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 .
DETAILED ACTION
Amendments
In the reply filed on 02/04/2026, Applicant has amended claims 1 and 24, and added new claims 25-28.
Claim Status
Claims 1, 3, 5-14 and 16-28 are pending and are considered on the merits
Withdrawn Claim Rejections - 35 USC § 103
The prior rejections of claims 1, 3, 5-14 and 16-24 under 35 U.S.C. 103 are withdrawn in light of Applicant’s amendment to claim 1 to recite new limitation “wherein the acoustic sonoporation pressure is at a subcavitation level to ensure that at least 50% of the microbubbles remain intact after irradiation”.
New 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.
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 1, 3, 5-7, 9-10 and 21-28 are rejected under 35 U.S.C. 103 as being unpatentable over Kodama et al., (Ultrasound in Med. & Biol. 2006; 32(6): 905-914. Prior art of record) in view of Zhou et al., (Theranostics. 2015; 5(4): p. 399-417. Prior art of record), Hensel et al., (2008 IEEE International Ultrasonics Symposium Proceedings, DOI: 10.1109/ULTSYM.2008.0408, p. 1671-1674) and Wamel et al., (J Control Release. 2006;112(2):149-55).
With respect to claims 1 and 3, Kodama teaches a method of combining ultrasound and ultrasound contrast agent in transfecting cells (see e.g., abstract), thus teaches the preamble an acoustic method for transfecting cells.
Regarding step (a) in claim 1, Kodama teaches CHO-E cells are seeded into wells alternately in 24-well (16-mm in diameter/well) plates in complete media (p. 907, left col, last para “Transfection”) containing plasmids with OptisonTM (consisting of octafluoropropane-filled albumin microbubbles, see p. 907, left col, para “Ultrasound contrast agents (UCAs)”), thus teaches providing a system that comprises (i) at least two reservoirs contained within a plurality of reservoirs (i.e., at least two wells in a 24-well plate), each containing in a fluid medium (i.e., complete media), gas filled microbubbles (i.e., OptisonTM consisting of octafluoropropane-filled microbubbles), host cells (i.e., CHO-E cells), and an exogenous material to be introduced into the host cells (i.e., plasmids). Kodama teaches the 24-well plates are located just above the ultrasound probe in a test chamber filled with water, as shown in Fig. 1, and exposed to ultrasound (p. 907, left col, last para “Transfection”), thus teaches (ii) an acoustic radiation generator external to the reservoirs (see the ultrasound generator in Fig 1a) and comprising a focusing element (i.e., the ultrasound probe in Fig 1a), to generate and direct focused acoustic radiation into the fluid medium (see Fig 1a-1b for diagram and see p. 908, left col, last para “Evaluation of ultrasound parameters” for acoustic radiation directed into the wells).
Regarding step (b) in claim 1, as stated supra, Kodama teaches the 24-well plates are located just above the ultrasound probe in a test chamber filled with water, as shown in Fig. 1, and exposed to ultrasound (p. 907, left col, last para “Transfection”), thus teaches (b) acoustically coupling the acoustic radiation generator to a first of the reservoirs (see Fig 1a-1b for the ultrasound probe placed underneath a well in the 24-well plate) via an acoustic coupling medium between the acoustic radiation generator and an external surface of the reservoir (i.e., see Fig 1a for water between the ultrasound probe and the bottom of the well, thus there is an acoustic coupling medium between the external surface of the well and the ultrasound probe connecting the ultrasound generator). Kodama teaches “[s]ince cells were seeded into wells alternately, neighboring wells were not exposed to ultrasound at the same time” (p. 907, left col, last para “Transfection”), thus teaches acoustically coupling a first reservoir without simultaneously acoustically coupling the acoustic radiation generator to any other of the reservoirs.
Regarding step (c) in claim 1, as stated supra, Kodama teaches the 24-well plates are exposed to ultrasound and the intensity of ultrasound in the well is determined (p. 907, left col, last para “Transfection” and p. 908, left col, last para “Evaluation of ultrasound parameters”). Kodama teaches ultrasound irradiation at an acoustic sonoporation pressure results in plasmid delivery into the cells (p. 909, left col, last para, see Fig 3a showing ultrasound and UCAs (filled circle) has higher luciferase activity than ultrasound alone (open circle) at medium height of 1-2 mm). Thus, Kodama teaches (c) activating the acoustic radiation generator to generate and direct the focused acoustic radiation at an acoustic sonoporation pressure into the first reservoir in a manner that acoustically activates the microbubbles, wherein acoustic radiation is transferred from the microbubbles to the host cells to provide sonoporated host cells (i.e., for plasmid transfer), thereby facilitating introduction of the exogenous material into the sonoporated host cells.
Regarding step (d), step (e) and step (f) in claim 1, and step (g) in claim 3, as stated supra, Kodama teaches CHO-E cells are seeded into wells alternately in 24-well plates and are exposed to ultrasound (p. 907, left col, last para “Transfection”). Kodama teaches “[s]ince cells were seeded into wells alternately, neighboring wells were not exposed to ultrasound at the same time” (p. 907, left col, last para “Transfection”). Kodama teaches the β-gal positive cell colonies are counted from triplicate wells in the 24-well plate (see Fig 4 legend). Thus, Kodama teaches cells are seeded into multiple wells in a well plate, each well is exposed to ultrasound individually while the neighboring wells are not affected, and the ultrasound treatment is performed in at least three wells. Thus, Kodama suggests a step (d) acoustically decoupling the acoustic radiation generator from the first reservoir (i.e., removing the probe from the first well), a step (e) acoustically coupling the acoustic radiation generator to a second of the reservoirs without simultaneously acoustically coupling the acoustic radiation generator to any other of the reservoirs (i.e., contacting the probe to the second well while neighboring wells are not exposed to ultrasound at the same time), a step (f) repeating step (c) with respect to the second reservoir (i.e., exposing the cells in the second well to ultrasound), and a step (g) in claim 3 acoustically decoupling the acoustic radiation generator from the second reservoir and thereafter repeating steps (b) through (f) of claim 1 with respect to additional reservoirs in the plurality of reservoirs (i.e., removing the probe from the second well, contacting the probe to the third well, exposing the cells in the third well to ultrasound, and so on).
However, regarding the new limitation in step (c), Kodama suggests that superposed ultrasound could result in cavitation and collapse of microbubbles and cell membrane damage and subsequent molecular delivery into cells (p. 911, right col, para 2, also see e.g., Fig 5 for luciferase activity and viability of cells being in an inverse correlation), but is silent on using an acoustic sonoporation pressure at a subcavitation level to ensure that at least 50% of the microbubbles remain intact after irradiation.
Hensel teaches a method of cell transfection using microbubbles and ultrasound (e.g., abstract). Hensel summarizes previous works (including Wamel as discussed below) demonstrating that the large scale oscillation of insonicated microbubbles (MB) is believed to be the primary effect in the creation of pores in cell membranes that allow the entering of molecules, thus the enhancement of gene therapy. MB destruction on the other hand is suspected to lead to lower transfection rates and increased cell damage (see e.g., abstract, and “Introduction” para 1). Thus, Hensel suggests using an acoustic sonoporation pressure at a subcavitation level to ensure that the microbubbles remain intact (i.e., are not destructed) after irradiation to reduce cell damage and increase transfection rate.
Wamel, referred to by Hensel, teaches ultrasound and microbubbles facilitate material delivery into cells (e.g., abstract and see Fig 4). Wamel teaches ultrasound waves cause the microbubbles to make repeated oscillations, and the vibrating microbubble induce cell deformation and cell membrane permeability (e.g., p. 153, “Results” section, also see Fig 3 and legend for pushing and pulling behavior of the oscillating microbubbles on nearby cells causing the deformation of the cell membrane, see Fig 8 for a proposed model of the oscillating microbubble enforced pore formation in the cell membrane). It is noted that Wamel uses an acoustic sonoporation pressure at a subcavitation level to ensure that at least 50% of the microbubbles remain intact after irradiation (see e.g., Fig 3B for imaging of vibrating microbubbles that remain intact after irradiation and see Fig 3C and Fig 8 for diagrams showing intact microbubbles during and after irradiation). Wamel teaches “in combination with microbubbles, ultra-short US field pulses … is below the threshold for irreversible damage, cells will remain viable” (p. 149, last para -p. 150, para 1).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the acoustic method for transfecting cells using an acoustic pressure that results in cavitation and collapse of microbubbles causing cell membrane damage disclosed in Kodama, by substituting with an acoustic sonoporation pressure at a subcavitation level to ensure that at least 50% of the microbubbles remain intact after irradiation as suggested by Hensel and Wamel with a reasonable expectation of success. Since Kodama suggests that the ultrasound could result in collapse of microbubbles and cell membrane damage (p. 911, right col, para 2, also see e.g., Fig 5 for luciferase activity and viability of cells being in an inverse correlation), and since Hensel suggests microbubble oscillation (i.e., of intact microbubbles) allows molecule entering cells for gene therapy while microbubble destruction leads to increased cell damage and lower transfection rates (see e.g., abstract, and “Introduction” para 1) and Wamel reduces to practice a method of using an acoustic sonoporation pressure below a threshold for irreversible cell damage to induce vibrating microbubble to cause cell deformation and cell membrane permeability (see above), one of ordinary skill in the art would have had a reason to substitute with an acoustic sonoporation pressure at a subcavitation level to ensure at least 50% of the microbubbles remain intact after irradiation as suggested by Hensel and Wamel in order to reduce cell damage and increase transfection rate.
However, Kodama is silent on the microbubbles being cationic and conjugated to the host cells in claim 1 (a), nor teach the conjugation by functionalizing and combining the microbubbles and host-cell-specific antibodies with binding moieties in claim 5.
Zhou teaches a method for transfecting a DNA plasmid into endothelial cells by ultrasound-mediated gene delivery (abstract, p. 404, right col, section “UTMD-mediated gene transfer in vitro”). Zhou teaches in preparation of a targeting microbubble “CMB105”, gas-filled cationic microbubbles (“CMB”, see p. 401, 2nd last para) is combined with streptavidin, and then biotinylated anti-CD105 antibody is added to the CMB to produce a CD105-targeting CMB (p. 401, last para, see Fig 1), and teaches CD105-expressing endothelial cells are observed with more targeted bound CMB105 microbubbles (p. 407, right col, para 1, see Fig 4C), thus teaches the microbubbles are gas-filled cationic and are conjugated to (i.e., targeted bound) the host cells in claim 1 (a), and teaches the conjugation is by functionalizing the microbubbles with a first binding moiety (i.e., streptavidin), functionalizing the host-cell specific antibody (i.e., anti-CD105 antibody) with a second binding moiety (i.e., biotin) that is configured to link to the first binding moiety (i.e., biotin-streptavidin link), and combining the microbubbles with the antibodies (i.e., to produce the CMB105) in claim 5.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the acoustic method for transfecting host cells using gas-filled microbubbles suggested by Kodama, by substituting the gas-filled microbubbles with gas-filled cationic microbubbles functionalized with binding moiety so as to be conjugated to the host cells mediated by host-cell-specific antibodies functionalized with linking moiety as taught by Zhou with a reasonable expectation of success. Since Wamel teaches only cells that are close enough to the vibrating microbubbles experience local forces and have deformation and induced permeability (p. 154, left col, para 2, see Fig 8), and since Zhou teaches CD105-expressing endothelial cells have more targeted bound CMB105 microbubbles (p. 407, right col, para 1, see Fig 4C), one of ordinary skill in the art would have had a reason to use gas-filled cationic host-cell-targeting microbubbles suggested by Zhou to conjugate the host cells to the microbubbles of Kodama in view of Hensel and Wamel so as to ensure the host cells experience local forces by the vibrating microbubbles to have deformation and induced permeability in order to improve the transfection efficiency.
However, Kodama, Hensel, Wamel, and Zhou do not specifically teach the reservoir-to-reservoir transition time.
Nevertheless, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have expected the reservoir-to-reservoir transition time of the probe from one well to an alternately seeded well in a 24-well plate (16-mm in diameter/well, i.e., about 32 mm between the centers of two alternately seeded wells) of Kodama would likely have been at most 0.1 seconds.
With respect to claim 6 directed to an isotonic buffer solution, as stated supra, Kodama teaches CHO-E cells are seeded in complete media (p. 907, left col, last para “Transfection”, see p. 906, right col, para “Cell preparation” for F-12 Nutrient Mixture media), which is an isotonic buffer solution.
With respect to claim 7 directed to the cells being plated on a surface of the reservoir, as stated supra, Kodama teaches CHO-E cells are seeded into wells alternately in 24-well plates and have a 24 h attachment period (p. 907, left col, last para “Transfection”). Wamel teaches bovine endothelial cell cultures (e.g., p. 150, right col, para 2.1 “Cell culture”). Thus, both teach the cells are plated on a surface of the reservoir.
With respect to claim 9 and claim 10 directed to the exogenous material comprising a nucleic acid or a DNA plasmid, as stated supra, Kodama teaches transfecting plasmids (pGL3-control or pCMVβ, see p. 907, left col, last para “Transfection”), thus teaches the exogenous material comprises a nucleic acid, a plasmid, and a DNA plasmid.
With respect to claim 21 directed to the plurality of reservoirs being arranged in an array, claim 22 directed to the reservoirs being contained within a substrate comprising an integrated multiple reservoir unit, and claim 23 directed to the integrated multiple reservoir unit comprising a well plate, as stated supra, Kodama teaches CHO-E cells are seeded into wells alternately in 24-well plates (p. 907, left col, last para “Transfection”), thus teach a well plate that is an integrated multiple reservoir unit within a substrate and that the reservoirs are arranged in an array in the well plate.
Claim 24 is directed to the reservoir-to-reservoir transition time being at most 0.001 seconds.
Kodama, Hensel, Wamel, and Zhou do not specifically teach the reservoir-to-reservoir transition time.
Nevertheless, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have practiced the reservoir-to-reservoir transition (i.e., to move the probe from one well to an alternately seeded well) in a manner that the transition time is at most 0.001 seconds with a reasonable expectation of success. One of ordinary skill in the art would have had a reason to do so in order to save time to perform the method. Since the distance between the wells is very short (16-mm in diameter/well, i.e., about 32 mm between the centers of two alternately seeded wells), one of ordinary skill in the art would have had a reasonable expectation of success in doing so.
With respect to claim 25 directed to the acoustic sonoporation pressure being in the range of 50% to 90% of a minimum acoustic pressure that would result in microbubble cavitation, as stated supra, Wamel teaches the acoustic sonoporation pressure is below the threshold for irreversible cell damage (p. 149, last para -p. 150, para 1), and teaches the ultrasound waves cause the microbubbles to make repeated oscillations (and thus are intact, e.g., p. 153, “Results” section, also see Figs 3 and 8 and legend for pushing and pulling behavior of the oscillating microbubbles), thus teaches the acoustic sonoporation pressure is below a minimum acoustic pressure that would result in microbubble cavitation.
Accordingly, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have chosen an acoustic sonoporation pressure being in the range of 50% to 90% of a minimum acoustic pressure that would result in microbubble cavitation in the method of Kodama in view of Hensel, Wamel, and Zhou with a reasonable expectation of success. Since Wamel reduces to practice an acoustic sonoporation pressure that is below the threshold for irreversible cell damage and does not cause microbubble cavitation, one of ordinary skill in the art would have had a reason to choose an acoustic sonoporation pressure being in the range of 50% to 90% of a minimum acoustic pressure that would result in microbubble cavitation in order to optimize the pressure to reduce cell damage and increase transfection efficiency.
With respect to claim 26 directed to the acoustic radiation having a wavelength selected so that the irradiated microbubbles vibrate at a frequency within 15% of an average resonance frequency of the microbubbles, it is noted that this wherein clause does not recite an active step in the claimed method, but only the results of the steps of (a), (b) and (c) as suggested by Kodama in view of Hensel, Wamel, and Zhou. MPEP 2111.04 I states a whereby clause (or a wherein clause) “in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.” Therefore, this wherein clause does not provide any patentable weight in determining patentability of the claimed method. Specifically, since Kodama in view of Hensel, Wamel, and Zhou, make obvious a method of generating acoustic radiation to irradiate microbubbles for transfecting cells wherein the acoustic radiation is selected so that at least 50% of the microbubbles remain intact after irradiation, the same method as recited in instant claim 1, thus, the method would have likely had the same intended result that the irradiated microbubbles vibrate at a frequency within 15% of an average resonance frequency of the microbubbles as recited in instant claim 26.
With respect to claim 27 directed to the microbubbles comprising an elastic shell material, Wamel teaches the irradiated microbubbles are compressed and expanded during irradiation (see e.g., Fig 3B for the imaging of different sizes of microbubbles and Fig 3C and Fig 8 for diagram), thus makes obvious to use microbubbles comprising an elastic shell material so as to be compressed and expanded under irradiation to induce cell deformation and permeability.
With respect to claim 28 directed to the acoustic sonoporation pressure being at a subcavitation level to ensure that 50% to 90% of the microbubbles remain intact after irradiation, as stated supra, Wamel uses an acoustic sonoporation pressure at a subcavitation level to ensure that about 50% to 90% of the microbubbles remain intact after irradiation (see e.g., Fig 3B for imaging of vibrating microbubbles that most remain intact after irradiation and see Fig 3C and Fig 8 for diagrams showing intact microbubbles during and after irradiation). Accordingly, one of ordinary skill in the art would have chosen an acoustic sonoporation pressure being at a subcavitation level to ensure that 50% to 90% of the microbubbles remain intact after irradiation as suggested by Wamel in the method of Kodama in view of Hensel, Wamel, and Zhou with a reasonable expectation of success. One of ordinary skill in the art would have had a reason to do so in order to reduce cell damage and increase transfection rate.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 02/04/2026 are acknowledged.
Applicant firstly argues that applicants are providing a high-throughput, extremely rapid transfection method that is automated. While in Kodama, the probe must evidently be moved manually from well to well. There could be no rapid reservoir-to-reservoir transition, a feature important to the claimed system (Remarks, p. 9).
Applicant’s arguments have been fully considered but they are not persuasive.
In response to Applicant's argument, it is noted that the features upon which applicant relies (e.g., high-throughput and automated) are not recited in the rejected claims. 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). See MPEP 2111.01 II.
Furthermore, as stated supra, one of ordinary skill in the art would have expected that the reservoir-to-reservoir transition time of the probe from one well to an alternately seeded well in a 24-well plate (16-mm in diameter/well, i.e., about 32 mm between the centers of two alternately seeded wells) of Kodama would likely have been at most 0.1 seconds, as recited in amended claim 1.
Applicant further argues that Kodama teaches ultrasound irradiation results in microbubble destruction (cavitation). By contrast, a goal of the presently claimed invention is to avoid cavitation. In the method of the invention, the microbubbles are irradiated to cause the microbubbles of the microbubble-host cell conjugates to vibrate; the vibrational energy is transferred to the host cells from the microbubbles, and it is this vibrational energy that causes the cell walls to become permeable and enable transfection. Cavitation would be counter-productive. (Remarks, p. 9-10).
Applicant’s arguments have been fully considered and they are persuasive. Therefore, the prior rejection over Kodama in view of Zhou has been withdrawn. However, as necessitated by amendment, a new ground of rejection has been made over Kodama in view of Hensel, Wamel, and Zhou. Specifically, as stated supra, Hensel teaches the large scale oscillation of insonicated microbubbles (MB) is believed to be the primary effect in the creation of pores in cell membranes that allow the entering of molecules, thus the enhancement of gene therapy. MB destruction on the other hand is suspected to lead to lower transfection rates and increased cell damage (see e.g., abstract, and “Introduction” para 1). Wamel, referred to by Hensel, teaches ultrasound waves cause the microbubbles to make repeated oscillations, and the vibrating microbubble induce cell deformation and cell membrane permeability (e.g., p. 153, “Results” section, also see Fig 3 and Fig 8). It is noted that Wamel uses an acoustic sonoporation pressure at a subcavitation level to ensure that at least 50% of the microbubbles remain intact after irradiation (see e.g., Fig 3B for imaging of vibrating microbubbles that remain intact after irradiation and see Fig 3C and Fig 8 for diagrams showing intact microbubbles during and after irradiation). Thus, Hensel and Wamel make obvious the vibrating microbubbles that are intact.
Applicant further argues that Zhou is specifically directed to ultrasound-targeted microbubble destruction and Zhou does not teach a transfection method with a rapid reservoir-to-reservoir transition as is necessary in a high throughput process (Remarks, p. 10).
Applicant’s arguments have been fully considered but they are not persuasive.
As a first matter, as stated supra, it is noted that the features upon which applicant relies (e.g., high-throughput) are not recited in the rejected claims. Additionally, one of ordinary skill in the art would have expected that the reservoir-to-reservoir transition time of Kodama would likely have been at most 0.1 seconds, as recited in amended claim 1.
Furthermore, in response to Zhou directed to ultrasound-targeted microbubble destruction, Applicant is reminded that a 35 U.S.C. § 103 based test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). In the instant case, as stated supra, Hensel and Wamel make obvious a method of ultrasound-induced microbubble oscillating to induce cell permeability. Wamel teaches only cells that are close enough to the vibrating microbubbles experience local forces and have deformation and induced permeability (p. 154, left col, para 2, see Fig 8). Zhou is cited to teach host-cell-targeting microbubbles by antibody that enhances cell-microbubble conjugation (p. 407, right col, para 1, see Fig 4C). Thus, one of ordinary skill in the art would have substituted with the conjugated microbubbles of Zhou in order to enhance the cell-microbubble conjugation and transfection efficiency.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Kodama et al., (Ultrasound in Med. & Biol. 2006; 32(6): 905-914. Prior art of record) in view of Zhou et al., (Theranostics. 2015; 5(4): p. 399-417. Prior art of record), Hensel et al., (2008 IEEE International Ultrasonics Symposium Proceedings, DOI: 10.1109/ULTSYM.2008.0408, p. 1671-1674) and Wamel et al., (J Control Release. 2006;112(2):149-55), as applied to claim 1 above, and further in view of Lee et al., (Ultrasound in Med. & Biol. 2007; 33(5): 734-742. Prior art of record).
Claim 8 is directed to the cells being non-adherent.
However, Kodama, Hensel, Wamel and Zhou do not specifically teach transfecting non-adherent cells using this acoustic method.
Lee teaches an acoustic resonance method for transfecting plasmid DNA/PEI complex into non-adherent K562 erythroleukemia cells (see e.g., abstract and Fig 1). Lee teaches studies show ultrasound standing wave fields bring suspended K562 cells and PEI/DNA complexes into close contact at the pressure nodal planes, yielding an approximately 10-fold increment of EGFP transgene expression compared with the group without ultrasonic treatment (see e.g., abstract).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the acoustic method for transfecting adherent cells suggested by Kodama in view of Hensel, Wamel and Zhou, by substituting the adherent cells with non-adherent cells suggested by Lee with a reasonable expectation of success. Since Kodama acknowledges the acoustic method can be used in transfecting suspended cells (p. 906, left col, para 3), since Wamel teaches only cells that are close enough to the vibrating microbubbles experience local forces and have deformation and induced permeability (p. 154, left col, para 2, see Fig 8), and since Lee suggests the mechanism of ultrasound-mediated transfection of non-adherent cells is to drive particles or cells in suspension to the pressure nodal planes, and nanometer-sized DNA vectors circulate between pressure nodal planes because microstreaming-induced drag force (p. 735, left col), one of ordinary skill in the art would have had a reason to substitute with non-adherent cells suggested by Lee in order to take advantage of Lee’s teaching of driving particles or cells in suspension to the pressure nodal planes so that the microbubbles (i.e., particles) and the non-adherent cells may be driven into close contact to have enhanced cell permeability.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 02/04/2026 are acknowledged.
Applicant argues that Lee does not use sonoporation but rather generates ultrasound standing wave fields in which cells move to the acoustic pressure nodes. No method described in Lee teaches microbubble-host cell conjugates and avoidance of cavitation are key. Lee teaches away from using microbubbles because, prior to the present invention, it was thought essential to burst microbubbles in order to induce transfection using ultrasound (see the first sentence of the Abstract) (Remarks, p. 10-11).
Applicant’s arguments have been fully considered but they are not persuasive. As a first matter, the prior art Zhou has taught microbubble-host cell conjugates, and Hensel and Wamel teach avoidance of cavitation by using microbubble oscillation instead (see discussion above). Lee is cited to teach using ultrasound method for transfecting non-adherent cells.
In response that Lee teaches away from using microbubbles because it was thought essential to burst microbubbles, the amended claims now recite the microbubbles to be intact, which was made obvious by prior art Hensel and Wamel. Thus, Lee clearly does not teach away the instant claims.
Claims 11-14 are rejected under 35 U.S.C. 103 as being unpatentable over Kodama et al., (Ultrasound in Med. & Biol. 2006; 32(6): 905-914. Prior art of record) in view of Zhou et al., (Theranostics. 2015; 5(4): p. 399-417. Prior art of record), Hensel et al., (2008 IEEE International Ultrasonics Symposium Proceedings, DOI: 10.1109/ULTSYM.2008.0408, p. 1671-1674) and Wamel et al., (J Control Release. 2006;112(2):149-55), as applied to claims 1 and 9 above, and further in view of Kim et al. (Genome Res. 2014; 24: 1012-1019. Prior art of record) and Togtema et al. (PLoS ONE. 2012; 7(11): e50730, p. 1-12. Prior art of record).
Claim 11 is directed to the exogenous material comprising a ribonucleoprotein. Claim 12 is directed to the ribonucleoprotein being capable of altering host cell nucleic acids. Claim 13 is directed to the ribonucleoprotein comprising a guide RNA and a CRISPR-associated nuclease protein. Claim 14 is directed to the ribonucleoprotein being the guide RNA and a Cas9 protein.
However, Kodama, Hensel, Wamel and Zhou are silent on a ribonucleoprotein in claims 11-14.
Nevertheless, Kodama acknowledges the acoustic method can be used in transfecting suspended cells, “however, when adherent cells are suspended, adhesion-generated signals would affect the regulation of signaling pathways, resulting in different cellular response. Therefore, adherent cells are preferable to be transfected in adhesion”. Thus, Kodama aims to improve and optimize the acoustic method for transfecting adherent cells in adhesion (p. 906, left col, para 3-4).
Kim teaches a method for targeted CRISPR/Cas9 mediated mutagenesis (i.e., altering host cell nucleic acid) in human cells (e.g. fibroblasts) using ribonucleoproteins (RNPs) comprising purified Cas9 protein and a guided RNA delivered by electroporation (abstract, p. 1013, left col, “Results” section, para 1), related to claims 11-14. Kim teaches ribonucleoproteins (RNPs) of RNA-guided engineered nucleases induce site-specific mutations in various species including model organisms (e.g., abstract and para 1), and RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target mutations associated with plasmid transfection (e.g., abstract). Kim teaches the cells are transfected by the Amaxa P3 Primary Cell 4D Nucleofector Kit (p. 1018, left col, para “Transfection”, which is an electroporation approach). One of ordinary skill in the art would immediately understand that the electroporation-mediated transfection requires the adherent cells (e.g., fibroblasts) to be in suspension.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the acoustic method for transfecting adherent cells in adhesion with plasmid DNA suggested by Kodama in view of Hensel, Wamel and Zhou, to substitute the plasmid DNA with a ribonucleoprotein comprising a Cas9 protein and a guide RNA as taught by Kim with a reasonable expectation of success. Since Kodama teaches adherent cells are preferable to be transfected in adhesion and has reduced to practice an improved and optimized acoustic method for transfecting adherent cells in adhesion (p. 906, left col, para 3-4), and since Kim teaches ribonucleoproteins (RNPs) of RNA-guided engineered nucleases induce site-specific mutations in various species including model organisms with reduced off-target mutations associated with plasmid transfection (e.g., abstract), but uses an electroporation method for transfecting adherent cells in suspension (p. 1018, left col, para “Transfection”), one of ordinary skill in the art would have had a reason to substitute with a ribonucleoprotein comprising a Cas9 protein and a guide RNA as taught by Kim in the acoustic transfection method of Kodama in view of Hensel, Wamel and Zhou in order to transfect adherent cells in adhesion with a ribonucleoprotein to induce site-specific mutations in various species including model organisms with reduced off-target mutations. Furthermore, since prior art Togtema teaches the acoustic method is suitable for transfecting large molecules such as a protein and other large molecules listed in Table 1 (such as a monoclonal antibody), one of ordinary skill in the art would have had a reasonable expectation of success in transfecting a Cas9/gRNA ribonucleoprotein of Kim by the acoustic method of Kodama in view of Hensel, Wamel and Zhou.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 02/04/2026 are acknowledged and have been discussed above.
Claims 16-17 are rejected under 35 U.S.C. 103 as being unpatentable over Kodama et al., (Ultrasound in Med. & Biol. 2006; 32(6): 905-914. Prior art of record) in view of Zhou et al., (Theranostics. 2015; 5(4): p. 399-417. Prior art of record), Hensel et al., (2008 IEEE International Ultrasonics Symposium Proceedings, DOI: 10.1109/ULTSYM.2008.0408, p. 1671-1674) and Wamel et al., (J Control Release. 2006;112(2):149-55), as applied to claim 1 above, and further in view of Karshafian et al. (Ultrasound in Med. & Biol., 2009; 35(5): 847-860. Prior art of record).
Claims 16 and 17 are directed to the parameters of ultrasound irradiation.
Kodama teaches the ultrasound is generated by a 1-MHz therapeutic ultrasound (duty cycle 20%, with a pulse repetition period of 10 ms) (p. 906, last sentence – p. 907, 1st sentence) with an exposure time being 20 seconds (see e.g., Figs 3-4 legend), thus teaches the irradiating the reservoir for 15 seconds to 40 seconds in claim 16.
However, Kodama, Hensel, Wamel and Zhou are silent on a rate of 10 to 25 cyclic tonebursts per second and a toneburst duration of less than 1 ms in claim 16 or each cyclic toneburst being an approximately 5-cycle to 10-cycle toneburst in claim 17.
Regarding the parameters of tonebursts in ultrasound irradiation, Karshafian teaches a method of tuning ultrasound exposure parameters by adjusting acoustic pressure, pulse repetition frequency (equivalent to a rate of cyclic tonebursts per second), pulse duration (equivalent to a cyclic toneburst duration), cycle number in pulse duration (equivalent to cycle number in each cyclic toneburst) and insonication time (equivalent to the irradiation duration) (see e.g., p. 856, Table 1 for the optimized parameters). To optimize the transfection, Karshafian tests an insonication time of 0.1 to 900 seconds, i.e., irradiating the reservoir for 0.1 second to 900 seconds (encompassing the range of 15 seconds to 40 seconds in claim 16); a pulse repetition frequency of 10 Hz to 3000 Hz, i.e., cyclic acoustic tonebursts at a rate of 10 to 3000 tonebursts per second (thus encompassing the claimed 10 to 25 tonebursts per second, see abstract); and a pulse duration of 4 to 32 µs (abstract) and 8 to 32 µs (p. 856, Table 1, thus teaches a toneburst duration of less than 1 ms). Thus, Karshafian teaches parameters encompass the claimed ranges in claim 16. Furthermore, Karshafian teaches the pulses being 4 cycles or 16 cycles in pulse duration (p. 856, Table 1), equivalent to a 4-cycle or 16-cycle toneburst, thus encompassing the claimed approximately 5-cycle to 10-cycle toneburst in claim 17. Thus, Karshafian teaches parameters encompass the claimed ranges in claim 17.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the acoustic method for transfecting cells suggested by Kodama in view of Hensel, Wamel and Zhou, by combining a method of optimizing the rate and duration of cyclic tonebursts and the number of cycles in each toneburst to arrive at the claimed ranges as taught by Karshafian with a reasonable expectation of success. Since Kodama aims to improve and optimize the acoustic method for transfecting adherent cells to achieve the best transfection/survival balance (p. 906, left col, para 3-4, see e.g. Fig 3 for transgene activity versus viability of cells), and since Karshafian reduces to practice a method of optimizing the rate and duration of tonebursts and the number of cycles in each toneburst to achieve the highest therapeutic ratio defined as the ratio of permeabilized to nonviable cells (abstract), one of ordinary skill in the art would have had a reason to combine Karshafian’s optimizing method in the acoustic transfection method of Kodama in view of Hensel, Wamel and Zhou in order to achieve the highest transfection/survival ratio balancing gene transfer and cell viability.
Furthermore, it is noted that 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). It is routine procedure to optimize component amounts to arrive at an optimal product that is superior for its intended use, since it has been held where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art. See M.P.E.P. §2144.05.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 02/04/2026 are acknowledged.
Applicant argues that Karshafian pertains to microbubble cavitation, i.e., microbubble destruction, and has been cited solely for its discussion of ultrasound exposure parameters and the effects of those parameters on microbubble destruction and cell permeability. The purpose is to ensure microbubble destruction, as explained throughout the reference and in contrast to applicants' claimed method (Remarks, p. 11-12).
Applicant’s arguments have been fully considered but they are not persuasive. Karshafian indeed pertains to sonoporation (see abstract, “ultrasound exposure parameters can be optimized for therapeutic sonoporation”), but not solely to microbubble cavitation. Karshafian actually teaches cell permeability and viability did not correlate with bubble disruption. Since Hensel and Wamel teach avoidance of cavitation by using microbubble oscillation instead (see discussion above), one of ordinary skill in the art would have had a reason to combine the teaching of Karshafian to optimize the ultrasound exposure parameters for therapeutic sonoporation.
Claims 18-20 are rejected under 35 U.S.C. 103 as being unpatentable over Kodama et al., (Ultrasound in Med. & Biol. 2006; 32(6): 905-914. Prior art of record) in view of Zhou et al., (Theranostics. 2015; 5(4): p. 399-417. Prior art of record), Hensel et al., (2008 IEEE International Ultrasonics Symposium Proceedings, DOI: 10.1109/ULTSYM.2008.0408, p. 1671-1674) and Wamel et al., (J Control Release. 2006;112(2):149-55), as applied to claim 1 above, and further in view of Deng et al (J. Controlled Release. 2012;157:103-111. Prior art of record).
Claims 18, 19 and 20 are directed to the acoustic radiation generator comprising a first transducer and a second transducer having the specified shapes, frequencies and functions.
However, Kodama, Hensel, Wamel and Zhou are silent on a dual transducer generator in claims 18-20.
Deng teaches a method of controlled permeation of cell membrane by microbubbles and acoustic pressure (title, abstract). Regarding the acoustic radiation generator, Deng teaches a dual-element dual-frequency ultrasound transducer (see Fig 1A) consists of two collinearly and con-focally aligned circular ultrasound transducers to generate ultrasound exposures at two different center frequency, duration, and amplitude to spatially control the location and cavitation of a single bubble (p. 104, section 2.2). Thus, Deng teaches the acoustic radiation generator comprises a first transducer (i.e., the inner transducer) and a second transducer (i.e., the outer transducer), wherein the second transducer comprises an annular transducer (i.e., donut-shaped) disposed around a first transducer (see Fig 1A for the outer transducer disposed around the inner transducer), wherein the first transducer is configured to operate at a different frequency than the second transducer (see p. 104, right col) in claim 18. Deng teaches the donut-shaped outer transducer is used to generate a focused ultrasound beam at center frequency of 1.5 MHz (p. 104, last para) and the central transducer is a broad-band transducer that has a center frequency 7.44 MHz (p. 104, 2nd to the last para), thus teaches the annular transducer (i.e., the donut-shaped outer transducer) operates at a frequency in the range of about 1 MHz to about 2.5 MHz, and the inner transducer operates at a frequency in the range of about 6 MHz to about 20 MHz in claim 19. Regarding the functions of the two transducers, Deng teaches the outer transducer is used to induce oscillation of a gas bubble (p. 104, last para) and the central transducer is used to push the bubble toward the cell membrane by the acoustic radiation of the pulses (p. 104, 2nd to the last para), thus teaches one of the two transducers primarily functions to supply the acoustic energy for sonoporation and the other transducer delivers acoustic energy to change the relative position of the microbubbles with respect to the host cells in claim 20.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the acoustic method for transfecting cells using an acoustic radiation generator suggested by Kodama in view of Hensel, Wamel and Zhou, by substituting with a dual-transducer acoustic radiation generator as taught by Deng with a reasonable expectation of success. Since Wamel teaches only cells that are close enough to the vibrating microbubbles experience local forces and have deformation and induced permeability (p. 154, left col, para 2, see Fig 8), and since Deng teaches the central transducer pushes the bubble toward the cell membrane and the outer transducer induces oscillation of a gas bubble (p. 104, last 2 para), one of ordinary skill in the art would have had a reason to substitute with the dual-transducer acoustic radiation generator in order to take advantage of the dual-transducer-mediated pushing the microbubbles toward the cell membrane and subsequently inducing oscillation to enhance the transfection efficiency.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 02/04/2026 are acknowledged.
Applicant argues that Deng's specific goal is to induce cavitation, and various parameters are evaluated in order to develop an optimal cavitation method. Deng thus teaches away from applicants' claimed method (Remarks, p. 12).
Applicant’s arguments have been fully considered but they are not persuasive. As stated supra, Hensel and Wamel teach avoidance of cavitation by using microbubble oscillation instead (see discussion above). Deng is cited to teach the use of dual-transducers. Deng does not negate the claimed microbubble oscillation method. Actually Deng specifically teaches the central transducer pushes the bubble toward the cell membrane and the outer transducer induces oscillation of a gas bubble (p. 104, last 2 para). Accordingly, one of ordinary skill in the art would have had a reason to substitute with the dual-transducer acoustic radiation generator in order to take advantage of the dual-transducer-mediated pushing the microbubbles toward the cell membrane and subsequently inducing oscillation to enhance the transfection efficiency.
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 extension fee 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 date of this final action.
No claims are allowed.
Examiner Contact Information
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jianjian Zhu whose telephone number is (571)272-0956. The examiner can normally be reached M - F 8:30AM - 4PM (EST).
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/JIANJIAN ZHU/Examiner, Art Unit 1631
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634
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