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 .
Election/Restrictions
The Office acknowledges the receipt of Applicant’s restriction election filed April 15, 2026. Applicant elected Group II, claims 69-84, directed to a method of biolistically delivering a substance to plant tissue including a heat treatment of the tissue. Applicant did not traverse the Unity of the invention Requirement. Accordingly, claims 1, 2, 4, 8-10, 12, 21, 23, 26, 30-32, and 85 are withdrawn from examination. The present Office Action is limited to examination of elected claim 69-84.
The restriction is made FINAL.
Claim Status
Claims 1, 2, 4, 8-10, 12, 21, 23, 26, 30-32, 69-85 are pending. Claims 1, 2, 4, 8-10, 12, 21, 23, 26, 30-32, and 85 are withdrawn from consideration. Claims 69-84 are examined on the merits.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 69 rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 69 is rejected as indefinite for the recitation “…. culturing … plant tissue on growth media in optimal incubation conditions for each substance…”. “optimal incubation conditions” fails to provide objective boundaries for the claimed method. Although the specification describes certain tested incubation conditions, the claim does not recite with incubation parameters are required or what objective standard determines whether a condition is “optimal”. The term “optimal” is a term of degree, and the claim does not specify whether optimality is measured by delivery efficiency, editing efficiency, transient expression, tissue viability, regeneration frequency, or any other endpoints. Further, “for each substance” makes the required incubation conditions vary depending on the selected substance without defining how those conditions are determined. Therefore, one of ordinary skill in the art would not be able to determine the scope of the claim with reasonable certainty.
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.
Claims 69-84 are rejected under 35 U.S.C. §103 as being unpatentable over Liang (Zhen Liang et. al., Nature Protocols (2018) VOL.13 NO.3, pp 413-430) in view of Kurokawa (Shuta Kurokawa et. al., Plant Cell Physiology (2021), 62(11): 1676–1686), and further in view of Bio-Rad (PDS-1000/He Biolistic Particle Delivery System manual, publicly available technical literature: https://www.bio-rad.com/webroot/web/pdf/lsr/literature/M1652249.pdf)
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Under BRI, clam 69 recites a biolistic delivery method with three main steps:
Step 1: applying rupture/bombardment pressure to a macrocarriter disk carrying at least one drop of microparticle suspension to bombard microparticles into plant tissue.
Step 2: Microparticle/substance limitations: the microparticles are coated with a “substance”, about 0.1 µm to about 2.0 µm in size, suspended in drops of about 0.1 µl to about 50 µl. Under BRI, the claimed “Substance” reasonably includes nucleic acids, proteins, RNPs, or mixtures thereof, particularly in view of claim 78.
Step 3: Post-bombardment, the bombarded tissue is subjected to heat treatment, and, the heat-treated bombarded tissue is cultured on growth media in “optimal incubation conditions for each substance”.
Liang teaches a method of biolistically delivering CRISPR/Cas9 in vitro transcripts or ribonucleoprotein complexes to plant tissue, including wheat immature embryos, by coating microparticles with the substance and bombarding the microparticles into the plant tissue (p413, Abstract). Liang further teaches culturing the bombarded plant tissue under tissue-culture/regeneration conditions after bombardment (p426 Tissue culture to regenerate testable seedlings). Liang therefore teaches biolistically delivering a substance coated on microparticles into plant tissue to form bombarded plant tissue and culturing the bombarded plant tissue.
Liang does not expressly teach subjecting the bombarded plant tissue to the presently claimed heat treatment. Kurokawa teaches that heat treatment of plant material after CRISPR/Cas delivery increases genome- editing efficiency, including heat treatment at elevated temperature, such as 37C for 24 hours (p1676, Abstract). Kurokawa further teaching optimizing treatment conditions, including temperatures and incubating duration for different CRISPR systems (p1683 both columns; p1684 both column). It would have been obvious to one of ordinary skill in the art to apply Kurokawa’s heat-treatment step to Lian’s biolistically bombarded plant tissue because both references concern CRISPR/Cas-mediated genome editing in plants, and Kurokawa teaches heat treatment as a predictable way to improve mutation/editing efficiency.
Bio-Rad teaches standard particle bombardment using a microcarrier disk carrying a suspension of coated microparticles, wherein helium rupture disks provide rupture/bombardment pressures, and wherein gold or tungsten microparticles in sizes including about 0.6 µm, 1.0 µm, and 1.6 µm are used (p34, Recommended starting particle size/type for bombardment of various cell types). The sizes fall within the claimed “about 0.1 µm to about 2.0 µm” range. Bio-Rad further teaches preparing microparticles on the microcarrier prior to bombardment (p21, 4 Microcarriers).
Bio-Rad teaches nine different rupture disks available ruptures at a specific pressure, ranging in rating from 450 to 2200 psi (p31, last paragraph). Bio-Rad further teaches different Helium Pressure recommendations to different biological materials, ranging from 1100-1300 psi (p31, Table 5.1). The pressures fall within the claimed “pressure of about 100 psi to about 3000 psi” ranges.
Bio-Rad teaches the protocol of preparing a suspension of microparticles (p22, Coating Washed Microcarriers with DNA). Liang also teaches adding 50 µl of gold nanoparticles to the mixture and spreading 15 µl of the mixture onto the central region of each macro-carrier (p426, line 2-3). The suspended volumes teaching by Liang and Bio-Rad fall within “suspended in drops of about 0.1 µl to about 50 µl” ranges.
It would have been obvious to use Bio-Rad’s known rupture-disk pressures, macrocarrier disk loading, droplet suspension, and microparticle sizes in liang’s biolistic plant delivery method because Bio-Rad provides standard operating conditions and components for carrying out particle bombardment, and because optimization of bombardment pressure, particle size, and suspension loading constituted routine optimization of result-effective variables in particle bombardment methods.
Therefore, claim 69 is obvious over Liang, in view of Kurokawa and Bio-Rad because the combined references teach or suggest biolistically bombarding coated microparticles into plant tissue, subjecting the bombarded tissue to heat treatment, and culturing the treated plant tissue after bombardment.
Claim 70 recites the method of claim 69, wherein the about 0.1 µl to about 50 µl volume of microparticle suspension is present as one to 100 drops on the macrocarrier. Claim 71 recites the method of claim 69, wherein the drops are dispersed over the macrocarrier disk surface. Claim 72 recites the method of claim 69, wherein the suspension of microparticles on the macrocarrier disk is dried or lyophilized prior to the application of the rupture or bombardment pressure.
Bio-Rad teaches preparing each microcarrier by removing 6 µl aliquots of microcarrier suspension and spreading the suspension evenly over the central area of the microcarrier using a pipette tip (p24, paragraph 5). The aliquots applied by pipette correspond to the claimed “drops”, and the disclosed application of aliquots/drops onto the microcarrier falls within the claimed range of one to 100 drops on the microcarrier.
Bio-Rad teaches that, after application of the microcarrier suspension to the microcarrier, “the ethanol should evaporate within 10 minutes to leave the DNA-coated microcarriers adhering to the microcarrier”, and that the loaded macrocarriers should be used within 2 hours (p24, paragraph 6).
Therefore, it would have been obvious to employ the known aliquot/drop application procedure in the biolistic delivery method of Liang to obtain proper loading and distribution of microparticles on the microcarrier prior to bombardment and drying the microparticle suspension on the microcarrier prior to bombardment.
For the same reasons set forth with respect to claim 69, claims 70—72 are prima facie obvious over Liang, in view of Kurokawa and Bio-Rad.
Claim 73 recites the method of claim 69, wherein the plant tissue is an immature embryo (IE),callus tissue, somatic embryo, zygotic embryo, meristem tissue, pollen, cotyledon, leaf, stem, or root tissue. Claim 74 recites the method of claim 69, wherein the plant is a crop plant.
Bio-Rad teaches plant embryos, callus or cell culture, tissues sections (p31, Table 5.1); and Liang teaches “immature embryos” (p424, Harvesting of immature embryos and biolistic delivery of CRISPR/Cas9IVTs). Liang further teaches bread wheat, a crop plant (p413, Abstract).
Accordingly, for the same reasons set forth with respect to claim 69, claims 73—74 are prima facie obvious over Liang, in view of Kurokawa and Bio-Rad.
Claim 75 recites the method of claim 69, wherein the microparticles are about 0.1 µm to about 2.0 µm in size. Claim 76 recites the method of claim 69, wherein the microparticle suspension is in a volume of about 1 µl to about 50 µl per bombardment. Claim 77 recites the method of claim 69, wherein the number of bombardments is about 1 and 20 per plant tissue sample.
Bio-Rad teaches gold or tungsten microparticles in sizes including about 0.6 µm, 1.0 µm, and 1.6 µm are used (p34, Recommended starting particle size/type for bombardment of various cell types); Bio-Rad further teaches preparing 6 µl aliquots of microcarrier suspension over the central area of the microcarrier using a pipette tip (p24, paragraph 5). Liang teaches spread 20 µl of the suspension onto the central region of a macro-carrier (p425, 37(A)(v)). Both Bio-Rad and Liang teach bombarding plant tissues (including immature embryos) with microparticles using biolistic. Under the broadest reasonable interpretation, s single bombardment constitutes “1” bombardment, which falls within the claimed range of “about 1 and 20 per plant tissue sample”.
Therefore, for the same reasons set forth with respect to claim 69, claims 75-77 are prima facie obvious over Liang, in view of Kurokawa and Bio-Rad.
Claim 78 recites the method of claim 69, wherein the substance on the microparticle comprises nucleic acids, a protein of interest or ribonucleoprotein (RNP), or a mixture thereof. Claim 79 recites the method of claim 69, wherein the culturing of the bombarded plant tissue is performed at about 25°C to about 28°C. Claim 80 recites the method of claim 69, wherein the culturing of the bombarded plant tissue is performed for about 1 hour or longer post-bombardment. Claim 81 recites the method of claim 69, wherein the culturing of the bombarded plant tissue is performed for about 24 hours to about 180 days post-bombardment.
Liang teaches DNA-free editing of bread wheat by delivering ribonucleoprotein complexes (RNPs) of CRISPR/Cas9 by particle bombardment (p413, Abstract).
Liang further teaches incubating bombarded embryos at 23 °C during recovering and regeneration (p426, Tissue Culture to Regenerate Testable Seedlings). Under broadest reasonable interpretation, 23 °C is sufficiently close to the claimed lower endpoint of “about 25°C” to fall within the scope of “about 25°C to about 28°C”, particularly where the specification does not define “about” or show criticality for the 25°C - 28°C range.
Liang teaches “incubate the bombarded embryos in high-osmotic medium overnight in the dark” (p425, 37(A)(vii)), which is covered within claimed “wherein the culturing of the bombarded plant tissue is performed for about 1 hour or longer post-bombardment”.
Liang also teaches that after RNP coating and delivery, plantlets regenerate in 6-8 weeks (p426, Tissue culture to regenerate testable seedlings ● timing 6–8 weeks). This post-bombardment culture/regeneration period falls within the claimed range of “about 24 hours to about 180 days”.
Accordingly, for the same reasons set forth with respect to claim 69, claims 78-81 are prima facie obvious over Liang, in view of Kurokawa and Bio-Rad.
Claim 82 recites the method of claim 69, wherein the heat treatment of the bombarded plant tissue is performed at about 28°C to about 40°C. Claim 83 recites the method of claim 69, wherein the heat treatment of the bombarded plant tissue is performed for about 1 hour to about 180 days post-bombardment. Claim 84 recites the method of claim 69, wherein the heat treatment of the bombarded plant tissue is performed for about 24 hours to about 30 days post-bombardment.
Kurokawa teaches one heat treatment at 37◦C for 24h increased the CRISPR/Cas9-mediated mutation efficiency (p1676, Abstract). The disclosed 37◦C falls within the claimed range of about 28°C to about 40°C, and the disclose 24-hour treatment falls within both the claimed range of about 1 hour to about 180 days and the claimed range of about 24 hours to about 30 days.
It would have been obvious to one of ordinary skill in the art to apply Kurokawa’s 37°C, 24-hour heat treatment to Liang’s RCISPR/Cas9 RNP-mediated biolistic delivery method because Kurokawa teaches that elevated heat treatment improves Cas9-mediated mutation efficiency, and improving genome-editing efficiency is a predictable and desirable goal in plant CRISPR systems. Temperature and duration of heat treatment would have been recognized as result-effective variables affecting editing efficiency, and selecting 37°C for 24 hours would have involved applying a known successful heat-treatment condition to a similar CRISPR/Cas9 plant editing method with a reasonable expectation of improving mutation efficiency. Therefore, for the same reasons set forth with respect to claim 69, claims 82-84 are prima facie obvious.
Conclusion
No claims are allowed.
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/YANXIN SHEN/ Examiner, Art Unit 1663
/WEIHUA FAN/ Primary Examiner, Art Unit 1663
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