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 .
Application Status
The amended claims were filed on 4/8/2026. Currently, claims 1-4, 7-19, 24-25 are under consideration. Claims 5,6, 20-23 have been cancelled.
In the prosecution of this application, a final Office action was sent on 12/8/2026. An Advisory action was sent on 4/7/2026 indicating, in-part that if the objected claims 11 and 12 were rewritten in independent form including all of the limitation of the base claim and any intervening claims, and presented as a separate amendment, the amendment would render the current art rejection moot. Upon further consideration, the finality of the previous final Office action of 4/8/2026 is being withdrawn and prosecution is being reopened.
Rejection under 35 USC § 112(b) is withdrawn: Claim 23 was cancelled.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
Claims 1-4, 7-11, 13-19, 24, 25 are rejected under 35 U.S.C. 103 as being unpatentable over Chen (US20180113137 A1, published Apr 26, 2018), in view of Kelly (WO 2018/204423 A1; published 11/8/2018), further in view of Beard (Beard, B. et al., 2014, Humana Press Pg. 321-344)
Re: Claim 1: A method for analysing insertion sites of an exogenous nucleotide sequence in a subject's genome, wherein the method comprises:(a) providing a sample from the subject comprising cell-free double-stranded DNA polynucleotides, wherein the sample is a fluid sample from the subject; (b) blunting the ends of the polynucleotides; (c) ligating an oligonucleotide to both ends of the polynucleotides; (d) amplifying polynucleotides comprising an insertion site; and(e) sequencing the product of step (d),wherein step (b) further comprises 5' phosphorylation and/or 3' adenylation, and wherein the exogenous nucleotide sequence is a viral vector, transposon or expression cassette.
Chen teaches a method for analyzing insertion sites of viral (exogenous) nucleic acids in subject’s genome by (a) providing a sample from the subject comprising circulating cell-free DNA sample from blood (i.e. fluid), (c) ligating at least one adapter, (d) amplifying and (e) sequencing product containing viral host junction (i.e. insertion site) (see Chen, claim 1)
Chen does not explicitly teach (b) blunting ends of the polynucleotides, followed by 5’ phosphorylation or 3’ adenylation.
Kelly similarly describes methods for the sample processing and sequencing of polynucleotides ([0003]). Kelly teaches the massively parallel sequencing of millions of DNA fragments using Illumina’s sequencing-by synthesis strategy ([0315]). Specifically, Kelly teaches (a) providing a sample of cfDNA ([0315]), (b) repairing the ends of the DNA fragments to produce blunt ends, including the addition of 5’ phosphorylated ends and addition of single A base to the 3’ end of the blunt phosphorylated DNA fragments ([0315]). Kelly further teaches (c) ligating adapters to the 3’ ends [0315]. Kelly specifically teaches how the adaptors are ligated to “both ends of the nucleic acid fragments” (see Figure 1A, 1B). Kelly further teaches (d) PCR amplifying the polynucleotides and (e) and sequencing the amplified products ([0315]).
Although Chen teaches that the exogenous nucleic acid sequence was a viral sequence, Chen does not teach that the exogenous nucleic acid was from a viral vector.
However, Beard teaches that retroviral gene therapy vectors can cause insertional mutagenesis in patients and teaches methods for detecting retroviral vector insertion sites (Abstract, inclusive of methods Pgs.321-343).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have processed the cell free DNA sample of Chen by using the process of Kelly because it would have merely amounted to a simple substitution of one known method for processing and sequencing of cell-free DNA sample for another to yield predictable results. Since each of Chen and Kelly similarly teach methods for processing and sequencing of cell-free DNA, then one would have had a reasonable expectation of success that the sample of Chen could have been processed using the method of Kelly. In doing so, one would have had a reasonable expectation of success that the insertion sites of the exogenous nucleic acids of Chen could have been detectable after the processing steps described by Kelly. In addition, it further would have been obvious to have done so to detect exogenous nucleic acids such as a retroviral vector sequences for the advantage of screening for potential unwanted side effects of insertional mutagenesis as described by Beard. Since Chen teaches that viral sequences are detectable in samples comprising cell-free DNA, then one of ordinary skill in the art would have had a reasonable expectation of success in detecting retroviral insertion sites as described by Beard in a sample comprising cell-free DNA.
Re: claim 2, identifying locations of insertion sites in subject genome and /or quantifying abundance,
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Chen further disclosed sequencing the target DNA with the viral-host junction sequence(s), each comprising viral and host genome sequence, and accumulating read number of the junctions and counting them (claim 1). In Table 1 Chen depicts the most abundant junction sequences detected from subject #1, which relays the host chromosome and integration position (aka insertion site) [0038]).
Re: claim 3, wherein the analysing further comprises determining the risk of insertional mutagenesis.
In evaluating the patentability of the claims presented in this application, the claims will be given their broadest reasonable interpretation, in view of the specification, and as set forth at MPEP§ 2111. “Risk of insertional mutagenesis” is not defined in the Specification. Pg 4 does recite: where analysing comprises determining this risk and Page 23, potential risk associated with integration is insertional mutagenesis ie mutations that arise as a consequence of the introduction of a nucleotide sequence. Therefore, determining risk of insertional mutagenesis occurs when insertion is detected, since this represents mutation.
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Chen further disclosed analyzing risk of insertional mutagenesis (detection and quantification of insertion sites) ([0021]([0025]).
Re: claim 4 and 7, The method of claim 1, wherein the subject has been administered gene therapy, wherein the vector is retroviral,
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Beard further disclosed that patients (subjects) globally had been administered (received) retroviral gene therapy (Pg 321, Abstract, Pg 322).
Previously discussed (re: claim 1) reason and motivation to combine addressed Beard’s contribution of evaluating retroviral gene therapy vectors can cause insertional mutagenesis, and applies here.
Re: claim 8, The method of claim 1, wherein the sample is plasma or serum,
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Chen discloses use of serum [0021].
Re: claim 9: The method of claim 1, wherein step (a) further comprises a step of purifying the polynucleotides,
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Chen further disclosed purified cf-DNA using MagNA Pure kits [0027].
Re: claim 10, The method of claim 1, wherein step (b) comprises end- repair or digestion of single-stranded overhangs, and claim 11, wherein step (b) further comprises 5' phosphorylation and 3' adenylation.
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Kelly further disclosed end repair, 5’ phosphorylation and 3’ adenylation ([0193], Fig 1C)
It would have been obvious to use Kelly, the obviousness and motivation discussed above as relevant to claim 1, which extends here, as these method steps are part of the same basic method of Kelly used for preparation for ligation of adapters, which the combination of Chen, Kelly, Beard performed.
Re: Claim 14, The method of claim 1, wherein the amplifying of step (d) is by PCR,
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Chen further disclosed performing PCR on polynucleotides comprising an insertion site ([0051][0054]).
Re: Claim 15, the method of claim 14, wherein the PCR comprises amplifying the product of step (c) using a primer that binds to a portion of the exogenous nucleotide sequence and a primer that binds to a portion of the oligonucleotide.
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Beard’s methods for detecting retroviral vector insertion sites additionally disclosed nested PCR (Pg 327) that similarly includes (a) providing a sample of DNA polynucleotides (see page 324, FIG 1), (b) use of a fragment end polishing kit such as “End-Repair kit” (page 323) (i.e. blunting the ends of the polynucleotides) (c) ligation of linkers (i.e. oligonucleotides) to each end of the DNA polynucleotides (see page 324, FIG 1), (d) amplifying polynucleotides comprising the insertion site (page 324, FIG 1), and (e) sequencing (see page 324, FIG 1). Regarding (d), Beard teaches amplification where one LTR (exogenous nucleotide) primer and one linker cassette-specific (oligonucleotide) were used (Pg 325 and 326). Beard’s prior contribution (to claim 1) was a demonstrated an effective method for detecting retroviral insertion sites, even an improvement over some methods (Pg 323 para 1).
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 modified the method of Chen by incorporating elements of Beard as it relates to the PCR amplification process because it would have merely amounted to a simple substitution of one known method of amplification for another to yield predictable results. Each of Chen, Kelly, and Beard describe similar methods of sample processing, ligating, amplification, and sequencing. Accordingly, it would have been predictable that one of ordinary skill in the art could have amplified the sample using Beard’s PCR amplification methodology and that the retroviral insertion sites would have been detectable by that method.
Re: claim 16, the method of claim 14, wherein the PCR comprises:(i) amplifying the product of step (c) using a first PCR step using a pair of outer primers to obtain a first PCR product; and (ii) amplifying the first PCR product using a second PCR step using a pair of inner primers to obtain a second PCR product.
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Beard’s methods for detecting retroviral vector insertion sites further disclosed a first PCR and a second, nested PCR (Pg 325-326).
Beard’s prior contribution (to claim 1) was a demonstrated an effective method for detecting retroviral insertion sites, even an improvement over some methods (Pg 323 para 1), motivating and making obvious the use of Beard’s method as disclosed in full, which included these steps.
Re: claim 17, The method of claim 16, wherein one primer of the pair of outer primers binds to a first portion of the exogenous nucleotide sequence and one primer of the pair of inner primers binds to a second portion of the exogenous nucleotide sequence.
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Beard’s methods further disclosed a first PCR uses one primer of the pair of outer primers binds to MSCV-LTR (exogenous sequence) and one primer of the inner primers binds to a second specific nested portion of MSCV (exogenous sequence) (Pg 325, step 7 then Pg 326, step 7).
Beard’s prior contribution (to claim 1) was a demonstrated an effective method for detecting retroviral insertion sites, even an improvement over some methods (Pg 323 para 1), motivating and making obvious the use of Beard’s method as disclosed in full, which included these steps.
Re: claim 18, The method of claim 16, wherein one primer of the pair of outer primers binds to a first portion of the oligonucleotide and one primer of the pair of inner primers binds to a second portion of the oligonucleotide.
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Beard additionally disclosed that one primer of the outer primers binds to a first portion of the linker cassette (oligonucleotide) and one primer of inner primers binds to specific nested portion of the linker cassette (Pg 325, step 7, Pg 326, step 7).
Beard’s prior contribution (to claim 1) was a demonstrated an effective method for detecting retroviral insertion sites, even an improvement over some methods (Pg 323 para 1), motivating and making obvious the use of Beard’s method as disclosed in full, which included these steps.
Re: claim 19, The method of claim 16, wherein the PCR is nested PCR, and wherein the nested PCR comprises:(i) amplifying the product of step (c) using a first PCR step using a first primer that binds to a first portion of the exogenous nucleotide sequence and a second primer that binds to a first portion of an oligonucleotide to obtain a first PCR product; and(ii) amplifying the first PCR product using a second PCR step using a third primer that binds to a second portion of the exogenous nucleotide sequence and a fourth primer that binds to a second portion of the oligonucleotide to obtain a second PCR product.
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Beard additionally teaches nested PCR and amplifies the product of step c using a first primer that binds MSCV (exogenous) nucleotide, second primer that binds (cassette specific) oligonucleotide to obtain first product (Pg 325 protocol, step 7 for primers), and amplifying then with second PCR with third primer to MSCV (exogenous) sequence and fourth primer to cassette specific (oligonucleotide) sequence (Pg 326 for protocol, step 7 for primers).
Beard’s prior contribution (to claim 1) was a demonstrated an effective method for detecting retroviral insertion sites, even an improvement over some methods (Pg 323 para 1), motivating and making obvious the use of Beard’s method as disclosed in full, which included these steps.
Re: Claim 24, the method of claim 1, wherein the method is for analyzing vector insertion site in a subject’s genome, wherein the insertion site of step (d) is a vector insertion site,
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Beard further disclosed analyzing (mapping) vector integration (insertion) sites in a patient’s (subject’s) genome (Abstract; Pg 328, para 3).
It would have been obvious to have analyzed vector insertion sites having employed the additional methods of Beard as applied to the methods of Chen in view of Kelly in view of Beard, given that a major purpose of the amplification of the vector insertion sites of Beard (and Chen’s in view of Kelly and Beard) was to have accurately evaluated retroviral insertion sites that presented the unwanted side effect of insertional mutagenesis, including as related to cancer, which both Beard and Chen demonstrated interest in viral integration sites and cancer (Beard, Abstract; Chen, [0025]). Given that Beard’s mapping was part of his complete methods and motivation to conduct these methods, it would have been obvious to combine this analysis step with the prior disclosure of Chen in view of Kelly in view of Beard, to yield predictable results. The motivation to do so would have come from the end goal of these works, inclusive of evaluating risks associated with viral insertion sites into host genome.
Re: Claim 25, the method of claim 1, used for identifying cancer cells,
Chen in view of Kelly in view of Beard has been discussed re: claim 1.
Chen further disclosed that sequencing chromosomal fragments indicated that circulating cell-free nucleic acids carry genetic alterations from cancer that, with conventional methods, are difficult to detect when cancers are not advanced [0005]-[0006]. FIGS. 2-4 depict obtaining cf-nucleic acids, viral-host junction and changes in amount of viral host junction before and after tumor resection; human patients with tumors are the subjects of the study where DNA of tumor cells is extracted for use in the methods taught which are used in evaluating cancer [0025]-[0027].
Claim 12 is rejected under 35 U.S.C. 103 as being unpatentable over Chen (US20180113137 A1, published Apr 26, 2018), in view of Kelly (WO 2018/204423 A1; published 11/8/2018), further in view of Beard (Beard, B. et al., 2014, Humana Press Pg. 321-344).
Re: claim 12, A method for analysing insertion sites of an exogenous nucleotide sequence in a subject's genome, wherein the method comprises: (a) providing a sample from the subiect comprising cell-free double-stranded DNA polynucleotides, wherein the sample is a fluid sample from the subiect; (b) blunting the ends of the polynucleotides;(c) ligating an oligonucleotide that is partially double-stranded to both ends of the polynucleotides;(d) amplifying polynucleotides comprising an insertion site; and(e) sequencing the product of step (d), wherein the exogenous nucleotide sequence is a viral vector, transposon or expression cassette.
Chen teaches detecting viral (exogenous) nucleic acids in subject’s genome by detecting circulating cell-free DNA sample from blood (i.e. fluid), ligating at least one adapter, amplifying and sequencing product containing viral host junction (ie insertion site) (see Chen, claim1)
Chen teaches ligating adapters to both ends, but does not explicitly teach ligating to partially double-stranded oligonucleotides
Kelly teaches cfDNA and ligation of adpaters to partially double stranded oligonucleotides ([0251][Fig 1c,2]).
Although Chen teaches that the exogenous nucleic acid sequence was a viral sequence, Chen does not teach that the exogenous nucleic acid was from a viral vector.
However, Beard teaches that retroviral gene therapy vectors can cause insertional mutagenesis in patients and teaches methods for detecting retroviral vector insertion sites (Abstract, inclusive of methods Pgs.321-343).
It would have been obvious to one of ordinary skill in the art to have processed the cell free DNA sample of Chen by using the process of Kelly because it would have merely amounted to a simple substitution of one known method for processing and sequencing of cell-free DNA sample for another to yield predictable results. Since each of Chen and Kelly similarly teach methods for processing and sequencing of cell-free DNA, then one would have had a reasonable expectation of success that the sample of Chen could have been processed using the method of Kelly. In doing so, one would have had a reasonable expectation of success that the insertion sites of the exogenous nucleic acids of Chen could have been detectable after the processing steps described by Kelly. In addition, it further would have been obvious to have done so to detect exogenous nucleic acids such as a viral vector sequence for the advantage of screening for potential unwanted side effects of insertional mutagenesis as described by Beard.
Claim 13 is rejected under 35 U.S.C. 103 as being unpatentable over Chen (US20180113137 A1, published Apr 26, 2018), in view of Kelly (WO 2018/204423 A1; published 11/8/2018), further in view of Beard (Beard, B. et al., 2014, Humana Press Pg. 321-344), further in view of MacConail (BMC Genomics, 2018 19:30).
Re: claim 13, The method of claim 1, wherein each oligonucleotide comprises:(i) a first portion comprising a random nucleotide sequence;(ii) a second portion comprising a barcode nucleotide sequence; and/or (iii) a third portion comprising a binding site for a primer for use in the sequencing of step (e).
Kelly teaches a barcode and a binding site for a primer for sequencing [0156].
Kelly does not teach the addition of a random sequence.
However, MacConaill et al teach the addition of a UMI (a random sequence) to tag each molecule prior to amplification (Abstract, Pg 2 right col, first full para).
It would have been obvious to have incorporated the additional random sequence of MacConaill into the methods of Chen in view of Kelly and Beard because a function of the random sequence was to identify library molecules to improve sensitivity, by reducing multiple error types, that are not addressed absent the addition of these random sequences (Pg 2, right col, first full para, final few lines; 3rd para). Accurate detection and sample categorization was imperative to Chen’s work addressing insertion sites. This would serve as motivation for inclusion of random sequences, as sensitivity was of value for Chen’s accurate identification of viral insertions sites and their relation to cancer. There would have been a reasonable expectation of success, absent evidence to the contrary, given MacConaill’s teaching relate to applications using the same basic adaptor methodology described previously for Chen in view of Kelly, and generally used for high throughput sequencing (MacConaill, Pg 2 right col).
Response to Remarks/Arguments
Applicant remarks submitted on 4/8/2026, after Advisory Action of 4/7/26:
Amendments to the claims: Per above, prosecution is reopened and claims are being reconsidered.
Claim 23 was cancelled rendering rejection moot.
Applicant’s remarks submitted on 3/6/2026, after Final Office action of 12/8/2025:
Amendments to the claims: Claim amendments are reconsidered in this Office action.
The indefiniteness rejection of claim 23 is now moot.
Conclusion
All claims are rejected.
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/LISA HORTH/Examiner, Art Unit 1636
/NEIL P HAMMELL/ Supervisory Patent Examiner, Art Unit 1636
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