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 .
This Office Action is in reply to Applicants' correspondence of 01/07/2026.
Applicants' remarks and amendments have been fully and carefully considered but are not found to be sufficient to put the application in condition for allowance. New grounds of rejection are presented in this Office Action. Any rejections or objections not reiterated herein have been withdrawn in light of the amendments to the claims or as discussed in this Office Action.
This Action is Non-Final.
Please note: The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim Status
Claims 74-79, 81-82, 87, 90, 92-98, 125, and 135 are pending and being examined on the merits.
Specification
Applicant’s amendments to the specification to properly note trade names or marks used in commerce is acknowledged.
Claim Objections
The objection to claims 92 and 95 is withdrawn in light of Applicant’s amendments of the claims.
Claim Rejections - 35 USC § 112b – Indefiniteness
The rejection of claims 79 and 92 under 35 U.S.C. 112(b) as noted in the Office Action of 7/29/2025 are withdrawn in light of Applicant’s amendments to the claims.
Claim Rejections - 35 USC § 112d – Failure to Further Limit
The rejection of claims 94 and 95 under 35 U.S.C. 112(d) is withdrawn in light of Applicant’s amendments to the claims.
Claim Interpretation
The preamble of claim 135 recites a “kit.” The specification, however, does not define this term, and so it is being interpreted to encompass any collection of reagents that includes all of the elements of the claim. Any further interpretation of the word is considered an “intended use” and does not impart any further structural limitation on the claimed subject matter.
New Claim Rejections - 35 USC § 103
Claims 74-78, 81-82, 87, 90, 92-95, and 125 are rejected under 35 U.S.C. 103 as being unpatentable over Yang (Yang et al., Pharmacogenomics, 2017; cited on PTO-892 of 7/29/2025) in view of Gilpatrick (Gilpatrick et al., bioRxiv, June 4, 2019; cited on IDS submitted on 12/5/2023), Mali (Mali et al., Science 2013), and NCBI Reference Sequence NC 000022.11 (Homo sapiens chromosome 22, GRCh38.p13 Primary Assembly, 9/9/2019).
Regarding claim 81: Yang teaches that CYP2D6 “metabolizes approximately 25% of commonly prescribed drugs, making it one of the most studied enzymes in the field of pharmacogenetics” and that clinical practice has begun to dictate determination of CYP2D6 status in relation to human drug metabolism (Abstract and CYP2D6: discovery & pharmacogenetic implications). However, Yang teaches that CYP2D6 “is a challenging region for short-read technologies due to repetitive elements, CNV, pseudogenes, and a high density of polymorphisms” (CYP2D6 interrogation & allele discovery - CYP2D6 second-generation short-read sequencing). Yang additionally highlights that enrichment-based techniques and short-read alignments are not specific enough to distinguish CYP2D6 from its two highly homologous pseudogenes CYP2D7 and CYP2D8 (CYP2D6 interrogation & allele discovery - CYP2D6 second-generation short-read sequencing).
Gilpatrick teaches analyzing a genetic locus (“ROI” in Figure 1) using a method comprising obtaining genomic DNA comprising said genetic locus (“DNA was extracted” from “cell lines”, Methods – Cell culture and DNA prep), contacting said genomic DNA with a CRISPR-associated endonuclease and two or more gRNAs to excise said genetic locus from said DNA (Methods – Cas9 Cleavage and Library Prep), wherein the two or more gRNAs each comprise nucleotide sequences that are substantially complementary to different nucleotide sequences that flank said genetic locus (“The gRNA duplex was designed to introduce cuts on complementary strands flanking the region of interest”, Methods – Guide RNA design), and then analyzing said genetic locus (claim 73; Figure 1, Results, and Methods – Sequencing and Analysis). Gilpatrick teaches that this methodology is useful for analyzing structural variation and mutations and employs a third-generation sequencing methodology, nanopore sequencing (Abstract and Discussion).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to have used the sequencing method taught by Gilpatrick to sequence the CYP2D6 genetic locus taught by Yang. The ordinary skilled artisan would have been motivated to sequence the CYP2D6 locus using the method of Gilpatrick because Yang teaches a clinical need for sequencing of the CYP2D6, but that conventional short-read sequencing and enrichment strategies such as PCR fail at capturing the complexity of this locus and its corresponding gene and pseudogenes. Gilpatrick teaches the advantages of their methodology when it comes to analyzing structural variations and mutations, namely long-read nanopore sequencing which enables advanced structural analysis of a locus and avoidance of error-prone PCR amplification (Discussion). Additionally, the ordinary artisan would have a reasonable expectation of success in using the method of Gilpatrick to sequence the CYP2D6 locus taught by Yang because Gilpatrick demonstrates usage of long-read sequencing, which Yang highlights as beneficial for analysis of the CYP2D6 locus.
Yang in view of Gilpatrick do not teach the specific gRNA sequences of SEQ ID NOs: 1-26. However, design of gRNAs for efficient cleavage of target sites (such as flanking genomic regions) is known in that art, as taught by Mali.
Mali teaches design of crRNA-tracrRNA fusion transcripts (gRNA) to site-specifically cleave regions in the genome (specifically NGG sites given the usage of the Cas9 enzyme in their system, pg 823 col 1-2). The gRNA sequences that Mali teaches have sequences that match the last 80nt of SEQ ID NOs: 1-13, with the first 20nt being the target-specific region of the gRNA and thus different between gRNAs depending on the sequence to be targeted (Figure 1A).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to have used the gRNA design method as taught by Mali in the design of flanking gRNAs for inclusion in the composition for study of the CYP2D6 locus, as taught by Yang in view of Gilpatrick. Based on the teaching of Mali, the gRNA sequence of SEQ ID NO: 1 would have been obvious given that the locus of CYP2D6, and in particular this gene sequence downstream of said locus, was known in the art as evidenced by the reference provided in the NCBI Reference Sequence NC_000022.11, available on 9/9/2019.
Regarding claims 74-76: Gilpatrick teaches analyzing the genetic locus by nanopore long-read sequencing (Abstract and Discussion).
Regarding claims 77-78: Gilpatrick teaches genotyping said genetic locus (Results - Single Nucleotide Variant Detection) and performing structural analysis of said genetic locus (Results - Structural Variation).
Regarding claim 82: Gilpatrick teaches analyzing a genetic locus that is at least 40 kilobases in length (Figure 3).
Regarding claim 87: Gilpatrick teaches that the CRISPR-associated endonuclease is Cas9 (Results and Figure 1).
Regarding claim 90: Gilpatrick teaches that the genomic DNA is not fragmented, digested, or sheared prior to obtaining the genomic DNA (Results and Methods).
Regarding claim 92: Gilpatrick teaches ligating one or more sequencing adapters to both ends of said genetic locus after said genetic locus is excised from said genomic DNA (Figure 1).
Regarding claims 93-95: Gilpatrick teaches a method that does not involve DNA amplification, does not involve polymerase chain reaction (PCR), and does not involve any one of multiple displacement amplification (MDA), strand displacement amplification (SDA), nucleic acid sequence based amplification (NASBA), loop-mediated isothermal amplification, rolling circle amplification (RCA), ligase chain reaction (LCR), helicase dependent amplification, or ramification amplification (Abstract and Results).
Regarding claim 125: Yang teaches that CYP2D6 “metabolizes approximately 25% of commonly prescribed drugs, making it one of the most studied enzymes in the field of pharmacogenetics” and that clinical practice has begun to dictate determination of CYP2D6 status in relation to human drug metabolism (Abstract and CYP2D6: discovery & pharmacogenetic implications). However, Yang teaches that CYP2D6 “is a challenging region for short-read technologies due to repetitive elements, CNV, pseudogenes, and a high density of polymorphisms” (CYP2D6 interrogation & allele discovery - CYP2D6 second-generation short-read sequencing). Yang additionally highlights that enrichment-based techniques and short-read alignments are not specific enough to distinguish CYP2D6 from its two highly homologous pseudogenes CYP2D7 and CYP2D8 (CYP2D6 interrogation & allele discovery - CYP2D6 second-generation short-read sequencing).
Gilpatrick teaches analyzing a genetic locus (“ROI” in Figure 1) using a method comprising obtaining genomic DNA comprising said genetic locus (“DNA was extracted” from “cell lines”, Methods – Cell culture and DNA prep), contacting said genomic DNA with a CRISPR-associated endonuclease and two or more gRNAs to excise said genetic locus from said DNA (Methods – Cas9 Cleavage and Library Prep), wherein the two or more gRNAs each comprise nucleotide sequences that are substantially complementary to different nucleotide sequences that flank said genetic locus (“The gRNA duplex was designed to introduce cuts on complementary strands flanking the region of interest”, Methods – Guide RNA design), and then analyzing said genetic locus (Figure 1, Results, and Methods – Sequencing and Analysis). Gilpatrick teaches that this methodology is useful for analyzing structural variation and mutations and employs a third-generation sequencing methodology, nanopore sequencing (Abstract and Discussion). Gilpatrick teaches a composition which comprises the two gRNAs flanking the genomic locus of interest and the CRISPR-associated endonuclease (claim 125; Methods - Ribonucleoprotein Complex Assembly).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to form a composition as taught by Gilpatrick with gRNAs designed to flank the CYP2D6 genetic locus taught by Yang. The ordinary skilled artisan would have been motivated to use gRNAs flanking the CYP2D6 locus in the composition of Gilpatrick because Yang teaches a clinical need for sequencing of the CYP2D6, but that conventional short-read sequencing and enrichment strategies such as PCR fail at capturing the complexity of this locus and its corresponding gene and pseudogenes. Gilpatrick teaches the advantages of their methodology when it comes to analyzing structural variations and mutations, namely long-read nanopore sequencing which enables advanced structural analysis of a locus and avoidance of error-prone PCR amplification (Discussion). Additionally, the ordinary artisan would have a reasonable expectation of success in using the composition of Gilpatrick to analyze the CYP2D6 locus taught by Yang because Gilpatrick demonstrates usage of their composition in long-read sequencing, which Yang highlights as beneficial for analysis of the CYP2D6 locus.
Yang in view of Gilpatrick do not teach the specific gRNA sequences of SEQ ID NOs: 1-26. However, design of gRNAs for efficient cleavage of target sites (such as flanking genomic regions) is known in that art, as taught by Mali.
Mali teaches design of crRNA-trcrRNA fusion transcripts (gRNA) to site-specifically cleave regions in the genome (specifically NGG sites given the usage of the Cas9 enzyme in their system, pg 823 col 1-2). The gRNA sequences that Mali teaches have sequences that match the last 80nt of SEQ ID NOs: 1-13, with the first 20nt being the target-specific region of the gRNA and thus different between gRNAs depending on the sequence to be targeted (Figure 1A).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to have used the gRNA design method as taught by Mali in the design of flanking gRNAs for inclusion in the composition for study of the CYP2D6 locus, as taught by Yang in view of Gilpatrick. Based on the teaching of Mali, the gRNA sequence of SEQ ID NO: 1 would have been obvious given that the locus of CYP2D6, and in particular this gene sequence downstream of said locus, was known in the art as evidenced by the reference provided in the NCBI Reference Sequence NC_000022.11, available on 9/9/2019.
Claim 79 is rejected under 35 U.S.C. 103 as being unpatentable over Yang (Yang et al., Pharmacogenomics, 2017; cited on PTO-892 of 7/29/2025) in view of Gilpatrick (Gilpatrick et al., bioRxiv, June 4, 2019; cited on IDS submitted on 12/5/2023), Mali (Mali et al., Science 2013), and NCBI Reference Sequence NC 000022.11 (Homo sapiens chromosome 22, GRCh38.p13 Primary Assembly, 9/9/2019) as applied to claims 74-78, 81-82, 87, 90, 92-95, and 125 above, and further in view of Gabrieli (Gabrieli et al., Nucleic Acids Research, 2018; cited on IDS submitted on 12/5/2023).
The teachings of Yang in view of Gilpatrick and Mali are outlined above. Relevant to the instantly rejected claims, Yang in view of Gilpatrick and Mali teaches using two gRNAs that flank the CYP2D6 locus in combination with Cas9 to excise and analyze the genetic locus of interest.
Yang in view of Gilpatrick and Mali does not teach, prior to performing analysis, isolating high molecular weight DNA comprising said genetic locus wherein said high molecular weight DNA is at least 10 kilobases in length (claim 79).
However, isolation of high molecular weight DNA containing a genomic region of interest was known in the art, as taught by Gabrieli.
Gabrieli teaches a method in which gRNAs flanking a region of interest are combined with Cas9 in order to facilitate excision of a target genomic region (Figure 1 and 3). Gabrieli teaches that the “genome was cleaved at two sites flanking the target according to the complementary gRNAs, and the fragment was isolated by PFGE” (Targeted enrichment of a large E. coli genomic region). Knowledge of the size of the genomic locus encompassed by the gRNAs allowed accurate isolation of the fragment containing the genomic region of interest (Figure 1). Gabrieli teaches isolating a high molecular weight fragment that is 200kb in length (which reads on at least 10 kilobases).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Yang in view of Gilpatrick and Mali to isolate high molecular weight DNA containing the genomic locus of interest prior to analysis, as taught by Gabrieli. One would be motivated to perform this isolation given the assertion by Gabrieli that this step, prior to sequencing analysis, leads to “∼21-fold enrichment” of the target region (Targeted enrichment of a large E. coli genomic region). One would have a reasonable expectation of success given that Gabrieli demonstrates successful use of Cas9/gRNA pair locus excision combined with isolation of high molecular weight DNA (Figure 1 and 2).
Claims 96-98 are rejected under 35 U.S.C. 103 as being unpatentable over Yang (Yang et al., Pharmacogenomics, 2017; cited on PTO-892 of 7/29/2025) in view of Gilpatrick (Gilpatrick et al., bioRxiv, June 4, 2019; cited on IDS submitted on 12/5/2023), Mali (Mali et al., Science 2013), and NCBI Reference Sequence NC 000022.11 (Homo sapiens chromosome 22, GRCh38.p13 Primary Assembly, 9/9/2019) as applied to claims 74-78, 81-82, 87, 90, 92-95, and 125 above, and further in view of Watson (Watson et al., Laboratory Investigation, 2020, Published July 4, 2019; cited on IDS submitted on 12/5/2023).
The teachings of Yang in view of Gilpatrick and Mali are outlined above. Relevant to the instantly rejected claims, Yang in view of Gilpatrick and Mali teaches analyzing a genetic locus of interest in genomic DNA obtained from cell culture (Gilpatrick, Methods - Cell culture and DNA prep).
Yang in view of Gilpatrick and Mali do not teach obtaining genomic DNA from a biological sample (claim 96) that is a body fluid or a solid tissue sample (claim 97), or that is a diagnostic sample (claim 98).
However, obtaining genomic DNA from a biological sample that is a bodily fluid/diagnostic sample is known in the art, as taught by Watson.
Watson teaches a method in which “DNA was isolated from peripheral blood lymphocytes” obtained from patients (Materials and methods). This reads on biological sample (claim 96) that is a body fluid (claim 97), and that is a diagnostic sample (claim 98).
It would have been prima facie obvious to one having ordinary skill in the art, before the effective filing date of the instant application, to have modified the method of Yang in view of Gilpatrick and Mali to apply to biological samples as taught by Watson. One would be motivated to perform the method of Yang in view of Gilpatrick and Mali on biological samples given the assertion by Yang that clinical practice is requiring testing of CYP2D6 locus genotyping in order to determine a patient’s ability to metabolize drugs. One would have a reasonable expectation of success given that Watson successfully applies a methodology similar to Gilpatrick (Cas9/gRNA enrichment and nanopore long-read sequencing, see Figures 1-3 of Watson) on genetic loci targeted in genomic DNA isolated from biological samples obtained from patients.
Claim 135 is rejected under 35 U.S.C. 103 as being unpatentable over Yang (Yang et al., Pharmacogenomics, 2017; cited on PTO-892 of 7/29/2025) in view of Gilpatrick (Gilpatrick et al., bioRxiv, June 4, 2019; cited on IDS submitted on 12/5/2023), Mali (Mali et al., Science 2013), NCBI Reference Sequence NC 000022.11 (Homo sapiens chromosome 22, GRCh38.p13 Primary Assembly, 9/9/2019), and Ahern (The Scientist, 1995; cited on PTO-892 of 7/29/2025).
Yang teaches that CYP2D6 “metabolizes approximately 25% of commonly prescribed drugs, making it one of the most studied enzymes in the field of pharmacogenetics” and that clinical practice has begun to dictate determination of CYP2D6 status in relation to human drug metabolism (Abstract and CYP2D6: discovery & pharmacogenetic implications). However, Yang teaches that CYP2D6 “is a challenging region for short-read technologies due to repetitive elements, CNV, pseudogenes, and a high density of polymorphisms” (CYP2D6 interrogation & allele discovery - CYP2D6 second-generation short-read sequencing). Yang additionally highlights that enrichment-based techniques and short-read alignments are not specific enough to distinguish CYP2D6 from its two highly homologous pseudogenes CYP2D7 and CYP2D8 (CYP2D6 interrogation & allele discovery - CYP2D6 second-generation short-read sequencing).
Gilpatrick teaches analyzing a genetic locus (“ROI” in Figure 1) using a method comprising obtaining genomic DNA comprising said genetic locus (“DNA was extracted” from “cell lines”, Methods – Cell culture and DNA prep), contacting said genomic DNA with a CRISPR-associated endonuclease and two or more gRNAs to excise said genetic locus from said DNA (Methods – Cas9 Cleavage and Library Prep), wherein the two or more gRNAs each comprise nucleotide sequences that are substantially complementary to different nucleotide sequences that flank said genetic locus (“The gRNA duplex was designed to introduce cuts on complementary strands flanking the region of interest”, Methods – Guide RNA design), and then analyzing said genetic locus (claim 73; Figure 1, Results, and Methods – Sequencing and Analysis). Gilpatrick teaches that this methodology is useful for analyzing structural variation and mutations and employs a third-generation sequencing methodology, nanopore sequencing (Abstract and Discussion).
Therefore, it would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to have used the sequencing method taught by Gilpatrick to genotype the CYP2D6 locus taught by Yang. The ordinary skilled artisan would have been motivated to genotype the CYP2D6 locus using the method of Gilpatrick because Yang teaches a clinical need for genotyping of the CYP2D6 locus, but that conventional short-read sequencing and enrichment strategies such as PCR fail at capturing the complexity of this locus. Gilpatrick teaches the advantages of their methodology when it comes to analyzing structural variations and mutations, namely long-read nanopore sequencing which enables advanced structural analysis of a locus and avoidance of error-prone PCR amplification (Discussion). Additionally, the ordinary artisan would have a reasonable expectation of success in using the method of Gilpatrick to sequence the CYP2D6 locus taught by Yang because Gilpatrick demonstrates usage of long-read sequencing, which Yang highlights as beneficial for analysis of the CYP2D6 locus.
Yang in view of Gilpatrick do not teach the specific gRNA sequences of SEQ ID NOs: 1-26. However, design of gRNAs for efficient cleavage of target sites (such as flanking genomic regions) is known in that art, as taught by Mali.
Mali teaches design of crRNA-tracrRNA fusion transcripts (gRNA) to site-specifically cleave regions in the genome (specifically NGG sites given the usage of the Cas9 enzyme in their system, pg 823 col 1-2). The gRNA sequences that Mali teaches have sequences that match the last 80nt of SEQ ID NOs: 1-13, with the first 20nt being the target-specific region of the gRNA and thus different between gRNAs depending on the sequence to be targeted (Figure 1A).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to have used the gRNA design method as taught by Mali in the design of flanking gRNAs for inclusion in the composition for study of the CYP2D6 locus, as taught by Yang in view of Gilpatrick. Based on the teaching of Mali, the gRNA sequence of SEQ ID NO: 1 would have been obvious given that the locus of CYP2D6, and in particular this gene sequence downstream of said locus, was known in the art as evidenced by the reference provided in the NCBI Reference Sequence NC_000022.11, available on 9/9/2019.
Yang in view of Gilpatrick and Mali does not teach providing the reagents necessary for genoptying of CYP2D6 in a kit.
However, providing reagents necessary for performing a methodology within a kit is well-known in the art, as taught by Ahern.
Ahern teaches aspects of kits of reagents. Ahern teaches that a kit supplies all of the necessary reagents for a particular application and provides detailed instructions to follow (p. 20 – The kit concept, Saving time and money).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to have provided the reagents necessary for performing the method Yang in view of Gilpatrick and Mali in a kit, as taught by Ahern. One would be motivated to include these reagents in a kit given the assertion by Ahern that inclusion of reagents in a kit saves time and money and is highly convenient (p. 20 – The kit concept, Saving time and money).
Response to Remarks
Applicant has amended the claims to include SEQ ID NOs: 1-26 in the method of analyzing a genetic locus (by cancelling claim 73 and including all limitations in claim 81, which originally claimed SEQ ID NOs: 1-26). The gRNA sequences were indicated as allowable subject matter in the previous office action. However, upon further consideration, the gRNA sequence of SEQ ID NO: 1 is considered obvious given the teachings of Mali and NCBI Reference Sequence NC 000022.11 regarding gRNA design for a region downstream of the CYP2D6 locus.
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.
Claims 74-78, 81, 87, 90, 92-98, and 135 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-5, 9-10, 16-17, 20-23, 28, 31, 33-34, 42-44, 46-48, 57, and 72 of copending Application No. 18/554,174 in view of Yang (Yang et al., Pharmacogenomics, 2017; cited on PTO-892 of 7/29/2025), Mali (Mali et al., Science 2013), and NCBI Reference Sequence NC 000022.11 (Homo sapiens chromosome 22, GRCh38.p13 Primary Assembly, 9/9/2019).
Although the claims are not identical, they are not patentably distinct from each other because both sets of claims are drawn to the same limitations. Any additional limitations of the ‘174 claims are encompassed by the open claim language “comprising” found in the instant claims.
Regarding claim 81: Claims 1 and 5 of ‘174 “A method of analyzing a genomic region of interest, said method comprising: a) contacting genomic DNA comprising said genomic region of interest with a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease and an outer pair of guide RNAs (gRNAs)” (claim 1) in which the outer pair of gRNAs comprise a first and second outer gRNA and “(ii) said first outer gRNA comprises a nucleotide sequence that is substantially complementary to a first nucleotide sequence present in said genomic DNA, and said second outer gRNA comprises a nucleotide sequence that is substantially complementary to a second nucleotide sequence present in said genomic DNA; (iii) said first nucleotide sequence and said second nucleotide sequence are different, and (iv) said first nucleotide sequence and said second nucleotide sequence flank said genomic region of interest” (claim 5). Claim 1 then teaches “analyzing said genomic region of interest”. Claims 1 and 5 of ‘174 do not explicitly teach providing or obtaining genomic DNA comprising said genetic locus, however for the method to be performed and analysis to be done genomic DNA necessarily had to be provided or obtained.
Claims 1 and 5 of ‘174 do not teach analyzing a genetic locus comprising CYP2D6, CYP2D7, and CYP2D8. However, Yang teaches the clinical need of sequencing CYP2D6 genomic locus and the need for methods that utilize long-read sequencing (as detailed above and repeated briefly below).
Yang teaches that CYP2D6 “metabolizes approximately 25% of commonly prescribed drugs, making it one of the most studied enzymes in the field of pharmacogenetics” and that clinical practice has begun to dictate determination of CYP2D6 status in relation to human drug metabolism (Abstract and CYP2D6: discovery & pharmacogenetic implications). However, Yang teaches that CYP2D6 “is a challenging region for short-read technologies due to repetitive elements, CNV, pseudogenes, and a high density of polymorphisms” (CYP2D6 interrogation & allele discovery - CYP2D6 second-generation short-read sequencing). Yang additionally highlights that enrichment-based techniques and short-read alignments are not specific enough to distinguish CYP2D6 from its two highly homologous pseudogenes CYP2D7 and CYP2D8 (CYP2D6 interrogation & allele discovery - CYP2D6 second-generation short-read sequencing).
The ordinary skilled artisan would have been motivated to sequence the CYP2D6 locus using the method ‘174 because Yang teaches a clinical need for sequencing of the CYP2D6 locus, but that conventional short-read sequencing and enrichment strategies such as PCR fail at capturing the complexity of this locus and its corresponding gene and pseudogenes.
Claims 1 and 5 of ‘174 in view of Yang do not teach that the sequence of at least one of the two outer gRNAs is selected from the group consisting of SEQ ID NOs: 1-26. However, design of gRNAs for efficient cleavage of target sites (such as flanking genomic regions) is known in that art, as taught by Mali.
Mali teaches design of crRNA-tracrRNA fusion transcripts (gRNA) to site-specifically cleave regions in the genome (specifically NGG sites given the usage of the Cas9 enzyme in their system, pg 823 col 1-2). The gRNA sequences that Mali teaches have sequences that match the last 80nt of SEQ ID NOs: 1-13, with the first 20nt being the target-specific region of the gRNA and thus different between gRNAs depending on the sequence to be targeted (Figure 1A).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to have used the gRNA design method as taught by Mali in the design of flanking gRNAs for inclusion in the composition for study of the CYP2D6 locus, as taught by Yang in view of Gilpatrick. Based on the teaching of Mali, the gRNA sequence of SEQ ID NO: 1 would have been obvious given that the locus of CYP2D6, and in particular this gene sequence downstream of said locus, was known in the art as evidenced by the reference provided in the NCBI Reference Sequence NC_000022.11, available on 9/9/2019.
Regarding claims 74-76: Claims 16 and 42-43 of ‘174 teach sequencing analysis (claim 16), specifically long-read sequencing (claim 42) that is accomplished by single-molecule real time sequencing or nanopore sequencing (claim 43).
Regarding claims 77-78: Claim 16 of ‘174 teaches genotyping and structural analysis of the genomic region of interest.
Regarding claim 87: Claim 28 of ‘174 teaches using Cas9.
Regarding claim 90: Claim 31 of ‘174 teaches “genomic DNA is not fragmented, digested, or sheared prior to a)”.
Regarding claims 93-95: Claims 22 and 44 teach “said method does not involve DNA amplification” (claim 22) and “wherein said method does not involve any one of polymerase chain reaction (PCR) or isothermal amplification” (claim 44).
Regarding claims 96-98: Claims 46-48 teach using a biological sample that is a body fluid or solid tissue sample and that is a diagnostic sample.
Regarding claim 135: Claim 57 of ‘174 teaches a kit for analyzing a genomic region of interest that includes a CRISPR-associated endonuclease and a flanking pair of gRNAs.
‘174 does not teach that these gRNAs are designed to be upstream and downstream of a genomic locus comprising CYP2D6, CYP2D7, and CYP2D8.
However, Yang teaches the clinical need of sequencing CYP2D6 genomic locus and the need for methods that utilize long-read sequencing (as detailed above and repeated briefly below).
Yang teaches that CYP2D6 “metabolizes approximately 25% of commonly prescribed drugs, making it one of the most studied enzymes in the field of pharmacogenetics” and that clinical practice has begun to dictate determination of CYP2D6 status in relation to human drug metabolism (Abstract and CYP2D6: discovery & pharmacogenetic implications). However, Yang teaches that CYP2D6 “is a challenging region for short-read technologies due to repetitive elements, CNV, pseudogenes, and a high density of polymorphisms” (CYP2D6 interrogation & allele discovery - CYP2D6 second-generation short-read sequencing). Yang additionally highlights that enrichment-based techniques and short-read alignments are not specific enough to distinguish CYP2D6 from its two highly homologous pseudogenes CYP2D7 and CYP2D8 (CYP2D6 interrogation & allele discovery - CYP2D6 second-generation short-read sequencing).
The ordinary skilled artisan would have been motivated to sequence the CYP2D6 locus using the method ‘174 because Yang teaches a clinical need for sequencing of the CYP2D6 locus, but that conventional short-read sequencing and enrichment strategies such as PCR fail at capturing the complexity of this locus and its corresponding gene and pseudogenes. One would thus be motivated to include the necessary gRNAs flanking said genomic locus in order to facilitate analysis.
Claim 56 of ‘174 in view of Yang do not teach that the sequence of at least one of the two outer gRNAs is selected from the group consisting of SEQ ID NOs: 1-26. However, design of gRNAs for efficient cleavage of target sites (such as flanking genomic regions) is known in that art, as taught by Mali.
Mali teaches design of crRNA-tracrRNA fusion transcripts (gRNA) to site-specifically cleave regions in the genome (specifically NGG sites given the usage of the Cas9 enzyme in their system, pg 823 col 1-2). The gRNA sequences that Mali teaches have sequences that match the last 80nt of SEQ ID NOs: 1-13, with the first 20nt being the target-specific region of the gRNA and thus different between gRNAs depending on the sequence to be targeted (Figure 1A).
It would have been prima facie obvious to one of ordinary skill in the art prior to the effective filing date to have used the gRNA design method as taught by Mali in the design of flanking gRNAs for inclusion in the composition for study of the CYP2D6 locus, as taught by Yang in view of Gilpatrick. Based on the teaching of Mali, the gRNA sequence of SEQ ID NO: 1 would have been obvious given that the locus of CYP2D6, and in particular this gene sequence downstream of said locus, was known in the art as evidenced by the reference provided in the NCBI Reference Sequence NC_000022.11, available on 9/9/2019.
Claim 79 is provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1-5, 9-10, 16-17, 20-23, 28, 31, 33-34, 42-44, 46-48, 57, and 72 of copending Application No. 18/554,174 in view of Yang (Yang et al., Pharmacogenomics, 2017; cited on PTO-892 of 7/29/2025), Mali (Mali et al., Science 2013), and NCBI Reference Sequence NC 000022.11 (Homo sapiens chromosome 22, GRCh38.p13 Primary Assembly, 9/9/2019) as applied to claims 74-78, 81, 87, 90, 92-98, and 135 above and further in view of Gabrieli (Gabrieli et al., Nucleic Acids Research, 2018; cited on IDS submitted on 12/5/2023) according to the citations and rationales provided above.
Claims 82 and 125 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1, 5, 16, 22-23, 28, 31, 42-44, 46-48, and 57 of copending Application No. 18/554,174 in view of Yang (Yang et al., Pharmacogenomics, 2017; cited on PTO-892 of 7/29/2025), Mali (Mali et al., Science 2013), and NCBI Reference Sequence NC 000022.11 (Homo sapiens chromosome 22, GRCh38.p13 Primary Assembly, 9/9/2019) as applied to claims 74-78, 81, 87, 90, 92-98, and 135 above and further in view of Gilpatrick (Gilpatrick et al., bioRxiv, June 4, 2019; cited on IDS submitted on 12/5/2023).
Regarding claim 82: Copending application ‘174 teaches analyzing a genomic region of interest. However, ‘174 does not teach wherein the genomic region of interest is at least 40 kilobases in length.
However, using flanking gRNA sequences and Cas9 to analyze a genomic region of interest at least 40kb in length is known in the art, as taught by Gilpatrick.
Gilpatrick teaches analyzing a genetic locus that is at least 40 kilobases in length (Figure 3).
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the instant application, to use the method of ‘174 to analyze a genomic locus that is at least 40kb in length, as taught by Gilpatrick. One would be motivated to analyze larger genomic loci given the assertion by Gilpatrick that this length allows for analysis of large structural variants in genomic loci (Results - Structural Variation). One would have a reasonable expectation of success given that Gilpatrick successfully analyzes a locus of interest over 40kb in length using flanking gRNAs/Cas9 (Figure 3).
Regarding claim 125: The claims of ‘174 do not teach a composition comprising the flanking gRNAs and CRISPR-associated endonuclease. However, inclusion of gRNAs and a CRISPR-associated endonuclease in a composition is known in the art, as taught by Gilpatrick.
Gilpatrick teaches a composition which comprises the two gRNAs flanking the genomic locus of interest and the CRISPR-associated endonuclease (claim 125; Methods - Ribonucleoprotein Complex Assembly).
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the instant application, to produce a composition as taught by Gilpatrick that could be used to perform the method of ‘174. One would be motivated to produce this composition given the teaching by Gilpatrick that this allows the assembly of the Ribonucleoprotein Complex prior to applying this complex to the genomic DNA (Methods - Ribonucleoprotein Complex Assembly). One would have a reasonable expectation of success given that Gilpatrick demonstrates successful generation of this composition and uses this composition to carry out a method similar to that taught by ‘174.
This is a provisional nonstatutory double patenting rejection.
Response to Remarks
As noted in the Response to Remarks of the 103 rejections above, upon further consideration of the SEQ ID NOs in claim 81, SEQ ID NO: 1 would have been obvious given the teachings of Mali and NCBI Reference Sequence NC 000022.11. Therefore, the above Double Patenting rejection has been amended to incorporate said teachings.
Conclusion
No claims are allowed.
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/KAILEY ELIZABETH CASH/Examiner, Art Unit 1683
/STEPHEN T KAPUSHOC/Primary Examiner, Art Unit 1683