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 .
Claim Interpretation
Based upon the Examiner’s understanding of the claimed invention:
Any 16S sequence, including those from E. coli and Salmonella, as well as any other E. coli or Salmonella sequence will be construed as reading upon the instant claims.
Claims 7 and 33 will be construed as introducing an additional fungal target nucleic acid sequence distinct from those that are specific to Psilocybe.
Claim Rejections
35 USC § 112(b)
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.
Claims 2, 27, 30, and 33 are 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.
Regarding claims 2 and 27, “16S nucleic acid sequences” are presented as an alternative to E. coli and Salmonella target nucleic acid sequences. However, 16S sequences are present in both claimed bacterial species, as described in Fukushima [abstract; Fukushima M et al. Journal of clinical microbiology. 2002 Aug;40(8):2779-85]. It is therefore unclear if the “16S target nucleic acid sequences” claimed by the applicant include or exclude those found in E. coli and Salmonella, rendering the metes and bounds of these claims indefinite.
Claims 30 and 33 depend from a rejected claim and are similarly rejected.
35 USC § 112(d)
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 7, 8, 12, and 33 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.
In these dependent claims, limitations regarding “a fungal target nucleic acid sequence” are introduced. The claim language does not specify that the fungal target nucleic acid sequence is in addition to the previously recited Psilocybe target nucleic acid sequence. Accordingly, it is unclear if the recited “a fungal target nucleic acid sequence” is intended to encompass the Psilocybe target sequence previously recited in claims 1 and 27 or instead refer to a fungal target sequence from some other fungus.
Under the first interpretation, dependent claims 7, 12, and 33 do not further limit the subject matter of the claims from which they depend because the independent claims already require a fungal target nucleic acid sequence (i.e., that of Psilocybe). While claim 8 would further limit, it depends from claim 7 which is rejected under this interpretation.
Under the second interpretation, the dependent claims broaden the scope of the claims upon which they depend by encompassing fungal targets other than Psilocybe.
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.
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 1, 5-17, 27, and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Savelkoul [US 2017/0349937 A1] in view of Zhang [Zhang X et al. Forensic Sci Res. 2021 Apr 13;7(3):490-7] and Abbas [Abbas AI et al. Oregon Psilocybin Advisory Board. 2021 Jul 30; p1-40].
Regarding claims 1, 15, 16, 17, and 27, Savelkoul discloses a method for detecting microbial blood infections using real-time PCR. An embodiment of this method comprises performing five separate multiplex PCR assays configured to detect the gene/gene product, or part thereof, of the following microbes and/or antibiotic-resistant genes [0027]:
(1) Aspergillus spp., gram positive bacteria, gram negative bacteria, Candida spp. and Candida glabrata
(2) Escherichia coli, Enterococcus faecium, Acinetobacter baumannii, and the mecA gene
(3) Staphylococcus aureus, Enterococcus faecalis, Pseudomonas aeruginosa, Candida krusei, and Streptococcus pneumoniae
(4) Staphylococcus spp., Enterococcus spp., Klebsiella spp. and Candida albicans
(5) vanA and ctxM
In this embodiment, the first multiplex reaction amplifies nucleic acids from both bacteria and fungi using target specific primers [0034-0038]. As such, Savelkoul demonstrates that multiplex PCR assays employing target-specific primers for both fungal and bacterial nucleic acids were known and routinely used for simultaneous detection of multiple classes of microorganisms. Savelkoul further discloses providing a kit adapted for performing this method comprised of a forward and reverse primer for amplifying each target, as well as fluorescently labeled probes which recognize the amplified regions of the target [Savelkoul, 0161, 0167].
However, Savelkoul does not disclose targeting nucleic acids originating from Psilocybe mushrooms.
Zhang discusses hallucinogenic mushrooms, defining them by their ability to produce psilocybin, and describes the need that currently exists in the art for accurate identification of these mushroom. According to Zhang, these mushrooms are often consumed recreationally and this consumption can lead to out-of-control/dangerous behavior and criminal activity. Traditional identification approaches have included study of mushroom morphology and cytology; however, these traditional methods suffer from a range of limitations as environmental factors can impart wide variability in their features [Zhang, p1].
On the other hand, DNA-based identification methods are widely applicable in food hygiene, forensic medicine, and other fields. While DNA barcoding methods of the internal transcribed spacer region of the fungal genome have been widely used for discrimination of P. cubensis, the universality of this region ultimately results in unclear species specificity. To combat this problem, additional regions of the fungal genome have been explored; however, no strategy yet exists for the specific identification of P. cubensis. Zhang attempts to fill this gap through the study of select markers in an attempt to design a species-specific panel for use in the forensic detection of P. cubensis [Zhang, p1-2].
In this study, four primer sets were designed to target four sequences related to P. cubensis: the largest subunit of RNA polymerase II (RPB1), psilocybin-related phosphotransferase gene, glyceraldehyde 3-phosphate dehydrogenase gene, and translation EF1α (tEF1α). Real-time PCR high resolution melting (HRM) was performed and the specificity, sensitivity, reproducibility, and performance in mixtures and multiplex PCR of the primer sets was evaluated [Zhang, p2-3].
To perform this evaluation, mushroom samples were collected and identified by their morphological characteristics and sequencing of their ITS region. Hallucinogenic mushroom samples (P. cubensis, P. merdaria, Panaeolus papilionaceus), as well as C. sativa samples, were obtained from forensic laboratories; non-hallucinogenic mushroom samples were obtained through foraging efforts. From these sample, genomic DNA was extracted and diluted to 1.0 ng/µL. Real-time PCR HRM analysis was conducted and the accumulation of amplified product was monitored using an intercalating dye. The results of this study showed that the primers were capable of distinguishing P. cubensis from 22 other species in pure and mixed samples and were amenable to multiplexing [Zhang, p2-3, 7].
In summary, Zhang discloses a method in which a sample containing nucleic acids from Psilocybe tissue was contacted with primers for amplifying Psilocybe target nucleic acid sequences, wherein the sequences were amplified when present, and wherein the amplification product was detected. However, Zhang does not disclose the inclusion of primers which would amplify a bacterial target nucleic acid sequence.
Abbas, as part of an advisory board for the state of Oregon, reviewed current literature regarding the use of Psilocybin in medicine and issued recommendations regarding the establishment of rules and regulations for legal use of the drug [Abbas, p4]. They found that psilocybin is efficacious in reducing depression and anxiety, increased spiritual well-being, and that study participants largely rated their experiences with psilocybin as highly meaningful. While the published clinical studies examined administered biosynthesized psilocybin, consumption of actual Psilocybe mushrooms, particularly Psilocybe cubensis, is the dominant method of administration in traditional and unsupervised settings and psilocybin and psilocin concentrations vary widely in dried mushrooms.
Abbas found that established techniques exist for analyzing commercial products for psilocybin presence, as well as for determining the concentration of psilocybin and contaminants in said products [Abbas, p3]. Accurate mushroom identification is vital as misidentification of visually similar fungi has several consequences, including death, gastrointestinal distresses, liver and kidney damage, and central nervous system malfunction. Even if appropriately identified, the presence of contamination is still a concern. Psilocybin can be obtained through cultivation of mushrooms/hyphae/sclerotia which can be consumed whole or subjected to laboratory extraction methods, artificial production via cell culture, or in vitro chemical biosynthesis. Each of these methods introduce their own risks, including potential carryover of harmful chemicals and solvents and the presence of pathogenic microbes (bacteria, viruses, parasites, fungi) from cultivation substrates. Therefore, in order to ensure consumer safety, detection of these contaminants is required [Abbas, p13-14].
As a result of their findings, Abbas issued eleven recommendations, three of which pertain to the aforementioned issues [Abbas, p17-18]:
Recommendation 8: Oregon Health Authority (OHA) should consider the range of research on cultivating and characterizing psilocybin-containing mushrooms (e.g., genotyping to confirm identity, methods for measuring psilocybin concentration) in developing a regulatory framework.
Recommendation 10: OHA should explore feasibility and capacity of employing modern DNA sequencing-based techniques to identify fungi and fungal tissues for use in production licensing and quality control.
Recommendation 11: OHA should facilitate the development of screening requirements for possible mushroom contaminants. These may include the following:
Residual solvents and/or disinfectants used in the extraction or sterilization processes
Toxic metals, pesticides, antibiotics, herbicides, livestock medications, and other potential bioaccumulation contaminants from growth substrates or direct application
Pathogenic microbes (bacteria, viruses, parasites, other fungi) and microbially produced toxins
The findings and recommendations of Abbas represent clear motivation for the development of testing methodologies capable of accurately distinguishing psilocybin-producing fungi from other potentially deadly fungi and detecting the presence of pathogenic microbes which may be present as a result of mushroom cultivation practices.
Savelkoul establishes that multiplex real-time PCR assays employing target-specific primers are capable of simultaneously detecting both fungal and bacterial nucleic acid targets within a single reaction. Zhang further demonstrates that primer sets designed to specifically target Psilocybe nucleic acid sequences are known in the art and capable of being incorporated into sensitive and specific multiplex PCR assays. Therefore, a person of ordinary skill in the art prior to the effective filing date of the claimed invention, would have been motivated to modify the multiplex real-time PCR assay of Savelkoul by incorporating at least one of the Psilocybe-specific primer sets taught by Zhang resulting in a multiplex assay capable of simultaneously detecting Psilocybe and bacterial target nucleic acid sequences in order to achieve the quality control goals of Abbas. Similarly, a kit adapted for performing the method of Savelkoul and Zhang would comprise primers and probes specific for both Psilocybe and bacterial nucleic acid sequences. A rationale to support a conclusion that a claim would have been obvious is that all the claimed elements were known in the prior art and one skilled in the art could have combined the elements as claimed by known methods with no change in their respective functions, and the combination would have yielded nothing more than predictable results to one of ordinary skill in the art. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395 (2007) (see MPEP §§ 2143, A. and 2143.02).
Regarding claims 5 and 6, the samples analyzed by Zhang originated from 22 different species including Psilocybe cubensis [Zhang, p2].
Regarding claim 7, Zhang designed primers to target P. cubensis, a fungus [Zhang, p3]. Savelkoul also discloses using target specific primers to target different fungal species (e.g., Aspergillus spp., Candida spp. and Candida glabrata in the first multiplex reaction as described above) [Savelkoul, 0027].
Regarding claim 8, Savelkoul designed primers to target the 18S-28S rRNA ITS regions of Aspergillus spp., Candida spp. and Candida glabrata in the first multiplex reaction as described above [Savelkoul, 0034, 0037, 0038].
Regarding claims 9, 10, 11, 13, and 14, Savelkoul states a “real-time PCR reaction” refers to a PCR amplification reaction in which a labeled probe or dye is added to generate a signal, the intensity of which increased as the amount of product increases. Detection of this signal in real-time allows for quantification of the starting material [0078]. Savelkoul explicitly describes using their method to quantitatively determine the amount of one or more of the targeted microorganisms in a sample [Savelkoul, 0193]. As the combined method of Savelkoul and Zhang involves real-time PCR analysis of samples using primers targeting Psilocybe and bacterial DNA in order to access the identity and purity of a sample, one of ordinary skill in the art would understand that positive quantification of said targets would equate to species confirmation and the detection of bacterial contamination, respectively.
Regarding claim 12 and 33, as previously mentioned, Savelkoul designed primers to target different fungal species which were then quantitated via real-time PCR [Savelkoul, 0034, 0037, 0038, 0078, 0193]. The inclusion of said fungal target in the combined method of Savelkoul and Zhang would have been of interest to the skilled artisan as Abbas explicitly discloses fungal contamination as one of their concerns [Abbas, p18]. Therefore, a skilled artisan would have understood that positive quantification of a fungal target would equate to the detection of fungal contamination. Similarly, a kit adapted for performing the method of Savelkoul and Zhang, wherein a fungal target is amplified and detected, would comprise primers and probes specific for that fungal target.
Claims 2-4 and 30 are rejected under 35 U.S.C. 103 as being unpatentable over Savelkoul, Zhang, and Abbas, as applied to claims 1 and 27 above, and further in view of Fricke [Fricke J et al. Angewandte Chemie International Edition. 2017 Sep 25;56(40):12352-5].
Savelkoul, Zhang, and Abbas are applied to the relevant teachings of claims 1 and 27 as discussed above and are incorporated herein by reference. Savelkoul further discloses targeting the 16S rDNA gene of both gram positive and gram negative bacteria in the first multiplex reaction of the previously discussed embodiment as well as the gadA and/or gadB gene of E. coli in the second multiplex reaction of the same embodiment [Savelkoul, 0028, 0035-0036]. Additional embodiments in which the 16S rRNA gene of E. coli is targeted in a multiplex reaction are also disclosed [Savelkoul, 0023].
However, none of the aforementioned references disclose using PsiK, PsiM, PsiH, or PsiD nucleic acid sequences as the Psilocybe target.
Fricke investigated the biosynthesis of Psilocybin and characterized four enzymes which play roles in this process: an L-tryptophan decarboxylase, a kinase, an S-adenosyl-L-methionine-dependent N-methyltransferase, and a monooxygenase. By sequencing the genomes of P. cubensis and P. cyanescens, a locus was identified that included genes for each of these enzymes, wherein the genes are referred to as psiD, psiK, psiM, and psiH, respectively [Fricke, abstract, p12352]. Further investigation into the pathway demonstrated that only PsiD, PsiK, and PsiM are required for in vitro psilocybin synthesis, although PsiH can play an ancillary role. This discovery laid the foundation for the production of psilocybin using engineered microbial hosts for pharmaceutical purposes [Fricke, abstract, p12352, 12355].
Fricke’s work demonstrates that the psiD, psiK, psiM, and psiH genes are directly associated with the production of psilocybin, providing biologically relevant and highly specific molecular targets for distinguishing psilocybin-producing organisms from non-producing organisms. Therefore, a person of ordinary skill in the art prior to the effective filing date of the claimed invention, would have been motivated to modify the combined method of Savelkoul and Zhang to target one or more of the PsiK, PsiM, PsiH, or PsiD nucleic acid sequences identified by Fricke as the Psilocybe target nucleic acid sequence. This modification represents the simple substitution of one known Psilocybe-specific target sequence for another known Psilocybe-specific target sequence to achieve predictable results and would have been obvious to try.
The Supreme Court decided that a claim can be proved obvious merely by showing that the combination of known elements was obvious to try. In this regard, the Supreme Court explained that, “[w]hen there is a design need or market pressure to solve a problem and there are a finite number of identified, predictable solutions, a person of ordinary skill in the art has a good reason to pursue the known options within his or her technical grasp.” An obviousness determination is not the result of a rigid formula disassociated from the consideration of the facts of the case. Indeed, the common sense of those skilled in the art demonstrates why some combinations would have been obvious where others would not. Therefore, choosing from a finite number of identified, predictable solutions, with a reasonable expectation for success, is likely to be obvious to a person if ordinary skill in the art. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, E.).
Finally, a kit adapted for performing the method of Savelkoul and Zhang, in which a bacterial nucleic acid sequence and at least one nucleic acid sequence specific for PsiD, PsiK, PsiM, or PsiH are targeted, would comprise primers and probes specific for those targets.
Conclusion
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/K.N.K./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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