DETAILED ACTION
Disposition of Claims
Claims 1-10 and 12-21 are pending.
Examiner’s Note
All paragraph numbers (¶) throughout this office action, unless otherwise noted, are from the US PGPub of this application US20250230430A1, Published 07/17/2025.
Applicant is encouraged to utilize the new web-based Automated Interview Request (AIR) tool for submitting interview requests; more information can be found at https://www.uspto.gov/patent/laws-and-regulations/interview-practice.
Optional Authorization to Initiate Electronic Communications
The Applicant’s representative may wish to consider supplying a written authorization in response to this Office action to correspond with the Examiner via electronic mail (e-mail). This authorization is optional on the part of the Applicant’s representative, but it should be noted that the Examiner may not initiate nor respond to communications via electronic mail unless and until Applicant’s representative authorizes such communications in writing within the official record of the patent application. A sample authorization is available at MPEP § 502.03, part II. If Applicant’s representative chooses to provide this authorization, please ensure to include a valid e-mail address along with said authorization.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 01/07/2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Notably, the disclosure statement filed lists a Search Report. The listing of the references cited in a Search Report itself is not considered to be an information disclosure statement (IDS) complying with 37 CFR 1.98. 37 CFR 1.98(a)(2) requires a legible copy of: (1) each foreign patent; (2) each publication or that portion which caused it to be listed; (3) for each cited pending U.S. application, the application specification including claims, and any drawing of the application, or that portion of the application which caused it to be listed including any claims directed to that portion, unless the cited pending U.S. application is stored in the Image File Wrapper (IFW) system; and (4) all other information, or that portion which caused it to be listed. In addition, each IDS must include a list of all patents, publications, applications, or other information submitted for consideration by the Office (see 37 CFR 1.98(a)(1) and (b)), and MPEP § 609.04(a), subsection I. states, "the list ... must be submitted on a separate paper." Therefore, the references cited in the Search Report have not been considered. Applicant is advised that the date of submission of any item of information or any missing element(s) will be the date of submission for purposes of determining compliance with the requirements based on the time of filing the IDS, including all "statement" requirements of 37 CFR 1.97(e). See MPEP § 609.05(a).
Note: If copies of the individual references cited on the Search Report are also cited separately on the IDS (and these references have not been lined-through) they have been considered.
Specification
Applicant is reminded of the proper language and format for an abstract of the disclosure.
The abstract should be in narrative form and generally limited to a single paragraph on a separate sheet within the range of 50 to 150 words in length. The abstract should describe the disclosure sufficiently to assist readers in deciding whether there is a need for consulting the full patent text for details.
The language should be clear and concise and should not repeat information given in the title. It should avoid using phrases which can be implied, such as, “The disclosure concerns,” “The disclosure defined by this invention,” “The disclosure describes,” etc. In addition, the form and legal phraseology often used in patent claims, such as “means” and “said,” should be avoided.
The abstract of the disclosure is objected to because of the use of implied phraseology (e.g. “The present disclosure belongs…” and “The detection method of the present disclosure has…”). A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01. See e.g. ¶[0125] “https://www.nibsc.org/products/brm_product_catalogue/detail_page.aspx?catid=18/144.”.
Claim Objections
Claim 1 is objected to because of the following informalities: the definition of the abbreviations “LTR” and “UMI” are not provided. For clarity, it is requested that the first recitation of an abbreviation within a claim set be preceded by its full-length name (i.e. … long terminal repeat (LTR)...).
Appropriate correction is required.
Claim 21 is objected to because of the following informalities: the definition of the abbreviations “ATCB”, “GAPDH”, and “ApoB” are not provided. For clarity, it is requested that the first recitation of an abbreviation within a claim set be preceded by its full-length name (i.e. … apolipoprotein B (ApoB)...).
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b); Second Paragraph
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 2 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 2 recites the limitation "the DNA fragment" in lines 1-2. There is insufficient antecedent basis for this limitation in the claim, as claim 1, from which claim 2 depends, recites “DNA fragments” and does not previously introduce a singular “DNA fragment.” It is therefore unclear which DNA fragment is intended by “the DNA fragment” and whether the recited limitation is intended to apply to one, some, or all of the DNA fragments produced according to claim 1, step 1).
For at least these reasons, the metes and bounds of claim 2 are unclear.
Claim 4 and dependent claim 19 thereof 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.
Claim 4 recites that the short strand of the asymmetric double-stranded adapter has a 5’ end provided with modification preventing primer extension. However, primer extension by a DNA polymerase proceeds from the 3’ end of a nucleic acid strand, and the specification describes the blocking modification as being located at the 3’ end of the short strand. Accordingly, it is unclear whether claim 4 is intended to require a modification at the recited 5’ end or instead is to require the art-recognized 3’-terminal blocking modification as described in the specification. Claim 19 does not cure this deficiency as it notes the modification as being either an inverted dT modification or a dideoxycytidine modification, but continues to depend from claim 4 and therefore retains the ambiguity as to whether the recited blocking modification is located at the 5’ or 3’ end of the short strand. For the purpose of prior art, claim 4 will be interpreted as “the 3’ end of the short-strand sequence is provided with a modification that prevents primer extension”.
Since a skilled artisan would not be reasonably apprised as to the metes and bounds of the claimed invention, instant claim 4 is rejected on the grounds of being indefinite. Claim 19 is also rejected since it depends from claim 4, but does not remedy these deficiencies of claim 4.
Claim 16 and dependent claim 18 thereof 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.
Claim 16 is drawn to a method for preparing an engineered immune cell, comprising the step of detecting the engineered immune cell according to the detection method according to claim 12. However, claim 12 is directed to detecting a retroviral integration site and does not recite a step of preparing or engineering an immune cell. Further, claim 16 does not recite a step of introducing a retroviral vector into an immune cell, selecting an engineered immune cell based on the detection result, releasing an engineered immune cell meeting a specified criterion, or otherwise acting on the detected cell in a manner that prepares the engineered immune cell. It is therefore unclear whether the phrase “for preparing an engineered immune cell” is intended merely to state the purpose or intended use of the detection method, whether detection itself is regarded as preparing the cell, or whether an unrecited selection or manufacturing step is required to satisfy the claim.
Since a skilled artisan would not be reasonably apprised as to the metes and bounds of the claimed invention, instant claim 16 is rejected on the grounds of being indefinite. Claim 18 is also rejected since it depends from claim 16, but does not remedy these deficiencies of claim 16.
Claim 20 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.
Regarding claim 20, the phrase "for example" renders the claim indefinite because it is unclear whether the limitation(s) following the phrase are part of the claimed invention. See MPEP § 2173.05(d).
Claim 21 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 21 provides that the housekeeping gene is one of ATCB, GAPDH, or ApoB. However, it is unclear what gene “ATCB” is meant to stand for, as the specification refers to gene “ATCB” as a housekeeping gene but does not provide a sequence, accession number, full gene name, or other identifying information that establishes what gene is intended by “ATCB”. In contrast, beta-actin is conventionally identified in the art as ACTB. Accordingly, it is unclear if the claim is meant to be drawn to beta-actin, ATCB, or some other housekeeping gene.
For at least these reasons, the metes and bounds of the claim are unclear.
Claim Interpretation
The claims in this application are given their broadest reasonable interpretation using the plain meaning of the claim language in light of the specification as it would be understood by one of ordinary skill in the art.
NB: “absolute quantitative detection method” is defined by the PCR art as a measurement of the exact copy number or concentration of a target nucleic acid sequence.
Claim 1 is drawn to a method for preparing a DNA library, comprising the steps of:
1) fragmenting a genomic DNA from a retrovirus-infected cell to obtain DNA fragments;
2) subjecting the DNA fragments to end-repair, A-tailing, and ligation with an adapter to obtain a ligation product, wherein the adapter is an asymmetric double-strand adapter comprising a long-strand sequence and a short-strand sequence, wherein the long-strand sequence sequentially comprises, from a 5' end to a 3' end, a fixed sequence, a random UMI sequence, and an amplification primer binding sequence, and the short-strand sequence comprises a sequence complementary to the fixed sequence;
3) performing a first round of PCR on the ligation product with primers for a long terminal repeat (LTR) sequence, primers for an internal reference gene sequence, and primers for an adapter sequence to obtain a first round of PCR products;
4) performing a second round of PCR on the first round of PCR products with primers for the LTR sequence, primers for the internal reference gene sequence, and primers for the adapter sequence which are different from those in step 3) to obtain a second round of PCR products; and
5) performing a third round of PCR on the second round of PCR products with primers having sequencing adapters to obtain a third round of PCR products as a DNA library.
Further limitations on the preparation method according to claim 1 are wherein in step 1), the DNA fragment is 500-1500 bp, 800-1200 bp, 900-1100 bp or 1000 bp in length (claim 2); wherein in step 2), the fixed sequence is 14-30 bases in length; and the random UMI sequence is 5-9 bases in length, wherein each base is independently A, G, C, or T (claim 3); wherein in step 2), the 3' end of the short-strand sequence is provided with a modification that prevents primer extension (claim 4), wherein the modification that prevents primer extension is an inverted dT modification or a dideoxycytidine modification (claim 19); wherein in step 4):
the primers for the LTR sequence in the second round of PCR are located downstream of the primers for the LTR sequence in the first round of PCR, the primers for the internal reference gene sequence in the second round of PCR are located downstream of the primers for the internal reference gene sequence in the first round of PCR, and the primers for the adapter sequence in the second round of PCR cover the primers for the adapter sequence in the first round of PCR and extend downstream (claim 5); wherein the total number of amplification cycles in the first round of PCR, the second round of PCR and the third round of PCR is 36-45 (claim 6), wherein:
(1) the number of cycles in the first round of PCR and the second round of PCR is 30 and 20, 15 and 20, 10 and 20, or 15 and 15, respectively;
(2) the number of cycles in the third round of PCR is 6-13, for example, 6, 10 or 13;
(3) the number of cycles in the first round of PCR, the second round of PCR and the third round of PCR is 15, 20 and 6, respectively, 10, 20 and 6, respectively, or 15, 15 and 6, respectively; or
(4) the number of cycles in the first round of PCR is 15-18, the number of cycles in the second round of PCR is 18-22, and the number of cycles in the third round of PCR is 6-8 (claim 20); wherein the ratio of the number of cycles in the first round of PCR to that in the second round of PCR is (1-3) : 2 (claim 7); wherein the step 3) further comprises the step of: performing screening to obtain the first round of PCR products having a fragment length of less than 1000 bp; the step 4) further comprises the step of: performing screening to obtain the second round of PCR products having a fragment length of less than 1000 bp; and the step 5) further comprises the step of: performing screening to obtain the third-round PCR products having a fragment length of less than 1000 bp (claim 8); wherein the retrovirus is a lentivirus or a gamma-retrovirus (claim 9); wherein the internal reference gene is a house-keeping gene (claim 10), wherein the house-keeping gene is gene ATCB, gene GAPDH or gene ApoB (claim 21)
Claim 12 is drawn to a method for detecting a retroviral integration site, comprising the method for preparing a DNA library according to claim 1, and further comprising the step of:
6) sequencing the resulting DNA library to obtain sequencing data.
Further limitations on the detection method according to claim 12 wherein the method is further comprising the step of:
7) performing bioinformatics analysis on the sequencing data to obtain integration site data (claim 13); wherein the detection method is an absolute quantitative detection method (claim 14); and wherein the retrovirus is a lentivirus or a gamma-retrovirus (claim 15).
Claim 16 is drawn to a method for detecting an engineered immune cell, comprising the step of detecting the engineered immune cell according to the method of claim 12.
Further limitations on the method for detecting an engineered immune cell according to claim 16 are wherein the engineered immune cell is obtained by a method comprising the steps of:
(1) isolating immune cells;
(2) constructing a recombinant retroviral vector comprising a nucleic acid encoding a chimeric antigen receptor; and
(3) introducing the recombinant retroviral vector obtained in step (2) into the immune cells (claim 17); and wherein the engineered immune cell is selected from a cytotoxic T cell, a helper T cell, a natural killer T cell, a yS T cell, an NK cell, a macrophage, a B cell, an antigen presenting cell, a dendritic cell and a stem cell-induced immune cell (claim 18).
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.
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 1-2, 5-10, 12-16, and 20-21 are rejected under 35 U.S.C. 103 as being unpatentable over Li et. al. (CN113046835A; Pub. 06/29/2021; Priority 12/27/2019; hereafter “Li”) in view of Paugh et. al. (Paugh BS, et. al. Sci Rep. 2021 Jan 11;11(1):389.; hereafter “Paugh”) and in further view of Dong et. al. (Dong J, et. al. Nature. 2015 Sep 3;525(7567):134-139. Epub 2015 Aug 26.; hereafter “Dong”) and DiGiusto et. al. (DiGiusto DL, et. al. Sci Transl Med. 2010 Jun 16;2(36):36ra43.; hereafter “DiGiusto”.)
The Prior Art
Li teaches a method for constructing a sequencing library for detecting lentivirus insertion sites from genomic DNA extracted from lentivirus-infected cells (entire document; see abstract; instant claims 9, 15.) Li teaches extracting genomic DNA from lentivirus-infected cells, fragmenting and processing the genomic DNA into suitable fragments in the form of linker connection, wherein linker connection comprises asymmetric double-stranded linkers are connected at both ends of the fragmented genomic DNA, which include long-chain sequences and short-chain sequences; amplifying the first round of PCR on the linker ligation product; and amplifying a second round of PCR amplification on the first round product (entire document; see abstract.) Li teaches that the linker connection on the fragmented genomic DNA may comprise the use of performing end repair ant A-tailing, and ligating an asymmetric double-stranded adapter to the fragmented DNA (abstract; reference claims 1-2). The long strand of the adapter sequentially contains a fixed sequence, a random UMI sequence, and an amplification-primer-binding sequence, while the short strand contains sequence complementary to the fixed sequence (reference claim 1). Li teaches a 10-base random UMI in which each position may be A, G, C, or T (reference claim 3). Li teaches a first round of PCR using a LTR-specific primer and an adapter-specific primer, followed by a second round of PCR using a different LTR-specific primer and a different adapter-associated primer (reference claims 1, 4-5; See Example 1 starting at p. 6 of the English translation of Li). The second primer binds downstream of the first LTR primer, thereby providing nested amplification for enrichment and specificity (p. 5, ¶3). Li teaches the random UMI sequence is 6-14 bases in length and each base is independently A, G, C, or T (p. 4, ¶13; reference claim 3). Li teaches the ratio for the number of cycles in the first round of PCR to that in the second round of PCR is 1:1 (Example 1; Tables 2-3, Sects. 5.1.2 and 5.2.2; instant claim 7). Li teaches a final bioinformatics analysis of viral insertion sites after obtaining the sequences (Sect. 8 in Example 1; instant claim 13).
Li therefore teaches the majority of instant claim 1, but fails to teach the inclusion of primers directed to an internal reference gene in both the first and second rounds of PCR, and it does not perform the recited separate third PCR to add sequencing adapters. However, such optimizations and steps were known in the art, as shown by the teachings of Paugh, Dong, and DiGiusto.
Paugh teaches analysis of integrated lentiviral DNA in lentivirally transduced Jurkat cells (immortalized T cells; instant claim 16) using host genomic reference genes to normalize the integrated lentiviral target (entire document; see abstract; instant claims 9, 15.) Paugh teaches simultaneous amplification of the integrated lentiviral GAG target and the host PTBP2 reference gene target in a single duplex reaction. A second duplex assay similarly amplifies the lentiviral RRE target together with RPL32, and the reference target is used to normalize the lentiviral target (p. 2, ¶4; instant claim 10). Paugh teaches that vector copy number is a critical attribute of gene modified cell products and calculates integrated vector copies relative to the genomic reference target. Paugh also uses next-generation sequencing (NGS) to map the proviral integration sites in the same gene modified cell lines (Fig. 2; instant claim 14).
Dong teaches in a high-throughput genomic junction sequencing library workflow, performing first- and second-round target amplification followed by a third round of tagging PCR to add Illumina Miseq-compatible adapters at 5’ and 3’ ends of the second round PCR product, wherein the third round was carried out for another 10 cycles with Phusion polymerase. Dong performs the third cycle and then performs sequencing the resulting library on an Illumina MiSeq instrument; Dong teaches PCR products were size-fractionated for DNA fragments between 300 1000bp on a 1% agarose gel and column purified before loading onto Illumina Miseq machine for sequencing (p.7; “High-throughput genome-wide translocation sequencing (HTGTS).”; instant claim 20).
DiGiusto teaches determining integrated lentiviral copy number in genetically modified human cells by quantitative PCR and normalizing the number of copies of the integrated vector to cell number using the housekeeping gene apolipoprotein B (ApoB)(entire document; see abstract; Fig. 2; instant claim 21). DiGiusto teaches that the ratio of copies of the lentiviral WPRE sequence to ApoB was used to determine the level of gene marking in lentivirally-modified cells (p. 3, rt. Col. ¶3).
It would have been obvious to one of ordinary skill to employ this known three-stage sequencing library workflow with the products of the Li method, thereby performing target enrichment in the first and second PCR rounds and adding the sequencing platform adaptors in a third PCR. Li recognizes that the particular fixed sequences introduced during library preparation depend upon the sequencing platform being used. Separating target enrichment from addition of sequencing platform adapters in the manner taught by Dong would have been a predictable alternative for producing a sequencing-ready library and would have yielded the claimed third round PCR without changing the underlying integration site detection principle. It would have been further obvious to include host reference sequences to permit normalization of the lentiviral measurement to the amount of host genomic DNA and to provide a more reliable quantitative measure of integrated vector DNA, as taught by Paugh. It would have been obvious to one of ordinary skill in the art, when incorporating a genomic reference gene into the lentiviral integration assay of Li, to select ApoB as that housekeeping reference gene because DiGiusto expressly teaches the use of ApoB for normalization of integrated lentiviral vector DNA in genetically modified cells. Given that Dong teaches size fractionization for DNA fragments as a result of the PCR between 300-100 bp, and given that Li teaches the use of downstream nested primers for the reactions, further dependent claims would be obvious in light of the combinations of these teachings. As Li teaches 15 PCR amplification cycles for rounds 1 and 2, and Dong teaches 10 amplification cycles for round 3, arriving at a final total round of PCR amplification at 40 amplification cycles would be obvious to a skilled artisan, as required by instant claim 6. Therefore, given the teachings of Li in view of Paugh, Dong, and DiGiusto, arriving at the limitations of instant claims 1-2, 5-10, 12-16, and 20-21 would have been obvious to a skilled artisan.
It would have been obvious to one of ordinary skill in the art to modify the methods taught by Li in order to add housekeeping genes and additional PCR steps to allow for efficient sequencing, thereby rendering the method of preparing a DNA library from a retrovirus infected cell obvious and allowing a skilled artisan to easily determine retrovirus integration sites from infected cells. One would have been motivated to do so, given the suggestion by Paugh that an integrated lentiviral target and a host genomic reference gene can be amplified in the same reaction to permit normalization of integrated vector DNA to genomic DNA, and given the teaching by DiGiusto that a housekeeping gene such as ApoB may be used to normalize integrated lentiviral vector copy number in genetically modified cells. One would have been further motivated to employ a third round of PCR, given the teachings of Dong that a third tagging PCR may be use to add sequence-compatible adapters to the products of the preceding amplification rounds. There would have been a reasonable expectation of success, given the knowledge that integrated lentiviral sequences and genomic reference sequences could be simultaneously amplified and quantitatively compared, as taught by Paugh, and also given the knowledge that sequencing adapters could be added in subsequent PCR without altering the underlying target enrichment function of the preceding amplification steps, as taught by Dong. Thus, the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time the invention was made.
Claims 4 and 19 are rejected under 35 U.S.C. 103 as being unpatentable over Li, Paugh, Dong, and DiGiusto as applied to claims 1-2, 5-10, 12-16, and 20-21 above, and further in view of Li et. al. (CN108456713A, Pub. 08/28/2018; Priority 11/27/2017; hereafter “Li-2017”.)
The Prior Art
The teachings of Li, Paugh, Dong, and DiGiusto have been set forth supra. While the preparation method of performing the PCR on fragmented DNA from infected cells is obvious, the use of a 3’ end modification on the short-strand sequence to prevent primer extension is not explicitly taught by any of Li, Paugh, Dong, and DiGiusto. However, the use of such modifications is known in the art, as evidenced by Li-2017.
Li-2017 teaches that a modification provided at the 3’ end of an adapter blocking sequence may be an inverted dT modification or a dideoxycytidine modification (entire document; see abstract; “Invention content” p. 2 of English translation; reference claim 1.) Li-2017 further teaches that 3’dideoxy-C and 3’-inverted dT modifications may be used to prevent extension of the DNA strand catalyzed by DNA polymerase ( “Invention content” p. 2 of English translation; Fig. 1 description.)
It would have been obvious to a skilled artisan to employ either of these known polymerase-blocking modifications at the 3’ end of the short adapter strand int eh method of the applied references because Li-2017 expressly teaches the modifications for preventing undesired polymerase extension during sequencing library preparation, thereby rendering the limitations of instant claims 4 and 19 obvious to a skilled artisan in light of the teachings of Li, Paugh, Dong, and DiGiusto.
It would have been obvious to one of ordinary skill in the art to modify the methods taught by Li, Paugh, Dong, and DiGiusto in order to provide the short strand of the adapter with a polymerase blocking modification such as a dT modification, thereby preventing extension of the short adapter strand during subsequent amplification reactions. One would have been motivated to do so, given the suggestion by Li-2017 that an inverted dT or dideoxycytidine modification may be provided at the terminal end of an adapter strand to prevent DNA polymerase-mediated extension and thereby maintain the desired adapter structure during library preparation. There would have been a reasonable expectation of success, given the knowledge that Li already employs an asymmetric double-stranded adapter in an amplification based sequencing library workflow, and this represents a predictable use of a known adapter modification for its established purpose. Thus, the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time the invention was made.
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Li, Paugh, Dong, and DiGiusto as applied to claims 1-2, 5-10, 12-16, and 20-21 above, and further in view of Read et. al. (Read DF, et. al. PLoS Pathog. 2019 Oct 4;15(10):e1007903.; hereafter “Read”.)
The Prior Art
The teachings of Li, Paugh, Dong, and DiGiusto have been set forth supra. While Li teaches the asymmetric double stranded adapter comprises a long strand sequence containing a fixed sequence, and teaches a random UMI sequence of 6-14 bases in length, Li does not expressly teach that the fixed sequence has a length of 14-30 bases. The teachings of Paugh, Dong, and DiGiusto fail to make up for this teaching. However, the fixed sequence length would be obvious to a skilled artisan, given the teachings of Read.
Read teaches a partially double stranded linker for HIV integration site analysis in which genomic DNA is sheared, end-repaired, A-tailed, and ligated to the linker before amplification using HIV LTR- and linker-specific primers. Read teaches a linker formed from a long oligonucleotide and the 15-nucleotide short oligonucleotide GTCCCTTAAGCGGAG, wherein the 15-nucleotide short strand is complimentary to a corresponding fixed region of the long strand and the long strand contains a non-paired dT overhang (“HIV integration-site sequencing” p. 18). Accordingly, Read teaches the use of a 15-base complementary fixed region in a partially double-stranded linker for retroviral integration site amplification, which falls within the claimed range of 14-30 bases as required by instant claim 3.
It would have been obvious to ordinary skill in the art to modify the fixed complementary sequence of the asymmetric adapter taught by Li to have a length of 15 bases, as taught by Read, thereby providing an adapter having a fixed sequence within the claimed range of 14-30 bases while retaining the 10 base random UMI of Li. One would have been motivated to do so because Read teaches use of a complementary linker region in the same technical context of linker-mediated amplification of retroviral integration sites from fragmented, end-repaired, A-tailed genomic DNA. There would have been a reasonable expectation of success because both Li and Read employ partially double-stranded linkers having a complementary region for ligation to genomic DNA followed by linker and LTR-specific amplification of retroviral integration sites. Accordingly, the selection of the known 15-base complementary linker region of Read for the corresponding fixed complementary region of Li would have been a predictable modification of the linker design. Thus, the invention as a whole was clearly prima facie obvious to one of ordinary skill in the art at the time the invention was made.
Claims 17-18 are rejected under 35 U.S.C. 103 as being unpatentable over Li, Paugh, Dong, and DiGiusto as applied to claims 1-2, 5-10, 12-16, and 20-21 above, and further in view of Shah et. al. (Shah NN, et. al. Blood Adv. 2019 Aug 13;3(15):2317-2322.; hereafter “Shah”); and Brogdon et. al. (US20140271635A1, Pub. 09/18/2014; hereafter “Brogdon”).
The Prior Art
The teachings of Li, Paugh, Dong, and DiGiusto have been set forth supra. Li, Paugh, Dong, and DiGiusto render obvious the retroviral integration-site detection method of instant claim 12 for the reasons set forth supra, and while the method teaches detection of lentivirus integration into an immune cell, none specifically alone or in combination teach detection of an engineered immune cell wherein said retroviral vector in said engineered immune cell encodes a chimeric antigen receptor (CAR). However, such a method would be obvious, given the further teachings of Shah and Brogdon.
Shah teaches applying quantitative lentiviral integration site analysis to engineered CAR T cells (entire document; see abstract; pp. 2318-9, ¶ bridging pages.) Shah explains that, because lentiviral vectors randomly integrate in the host genome and the integration site can serve as a unique molecular barcode to track the clonal abundance, quantitative vector integration site analysis was performed to determine if the vector insertion site potentially contributed to the unexpected secondary clonal expansion of the CAR T cells (pp. 2318-9, ¶ bridging pages.) Shah teaches the genomic DNA was randomly sheared, end-repaired, ligated to a linker, amplified using a lentivector-LTR-specific primer and a linker-specific primer, and subjected to high throughput Illumina sequencing (pp. 2319-20; “Integration site analysis”.) Shah further teaches applying integration site analysis to samples from additional patients receiving lentiviral vector modified CAR T cells and explains that integration site analysis is relevant to evaluation of insertional mutagenesis and clonal expansion in gene-modified T cel products (“Results and Discussion”, p. 2320).
Brogdon teaches preparation of engineered immune cells using recombinant lentiviral vectors comprising nucleic acid encoding a chimeric antigen receptor (entire document; see abstract). Brogdon teaches obtaining T cells from a subject, and may include lymphocytes, including T cells, monocytes, granulocytes, B cells, other nucleated white blood cells, red blood cells, and platelets (¶[0268]), but would preferably include T cells (¶[0269-0272]), such as helper T cells (¶[0287]). Brogdon teaches these cells can be manufactured to express the CAR by purifying the T cells from the peripheral blood mononuclear cells, transducing the purified T cells with a CAR-encoding lentiviral vector, expanding the transduced cells, and preserving the resulting CAR-T cell product (¶[0437]).
It would have been obvious to one of ordinary skill in the art to modify the methods of Li, Paugh, Dong, and DiGiusto by applying the resulting retroviral integration site detection method to lentiviral vector engineered CAR immune cells as taught by Shah, preparing those engineered cells using known CAR-vector transduction methods taught by Brogdon, and selecting known CAR-expressing cells, such as helper T cells expressing the CAR, as taught by Brogdon, thereby rendering obvious the limitations of instant claims 17-18.
One would have been motivated to do so, given the suggestion by Shah that lentiviral integration site analysis is useful for evaluating the integration pattern and clonal behavior of engineered CAR T cells, and given the teaching by Brogdon that such CAR T cells are conventionally prepared by obtaining T cells and introducing CAR-encoding lentiviral vectors into those cells. There would have been a reasonable expectation of success, given the knowledge that engineered CAR T cells containing integrated lentiviral vectors were routinely produced by lentiviral transduction, as taught by Brogdon, and also given the knowledge that the resulting lentiviral integration sites could be amplified, sequenced, and quantitatively analyzed in CAR T-cell genomic DNA, as taught by Shah. Therefore, the invention as a whole was prima facie obvious at the time the invention was made.
Conclusion
No claims are allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure and is listed below.
US2020008737A1. Teaches CAR-T manufacturing and analysis of lentiviral vector integration sites from genomic DNA. Not utilized as rejection would be redundant to those set forth supra.
US20210380972A1. Teaches adapter/library limitations in that it teaches formation of asymmetric sequencing library constructs. Not utilized as rejection would be redundant to those set forth supra.
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/RACHEL B GILL/
Primary Examiner, Art Unit 1671
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