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 .
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 06/02/2023 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner.
Objection to Specification
The disclosure is objected to because para. [0094] lines 8, 12 of the specification 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.
Status of Claims
This office action is in response to Applicant's Response to Election / Restriction filed on August 21, 2026. No claims amendment are made in the response filed on August 21, 2026.
Claims 39-58 are currently pending, with claims 49-50 and 54-58 withdrawn.
Claims 39-48 and 51-53 are under examination. This is the first action on the merits.
Election/Restrictions
Applicant’s election without traverse of Group I (claims 39-57) in the reply filed on August 21, 2026 is acknowledged 1.
Applicant’s election without traverse of the following species in the reply filed on August 21, 2026 is acknowledged:
Species of pooled library: A) the pooled library is a gene targeting library (e.g., CRISPR-based targeting library) (claim 39; spec [0060]) 2;
Species of hybrid capture: D) hybrid capture is performed in a solution (claim 48)3;
Species of probe: F) RNA probe (claim 46);
Species of hybrid capture buffer: N) buffer comprises a buffering agent, a salt, a denaturing agent, and a chelating agent (claim 51)4.
Claims 49-50 and 54-58 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention.
Examination on the merits commences on claims 39-48 and 51-53.
Priority
The priority date of the instant claims 39-40, 42-46, 48 and 53 is 10/16/2017, filling date of the US provisional application NO. 62/573,061.
Regarding claims 41, 47, 51-52, the earliest priority is 10/15/2018 because the priority document (PCT/US2018/000383) filed that date is the first to disclose the features recited by these claims, including "wherein the constant region is a promoter, selectable marker, origin of replication, Cas9 gene, a viral vector backbone, a nucleic acid encoding a fluorescent protein, a nucleic acid encoding a peptide tag, a minimal promoter region, a minimal enhancer region, a minimal splice site region, a minimal 5' or 3' untranslated region, or a fragment of each thereof";
"wherein the probe comprises 10 to 3000 nucleotides"; and
"wherein the hybridization buffer comprises a buffering agent, a salt, a denaturing agent, and a chelating agent."
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 39, it recites a "method of preparing a pooled gene targeting library for high throughput sequencing" in the preamble, this claim language is interpreted as descriptive language that does not distinguish the claimed method from prior art methods that disclose all the claimed steps.
MPEP§ 2111.04 states: "Claim scope is not limited by claim language that suggests or makes optional but does not require steps to be performed, or by claim language that does not limit a claim to a particular structure."
Here, the claim body does not include any step that requires "preparing a pooled gene targeting library" or performing "high throughput sequencing." In fact, the claim only recites steps of performing hybrid capture of nucleic acids in "a pooled library" to isolate nucleic acid for subsequent amplification. In other words, the preamble at most states the intended outcome of the claimed method and makes no manipulative difference. Therefore, the claimed method does not distinguish itself from prior art that discloses all the required steps.
For the purpose of applying prior art, claim 39 recites "hybrid capture," which is defined in the specification as follows:
"As used herein, the term “hybrid capture” refers to a quantitative nucleic acid test that uses an efficient signal amplification strategy. " ([0057])
Accordingly, under BRI and in light of the specification, "hybrid capture" is interpreted to encompass any quantitative nucleic acid analysis approach involving signal amplification, such as PCR amplification.
For the purpose of applying prior art, claim 40 recites a "nucleic acid constant region," which is defined in the specification as follows:
"The term “constant region” as used herein refers to any nucleic acid sequence or region in a library or pooled library that does not vary between clones. For example, in a library that comprises cloning vectors, the sequence of the cloning vector backbone is constant while the sequence of the insert (e.g., a cDNA or gene) is variable. Thus, in some embodiments, a suitable constant region can comprise any non-variable sequence within a vector backbone." ([0040])
For the purpose of applying prior art, claim 42 recites "high throughput sequencing," which is not expressly defined in the application's disclosure.
Under BRI and based on the commonly understood meaning by one of ordinary skill in the art (see Logares5, p. 107, introduction, para 2), the term "high throughput sequencing" is interpreted to encompass any sequencing approach having higher throughput than traditional Sanger sequencing, such as Illumina sequencing.
For the purpose of applying prior art, claim 44 recites "the targeting nucleic acids are stably integrated into the genomic DNA of the sample."
The phrase "stably integrated" is defined in the specification as follows:
"As used herein, the term “stably integrated” refers to a polynucleotide that is incorporated into a locus in the genome of a cell or organism, and this incorporation is durable (i.e. the polynucleotide remains integrated in the genomic locus throughout the cell cycle including through DNA replication and mitosis)." ([0064])
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.
Claims 44, 46-47 and 51-52 are rejected under 35 U.S.C. 112(b), 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.
A) Regarding claim 44, it depends from claim 41/40/39 and recites "the targeting nucleic acids," which lacks antecedent basis.
Neither claim 44 nor any of its base claims previously recites "targeting nucleic acids." Although this term is introduced in claim 43, claim 44 does not depend from claim 43.
B) Regarding claim 46, it depends from claim 43/39 and recites "the probe," which lacks antecedent basis. Neither claim 46 nor any of its base claims previously recites a "probe."
Although claim 45 recites "one or more probes," claim 46 does not depend from claim 45.
C) Regarding claim 47, it depends from claim 43/39 and recites "the probe," which lacks antecedent basis. Neither claim 47 nor any of its base claims previously recites a "probe."
Although claim 45 recites "one or more probes," claim 47 does not depend from claim 45.
D) Regarding claim 51, it recites "the hybridization buffer," which lacks antecedent basis.
Neither claim 51 nor its base claim 39 previously recites a "hybridization buffer."
Claim 52 is rejected for depending from claim 51 and not remedying the indefiniteness.
Claim Rejections - 35 USC § 102
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 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.
Claims 39-48 and 51-53 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Brummelkamp (WO2016190743A1 - Assays to identify genetic elements affecting phenotype; published 2016-12-01), as evidenced by
Sakuma (Sakuma et al. Lentiviral vectors: basic to translational. Biochem J. 2012 May 1;443(3):603-18. doi: 10.1042/BJ20120146. PMID: 22507128.) and
Genbank (Retroviral vector pSF71 (GRS), 5' untranslated region; GenBank: AJ132036.1; 27-APR-1999).
Brummelkamp teaches genetic screening of a pool of mutagenized cells and sequencing of mutagenesis sites (Figure 1, 2; pp. 13-20; p. 27, lines 20-27; p. 29-32), which allows the identification of one or more genetic element from the pool of cells that have subjected to the mutagenesis treatment, wherein each genetic element alone, or in combination, affects a phenotype.
Regarding claim 39, Brummelkamp teaches a method comprising:
(a) performing hybrid capture of nucleic acids in a sample comprising a pooled library (p. 30, lines 10-15, a mutant library was generated and expanded; p. 31, lines 8-13, the mutagenized cells are sorted, pooled into pellets for DNA isolation; p. 31, lines 14-17, LAM-PCR used to capture insertion sites, which involves hybrid capture of insertion sites in the cell genome using a biotinylated primer);
(b) isolating the captured nucleic acids (p. 31, lines 20-23, isolating biotinylated extension product comprising the insertion sites via streptavidin-coated magnetic beads); and
(c) amplifying the isolated, captured nucleic acids (p. 32, lines 9-12, PCR amplification prior to sequencing).
Regarding claims 40-41, they are anticipated by Brummelkamp because they recite descriptive statements that do not further distinguish the claimed method from prior art method.
Claims 40-41 recite the following:
40. The method of claim 39, wherein the pooled gene targeting library comprises a nucleic acid constant region.
41. The method of claim 40, wherein the constant region is a promoter, selectable marker, origin of replication, Cas9 gene, a viral vector backbone, a nucleic acid encoding a fluorescent protein, a nucleic acid encoding a peptide tag, a minimal promoter region, a minimal enhancer region, a minimal splice site region, a minimal 5' or 3' untranslated region, or a fragment of each thereof.
Therefore, claims 40-41 describe the “the pooled gene targeting library,” which is merely the intended result of the claimed method, only appearing in the preamble of the base claim 1, which does not distinguish the claimed method from prior art method as it does not make manipulative difference.
Per MPEP 2111.04, a wherein clause can limit a method claim if it contributes meaning and purpose to the manipulative steps.
Here, claims 40-41 do not introduce any additional active steps, nor do they further modify any existing steps. The base claim 39 does not recite any steps of yielding a pooled gene targeting library for high throughput sequencing, or any sequencing steps. As such, the wherein clauses in claims 40-41 merely describe an intended result, without modifying any step of the claimed method. Therefore, this claim language is interpreted as descriptive statement without any associated active steps and do not distinguish the claims from the prior art.
Regarding claim 42, Brummelkamp teaches further comprising (d) performing high throughput sequencing analysis of the amplified nucleic acids produced in step (c) (p. 32, 13-15, Illumina sequencing is high throughput sequencing).
Regarding claim 43, Brummelkamp teaches wherein the pooled library comprises one or more targeting nucleic acids which are guide RNAs (p. 23, lines 4-7, “library may consist of a CRISPR library of guide RNA sequences, wherein each guide RNA targets a limited amount of sequences/positions in the genome of the cell in the pool, but wherein the library consists of different guide RNA's each targeting a specific sequence in the cells ” ; p. 38, lines 10-19; p. 27, lines 32-37 to p. 28, lines 1-2).
Regarding claim 44, Brummelkamp teaches stably integrating a CRISPR library of guide RNA sequences into the genome of cells via lentiviral transduction (p. 32, lines 35-37 to p. 33, lines 1-8 ; p. 23, lines 4-7.) Lentiviral transduction achieves stable vector integration into the cellular genome, as evidenced by Sakuma (Abstract).
Regarding claim 45, Brummelkamp teaches performing targeted amplification of mutagenesis insertion sites using LAM-PCR, which involves hybrid capture with a biotinylated primer (p. 31, lines 14-17, “5'-/double biotin/ggtctccaaatctcggtggaac-3')(SEQ ID NO: 1)”) targeting a nucleic acid constant region of the retroviral vector, as evidenced by Genbank.
The biotinylated primer in Brummelkamp hybridizes to the 5’ untranslated region of retroviral vector.
PNG
media_image1.png
179
624
media_image1.png
Greyscale
Regarding claim 46, Brummelkamp teaches probe comprises one or more biotinylated nucleotides (p. 31, lines 14-17, “5'-/double biotin/ggtctccaaatctcggtggaac-3')(SEQ ID NO: 1)”).
Regarding claim 47, Brummelkamp teaches probe comprises 10 to 3000 nucleotides (p. 31, lines 14-17, “5'-/double biotin/ggtctccaaatctcggtggaac-3')(SEQ ID NO: 1)”).
Regarding claim 48, Brummelkamp teaches the hybrid capture is performed in a solution ( p. 31, lines 20-23, “To capture biotinylated single-stranded DNA (ssDNA) products, PCR reactions were combined with M270 streptavidin in-coated magnetic beads (Life technologies) in 2x binding buffer (6 M LiCI, 10 mM Tris, 1 mM EDTA, pH=7.5) for 2 hours at room temperature and subsequently captured using a magnet. ”).
Regarding claims 51-52, they recite:
51. The method of claim 39, wherein the hybridization buffer comprises a buffering agent, a salt, a denaturing agent, and a chelating agent.
52. The method of claim 51,
wherein the buffering agent is selected from the group of TRIS, HEPES, PIPES, PBS, MES, and MOPS;
wherein the salt is selected from the group of NaCl, LiCl, KCl, and NH4Cl;
wherein the denaturing agent is Urea;
wherein the chelating agent is selected from the group of EDTA, citric acid, EGTA, and NTA; or
wherein the hybridization buffer further comprises one or more ionic detergents, non-ionic detergents, and/or reducing agents.
These claims are anticipated by Brummelkamp because they recite descriptive statements that do not further distinguish the claimed method from prior art method.
Per MPEP 2111.04, a wherein clause can limit a method claim if it contributes meaning and purpose to the manipulative steps.
In this instant case, the "wherein" clauses describe a “hybridization buffer.” However, such a “hybridization buffer” lacks antecedent basis and is not clearly required by any of the steps in the claimed method. Therefore, these claim languages are interpreted as descriptive statements without any associated active steps and do not distinguish the claims from the prior art.
Regarding claim 53, Brummelkamp teaches adding at least one adapter to the isolated, captured nucleic acids (p. 32, lines 5-7, PCR reaction was performed that introduced the adaptors sequences required for Illumina sequencing).
Prior Art
Below are relevant prior art not used in rejection but pertinent to the claims or disclosure.
Performing hybridization capture followed by sequencing is a well-known approach in the field of genetic functional screening via mutagenesis:
Ranzani et al., Lentiviral vector-based insertional mutagenesis identifies genes involved in the resistance to targeted anticancer therapies. Mol Ther. 2014 Dec;22(12):2056-2068. doi: 10.1038/mt.2014.174. Epub 2014 Sep 8. PMID: 25195596; PMCID: PMC4429698;
Joung et al. Genome-scale CRISPR-Cas9 knockout and transcriptional activation screening. Nat Protoc 12, 828–863 (2017). doi.org/10.1038/nprot.2017.016; published March 2017;
Mettananda et al. Editing an α-globin enhancer in primary human hematopoietic stem cells as a treatment for β-thalassemia. Nat Commun 8, 424 (2017). https://doi.org/10.1038/s41467-017-00479-7;
US20140357530A1--Functional genomics using crispr-cas systems, compositions, methods, knock out libraries and applications thereof.
Hybridization capture using a denaturant-containing buffer to optimize for probe hybridization stringency is known in the art:
Yilmaz et al., Modeling formamide denaturation of probe-target hybrids for improved microarray probe design in microbial diagnostics. PLoS One. 2012;7(8):e43862. doi: 10.1371/journal.pone.0043862. Epub 2012 Aug 27. PMID: 22952791; PMCID: PMC3428302.;
Simard et al., Urea substitutes toxic formamide as destabilizing agent in nucleic acid hybridizations with RNA probes. Electrophoresis. 2001 Aug;22(13):2679-83. doi: 10.1002/1522-2683(200108)22:13<2679::AID-ELPS2679>3.0.CO;2-L. PMID: 11545392.
Basicmedicalkey (Nucleic Acid–Based Analytic Methods for Microbial Identification and Characterization; Aug 25, 2016).
Conclusion
No claims are allowed.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to TIAN NMN YU whose telephone number is (703)756-4694. The examiner can normally be reached Monday - Friday 8:30 am - 5:30 pm.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Gary Benzion can be reached at (571) 272-0782. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/TIAN NMN YU/Examiner , Art Unit 1681
1 Claim 58 is withdrawn as being drawn to non-elected group II.
2 Claims 54-57 are withdrawn as being drawn to non-elected species C.
3 Claim 49 is withdrawn as being drawn to non-elected species E.
4 Claim 50 is withdrawn as being drawn to non-elected species I-M.
5 Logares, Ramiro, et al. "Environmental microbiology through the lens of high-throughput DNA sequencing: synopsis of current platforms and bioinformatics approaches." Journal of microbiological methods 91.1 (2012): 106-113.
“During the last five years, the popularization of High Throughput Sequencing (HTS) techniques has revolutionized the field of microbial ecology, promoting multiple research lines. The most important difference between HTS and traditional Sanger sequencing is throughput. While a typical Sanger run would generate 102 sequences (600–900 bp of length), HTS (e.g. 454 and Illumina) can potentially generate 106–109 sequences (100–700 bp) per run (Glenn, 2011, Scholz et al., 2011). Such high-throughput is achieved by massive parallel sequencing, which is carried out differently in the available platforms (Glenn, 2011). One typical trade-off of this increased throughput is reduced read length, although newer versions of the HTS platforms provide longer reads.” (Logares, p. 107, introduction, para 2)
Prosecution Insights