DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
The rejection of claims 1, 5 & 7 under 35 U.S.C. 103 as being unpatentable over Aguilar et al (2010) J Sci Food Agric. 90: 1385–1392, hereafter Aguilar; in view of Mustafa et al (2011) Nature protocols, 6(6), 715-742, hereafter Mustafa (previously disclosed by Applicant), and further in view of Kim et al (2015) PloS one, 10(6), e0129760, hereafter Kim; Rider et al (2016). Scientific reports, 6(1), 1-14, hereafter Rider; Yamawaki et al (US 2018/0275029 A1, published 9/27/2018, hereafter Yamawaki), and Burgess. Protocols.io. (1/23/2019, https://www.protocols.io/view/murashige-and-skoog-ms-agar-5qpvonpn9l4o/v1), hereafter Burgess; and in further light of the VWR Cellector™ Tissue sieve listing (https://uk.vwr.com/store/product/11038246/homogenisers-cellector-tissue-sieve, accessed 10/10/2024) and a Usenet forum post (https://groups.google.com/g/bionet.genome.arabidopsis/c/bb9slBa8z6w, from 1996) is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 5-6 & 8 under 35 U.S.C. 103 as being unpatentable over Aguilar in view of Mustafa, Burgess, Kim, Rider, Yamawaki, the VWR Cellector™ listing, and the Usenet forum post from 1996, and further in view of Lukatkin et al (2007) Med. Aromat. Plant. Sci. Biotechnol, 107-111 is withdrawn in light of Applicant’s amendments.
The rejection of claims 1, 5 & 7-8 under 35 U.S.C. 103 as being unpatentable over Aguilar in view of Mustafa, Burgess, Kim, Rider, Yamawaki, the VWR Cellector™ listing, and the Usenet forum post from 1996, and further in view of Charoenviriyakul et al (2018) International journal of pharmaceutics, 553(1-2), 1-7 is withdrawn in light of Applicant’s amendments.
Status of Claims
Claims 1 & 5-8 are under examination on the merits.
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.
Claims 1, 5 & 7 are rejected under 35 U.S.C. 103 as being unpatentable over Aguilar et al (2010) J Sci Food Agric. 90: 1385–1392, hereafter Aguilar; in view of Mustafa et al (2011) Nature protocols, 6(6), 715-742, hereafter Mustafa (previously disclosed by Applicant), and further in view of Kim et al (2015) PloS one, 10(6), e0129760, hereafter Kim; Rider et al (2016). Scientific reports, 6(1), 1-14, hereafter Rider; Mu et al (2014) Mol. Nutr. Food Res. 2014, 58, 1561–1573 (published 5/19/2014, hereafter Mu), Yamawaki et al (US 2018/0275029 A1, published 9/27/2018, hereafter Yamawaki), and Burgess. Protocols.io. (1/23/2019, https://www.protocols.io/view/murashige-and-skoog-ms-agar-5qpvonpn9l4o/v1), hereafter Burgess; and in further light of the VWR Cellector™ Tissue sieve listing (https://uk.vwr.com/store/product/11038246/homogenisers-cellector-tissue-sieve, accessed 10/10/2024) a Usenet forum post (https://groups.google.com/g/bionet.genome.arabidopsis/c/bb9slBa8z6w, from 1996).
This is a new rejection based on Applicant’s amendments. Applicant' s arguments filed 10/17/2025 have been fully considered as they pertain to the instant rejection but they are not persuasive.
Claim 1 is drawn to a method of producing plant-derived exosomes from a cell suspension culture obtained by wounding plants to make a regularly subcultured callus ready to be transferred to liquid culture within 2 to 3 weeks, dividing the callus into pieces 1-5 mm and placing into Erlenmeyer flasks so the flasks are 10-50% full, preparing a liquid culture medium in the flasks containing 20-30 g/L sucrose, 6-benzylaminopurine, 1-4 mg/L 1-Naphthaleneacetic acid, and a Murashige & Skoog vitamin-containing salt mixture, maintaining the liquid culture medium continuously under light, at 20-26°C, agitating at a speed of 80-120 rpm, performing a sub-culture via vacuum filtration at 5-10 days, and straining through a sterile steel sieve every 3-5 subcultures.
Claim 1 is drawn to mixing the plant cell suspension culture with an isolation solution at a ratio of 1:1 by inverting 20 times, then centrifuging the mixture at 1500 g for 10 minutes at 4°C, afterwards obtaining two separate phases, wherein the supernatant comprises protein and cellular wastes and the infranatant comprising exosomes is 10% of the combined phases. Claim 1 is drawn to pulling and discarding the supernatant and transferring the infranatant to a clean tube. Claim 1 is drawn to diluting isolation solution with water at a 1:1 ratio and centrifuging at 1000 g for 10 minutes to obtain a supernatant as a solution, then adding the solution at a 1:1 ratio to the exosome infranatant and inverting 10 times to mix. Claim 1 is drawn to centrifuging this mixture at 12000-14000 g for 10 minutes at 4°C and collecting a supernatant to obtain plant-derived exosomes that have a distribution of sizes between 84 nm and 422 nm. Finally claim 1 is drawn to storing the exosomes as a final product. Claim 7 is drawn to the method of claim 1 wherein the exosomes are the final product, aliquoted and stored at -80°C for up to 12 months. Claim 5 is drawn to the method of claim 1 wherein tobacco leaves are used and the liquid culture medium comprises 0.1-0.8 mg/L 6-Benzylaminopurine.
Limitations introduced by amendment have been underlined above.
Aguilar teaches a method of isolating plant cells, organelles, and membrane fragments using PEG/dextran aqueous two-phase partitioning systems (ATPS), including microsomal membranes from suspension-cultured tobacco cells and plasma membrane vesicles from maize (page 1388, paragraph 4 and table 2). Aguilar teaches a motivation of using the method in studying plant metabolism. Aguilar teaches a motivation to use a PEG/dextran aqueous two-phase system for the partitioning of plant cells because it is efficient, feasible to scale up, and can be integrated with other processes (abstract), leading to lower labor costs and lower energy and resource demand (page 1391, paragraph 1). Aguilar teaches that ATPS works by mixing water-soluble polymers in aqueous solution at a specific proportion (page 1386, paragraph 1). Aguilar teaches a motivation for additional “polishing” steps of isolated membrane and organelle particles (figure 1) or for combining additional extraction procedures in order to improve resolution between cell components with small differences in surface properties (page 1388, paragraph 4).
Aguilar does not teach a method of obtaining a plant tissue culture-based cell suspension culture. Aguilar does not teach mixing the cell suspension culture with an isolation solution at a 1:1 ratio by inverting 20 times, centrifuging at 1500 g for 10 minutes at 4°C, obtaining two separated phases with the supernatant comprising protein and the infranatant comprising exosomes, and then pulling and discarding the supernatant and transferring the infranatant to a clean tube.
Aguilar does not teach diluting isolation solution with water at a 1:1 ratio, centrifuging at 1000 g for 10 minutes, and obtaining the supernatant as a second supernatant, then adding the second supernatant at a 1:1 ratio to the infranatant of the previous step, mixing by inverting 10 times, and centrifuging at 12000-14000 g for 10 minutes at 4°C. Aguilar does not teach collecting the supernatant to obtain the exosomes and then storing the exosomes as a final product.
Mustafa teaches a method for obtaining a plant tissue culture-based cell suspension culture wherein callus culture is started from plant tissue by cutting (page 715, paragraph 4), including from leaves (page 726, paragraph 7), and subcultured every 4-6 weeks (page 731, paragraph 7). Mustafa teaches that the callus culture will be ready to be transferred to a liquid medium in 2-6 weeks (page 716, figure 1). Mustafa teaches cutting the callus into 2-3mm length fragments and placing in an Erlenmeyer flask that is 15%-25% full of liquid medium (page 731, paragraph 9).
Mustafa teaches that the growth medium for the plant cell culture contains a carbon source, such as sucrose (page 721, paragraph 4), and growth hormones. Mustafa teaches six commonly used growth hormones, including 1-naphthaleneacetic acid and benzylaminopurine (page 721, paragraph 8). Mustafa teaches that Murashige and Skoog is one of the most common media for plant cell cultures when further optimized with growth hormones (page 721, paragraph 9). Mustafa teaches a method for preparing a liquid medium comprising 30g sucrose and 1-2mg of NAA in a final volume of water (page 729, left column, paragraph 5). Mustafa teaches an example of a culture medium comprising 2mg/L NAA and 0.2mg/L BAP (table 3).
Mustafa teaches maintaining the liquid cell culture under continuous light as a growth condition, and that permanent light is easiest as full darkness is difficult to achieve (page 723, paragraphs 2-3) and light is required for some biosynthetic pathways (page 724, paragraph 2). Thus, Mustafa teaches a motivation for maintaining the cell culture under light because the cell culture would be more reproducible across batches.
Mustafa teaches maintaining the liquid cell culture at 24-25°C in a shaker at 90-125 rpm (page 723, paragraph 2). Mustafa teaches subculturing the liquid cell culture every 5 days or 1-4 weeks (page 724, paragraph 4) and subculturing cell suspension culture with a vacuum filter (page 732, paragraphs 1-2).
Finally, Mustafa teaches straining the cell suspension culture through a sterile metal sieve in order to separate fine cells from larger cell aggregates (page 731, paragraph 12) once the suspension culture becomes dense (page 731, paragraph 11). Mustafa teaches the rationale for straining in that fine cell suspension is more convenient to work with, more homogenous, and provides higher reproducibility (page 715, paragraph 5). Thus, Mustafa teaches a rationale for straining through a sterile sieve at least as often as 3-5 cultures. Mustafa teaches that the filtering step takes time: 15 minutes (page 716 figure 1). Mustafa teaches a motivation to use tobacco as the plant tissue for the cell culture suspension, because tobacco cell culture is fast growing (page 726, paragraph 1). Mustafa teaches an example sieve from VWR with part number 62399-918 (page 727, paragraph 40).
Burgess teaches a protocol for Murashige and Skoog agar that comprises 4.33g of Murashige and Skoog basal salt medium in a total volume of 1L (steps 1 and 2).
Kim teaches a method to isolate extracellular vesicles in mice using a two-phase system similar to the method described by Aguilar with additional steps to remove protein contaminants. Kim teaches a method to isolate extracellular vesicles in by mixing samples with a polyethylene glycol (PEG) and dextran solution (page 2, paragraph 10). The solution is added at a 1:1 ratio by volume to the samples (500ul and 500ul: page 2, paragraph 10 & table 1). The mixture is shaken at 200 rpm at 4°C to ensure polymers are dissolved. After mixing, Kim teaches that the samples are centrifuged for 10 min. Kim teaches that the infranatant phase (the DEX-phase) is collected for further analysis (page 2, paragraph 10). The dextran:polyethylene glycol ratios taught in the solution used by Kim (see system A: page 2, table 1) would result in two phases after the first centrifugation step that are supernatant comprising protein and cellular waste and infranatant comprising plant-derived exosomes. Kim teaches that the solution for the partitioning comprises PEG of a molecular weight of 25-45 kDa at 3.5-4.5% (w/w) and dextran of a molecular weight of 450-650 kDa at 1.5% (w/w) in PBS (page 2 paragraph 8; figure 1b & table 1). The systems taught by Kim (table 1) range from 11% dextran (55uL/(445 + 55uL)) to 14% dextran (70/430+70uL). Kim teaches that the PEG concentration at which the transition between two-phase and on-phase system occurred decreased as proportion of DEX increased (page 4 paragraph 2), providing a motivation to alter and optimize dextran proportion based on PEG concentration.
Kim teaches an additional step to enhance purity of extracted extracellular vesicles by dissolving PEG and dextran in PBS in the same composition used for isolation (described on page 2, paragraph 8), centrifuging this mixture at 1000g for 10 min, removing the supernatant of the centrifuged mixture, adding this PEG-phase supernatant to the infranatant of the isolation step, mixing, and centrifuging again for 10 min (page 3, paragraph 1). In this washing step, Kim teaches that the supernatant of the first phase separation is removed (page 3, paragraph 1). Kim teaches that washing via the described batch procedure lowers contamination by protein in the extracellular vesicle sample (figure 5). Kim also teaches that a two-phase method similar to the one they describe had successfully purified plasma membranes in plants (page 1, paragraph 4). Kim also teaches a variety of characterization analyses performed on the isolated extracellular vesicles, including Western blot, Bradford method protein quantification, and transmission electron microscopy (page 3, paragraphs 3, 6, 7).
Rider teaches a method to enrich for extracellular vesicles from cell culture that involves a second washing step with a water-based PEG solution. Specifically, Rider teaches centrifuging a cell culture at 500 g for five minutes followed by 2000 g for thirty minutes at 4°C to remove cellular debris (page 10, paragraph 5). Then, Rider teaches mixing the centrifuged media via inversion (page 10, paragraph 5) with equal volume of a PEG and water stock solution (page 10, paragraph 3). After incubation, the mixture was centrifuged at maximum speed, decanted, and drained (page 10, paragraph 5). A second washing step was performed either by resuspending the pellet and adding an equal volume of the PEG solution to a lower final concentration of PEG or by adding PBS and ultracentrifuging at high speed (100,000 g) (page 10, paragraph 5). The exosomes were stored at −80°C (page 10, paragraph 5). Rider also teaches a method of harvesting vesicles using differential centrifugation that comprises a step of centrifugation at 10,000g for 30 minutes to deplete large microvesicles (page 10, paragraph 2). Thus, Rider teaches that a centrifugation step to remove cellular debris from cell culture in the isolation of exosomes can be higher than 1000 g (for example, 1500g in claim 1, line 21) and is optimally performed at 4°C. Rider teaches that the isolation solution can contain water, and teaches diluting the isolation solution so that the PEG concentration is lower.
Mu teaches plant derived exosome-like nanoparticles from grape, grapefruit, carrots, and ginger and a method of isolating exosome-like nanoparticles by sequential centrifugation and sucrose step gradient ultracentrifugation (page 1562, left column, paragraph 4), a method described by Mu as mammalian exosome purification techniques (page 1564, right column, paragraph 2). Mu teaches that the recovered exosome-like particles varied in size within and between plant species (figure 1 A & B), and teaches a motivation to exclude particles from different bands of the sucrose gradient because their role in inducing expression of genes could not be determined (page 1564, right column, paragraph 2). Mu also teaches that heterogeneity in diameter and size of exosome-like nanoparticles decreased in solutions resembling stomach or intestinal conditions as compared to water, and that these changes differed across species (page 1566, left column, paragraph 2-page 1657, left column, figure 2A). The distribution of exosome-like nanoparticles varied in size from <100nm to >1000nm depending on species and solution (figure 2A).
Yamawaki teaches a method of aqueous two-phase partitioning of extracellular vesicles using PEG and dextran (figure 1A, paragraph [0003]). Yamawaki teaches a motivation to centrifuge the two-phase separation system because the step of centrifuging the mixture causes clearer phase separation between at least the first fraction and the second fraction (paragraph [0044]). Yamawaki teaches that phase separation can be achieved at less than 10,000g or “a lower centrifugal acceleration than ever before” (paragraph [0044]). Yamawaki also teaches ratios between upper and lower layers after phase separation in different solutions, including a ratio of 10% lower layer (30uL/300uL) in PBS (paragraphs [0066-0075], table 5) and even lower in culture medium. Yamawaki teaches that the volume of the lower layer tended to decrease as concentration of the polyether increased (paragraph [0075]).
A Usenet post from 1996 (attached) teaches that VWR part number 62399-918 corresponds to the Cellector™ tissue sieve.
VWR Cellector™ listing webpage (attached) teaches that the Cellector™ tissue sieve is made of stainless steel.
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to modify the aqueous two-phase isolation method of producing plant-derived plasma membrane vesicles or microsomal membranes from a cell suspension culture of tobacco taught by Aguilar to incorporate the method of obtaining a plant cell suspension culture as described by Mustafa. One of ordinary skill in the art would have been motivated to do so because although Aguilar does not elaborate on the exact process of generating the suspension culture, the process described by Mustafa is one that one having ordinary skill in the art could use to generate the suspension culture used as a starting material in a microsome isolation method. It would have further been obvious to one of ordinary skill in the art that the sieve described in the method of Mustafa is a steel sieve, based on the Usenet post and the VWR Cellector™ listing webpage.
Mustafa’s method of plant callus culture started by cutting (page 715, paragraph 4) from leaves (page 726, paragraph 7) reads on a callus culture made by a wounding method. Additionally, Mustafa’s transferring to a liquid medium in 2-6 weeks (page 716, figure 1) includes the 2-3 week range of the instant claim. Cutting callus into 2-3mm length fragments (page 731, paragraph 9) is within the range required by claim 1 and placing in a 15-25% Erlenmeyer flask filled with medium reads on placing callus pieces into flasks that are 10-50% full.
Mustafa teaches preparing a liquid culture medium in the Erlenmeyer flasks such that the medium contains sucrose, 6-benzylaminopurine, 1-Naphthaleneacetic acid, and a Murashige & Skoog vitamin-containing salt, and teaches medium that falls within the range of 20-30 g/L sucrose and 1-3 mg/L of NAA, as well as medium within the range of 0.1-0.8 mg/L BAP. Although Mustafa does not teach the exact concentration of 3.5-4.5 g/L of a M&S vitamin-containing salt mixture, it would have been obvious to combine the protocol of Burgess using this concentration of M&S salts with the medium taught by Mustafa comprising M&S salts under the rationale of combining prior art elements according to known methods to yield predictable results. It would likewise be obvious to modify the method of Murashige & Skoog using medium comprising 0.2 mg/L BAP that had been used for Tabernaemontana divaricata (figure 8, table 3) for culturing tobacco cells (as in Aguilar or other Mustafa examples) as part of routine optimization of cell culture medium, and one would have been motivated to combine this medium with the tobacco cells because Mustafa teaches different growth hormones cause different effects on biomass and optimum concentrations must be established experimentally for each species (page 721, right column, paragraph 6).
Mustafa’s method of maintaining the liquid cell culture under continuous light and at 24-25°C in a shaker at 90-125 rpm reads on agitating the liquid culture at a speed of 80-120 rpm at a temperature of 20-26°C. Mustafa teaches subculturing the liquid cell culture every 5 days or 1-4 weeks (page 724, paragraph 4) and subculturing cell suspension culture with a vacuum filter (page 732, paragraphs 1-2), which reads on claim 1 lines 14-17.
Finally, Mustafa teaches straining the cell suspension culture through a sterile metal sieve in order to separate fine cells from larger cell aggregates (page 731, paragraph 12) once the suspension culture becomes dense (page 731, paragraph 11). Mustafa teaches a rationale for straining through a sterile sieve at least as often as 3-5 cultures (because fine cell suspension is more convenient to work with, more homogenous, and provides higher reproducibility (page 715, paragraph 5)). The instant application does not teach a rationale for subculturing less frequently or why the results of subculturing less frequently could be expected to differ from more frequent subculturing. Moreover, it would be obvious to one skilled in the art to filter less frequently if the results were equivalent to filtering at every subculture, because not filtering would save time (15 minutes, Mustafa page 716 figure 1). Applicant would need to demonstrate subculturing less frequently would produce unexpected results to one of ordinary skill in the art in order for the instantly claimed frequency of filtering to be non-obvious. Mustafa teaches straining through a sterile sieve as in claim 1 line 17, and an example sieve from VWR with part number 62399-918.
Thus, the first step of instant claim 1 (lines 4-17) of obtaining a plant tissue culture-based cell suspension culture is obvious in light of Mustafa, Burgess, a Usenet archive, and the VWR website.
Before the effective filing date of the claimed invention, it would have further been obvious to one of ordinary skill in the art to further modify the aqueous two-phase isolation method taught by Aguilar to incorporate the teachings of Kim for isolating extracellular vesicles using a two-phase system with additional steps to remove protein contaminants. One of ordinary skill in the art would have been motivated to do so because Aguilar taught a motivation for additional polishing steps to improve resolution between cell components.
Kim’s method to isolate extracellular vesicles (defined by Kim as 50–1000 nm lipid bilayer spheres that enclose components from their mother cell, page 1, paragraph 1; and thus reading on exosomes defined in the instant specification as containing lipid bilayer vesicles of different sizes, page 1, paragraph 3) teaches mixing samples with a polyethylene glycol (PEG) and dextran solution at concentrations that fall within the claimed isolation solution requirements, which reads on claim 1 line 18-21). Kim teaches adding the solution at a 1:1 ratio by volume to the samples (500ul and 500ul: page 2, paragraph 10 & table 1), which reads on claim 1 line 19. Kim teaches shaking the mixture at 200 rpm at 4°C to ensure polymers are dissolved (page 2, paragraph 10), which makes obvious a method of mixing of the instant application to invert 20 times to obtain a first resulting mixture (claim 1 line 19). Before the time of filing, it would have been obvious to one having ordinary skill in the art to perform routine optimization and try different centrifugation speeds relative to the 1000 g for 10 min taught by Kim. Thus, performing the centrifugation step at 1500 g rather than 1000g would have been obvious at the time of filing.
The dextran:polyethylene glycol ratios taught in the solution used by Kim would result in two phases after the first centrifugation step that read on the recited supernatant comprising protein and cellular waste and infranatant comprising plant-derived exosomes (claim 1, lines 23-25). Although the systems taught by Kim (table 1) comprise dextran as 11% of the total volume rather than an infranatant of 10% of combined phases (claim 1, line 25), one of ordinary skill in the art would have been motivated to optimize the volume of dextran and thus the proportion of infranatant recovered because the proportion of dextran influenced the PEG concentration at which transition between two-phase and on-phase system occurred (Kim page 4, paragraph 2). One of ordinary skill in the art would have been motivated to try different dextran proportions, including 10%, in order to optimize the system. One of ordinary skill in the art would have had reasonable expectation of success, because altering phase volumes for two-phase separation was a known laboratory optimization prior to the filing of the instant application (see Yamawaki tables 4-6).
After centrifugation, Kim teaches that the infranatant phase (the DEX-phase) was collected for further analysis (page 2, paragraph 10), which reads on transferring the infranatant (claim 1, line 26). Although Kim does not teach transferring the infranatant to a clean tube, it would have been obvious before the time of filing to one of ordinary skill in the art to transfer the infranatant to a tube that is clean, because a dirty tube would introduce contamination.
The additional purity enhancing step of Kim by dissolving PEG and dextran in PBS in the same composition used for isolation, centrifuging this mixture at 1000g for 10 min, removing the supernatant of the centrifuged mixture, adding this PEG-phase supernatant to the infranatant of the isolation step, mixing, and centrifuging again for 10 min (page 3, paragraph 1) provides a motivation for an additional step of claim 1 lines 28-33 to enhance purity of extracted extracellular vesicles. Although Kim does not specify a method of mixing the samples after adding the supernatant to the infranatant containing extracellular vesicles, mixing vigorously could encompass inverting 10 times as recited in claim 1 line 33. Kim’s method reads on claim 1 lines 28-35 wherein a solution as a supernatant of an aqueous two-phase system is obtained. In this washing step, Kim teaches that the supernatant of the first phase separation is removed (page 3, paragraph 1), which reads on pulling and discarding the supernatant (claim 1 line 25). One of ordinary skill in the art would have been motivated to use the washing step for exosome isolation taught by Kim, because washing via the described batch procedure lowers contamination by protein in the extracellular vesicle sample. One of ordinary skill in the art would have had reasonable expectation of success, because the method of Kim recovered high-purity extracellular vesicles.
Yamawaki teaches that higher centrifugation speeds are permissible for the isolation of extracellular vesicles, such as exosomes, in a ATPS in order to achieve clear phase separation. Yamawaki teaches phase separation “at a lower centrifugal acceleration than ever before” (paragraph [0044]), and that centrifugal acceleration may be less than 10,000g (paragraph [0044]). One of ordinary skill in the art would reasonably expect from Yamawaki’s teachings that centrifugal acceleration of 10,000g may be used in ATPS in the isolation of extracellular vesicles. In addition, Rider teaches that differential centrifugation at 10,000 g depletes large microvesicles (page 10, paragraph 2). Mu teaches that the size and heterogeneity of recovered plant exosome-like particles can vary across species and the solution they are within, and the range of sizes of plant exosome-like particles (<100 nm to >1000) overlaps with the distribution of sizes between 84 nm and 422 nm of instant claim 1. Therefore, it would have been obvious to one having skill in the art 1) that the obtained plant-derived exosomes could have a distribution of sizes between 84 nm and 422 nm and 2) that the final centrifugation step could be increased to 12,000-14,000 g for 10 minutes because speeds of at least 10,000g were known in the art to be effective for isolating extracellular vesicles via ATPS. Trying centrifugation speeds up to 12,000g from 10,000g would be routine optimization. One of ordinary skill in the art would have been motivated to try different centrifugation speeds to deplete larger microvesicles and optimize exosome size across different species.
Because Kim teaches maintaining the mixture with the extracellular vesicles at 4°C for other steps, namely the mixing step, it would have been obvious to a person of ordinary skill in the art to control the temperature of the sample during a heat-generating process like centrifugation.
Thus, Kim teaches a method of isolation of extracellular vesicles that reads on claim 1 lines 18-25 & 27 and a method of washing that reads on claim 1 lines 26 & 28-34 in view of Yamawaki and Rider.
It would have further been obvious to one of ordinary skill in the art to modify the two-phase isolation method of Kim to incorporate the methods of Rider for a second washing step wherein an equal volume of the PEG solution is added to a lower final concentration of PEG or PBS is added and the sample is centrifuged at high speed (page 10, paragraph 5). Rider teaches that the isolation solution can contain water, and teaches diluting the isolation solution so that the PEG concentration is lower. It would have been obvious to one having ordinary skill in the art that the PEG solution, equivalent to the supernatant of the two-phase system of Kim, could be diluted with water at an equal volume, because a PEG/water solution is what Rider teaches as a secondary wash step for purification of extracellular vesicles. One combining the method of Kim with the method of Rider would be combining two known methods to accomplish the same effect (isolation and purification of extracellular vesicles) and so could have been confident of success. The lower concentration taught by Rider could be achieved by diluting with ratio of 1:1 (from between 5 and 15% PEG in the first mixture to 5% PEG in the second mixture). One of ordinary skill in the art would have been motivated to perform the second, lower PEG concentration washing step because the second, diluted washing step produced stronger evidence of exosomes on a Western blot than the protocol without an extra step. This aligns with the motivation of Kim to have high purity extracellular vesicles.
In view of Rider, it would have been obvious to use inversion to mix the solutions. This would be exchanging a known method of mixing (via shaker) for another (inversion). The exact number of inversions (10 vs 20 vs another number) has not been shown by the specification to have a differentiable effect on the final mixture than another number and would fall into routine optimization. Applicant would need to demonstrate that the number of inversions would produce unexpected results to one of ordinary skill in the art in order for the instant limitation to be non-obvious over the art.
Finally, in view of Rider, storage of subsets of the extracellular vesicles (which read on exosomes) would have been obvious (claim 1, line 36), including at −80°C (page 10, paragraph 5). Making subsets of the extracellular vesicles (Rider page 10, paragraph 5) reads on aliquoting (claim 7 line 2). Rider is silent as to how long the subsamples were stored at -80°C before being further analyzed via mass spectrometry, transmission electron microscopy, or another method (page 11), but it would have been obvious, routine, and desirable to one skilled in the art to analyze one’s scientific samples via at least one method in less than 12 months’ time. Therefore, the method of Rider teaches the aliquoting and storing extracellular vesicles (which reads on exosomes) at -80°C for up to 12 months as recited in claim 7.
Claims 1, 5 & 7 are obvious in view of Aguilar, Mustafa, Burgess, Kim, Rider, Yamawaki, Mu, the VWR Cellector™ listing, and the Usenet forum post from 1996.
Applicant urges that independent claim 1 recites the distinguishing limitation that the obtained plant-derived exosomes have a distribution of sizes between 84 nm and 422 nm. Aguilar, Kim, Rider, Mustafa, Burgess, Yamawaki, Lukatkin, and the other references do not teach, suggest, provide motivation for or reasonable expectation of isolation of plant-derived exosomes with the specific size distribution. In contrast, Applicant urges that Applicant’s claimed process steps results in exosomes with the claimed size distribution (Remarks, page 5, paragraph 6-page 6, paragraph 1).
This argument is unpersuasive, because the range of obtained plant-derived exosomes between 84 nm and 422 nm overlaps with the range of obtained plant-derived exosome like nanoparticles known in the prior art, and the size and heterogeneity of recovered exosome-like particles was known in the art to depend on the species of plant. Therefore, it would have been obvious to one of skill in the art to obtain plant-derived exosomes with a distribution of sizes within the range recited by instant claim 1.
Applicant urges that the amended claims require a step of obtaining two separated phases wherein the infranatant is 10% of the combined phases, which would not be obvious in consideration of the cited prior art because Kim does not disclose that DEX phase would be 10% of the combined phases. The examples of phase volumes provided by Kim vary and are not tied to the outcome of isolating plant-derived exosomes. The other cited references are silent with respect to an infranatant that constitutes 10% of the combined phases. Applicant urges that this limitation is not a result that would be expected or inherent but rather the result of unique combination of steps including defined concentrations of PEG and dextran, mixing and centrifugation, and use of tissue culture-based cell suspension cultures. Furthermore, Applicant urges that obtaining an infranatant that is 10% of the combined phases is critical for downstream processing, scalability, and product consistency (Remarks, page 6, paragraph 2-page 7, paragraph 1).
This argument is unpersuasive, because Kim taught a motivation to use different ratios of dextran/PEG to optimize proportions based on PEG concentration for phase transition. Therefore, an infranatant dextran phase comprising 10% of the combined phases would be obvious over the 11% dextran by volume in the method used by Kim in light of routine optimization of concentrations and proportions of PEG/Dextran phases. Yamawaki, notably, teaches phase separations that resulted in lower layers constituting 10% or less of the mixture.
The fact that the methods of Kim and Yamawaki are not tied to plant-derived exosomes does not make the method non-obvious, because Mu teaches mammalian exosome purification techniques are effective in deriving exosome-like nanoparticles from plants.
In addition, if the specific ratio of 10% of the combined phases is a result of the combination of steps recited in claim 1, as Applicant urges, then the 10% of the combined phases being the infranatant would result from the active steps of the method which are obvious over the prior art for the reasons presented above.
Applicant urges that the cited references of Aguilar, Mustafa, Kim, Rider, Yamawaki, and Burgess do not teach or suggest centrifuging a PEG/dextran-containing second mixture at 12,000-14,000 g for 10 minutes at 4°C to isolate exosomes. Applicant urges that Rider uses 3,214 g for one hour for PEG precipitation and reserves centrifugation at higher speeds for differential centrifugation or ultracentrifugation. Applicant urges that the teaching of 3,214 g centrifugation for 1 hour for PEG precipitation would not suggest centrifuging PEG/dextran mixture at 12,000-14,000g for 10 minutes and the prior art does not provide a motivation or rational to increase speed or provide data supporting such a modification. Applicant urges that the prior art teaches away from combining certain known elements (Remarks, page 8, paragraph 1-page 9, paragraph 2).
This argument is unpersuasive, because the teaching of a different method does not constitute teaching away. Rider does not teach that centrifugation of a PEG/dextran mixture at speeds above 3,214 g would result in an inoperable process. Rather, Rider teaches the 3,214 g centrifugation speed for PEG enrichment of extracellular vesicles as the maximum speed of the tabletop centrifuge (page 10, paragraph 5), not a limit on the process of PEG precipitation of extracellular vesicles. Additionally, Rider teaches a motivation to centrifuge a PEG solution at a higher speed, namely to select for the size of recovered microvesicles. Yamawaki suggests centrifuge speeds of “less than” 10,000g in a method of concentrating extracellular vesicles, but Yamawaki also does not teach that higher centrifuge speeds would render the method inoperable. Therefore, the prior art does not teach away from centrifuging a PEG/dextran-containing second mixture at 12,000-14,000g for 10 minutes to isolate exosomes.
Applicant urges that claim 1 recites additional, patentable limitations because the inventor identified a problem that was not previously recognized in the field and required specific steps even though this increased the cost and complexity of producing plant-derived exosomes. Applicant urges that prior to Applicant’s invention, the field focused on extracting exosomes from immature plant tissues which necessitated complicated purification and reduced exosome homogeneity. Applicant urges that a person of skill would not have had a reason to combine tobacco or stevia suspension culture with an aqueous PEG/Dextran two-phase system, as this would increase cost and complexity of preparing and maintaining suspension cultures. Applicant urges that the prior art did not recognize the problem of phytochemical contamination nor did it suggest that the solution lay in combining cell suspension cultures with two-phase separation system (Remarks, page 9, paragraph 3-page 10, paragraph 2).
This argument is unpersuasive, because aqueous PEG/dextran two-phase systems had been used by the prior art for the recovery of vesicles of vacuolar and plasma membranes from suspension-cultured tobacco cells (Aguilar, table 2). Therefore, the combination of tobacco plant cell suspension culture with PEG/dextran aqueous two-phase system extraction of vesicles of plasma membranes, which read on the exosomes of the present invention, was already known in the art prior to the filing of the instant application. The steps and limitations of the plant cell suspension culture and the PEG/dextran aqueous two-phase system isolation methods of the instant application fall within ranges or within routine optimization of methods known in the prior art. The motivation to combine references does not need to be Applicant’s own in order to be obvious.
Applicant urges that the rejection is based on an impermissible use of hindsight because the proposed combination of references does not provide teaching, suggestion, or motivation to arrive at the claimed invention without relying on knowledge gleaned from Applicant’s disclosure. Applicant urges that because Aguilar is silent on exosome isolation from plant cell suspension cultures with the specific size and morphological uniformity achieved by the claimed method or the specific sequence of steps required to achieve these results, and because Kim and Rider are directed to animal-derived extracellular vesicles, the rationale for combining animal EV isolation protocols with plant cell culture methos I based on Applicant’s own disclosure. Applicant urges there is no evidence that a person of ordinary skill would have expected that animal EV isolation protocols could be directly and successfully applied to plant cell cultures. Applicant urges that the specific sequence of steps is not taught or suggested by any single reference or by the combination of references and that the cited references do not teach the critical steps of centrifuging a second resulting mixture containing PEG and dextran at 12000-14000g for 10 minutes to obtain plant-derived exosomes. Applicant urges that the only source for this specific combination and optimization is Applicant’s own disclosure (Remarks, page 10, paragraph 3-page 12, paragraph 2).
This argument is unpersuasive, because although Aguilar is silent on the size of vesicles achieved, Mu teaches that exosome-like nanoparticles derived from plants can be expected in a distribution of sizes that encompasses or overlaps with the range recited in claim 1. Mu furthermore teaches that methods for isolating animal-derived extracellular vesicles would have been expected prior to the filing of the instant application to function to isolate plant-derived exosome-like vesicles.
The specific sequence of steps does not need to be taught by a single reference in order for the combination of steps to be obvious over the prior art.
The cited references teach that extraction of vesicles using two-phase separation at speeds up to at least 10,000g is known (Yamawaki). The cited references teach that centrifugation at higher speeds can deplete larger microvesicles (Rider). Centrifuging a mixture at 12,000g is not beyond routine optimization of centrifuging a mixture at 10,000g, and would be well within the skill of one of ordinary skill in the art. One of skill in the art would have been motivated to optimize centrifugation steps in the isolation of exosomes in order to select for certain sizes. Thus, the steps of the instantly claimed method are obvious over the prior art. “[a]ny judgement on obviousness is in a sense necessarily a reconstruction based on hindsight reasoning, but so long as it takes into account only knowledge which was within the level of ordinary skill in the art at the time the claimed invention was made and does not include knowledge gleaned only from applicant’s disclosure, such a reconstruction is proper.” In re McLaughlin 443 F.2d 1392, 1395, 170 USPQ 209, 212 (CCPA 1971).
Applicant urges that the claimed invention provides unexpected advantageous properties in comparison to Aguilar, because Aguilar is silent on the ability to produce plant-derived exosomes with a highly uniform size distribution and morphology in a defined nanometer size range from plant cell suspension cultures. Applicant urges that the instant disclosure demonstrates that produced exosomes are within a narrow and well-defined size distribution with uniform morphologies and sizes. Applicant urges that the unexpectedly good results only need to be shown versus the closest subject matter in the prior art (MPEP 716.02(e)(III)). (Remarks, page 12, paragraph 3-page 13, paragraph 2).
This argument is unpersuasive, because the silence of Aguilar with respect to size and uniformity of produced exosomes does not demonstrate that the achieved morphology, size, and size distribution of the methods of the instant claims are unexpected to one of skill in the art. In addition, Applicant has not presented data comparing the instant results to those of the closest subject matter, nor statistical analysis comparing the results. Even if the data relates to the unexpected results of the claimed invention it cannot obviate the instant rejection as it would not be viewed as persuasively presented by a person of ordinary skill in the art. The evidence relied upon should establish "that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance." Ex parte Gelles, 22 USPQ2d 1318, 1319 (Bd. Pat. App. & Inter. 1992) (MPEP 716.02(b).
Applicant urges that claims 5-8 depend on claim 1, and because claim 1 provides the limitation of infranatant being 10% of the combined phases and the obtained plant-derived exosomes having a distribution of sizes between 84 nm and 422 nm, the dependent claims are also non-obvious (Remarks, page 13, paragraph 3-page 14, paragraph 2).
This argument is unpersuasive, because claim 1 is not non-obvious over the prior art for the reasons provided above.
Applicant urges that Aguilar and Mustafa focus on basic biomolecule isolation and culture preservation and are quite limited and outdated and have not developed a method for isolating exosomes (Declaration, paragraph 6).
This argument is unpersuasive, because the instant application describes exosomes as vesicles of different sizes containing a lipid bilayer (page 1, paragraph 3). In this regard, the recovery of vesicles of vacuolar and plasma membranes from suspension-cultured tobacco cells (Aguilar, table 2) reads on a method of isolating exosomes. The limitations of the instant claims taught by Aguilar and Mustafa are not non-obvious simply because newer methods are known in the art.
Applicant urges that the method of Kim targets proteins not exosomes and the conditions were not optimized to preserve exosome integrity and functionality, while the ATPS parameters used in the instant method provides an unparalleled advantage in preserving yield and function integrity (Declaration, paragraph 7).
This argument is unpersuasive, because Applicant has not provided data, nor statistical analysis, to compare the results of the instantly claimed method to those of methods known in the art. The declaration, without data, cannot obviate the instant rejection as it would not be viewed as persuasively presented by a person of ordinary skill in the art. The evidence relied upon should establish "that the differences in results are in fact unexpected and unobvious and of both statistical and practical significance." Ex parte Gelles, 22 USPQ2d 1318, 1319 (Bd. Pat. App. & Inter. 1992) (MPEP 716.02(b).
In order to show that the instantly claimed methods provide unexpected results to one of ordinary skill in the art, Applicant would need to present data comparing the results of the instantly claimed method to those of comparable methods known in the art. Applicant is reminded that evidence after final including secondary references is not considered unless Applicant can provide sufficient evidence why such material could not have been earlier provided.
Applicant urges that the methods disclosed in Rider used only a PEG-based precipitation method which cannot be compared to ATPS because it does not contain a second phase such as dextran. Applicant urges that a direct comparison between Rider and the instant invention is technically flawed (Declaration, paragraph 8).
This argument is unpersuasive, because Yamawaki teaches centrifugation at speeds up to 10,000 in a two-phase separation method for extracellular vesicles. Rider, on the other hand, teaches a motivation to centrifuge at different speeds in order to deplete large microvesicles, while Mu teaches that plant-derived exosome-like particles are obtained across a range of sizes. Thus, one of skill in the art would have had motivation to centrifuge the mixture comprising a PEG layer at a higher speed in order to deplete larger microvesicles and arrive at an appropriate size distribution.
Applicant urges that the instant applicant describes optimized conditions specifically developed for exosome isolation from plant cell cultures not a purification method. Applicant urges that the instant invention is novel because prior art has primarily focused on exosomes isolated from animal cells or serum. Furthermore, Applicant urges that the instant application provides unexpected advantages and differs from the prior art by enabling collection of higher amounts of exosomes, preserving biological activity, ensuring integrity of RNA and protein cargo transport, and providing industrial-scale reproducible process (Declaration, paragraphs 9-10).
This argument is unpersuasive, because Applicant has not provided evidence in the form of data to demonstrate that the instantly claimed method provides results that would be unexpected to one of ordinary skill in the art. The declaration, without data comparing to methods known in the art, cannot obviate the instant rejection as it would not be viewed as persuasively presented by a person of ordinary skill in the art.
Exosomes isolated from plants were known in the art prior to the filing of the instant application. The relative similarity in the art of methods in animal cells or serum does not make methods of exosome isolation in plants non-obvious.
Applicant urges that claims 5-8 depend on claim 1, and because claim 1 provides the limitation of infranatant being 10% of the combined phases and the obtained plant-derived exosomes having a distribution of sizes between 84 nmand 422 nm, the dependent claims are also non-obvious (Remarks, page 13, paragraph 3-page 14, paragraph 2).
This argument is unpersuasive, because claim 1 is not non-obvious over the prior art for the reasons provided above.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over Aguilar in view of Mustafa, Burgess, Kim, Rider, Mu, Yamawaki, the VWR Cellector™ listing, and the Usenet forum post from 1996, as applied to claims 1, 5 &7 above, and further in view of Lukatkin et al (2007) Med. Aromat. Plant. Sci. Biotechnol, 107-111, hereafter Lukatkin).
This is a new rejection based on Applicant’s amendments. Applicant' s arguments filed 10/17/2025 have been fully considered as they pertain to the instant rejection but they are not persuasive.
Claim 6 is drawn to the method of claim 1 wherein stevia leaves are used and the liquid culture medium comprises 1-4 mg/L 6-Benzylaminopurine.
The teachings of Aguilar in view of Mustafa, Burgess, Kim, Rider, Mu, Yamawaki, the VWR Cellector™ listing, and the Usenet forum post from 1996 are discussed above. They do not teach stevia leaves used as the plant tissue and the liquid culture medium comprising 1-4 mg/L 6-Benzylaminopurine.
Lukatkin teaches tissue culture of stevia in various media. Lukatkin teaches a motivation for culturing stevia: stevia produces substances with sweetening ability that do not have secondary effects in human diets (page 107 final paragraph – page 108 first paragraph). Lukatkin teaches variations of media for growing stevia callus comprising 6-Benzylaminopurine, naphthylene acetic acid at 1 mg/L, Murashige & Skoog salts, and sucrose at a concentration of 30g/l (page 108, paragraph 6 & table 3). Lukatkin tests media with different concentrations of 6-Benzylaminopurine (0.1-1 mg/L, page 108, paragraph 6) and shows that stevia callus is able to grow on media comprising 1mg/L of 6-Benzylaminopurine (table 3). Lukatkin also teaches that stevia in vitro culture had been performed on both solid and liquid media (page 108, paragraph 3).
Before the effective filing date of the claimed invention, it would have been obvious to one of ordinary skill in the art to modify the process taught by Aguilar in view of Mustafa, Burgess, Kim, Rider, Mu, Yamawaki, Burgess, the VWR Cellector™ listing, and the Usenet forum post from 1996 discussed above to incorporate a medium as described in Lukatkin and to culture stevia cells.
The media taught by Lukatkin comprises ingredients in concentrations that encompass the ranges of concentrations in claim 6. Arriving at the exact concentrations in the instant application for stevia would have been routine optimization. It would have been routine optimization to one skilled in the art at the time of filing of the instant application to try a culture medium comprising 1mg/L of 6-Benzylaminopurine to culture stevia. It would have been obvious to one skilled in the art that the solid media described by Lukatkin for stevia callus culture would have been suitable for stevia liquid suspension culture if the agar had been removed. This would have been simple substitution of one known element for another to obtain predictable results.
Therefore, claims 1 & 5-7 are rejected as being obvious in view of Aguilar, Mustafa, Kim, Rider, Mu, Burgess, Yamawaki, the VWR Cellector™ listing, and the Usenet forum post from 1996, and in further view of Lukatkin.
Applicant urges that claims 5-8 depend on claim 1, and because claim 1 provides the limitation of infranatant being 10% of the combined phases and the obtained plant-derived exosomes having a distribution of sizes between 84 nmand 422 nm, the dependent claims are also non-obvious (Remarks, page 13, paragraph 3-page 14, paragraph 2).
This argument is unpersuasive, because claim 1 is not non-obvious over the prior art for the reasons provided above.
Claim 8 is rejected under 35 U.S.C. 103 as being unpatentable over Aguilar in view of Mustafa, Burgess, Kim, Rider, Mu, Yamawaki, the VWR Cellector™ listing, and the Usenet forum post from 1996, as applied to claims 1, 5 & 7 above, and further in view of Charoenviriyakul et al (2018) International journal of pharmaceutics, 553(1-2), 1-7, hereafter Charoenviriyakul.
This is a new rejection based on Applicant’s amendments. Applicant' s arguments filed 10/17/2025 have been fully considered as they pertain to the instant rejection but they are not persuasive.
Claim 8 is drawn to the method of claim 1 wherein in the exosomes are the final product, lyophilized, and stored at 4°C for up to 36 months in powder form.
The teachings of Aguilar in view of Mustafa, Burgess, Kim, Rider, Mu, Yamawaki, the VWR Cellector™ listing, and the Usenet forum post from 1996 are presented above. These references do not teach the exosomes lyophilized and stored at 4°C for up to 36 months in powder form.
Charoenviriyakul teaches a method of lyophilization of exosomes and storage for 1-4 weeks (page 2, paragraph 4). Charoenviriyakul teaches that the exosomes stored in this way were rehydrated with water (page 2, paragraph 4), which reads on the exosomes being in a powder form. Charoenviriyakul teaches the lyophilized exosomes being stored at room temperature (page 2, paragraph 4), but also teaches exosomes being stored at colder temperatures, including -80°C and 4°C (page 1, paragraph 2) and that lyophilization may be able to improve the preservation stability of exosomes (page 1, paragraph 3).
Before the time of filing, it would have been obvious to one having ordinary skill in the art to modify the method of Aguilar in view of Mustafa, Burgess, Kim, Rider, Mu, Yamawaki, the VWR Cellector™ listing, and the Usenet forum post from 1996 to incorporate the lyophilization storing method of Charoenviriyakul. One would have been motivated to do so because Aguilar teaches the motivation to lower costs with two-phase isolation processes (page 1391, paragraph 1), and storage at -80°C as described by Rider is costly. One would have reasonable expectation of success because Charoenviriyakul showed the lyophilization method to be suitable for the preservation and storage of exosomes. It would also have been obvious to combine the preservation method of lyophilization with the preservation method of cool temperatures (4°C) described by Charoenviriyakul under the rationale of combining prior art elements for preservation according to known methods to yield predictable results.
Thus, claims 1, 5 & 7-8 is rejected as being obvious in light of Aguilar, Mustafa, Burgess, Kim, Rider, Mu, Yamawaki, the VWR Cellector™ listing, the Usenet forum post from 1996, and Charoenviriyakul.
Applicant urges that claims 5-8 depend on claim 1, and because claim 1 provides the limitation of infranatant being 10% of the combined phases and the obtained plant-derived exosomes having a distribution of sizes between 84 nmand 422 nm, the dependent claims are also non-obvious (Remarks, page 13, paragraph 3-page 14, paragraph 2).
This argument is unpersuasive, because claim 1 is not non-obvious over the prior art for the reasons provided above.
Conclusion
No claims are allowed.
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 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.
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/VICTORIA L DELEO/Examiner, Art Unit 1662
/Anne Kubelik/Primary Examiner, Art Unit 1663
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