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 .
Objections to the Specification
The use of multiple trade names or marks used in commerce, such as “SureSelectXT” on page 28 of the specification, has been noted in this application. The terms should be accompanied by the generic terminology. Furthermore, the terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM, or ® following the terms.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
The lengthy specification has not been checked to the extent necessary to determine the presence of all possible minor errors. Applicant’s cooperation is requested in correcting any errors of which applicant may become aware in the specification.
Objections to the Claims
Applicant is advised that should claim 129 be found allowable, claim 132 will be objected to under 37 CFR 1.75 as being a substantial duplicate thereof. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m). While the term “microarray” is explicitly defined in the specification to mean “a collection of nucleic acid molecules or polypeptides from one or more organisms arranged on a solid support”, the term “array” is not explicitly defined, and can be broadly construed to simply mean “a collection” or “panel” of probes.
The following claims are objected to because of the informalities described below:
Claim 110, last line: “gene cluster” should be “cluster” for consistency with the remainder of the claim, as well as claim 125.
Claim 116: “the sample” should be “the complex biological sample” for consistency with the remainder of the claims.
Claim 122: each of the strains presented as abbreviations should be spelled out in full at the first recitation.
Claim 128, last line of (a): the second recitation of organism should be plural.
Appropriate correction is required.
Claim Interpretation
Based upon the Examiner’s understanding of the following claims in light of the accompanying rejections under 112(b) and/or 112(d), for the purposes of compact prosecution they are being construed as follows:
Claim 111 will be assumed to depend from claim 110 (i.e., “the set of probes generated in claim 110”).
Claim 114 will be assumed to depend from claim 111.
Claims 117-122, and 125 will be assumed to depend from, or ultimately depend from, claim 110.
Claim 124 will be interpretated to mean that the polynucleotide-probe complexes result in an unbiased representation of the sequence diversity of the targeted organisms in the complex biological sample.
Additionally:
Claim 128 (b) is being interpreted to require only that a plurality of gene clusters is present across all orthogroup clusters, not that each orthogroup cluster is comprised of a plurality of gene clusters.
Claims 131 and 132 are product-by-process claims. As such, for claim 131, any set of enriched polynucleotide sequences, regardless of enrichment method, can be construed as anticipating the enriched polynucleotide sequences of the instant claim. Similarly, for claim 132, any panel of probes, regardless of manufacturing method, can be construed as anticipating the panel of probes of the instant claim. "[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985) (citations omitted). See MPEP 2113.I.
Claim 132 “a panel of probes” is being interpreted as any configuration of probes regardless of immobilization status.
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 110-133 are rejected under 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, 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.
Claims 114, 117-119, 121, 122, 125, 126, and 128 recite the following limitations for which there is insufficient antecedent basis:
Claim 114: “the complex biological sample”, “the enriched polynucleotides”, and “the organism of interest”.
Claim 117: “the set of genomes”
Claim 118: “the set of genomes”
Claim 119: “the organisms”
Claim 121: “the organisms”
Claim 122: “the organisms”
Claim 125: “the clusters”
Claim 126: “the subject” and “the organisms”
Claim 112 contains the trademark/trade name “FACS” and “NanoString”. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or pre-AIA 35 U.S.C. 112, second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a characterization method and, accordingly, the identification/description is indefinite.
The following claims have additional issues of indefiniteness:
In Claim 110, the recitation of “the set” in the first line of (b) is indefinite with regards to which set it is referring to, the “set of genomes” in the first line of (a) or the “set of probes” in the preamble and first line of (b).
In claim 128, (c), line 1 and 4, recitations of “the set” suffer from the same indefiniteness.
Claims 111 and 113 are indefinite as they both require the use of the probe set of claim 110 (for claim 111, more specifically, “the set of probes generated in claim 111”, which is presumed to have intended to say “the set of probes generated in claim 110”). However, claim 110 is directed towards a method, not a product or composition. As such, it is unclear if claims 111 and 113 require performing the method of claim 110, or merely require using probes that could be generated using the method of claim 110 even if such probes were generated by a substantially different method.
While claim 129 is directed towards a polynucleotide array, it similarly requires the probes of claim 110 and suffers the same indefinite issues of claims 111 and 113.
Claim 124 is indefinite because it is unclear how the method of claim 111 (from which claim 124 ultimately depends) results in a “non-biased representation of sequence diversity in the complex biological sample.” This would imply that all sequences present in the sample are equally represented by the polynucleotide-probe complexes. However, the purpose of the method is to enrich a subset of the sequences present in the sample through the use of targeted probes which inherently introduces bias for the targeted sequences and against the non-targeted sequences.
Claims rejected under 35 USC § 112(b) for which no explicit rationale is provided are rejected by virtue of their dependency upon an explicitly rejected claim.
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 111, 112, 114-127 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.
Claim 111, as currently presented, requires the use of “probes generated in claim 111.” As such, claim 111 depends from itself and cannot further limit itself.
Claims 114, 117-122, 125-127 do not further limit the subject matter of the claims upon which they depend by expanding their scopes through the introduction of unsupported limitations.
Claims rejected under 35 USC § 112(d) for which no explicit rationale is provided are rejected by virtue of their dependency upon an explicitly rejected claim.
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 § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 129-133 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a natural phenomenon (including a product of nature) without significantly more.
Subject Matter Eligibility Test (see MPEP § 2106):
Step 1: Are the claims directed towards a process, machine, manufacture, or composition of matter?
Yes. The claims are directed towards a manufacture and/or composition of matter:
Claim 129: “A polynucleotide array…”
Claim 130: “A set of probes…”
Claim 131: “A set of enriched polynucleotide sequences…”
Claims 132 and 133: “A panel of probes…”
Step 2A: Are the claims directed to a judicial exception?
Prong 1: Do the claims recite an abstract idea, law of nature, or natural phenomenon?
The array/probe set/probe panel of claims 129, 130, 132, and 133 are comprised of probes which are at least 80% complementary to target sequences present in the genomes of a set of organisms. This includes probes which are 100% complementary to the targeted sequences and thus would be indistinguishable from fragments of the naturally occurring complementary strands of said targets. Therefore, these claims recite a natural phenomenon (i.e., a product of nature).
The enriched polynucleotide sequences of claim 131 refer to the nucleic acids already present in a sample which were targeted by the aforementioned probes and thus enriched. No structural modifications occurred to the polynucleotide sequences in the course of enrichment and thus this claim recites a natural phenomenon (i.e., a product of nature).
Prong 2: Do the claim recite additional elements that integrate the judicial exception into a practical application?
Claim 129 includes a limitation of an array. However, the term “array” is not explicitly defined in the specification (as opposed to the term “microarray”), and would therefore encompass a mere “set” or “collection” of the probes.
Claim 130 includes additional limitations which dictate the makeup of the probes. However, these limitations require that the probes share at least 80% sequence identity across at least 65 nucleotides to a sequence selected from SEQ ID Nos: 1 to 892,415. These limitations do no more than dictate the targets of the probes and is considered insignificant extra-solution activity which is merely a nominal or tangential addition to the claim. Additionally, a search of SEQ ID NO: 1 returned a 100% match to a portion of the E. coli genome (Loukiadis et al. J Bacteriol. 2008 Jan; 190(1):275-85). As such, these limitations do not meaningfully alter the probes in a way that would differentiate them from the natural product they are targeting. Therefore, the judicial exception is not integrated into a practical application.
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Claims 131 and 132 do not recite any additional elements; therefore, the judicial exception is not integrated into a practical application.
Claim 133 includes a limitation that each probe comprises a unique molecular identifier, a barcode, a detectable moiety, and/or a binding member. However, any of “a unique molecular identifier”, “a barcode”, “a detectable moiety” and “a binding member” can simply be a nucleotide sequence, which would encompass any naturally occurring adjacent nucleotide sequence found in the genome from which the probes were derived.
Step 2B: Do the claims recite additional elements that amount to significantly more than the judicial exception?
As discussed in step 2A prong 2, claims 131 and 132 do not recite any additional elements.
The additional limitations of claims 129, 130, and 133 amount to insignificant extra-solution activity and/or merely linking a judicial exception to a particular technological environment. They further represent well-understood and conventional activity in the field as evidenced by Metsky (cited below) who generated probes capable for use as an array, coupled said probes to binding members, and further indicated benefits of including a unique molecular identifier in the probes.
Therefore, as the inclusion of these additional elements of claims 129-133 do not amount to significantly more than the judicial exception, these claims do not contain eligible subject matter.
35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim 110 is rejected under35 U.S.C. 102(a)(2) as being anticipated by Bergeron (US 2003/0049636 A1).
Bergeron disclosed a method for determining the presence and/or amount of nucleic acids from any bacterial species using probes and/or amplification primers which are hybridizable to selected target regions within the bacterial nucleic acids [Bergeron, 0025-0030]. First, all sequence data for the targeted organisms were analyzed to determine which gene sequences offered the most diagnostic utility based upon sequence information and comparison with the corresponding gene in non-targeted organisms [Bergeron, 0049]. Based upon this analysis, sequences were selected which was specific and ubiquitous for the targeted organisms [Bergeron, 0049]. Probes/primers were evaluated for their ability to hybridize to the selected sequences and then synthesized. The probes/primers may be of any suitable length, however, using single-stranded oligonucleotides which are less than 100 nucleotides in length is advantageous due the ease of synthesis in large quantities, batch to batch consistency, and chemical stability. Furthermore, the probes/primers may be selected from anywhere within database sequences suitable for organism detection. Bergeron also acknowledges strain-level variation can occur within the same species which can be detected by sequencing the PCR product that results from amplification using the selected primers [Bergeron, 0052-0054, 0080].
As an example, Bergeron determined the sequences of a portion of the tuf gene from a variety of bacterial and fungal species. Alignment showed that the length of the sequenced portion varied and allowed for the selection of PCR primers which genera-specific by illuminating which sequences were conserved within the target genus but which discriminated sequences from other closely related genera [Bergeron, 0102]. Probe sequences were similarly selected for their ability to hybridize to the amplification products of the selected primers and tested for specificity and ubiquity. Probes were determined to be specific if they hybridized only to their targeted organisms which also indicated a high degree of sequence similarity between the probe and the target due to the stringency of the hybridization conditions used by Bergeron. Probes were determined to be ubiquitous if they hybridized to at least 80% of all tested DNA from the targeted organisms [Bergeron, 0081-0084].
By following Bergeron’s method, gene sequences from multiple organisms which share regions of 100% sequence identity would be clustered. Probes of any length could then be developed to target these shared regions with high hybridization stringency so that the probes are capable of hybridizing to at least 80% of all the shared sequences.
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 110-115, 117-119, 123-126, 128-133 are rejected under 35 U.S.C. 103 as being unpatentable over Metsky (Metsky HC et al. Nature biotechnology. 2019 Feb;37(2):160-8) in view of Dickson (Dickson ZW et al. Cell Press Sneak Peek. 2020 Dec 23; 32pages. Available at SSRN: https://ssrn.com/abstract=3754546).
Regarding claims 110 and 128, Metsky introduced a novel computational method which designs optimal probes sets of a specified number capable of achieving full coverage of known sequence diversity in an attempt to enhance nucleic acid capture for enrichment of diverse microbial taxa during metagenomic sequencing [Metsky, abstract]. This method, called CATCH or compact aggregation of targets for comprehensive hybridization, is both scalable and capable of designing probes for any collection of target sequences [Metsky, p161].
To design the probes, any collection of sequences a user wishes to target is inputted into CATCH. CATCH then searches for an optional probe set given a desired number of oligonucleotides to output and incorporates into this search various parameters that govern hybridization, such as sequence complementarity between probe and target. CATCH offers additional flexibility in probe design by allowing the user to customize the hybridization model used, specify a fraction of each target genome to capture, specify targets of the genome which would distinguish similar but distinct subtypes, or blacklist specific sequences, such as highly abundant rRNA sequences, to prevent capture by the probes [Metsky, p161].
Below is a list of the parameters which are customizable by the user [Metsky, GitHub]:
Probe length (Default: 100 nucleotides)
Probe stride (Default: 50 nucleotides)
Mismatches (Default: 0):
How many mismatches are tolerated when determining if a probe covers a target sequence. Higher values lead to fewer probes.
“-l/--lcf -thres” (Default: probe length)
Requires that there be no more than a specified number of mismatches within a specified length between a portion of the probe and a portion of the target.
“--island-of-exact-match”
Requires that there be an exact match of a specified length between a portion of the probe and a portion of the target.
Coverage (Default: 1.0 – i.e., whole genome)
Represents the fraction of a genome, or the number of nucleotides, which is captured by the probes.
Cover Extension (Default: 0):
The number of nucleotides on each side of the target region which is captured by the probe. Accounts for the fact that library fragments are generally longer than the capture probes. Higher values lead to few probes, whereas lower values are more stringent in modeling capture.
Identify:
Probes for an inputted dataset are designed so that they are unlikely to capture another inputted dataset. Typically used with small coverage values.
Blacklist genomes dataset:
Allows the user to input a dataset of genomes they do not want the probes to capture.
Within this function, the number of mismatches tolerated, the length of complementarity/maximum allowed similarity tolerated, and the island of exact match tolerated can be customized.
The following is an example input provided by Metsky which would result in CATCH designing probes that:
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Therefore, Metsky clearly teaches that probe length, probe coverage, length of a fully complementary hybridization site between probe and target, the exclusion of probes which could have contiguous sequences identical to a set of reference sequences, and cover extension are result effective parameters that a person having ordinary skill in the art would routinely optimize. It would have been customary for an artisan of ordinary skill to determine the specifics of each parameter to obtain a probe set suitable for their specific use. “[W]here the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation,” (In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955)). See MPEP § 2144.05.
Metsky does not teach identifying clustering gene sequences from a set of genomes first by orthogroup and then by gene with at least 80% shared sequence identity prior to probe set generation.
Dickson describes how the advent of next generation sequencing has allowed for rapid identification of organisms and characterization of diverse nucleic acids within heterogenous samples. However, sequencing rare organisms remain a challenge due to the masking effects of host or environmental DNA as well as the difficultly in discerning true low-level signals from contaminants [Dickson, p3]. Similar to Metsky, Dickson presents targeted enrichment through the use of oligonucleotide probes as a solution to this problem in a novel computational method called the HUBDesign pipeline [Dickson, abstract]. Dickson acknowledges the similarities between their method and Metsky’s method and points out specific differences, such as CATCH’s large computational requirements (i.e., computer memory) and long run-times when applied to larger genomes [Dickson, p12]. Dickson attributes this difference to their method’s consideration of sequence homology of related organisms, which allows for design of more efficient probes capable of specifically and simultaneously capturing known and novel members of a group of organisms, while also reducing the overall computational effort of the probe design process [Dickson, p4-5].
Dickson begins by grouping homologous genes which are at most 15% divergent (i.e., more than 85% similar) from multiple annotated genomes into gene families. These genes are aligned and a phylogenic tree is generated. Specific nodes are selected based off of predetermined criteria and a representative consensus sequence is generated for each cluster of descendants. Therefore, these representative sequences represent a group of organisms who share a common ancestor, consistent with the applicant’s definition of an orthogroup. All representative sequences for a given node are then combined to form a pseudo-genome for that organism. Dickson’s figure 1 (replicated below) describes this process, showing the plurality of genes identified across the plurality of orthogroups. These pseudo-genomes are inputted into a program to perform the next stages of probe generation.
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Therefore, one of ordinary skill in the art prior to the effective filling date of the claimed invention and looking to reduce the computational burden of Metsky’s CATCH probe design while maintaining the ability to reliably and specifically enrich polynucleotides from a targeted set of organisms, would be motivated to first perform the clustering process of Dickson. As Dickson’s clustering process outputs a set of pseudo-genomes and CATCH is capable of designing probes for any collection of sequences, the systems are clearly compatible with one another. The use of a known technique to improve similar devices (methods or products) in the same way is likely to be obvious. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, C.).
Finally, while the disclosures of Metsky and Dickson largely pertain to the enrichment of viral genomes, both indicate their suitability for a wider range on sequence inputs. Metsky indicates that CATCH is well suited to design probes which target any microbes that have a high degree of diversity, including bacteria [Metsky, p167] whereas Dickson’s HUBDesign pipeline was actually tested against four different input sets which separately contained the genomes of coronaviruses (56), respiratory viruses (110), sepsis bacteria (1926), and gut bacteria (1473), thereby explicitly showing its ability to be used against non-viral organisms [Dickson, p12, Table 5].
Regarding claims 111, 112, and 113, Metsky used CATCH to design, synthesize, and validate multiple probe sets. Included in this process was a method in which 356 viral species were targeted [Metsky, abstract]. Human patient and environmental samples were collected which encompassed a range of source materials including plasma, serum, buccal swabs, urine, avian swabs, and mosquito pools [Metsky, p181-182]. These samples were treated to remove contaminating DNA followed by synthesis of double stranded cDNA and library preparation. In-solution hybridization of the prepared libraries was performed using biotinylated ssDNA probes designed by the CATCH method and synthesized using the SeqCap Ez Developer platform. As biotin is identified by the Applicant as a suitable binding member, the hybridization complex that results from the capture of the prepared libraries would be equivalent to the polynucleotide/probe complex described by the Applicant. These hybridization complexes were then captured on streptavidin beads, which are equivalent to the capture molecule fixed to a solid support as defined by the Applicant, resulting in the enrichment of the targeted viral content in the samples. Finally, the samples were amplified and sequenced on either the Illumina MiSeq or HiSeq 2500 platforms [Metsky, PDF p11].
Regarding claim 114, Metsky states that they explored the impact of capture on the complete metagenomic diversity within each sample. While metagenomic sequencing will generate reads from both the host genome and background contaminants, they found that the fraction of human and non-targeted taxa sequences in patient samples were decreased while the fraction of targeted viral sequences was enriched [Metsky, p163].
Regarding claim 115, as previously discussed, Metsky’s samples included body fluid and avian samples (i.e., biological samples) as well as mosquito pools (i.e., environmental samples) [Metsky, p161-162].
Regarding claim 117, Metsky developed probes in which the whole genomes of 356 viral species were targeted [Metsky, abstract].
Regarding claim 118, Metsky provides the accession numbers for the viral genomes used in the design of the targeted probe sets in Supplementary Table 2, which shows the genomes of multiple strains for a single species were used (e.g., MUV: MF965227, MF965277, MF965239, MF965226).
Regarding claim 119, Metsky states that CATCH can be used to quickly design focused probe sets providing flexibility for targeting pathogens associated with specific clinical syndromes or for outbreak response, as they did when analyzing patient samples from a Lassa outbreak in Nigeria [Metsky, p165-166].
Regarding claim 123, Metsky observed a median enrichment of 18x [Metsky, abstract].
Regarding claim 124, Metsky states that the probes designed by CATCH enable more efficient and sensitive sequencing that is only biased by the extent of known diversity, which would mean that their method of probe design and enrichment does not introduce any bias itself [Metsky, p166]. As such, the polynucleotide-probe complexes which result from enrichment by these methods would represent an un-biased representation of sequence diversity of the targeted viral genomes in the complex biological sample.
Regarding claim 125, CATCH allows the user to determine the number of mismatches allowed between a probe and its target sequence [Metsky, Figure 1]. This allows for any amount of shared sequence identity between the probe and its target, including 90%.
Regarding claim 126, Metsky analyzed 23 samples from individuals who were clinically confirmed to have Lassa fever with their method/probes [Metsky, p165].
Regarding claims 129 and 132, as proof of concept, Metsky designed a probe set to target all viral species reported to infect humans (VALL), as well as three focused probe sets for commonly co-circulating viral infection: measles and mumps viruses (VMM), Zika and chikungunya viruses (VZC), and a panel of 23 species circulating in West Africa (VWAFR) [Metsky, p161]. The VALL probes were synthesized on an array using the SeqCap EZ Developer platform from Roche. The focused probe sets were synthesized on a 12k or 90k array [Metsky, PDF p11].
Regarding claim 130, the method of claim 111 requires probes generated by the method of claim 110. Therefore, the probes generated by the combined methods of Metsky and Dickson, as previously described in the rejection of claim 110, which are capable of use in the virus detection method of Metsky, as previously described in the claim 111 rejection, could be considered equivalent to the probes of claim 130. As established in the claim 110 rejection, probe length and shared sequence identity to a target (i.e., allowed number of mismatches) are user-defined inputs and considered to be result effective parameters that a person having ordinary skill in the art would routinely optimize. As CATCH allows for the input of any collection of sequences a user wishes to target, the skilled artisan would be able to input SEQ ID NOs: 1 to 892,415 into CATCH and create 65nt-long probes which share at least 80% sequence identity to the inputted sequences thereby arriving at the probe set of claim 130.
Regarding claim 131, Metsky performed in-solution hybridization and capture of targeted viral capture, resulting in a set of enriched polynucleotides sequences [Metsky, PDF p11].
Regarding claim 133, Metsky teaches that the inclusion of unique molecular identifies could improve determination of unique fragments. Additionally, all of Metsky’s probes were biotinylated; biotin is suggested by the Applicant as a suitable binding member [Metsky, p166, PDF p11].
Claims 120 and 127 are rejected under 35 U.S.C. 103 as being unpatentable over Metsky and Dickson, as applied to claims 110, 111, and 119 above, and further in view of van Dijk (van Dijk et al. bioRxiv. 2021 May 29; 32pages. Available at https://doi.org/10.1101/2021.02.14.431013).
Metsky and Dickson are applied to the relevant teachings of claims 110, 111, and 119 as discussed previously and are incorporated herein by reference. While Metsky and Dickson both disclose the suitability of their systems for developing probes for bacteria genomes, only Dickson actually performed this testing and did not disclose the included bacterial genera or species. As such, while there is a clear capability, neither Metsky or Dickson disclose the application of CATCH or HUBDesign to probe generation and subsequent polynucleotide enrichment of the genomes of the organisms specified in claim 120. Furthermore, neither disclose performance of the enrichment method on samples collected from individuals suffering from a chronic or recurring infection associated with a targeted organism as recited in claim 127.
van Dijk teaches that study of strain-level variation of complex community samples has been largely unexplored due to analytical difficulties. Culture-based approaches are limited in their ability to accurately represent the true diversity of species present in cultures. While whole metagenomic shotgun sequencing approaches offer a solution to this problem, they suffer from their own limitations. Namely, sequencing mixed-strain samples require analytical tools capable of deconvoluting the sequence results between the different strains. However, the tools available often struggle to distinguish similar strains at the low coverages at which clinically relevant organisms in metagenomic samples are typically found, such as Escherichia coli in the human gut. The Strain Genome Explorer (StrainGE) toolkit is presented as a solution enabling deconvolution of stain mixtures comprised of low abundance strains and characterization of said strains across samples [van Dijk, p3]. While designed to be broadly applicable across different bacterial genera and species, E. coli was used to test the toolkit because it represents a clinically relevant challenge as it is an opportunistic pathogen often found at low relative abundance in diverse strain mixtures in human guts [van Dijk, p5-6].
Before testing, a reference database for E. coli was developed containing 361 complete genomes representing all eight phylogroups (A, B1, B2, C, D, E, F, and G) and listed in Supplemental Table 2 [van Dijk, p6]. Once the reference database was complied, StrainGE was tested against multiple datasets. In one test, metagenomic data obtained from longitudinal stool samples from a woman with recurrent urinary tract infections (UTIs) was examined and five distinct strains were identified, including one which persisted across all samples suggesting that its persistence in the gut may be an important mechanism for UTI recurrence [van Dijk, p14].
The teachings of Metsky/Dickson and van Dijk represent different methods to address the same problem, namely the difficulty that arises in attempting to detect strain-level variation in complex biological samples in which some strains are present in low abundance. While Metsky/Dickson approach this problem from a biological standpoint (i.e., developing probes to enrich the sample for the target DNA prior to sequencing), van Dijk uses a computational approach (i.e., a method of analyzing sequencing data). van Dijk further provides motivation for targeting E. coli strains specifically, teaching the historical difficulties with strain-level analysis of this species and the clinical benefit that could arise from being able to do so. As the methods of Metsky/Dickson are suitable for use with bacterial genomes, one of ordinary skill in the art prior to the effective filling date of the claimed invention, would recognize the compatibility of these teachings and be motivated to combine them resulting in a method in which a sample is enriched for targeted organisms (i.e., E. coli) via the method of Metsky/Dickson and the resultant sequencing data analyzed by the method of van Dijk thereby maximizing the strain information obtainable from a complex sample. The combination of familiar elements is likely to be obvious when it does no more than yield predictable results. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, 82 USPQ2d 1385, 1395 – 97 (2007) (see MPEP § 2143, A.).
Claim 122 is rejected under 35 U.S.C. 103 as being unpatentable over Metsky, Dickson and van Dijk, as applied to claim 120 above, and as evidenced by Croxen (Croxen MA et al. Clinical microbiology reviews. 2013 Oct;26(4):822-80).
Metsky, Dickson, and van Dijk are applied to the relevant teachings of claim 120 as discussed previously and are incorporated herein by reference. None disclose using genomes from a strain of E. coli as specified in claim 122. However, the van Dijk taught that their reference database represented all eight phylogroups of E. coli (A, B1, B2, C, D, E, F, and G) and listed the specific strains in Supplemental Table 2 [van Dijk, p6].
Croxen teaches other methods of E. coli classification including by pathotype (i.e., tEPEC, aEPEC, STEC, EIEC/Shigella, EAEC, ETEC, DAEC, and AIEC) [Croxen, Table 1]. Croxen further shows in their Figure 3 the phylogenetic tree of a selection of E. coli strains and indicates their respective pathotype to show that pathotypes do not always group together in the same phylogroup. A comparison of Croxen’s Figure 3 with the genomes listed in van Dijk’s Supplemental Table 2 provides the pathotype for a subset of the strains used by van Dijk in this reference database: EAEC (55989 and 042), STEC (12009), ETEC (E24377A and UMNK88), AIEC (UM146 and LF82), and EPEC (E2348_69).
Claim 116 is rejected under 35 U.S.C. 103 as being unpatentable over Metsky, Dickson and van Dijk, as applied to claim 120 above, and further in view of Werneburg (Werneburg GT et al. Journal of Urology. 2020 Feb 1; 203(2):357-64).
Metsky and Dickson are applied to the relevant teachings of claims 111 and 115 as discussed previously and are incorporated herein by reference. However, neither teach that the sample in which polynucleotides are enriched is collected from the surface of a medical device.
Werneburg teaches that approximately 80% of urinary tract infections are associated with indwelling urethral catheters leading to an increased morbidity and mortality. The formation of bacterial biofilms leads to a survival advantage for pathogenic bacteria resulting in persistent and antibiotic-resistant infections. Werneburg hypothesized that the surface of catheters would be rapidly colonized and used next generation sequencing to characterize the multispecies biofilms present on their surface [Werneburg, p357-358]. They found that all of the catheter samples comprised uropathogenic bacteria, half of which contained antibiotic resistance genes. This characterization may allow for improved antimicrobial strategies for catheters, maximizing efficacy, minimizing cost, and ultimately resulting in improved patient outcomes [Werneburg, p362].
As Werneburg provides express motivation for the accurate characterization of biofilm bacteria present on the surface of catheters, a person of ordinary skill in the art prior to the effective filling date of the claimed invention, would have been motivated to use the polynucleotide enrichment method of Metsky/Dickson on samples obtained from catheters to obtain more accurate strain-level data allowing for more informed decisions as it relates to antimicrobial strategies.
Claim 121 is rejected under 35 U.S.C. 103 as being unpatentable over Metsky and Dickson, as applied to claim 110 above, and further in view of Kameoka (Kameoka S et al. BMC genomics. 2021 Jul 10;22(1):527).
Metsky and Dickson are applied to the relevant teachings of claim 110 as discussed previously and are incorporated herein by reference. However, neither teach that the targeted organisms comprise a species belonging to the genus Akkermansia and/or Bifidobacterium.
Kameoka compared fecal 16S rRNA sequencing data obtained by using two different primer sets in order to obtain an optimized gut microbiota analysis protocol, finding that one of the primer sets resulted in inaccurate abundance determinations when compared to the results of qPCR assays, specifically for Bifidobacterium and Akkermansia [Kameoka, abstract]. Accurate detection of Akkermansia, which is present in low abundance in the human gut, is particularly important as it is considered to be an important health indicator due to its associations with obesity and diabetes [Kameoka, p7]. As such, Kameoka’s work suggests an overall desire within the art to more accurately and sensitively detect specific bacteria present in the human gut. Therefore, a person of ordinary skill in the art prior to the effective filling date of the claimed invention, would have been motivated to design probes for the enrichment of these gut bacteria in complex samples in order to obtain more accurate information regarding the bacterial composition of the gut microbiome, particularly as it relates to Akkermansia.
Conclusion
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/K.N.K./Examiner, Art Unit 1681
/SAMUEL C WOOLWINE/Primary Examiner, Art Unit 1681
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