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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 1/28/2026 has been entered.
Claims 16-17 and 20-22 remain withdrawn. Claims 12-15, 18-19, and 25-35 are pending and are examined on the merits herein.
Response to Applicant’s Arguments and Amendments
Regarding the 35 USC 103 Rejections presented in the Final Rejection mailed 9/2/2025, Applicant argues that the combination of references does not arrive at the claimed method, particularly because the combination results in nucleotides outside the GUV, and said nucleotides would not be inside the GUV for amplification. Applicant argues that the combination of references does not teach any nucleotides on the inside of any droplet emulsion or GUV, and particularly not “a GUV with nucleotides on the inside at the time that it was created from the double-emulsion microdroplet,” (Remarks, pages 6-8).
Applicant has amended instant claim 12 to now contain two sets of amplification reagents that both contain nucleotides – amplification reagents that are initially found in the miscible phase droplets and thus are eventually incorporated into the GUV, and carrier fluid amplification reagents that surround the double-emulsion microdroplet and eventual GUV, and then diffuse into said GUV.
In the instant claims, the diffusion of the carrier fluid amplification reagent nucleotides through the GUV membrane is due to subjecting the GUV to sufficient amplification conditions, where there is no specific indicator for what those conditions may be. In the instant specification, no specific amplification conditions are provided either. Para. 151 notes that emulsion membranes may naturally be permeable to particular molecules, allowing their diffusion in or out, and notes that emulsions may undergo shrinking or growing by allowing diffusion of a solvent in or out. Para. 311 notes that GUVs may be selectively permeable, allowing nucleotides to pass through and not macromolecules such as DNA and RNA. Thus, the ability of the nucleotides to diffuse into the GUV as claimed does not appear to rely on anything other than the use of non-specific amplification conditions and the permeability of the GUV membrane.
Regarding the placement of the nucleotides in the combination of references presented in the Final Rejection, the following is quoted from the Advisory Action mailed 1/7/2026:
“In the "Response to Applicant's Arguments" section of the Final Rejection, paras. 8 and 9 describe that in the combination of references used, nucleotides in the aqueous carrier would be surrounding the GUV, and that the GUV is permeable, as taught by Lorenceau. While the permeability teachings of Lorenceau do focus on osmotic pressure differences and shrinking of the polymerosomes, shrinking is not prohibited by the instant claims, nor is an addition of solute to the environment surrounding the GUV. Additionally, Lorenceau does not require the collapse of GUVs, but merely shows the point at which collapse may occur given a particular osmotic pressure shock and sufficient time (Figure 5). Furthermore, Walde also provides teachings regarding GUV permeability (page 849, information box and column 1, para. 1 and Table 2), and in particular permeability while still trapping nucleic acids (page 860, column 1, para. 3). Lim 2 also discusses selective diffusion across membranes and the alteration of such diffusion (page 4568). Lim 1 also notes that altering permeabilization of membranes is possible, though it may require some trial-and-error optimization (page 2, para. 3). Thus, in examining the combination of references, the ordinary artisan, utilizing ordinary skill and creativity, would recognize that permeable membranes for droplet emulsions and GUVs are known in the art, and that said permeability can be selective. Given that in the combination of Lim 1, Lim 2, Abate, Lorenceau, Walde, and Link, the nucleotides in the aqueous carrier would be surrounding the GUV, in order to accomplish amplification of the linear nucleic acid within the GUV, the nucleotides would logically need to diffuse into the GUV. The Final Rejection does not specifically state that the methods of Lorenceau involving glucose must be used, but simply states that the reference teaches the use of polymerosomes with permeable membranes. The ordinary artisan would thus be able to use the guidance and techniques described in the prior art to successfully perform amplification within the GUV as claimed, particularly in light of the fact that any combined amplification/ diffusion method that results in successful amplification within the GUV would read on the claimed method.”
These arguments rely on the ordinary knowledge, skill, and creativity available to a person of ordinary skill in the art. Applicant does not appear to directly address these teachings or arguments in their Remarks, and argues that the combination of references does not teach that nucleotides enter the GUV (see the diagram on page 7 of Applicant’s Remarks). This argument is not persuasive in view of the argument above.
In the Final Rejection, a summary of the references and their combination/teachings is as follows:
Lim 1 teaches a method for amplifying target genes in emulsion droplets, where the materials needed for amplification are within the droplets. Lim 1 mentions that double emulsification methods may be used, and Lim 2 provides details on this method. Together, these references teach the creation of a double-emulsion with amplification methods, where the nucleotides are within the emulsion.
Abate is then used to teach envelopment methods to render obvious separately adding the nucleotides to the reagent around the double-emulsion, a method also compatible with triple emulsions.
Lorenceau and Walde are then used to teach using GUVs rather than triple emulsions in the method of Lim 1, in view of Lim 2, and in view of Abate.
Link is then used to teach the use of linear genomic DNA in the method of Lim 1, in view of Lim 2, in view of Abate, in view of Lorenceau, and in view of Walde.
It is noted that Applicant does not point out any supposed deficiencies in these combinations of references, except in reference to the newly amended claim limitations, where Applicant states, “For example, as discussed above, the Office stated that separating the nucleotides from the other amplification reagents would provide a technical advantage by ensuring that no errant hybridization or extension occurred prematurely. This technical advantage would be lost if nucleotides were present on the inside, i.e. because this would allow accidental hybridization or extension according to the Office,” (Remarks, page 8). The Examiner agrees that it would not be prima facie obvious to include nucleotides both inside and outside the droplets, where the latter nucleotides later diffuse into the GUV, in view of the teachings of the references cited above.
Thus, the previous 35 USC 103 Rejections have been withdrawn, and new grounds of rejection are provided below.
Claim Objections
Claim 12 is objected to because of the following informalities: in (i) of step (b), it is recommended to state “the amplification reagents” for clarity, as these reagents are first described in step (a). Appropriate correction is required.
Claim 25 is objected to because of the following informality: “polyethyleneglycol” should be written as “polyethylene glycol”. Appropriate correction is required.
Claims 31-33 are objected to because of the following identical informalities: “polyethylene-glycol” should be written as “polyethylene glycol.” It is also recommended to include the abbreviation for polyethylene glycol (i.e. (PEG)), as is done in previous claims that recite this chemical. Appropriate correction is required.
Claim Interpretation
Regarding the diameter ranges for the double-emulsion microdroplets and GUVs described in instant claims 12 and 30, in the instant specification, diameter ranges of 0.1-1000 µm are recited for double-emulsions, along with many narrower ranges (see para. 15). Para. 16 notes a similar 0.1-1000 µm range for GUVs. Para. 197 describes producing droplets of different sizes, where droplets can then be sorted based on size, but the particular diameters chosen do not appear to be critical. Similar teachings are shown in paras. 307 and 313, where the latter paragraph notes the ability to generate monodisperse droplets. Para. 321 states that even though generation of small droplets is possible, the practical limit so that enzymes and reaction reagents can function as intended in 50 µm, which is at the higher end of the claimed ranges of diameters. However, Applicant does not describe that their claimed diameter ranges are critical or unexpected. MPEP 2144.05 I states, “In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990).” Thus, prior art which overlaps with the claimed diameter range(s) will be considered to render obvious said ranges.
Claim Rejections - 35 USC § 103
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 12-15, 18-19, and 25-35 are rejected under 35 U.S.C. 103 as being obvious over Abate et al. (WO 2016/126865 A1).
The applied reference has a common Applicant and joint inventors with the instant application. Based upon the earlier effectively filed date of the reference, it constitutes prior art under 35 U.S.C. 102(a)(2).
This rejection under 35 U.S.C. 103 might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C.102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B); or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement. See generally MPEP § 717.02.
Abate teaches multiple-emulsion nucleic acid amplification that allows biological systems to be detected (Abstract). In particular, the reference teaches the use of giant unilamellar vesicles (GUVs; para. 4). Figure 1 and paras. 6 and 60 detail the formation of double-emulsion droplets via microfluidics methods, where a water-in-oil-in water droplet can be made utilizing a miscible phase fluid-in immiscible phase fluid-in miscible phase fluid. This second miscible phase fluid is also called a carrier fluid by Abate (e.g. paras. 122, 124, and 131). After the double emulsion is made, Abate describes the formation of GUVs – “the double emulsion microdroplets may be subjected to dewetting conditions, forming GUVs, in which the immiscible phase fluid of the double emulsion is expunged from the shell, leaving behind a membrane of surfactant, with a small immiscible phase droplet adhered to the outside of the membrane,” (para. 60). Figure 1 notes that within the initial droplet formation, target nucleic acids and amplification reagents can be captured, and that between single and double-emulsion formation, droplets must collected and then reentered into a microfluidics device. Para. 119 teaches that surfactants can be used to stabilize the microdroplets, including in the case of single emulsions. Paras. 273 and 305 note the formation of single emulsions in which the initial mixture contains the target DNA, PCR Master Mix, and a surfactant (i.e. Tween-20, also known as polysorbate 20). Para. 99 states that digital PCR methods can be utilized, where target nucleic acids from a solution are diluted so that when the sample is isolated in components (such as droplets), the compartments include either none or a single target molecule. The methods of Abate can be used on linear nucleic acids specifically (e.g. para. 173 notes the use of small, linear viral genomes).
Para. 75 states that amplification reagents can be introduced into GUVs, at least in part, by including said reagents in the second miscible phase carrier fluid, where said reagents can then diffuse into the center of the GUVs (it is noted that the same diffusion can also apply to double emulsions). Paras. 289-291 describe an embodiment of Abate in which GUVs were generated with PCR reagents (including dNTPs) included within the emulsion/GUV, and then dNTPs were additionally added in the carrier phase fluid – these dNTPs could then diffuse into the GUVs and “improve [PCR] efficiency as reagents are consumed by the reaction.”
Paras. 13-14 note that the multiple emulsion droplets and GUVs of Abate can be about 0.1-1000 µm in diameter, overlapping with the diameter ranges described in instant claims 12 and 30 and rendering these ranges obvious (see the “Claim Interpretation” section above).
Abate also teaches that PCR products may be detected (e.g. paras. 5, 12, 170, and 293).
Though these teachings of Abate are not all present in the same embodiment, it would be prima facie obvious to utilize these methods together to arrive at the inventions of instant claims 12, 15, 25, and 30. This would involve the formation of GUVs to PCR amplify and detect single, linear nucleic acids (where dNTPs can be in the initial miscible phase fluid and the carrier miscible phase fluid), the use of Tween 20 as a surfactant in the miscible phase fluid, and the GUV formation methods described by Abate (i.e. the double-emulsion dewetting methods that then form a surfactant membrane). This would create a method capable of detecting individual linear nucleic acids within single droplets/GUVs, which could be useful in sorting through a sample to determine if particular pathogenic or disease causing mutants are present, which would have clinical utility. Additionally, Abate teaches that the addition of dNTPs in the carrier fluid as well as the initial miscible phase fluid allows for more efficient PCR and ensures that reactions can proceed to their desired endpoint. There would be a reasonable expectation of success in utilizing these methods together because none of the individual teachings of Abate are being altered, and these teachings are all taught to be compatible with GUV usage and manipulation.
Thus, claims 12, 15, 25, and 30 are prima facie obvious over Abate.
Regarding claims 13 and 14, Abate teaches that the miscible phase carrier fluid can be a buffered aqueous phase carrier fluid (para. 77 and embodiment 2 on page 81, for example), and that the first and second miscible phase fluids can be the same (para. 77 and embodiment 3 on page 81, for example). The ordinary artisan would additionally be motivated to include these features so that the carrier fluid is able to function and flow as intended (e.g. including buffers can allow for a slower flow than using water alone, allowing time for droplet formation, or can strengthen microdroplet shells), and so that the method would require less distinct reagents and components overall (in the case where the fluids are the same), increasing efficiency and reducing resource costs.
Regarding claim 18, Abate teaches that detectably labeling the amplification product can occur subsequent to amplification (embodiment 37 on page 86). Para. 94 also states that labeled probes can be introduced to GUVs after primers. Embodiments 7 (page 82), 22 (page 84), and 50 (page 88) also detail labeling after amplification.
Regarding claim 19, Abate teaches that targets can be detectably labeled utilizing TaqMan PCR (para. 5) and PCR generally (para. 9). Labeled probes are specifically recited, and embodiment 38 (on page 86 of the reference) notes the use of detectably labeled probes as amplification reagents present in the initial miscible phase fluid (see also embodiment 31 on page 85).
Regarding claim 26, Abate teaches that thickening agents may be used to induce GUV formation from double-emulsions, such as PEG, alginate, and glycerol (para. 137). Para. 283 also lists these thickening agents. Para. 137 states that, “The tendency of a double emulsion to de-wet depends on the properties of the different solutions and surfactants, especially the interfacial tensions of the different phases with respect to one another.” Para. 279 also states that, “The challenge to performing reactions in GUVs, however, is that they are fragile, consisting of a membrane a few molecular layers thick that can easily rupture due to mechanical stress or heat. To perform PCR in GUVs, a membrane composition must be identified that can withstand repeated thermalcycling up to 95°C.” However, it is unclear if these thickening reagents are specifically taught to be in the initial miscible phase solution. As noted above in the rejection of claim 14, Abate also teaches that the two miscible phase solutions can be the same, and that performing the methods of Abate in this way would increase efficiency and reduce resource costs. Thus, it would be prima facie obvious to include the thickeners described by Abate in both miscible phase solutions, both to retain the benefits described above by utilizing the same miscible phase solution throughout the method, and to provide reagents that would allow for the successful formation of GUVs. As Abate points out in the teachings described earlier in this paragraph, GUV formation, particularly for the purposes of amplifying nucleic acids sequences, can involve particular challenges (e.g. ensuring proper de-wetting and thermostability concerns). Thus, the ordinary artisan would be motivated to use the fluids and additives (such as the thickeners PEG, alginate, and glycerol) already shown by Abate to result in successful GUV formation.
Regarding claims 27-29, Abate notes that crosslinking can render shells of emulsions thermostable, and that they may be less prone to rupture or coalescence (paras. 141-142). Abate teaches that surfactants or chemicals such as polydimethylsiloxane can be cross-linked for such purposes, and notes that cross-linking can occur via reactions between streptavidin and biotin moieties on polymers (paras. 141-142). This increased thermostability would motivate the ordinary artisan to create such membranes on the GUVs of the invention of Abate described above in the rejection of claim 12, as these GUVs are subjected to PCR amplification conditions, which involve temperature changes. If the membranes were not thermostable, they would risk rupturing before amplification is complete, potentially leading to lost sample and wasted reagents. As Abate teaches particular ways to accomplish such cross-linking (i.e. cross-linking surfactants and using biotin/streptavidin interactions), the ordinary artisan would be motivated to use these methods to ensure that the advantages described by Abate are applicable. Though Abate only teaches these methods for double-emulsions, the reference does note that thermostable GUVs are also desirable, and describes similar surfactants for use in creating both double-emulsions and GUVs (paras. 138-139), providing a reasonable expectation of success for cross-linking surfactants when creating GUVs as well.
Regarding claims 31-34, para. 284 of Abate teaches that in the aqueous phases of GUV formation, fluorinate surfactants can be used, and specifically fluorine end-capped homopolymers of hexafluoropropylene epoxide-polyethylene-glycol surfactants with varying molecular weights. This paragraph goes on to specifically state molecular weights of 6,000-10,000 for the hexafluoropropylene epoxide blocks and 600-800 for the PEG block. The addition of bovine serum albumin (BSA) is also recited. This paragraph is used to detail teachings that will generate thermostable GUVs that have high melting temperatures. As the GUVs in the methods of Abate described above in the rejection of claim 12 are being used with PCR amplification (and thus, will undergo thermocycling), it would be prima facie obvious to utilize thermostable surfactants and additives as described by Abate for the formation and use of GUVs in these methods as well, to reduce risk of the GUVs rupturing before amplification can be completed.
Regarding claim 35, Abate teaches that GUVs can be ruptured with a buffer containing HFE-7500 and perfluorooctanol (para. 296). This can allow for the recovery of molecules within the GUVs for use in downstream sequencing methods. Thus, it would be prima facie obvious to use such a buffer in the invention of Abate described above in the rejection of claim 12 in order to incorporate sequencing techniques into the method.
Conclusion
No claims are currently allowable.
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/FRANCESCA FILIPPA GIAMMONA/Examiner, Art Unit 1681