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, claims 1-4, 10, 12, 14-17, 20-24, 30-32, and 36 in the reply filed on 11/24/2025 is acknowledged.
Claim 29 is withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 11/24/2025.
Status of Claims
Applicant’s amendment filed 11/24/2025 is acknowledged. Claims 5-9, 11, 13, 18-19, 25-28, and 33-35 have been cancelled. Claims 1-4, 10, 12, 14-17, 20-24, 29-32, and 36 are pending in the instant application and claims 1-4, 10, 12, 14-17, 20-24, 30-32, and 36 are the subject of this non-final office action.
Claim Objections
Claim 3 is objected to because of the following informalities: The claim recites a list of seemingly alternative numbers but lacks the conjunction “or”.
Appropriate correction is required.
Claim Interpretation
In evaluating the patentability of the claims presented in this application, claim terms have been given their broadest reasonable interpretation (BRI) consistent with the specification, as understood by one of ordinary skill in the art, as outlined in MPEP 2111.
Regarding claim 1, the claim recites “binding a blocking element to said one or more non-fusion circular template polynucleotides … hybridizing a first primer and a second primer … to generate a first number of non-fusion … amplification products and a second number of fusion … amplification products, wherein said first number is detectably less than said second number; thereby differentially amplifying the polynucleotide comprising the fusion gene.”
The specification defines “differentially amplifying” in para [0043] as “amplification of a gene of interest to a greater degree than amplification of a reference gene thereby resulting in a greater number of amplification products from the gene of interest relative to the number of amplification products from the reference gene”. Given the claim and the definition, this encompasses amplification differences that flow from template quantity differences (e.g., cleavage of undesired templates or naturally lower level of one starting template compared to another) as well as methods that include enrichment during the amplification (e.g., blocking oligonucleotide interfering with extension of one template).
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 1-14, 10, 12, 14-17, 20-24, and 36 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 1. 15-17, and 20-21, first, one or both of “non-fusion circular template polynucleotides” and “fusion circular template polynucleotides” are recited in claim 1 steps (ii) and (iii), claims 15-17 and 20-21. There is insufficient antecedent basis for these limitation(s) in the claim(s) and these are unclear as pluralities of circular template polynucleotides are recited. Step (i) of claim 1 recites “non-fusion gene circular template polynucleotides” and “fusion gene circular template polynucleotides”.
Claims 2-14, 10, 12, 14-17, 20-24, and 36 are indefinite for depending from claim 1 and not rectifying the deficiency.
Regarding claims 1 and 20, claim 1 recites “hybridizing “a first primer and a second primer to said … circular template polynucleotides and extending with a polymerase to generate a … number of … amplification products”.
It is not clear if the claim is intended, in the case of single-stranded circular template polynucleotides, to require hybridization of the first primer and the second primer to the circular template polynucleotide itself or to encompass hybridization to an extended polynucleotide as part of an amplification process (e.g., para [0009]: “Figs 2A-2B … blocked inverse PCR…outward facing inverse PCR primer pair”; [00361-362]; para [0167]: “amplifying includes … eRCA … second primer having a sequence identical to at least a portion of the circular template … HRCA … second primer complementary to the first amplification product”). See also the 112(b) rejections of claims 15 and 16 below for further citations of single-stranded and double-stranded circular template polynucleotides descriptions in the instant specification relevant to the instant claim.
Claim 21 similar recites “hybridizing a third primer and a fourth primer to said one or more non-fusion … [and] fusion circular template polynucleotides”. The claim is unclear for the same reasons.
Claims 2-14, 10, 12, 14-17, 20-24, and 36 are indefinite for depending from claim 1 and not rectifying the deficiency.
Regarding claims 2 and 17, claim 2 recites “the binding the blocking element upstream of the first primer”. The template to which the first primer is binding is circular, as recited in claim 1. It is not clear at what point along the template in the same sequence “plane” of the first primer that a blocking element would switch from being “upstream” to “downstream” and vice versa as these terms are only definite for orientation by themselves in a linear plane. Thus, the metes and bounds are not clear to one of ordinary skill in the art.
Similarly, claim 17 recites “binding a second blocking element downstream relative to the second primer … wherein the second blocking element binds about 100 to about 300 nucleotides downstream relative to the second primer”. It is not clear if the claim intends to limit the binding beyond about 100 to about 300 nucleotides downstream relative to whichever primer is chosen to be the “second primer”, i.e., to whatever was intended by the broader “downstream”, for the same reasons as above. For example, for smaller circularized nucleic acids, about 100-300 nt downstream may be overlapping at least a portion the second primer—is this sufficient to meet the intended limitations of the claim? Thus, the metes and bounds are not clear to one of ordinary skill in the art.
Regarding claims 2, 17, and 31, the claims 2 and 31 recite “binding the blocking element upstream of the first primer”. The blocking element blinding is not limited in time in claims 1 and 30, respectively. It is not clear if the claims intend to limit the binding in time to after the first primer is bound (i.e., contrary to what is implied by the numerals) or if the claims intend to refer to the hybridization site of the first primer.
Claim 17, which recites “binding a second blocking element downstream relative to the second primer”, is similarly unclear given the ambiguous use of the relative position and timing in these other claims.
Claim 32 is indefinite for depending from claim 31 and not rectifying the deficiency.
Regarding claim 4, the claim recites “wherein the second number is about 2-fold, at least about 1.5-fold, at least about 2.0-fold, … , or more than about 10-fold than said first number”.
There appears to be a typo, wherein it appears the claim was intended to recite “more than said first number” when describing the fold change, as fold change requires a directionality, instructions for division, etc. Accordingly, it is unclear whether it was intended for “about 10-fold” to be “at least about 10-fold” or merely “about 10-fold”. Alternatively, it would be unclear what the fold would be inference to aside from the last value in the list, and “10-fold than said first number” still appears to contain a typo, wherein it is unclear whether “more” or “less” (or synonyms thereof) is missing or something else.
Regarding claim 15, the claim recites “wherein the first primer hybridizes to one or more fusion circular template polynucleotides … downstream relative to a fusion junction”. Claim 1 does not limit the position of the first primer as that claim only requires extension (wherein the primer extended is not named) and generation of named products.
It is unclear whether the claim is intended to limit the fusion circular template polynucleotides to single-stranded nucleic acids or whether they maybe double-stranded, in which case it would be unclear which strand is being referred to regarding the orientation “downstream”. It is noted that while many examples recite single-stranded DNA (see citations in the 112(b) of claim 16 below), the specification also recites double-stranded ligation enzymes, e.g., para [0144]. See also para [0033].
What is intended by “downstream” is also not clear in the circular context for the same reasons as in the rejection of claims 2 and 17 above, particularly for the longer portion of the range.
For these reasons, the metes and bounds are not clear to one of ordinary skill in the art.
Regarding claim 16, the claim recites “wherein the first primer and the second primer hybridize to complementary sequences of the one or more fusion circular template polynucleotides and the one or more non-fusion circular template polynucleotides …”
It is unclear whether the claim is intended to limit the circular template polynucleotides of claim 1 to double-stranded nucleic acids or whether the claims are intended to encompass primers hybridizing to elongated products of said circular template polynucleotides.
It is noted that the instant specification explicitly recites ssDNA in Fig. 1 and Example 1, as recited para [00143], Example 4, and throughout the blocking element descriptions (para [00128-130], [0369-371])). It is also noted that, while not in the dependency chain, claim 15 recites a distance downstream relative to a fusion junction, wherein such would be unclear in the context of a double-stranded molecule.
Regarding claim 22, the claim recites “the third primer hybridizes upstream of a target sequence, and the fourth primer hybridizes downstream of a target sequence”.
As in the rejections of claims 15 and 16 above, the circular context of the template polynucleotides renders upstream and downstream insufficient for the artisan to clearly distinguish the metes and bounds of the claim.
Claim Rejections - 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.
Claim 24 is 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. 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.
Regarding claim 24, the claim recites “wherein sequencing the non-fusion polynucleotide amplification products and the fusion polynucleotide amplification products produces one or more sequencing reads”.
The claim does not require sequencing (i.e., does not state “ The method … comprising sequencing, wherein the sequencing …” and the limitation of producing one or more sequencing reads recites nothing more than an inherent feature of sequencing.
Thus, the claim fails to further limit the claim on which it depends and, therefore, fails to comply with the requirements of 35 USC 112(d).
Claim Rejections - 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)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(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(s) 1-2, 4, 10, 12, 14-16, and 23-24 is/are rejected under 35 U.S.C. 102(a)(1) and 102(a)(2) as being anticipated by Wang (US 2021/0147834 A1; published 05/20/2021), as evidenced by BLAST (Blast: Basic local alignment search tool [Internet]. U.S. National Library of Medicine; [cited 2026 Mar 12]. Available from: https://blast.ncbi.nlm.nih.gov/Blast.cgi) and NCBI (Homo sapiens partial mrna for BCR-ABL1 E6A2 chimeric protein - nucleotide - NCBI [Internet]. U.S. National Library of Medicine; 2016 [cited 2026 Mar 15]. Available from: https://www.ncbi.nlm.nih.gov/nuccore/AM491362.1).
Regarding claims 1, 4, and 12, Wang teaches a method of amplifying a polynucleotides comprising a fusion gene and not comprising a fusion gene (entire document, e.g., claims 1-19; Summary of the Invention; Examples; Fig. 1-3), said method comprising:
circularizing a plurality of linear nucleic acid molecules to form a plurality of circular template polynucleotides comprising the fusion gene and not comprising the fusion gene (Fig. 1; Fig. 2A-D; claim 1; para [0022-23]);
binding a blocking element to non-fusion circular polynucleotides (para [0024-28]; Fig. 2A-D; claim 1);
hybridizing a first and a second primer to each of the non-fusion gene circular template polynucleotides and fusion gene circular template polynucleotides and extending with a polymerase to generate a first number of non-fusion polynucleotide amplification products and a second number of fusion polynucleotide amplification products, wherein said first number is detectably less than said second number (Fig. 3C-D, 5B; para [0043]: “the amplification of wild type ABL1 gene disappeared after enzyme digestion”, i.e., more than about 10-fold greater than said first number; instant claim 4)
It is noted, regarding instant claim 12, that the blocking element of Wang may be considered to be the restriction enzyme and/or the combination with the circle cleaving primer, as cited in step (ii). For the sake of compact prosecution, is also noted that Wang teaches an embodiment where the primer comprises a moiety to block primer extension (para [0028]). It is also noted that while Wang teaches the cleaving prior to amplification, such is not required by the claim language and lack of nexus to the extension/amplification.
Regarding claims 2, 14 and 15, Wang teaches binding a blocking element upstream of its primer binding sites (Fig. 2A, 2C, 3C-D, and Fig. 4; para [0024-29]; instant claim 2), wherein the blocking element may be considered to comprise the endonuclease and/or the combination with the circle cleaving primer.
While Wang does not explicitly teach a binding distance between a primer and the blocking element and the fusion junction, they are implicitly taught because Wang teaches primer sequences in Fig. 4 bind to the ABL1 transcript and at least the Bcr-ABL1 exon 6 to exon 2 fusion transcript (para [0033]). These primers bind at the following positions in of the NM_007313.3 ABL1 transcript and AM491362.1 Bcr-ABL1 e6a2, as evidenced by BLAST. The fusion junction is located at position 1097/1098 in AM491362.1, as evidenced by NCBI.
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If the combined endonuclease/primer is considered the blocking element for the purposes of instant claim 14, the cleaving primer SEQ ID NO: 4 binds about 95 bases upstream of the ABL1 reverse primer SEQ ID NO: 10, as evidenced by the above. For the sake of compact prosecution, it is noted that the blocking element may also be considered to be the endonuclease itself or a modified primer, as taught by Wang. See the 103 rejection of claim 17 below. As elsewhere, the term “first primer” is not limited and may be broadly interpreted to include either.
ABL1 reverse primer SEQ ID NO: 10 also binds about 19 bases downstream relative to the fusion junction (instant claim 15), as evidenced by the above.
Regarding claim 10, Wang teaches the Bcr-Abl fusion originates from an interchromosomal translocation (para [0033]).
Regarding claim 16, Wang teaches amplifying by a PCR method using an ABL1 primer set located at exon 2 of the ABL1cDNA with a forward primer complementary to the sequence of the RT primer and a reverse primer identical to the cDNA downstream of the flanking sequence of the RT primer (para [0036]).
The sequences the two primers SEQ ID NO: 9 and 10 (see Fig. 4) were aligned to NM.005157.6 ABL1 transcript variant a mRNA. While Wang does not explicitly teach the distance the first and second primer are separated by, it is inherently taught because SEQ ID NO: 9 and 10 bind 1 nucleotide apart on the ABL1 transcript, as evidenced by BLAST.
Regarding claims 23 and 24, Wang teaches detecting a length of the amplification products (Fig. 6).
It is noted claim 24 does not require products to be sequenced and Wang does not teach sequencing the non-fusion amplification products.
For at least these reasons, claims 1-2, 4, 10, 12, 14-16, and 23-24 are anticipated by Wang, as evidenced by BLAST and NCBI.
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.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim(s) 1-2, 4, 10, 12, 14-17, 20, 23-24 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang (US 2021/0147834 A1), as evidenced by BLAST (Blast: Basic local alignment search tool [Internet]. U.S. National Library of Medicine; [cited 2026 Mar 12]. Available from: https://blast.ncbi.nlm.nih.gov/Blast.cgi), NCBI (Homo sapiens partial mrna for BCR-ABL1 E6A2 chimeric protein - nucleotide - NCBI [Internet]. U.S. National Library of Medicine; 2016 [cited 2026 Mar 15]. Available from: https://www.ncbi.nlm.nih.gov/nuccore/AM491362.1), and Wikipedia (HindIII [Internet]. Wikimedia Foundation; 2025 [cited 2026 Mar 15]. Available from: https://en.wikipedia.org/wiki/HindIII).
Regarding claims 1, 4, and 12, Wang teaches a method of amplifying a polynucleotide comprising a fusion gene and not comprising a fusion gene (entire document, e.g., claims 1-19; Summary of the Invention; Examples; Fig. 1-3), said method comprising:
circularizing a plurality of linear nucleic acid molecules to form a plurality of circular template polynucleotides comprising the fusion gene and not comprising the fusion gene (Fig. 1; Fig. 2A-D; claim 1; para [0022-23]);
binding a blocking element to non-fusion circular polynucleotides (para [0024-28]; Fig. 2A-D; claim 1);
hybridizing a first and a second primer to each of the non-fusion gene circular template polynucleotides and fusion gene circular template polynucleotides and extending with a polymerase to generate a first number of non-fusion polynucleotide amplification products and a second number of fusion polynucleotide amplification products, wherein said first number is detectably less than said second number (Fig. 3C-D, 5B; para [0043]: “the amplification of wild type ABL1 gene disappeared after enzyme digestion”, i.e., more than about 10-fold greater than said first number; instant claim 4)
It is noted, regarding instant claim 12, that the blocking element of Wang may be considered to be the restriction enzyme and/or the combination with the circle cleaving primer, as cited in step (ii). For the sake of compact prosecution, is also noted that Wang teaches an embodiment where the primer comprises a moiety to block primer extension (para [0028]). It is also noted that while Wang teaches the cleaving prior to amplification, such is not required by the claim language and lack of nexus to the extension/amplification.
Regarding claims 2, 14 and 15, in the method of Wang, Wang teaches binding a blocking element upstream of its primer binding sites (Fig. 2A, 2C, 3C-D, and Fig. 4; para [0024-29]; instant claim 2), wherein the blocking element may be considered to comprise the endonuclease and/or the combination with the circle cleaving primer.
While Wang does not explicitly teach a binding distance between a primer and the blocking element and the fusion junction, they are implicitly taught because Wang teaches primer sequences in Fig. 4 bind to the ABL1 transcript and at least the Bcr-ABL1 exon 6 to exon 2 fusion transcript (para [0033]). These primers bind at the following positions in of the NM_007313.3 ABL1 transcript and AM491362.1 Bcr-ABL1 e6a2, as evidenced by BLAST. The fusion junction is located at position 1097/1098 in AM491362.1, as evidenced by NCBI.
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If the combined endonuclease/primer is considered the blocking element for the purposes of instant claim 14, the cleaving primer SEQ ID NO: 4 binds about 95 bases upstream of the ABL1 reverse primer SEQ ID NO: 10, as evidenced by the above. For the sake of compact prosecution, it is noted that the blocking element may also be considered to be the endonuclease itself or a modified primer, as taught by Wang. See the 103 rejection of claim 17 below. As elsewhere, the term “first primer” is not limited and may be broadly interpreted to include either.
ABL1 reverse primer SEQ ID NO: 10 also binds about 19 bases downstream relative to the fusion junction (instant claim 15), as evidenced by the above.
The relative positions and lengths of primers are subject to routine optimization, for example to optimize a Tm between primer pairs, to avoid GC rich areas, etc. See MPEP 2144.05.
Regarding claim 10, in the method of Wang, Wang teaches the Bcr-Abl fusion originates from an interchromsomal translocation (para [0033]).
Regarding claim 16, in the method of Wang, Wang teaches amplifying by a PCR method using an ABL1 primer set located at exon 2 of the ABL1cDNA with a forward primer complementary to the sequence of the RT primer and a reverse primer identical to the cDNA downstream of the flanking sequence of the RT primer (para [0036]).
The sequences the two primers SEQ ID NO: 9 and 10 (see Fig. 4) were aligned to NM.005157.6 ABL1 transcript variant a mRNA. While Wang does not explicitly teach the distance the first and second primer are separated by, it is inherently taught because SEQ ID NO: 9 and 10 bind 1 nucleotide apart on the ABL1 transcript, as evidenced by BLAST.
The separation distance of primers would also be matter of routinely optimization dependent on, for example, the sequence context and desire to match the respective Tm of the primers. See MPEP 2144.05.
Regarding claim 17, in the method of Wang, Wang does not explicitly teach binding a second blocking element wherein said blocking element “reduces and/or inhibits nucleotide incorporation” (instant para [0097]).
Wang rectifies this by teaching that the circle cleaving primer may be modified with a moiety to block primer extension (para [0028]), such that the circle cleaving primer may itself be considered a blocking element, outside of the combination with the endonuclease of Wang. Wang teaches such modifications had the effect of further blocking extension of polymerase (para [0033]; Fig. 4), and the purpose of the endonuclease cleavage reduce amplification of the non-fusion circle (para [0008]).
It is noted that either primer may be considered to be the “first primer” or the “second primer”, as such is a matter of nomenclature. See also 112(b) rejections regarding the “upstream” and “downstream” interpretations given the circular nature of the template molecule.
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The cartoon to the left was produced based on the BLAST alignments for discussion purposes (above; query names replaced for clarity and the HindIII recognition site marked with lines) of the SEQ IDs of Wang. HindIII has a binding site of AAGCCT, as evidenced by Wikipedia. There are 91 bases from the midpoint of the HindIII restriction recognition sequence to the 3’ of the SEQ ID NO: 9 ABL1 F primer, i.e., about 100 nt downstream relative to the “second primer” (instant claim 17). It is also noted that Wang teaches that the molecule of the wild type ABL1 product is 190 bp (para [0039]) and, as the term about has not been defined, either the cleaving primer (i.e., modified to be extension blocking) or the restriction enzyme may be considered “about 100” nucleotides downstream.
Thus, while Wang does not explicitly teach a second blocking element binding about 100 nucleotides downstream relative to a second primer, the location the element is inherently taught, as evidenced by Wikipedia and BLAST.
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized a blocking moiety in the circle cleaving primer, thus rendering it a blocking element itself. The artisan would have been so motivated to combine because they were equivalents known for the same purpose. See MPEP 2144.06. Thus it would follow that the endonuclease may be considered the second blocking element located about 100 nucleotides downstream of “the second primer”, as discussed above.
The blocking element binding sites distance relative to a primer also would be a matter of routinely optimization, at least because restriction enzymes are dependent on the presence of a particular sequence, wherein another choice of template or restriction enzyme may alter the distance, and also because the position and length of the primers is also subject to routine optimization (e.g., to optimize for a compatible Tm relative to each other and, for example, any other primers utilized in a multiplex system). See MPEP 2144.05.
Regarding claim 20, in the method of Wang, Wang doesn’t explicitly teach a single method of further amplifying said circular template polynucleotides to generate substantially the same number.
The term(s) “said one or more … circular template polynucleotides” are interpreted broadly to encompass aliquots of the ligation product. See instant para [0363].
Wang teaches performing amplification on an aliquot of the ligation reaction containing said circular template polynucleotides (para [0034-0035]), performing separate amplifications with and without the blocking element Hind III enzyme (Fig. 6). Wang teaches that the Bcr-ABL fusion and ABL no fusion amplification products in the undigested reaction amplify to about the same number (Fig. 6).
Therefore, it would have been obvious one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized a single method utilizing the aliquot splitting of Wang in the split amplification with and without digestion of Wang, motivated by the desire to have a control to assess the level of enrichment, as shown by Wang, wherein it would have been necessary to separate into aliquots to effectively act as a control for the digestion. There would have been a strong expectation of success as Wang teaches all of the steps and expected outcome.
It is noted for the sake of compact prosecution that the third and fourth numbers being substantially the same is a matter of routine optimization, dependent at least on the choice of template (e.g., expression if mRNA), efficiency of primer set binding (e.g., to SNPs that may be present in either template), impacts of the length of amplicons on efficiency, etc.
Regarding claims 23 and 24, in the method of Wang, Wang teaches detecting a length of the amplification products (Fig. 6).
It is noted claim 24 does not require products to be sequenced and Wang does not teach sequencing the non-fusion amplification products.
Claim(s) 3 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang (US 2021/0147834 A1; published 05/20/2021 as applied to claim 1 above, and further in view of Thermo Fisher (Restriction Enzyme Digestion [Internet]. Thermo Fisher Scientific; 2017 [cited 2026 Mar 16]. Available from: https://web.archive.org/web/20170607192023/https://www.thermofisher.com/us/en/home/life-science/cloning/cloning-learning-center/invitrogen-school-of-molecular-biology/molecular-cloning/restriction-enzymes/restriction-enzyme-key-considerations.html).
Regarding claim 3, in the method of Wang, Wang fails to teach a second number about 1% to about 75% more than said first number.
Thermo Fisher teaches that one unit of restriction enzyme cleaves 1 ug of a substrate to completion in 1 hour in 50 uL under optimal conditions, but that various DNA substrates in the presence of the same amount of restriction enzyme may have different optimal requirements based on the types of substrate, such that 5- to 20-fold excess may be required for a complete digestion (para 2).
Thermo Fisher teaches that incomplete digestion is frequently encountered, and may occur with too much or too little enzyme; contaminant; or suboptimal buffer, incubation time, or temperature (Incomplete digestion, para 1). Thermo Fisher teaches that some supercoiled [i.e., some circular] molecules are more challenging to cleave than linear counterparts (Incomplete digestion, para 3).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have achieve a second number between about 1% to about 75% more than the first number, in view of Thermo Fisher, given an incomplete digestion in the method of Wang. Such digestion efficiency is a matter of routine optimization for the reasons detailed in Thermo Fisher above, wherein the blocking of amplification and reduction of templates would flow from the cleavage efficiency. Both are directed to methods of cleavage of nucleic acids.
Claim(s) 20-24 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang (US 2021/0147834 A1; published 05/20/2021 as applied to claims 1 or 20 above, and further in view of Chenchik (US 2020/0063190 A1; published 02/27/2020), as evidenced by NCBI (Homo sapiens partial mrna for BCR-ABL1 E6A2 chimeric protein - nucleotide - NCBI [Internet]. U.S. National Library of Medicine; 2016 [cited 2026 Mar 15]. Available from: https://www.ncbi.nlm.nih.gov/nuccore/AM491362.1).
Regarding claim 20-24, in the method of Wang, Wang teaches that the amplified Bcr-ABL1 fusion comprises partial sequences of exons 2 of ABL1 (para [0039]), and targeting the e6a2 fusion of Bcr-ABL1 (para [0033]).
Wang teaches analyzing the circle cDNA by sequencing (para [0007], [0030]) and utilizing a group of RT primers complementary to different regions of a targeted gene (para [0022]).
Wang fails to explicitly teach an embodiment comprising a third and fourth primer wherein the amplification products would be anticipated to be the same, the relative locations of said additional primers (instant claims 21-22). Wang fails to explicitly teach sequencing non-fusion and fusion amplification products (instant claims 23-24).
While Wang does not explicitly teach variants in the fusion gene, the possibility of their presence is inherently taught, evidenced by NCBI. NCBI teaches that both the Bcr and ABL1 portion of the e6a2 Bcr-ABL1 fusion transcript each comprise a variant.
Chenchik teaches a method of amplifying from a circular nucleic acid intermediate molecule using a forward gene-specific primer and a primer the hybridizes to part of the reverse transcriptase primer sequence (para [0070-72]; Fig. 2; instant claim 21). Chenchick teaches adding a barcode and linker (para [0070-72]; Fig. 2), wherein Chenchick teaches that one of the best strategies of processing large numbers of biological samples is labeling a target nucleic acid with unique sample-specific barcodes to denote the source of each particular sample, for example to use for deconvolution of final multiplex profiling data (para [0004]).
Chenchik teaches profiling clinically actionable mutations in a set of human genes including ABL1 using a set of multiplex PCR primers designed to amplify the set of overlapping amplicons that cover the whole mRNA sequence of the target genes (para [0049]). Chenchik teaches point mutations, insertions, and deletions (para [00115]; instant claim 22). It is noted that a barcode may be considered an insertion under the broadest reasonable interpretation.
Chenchik also teaches preparation of target nucleic acids using an initial nucleic acid composition with a calibration control template composition comprising an amplicon structure with two primer binding sites and that have one or more point mutations, or alternatively deletion or insertions, (instant claim 22) downstream of primer binding sites allowing for identification and differentiation via NGS analysis (instant claims 23-24), wherein the calibration control templates may be spiked in at different amounts an used as internal calibration standards to calibrate the actual concentration of natural target mRNA template (para [0115]; instant claim 20). Chenchik also teaches the calibration control templates may be synthetic, naturally occurring, or a combination thereof (para [00115]).
Chenchik teaches that combination of multiplex PCR assays with sample-specific barcodes is the next frontier in reducing cost of the analysis of multiple biological samples for clinical and experimental research applications (para [0007]).
Chenchik teaches that NGS is a powerful tool that can unambiguously define a unique gene expression signature for each tumor that can be useful in disease classification and prognosis (para [0217]). Chenchik teaches predesigned targeted NGS gene panels that conserve sequencing resources and allow focusing on genes most likely to be involved in cellular processes and minimize data analysis considerations (para [0218]).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized the barcoding method of Chenchik comprising inward-facing gene-specific primer + RT anchor primer to have amplified a set of overlapping amplicons of ABL1, including the exon 2 portions of the fusion gene (which may comprise target mutations) and the barcode (which may be considered an insertion), as part of a multiplex sequencing panel. The artisan would have been so motivated by the desire to identify clinically actionable mutations that may occur in ABL1, such as those that may occur in the transcripts targeted by Wang, evidenced by NCBI, while enabling the ability to define a signature useful in disease classification and prognosis, and/or taking advantage of the ability of sample-specific barcodes to improve the cost of processing large numbers of biological samples, as taught by Chenchik. As Wang teaches substantially the same number of Bcr-ABL1 fusion vs. non-fusion ABL1 amplicon numbers from its primer set (Fig. 6), it would follow that a primer set from the combined method would also produce a substantially similar number of amplicons from the fusion and non-fusion templates.
Further, it would have been obvious to have utilized the calibration control template composition method of Chenchik wherein mutations in the combined method, motivated by the desire to calibrate the actual concentration of natural target mRNA templates, e.g., in applying the method more broadly in a multiplex analysis, which would further be obvious from the teachings of Chenchik to reduce cost.
There would be a strong expectation of success as both are directed to circularization of mRNA/cDNA, amplification of clinically relevant mutations, overlapping genes, and downstream sequencing.
It is noted that the primers binding upstream and downstream of a target sequence in instant claim 22 is interpreted to encompass the inward-facing primers of Chenchik. See also the 112(b) rejection and instant Fig. 10-11.
It is also noted that such would be obvious under and compatible with either the aliquot (Wang claim 20) or a no aliquot method. The artisan would be capable of designing such inward-facing primers to produce amplicons would not cross a cut site of the blocking element(s) of Wang in such a combination (e.g., the portions of the fusion gene, as discussed above).
Claim(s) 36 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang (US 2021/0147834 A1; published 05/20/2021 as applied to claim 1, and further in view of Wangh (US 9,758,813 B2; published 09/12/2017).
Regarding claim 36, in the method of Wang, Wang teaches that the circle cleaving primer may be modified with a moiety to block primer extension (para [0028]) and that said primer may be a mismatch primer that can contain one or more nucleotides that mismatch with a wild type sequence (para [0026]).
Wang fails to teach a blocking element comprising dsDNA.
Wangh rectifies this by teaching that double-stranded DNA oligonucleotides can inhibit mispriming that occurs at or just below the optimal annealing temperature of symmetric PCR reactions and thereby prevent amplification of non-specific products (col 5, para 1). Wangh teaches double-stranded oligonucleotides that, when included in a PCR, are effective for increasing polymerase selectivity against hybrids having recessed 3’ terminal sequences that are not perfectly complementary, increasing polymerase selectivity (col 5, Summary, para 1, spanning col 6; Fig. 18). Wangh taches the primer-dependent amplification of DNA may be directed to cDNA and include RT (col 6, para 3).
Wangh teaches suppression of Type I (col 23, para 1), Type II (col 25-26), and Type III mispriming (col 27) and inhibition of 5’ exonuclease activity (col 32-33).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized the double-stranded oligonucleotides of Wangh and to make them DNA as a modification of the cleaving primer of Wang. The artisan would have been so motivated by the diverse suppression of blocking performed by the oligonucleotides, including suppression of mispriming, as taught by Wang, that would be able to supplement endonuclease activity. Further, both are taught for the same purpose. See MPEP 2144.06. There would have been a strong expectation for success as both are directed to methods that enrich for products in amplification and the artisan would have been capable of designing such double-stranded molecules to comprise the endonuclease sites of Wang.
Claim(s) 30-32 is/are rejected under 35 U.S.C. 103 as being unpatentable over Wang (US 2021/0147834 A1) in view of Ochman (Ochman H., et al. Use of polymerase chain reaction to amplify segments outside boundaries of known sequences. Methods Enzymol. 1993;218:309-21), as evidenced by NEB (DNA polymerase strand displacement activity [Internet]. New England Biolabs; [cited 2026 Mar 16]. Available from: https://www.neb.com/en-us/products/pcr-qpcr-and-amplification-technologies/dna-polymerase-strand-displacement-activity).
Regarding claims 30-32, Wang teaches a method of amplifying a polynucleotides comprising a fusion gene and not comprising a fusion gene (entire document, e.g., claims 1-19; Summary of the Invention; Examples; Fig. 1-3), said method comprising:
circularizing a plurality of linear nucleic acid molecules to form a plurality of circular template polynucleotides comprising the fusion gene and not comprising the fusion gene (Fig. 1; Fig. 2A-D; claim 1; para [0022-23]; instant claim 32);
binding a blocking element to non-fusion circular polynucleotides (para [0024-28]; Fig. 2A-D; claim 1);
hybridizing a first and a second primer to each of the non-fusion gene circular template polynucleotides and fusion gene circular template polynucleotides and extending with a polymerase to generate a fusion polynucleotide amplification product (Fig. 3C-D, 5B; para [0043]);
wherein the blocking element may be considered to have been bound upstream of the first primer binding site (Fig. 2A, 2C, 3C-D, and Fig. 4; para [0024-29]; instant claim 31). See also the discussion in the rejection of claim 2.
Wang teaches that amplification may be PCR (claim 18, Fig. 3C-D, para [0039]), but fails to teach a specific polymerase.
Ochman teaches Taq polymerase for inverse PCR (pg. 316, para 4), wherein the Taq polymerase introduces nicks to facilitate amplification of circular templates (pg. 317, para 1). Ochman teaches selecting for amplicons of the anticipated size for downstream applications (pg. 317, para 1).
Ochman discusses inverse PCR discusses applications of inverse PCR to various templates including cDNA (pg. 313, para 1).
While Ochman does not explicitly teach that Taq polymerase is non-strand displacing, it is inherently taught because Ochman teaches Taq, evidenced by NEB (para 2: “In contrast to strand displacement … Taq DNA polymerase … degrade[s] an encountered downstream strand via 5’-3’ endonuclease activity”).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized non-strand displacing Taq polymerase in the method of Wang, motivated by the desire to facilitate the amplification of circular templates and, in contrast to the RCA methods of Wang, to enable selecting for a particular amplicon size for downstream applications, as taught by Ochman. Further, such a combination/substitution would have been obvious as the polymerase was known for the purpose of PCR/inverse PCR. See MPEP 2144.06. There would have been a strong expectation of success the methods are directed to outward facing primers for circular templates that may be cDNA.
Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claim 1-4, 10, 12, 14-17, 20-24, 30-32, and 36 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-20 of copending Application No. 18/183,818 in view of Wang (US 2021/0147834 A1), Chenchik (US 2020/0063190 A1; published 02/27/2020), Wangh (US 9,758,813 B2; published 09/12/2017, and Ochman (Ochman H., et al. Use of polymerase chain reaction to amplify segments outside boundaries of known sequences. Methods Enzymol. 1993;218:309-21), as evidenced by BLAST (Blast: Basic local alignment search tool [Internet]. U.S. National Library of Medicine; [cited 2026 Mar 12]. Available from: https://blast.ncbi.nlm.nih.gov/Blast.cgi), NCBI (Homo sapiens partial mrna for BCR-ABL1 E6A2 chimeric protein - nucleotide - NCBI [Internet]. U.S. National Library of Medicine; 2016 [cited 2026 Mar 15]. Available from: https://www.ncbi.nlm.nih.gov/nuccore/AM491362.1), Wikipedia (HindIII [Internet]. Wikimedia Foundation; 2025 [cited 2026 Mar 15]. Available from: https://en.wikipedia.org/wiki/HindIII), and NEB (DNA polymerase strand displacement activity [Internet]. New England Biolabs; [cited 2026 Mar 16]. Available from: https://www.neb.com/en-us/products/pcr-qpcr-and-amplification-technologies/dna-polymerase-strand-displacement-activity). This is a provisional nonstatutory double patenting rejection.
Both sets of claims are directed to detecting a fusion gene by circularizing fusion linear nucleic acid molecules and non-fusion linear nucleic acid molecules to form circular template polynucleotides, binding a blocking element to non-fusion circular template polynucleotides, and hybridizing a first primer to said fusion gene circular template polynucleotide and a second primer to an extension product of said fusion gene circular template polynucleotide.
Claims 15 and 16 of ‘818 recite a second sample obtained obtaining samples at different time points and detecting a fusion gene, i.e., performing a second amplification, wherein the numbers may have changed (e.g., to be substantially the same; instant claim 20).
Claim 18 of ‘818 recites a CRISPR-Cas9 complex with a guide RNA molecule, i.e., a two-component blocking element that may broadly be considered two blocking elements.
Claim 2 of ‘818 recites positions of the blocking element relative to the first primer. Claim 10 of ‘818 recites that the circularize molecules be 500 bp or less as a result of the ligase.
‘818 fails to explicitly recite: that the gene fusions may be interchromsomal that there would be a detectable difference and by what percentages/fold such a difference would occur; all the claimed positions of the primer(s) and blocking elements relative to each other and the fusion junction; amplifying a third and fourth number, including with a third and fourth primer targeting a particular sequence; that the polymerase may be non-strand displacing; and that the blocking element may comprise dsDNA.
‘818 fails to explicitly recites hybridizing primers to the non-fusion circular template polynucleotides.
It is also noted that while ‘818 recites hybridizing the first primer to the fusion circular template and the second primer to extension product, the instant method appears to encompass this. See the discussion including the 112(b) rejection of instant claim 16. Alternatively, it is noted the linear nucleic acid molecules of ‘818 would include double stranded species and in hybridizing to a first extension product, as sequence would be expected to be present in the complementary strand of the circularized double-stranded molecule, such would encompass hybridizing to the circular template as well.
As discussed and cited in the 103 rejection above, Wang, evidenced by BLAST, NCBI, and Wikipedia, teaches and/or suggests: that the circular polynucleotides may originate from interchromsomal gene fusion nucleic acids (instant claims 1 and 10); positions of the primers and blocking elements relative to each other and the gene fusion (instant claims 2 and 14-17), including that the two primers bind to the non-fusion circular polynucleotides, as well; two-part blocking elements that may separately be considered blocking elements (instant claim 17); and relative changes in enrichment of amplicons after application of blocking element(s) (instant claim 4).
Wang teaches designing primers from a known sequence of a wild type target gene (para [0007]; Fig. 3C-3D).
Wang teaches that gene fusions are one of the chromosome abnormalities that causes various genetic disorders, but unlike other types of mutations that may have “hot spot characteristics”, each case can have a different gene fusion partner or the same fusion partner with a different breakpoint, making it difficult to design probes and primers for all of the partners (para [0003]), wherein such gene fusions comprise interchromosomal gene fusions (para [0033]). Wang teaches the usefulness of identification of fusion partners for disease diagnosis, treatment monitoring, and drug development and that there is no method that can analyze these fusion partners with a simple test in a short period of time (para [0005]). Wang teaches that standard long-distance inverse PCR generates a large amount of non-fusion circle, which can cause the failure of amplification and sequencing, restricting the method for clinical applications (para [0003]).
As discussed and cited in the 103 rejection above, Thermo Fisher teaches optimizing the use of restriction enzymes given that incomplete digestion is frequently encountered, particularly in supercoiled [i.e., some circular] templates.
As discussed and cited in the 103 above, Chenchik, evidenced by NCBI, teaches amplifying a third and fourth number targeting particular mutations and/or barcode insertions on calibration control template molecules where the concentration is controlled such that the artisan could optimize to get substantially the same amplification (instant claims 20-22).
Chenchik teaches that combination of multiplex PCR assays with sample-specific barcodes is the next frontier in reducing cost of the analysis of multiple biological samples for clinical and experimental research applications (para [0007]). Chenchik teaches calibration control templates may be spiked in at different amounts and used as internal calibration standards to calibrate the actual concentration of natural target mRNA template (para [0115]).
As discussed and cited in the 103 above, Wangh teaches double-stranded oligonucleotide, including DNA, blocking elements.
Wangh teaches suppression of Type I (col 23, para 1), Type II (col 25-26), and Type III mispriming (col 27) and inhibition of 5’ exonuclease activity (col 32-33).
As discussed and cited in the 103 above, Ochman, evidenced by NEB, teaches that the polymerase may be non-strand displacing (instant claims 30-32).
Ochman teaches Taq polymerase introduces nicks to facilitate amplification of circular templates (pg. 317, para 1). Ochman teaches selecting for amplicons of the anticipated size for downstream applications (pg. 317, para 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined the method of ‘818 with Wang, evidenced by BLAST, NCBI, and Wikipedia, motivated by the desire to address the need taught by Wang to analyze gene fusion abnormalities for disease diagnosis, treatment monitoring, and/or drug development. It further would have been obvious to model the positioning off Wang, motivated by the desire to have efficient amplification. Alternatively or additionally, it would have been obvious to try relative positions upstream or downstream and/or on the same strand or opposite strand (instant claim 16), given that there are two relative options, and to further optimize positions and lengths (which change the relative distance) where claimed, dependent on the sequence context, respective Tm, etc. Likewise, it would have been obvious to utilize the blocking elements of Wang on the species of interchromosomal gene fusions and the PCR primer sets directed to known portions of the wild-type gene that are shared with non-fusion circular polynucleotides given that they are taught for the same purpose and further motivated by their ability to still enrich completely, as shown in Fig. 6 of Wang, and or apply to the clinical applications taught by Wang. See MPEP 2144.06.
There would have been a strong expectation for success as both are directed to means of amplifying circular molecules may include means of enriching for one over the other.
Additionally, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have achieve a second number between about 1% to about 75% more than the first number, in view of Thermo Fisher, given an incomplete digestion in the combined method. Such digestion efficiency is a matter of routine optimization for the reasons detailed in Thermo Fisher above, wherein the blocking of amplification and reduction of templates would flow from the cleavage efficiency. Both are directed to methods of cleavage of nucleic acids.
Further, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have further utilized the barcoding, with amplification thereof, with the calibration control templates of Chenchik in a secondary amplification with a third and fourth primer targeting SNVs/the barcode insertion such that the amplicons were substantially the same, motivated by the desire to reduce the cost of the analysis for multiple biological samples and so as to calibrate the actual concentration of the natural target mRNA template, as taught by Chenchik. There would have been a strong expectation of success as the method is also directed to amplifying of circular nucleic acids, where readouts include sequencing.
It also would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized the double-stranded oligonucleotides of Wangh and to make them DNA, including as a modification of the cleaving primer of the combined method. The artisan would have been so motivated by the diverse suppression of blocking performed by the oligonucleotides, including suppression of mispriming, as taught by Wang, that would be able to supplement endonuclease activity. Further, both are taught for the same purpose of blocking amplification of the template. See MPEP 2144.06. There would have been a strong expectation for success as the methods are directed to methods that enrich for products in amplification.
Finally, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized non-strand displacing Taq polymerase in the combined method, motivated by the desire to facilitate the amplification of circular templates and to enable selecting for a particular amplicon size for downstream applications, as taught by Ochman. Further, such a combination/substitution would have been obvious as the polymerase was known for the purpose of PCR/inverse PCR. See MPEP 2144.06. There would have been a strong expectation of success the methods are also directed to amplification of circular molecules.
Claim 1-4, 10, 12, 14-17, 20-24, 30-32, and 36 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claim 1-20 of copending Application No. 19/042,521 in view of Wang (US 2021/0147834 A1), Chenchik (US 2020/0063190 A1; published 02/27/2020), and Ochman (Ochman H., et al. Use of polymerase chain reaction to amplify segments outside boundaries of known sequences. Methods Enzymol. 1993;218:309-21), as evidenced by BLAST (Blast: Basic local alignment search tool [Internet]. U.S. National Library of Medicine; [cited 2026 Mar 12]. Available from: https://blast.ncbi.nlm.nih.gov/Blast.cgi), NCBI (Homo sapiens partial mrna for BCR-ABL1 E6A2 chimeric protein - nucleotide - NCBI [Internet]. U.S. National Library of Medicine; 2016 [cited 2026 Mar 15]. Available from: https://www.ncbi.nlm.nih.gov/nuccore/AM491362.1), Wikipedia (HindIII [Internet]. Wikimedia Foundation; 2025 [cited 2026 Mar 15]. Available from: https://en.wikipedia.org/wiki/HindIII), and NEB (DNA polymerase strand displacement activity [Internet]. New England Biolabs; [cited 2026 Mar 16]. Available from: https://www.neb.com/en-us/products/pcr-qpcr-and-amplification-technologies/dna-polymerase-strand-displacement-activity). This is a provisional nonstatutory double patenting rejection.
Both sets of claims are directed to circularizing polynucleotides (claims 9-11), amplifying [i.e., extending a polymerase from a primer; see claims 17 and 18] two circular polynucleotides to generate two numbers of sequences, wherein one is detectably less than the other (claim 1).
It is noted that instant claim 1 may be interpreted broadly as it does not explicitly require the binding of both the primers to each template, nor ‘521 preclude the binding of two primers to each circular template.
Claim 1 of ‘521 recites that the first circular polynucleotide is hybridized to a first nucleic acid molecule, wherein said first circular polynucleotide comprises a retarding agent [i.e., blocking agent]. See also claims 2 and 7 for particular types of blocking elements, including hairpin oligonucleotides and aptamers [i.e., comprising dsDNA]. It follows that the hybridization (binding in claim 7) must have occurred as an inherent step, i.e., the blocking element is bound/hybridized to said circular polynucleotide.
Claim 1 of ‘521 recites the first nucleic acid molecule is attached to a protein-specific binding agent, wherein the protein-specific binding agent may be an aptamer (claim 8), which is also taught among the species of retarding agents (claim 2). It may therefore be broadly considered to be a second blocking element.
Claims 13 and 14 of ‘521 recites percentages and fold, respectively, of the second number of relative to the first. Claim 15 teaches sequencing the [i.e., both] amplification products.
Claim 12 of ‘521 recites the circular polynucleotides are each about 50 to 500 polynucleotides.
‘521 fails to explicitly recite: that the nucleic acid molecules comprise a fusion gene; positions of the primer(s) and blocking elements relative to each other and the fusion junction; amplifying a third and fourth number, including with a third and fourth primer targeting a particular sequence; and that the polymerase may be non-strand displacing.
As discussed and cited in the 103 above, Wang, evidenced by BLAST, NCBI, and Wikipedia, teaches: that the circular polynucleotides may originate from interchromosomal gene fusion nucleic acids (instant claims 1 and 10); positions of the primers and blocking elements relative to each other and the gene fusion (instant claims 2 and 14-17).
Given that ‘521 also teaches that the circular polynucleotides may be 50-500 nucleotides, overlapping the product lengths taught by Wang, the positions and the optimization thereof apply for the same reasons discussed above.
Wang teaches that gene fusions are one of the chromosome abnormalities that causes various genetic disorders, but unlike other types of mutations that may have “hot spot characteristics”, each case can have a different gene fusion partner or the same fusion partner with a different breakpoint, making it difficult to design probes and primers for all of the partners (para [0003]) wherein such gene fusions comprise interchromosomal gene fusions (para [0033]). Wang teaches the usefulness of identification of fusion partners for disease diagnosis, treatment monitoring, and drug development and that there is no method that can analyze these fusion partners with a simple test in a short period of time (para [0005]).
As discussed and cited in the 103 above, Chenchik, evidenced by NCBI, teaches amplifying a third and fourth number targeting particular mutations and/or barcode insertions on calibration control template molecules where the concentration is controlled such that the artisan could optimize to get substantially the same amplification (instant claims 20-22).
Chenchik teaches that combination of multiplex PCR assays with sample-specific barcodes is the next frontier in reducing cost of the analysis of multiple biological samples for clinical and experimental research applications (para [0007]). Chenchik teaches calibration control templates may be spiked in at different amounts and used as internal calibration standards to calibrate the actual concentration of natural target mRNA template (para [0115]).
As discussed and cited in the 103, Ochman, evidenced by NEB, teaches that the polymerase may be non-strand displacing (instant claims 30-32).
Ochman teaches Taq polymerase introduces nicks to facilitate amplification of circular templates (pg. 317, para 1). Ochman teaches selecting for amplicons of the anticipated size for downstream applications (pg. 317, para 1).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined the method of ‘521 with Wang, evidenced by BLAST, NCBI, and Wikipedia, motivated by the desire to address the need taught by Wang to analyze gene fusion abnormalities for disease diagnosis, treatment monitoring, and/or drug development. It further would have been obvious to model the positioning off Wang, motivated by the desire to have efficient amplification. Alternatively or additionally, it would have been obvious to try relative positions upstream or downstream and/or on the same strand or opposite strand (instant claim 16), given that there are two relative options, and to further optimize positions and lengths (which change the relative distance) where claimed, dependent on the sequence context, respective Tm, etc. There would have been a strong expectation for success as both are directed to means of amplifying circular molecules may include means of enriching for one over the other.
Further, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have further utilized the barcoding, with amplification thereof, with the calibration control templates of Chenchik in a secondary amplification with a third and fourth primer targeting SNVs/the barcode insertion such that the amplicons were substantially the same, motivated by the desire to reduce the cost of the analysis for multiple biological samples and so as to calibrate the actual concentration of the natural target mRNA template, as taught by Chenchik. There would have been a strong expectation of success as the method is also directed to amplifying of circular nucleic acids, where readouts include sequencing.
Finally, it would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have utilized non-strand displacing Taq polymerase in the combined method, motivated by the desire to facilitate the amplification of circular templates and to enable selecting for a particular amplicon size for downstream applications, as taught by Ochman. Further, such a combination/substitution would have been obvious as the polymerase was known for the purpose of PCR/inverse PCR. See MPEP 2144.06. There would have been a strong expectation of success the methods are also directed to amplification of circular molecules.
Conclusion
No claims are allowed.
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/EMMA R HOPPE/ Examiner, Art Unit 1683 /ANNE M. GUSSOW/Supervisory Patent Examiner, Art Unit 1683