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 .
Election/Restrictions
Applicant’s election without traverse of Group III, claims 8-15 drawn to a method for delivering material into cells comprising: generating droplets of a hydrophilic solution containing cells and material to be delivered in a hydrophobic solution and deforming the droplets via transportation through a channel wherein the deformation creates nanopores in the cell allowing delivery of the material into the cell, in applicant’s response to the restriction requirement filed on May 28, 2025 is acknowledged.
Claims 1-7 and 16-18; Groups I, II, and IV; are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected inventions, there being no allowable generic or linking claim.
Status of the Claims
Applicant’s submission filed on October 28, 2025 has been entered and considered. Claims 1-7 and 16-18 were previously withdrawn after the finalization of the restriction requirement in the Office Action dated April 21, 2025. Rejections and/or objections not reiterated from the previous action mailed August 11, 2025 are hereby withdrawn. The following rejections and/or objections are either newly applied or are reiterated and are the only rejections and/or objections presently applied to the instant application. The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office Action.
The amended claims filed on October 28, 2025 are acknowledged. Claims 8 and 12 have been amended.
Claims 8-15 are examined on the merits.
Priority
The instant application is a 35 U.S.C 371 national stage filing of the International Application No. PCT/KR2020/015261 filed on November 4, 2020. The instant application claims foreign priority under 35 U.S.C 119(a)-(d) to Korean Patent Applications KR10-2019-0140657, filed on November 6, 2019, and KR10-2020-0129659, filed on October 7, 2020. Receipt is acknowledged of a certified copy of the foreign patent application in the original language as required by 37 CFR 1.55. Thus, the earliest possible priority for the instant application is November 6, 2019.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on May 5, 2022; March 28, 2023; April 27, 2023; and June 15, 2023 are in compliance with the provisions of 37 CFR 1.97 and have been considered by the examiner.
Withdrawn Objections to the Specification
In view of the amendment to the Specification to correct the reference to FIG. 12 and the filing of amended drawings for FIGS 1, 2, 6, and 7, the objections to the specification have been withdrawn.
Withdrawn Claim Rejections - 35 USC § 112
In view of the amendment to claim 8 in the response filed October 28, 2025, wherein claim 8 has been amended to recite “a first channel” and “a second channel”, the rejection of claim 8 has been withdrawn.
Claim Rejections - 35 USC § 103
Claims 8-15 are rejected under 35 U.S.C. 103 as being unpatentable over Sharei et al. (US 2014/0287509A1, found in IDS dated 05/05/2022, hereafter “Sharei”) in view of Lee et al. (KR 101605701-1B, found in IDS dated 05/05/2022 and Espacenet translation attached for clarity, hereafter “Lee”).
This is a new rejection. However, this rejection is based on a similar interpretation of Sharei in view of Lee as set forth in the office action dated August 11, 2025. The following new rejection provides additional interpretation of the claims which is necessitated by Applicant’s amendment in the response filed October 28, 2025. Any aspect of Applicant’s traversal that pertains to the rejection as newly set forth with be provided following the new statement of rejection.
With regard to claim 8, Sharei teaches a method of delivering a compound into a cell, the method comprising “providing the cell in a suspension or suspending a cell and a payload in a solution, passing the solution through a microfluidic channel that includes a constriction…such that a pressure is applied to the cell causing perturbations of the cell large enough for the payload to pass through” (Para. [0020], lines 2-10) wherein “payload” is defined as a compound or composition to be delivered into a cell (Para. [0007], lines 1-2). Sharei teaches that the constriction applies mechanical compression to the cell, thereby “squeezing” the cell (Para. [0091], lines 10-12 and Fig. 1B), which is considered to read on primary deformation, and that “once the cell passes through the constriction…the cell membrane recovers over time.” (Para. [0091], lines 15 and 18-19 and Fig. 1B), which is considered to read on secondary deformation interpreted as detailed above. Additionally, Sharei teaches an embodiment wherein, in the primary deforming, the diameter of the channel constriction can be configured such that the channel applies pressure to a cell without the cell touching the walls of the channel (Para. [0093], lines 13-14). Sharei also teaches wherein the cells are not in direct contact with the wall of the channel having the larger diameter (See Figs 1A & 1B, 2A, 3, and 39), which is considered to reasonably read on cells which are not in direct contact with the wall of the first channel.
Sharei does not teach generating droplets of a hydrophilic solution containing cells and material to be delivered into cells in a hydrophobic solution prior to flow of droplets through a channel and droplet deformation.
Lee teaches a method of microdroplet based assay comprising initial steps of introducing an aqueous fluid, which is considered to read on a hydrophilic solution, containing cells and PCR reagents in a first channel (Para. [0022]) and forming aqueous microdroplets within a carrier fluid wherein the carrier fluid is “immiscible” with the aqueous fluid, which is considered to read on a hydrophobic solution, (Para. [0023]) and wherein the formed aqueous microdroplets contained in the immiscible carrier fluid subsequently flow through a channel (Para. [0024]). Additionally, Lee teaches that the microdroplet formation method is advantageous as it allows use of small reaction volumes to increase reaction rates and forms more concentrated products, thereby reducing costs (Para. [0008], lines 11-12 and 15) and that the process allows for controlling the number of cells captured within the microdroplets as well as produces microdroplets of uniform size (Para. [0093], lines 6-7 and 10).
Therefore, it would have been obvious to one having ordinary skill in the art, before the effective filing date of the claimed invention, to modify the method of delivering a material into cells, the method comprising suspending a cell and a “payload” in a solution, passing the solution through a microfluidic channel including a constriction wherein the pressure caused by the constriction causes holes in the cell membrane such that the “payload” is able to enter the cell as taught by Sharei with an initial step of generating microdroplets comprising cells and other materials contained in an aqueous solution within a hydrophobic solution as taught by Lee. As Lee discloses that the steps of microdroplet generation result in the benefits of use of smaller reaction volumes thereby reducing cost and increased control of microdroplet contents and size (as detailed above) and Sharei discloses embodiments wherein the structural aspects of the microfluidic channels are altered in order to reduce clogging of the channel (Para. [0021], line 9), a skilled artisan would have been motivated to combine the initial step of generating microdroplets as taught by Lee with the method of cellular delivery using droplet deformation as taught by Sharei in order to increase efficiency while reducing costs and to help to reduce clogging of the channel by controlling and standardizing microdroplet size. One having ordinary skill in the art would have had a reasonable expectation of success as both Sharei and Lee teach methods of using microfluidic devices to perform reactions in cells.
With regard to claim 9, Sharei teaches that the cells in the solution are squeezed which causes “perturbations (e.g., holes…) in the cell membrane”, which are considered to read on nanopores (Para. [0091] lines 13-160 and Figs. 3 and 4).
With regard to claim 10, Sharei teaches that once the cell passes through the constriction, the cell membrane recovers over time (which is considered to read on returning to a shape before the constriction/primary deformation), and also that subsequent to passage through the constriction, the cell begins to uptake delivery material in the surrounding solution buffer through holes in the cell membrane (created by the deformation) and at least a portion of the delivery material remains trapped in the cell (Para. [0091], lines 15-20, Figs. 1B, 3, and 4).
With regard to claim 11, Sharei teaches that after the cells pass through the constriction, which is considered to read on secondary deformation wherein the deformation is returning to a shape prior to constriction, intake of delivery materials from the surrounding solution buffer is “diffusion-based” (Para. [0127], lines 1-6) and can include other conditions such as “post-constriction convective delivery mechanisms” (Para. [0127], line 17). Further, Sharei teaches that supplementation of diffusive delivery with convective delivery would enable higher concentrations of delivery material to enter the cell (Para. [0113], lines 14-15) which is considered to read on increased delivery efficiency of the material to be delivered.
With regard to claim 12, Sharei teaches that “[m]ultiple constrictions can be placed in parallel and/or in series.” (Para. [0090], line 22).
With regard to claim 13, Lee teaches generation of microdroplets wherein the aqueous liquid containing cells and PCR reagents flows in a first channel and the immiscible carrier fluid flows in second channels and wherein the first and second channels are arranged in an intersecting manner and wherein the arrangement may be in various shapes such as a Y shape or T shape (Para. [0052], Fig. 6A). This is considered to read on a hydrophilic solution flowing in one direction and a hydrophobic solution flowing in a second direction such that the hydrophilic and hydrophobic solutions come in contact with each other.
With regard to claim 14, Sharei teaches that the velocity at which the cells pass through the channel can be adjusted to control material delivery to the cells (Para. [0096], lines 1-3) and that the cells of Sharei’s method can be pushed through the microfluidic channels via the application of pressure (Para. [0011], lines 13-15). While Sharei does not specifically disclose application of a secondary solution in the method, one having ordinary skill in the art would recognize that application of additional solution to a contained system would increase the pressure and therefore the velocity, or acceleration, of the original solution in the microfluidic device.
With regard to claim 15, Sharei teaches that the payload is a compound or composition to be delivered into a cell, e.g., proteins, RNA and/or DNA molecules, other macromolecules, nanoparticles, compositions of matter (Para. [0007]), plasmids (Para. [0028], line 4), and transcription factors (Para. [0196].
Response to Applicant’s Traversal
Applicant’s traversal filed on October 28, 2025 is acknowledged and has been fully considered but is not persuasive.
First, Applicant asserts in section (1) on Pgs. 10-11 that modification of Sharei to include encapsulation of cells in droplets would destroy the core principle of Sharei’s system thus rendering the primary reference inoperative. Applicant asserts that in the primary reference of Sharei, the core mechanism responsible for material delivery relies on deformation of the cell caused by the cell’s passage through a constriction and requires that the cell must be in direct contact with the channel wall such that transient pores are formed by shear stress. Applicant asserts that, in the instant invention, “it is the droplet, not the cell that is deformed as it passes through the constriction” and that material delivery occurs via diffusion and convection within the droplet and not via deformation of the membrane. Applicant asserts that encapsulation of droplets in the system taught by Lee would render Sharei’s system inoperative as the cell would no longer deform and thus no pores in the membrane would form.
Applicant’s traversal has been fully considered but is not found persuasive.
With regard to Applicant’s assertion that the droplets, but not the cells suspended in the droplets, are deformed in the instant invention, based on Applicant’s disclosure, it appears that the instantly claimed invention relies on deformation of the cell in order to deliver material to the inside of the cell, similar to the method of Sharei. Para. [0019] of the specification states, “…material delivery into the cells is possible by deforming the cells in the droplets…”, and instant FIGS 8 and 13 appear to depict deformation of both the droplet and the cell suspended in the droplet. Additionally, Applicant’s specification refers to the cell being restored to the original shape (Para. [0019]), cell deformation (Paras. [0049], [0056]), deforming the cell membrane (Paras. [0053], [0055]), and indicates that “secondary deformation occurs in the droplet…and the cell…so the cell is restored to an original size” (Para. [0071]). Thus, one having ordinary skill in the art would understand, based on Applicant’s specification, that the instant invention requires cell deformation inside the droplet. Further, Applicant’s specification indicates that squeezing of the droplets (and cells) during primary deformation generates nanopores in the cell membrane which allows material delivery into the cell (Paras. [0036], [0068]). Thus, one having ordinary skill in the art would understand, based on Applicant’s specification, that the material delivery requires generation of nanopores via plasma membrane deformation, which is taught by Sharei. Although Applicant asserts that use of droplet encapsulation in Sharei’s system would eliminate cell deformation and generation of nanopores, the instant specification provides evidence that both cell deformation and nanopore generation occur when using droplet encapsulation and that these features are required in order for successful material delivery.
With regard to Applicant’s traversal pertaining to inoperability, MPEP 716.01(c)(II) states:
Arguments presented by the applicant cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965) and In re De Blauwe, 736 F.2d 699, 705, 222 USPQ 191, 196 (Fed. Cir. 1984). Examples of statements which are not evidence and which must be supported by an appropriate affidavit or declaration include statements regarding unexpected results, commercial success, solution of a long-felt need, inoperability of the prior art, invention before the date of the reference, and allegations that the author(s) of the prior art derived the disclosed subject matter from the inventor or at least one joint inventor.
Further, one having ordinary skill in the art has every reason to believe that droplet encapsulation of cells would allow the method of Sharei to function as taught. A skilled artisan could easily envision wherein droplets comprising cells were passed through a channel comprising a constriction such that both the droplets and cells inside the droplets are deformed, similar to the method of Sharei, and would have no reason to believe that addition of droplets would render the method of Sharei inoperable. See modified Fig. 13 of Sharei below which has been modified to include envisioned droplet encapsulation.
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Second, Applicant asserts in section (2) on Pg. 11 that the cited combination of Sharei and Lee fails to disclose or suggest the key feature that cells are not in direct contact with a wall of the first channel and references FIG 1B of Sharei as showing a cell in contact with a channel wall. Applicant further asserts that Lee does not teach deformation of droplets for cell delivery nor discloses wherein cells are not in contact with a wall of a channel during deformation.
Applicant’s traversal has been fully considered but is not found persuasive.
As detailed in the rejection above, Sharei does teach that the constriction (i.e., the second channel) may be larger than the diameter of the cell and configured to apply pressure to the cell without the cell touching the walls of the channel (Para. [0127]). However, as instantly claimed, the term “first channel” is used to define a channel with the larger diameter and the term “the second channel” is used to define a channel with a smaller diameter than the first channel (i.e., the constriction) through which the cells and droplets are passed. Thus, the amended instant claims are directed toward the feature of the cell not being in contact with the wall of the first channel which has the larger diameter. FIGs 1A & 1B, 2A, 3, and 39 of Sharei show a cell which is not in direct contact with the wall of the channel having the larger diameter both prior to and after the deformation step.
Third, Applicant asserts in section (3) on Pg. 12 that the claimed invention achieves alleged unexpected results over the closest prior art. Applicant asserts that the instant invention achieves alleged superior performance based on delivery of material via both diffusion and convection compared to the invention of Sharei wherein the material delivery occurs only through diffusion resulting in allegedly unexpected enhanced delivery and improved cell viability.
Applicant’s traversal has been fully considered but is not found persuasive.
Although Sharei’s preferred embodiment teaches cellular delivery of material via diffusion, Sharei also teaches that supplementation of the diffusion-based delivery with a convective component would provide enhanced cellular delivery of materials and the advantage of conserving delivery materials by reducing concentration of delivery materials required in the surrounding buffer. Sharei suggests a localized delivery method that exposes cells to a concentrated “cloud” of delivery materials after nanopore generation in the cell membrane (Para. [113]). Thus, Sharei contemplates use of both diffusion and convection based delivery. Lee teaches that encapsulation of cells and materials allows for more concentrated reaction volumes and increased reaction rates (Para. [0008]). Therefore, a skilled artisan would recognize, based on the teachings of Lee, that encapsulation of cells and delivery materials inside droplets would allow for maintained increased concentration of materials in the immediate area surrounding the cell and would prevent delivery materials, which are generally small molecules, from being pushed away from the cell during constriction allowing for increased delivery. Based on Pascal’s principle, when pressure is applied to any point in a confined fluid (the droplet), there is an equal increase in pressure at every other point in the fluid. Therefore, in the instant case, compression of the droplet would also compress the cell and the fluid in the droplet which contains a payload to be delivered would be forced into the cell via the generated nanopores.
With regard to Applicant’s assertion to alleged unexpected results, unexpected results are based on objective scientific evidence (See MPEP 2145). Sharei teaches a typical cell viability of 90% (Para. [0006]) which is supported by FIGs. 20B and 40. Thus, it appears that the cell viability of the instant invention is similar to the cell viability as taught by Sharei. Sharei also teaches delivery efficiency of Dextran greater than 90% (FIG. 40) in an embodiment wherein the channel prior to the constriction (i.e., the first channel) is larger than the cell such that the cell would not be in contact with the wall of the first channel (See FIG. 39) which is similar to the instantly disclosed delivery efficiency. Thus, it appears that the delivery efficiency of the instant invention is similar to the delivery efficiency as taught by Sharei. Additionally, although the instant claims are directed to intracellular delivery of a large genus of materials, the working examples indicating improved cell viability and delivery are directed toward delivery of a single material, Dextran (instant FIG. 15). Further, as supported by Sharei, intracellular delivery of material could be affected by additional parameters including the solution or buffer surrounding the cell (Para. [100]), the size of the compound to be delivered (FIG. 8A), temperature (Para. [106]), concentration of the compound surrounding the cell (Para. [102]), the type of cell (FIG. 30), flow velocity, number of cells per droplet, etc. Therefore, the alleged improved delivery efficiency as shown in Applicant’s working examples is not commensurate in scope with the large genus of materials to be delivered, large genus of method parameters, and large genus of cells as instantly claimed.
Conclusion
No claims are 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.
/ERIN V PAULUS/Examiner, Art Unit 1631
/ARTHUR S LEONARD/Examiner, Art Unit 1631