DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group I, a method for detecting a fusion gene in a sample from a subject, in the reply filed on 11/24/25 is acknowledged.
Claims 1-20 are pending. Claims 19-20 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 11/24/25.
Claims 1-18 are currently under examination.
Priority
The instant application 18/183,818 filed on 3/14/23 claims domestic priority to provisional applications 63/356,901 filed on 6/29/22; 63/348,947 filed on 6/3/22; and 63/319,521 filed on 3/14/22. The priority date is determined to be 3/14/22.
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 14 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.
Claim 14 recitation of “nucleotide analogue” in lines 3-4 is vague and indefinite because it is unclear how far one can deviate from the parent molecule without the “analogue” being so far removed therefrom as to be a completely different molecule. Regarding the limitation “analogue”, it is noted that the spontaneously occurring deamination of a methylated cytosine in a 5’-CpG-3’ site results in a thymine, and cytosine (C) and thymine (T) are two totally different pyrimidine bases of DNA. Although there are examples in the specification of derivatives – see paragraph 0050 – these are not limiting and the specification fails to provide a limiting definition of the above phrase.
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
Claims 1-17 are rejected under 35 U.S.C. 103 as being unpatentable over Weng et al. (2020; US 2020/0080141 A1).
Relevant to claim 1, Weng et al. teaches "Recognized herein is a need for alternative and/or robust methods and compositions of detecting rare sequence variations, particularly rare sequence changes and gene fusion events. The compositions and methods of the present disclosure address this need, and provide additional advantages as well" (paragraph 0007).
This teaching reads on claim 1 A method for detecting a fusion gene in a sample from a subject, said method comprising.
Further relevant to claim 1, Weng et al. teaches "In an aspect, a method for enriching amplicons comprising a concatemer of at least two or more copies of a target polynucleotide is disclosed. The method comprises (a) generating a concatemer comprising a single-stranded polynucleotide from a circular target polynucleotide by extension of a first primer" (paragraph 0009).
This teaching reads on claim 1 circularizing a fusion linear nucleic acid molecule of the sample to form a fusion gene circular template polynucleotide, wherein said fusion linear nucleic acid molecule comprises a fusion gene… hybridizing a first primer to said fusion gene circular template polynucleotide and extending the first primer with a polymerase thereby generating a first fusion extension product.
Further relevant to claim 1, Weng et al. teaches "In another aspect, the disclosure provides a method of identifying a sequence variant, such as in a nucleic acid sample. In some embodiments, each polynucleotide of the plurality has a 5' end and a 3' end, and the method comprises: (a) circularizing individual polynucleotides of said plurality to form a plurality of circular polynucleotides, each of which having a junction between the 5' end and 3' end" (paragraph 0010).
The skilled artisan would recognize that not all of the polynucleotides of the plurality would each contain the target fusion gene, thus allowing for circularization of non-target (or non-fusion) nucleic acid molecules. This is supported by the Weng et al. teachings of "FIGS. 18A and 18B illustrate results of an analysis of ligation efficiency and on-target rate of an embodiment of the disclosure" (paragraph 0050) and "As shown in FIG. 18B, the amplification curves of 10 ng of WGA product and reference genomic DNA (gDNA) (12878, 10 ng) virtually overlap with each other. The average Ct for the WGA sample was 26.655, while that of the gDNA sample was 26.605, indicating a high on-target rate of over 96%" (paragraph 0215). The non-100% rate of 96% indicates that non-target (such as non-fusion) nucleic acid molecules are included within the methodology.
These teachings read on claim 1 circularizing a non-fusion linear nucleic acid molecule of the sample to form a non-fusion circular template polynucleotide, wherein said non-fusion linear nucleic acid molecule does not comprise the fusion gene.
Further relevant to claim 1, Weng et al. teaches "FIGS. 6A and 6B illustrate further non-limiting example methods of circularizing polynucleotides, such as single-stranded DNA. The adapter can be asymmetrically added to either the 5' or 3' end of a polynucleotide. As shown in FIG. 6A, the single-stranded DNA (ssDNA) has a free hydroxyl group at the 3' end, and the adapter has a blocked 3' end such that in the presence of a ligase, a preferred reaction joins the 3' end of the ssDNA to the 5' end of the adapter" (paragraph 0090).
This teaching reads on claim 1 binding a blocking element to said non-fusion circular template polynucleotide.
Further relevant to claim 1, Weng et al. teaches "(b) generating a plurality of extension products containing one or more copies of the target polynucleotide by extension of a second primer comprising a second 3' end that specifically hybridizes to the concatemer" (paragraph 0009).
This teaching reads on claim 1 hybridizing a second primer to said first extension product and extending said second primer with a polymerase thereby generating a second extension product.
Further relevant to claim 1, Weng et al. teaches "In some embodiments, the method further comprises sequencing the plurality of amplicons produced in step (c)" (paragraph 0009).
This teaching reads on claim 1 sequencing the second extension product, or a complement thereof, thereby detecting the fusion gene.
Relevant to claims 2-3, Weng et al. teaches "According to some embodiments, polynucleotides among the plurality of polynucleotides from a sample are circularized. Circularization can include joining the 5' end of a polynucleotide to the 3' end of the same polynucleotide, to the 3' end of another polynucleotide in the sample, or to the 3' end of a polynucleotide from a different source (e.g. an artificial polynucleotide, such as an oligonucleotide adapter)" (paragraph 0083).
Relevant to claim 2, Weng et al. teaches "(b) a first primer comprising sequence A', wherein the first primer specifically hybridizes to sequence A of the target sequence via sequence complementarity between sequence A and sequence A'…(d) a polymerase that extends the first primer and the second primer to produce amplified polynucleotides; wherein the distance between the 5' end of sequence A and the 3' end of sequence B of the target sequence is 75 nt or less" (paragraph 0012).
The skilled artisan would recognize that the blocking element (Weng et al. oligonucleotide adapter with a blocked end, see rejection of claim 1) bound to the end of the sequence would be within 75 nt or less distance from the first primer (Weng et al. A/A' region).
Relevant to claim 4, Weng et al. teaches "(c) amplifying the plurality of extension products of step (b) under conditions to generate a plurality of amplicons, wherein amplicons comprising at least 2 or more copies of the target polynucleotide are enriched" (paragraph 0009).
Further relevant to claim 4, Weng et al. teaches "In some embodiments, amplifying is effected by using a polymerase having strand-displacement activity, such as in rolling-circle amplification (RCA)" (paragraph 0011).
Relevant to claim 5, Weng et al. teaches "The target sequence may be a portion of a gene, a regulatory sequence, genomic DNA…" (paragraph 0070).
Relevant to claim 6, Weng et al. teaches "Where polynucleotides are isolated from a sample without a cellular extraction step, polynucleotides will largely be extracellular or 'cell-free' polynucleotides, which may correspond to dead or damaged cells" (paragraph 0080).
Relevant to claims 7 and 9, Weng et al. teaches "A sample may be a fresh sample, or a sample subjected to one or more storage processes (e.g. paraffin-embedded samples, particularly formalin-fixed paraffin-embedded (FFPE) sample)" (paragraph 0158).
Relevant to claim 8, Weng et al. teaches "Examples of cancers that may be diagnosed based on calling one or more sequence variants in accordance with a method disclosed herein include… Hodgkin Lymphoma, Hodgkin's lymphoma…" (paragraph 0174).
Relevant to claims 10-12, Weng et al. teaches "A variety of methods for circularizing polynucleotides are available. In some embodiments, circularization comprises an enzymatic reaction, such as use of a ligase (e.g. an RNA or DNA ligase). A variety of ligases are available, including, but not limited to… [various ligases]" (paragraph 0087).
Relevant to claims 13-14, Weng et al. teaches "According to some embodiments, circularized polynucleotides (or amplification products thereof, which may have optionally been enriched) are subjected to a sequencing reaction to generate sequencing reads… In some embodiments, sequencing comprises a sequencing by synthesis process, where individual nucleotides are identified iteratively, as they are added to the growing primer extension product. Pyrosequencing is an example of a sequence by synthesis process that identifies the incorporation of a nucleotide by assaying the resulting synthesis mixture for the presence of by-products of the sequencing reaction, namely pyrophosphate. In particular, a primer/template/polymerase complex is contacted with a single type of nucleotide. If that nucleotide is incorporated, the polymerization reaction cleaves the nucleoside triphosphate between the a and 13 phosphates of the triphosphate chain, releasing pyrophosphate. The presence of released pyrophosphate is then identified using a chemiluminescent enzyme reporter system that converts the pyrophosphate, with AMP, into ATP, then measures ATP using a luciferase enzyme to produce measurable light signals. Where light is detected, the base is incorporated, where no light is detected, the base is not incorporated" (paragraph 0102).
Relevant to claims 15-17, Weng et al. teaches "In some embodiments of any of the various aspects disclosed herein, the methods, compositions, and systems have therapeutic applications, such as in the characterization of a patient sample and optionally diagnosis of a condition of a subject. Therapeutic applications may also include informing the selection of therapies to which a patient may be most responsive (also referred to as 'theranostics'), and actual treatment of a subject in need thereof, based on the results of a method described herein… In some embodiments, a subject is monitored for treatment efficacy. For example, by monitoring ctDNA over time, a decrease in ctDNA can be used as an indication of efficacious treatment, while increases can facilitate selection of different treatments or different dosages" (paragraph 0154).
Weng et al. does not teach a specific embodiment having all the claimed elements. That being said, however, it must be remembered that "[w]hen a patent simply arranges old elements with each performing the same function it had been known to perform and yields no more than one would expect from such an arrangement, the combination is obvious." KSR v. Teleflex, 127 S.Ct. 1727, 1740 (2007) (quoting Sakraida v. AG. Pro, 425 U.S. 273, 282 (1976)). "[W]hen the question is whether a patent claiming the combination of elements of prior art is obvious," the relevant question is "whether the improvement is more than the predictable use of prior art elements according to their established functions." (Id.). Addressing the issue of obviousness, the Supreme Court noted that the analysis under 35 USC 103 "need not seek out precise teachings directed to the specific subject matter of the challenged claim, for a court can take account of the inferences and creative steps that a person of ordinary skill in the art would employ." KSR at 1741. The Court emphasized that "[a] person of ordinary skill is... a person of ordinary creativity, not an automaton." Id. At 1742.
Consistent with this reasoning, it would have been prima facie obvious to have selected various combinations of various disclosed elements — including blocking elements, ligases, amplification, and sequencing — for a method for detecting a fusion gene in a sample from a subject, to arrive at compositions "yielding no more than one would expect from such an arrangement."
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over Weng et al. (2020; US 2020/0080141 A1) as applied to claims 1-17 above, and further in view of Gu et al. (2016; "Depletion of Abundant Sequences by Hybridization (DASH): using Cas9 to remove unwanted high-abundance species in sequencing libraries and molecular counting applications"; Genome Biology 17:41; DOI 10.1186/s13059-016-0904-5).
The teachings of Weng et al. are applied to instantly rejected claim 18 as they were applied to claims 1-17 as rendering obvious a method for detecting a fusion gene in a sample from a subject.
Weng et al. is silent to specifics regarding a CRISPR-Cas9 complex with a guide RNA molecule blocking element. However, these limitations were known in the prior art and taught by Gu et al.
Relevant to claim 18, Gu et al. Abstract teaches "Next-generation sequencing has generated a need for a broadly applicable method to remove unwanted high-abundance species prior to sequencing. We introduce DASH (Depletion of Abundant Sequences by Hybridization). Sequencing libraries are ‘DASHed’ with recombinant Cas9 protein complexed with a library of guide RNAs targeting unwanted species for cleavage, thus preventing them from consuming sequencing space… We also demonstrate an application of DASH in cancer. This simple method can be adapted for any sample type and increases sequencing yield without additional cost."
Although Weng et al. does not include a CRISPR-Cas9 complex with a guide RNA molecule blocking element, it would have been prima facie obvious to the skilled artisan. It is noted that Weng et al. and Gu et al. are analogous disclosures to the instant method of nucleic acid detection.
The skilled artisan would be motivated to include the Gu et al. CRISPR-Cas9 complex with a guide RNA molecule blocking element within the Weng et al. methodology because Weng et al. allows for enzymatic removal of blocking moieties ("Once the linear ligation is accomplished, the ligated pieces can be treated with an enzyme to remove the blocking moiety, such as through the use of a kinase or other suitable enzymes or chemistries" (Weng et al. paragraph 0090)) and Gu et al. Abstract teaches that the CRISPR-Cas9 complex with a guide RNA molecule blocking element is a “simple method can be adapted for any sample type and increases sequencing yield without additional cost.”
The skilled artisan would have a reasonable expectation of success based on the disclosure of Weng et al., and further in view of Gu et al., as discussed in the preceding paragraphs.
Conclusion
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/SARAH JANE KENNEDY/Examiner, Art Unit 1682
/WU CHENG W SHEN/Supervisory Patent Examiner, Art Unit 1682