitro assay.
Response to Arguments
Applicant argues: The cited references do not teach or suggest that using Hep-G2 specifically on collagen or lysine-coated plates is critical for mRNA-LNP potency assays. The instant claimed method is based, in part, on the recognition that LNP uptake variability between cell types and adhesion control issues resulted in non-linear assay behavior. Hep-G2 cells exhibit variable adherence and polarization that affect uptake pathways. The combination with collagen or lysine coating stabilizes adhesion, reduces detachment during wash steps in immunodetection, and normalizes membrane dynamics relevant to LNP endocytosis. Indeed, Example 3 of the application as filed demonstrates that the use of a coated culture plate in the method results in improved sensitivity and precision of the assay, and uptake kinetics, compared to when an un-coated cell culture plate is used.
In response: Applicant’s arguments have been fully considered, but are not persuasive. In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., the criticality of plating Hep-G2 cells on collagen or lysine-coated plates for mRNA-LNP potency assays) are not recited in the rejected claim(s). Claim 1 merely recites the limitation of “seeding a population of Hep-G2 cells o a cell culture plate comprising wells coated with collagen or lysine,” which is rendered obvious over Van Gaal, in view of Fenton, Youn, ThermoFisher, and Morozov, as outlined above. Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Applicant argues: While coated plates may have been known to improve cell properties in diverse contexts, the cited references do not link this to resolving LNP uptake variability in Hep-G2 cells for quantitative potency. Routine optimization would not necessarily converge on Hep-G2 cells with collagen or lysine coating as the standard for potency. The combination of Hep-G2 and coating, used specifically for relative potency rather than general expression detection, is not taught. The cited references lack any motivation to adopt a multi-step immunodetection method specifically to solve linearity and reproducibility constraints in LNP potency assays and do not provide a reasonable expectation of success for achieving a robust, reproducible relative potency assay for mRNA-LNPs.
In response: Applicant’s arguments have been fully considered, but are not persuasive. First, the claims are drawn to a method for determining relative potency, not for achieving relative potency or relative potency, per se. Thus, it is unclear what is meant by the applicant’s argument that “The combination of Hep-G2 and coating, used specifically for relative potency rather than general expression detection, is not taught” (emphasis added). Therefore, the feature argued by the applicant (i.e., “resolving LNP uptake variability in Hep-G2 cells for quantitative potency”) is not commensurate with the scope of the claim.
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., a multi-step immunodetection method specifically to solve linearity and reproducibility constraints in LNP potency assays) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
Conclusion
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Risa Takenaka whose telephone number is (571)272-0149. The examiner can normally be reached M-F, 12-7 EST.
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/RISA TAKENAKA/Examiner, Art Unit 1632
/TITILAYO MOLOYE/ Primary Examiner, Art Unit 1632