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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 6/29/26 has been entered.
Claim Status
Claims 1-11, 13, 14 and 18 are cancelled.
Claims 23-24 are new.
Claims 12, 15-17 and 19-24 are pending.
Withdrawn rejections
Applicant's amendments and arguments filed 6/8/26 are acknowledged and have been fully considered. The Examiner has re-weighed all the evidence of record. Any rejection and/or objection not specifically addressed below is herein withdrawn. Claims 12, 15-17 and 19-22 were rejected under 35 U.S.C. 103 as being unpatentable over Sakimura et al. (WO2012132141) in further view of Xu et al. (Int J Mol Sci 2011;12:462-475) and Sirsi et al. (Bubble Sci Eng Technol 2009;1(1-2):3-17) and Schneider et al. (US20030185759). This rejection is withdrawn in favor of the rejection to follow.
The following rejections and/or objections are either reiterated or newly applied. They constitute the complete set of rejections and/or objections presently being applied to the instant application.
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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied 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.
Claims 12, 15-17 and 19-22 are rejected under 35 U.S.C. 103 as being unpatentable over Sakimura et al. (WO2012132141) in further view of Xu et al. (Int J Mol Sci 2011;12:462-475) and Sirsi et al. (Bubble Sci Eng Technol 2009;1(1-2):3-17) and Schneider et al. (US20030185759) and Merkus, HG. (Particles Size Measurements Fundamentals, Practice, Quality. 2009; Springer: 12 pages).
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.
Applicant claims, for example:
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Claim interpretation: the limitation of “positively-charged” means that the ultrafine bubbles are in an aqueous solution of pH 1-4 (Specification [0031]).
Level of Ordinary Skill in the Art
(MPEP 2141.03)
MPEP 2141.03 (I) states: “The “hypothetical ‘person having ordinary skill in the art’ to which the claimed subject matter pertains would, of necessity have the capability of understanding the scientific and engineering principles applicable to the pertinent art.” Ex parte Hiyamizu, 10 USPQ2d 1393, 1394 (Bd. Pat. App. & Inter. 1988). The level of skill is that of a cellular transfection research scientist, as is the case here, then one can assume comfortably that such an educated artisan will draw conventional ideas from cell transfection technology and techniques, cellular physiology and pharmaceutical chemistry— without being told to do so.
In addition, the prior art itself reflects an appropriate level (MPEP 2141.03(II)).
Determination of the scope and content of the prior art
(MPEP 2141.01)
Regarding claims 12, 17, 20 and 21, Sakimura et al. teaches compositions and methods for delivering a drug into a cell (Abstract; claim 1; page 22 bottom) comprising microbubbles1 generated by physical stimulation (Claim 2) of ultrasonic radiation (Claim 3) and peptide, antibody, gene, oligonucleotide (RNA, DNA), siRNA, viral vector or a low-molecular weight drug compound (Claim 7) and not containing phospholipid and methods of contacting cells (Claim 13) and methods of introducing a drug into a cell (Claim 14) wherein the drug is a peptide, antibody, gene oligonucleotide, siRNA, viral vector, plasmid or low molecular weight organic compound (Claim 16). Cells were exposed to ultrasonic waves (Examples 5-8 and 11, for example). Sakimura et al. also teach exposing tissue in which a target cell for performing drug introduction is present with physical stimulation (Page 7, 3rd paragraph from bottom). The limitation of “wherein immune cells are excluded” is met by Sakimura et al. not delivering a nucleic acid, protein or low-molecular weight compound into an immune cell. Sakimura et al. teach applying ultrasonic irradiation at an intensity of 0.03 to 5 W/cm2 (Claim 4) or even to 0.06 to 0.1 W/cm2 or 0.03 to 1 W/cm2, which are equivalent to 30 to 5000 mW/cm2, 60 to 100 mW/cm2, 30 to 1000 mW/cm2 respectively, which the person skilled in the art can appropriately set (Page 6, 2nd to 3rd paragraphs from top; page 4 last line; “A person skilled in the art can appropriately set the intensity and frequency of the ultrasonic stimulation.”), and overlap the claimed range of 250-500 mW/cm2, thus rendering the claimed range obvious. Sakimura et al. also teach examples employing 240 mW/cm2 (Example 10) and 500 mW/cm2 (Example 11), thereby establishing a range of 240-500 mW/cm2 as within the scope of Sakimura et al. and obvious to the ordinary artisan. See MPEP 2144.05(I): 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). Sakimura et al. also teach that it is desirable to use lower ultrasonic intensity to reduce adverse effects on cells (Example 10). Sakimura et al. also teach applying a frequency of 1.0 to 3.4 MHz (Claim 5), or even in the range of 50KHz to 3.4 MHz or 1.0 MHz to 3.4 MHz (Page 6, paragraphs 3-6), which overlaps the claimed range of 0.5 to 10 MHz. An ultrasound generator is necessarily present to generate the ultrasonic radiation. The size of microbubbles can be from 1 nm to 1000 microns; 10 nm to 100 microns; 100 nm to 10 microns or 20 microns or less (Page 6 of 19; 6th paragraph), which overlaps or lies within the claimed range of not more than 200 nm or 50-200 nm and reads on nanobubbles. Therefore, the system of Sakimura et al. is implicitly configured for delivering a target substance into a cell, wherein immune cells are excluded. Methods of contacting a cell with the composition and applying ultrasonic radiation are taught (Claims 13-19), thus increasing the delivery of a nucleic acid, protein or a low-molecular weight compound into a cell. Sakimura et al. teach adjusting the pH to 2.2 (Page 15, Example 4 Generation of microbubbles under acidic conditions) and suggest pH adjuster such as hydrochloric acid, citric acid and the like (Page 12, last paragraph). Thus, the microbubbles will be inherently positively charged as taught by Applicant and noted above because such a charge naturally flows from the pH of the solution.
Regarding claim 15, Sakimura et al. teach using surfactants, albumin, gamma globulin, protein and fatty acids (Page 7, 6th paragraph) including stearic acid, which is an anionic surfactant (Example 3) as well as buffers (Example 1) and buffering agents (Page 11 of 19, 5th paragraph).
Regarding claim 16, Sakimura et al. teach using fluorocarbons and air (Claim 9; Example 9) where the fluorocarbons include CF4, C2F6, C3F8, etc…(Page 6, 1st paragraph), which are perfluorohydrocarbons because all the hydrogen atoms have been replaced with fluorine, and would consist of perfluorohydrocarbons or air.
Regarding claim 19, Sakimura et al. teach a density of 1 X 106 to 1 X 109 (Page 6 of 19, 7th paragraph), thus overlapping the claimed range of not less than 1.0 X 108.
Regarding claim 22, Sakimura et al. teach that the cells can be skeletal muscle cells and brain cells (Page 7 paragraphs 4 and last paragraph), which would read on nerve cells.
Regarding claims 12 and 15, Sirsi et al. teach microbubble compositions stabilized with a shell comprised of proteins, lipids or polymers (Abstract) including surfactant shells of non-ionic surfactants SPAN and TWEEN (page 2 bottom through page 3) and non-ionic polyvinyl alcohol (Page 5 top) for gene and drug delivery (Pages 9-16). Xu et al. teach anionic, neutral and cationic biosurfactants for microbubble preparation and include polysaccharides (Page 464, Types of Biosurfactants). Schnieder et al. teach microbubble suspensions in aqueous phases that contain surfactants and hydrophilic stabilizers (Abstract) wherein the surfactant is one or more surfactants selected from the group consisting of lipid, phospholipids, phosphatidylserine, lecithins, cholesterol, free fatty acids, esters of fatty acids with polyoxyalkylene compounds, ethers of fatty acids with polyoxyalkylene glycols, esters of fatty acids with polyoxy alkylated sorbitan2, soaps, glycerol-polyalkylene stearate, glycerol-polyoxyethylene ricinoleate, homo and copolymers of polyalkylene glycols, polyethoxylated soya-oil and castor oil and hydrogenated derivatives, ethers and esters of sucrose and other carbohydrates with fatty acids or fatty alcohols, mono, di and triglycerides of saturated and unsaturated fatty acids, fatty alcohols, glycerides of soya-oil and sucrose (Claim 1) with a stabilizing polymer of one or more polymers selected from the group consisting of hydrophilic polymers, starch, dextran, polyvinyl alcohol, polyvinyl-pyrrollidone, dextrin, xanthan, partly hydrolyzed cellulose oligomers, protein, human serum albumin, gelatin, synthetic polymers and polypeptides (Claims 13 and 17; [0173]). The instant specification teaches that polyvinyl alcohol (PVA) is a hydrophilic resin. PVA is also non-ionic.
Regarding claim 12, Merkus teaches that a particle size distribution width D90/D10 is considered to represent:
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(Page 12 of 12). Thus, a lower ratio represents a more uniform homogenous bubble preparation where an ideal ratio of 1.00 indicates that all the bubbles are the same size resulting in maximum uniformity.
Ascertainment of the difference between the prior art and the claims
(MPEP 2141.02); and Finding of prima facie obviousness
Rational and Motivation (MPEP 2142-2143)
The difference between the instant application and Sakimura et al. is that Sakimura et al. do not expressly teach a method of first preparing an ultrafine bubble aqueous solution comprising positively-charged ultrafine bubbles as claimed, secondly contacting a cell to be transfected with the ultrafine bubble aqueous solution and thirdly exposing the ultrafine bubble aqueous solution and the cell to ultrasound or a d90/d10 ratio of not more than 5. However, it would have been obvious to one of ordinary skill in the art before to the effective filing date of the claimed invention to perform the method of Sakimura et al. by preparing an ultrafine bubble aqueous solution comprising positively-charged ultrafine bubbles as claimed, contacting a cell to be transfected with the ultrafine bubble aqueous solution and exposing the ultrafine bubble aqueous solution and the cell to ultrasound or a d90/d10 ratio of not more than 5, as suggested by Merkus, and produce the instant invention.
One of ordinary skill in the art would have been motivated to do this because of the following rational. As discussed above, Sakimura et al. teach contacting a cell with an aqueous solution in which the drug is dissolved in gaseous supersaturated water and providing a physical stimulus of ultrasonic waves. Sakimura et al. does not first generate the microbubbles but appears to do so simultaneously when in contact with the cells and exposed to ultrasonic waves. However, the net result is accomplished is substantially the same: the cells are transfected. Consequently, whether the microbubbles are formed first or formed simultaneously upon exposure of the cells to physical stimulating ultrasound waves produces the same result of cell transfection and would be obvious to the ordinary artisan in this art. "Where the result accomplished is substantially the same, steps taken concurrently or simultaneously are the equivalent of and not patentable over steps taken successively" New Wrinkle, Inc. v. Watson, Comr. Pats., 96 USPQ 436, 437 (D.C. Cir. 1953).
Concerning the limitation of a d90/d10 ratio of not more than 5, as taught by Merkus, a low ratio such as less than 5 represents more uniform particle size distribution and thus a more homogenous set of particles that will have similar properties. The ordinary artisan would desire the most uniform and homogenous particle size distribution that have predictable and consistent properties and strive for a d90/d10 ratio of not more than 5 to avoid broad particle size distribution with a heterogeneous particle population, which does not have a uniform composition throughout and could affect the properties of the particles in unpredictable ways.
Sakimura et al. do not expressly teach an ultrafine bubble aqueous solution with a nonionic surfactant and/or a hydrophilic resin for use in a method for increasing the delivery of a nucleic acid, a protein or a low-molecular-weight compound into a cell. However as discussed above, Sirsi et al. teach microbubble compositions stabilized with a shell comprised of proteins, lipids or polymers (Abstract) including surfactant shells of non-ionic surfactants SPAN and TWEEN-40 (page 2 bottom through page 3) and non-ionic polyvinyl alcohol (Page 5 top) for gene and drug delivery (Pages 9-16). Xu et al. teach anionic, neutral and cationic biosurfactants for microbubble preparation and include polysaccharides (Page 464, Types of Biosurfactants). Schnieder et al. teach microbubble suspensions in aqueous phases that contain a variety of surfactants including esters of fatty acids with polyoxy alkylated sorbitan and hydrophilic stabilizers with a stabilizing polymer of one or more polymers selected from the group consisting of hydrophilic polymers, starch, dextran, polyvinyl alcohol, polyvinyl-pyrrollidone, dextrin, xanthan, partly hydrolyzed cellulose oligomers, protein, human serum albumin, gelatin, synthetic polymers and polypeptides (Claims 13 and 17; [0173]). The instant specification teaches that polyvinyl alcohol (PVA) is a hydrophilic resin. PVA is also non-ionic.
It would have been obvious to one of ordinary skill in the art prior to the effective filing date of the claimed invention to perform the method of Sakimura et al. with the claimed one or more surfactants selected from an anionic surfactant, a cationic surfactant and an amphoteric surfactant, as suggested by Schnieder et al., Xu et al. and Sirsi et al., and produce the instant invention. One of ordinary skill in the art would have been motivated to do this because Sakimura et al. teach adding a surfactant and the ordinary artisan in the microbubble art understands that all sorts of surfactants can be used to make microbubbles including anionic surfactants, non-ionic surfactants, cationic surfactants, amphoteric surfactants and hydrophilic resin surfactants. It is then merely a matter of judicious selection of conventional known surfactants such as a non-ionic surfactant and/or a hydrophilic resin such as PVA by the ordinary artisan with a reasonable expectation of success in producing the bubbles.
In light of the forgoing discussion, the Examiner concludes that the subject matter defined by the instant claims would have been obvious within the meaning of 35 USC 103.
From the combined teachings of the references, it is apparent that one of ordinary skill in the art would have had a reasonable expectation of success in producing the claimed invention. Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the time the invention was made, as evidenced by the combined references, especially in the absence of evidence to the contrary.
Response to Arguments:
Applicant’s arguments filed 6/8/26 have been carefully considered but are not persuasive.
Applicant asserts that: “the fundamental distinction between the presently claimed methods and Sakimura is that Sakimura does not teach, suggest, or even contemplate the technical concept of preparing and utilizing nanobubbles whose physical properties are pre-controlled and pre-characterized. By contrast, Sakimura merely utilizes bubbles transiently generated during ultrasound irradiation and provides no teaching regarding pre-defining, evaluating, or controlling bubble size or particle size distribution of the bubbles… Furthermore, in the system of Sakimura, bubble generation and bubble collapse occur simultaneously during ultrasound irradiation.” The Examiner has carefully considered this argument but does not find it persuasive. As explained above, the net result is the same from performing the method of Sakimura et al. and the instantly claimed method: cells are transfected. Sakimura et al. teach and suggest the same ultrasound output intensity and ultrasound frequency to generate ultrafine bubbles having an average diameter overlapping the claimed range. It does not appear to matter at all if the bubbles are made first and characterized as claimed by Applicant or as produced by Sakimura et al. with regard to increasing the delivery of a nucleic acid, protein or low-molecular weight compound into a cell. Applicant has not demonstrated any structural difference between the ultrafine bubbles claimed and the bubbles of Sakimura et al. or any additional unexpected benefit(s) of characterizing the bubbles prior to cell transfection. From the Examiner’s perspective, it does not appear to require any inventive skill to characterize the bubbles produced by Sakimura et al. first and then use those bubbles to transfect a cell. The same ultrasound output intensity and ultrasound frequency employed by Sakimura et al. ensures that the bubbles produced have a controlled average diameter and D90/D10 ratio of not more than 5 as claimed. The Examiner has explained why a D90/D10 ratio of no greater than 5 is desirable to the ordinary artisan and thus the artisan would control bubble size for uniformity. There does not appear to be any evidence in the record that prior characterization of the nanobubbles before transfection enhances the transfection at all. Consequently, the claimed method remains obvious over the combined references without more.
Applicant argues that: “Sakimura provides no teaching or suggestion as to which particle diameter range should be selected, which particle size distribution is preferable, or which ultrasound intensity should be combined therewith.” Respectfully, the Examiner does not agree because each and every limitation claimed has been met by the combined references to render them obvious as explained in detail above. Sakimura et al. provides for a reasonable expectation of success in transfecting cells and is not fundamentally different at all. Rather, it is Applicant that must show a criticality of the range. See MPEP 716.02(d)(II): To establish unexpected results over a claimed range, applicants should compare a sufficient number of tests both inside and outside the claimed range to show the criticality of the claimed range. In re Hill, 284 F.2d 955, 128 USPQ 197 (CCPA 1960). From MPEP 2144.05(III)(A): “Applicants can rebut a prima facie case of obviousness by showing the criticality of the range…the applicant must show that the particular range is critical, generally by showing that the claimed range achieves unexpected results relative to the prior art range.” In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990).” In the present case, both the instant method and the method of Sakimura et al. transfects cells. Applicant has not at this time shown any unexpected results. While Applicant states: “The present application demonstrates that the claimed methods achieve an unexpectedly remarkable effect on delivery of a target substance into living cells”; the inventors do not appear to characterize any results as unexpected. Thus, the characterization of these results as "unexpected" is unsupported attorney argument. See In re Geisler, 116 F.3d 1465, 1471 (Fed. Cir. 1997). Also see MPEP 716.01(c) II: The arguments of counsel cannot take the place of evidence in the record. In re Schulze, 346 F.2d 600, 602, 145 USPQ 716, 718 (CCPA 1965). The secondary references are relied upon as characterized by the Examiner and not as characterized by Applicant. Respectfully, Applicant’s arguments are not persuasive.
Claims 23 and 24 are rejected under 35 U.S.C. 103 as being unpatentable over Sakimura et al. (WO2012132141) in view of Xu et al. (Int J Mol Sci 2011;12:462-475) and Sirsi et al. (Bubble Sci Eng Technol 2009;1(1-2):3-17) and Schneider et al. (US20030185759) and Merkus, HG. (Particles Size Measurements Fundamentals, Practice, Quality. 2009; Springer: 12 pages), as applied to claims 12, 15-17 and 19-22 above, in view of Unger et al. (HRP970328A2; English translation provided).
Applicant claims:
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The references of Sakimura et al., Sirsi et al., Xu et al., Schneider et al. and Merkus are discussed in detail above.
The combined references do not expressly teach the non-ionic surfactant is polysorbate 80. The Examiner notes that Sirsi et al. teach TWEEN-40 (Page 2, surfactant shells), which is polysorbate 40 and Schneider et al. teach TWEEN 20 [0239], which is polysorbate 20. However, in the field of ultrasound to facilitate the uptake of compounds into the cell (Unger et al. Page 1, Field of the Invention), solubility agents such as polysorbate 20, polysorbate 40 and polysorbate 80 (TWEEN 20, TWEEN 40 and TWEEN 80) are functionally equivalent (page 16, last paragraph through page 17 first paragraph). Accordingly, it would be obvious to employ polysorbate 20 and/or polysorbate 80 as the non-ionic surfactant in the method of Sakimura et al. with a reasonable expectation of success. "The combination of familiar elements according to known methods is likely to be obvious when it does no more than yield predictable results." KSR Int'l Co. v. Teleflex Inc., 550 U.S. 398, 417 (2007). Moreover, “Where two known alternatives are interchangeable for a desired function, an express suggestion to substitute one for the other is not needed to render a substitution obvious." In re Fout 675 F.2d 297, 301 (CCPA 1982).
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to ERNST V ARNOLD whose telephone number is (571)272-8509. The examiner can normally be reached M-F 7-3:30.
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/ERNST V ARNOLD/Primary Examiner, Art Unit 1613
1 The term “microbbubles” includes nano-sized bubbles as defined by Sakimura et al. teaching: “The size of microbubbles can be from 1 nm to 1000 microns; 10 nm to 100 microns; 100 nm to 10 microns or 20 microns or less” (Page 6 of 19; 6th paragraph).
2 Universally known as polysorbates.
Prosecution Insights