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 of group I, a method of producing Nano ghost particles in the reply filed on 5/8/2026 is acknowledged. Because applicant did not distinctly and specifically point out the supposed errors in the restriction requirement, the election has been treated as an election without traverse (MPEP § 818.01(a)).
Claims 23-26,31 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected group II/III, a composition of spherical Nano ghost particles, and a method of producing a pharmaceutical composition comprising the Nano ghost particles, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 5/8/2026.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-2,6,9,13-14,16-20,22 are rejected under 35 U.S.C. 103 as being unpatentable over Furman NET et al 2013, Roach P et al 2008, Huang X et al 2019, Talsma H et al 1989, Boone et a 1969, Yusuf et al 2014, Nordin JZ et al 2019, Böing AN et al 2014, Chen T et al 2018, Shiromizu et al 2017, and Chomistek J et al. 2010.
Furman NET et al 2013 disclose a method of producing spherical particles from mesenchymal stem cells (MSCs) by subjecting the cells to hypotonic conditions, subjecting the cells to mechanical stress to rupture cells while avoiding nuclei lysis, removing nuclei, subjecting the homogenized solution to sonication, then nano ghost particles were purified by ultracentrifugation. The spherical particles averaged about 180nm diameter. Specifically, “NGs were prepared from the cytoplasmatic membranes of human and rat MSCs…the cells (Figure 1a) are harvested and hypotonically treated with tris magnesium buffer followed by mild homogenization to allow cytosol removal without substantially disrupting cell membrane. Cells are centrifuged, precipitated, and washed several times to remove most nucleic matter…The homogenized cytoplasm-free cells (termed ghost cells or ghosts) are then mildly sonicated and washed again (Figure 1c)…The sonicated ghosts were extruded into NGs in a medium containing sTRAIL and retrieved by ultracentrifugation. The NGs exhibited…average diameters of ∼180 nm (Figure 1d).” (paragraph 1 of “Physical Characterization of NGs” section). Importantly they clarify, “MSC-NGs are manufactured …by isolating intact MSC cell membranes (ghost cells), and homogenizing them into nanosized vesicles (nanoghosts). (paragraph 2 on page 1).
Additionally, they disclose the standard in-vitro culture of the MSC cells prior to Nano ghost extraction. Specifically, “Human bone marrow derived mesenchymal stem cells (MSCs, Lonza™, Basel, Switzerland) were cultured in alpha-MEM (Sigma-Aldrich™, St. Louis, MO) supplemented with 5 ng/ml basic fibroblast growth factor (bFGF).” (supplementary experimental procedures sheet under “Cells and media”). It is important to note that homogenization is conducted during hypotonic treatment, and therefore the hypotonic treatment is under dynamic conditions.
Furman NET et al 2013 does not disclose the use of flow shearing to obtain ruptured cells, they do not remove nuclei using filtration, they do not conduct size separation of ruptured-nuclei free preparations. Finally, they do not use homogenization or microfluidizer to downsize ghosts.
Huang X et al 2019 discloses the use of channel-based flow shearing to rupture cells without destroying nuclei at the same time. Therefore, the isolation of a ruptured cell composition from nuclei contamination is easy and effective. Specifically, “are arranged in a cascade along microfluidic channels and can effectively rupture cells delivered in a pressure-driven flow… More importantly, many intact nuclei are found in the lysates with a relatively high nuclei-isolation efficiency from a four-constriction treatment.” (abstract).
Roach P et al 2008 discloses the use of filters/porous membranes with pore sizes designed to selectively remove different components (aka nuclei) of disrupted-cell solutions based on their size. Specifically, “the objective of TFF was to purify membranes by passing them over a filtration membrane with a pore size sufficient to allow ready passage of cytosolic proteins but prevent passage of membrane vesicles and other membrane fragments.” (paragraph “Tangential flow filtration preparation” in results section). They show that filtration is superior to ultracentrifugation in speed, yield and scalability when separating membranes from other cellular components in solution.
Böing AN et al 2014 discloses a size exclusion chromatography technique to isolate extracellular vesicles from cellular debris (proteins and lipoproteins) as well as other cells. The technique separates components based on their size. Specifically, “We demonstrate that vesicles can be purified from human platelet-free supernatant of platelet concentrates by sepharose CL-2B SEC.” (paragraph 1 of discussion section). Additionally, “The principle of SEC is separation based on a difference in size.”(5th paragraph of discussion). Notably, the applicant suggests this exact column, sepharose CL-2B SEC, to be used in size separation in the specification.
Talsma H et al 1989 discloses the use of a microfluidizer to reduce the size of liposomes in a controlled and reproducible way. Specifically, “A high pressure homogenizer (microfluidizer), was tested for its ability to produce liposomal distributions with narrow size distributions...With the microfluidizer it weas possible to produce reproducible dispersions with a mean particle size of [50-250nm].” (abstract).
It would be obvious to a person of ordinary skill in the art, before the effective filing date, to modify the MSC-derived nano ghost preparation method (wherein MSC cells are cultured under normal conditions before hypotonic treatment) disclosed by Furman NET et al 2013, by replacing homogenization-based cell rupture for the channel-based flow shearing cell rupture techniques as disclosed in Huang X et al 2019. It is known in the field that liquid mechanical shear can selectively disrupt plasma membranes and preserve nuclei, as supported by Boone et a 1969. Huang X et al 2019 teaches that forcing cells to flow through narrow paths results in the selective rupture of cellular plasma membranes, while preserving nuclei. Importantly, homogenization and flow shear both apply mechanical force/shear and result in selective rupture of plasma membranes while preserving nuclei. Therefore, using flow shear techniques as an alternative to homogenization when selectively disrupting cell plasma membranes amounts to the substitution of one known element for another to achieve predictable and identical results. Flow shear is also superior to homogenization for this purpose because it provides scalability, and improved reproducibility, as supported by Chomistek J et al. 2010.
It would be obvious to a person of ordinary skill in the art, before the effective filing date, to replace centrifugation-based separation steps (aka separation of nuclei from solution, and the subsequent size separation to obtain ghosts from other debris) in the MSC-derived nano ghost preparation method detailed above, with one of the known filtration techniques from Böing AN et al 2014 or Roach P et al 2008. Both the chromatography-based filtration method disclosed by Böing AN et al 2014 and, the tangential flow filtration membrane-based approach disclosed by Roach P et al 2008 are known to be alternative size separation techniques for centrifugation, as supported by Nordin JZ et al 2019. Importantly, they have been used on disrupted cellular solutions to purify membrane vesicles while controlling for particle size distribution. Therefore, this a case of the substitution of one known element for another to achieve predictable results. Additionally, both the aforementioned techniques improve scalability and improved reproducibility.
Furthermore, it would be obvious to a person of ordinary skill in the art, before the effective filing date, to modify the aforementioned protocol/methodology designed to create spherical particles/ nano ghosts, by replacing the sonication-based method of downsizing ghosts with the high-pressure homogenizer (microfluidizer) based method disclosed by Talsma H et al 1989. A skilled artisan would understand that this replacement is a simple substitution of one known element for another to achieve predictable results, as supported by Chen T et al 2018. Additionally, the microfluidizer exhibits improved scalability and reproducibility when compared to sonication, as supported by Talsma et al 1989.
The aforementioned prior art disclosures and teachings reveal that claim 1’s theoretical design, methodology and sequence of events is virtually identical to the prior art. However, while claim 1 follows the same steps, it employs different techniques for accomplishing the same steps. Importantly, the techniques employed by the claims are well known alternatives to the techniques used in the prior art. Both techniques are known to yield similar and predictable results. Therefore, these disclosures and rationales render claims 1-2,6,9,13-14,17-18 obvious.
The limitations recited in claims 16,22 (pore sizes), claims 19-20 (bar values for high shear homogenizer), are ordinary and well understood optimizable parameters and therefore do not add patentable weight to the claims.
For claims 16,22, the use of a filter in with a cutoff size within the claimed range of 1.2um-10um for nuclei removal (claim 16) and 0.22 for purifying particles (claim 22) are obvious optimization parameters of known size-based separation techniques. Prior art recognizes that a 5um pore size is sufficient to separate nuclei from disrupted cellular preparations, and thus teaches different cellular components and materials can be separated based on their relative sizes. (see Yusuf et al 2014). Additional art also teaches that the use of filters (0.22um-0.1um pore size) during isolation/purification of other subcellular membrane enclosed structures (See Shiromizu et al 2017 for extracellular vesicles). This reinforces the use of size-based filtration for separation of subcellular membrane enclosed structures from known contaminants. Therefore, it would be obvious to a person of ordinary skill in the art, before the effective filing date, to select the appropriate cutoff within in the claimed range to remove nuclei and to purify nano ghosts based on the desired composition components.
For claims 19-20 it is known that pressure settings on the high-pressure homogenizer directly affect the degree of particle size reduction and the particle size distribution (see Talsma et al 1989). Therefore, it would be obvious to a person of ordinary skill in the art, before the effective filing date, that alteration of homogenizer pressure predictably alters particle size. Therefore, they would select an appropriate pressure to achieve a desired particle size distribution.
Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over Furman NET et al 2013, Roach P et al 2008, Huang X et al 2019, Talsma H et al 1989, Boone et a 1969, Yusuf et al 2014, Nordin JZ et al 2019, Böing AN et al 2014, Chen T et al 2018, and Chomistek J et al. 2010as applied to claim 1 above, and further in view of Thery C et al 2006.
In the previous rejection a combination of sources is used to show the method of claim 1 only differs from the prior art by employing different techniques for accomplishing the same steps. Importantly, the techniques employed by the claims are well known alternatives to the techniques used in the prior art.
However, the aforementioned art does not teach or suggest sequential size exclusion of preparations
Thery C et al 2006 uses the size exclusion technique called differential centrifugation or sequential ultracentrifugation for the sequential size exclusion of subcellular membrane enclosed bodies and vesicles from various other cellular contaminates of varying sizes. Specifically, “The general idea of exosome purification by ultracentrifugation is depicted in Figure3.22.1. The first steps are designed to eliminate large dead cells and large cell debris by successive centrifugations at increasing speeds (steps 1 to 5 below). At each of these steps, the pellet is thrown away, and the supernatant is used for the following step (Fig.3.22.1). The final supernatant is then ultracentrifuged at 100,000 × g to pellet the small vesicles that correspond to exosomes. (“PURIFICATION OF EXOSOMES BY DIFFERENTIALULTRACENTRIFUGATION” paragraph)
It would be obvious to a person of ordinary skill in the art, before the effective filing date, to reperform a size separation method after completing the microfluidizer-based downsizing step of the aforementioned method. Considering the microfluidizer works by forcing suspensions through channels at high pressures, the production of unwanted debris, altered structures, and fragments is common. This requires clean-up/purification steps (aka size exclusion). Sequential size exclusion using centrifugation (aka differential centrifugation or sequential ultracentrifugation) is a classical technique for sequential size exclusion (Thery C et al 2006). Therefore, claim 21 is obvious.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Adam M Smith whose telephone number is (571)272-7517. The examiner can normally be reached Monday- Friday 10:30AM-5PM.
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/Tracy Vivlemore/Supervisory Primary Examiner, Art Unit 1638
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