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 .
Status of Claims
Claims 1-3, 5-12, 14-15, & 17-22 are under examination on the merits.
The objections to claims 1, 7 & 14 are withdrawn in light of Applicant’s amendments.
The rejection of claims 1-20 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, is withdrawn in light of Applicant’s amendments.
The rejection of claims 1-20 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement is withdrawn in light of Applicant’s amendments.
The rejection of claims 1-11 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, for scope of enablement is withdrawn in light of Applicant’s amendments.
Information Disclosure Statement
The information disclosure statement filed 1/2/2026 fails to comply with 37 CFR 1.98(a)(2), which requires a legible copy of each cited foreign patent document; each non-patent literature publication or that portion which caused it to be listed; and all other information or that portion which caused it to be listed. It has been placed in the application file, but the information referred to therein has not been considered. Specifically, the IDS filed 1/2/2026 lists foreign patent FR3049868. An English translation of FR3049868 has been provided appended to the French language foreign patent FR3049867. No copy of FR3049868 has been provided.
An IDS listing the foreign patent document and providing the missing copy of the cited foreign patent document would comply with 37 CFR 1.98(a)(2).
Claim Interpretation
The instant specification has defined “attenuated gene expression” to encompass not only a reduced amount or rate of transcription and translation but also disruption in proper translation, folding, activity, and/or assembly of a protein such as by an unnatural mutation in the gene (page 8, lines 3-21). The claims, especially claims 7 & 8, have been examined under Applicant’s definition for attenuated gene expression.
The claims recite the species Aloe vera. Aloe vera L. is considered synonymous with Aloe barbadensis Mill. (see Sung (2006). The history of Aloe. In New perspectives on Aloe. 7-17. page 10, paragraph 5), and has been treated as the same species for the purpose of examination.
Claim Objections
Claims 9, 14 & 20 are objected to because of the following informalities:
Claim 9 (line 3-4) recites “transforming bacterial cells with an endonuclease configured to cleave pks1 and/or pks2 in the obtained plant cells”. Wording such as --transforming bacterial cells with a plasmid encoding an endonuclease configured to cleave pks1 and/or pks2 in the obtained plant cells-- would clarify that the bacterial cells and any endogenous pks1/pks2 genes are not transformed by the endonuclease.
Claim 14 (lines 2-3): “an amount of mRNA transcribed by pks1 and/or pks2” should read --the amount of mRNA transcribed from pks1 and/or pks2--.
Claim 20 (lines 1 & 2): “an amount of aloin” should read --the amount of aloin-- in both uses.
Appropriate correction is required.
Claim Rejections - 35 USC § 112
Indefiniteness
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 15 is 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. This is a new rejection.
Claim 15 is drawn to the mutant Aloe vera of claim 12, wherein the mutant Aloe vera comprises a mutant Aloe vera cell or cell culture. It is unclear whether this limitation requires the mutant Aloe vera to be a cell or whether this limitation merely requires that the Aloe vera comprises a cell. The first interpretation would be reasonable because it further limits independent claim 12, but the second interpretation is a plain language reading of the claim. Because the claim can have two distinct interpretations with different scope, claim 15 is indefinite.
Improper Dependency
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 15 and 17 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Due to Applicant' s amendment of the claims, the rejection is modified from the rejection as set forth in the Office action mailed 10/1/2025 as applied to claims 4, 17 & 20. Applicant' s arguments filed 1/2/2026 have been fully considered but they are not persuasive.
Claim 17 is drawn to the mutant Aloe vera of claim 12, wherein the Aloe vera constitutes a component of a bioproduct. Claim 17 is not drawn to a bioproduct but rather the mutant Aloe vera. The Aloe vera being present as a component of a bioproduct would not change the identity of the mutant Aloe vera nor its structural or biological features. Thus, claim 17 fails to further limit claim 12 on which it depends.
Claim 15 is drawn to the mutant Aloe vera of claim 12 wherein the mutant comprises a mutant Aloe vera cell or cell culture. It is indefinite whether this limitation is intended to require that the mutant Aloe vera is a mutant cell or merely comprises a mutant cell. A mutant plant would necessarily comprise at least one mutant cell. Thus, under the second possible interpretations, claim 15 would not further limit the mutant Aloe vera of claim 12.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Applicant urges that claims 17 & 20 have been amended to properly limit the dependent claims from which they depend (Remarks, page 8, paragraphs 3-5).
This argument is unpersuasive, because amended claim 17 requires that the mutant Aloe vera constitute a component of a bioproduct. The Aloe vera being a component of a bioproduct does not impart structural or biological features on the Aloe vera. The Aloe vera that is a component of a bioproduct is not patentably distinct from the Aloe vera that is not a component of a bioproduct, and so claim 17 does not properly limit claim 12.
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.
Claim(s) 12, 14, 15 & 17-20 are rejected under 35 U.S.C. 103 as being unpatentable over Shi et al (2018) Plant Science. 277: 145-154 (available online 8/22/2018, hereafter Shi) in view of Liu et al (2020). Journal of Asian Natural Products Research. 22(5). 434–443. (published online 12/3/2019, hereafter Liu) and in view of Boudreau et al (2017). Toxicological Sciences. 158(2). 302-318. (published 5/19/2017, hereafter Boudreau).
This is a new rejection.
Claims 12, 14, 15 & 17-20 are drawn to a mutant Aloe vera that has attenuated expression of at least one pks gene comprising pks1 and/or pks2.
Shi teaches a polyketide synthase 1 (pks1) gene knockout in rice obtained by Crispr-Cas9 mediated mutation (page 146, right column, paragraph 3). Shi teaches the identification of target sites in the first and second exons using designer software, then introduction of plasmids containing the recognition sites and Crispr-Cas9 into rice via Agrobacterium tumefaciens EHA105 (page 146, right column, paragraph 3). Sequencing confirmed mutations in the OsPKS1 gene (figure 1). The mutations included one and two base deletions and led to complete male sterility and were characterized as knockouts (page 148, left column, paragraph 3). Shi suggests that enzymatic activity of PKS in dicots and monocots is evolutionarily conserved (page 152, left column, paragraph 2).
Shi does not teach a mutant aloe comprising attenuated expression of at least one pks gene.
Liu teaches that aloesin (a heptaketide) and aloin (an octaketide) are important bioactive constituents of Aloe barbadensis that are biosynthesized by type III PKSs (page 435, paragraph 2). Liu teaches a PKS1 and PKS2 protein in Aloe barbadensis involved in biosynthesis of heptaketides and octaketides (page 435, paragraph 2). Liu teaches that the full-length cDNA sequence of PKS1 is GenBank MK801868 and the full-length cDNA sequence of PSK2 is GenBank MK801869 (page 436, paragraph 3). PKS1 was able to catalyze condensations to produce aleosone and heptaketides TW93a, while PKS2 catalyzed condensations to produce octaketides and might be involved in biosynthesis of aloin (page 436, paragraph 3-page 437, paragraph 1).
Boudreau teaches that Aloe vera is a popular herbal remedy and dietary supplement (page 302, left column). Boudreau teaches that increasing concentration of aloin in water administered to male rates increased incidence of hyperplasias and incidence and severity of chronic-active inflammation (table 1 & 2, page 308, right column, paragraph 2). Boudreau teaches that chronic-active inflammatory conditions resulting in chronic diseases alter the gut microbiota (page 314, right column, paragraph 2). Boudreau also teaches that anthraquinones, in particular hydroxyanthraquiones and dihydroxyanthraquinones, have mutagenic and genotoxic activities and have exhibited tumor-promoting activities (page 316, left column, paragraph 1). Boudreau suggests aloin and Aloe vera leaf extract induce colon cancer in rates (page 316, left column, paragraph 3). Boudreau teaches that the IASC sets a maximum level of aloin in Aloe vera products for human consumption (page 316, left column, paragraph 2).
Before the time of filing of the instant application, one of ordinary skill in the art would have been motivated to modify the Crispr-Cas9 PKS1 knockout method used to knockout PKS1 in rice to instead knockout PKS1/PKS2 in Aloe barbadensis identified by Liu. One of ordinary skill in the art would have been motivated to knockout PKS1/PKS2 in Aloe, because at least PKS2 was believed to be involved in the biosynthesis of Aloin, and the IASC sets an upper limit for aloin in products for human consumption. One of ordinary skill in the art would have had reasonable expectation of success, because Aloe barbadensis had been genetically modified before the filing of the instant application, and both rice and aloe are monocots. Thus, a mutant Aloe vera with attenuated expression of at least one pks gene of pks1 or pks2 (instant claim 12) would have been obvious in light of Liu, Boudreau, and Shi. A mutant plant would comprise cells, so claim 15 (wherein the mutant comprises a mutant cell) would also be obvious.
It would have been obvious to use the mutant Aloe vera in an edible food because Aloe vera is a popular dietary supplement (instant claim 17).
Although Shi, Liu, and Boudreau are silent about the transcriptional levels of the mutant pks gene, it would have been obvious, before the filing date of the instant application, to recover mutant Aloe vera plants with reduced expression. Some of the plants generated would be likely to have less than 10% of an amount of mRNA transcribed by the genes in wild-type Aloe vera. Thus, it would have been obvious to one of ordinary skill in the art to recover a mutant aloe with mRNA transcribed from pks1 and pks2 less than 10% of the amount transcribed in a wild-type plant (instant claim 14).
Likewise, it would have been obvious to recover mutant Aloe vera plants producing reduced amounts of aloin because the IASC sets an upper limit to the amount of aloin in aloe products for human consumption. Some of the plants generated would likely have less than 10% of the amount of aloin produced in wild-type Aloe vera or even no aloin. Thus, it would have been obvious to one of ordinary skill in the art to recover a mutant Aloe vera wherein the amount of aloin produced in the mutant is less than 10% of the amount of aloin produced in wild-type Aloe vera (instant claim 20) or wherein the mutant exhibits an absence of aloin (instant claims 18 & 19).
Claim(s) 1, 2, 5-10 & 21-22 are rejected under 35 U.S.C. 103 as being unpatentable over Shi, Liu, and Boudreau as applied to claims 12, 14, 15 & 17-20 above, and further in view of He et al (2007) Journal of Genetics and Genomics. 34(12): 1053-1060 (available online 12/31/2007, hereafter He), GenBank reference sequence MK801869 (available 5/16/2020), and GenBank reference sequence MK801868 (available 5/16/2020).
Claims 1, 2, 5-10 & 21-22 are drawn to a method of producing a mutant aloe comprising attenuating the expression level of at least one protein comprising PKS1 and/or PKS2.
The teachings of Shi, Liu, and Boudreau are presented above. Shi teaches that designer software can be used to identify target sites in exons of a pks gene and that recognition sites can be introduced via plasmids into Agrobacterium (page 146, right column, paragraph 3). Shi teaches one of the gRNAs recognizing the editing site with a sequence of AGCAGGAGCCAATGCTCCAGGGG (figure 1a). This sequence has 30% sequence identity to instant SEQ ID NO: 8 (see alignment below, SEQ ID NO: 8 is on the top).
Score
Expect
Identities
Gaps
Strand
14.4 bits(7)
0.017
7/7(100%)
0/7(0%)
Plus/Plus
Query 16 GCAGGAG 22
|||||||
Sbjct 2 GCAGGAG 8
They do not teach a method of obtaining plant cells derived from Aloe vera and culturing mutant plant cells under a set of culture conditions.
He teaches a motivation for genetic improvement of aloe through genetic engineering because conventional methods are not easy because the plant does not produce vigorous seeds by self-crossing and cross pollination (page 1053, left column-right column, paragraph 1). He teaches a method of transforming aloe plants by harvesting aloe plants and cutting pieces of aloe stem, sheaths, and leaves and then culturing the pieces in shoot induction medium. He further teaches infecting the cultured aloe with Agrobacterium carrying plasmids with genes for resistance to antibiotics (page 1054, left column, paragraph 2-page 1055, left column, paragraph 2). Infected explants were incubated on induction medium with antibiotics and then moved onto selection medium to regenerate shoots with resistance to the selective reagent (page 1055, left column, paragraph 4).
He teaches that although plant regeneration can be theoretically obtained in vitro from any living cells, regeneration of leaf was not successful but regeneration from stem and sheath was (page 1058, left column, paragraph 3-right column, paragraph 1). However, He teaches that regeneration plants have been obtained from leaf in certain genotypes by others although division and growth is less feasible with more differentiated cells (page 1058, right column, paragraph 1).
GenBank reference MK801869 teaches that the Aloe vera PKS2 gene of Liu encodes an amino acid sequence comprising 100% identity to instant SEQ ID NO: 2. See alignment of SEQ ID NO: 2 (top) to translated sequence (bottom) below. GenBank reference MK801869 teaches that the Aloe vera PKS2 gene of Liu comprises a sequence with 100% identity to instant SEQ ID NO: 8. See second alignment below.
Score
Expect
Method
Identities
Positives
Gaps
Frame
842 bits(2175)
0.0
Compositional matrix adjust.
403/403(100%)
403/403(100%)
0/403(0%)
+1
Query 1 MGSLSNYSPVMEDVQAIRKAQKADGTATVMAIGTAHPPHIFPQDTYADFYFRATNSEHKV 60
MGSLSNYSPVMEDVQAIRKAQKADGTATVMAIGTAHPPHIFPQDTYADFYFRATNSEHKV
Sbjct 1 MGSLSNYSPVMEDVQAIRKAQKADGTATVMAIGTAHPPHIFPQDTYADFYFRATNSEHKV 180
Query 61 ELKKKFDRICKKTMIGKRYFNYDEEFLKKFPNITSFDEPSLNDRQDICVPGVPALGAEAA 120
ELKKKFDRICKKTMIGKRYFNYDEEFLKKFPNITSFDEPSLNDRQDICVPGVPALGAEAA
Sbjct 181 ELKKKFDRICKKTMIGKRYFNYDEEFLKKFPNITSFDEPSLNDRQDICVPGVPALGAEAA 360
Query 121 VKAIA EWGRPKSEITHLVFCTSCGVDMPSADFQCAKLLGLRANVNKYCVYMQGCYAGGTV 180
VKAIA EWGRPKSEITHLVFCTSCGVDMPSADFQCAKLLGLRANVNKYCVYMQGCYAGGTV
Sbjct 361 VKAIA EWGRPKSEITHLVFCTSCGVDMPSADFQCAKLLGLRANVNKYCVYMQGCYAGGTV 540
Query 181 MRYAKDLAENNRGARVLVVCAELTIIGLRGPNESHLDNAIGNSLFGDGAAALIVGSDPII 240
MRYAKDLAENNRGARVLVVCAELTIIGLRGPNESHLDNAIGNSLFGDGAAALIVGSDPII
Sbjct 541 MRYAKDLAENNRGARVLVVCAELTIIGLRGPNESHLDNAIGNSLFGDGAAALIVGSDPII 720
Query 241 GVEKPMFEIVCAKQTVIPNSEDVIHLHMREAGLMFYMSKDSPETISNNVEACLVDVFKSV 300
GVEKPMFEIVCAKQTVIPNSEDVIHLHMREAGLMFYMSKDSPETISNNVEACLVDVFKSV
Sbjct 721 GVEKPMFEIVCAKQTVIPNSEDVIHLHMREAGLMFYMSKDSPETISNNVEACLVDVFKSV 900
Query 301 GMTPPEDWNSLFWIPHPGGRAILDQVESRLKLRPEKFGATRTVLWDCGNMVSACVLYILD 360
GMTPPEDWNSLFWIPHPGGRAILDQVESRLKLRPEKFGATRTVLWDCGNMVSACVLYILD
Sbjct 901 GMTPPEDWNSLFWIPHPGGRAILDQVESRLKLRPEKFGATRTVLWDCGNMVSACVLYILD 1080
Query 361 EMRRKSAADGLATYGEGLEWGVLLGFGPGMTVETILLHSLPTV 403
EMRRKSAADGLATYGEGLEWGVLLGFGPGMTVETILLHSLPTV
Sbjct 1081 EMRRKSAADGLATYGEGLEWGVLLGFGPGMTVETILLHSLPTV 1209
Score
Expect
Identities
Gaps
Strand
46.1 bits(23)
3e-10
23/23(100%)
0/23(0%)
Plus/Minus
Query 1 AGGCATGTCGACACCGCAGGAGG 23
|||||||||||||||||||||||
Sbjct 444 AGGCATGTCGACACCGCAGGAGG 422
GenBank reference MK801868 teaches the coding sequence of Aloe vera PKS1, comprising a sequence with 94% identity to a sequence found in instant SEQ ID NO: 8. See first alignment below. GenBank reference MK801868 also teaches that the encoded protein has an amino acid sequence with 100% sequence identity to instant SEQ ID NO: 1. See second alignment below.
Score
Expect
Identities
Gaps
Strand
26.3 bits(13)
2e-04
16/17(94%)
0/17(0%)
Plus/Minus
Query 1 AGGCATGTCGACACCGC 17
||||||||| |||||||
Sbjct 444 AGGCATGTCAACACCGC 42
Score
Expect
Method
Identities
Positives
Gaps
839 bits(2168)
0.0
Compositional matrix adjust.
403/403(100%)
403/403(100%)
0/403(0%)
Query 1 MGSLSDSTPLMKDVQGIRKAQKADGTATVMAIGTAHPPHIISQDSYADFYFRVTNSEHKV 60
MGSLSDSTPLMKDVQGIRKAQKADGTATVMAIGTAHPPHIISQDSYADFYFRVTNSEHKV
Sbjct 1 MGSLSDSTPLMKDVQGIRKAQKADGTATVMAIGTAHPPHIISQDSYADFYFRVTNSEHKV 60
Query 61 ELKKKFDRICKKTMIGKRYFNFDEEFLKKYPNITSFDKPSLNDRHDICIPGVPALGAEAA 120
ELKKKFDRICKKTMIGKRYFNFDEEFLKKYPNITSFDKPSLNDRHDICIPGVPALGAEAA
Sbjct 61 ELKKKFDRICKKTMIGKRYFNFDEEFLKKYPNITSFDKPSLNDRHDICIPGVPALGAEAA 120
Query 121 VKAIEEWGRPKSEITHLVFCTSGGVDMPSADFQCAKLLGLRTNVNKYCIYMQGCYAGGTV 180
VKAIEEWGRPKSEITHLVFCTSGGVDMPSADFQCAKLLGLRTNVNKYCIYMQGCYAGGTV
Sbjct 121 VKAIEEWGRPKSEITHLVFCTSGGVDMPSADFQCAKLLGLRTNVNKYCIYMQGCYAGGTV 180
Query 181 MRYAKDLAENNRGARVLMVCAELTIIALRGPNDSHIDNAIGNSLFGDGAAALIVGSDPII 240
MRYAKDLAENNRGARVLMVCAELTIIALRGPNDSHIDNAIGNSLFGDGAAALIVGSDPII
Sbjct 181 MRYAKDLAENNRGARVLMVCAELTIIALRGPNDSHIDNAIGNSLFGDGAAALIVGSDPII 240
Query 241 GVEKPMFEIVCAKQTVIPNSEDVIHLHLRESGLMFYMTKDSAATISNNIEACLVDVFKSV 300
GVEKPMFEIVCAKQTVIPNSEDVIHLHLRESGLMFYMTKDSAATISNNIEACLVDVFKSV
Sbjct 241 GVEKPMFEIVCAKQTVIPNSEDVIHLHLRESGLMFYMTKDSAATISNNIEACLVDVFKSV 300
Query 301 GMTPPEDWNSLFWIPHPGGRAILDQVEAKLKLRPEKFSATRTVLWDYGNMISACVLFILD 360
GMTPPEDWNSLFWIPHPGGRAILDQVEAKLKLRPEKFSATRTVLWDYGNMISACVLFILD
Sbjct 301 GMTPPEDWNSLFWIPHPGGRAILDQVEAKLKLRPEKFSATRTVLWDYGNMISACVLFILD 360
Query 361 EMRRKSAAEGLETYGEGLEWGVLLGFGPGMTIETILLHSLPPV 403
EMRRKSAAEGLETYGEGLEWGVLLGFGPGMTIETILLHSLPPV
Sbjct 361 EMRRKSAAEGLETYGEGLEWGVLLGFGPGMTIETILLHSLPPV 403
Before the filing date of the instant application, it would have been obvious to one of ordinary skill in the art to modify the method of knocking out a pks gene in rice to instead knock out (which reads on attenuating the expression level of) a pks1 gene in aloe. In the transformation of aloe, it would have been obvious to obtain plant cells derived from Aloe vera and culture mutant plant cells under a set of culture conditions, as taught by He. One of ordinary skill in the art would have been motivated to transform cultured aloe cells following the method of He, because conventional methods of plant improvement are not easy in aloe. One of ordinary skill in the art would have had reasonable expectation of success, because He successfully transformed aloe plants in in vitro culture using Agrobacterium, and Shi also used Agrobacterium transformation. Thus, instant claim 1 is obvious.
Furthermore, He teaches regeneration of aloe plants has been successful in some genotypes from leaf, sheath and stem of aloe despite the differentiation of the cells. Regeneration of plants from differentiated cells like leaf reads on converting obtained cells into plant stem cells (instant claim 2) and on regenerating an Aloe vera plant using the mutant plant cells (instant claim 5). It would have been obvious to one of ordinary skill in the art to convert the obtained plant cells into plant stem cells to regenerate aloe plants for genetic improvement. One of ordinary skill in the art would have had reasonable expectation of success, because the prior art had reported successful regeneration from leaf cells.
Regarding claim 6, He teaches culturing the plant cells after agrobacterium infection on induction medium with antibiotics. This reads on selecting mutant plant cells by applying an antibiotic before culturing the mutant plant cells under a set of culture conditions (instant claim 6).
The CRISPR Cas9 method taught by Shi to knockout pks1 activity by causing deletion mutation in exons reads on a method wherein attenuating the expression of a protein comprises disrupting an enzymatic activity of the at least one protein (instant claim 7), because the resulting phenotype is described as knockout. The pks1 or pks2 gene in aloe read on proteins required for biosynthesis of a toxin because Liu’s pks2 likely is involved in biosynthesis of aloin, and aloin consumption is linked to inflammation.
Disrupting the expression of the wild-type PKS1 or PKS2 protein reads on disrupting expression of the pks1 and/or pks2 gene (instant claim 8). Claim 8 does not require that transcription levels are attenuated. The method of He and the method of Shi both comprise transforming agrobacterium (which read on bacterial cells) with a construct in order to transform plants, and so the method reads on instant claim 9. Although, Shi, Liu, Boudreau, and He do not teach a guide RNA sequence of instant SEQ ID NO: 3-10, it would have been obvious to one of ordinary skill in the art, and one of ordinary skill in the art would have been motivated, to include a guide RNA with a sequence of at least 55% identity to SEQ ID NO: 8 because SEQ ID NO: 8 is identical to a sequence found in the known pks2 gene. One of ordinary skill in the art would have had reasonable expectation of success because CRISPR/Cas9 editing and guide RNA design was routine at the time of filing of the instant application and the sequence of the target gene was known.
Because the aloe pks2 taught by Liu encodes an amino acid sequence with 100% identity to instant SEQ ID NO: 2, instant claim 22 is also obvious.
Finally, although one of ordinary skill in the art would have been motivated to target aloe pks2 to reduce aloin content, a gRNA of instant SEQ ID NO: 8 comprises a sequence with 94% identity to a 17 base polynucleotide sequence within the coding sequence of aloe pks1. A method of attenuating the expression level of PKS2 in aloe using a gRNA target sequence of SEQ ID NO: 8 would likely have off-target effects due to the high similarity with the gene encoding PKS1, leading to attenuation of expression of the PKS1 with an amino acid sequence of SEQ ID NO: 1 (instant claim 21).
Claims 1, 2, 5-10, 12, 14-15, & 17-22 are obvious over Shi, Liu, Boudreau, He, GenBank reference MK801869, and GenBank reference MK801868.
Claim(s) 11 is rejected under 35 U.S.C. 103 as being unpatentable over Shi, Liu, Boudreau, He, GenBank reference MK801869, and GenBank reference MK801868 as applied to claims 1, 2, 5-10, 12, 14-15, & 17-22 above, and further in view of Zhang et al (2019) Nature Plants. 5. 778-794 (published 7/15/2019, hereafter Zhang).
Claim 11 is drawn to a method wherein disrupting expression of pks1 and/or pks2 comprises delivering an endonuclease and/or ribonucleoprotein via particle bombardment.
The teachings of Shi, Liu, Boudreau, He, GenBank reference MK801869, and GenBank reference MK801868 are presented above. They do not teach delivery of an endonuclease and/or ribonucleoprotein via particle bombardment, carbon nanotubes, silicon carbide whiskers, or direct injection.
Zhang reviews CRISPR applications in plants, including gene expression knockdown using LwaCas13a in rice protoplasts (page 5, right column, paragraph 1). Zhang teaches that Cas13a can target multiple RNAs simultaneously (page 5, right column, paragraph 1).
Zhang teaches that CRISPR-Cas systems can alter gene expression by editing regulatory elements and also by inhibiting initial binding of transcription machinery or blocking RNA polymerase elongation (page 9, left column, paragraph 2-3). Zhang teaches that gene expression levels can also be regulated post-transcriptionally by RNA-targeting CRIPSR systems such as Cas13 and RNA-targeting Cas9s (page 9, right column, paragraph 3).
Zhang teaches a motivation to deliver RNPs into plant tissues that are used for plant regeneration, such as callus, by particle bombardment, because RNP delivery eliminates introduction of foreign DNA and shortens exposure of genomic DNA to CRISPR reagents, minimizing off-target effects (page 9, right column, paragraph 7).
Before the filing of the instant application, it would have been obvious to one of ordinary skill in the art to use particle bombardment to deliver RNP targeting pks1 and/or pks2 rather than agrobacterium transformation. One of ordinary skill would have been motivated to modify the method of Shi or He in order to eliminate introduction of foreign DNA and minimize off-target effects. One of ordinary skill in the art would have had reasonable expectation of success because particle bombardment for plant transformation was well known in the art prior to the filing of the instant application.
Claims 1, 2, 5, 6-12, 14-15, 17-20 & 22 are obvious over Shi, Liu, Boudreau, He, GenBank reference MK801869, GenBank reference MK801868, and Zhang.
Claim(s) 3 is rejected under 35 U.S.C. 103 as being unpatentable over Shi, Liu, Boudreau, He, GenBank reference MK801869, and GenBank reference MK801868 as applied to claims 1, 2, 5-10, 12, 14-15, & 17-22 above, and further in view of Lee. 2014. Improvement of secondary metabolite production with anti-inflammatory activity via adventitious root culture of Aloe vera. Doctoral dissertation from the graduate school of Seoul National University. (Relevant pages of Introduction, Literature Revies, and Chapter III have been provided with the Office Action. hereafter, Lee).
Claim 3 is drawn to a method wherein cultured mutant plant cells are grown in a bioreactor and an Aloe vera bioproduct is harvested from the cells.
The teachings of Shi, Liu, Boudreau, He, GenBank reference MK801869, and GenBank reference MK801868 are presented above. They do not teach growing mutant plant cells in a bioreactor and harvesting a bioproduct.
Lee teaches a motivation to produce secondary metabolites using plants over mammalian or microbial cell culture systems to eliminate a purification step when the plant cells are used as food (page 3, paragraph 2-page 4, paragraph 1). Lee teaches a method of culturing Aloe vera adventitious roots in suspension culture on an orbital shaker and harvested every 7 days, while aloe emodin and chrysophanol were extracted from the culture medium (page 72, paragraph 1-page 74, paragraph 1).
Before the time of filing of the instant application, one of ordinary skill in the art would have been motivated to culture mutant Aloe vera cells in a suspension culture on an orbital shaker as taught by Lee. One of ordinary skill in the art would have been motivated to use this culturing method in order to eliminate a purification step in harvesting cells as a food product. One of ordinary skill would have had reasonable expectation of success, because Lee used this culture system for Aloe vera cells to extract aloe emodin and crysophanol.
The suspension cultures of aloe on an orbital shaker read on a bioreactor, which the instant specification defines as an apparatus configured to support and expand a population of mutant aloe plant cells by maintaining an internal environment conducive to cell viability and growth (page 21, lines 13-15). Extracting aloe emodin and crysophanol from cell culture and/or harvesting cells for a food product would both read on harvesting a bioproduct, because the instant specification defines “bioproduct” as a product comprising or derived from the mutant cells and can comprise various amounts of a molecule (page 21, line 25-page 22, line 11). Thus, instant claim 3 would have been obvious.
Claims 1-3, 5-10, 12, 14-15, & 17-22 are obvious over Shi, Liu, Boudreau, He, GenBank reference MK801869, GenBank reference MK801868, and Lee.
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Victoria L DeLeo whose telephone number is (703)756-5998. The examiner can normally be reached M-F 8:00am-4pm EDT.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Bratislav Stankovic can be reached at (571) 270-0305. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/VICTORIA L DELEO/Examiner, Art Unit 1662
/Anne Kubelik/Primary Examiner, Art Unit 1663
Prosecution Insights