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 .
Priority
Applicant’s claims of priority from US Provisional Applications Nos. 62/008,832 filed 06/06/2014 and 62/017,916 filed 06/27/2014 are hereby acknowledged.
Application Status
Amendments to claims filed 04/27/2026 are hereby acknowledged. Claims 1 and 7 are currently amended. Claims 4, 6, 8, 10 and 12 are cancelled.
Claims 13 and 24-25 are still withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected Species.
Therefore, claims 1-3, 5, 7, 9, 11, 13-37 are pending and claims 1-3, 5, 7, 9, 11, 14-23 and 26-37 are under consideration in this office action.
Any objection or rejection not reiterated herein has been overcome by Applicant’s amendments and is therefore withdrawn.
The following rejections are new ground of rejections as necessitated by Applicant’s amendments:
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 non-obviousness.
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, 7, 11, 14, 16, 21, 27, 28, 29, and 32-37 are rejected under 35 U.S.C. § 103 as being unpatentable over Collin (Collin, J. et al. “Concise review: Putting a finger on stem cell biology: Zinc Finger Nuclease-driven targeted genetic editing in human pluripotent stem cells”. Stem Cells, Vol. 29 (2011), pp: 1021-1033; previously cited) in view of Zhang (Zhang, F. US Patent No. 8,795,965 B2, published August 5, 2014, benefitting from priority of US Application No. 14/183,486 filed February 18, 2014; previously cited) and Raynal (Raynal, A. et al. “Excisable cassettes: New tools for functional analysis of Streptomyces genomes”. Applied and Environmental Microbiology, Vol. 72, No. 7 (2006), pp: 4839-4844).
Regarding claim 1(a), Collin teaches a method for serial modifications of a target locus in a cell comprising a target locus with a gene which comprises recognition sites for a ZFN for targeted genomic modifications (see title and abstract). Collin teaches that the first nuclease recognition site is located in a coding region of a first selection marker (see Figure 2). Collin teaches ZFN-mediated targeted gene editing (see title, abstract and page 1022, right column, and page 1024, Figure 2B). In Figure 2B, Collin teaches the use of ZFN nuclease to correct a mutation in exon 1 of a gene, i.e. a target locus. This mutated exon can be then targeted for repair by Homologous recombination (HDR), using a targeting vector comprising homology arms flanking a cassette comprising a selection marker, i.e. puromycin. See below:
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In Figure 2B, Collin teaches the disruption and knock-out of exon 1 of a gene by inserting a selectable marker, using ZFNs. In Figure 2C, Collin teaches mutagenesis of exon 1 of a gene in a cassette to reproduce a mutated allele and the targeting of the cassette using a secondary targeting cassette comprising a selection marker, also using ZFNs.
Collin teaches a polynucleotide comprising the polynucleotide encoding a first nuclease agent that is introduced into the cells.
Regarding claim 1(b)(i), Collin teaches the nuclease agent ZFN induces a double-strand break at the first nuclease recognition site (see Figure 1).
Regarding claim 1(b)(ii), Collin teaches a targeting vector comprising an insert polynucleotide flanked by an upstream homology arm corresponding to an upstream target site and a downstream homology arm corresponding to a downstream target site located in the target locus (see Figure 2’s legend, on page 1023; see page 1024, left column), wherein the insert polynucleotide encodes a selection marker operably linked to a promoter (see Figure 2B, C and D; see Table 2 and page 1028, left column).
Applicant does not disclose the definition of a selection marker, only some embodiments, therefore Examiner interprets “selection marker” as encompassed by the BRI (Broad and Reasonable Interpretation) of the terms, which is any marker allowing distinction and selection between non-modified and modified cells.
Regarding claim 1(b)(ii) reciting “wherein the first selection marker and the second selection marker are different”, Collin teaches puromycin or EGFP as selection markers (see Figures 2B, C and D).
Collin also teaches using multiple ZFNs. Collin also teaches the targeting of both alleles at a AAVS1 locus using AAVS1-ZFNs and two different donor DNAs, with two promoter-less resistance markers driven by the PPP1R12C promoter and two separate promoter-transcription units (see page 1029, left column, first paragraph). Examiner interprets that the need for multiple ZFNs means that they need to be different. Figure 1 shows that ZFNs work in pairs; Panel A shows a module consisting in four ZFPs (Zinc Finger Proteins) which recognize a composite site of 24 bp.
In claim 1(c ), regarding the recitation “identifying a modified cell comprising the insert polynucleotide integrated at the target locus, wherein the modified cell has the activity of the second selection marker but does not have the activity of the first selection marker”, Collin does not teach identifying a modified cell comprising the insert polynucleotide integrated at the target locus, wherein the modified cell has the activity of the second selection marker but does not have the activity of the first selection marker.
Although Collin teaches a recognition site within the first selection marker, Collin do not teach the limitation recited in claim (b)(ii): “wherein the insert polynucleotide encodes …a second nuclease recognition site for a second nuclease agent, wherein the second nuclease recognition site is located within a coding region of the second selection marker”.
However, Zhang teaches serial modifications, i.e. “sequential introduction of mutations” by CRISPR-mediated genome editing (see column 14, lines 21-45 and Figure 34A-D).
Regarding claim (b)(ii): “wherein the insert polynucleotide encodes …a second nuclease recognition site for a second nuclease agent, wherein the second nuclease recognition site is located within a coding region of the second selection marker”, Zhang teaches the introduction of mutation or deletion of sequences within a gene marker and using the CRISPR- Cas system, for example introduction of amino acid substitutions R>A and NE>AA mutations into β-galactosidase (bgaA) (see Figure 25c and column 13, lines 11-13) and deletion of 6664bp within bga A ORF (see figures 25c and 25f; column 13, lines 14-16). Therefore, Zhang teaches mutating a selection marker (see Figure 25e, Editing template 2) and selection with Kanamycin (see Figure 25e).
Regarding claim 1(c ) reciting “identifying a modified cell comprising the insert polynucleotide integrated at the target locus, wherein the modified cell has the activity of the second selection marker but does not have the activity of the first selection marker”, Zhang teaches (see Figures 34 A-D) that “First R6 is engineered to generate crR6Rk” (see column 14, line 24). Zhang teaches that R6 is the name of the Streptococcus pneumoniae strain used (see ref# 39, column 140); crR6 is a strain where IS1167 element of S. pneumoniae is replaced by the CRISPR01 locus of S. pyogenes SF370 strain using a first targeting vector; then crR6M is further engineered to contain a minimal functional CRISPR system without cas1, cas2 and Csn2 (see column 12, lines 46-55); crR6Rk is engineered from crR6M to contain tracrRNA , Cas9 and only one repeat of the CRISPR array followed by Kanamycin resistance marker (aphA-3) after another targeting and recombination event (see column 13, lines 39-42).
Zhang teaches that crR6Rk is then co-transformed with a srt.4-targeting construct fused to CAT for chloramphenicol selection of edited cells, along with an editing construct for a ΔsrtA in-frame deletion (column 14, lines 29-32). Zhang teaches that srtA is a sortase allowing proteins to remain anchored on cell surface, located in srt.4 locus; srtA gene was engineered to incorporate a protospacer from streptococcal bacteriophage; deleting srtA in the engineered strain crR6 results in release of β-galactosidase in the cell medium (column 14, lines 36-42).
Zhang teaches that strain crR6 ΔsrtA is generated by selection on chloramphenicol. Subsequently, the ΔsrtA strain is co-transformed with a bgaA-targeting construct fused to aphA-3 for kanamycin selection of edited cells, and an editing construct containing a ΔbgaA in-frame deletion.” (see column 14, lines 21-32 and Figure 34A-D). [ bgaA is β-galactosidase gene (column 13, line 13).]
Zhang teaches that “such a sequential selection can be iterated as many
times as required to generate multiple mutations”. (see column 90, lines 4-18).
Regarding claim 1(c ) and claim 5, Zhang teaches isolating individual clone cells , then expanding the cells for genotype analysis using PCR to detect the presence or absence of deleted sequences (see column 17, lines 31-34, and lines 42-45). Zhang teaches that the cells can be isolated, their nuclei further isolated for detection using assays such as DNA cleavage or mutation at the target sequence, or assay for altered gene expression activity as compared to control (column 28, lines 10-21).
Therefore, Zhang teaches CRISPR-Cas system with guide RNA and targeting constructs transfected in stable transformants selected a first time with a first selection marker flanked by two sgRNAs recognition sites (see Figs.34A-D). Zhang teaches replacement of a first selection marker with a second selection marker and selected cells using the second selection marker (Figs. 34A-D and column 14, lines 21-45).
In conclusion, Zhang teaches that “The understanding of gene function depend in the possibility of altering DNA sequences within the cell in a controlled fashion. Site-specific mutagenesis in eukaryotes is achieved by the use of sequence-specific nucleases that promotes homologous recombination of a template DNA containing the mutation of interest. Zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and homing meganucleases can be programmed to cleave genomes in specific locations, but these approaches require engineering of new enzymes for each target sequence. In prokaryotic organism, mutagenesis methods either introduce a selection marker in the edited locus or require a two-step process that includes a counter-selection system….Therefore, new technologies that are affordable, easy to use and efficient are still in need for the genetic engineering of both eukaryotic and prokaryotic organisms” (see Example 5 “RNA-guided editing of Bacterial genomes using CRISPR-Cas systems”, column 86, lines 41-63).
It would have been obvious to one with ordinary skills before the effective filing date of the claimed invention, to have combined the teachings of Collin and Zhang, and performed serial modifications as taught by Collin, and using the CRISPR-Cas system and selection markers taught by Zhang. One with ordinary skill in the art could have performed this modification replacing the ZFNs taught by Collin with a CRISPR-Cas system taught by Zhang, since using the most recent engineering advances leads to more specificity and precision. One with ordinary skills in the art, could have inserted the sgRNA recognition sequence within a selectable marker coding sequence, and deactivated the marker as taught by Collin and Zhang, and using the CRISPR-Cas system taught by Zhang. One motivated in serial modification of a target locus, and motivated in practical and affordable methods, and in increasing efficiency and specificity in homologous recombination to engineer specific alterations, could have use the system taught by Zhang and performed this modification with a reasonable expectation of success and arrived at the claimed invention.
The combination of Collin and Zhang does not teach targeting specifically a first selection marker wherein the first selection marker imparts resistance to a first antibiotic, nor targeting a second selection marker wherein the second selection marker imparts resistance to a second antibiotic.
However, Raynal teaches using selectable antibiotic resistance markers in cassettes that were used to inactivate genes of interest through insertional mutagenesis (see page 4839, left column, second paragraph). Raynal further teaches that these markers are widely used to select genetic transformants, and that they may have unintended detrimental environmental impact, i.e., spreading antibiotic resistance. Raynal also teaches that there are other reasons for removing and/or recycling selective markers, especially when several modifications are required. Raynal states: “If every genetic modification in a given strain results in an antibiotic marker being retained in the modified organism, and if the combination of several mutations needs to be analyzed, it becomes progressively more difficult to find new antibiotic resistance genes to use. Moreover, this situation prevents a plethora of selectable markers from being used in subsequent rounds of gene modification in the same host (see page 4839, left column, second paragraph).
Therefore, Raynal teaches removing and replacing selectable antibiotic resistance genes in a host, via steps including a step of removing the cassette via a site-specific excision event promoted by the expression of other genes in trans (see page 4839, right column, last sentence, and page 4840, left column, lines 1-2, and see figure 2).
Raynal’s teachings therefore suggest that a problematic antibiotic resistance gene could be targeted and removed using a system for serial genetic modifications, and replaced with another antibiotic resistance gene of interest.
It would have been obvious to one with ordinary skills in the art, before the effective filing date, to have used the system as taught by Collin modified by Zhang, and targeted an antibiotic resistance gene / selection marker, to remove and replace the antibiotic resistance gene / selection marker, as suggested by the teachings of Raynal. One with ordinary skills in the art, motivated in preserving the use of a specific host through serial modifications and recycling selectable markers, could have performed this modification with a reasonable expectation of success and arrived at the claimed invention.
Collin also teaches limitations in dependent claims:
Regarding claim 2, Collin teaches a method, using, wherein a polynucleotide encoding a selection marker is flanked by upstream target site and downstream target site (see Figure 2B, C and D).
Regarding claim 7, Collin teaches a selection marker that can be an antibiotic (i.e. puromycin) (see Figure 2B and C).
Regarding claim 32, Collin teaches loss of the first selection marker’s activity after ZFN-induced double strand break (see Figure 2).
Regarding claim 33, Collin teaches inserting an insert polynucleotide within a first gene, that can be a selectable marker and introducing a second selectable marker in a cassette (see figure 2B).
Regarding claims 34-37, Collin teaches eukaryotic cells, mammalian cells (mammalian cells: human embryonic stem cells; see Table 2) and ES cells from mice (see page 1021, right column, first paragraph; page 1024, right paragraph).
Zhang also teaches limitations in dependent claims:
Regarding claim 11 (III), Zhang teaches an expression construct comprising a DNA sequence encoding the Cas9, wherein the DNA sequence encoding the Cas9 is operably linked to a promoter active in the cell (see column 3, lines 21-35; column 4, line 65 to column 5, line 6). Zhang also teaches an expression construct comprising a DNA sequence encoding the gRNA, wherein the DNA sequence encoding the gRNA is operably linked to a promoter active in the cell (see column 5, lines 30-60).
Regarding claim 14, Zhang teaches a CRISPR-Cas protein and a guide RNA that can be used as first and second nuclease agents in sequential mutagenesis (see column 14, lines 21-45, column 90, lines 17-18 and Figure 34A-D).
Zhang discloses PAM sequence (See Figure 24 B) and CRISPR target site and the design of gRNA sequence comprising CRISPR RNA and tracrRNA (see column 24, lines 4-15; Figures 31, 34A-D, 36).
Regarding claim 16, Zhang teaches a first and a second gRNA that are different ( see column 25, lines 39-53).
Regarding claim 21, Zhang teaches a polynucleotide of interest comprises an exogenous nucleic acid sequence, in the form of a Cas9 expressing cassette (see Figure 34A-D) or a nucleic acid sequence that is homologous to a nucleic acid sequence in the genome of the cell (see column 7, lines 11-30 and column 30 line 60 to column 31, line 14).
Regarding claim 28, Zhang teaches the replacement of the IS1167 element of S. pneumoniae R6 with the CRISPR01 locus of S. pyogenes SF370 to generate crR6 strain (see Figures 28A-B and column 12, lines 47-51). Zhang also states that “To introduce specific changes in the genome, one must use an editing template carrying mutations that abolish Cas9-mediated cleavage therefore preventing cell death. This is easy to achieve when the deletion of the target or its replacement by another sequence (gene insertion) is sought” (see column 88, lines 13-17).
Regarding claim 27, Zhang teaches gene swap, point mutation, Knock-out or knock-in using integration of the insert polynucleotide ( see column 55, lines 1-4; see column 119, lines 44-49; see column 88, lines 13-17).
Regarding claim 29, Zhang teaches that reporter gene integrated within the mouse genome (see page 2, “Introduction” section, third paragraph). Therefore, the target locus is in the genome of the cell.
The obviousness of combining the references Collin, Zhang and Raynal, is described above; The references teach elements of claims 2, 6-7, 11, 14, 16, 21, 27-28 and 32-37 as described above. Therefore, the combination of references also renders the said claims obvious, and the claims 2, 6-7, 11, 14, 16, 21, 27-28 and 32-37 are rejected as well.
Claims 19, 20 and 26 are rejected under 35 U.S.C. § 103 as being unpatentable over Collin (Collin, J. et al. “Concise review: Putting a finger on stem cell biology: Zinc Finger Nuclease-driven targeted genetic editing in human pluripotent stem cells”. Stem Cells, Vol. 29 (2011), pp: 1021-1033; previously cited), in view of Zhang (Zhang, F. US Patent No. 8,795,965 B2, published August 5, 2014, benefitting from priority of US Application No. 14/183,486 filed February 18, 2014; previously cited) and Raynal (Raynal, A. et al. “Excisable cassettes: New tools for functional analysis of Streptomyces genomes”. Applied and Environmental Microbiology, Vol. 72, No. 7 (2006), pp: 4839-4844) , as applied to claim 1 above, and in further view of Hall (Hall, B. et al. “Overview: generation of gene knockout mice”. Current Protocol in Cell Biology, Vol. 44 (2009), pp: 19.12.1-19.12.17; previously cited).
Regarding claim 1, its rejection has been described above. The elements of claim 1 are rendered obvious by the combination of Collin, Zhang and Raynal.
Zhang’s teachings further suggest elements of claim 20, without teaching them directly: Regarding claim 20 (iv), Zhang also teaches that one can take advantage of highly evolved processes for targeting a virus to specific cells in the body, as gene transfer system, since retroviral vectors are comprised of cis-acting long terminal repeats with packaging capacity for up to 6-10 Kb of foreign sequence (see column 32, lines 49-57).
However, the combination of references fails to disclose the elements claimed in claims 19, 20 and 26.
Hall reviews the technique for obtaining a knock-out mice (see title). Hall teaches that the initial step for the generation of a mouse with a targeted mutation is the construction of an efficient targeting vector that will be introduced into the murine embryonic stem (ES) cells (see abstract).
Regarding claims 19 and 20, Hall teaches strategies wherein the upstream and downstream homology arms can range from 1-2 kb to up to 110 kb (see page 19.12.11, right column, second paragraph). Hall teaches that 2kb of sequence homology is usually required for designing a targeting vector for recombination to occur at all in a cell. However, 6 to 14 kb of sequence homology is typical usually for targeting vectors (see page 19.12.3; right column, last paragraph).
It would have been obvious to one of ordinary skills in the art, before the effective filing date of the claimed invention to have designed a targeting vector as taught by Collin modified by Zhang and Raynal, and added at least 6 kb of homolog sequence for the homology arms flanking the target site in the cassettes taught by Zhang. One with ordinary skills in the art motivated in optimizing and ensuring recombination in a cell, could have performed this modification with a reasonable expectation of success, since Zhang teaches the packaging capacity of a viral-like particle to reach 10Kb, and arrived at the claimed invention.
Regarding claim 26, Hall teaches that up to 15kb of sequence was deleted with a replacement vector to generate the T cell-receptor knock-out mouse (see page 19.12.12, left column, “Designing a knockout targeting construct (replacement vector)” section, lines 19-22).
The obviousness of combining the references is described above.
Claim 3 is rejected under 35 U.S.C. § 103 as being unpatentable over Collin (Collin, J. et al. “Concise review: Putting a finger on stem cell biology: Zinc Finger Nuclease-driven targeted genetic editing in human pluripotent stem cells”. Stem Cells, Vol. 29 (2011), pp: 1021-1033; previously cited), in view of Zhang (Zhang, F. US Patent No. 8,795,965 B2, published August 5, 2014, benefitting from priority of US Application No. 14/183,486 filed February 18, 2014; previously cited) and Raynal (Raynal, A. et al. “Excisable cassettes: New tools for functional analysis of Streptomyces genomes”. Applied and Environmental Microbiology, Vol. 72, No. 7 (2006), pp: 4839-4844), as applied to claim 1 above, and in further view of Bӧttcher (Bӧttcher, R. et al. Nucleic acids Research, Vol. 42 (April 19, 2014), p: e89; previously cited).
The rejection of claim 1 is described above. The combination of references Collin, Zhang and Raynal renders obvious elements of claim 1.
However, regarding claim 3, The combination of Collin, Zhang and Raynal does not render obvious elements of claim 3. The combination does not teach an identifying step carried out via a modification of allele (MOA) assay.
Bӧttcher teaches a first nuclease recognition site, i.e. homing nuclease SCe-1 recognition site, and modification to add a second recognition site, i.e. PAM sequence for gRNA, for a second nuclease, i.e. Cas9 effector protein.
Bӧttcher teaches a targeting vector for site-specific integration and homologous recombination (see “Abstract, and page 8, left column), containing a polynucleotide comprising an insert (PGK gene and tag) and a selection marker, i.e. Blasticidin resistance gene that is flanked by Cas9-gRNA recognition sites, i.e. an upstream target site and a downstream target site, and flanked by an upstream and a downstream homology arms (see figure 3A).
Bӧttcher also teaches a myc-Cas9 transgene in plasmid pRB14 derived from pKF257 which has an operably linked α-tub84 promoter (see page 2, right column, lines 10-24).
Bӧttcher teaches the modification of a construct / a targeting vector comprising GFP and a I-Sce I recognition site by adding nucleotide at the 3’ end of the site to allow recognition by CRISPR RNA (PAM site) (see Figure 2A). Bӧttcher teaches that cleavage by the nuclease occurs once the cells are also modified by transfecting a myc-Cas9 encoding plasmid vector (see page 2, right column, lines 13-24). Bӧttcher teaches inducing cleavage using the CRISPR-Cas9 system (see figure 1C, and page 7, right column, lines 8-17).
Regarding claim 3, Bӧttcher teaches a specific assessment method to calculate the targeting efficiency measuring the percentage of GFP-positive cells according to the amount of targeting product transfected (see Figure 3B).
Therefore, it would have been obvious to one with ordinary skills in the art, before the effective filing date of the claimed invention, to have considered the teachings of Bӧttcher. One with ordinary skills in the art interested in precision and specificity in homologous recombination, would consider the teachings of Bӧttcher using a CRISPR-Cas system for precise and specific homologous recombination.
After serial modifications as taught by Collin, with a CRISPR-Cas system taught by Zhang modified with Raynal, one with ordinary skills in the art motivated in assessing the efficiency of the method could have performed a MOA assay and added it to the method of identifying, as taught by Bӧttcher. One with ordinary skills in the art could have performed this modification with a reasonable expectation of success and arrived at the claimed invention.
Claim 9 is rejected under 35 U.S.C. §103 as being unpatentable over Collin (Collin, J. et al. “Concise review: Putting a finger on stem cell biology: Zinc Finger Nuclease-driven targeted genetic editing in human pluripotent stem cells”. Stem Cells, Vol. 29 (2011), pp: 1021-1033; previously cited), in view of Zhang (Zhang, F. US Patent No. 8,795,965 B2, published August 5, 2014, benefitting from priority of US Application No. 14/183,486 filed February 18, 2014; previously cited) and Raynal (Raynal, A. et al. “Excisable cassettes: New tools for functional analysis of Streptomyces genomes”. Applied and Environmental Microbiology, Vol. 72, No. 7 (2006), pp: 4839-4844), as applied to claim 1 above, and in further view of Hasty (Hasty, P. et al. Molecular and Cellular Biology, Vol. 12 (1992), pp: 2464-2474; previously cited).
Regarding claim 9, the combination of Collin, Zhang and Raynal renders elements of claim 1 obvious.
Collin, Zhang and Raynal do not specifically teach a method wherein the combined use of the first targeting vector with the first nuclease agent results in an increased targeting efficiency that is at least two-fold compared to the use of the first targeting vector alone.
However, Hasty teaches that an endonuclease site in the targeting vector produces linearized targeting vector, with an increase in targeting efficiency, five to twelve fold (see page 2467, right column, second ¶ and Table 1, experiment A).
Therefore, it would have been obvious to one with ordinary skills in the art before the effective filing date of the claimed invention, to have combined the teachings of Collin, Zhang and Raynal, with the teachings of Hasty and used an expression construct, expressing a Cas nuclease, and transfect before or at the same time than transfecting the targeting vector for an increased targeting efficiency, as taught by Hasty. One motivated in increasing the targeting efficiency of the targeting vector by at least two-fold could have performed this modification with a reasonable expectation of success and arrived at the claimed invention.
Claim 15 is rejected under 35 U.S.C. §103 as being unpatentable over Collin (Collin, J. et al. “Concise review: Putting a finger on stem cell biology: Zinc Finger Nuclease-driven targeted genetic editing in human pluripotent stem cells”. Stem Cells, Vol. 29 (2011), pp: 1021-1033; previously cited), in view of Zhang (Zhang, F. US Patent No. 8,795,965 B2, published August 5, 2014, benefitting from priority of US Application No. 14/183,486 filed February 18, 2014; previously cited) and Raynal (Raynal, A. et al. “Excisable cassettes: New tools for functional analysis of Streptomyces genomes”. Applied and Environmental Microbiology, Vol. 72, No. 7 (2006), pp: 4839-4844) , as applied to claims 1 and 14 above, and in further view of Bӧttcher (Bӧttcher, R. et al. Nucleic acids Research, Vol. 42 (April 19, 2014), p: e89; previously cited) and Evans (Evans, G.A. WO 99/14318 A1, published March 25, 1999; previously cited).
Regarding claim 15, the combination of Collin, Zhang and Raynal renders the elements of claims 1 and 14 obvious as required for claim 15. The rejections of claims 1 and 14 are described above.
Zhang teaches PAM sequences NGG that may be identified by searching for:
5’-Nx—NGG-3’ (see example 3, “sample Target sequence selection Algorithm”, column 67, line 18). Zhang also teaches 5’-N20-NGG-3’ (see column 69, line 37).
However, the combination of references does not render all elements of claim 15 obvious, specifically, the sequence set forth in SEQ ID NO: 1 (GN19NGG).
Bӧttcher teaches a target locus comprising the nucleotide sequence of SEQ ID NO: 1 (GN19NGG) (see Supplemental pdf-2 page 17).
Collin in view of Zhang modified with Raynal teach serial modifications of a target locus using a CRISPR-Cas system. The claimed PAM element is taught by Bӧttcher.
Therefore, it would have been obvious to one with ordinary skills in the art, before the effective filing date to have substituted the PAM elements taught by Collin/Zhang/Raynal with a PAM as taught by Bӧttcher, since the specific PAM is similar to the general formula taught by Zhang. One with ordinary skills in the art motivated in optimizing PAM sequences for specific gene of interest, could have performed this modification with a reasonable expectation of success and arrived at the claimed invention.
Evans teaches a sequence that comprises SEQ ID NO: 13 in oligonucleotide R28 (oligoR28) in Figure 5D. This oligonucleotide used as a primer to amplify and construct a synthetic plasmid comprising a sequence for Kanamycin/neomycin phosphotransferase selection marker.
This oligoR28 taught by Evans is a 50 nt long and encompasses SEQ ID NO: 3 and is a 100% match to SEQ ID NO: 3, therefore it is inherently a sequence to target when using a neomycin resistance selection marker.
See alignment below (Qy= query: SEQ ID NO: 3; Db= database: OligoR28)
Query Match 100.0%; Score 20; Length 50;
Best Local Similarity 100.0%;
Matches 20; Conservative 0; Mismatches 0; Indels 0; Gaps 0;
Qy 1 TGCGCAAGGAACGCCCGTCG 20
||||||||||||||||||||
Db 24 TGCGCAAGGAACGCCCGTCG 43
The combination of Collin, Zhang modified by Raynal teaches a method for serial modifications of a target locus using a CRISPR-Cas system and different selection markers / antibiotic resistance gene at each step. The claimed PAM element is taught by Bӧttcher, and Evans teaches SEQ ID NO: 3, the gRNA sequence for the neomycin gene resistance.
Therefore, it would have been obvious to one with ordinary skills in the art, before the effective filing date of the claimed invention, to have substituted the kanamycin resistance gene taught by Zhang in the system taught by Collin modified by Zhang/Raynal, with a neomycin resistance cassette and use the sequence taught by Evans to design a specific gRNA for the selection marker. The selection markers are equivalent in their function and neomycin resistance is well known and largely used in the art. One with ordinary skills in the art could have performed this modification with a reasonable expectation of success and arrived at the claimed invention.
Claims 22-23, 30 and 31 are rejected under 35 U.S.C. §103 as being unpatentable over Collin (Collin, J. et al. “Concise review: Putting a finger on stem cell biology: Zinc Finger Nuclease-driven targeted genetic editing in human pluripotent stem cells”. Stem Cells, Vol. 29 (2011), pp: 1021-1033; previously cited), in view of Zhang (Zhang, F. US Patent No. 8,795,965 B2, published August 5, 2014, benefitting from priority of US Application No. 14/183,486 filed February 18, 2014; previously cited) and Raynal (Raynal, A. et al. “Excisable cassettes: New tools for functional analysis of Streptomyces genomes”. Applied and Environmental Microbiology, Vol. 72, No. 7 (2006), pp: 4839-4844), as applied to claims 1, 21 and 29 above, and in further view of Mombaerts ( Mombaerts, P. et al. Nature, Vol. 360 (1992), pp: 225-231; previously cited) and Kimura (Kimura, N. et al. European Journal of Immunology, Vol. 17 (1987), pp: 375-383; previously cited).
The rejection of claims 1, 21 and 29 based on obviousness of the combination of Collin, Zhang and Raynal have been described above.
Regarding claims 22-23, 30 and 31, the combination of Collin, Zhang and Raynal renders obvious the elements of claims 1, 21 and 29, as presented above, and as required by the claims.
The combination of Collin, Zhang and Raynal does not render obvious a polynucleotide of interest comprising a human polynucleotide, the targeting of a T cell receptor alpha locus or a region encoding a region of a T cell receptor (claim 22), using a first polynucleotide that comprises a joining region gene segment of a T cell receptor alpha locus (claims 23, 30-31).
However, Mombaerts teaches the targeting of a TCR alpha locus. Mombaerts teaches a targeting vector pPMKO-1 that comprises 3.9 kb of homologous DNA sequence from TCR α locus in the first exon of TCR Cα and a pgk-neo selectable marker (see figure 1a).
Mombaerts teaches that two ES clones were produced carrying a genomic deletion of 15 kb that encompasses the TCR-β (see page 229, Figure 4, right column, lines 5-7).
It would have been obvious to one with ordinary skills in the art, before the effective filing date of the claimed invention, to have substituted any gene of interest within the targeting vector of the system taught by Collin modified by Zhang, for a TCR Cα gene and homology arms as taught by Mombaerts. One with ordinary skills in the art, motivated in targeting the TCRα for obtaining a targeting vector usable in gene therapy for immune disorders involving the TCR, could have made this modification with a reasonable expectation of success.
Although the combination of Collin/ Zhang/ Raynal and Mombaerts does not teach specifically a variable region gene segment and/or a joining region gene segment of a T cell receptor alpha locus, Kimura does teach the sequencing and cloning of variable regions and joining region gene segments in human (see title)(claim 23). Kimura teaches that a joining region is located upstream of the Cα (see figure 1a). Kimura teaches genomic clones that contain both Jα and a Cα genes (see figure 4).
Therefore, it would be obvious to extend the homology arms taught by Mombaerts to comprises a human Jα (joining region) gene segment as taught by Kimura, and include the resulting sequence into the targeting vector from the system as taught by Collin/Zhang. The level of skills in the art is high. One with ordinary skills in art, motivated in targeting a region with high genetic diversity and recombination important in human diseases, could have performed this modification with a reasonable expectation of success and arrived at the claimed invention.
Response to Arguments
Applicant’s arguments with respect to claims 1-3, 5, 7, 9, 11, 14-23 and 26-37
have been considered but are moot because the new ground of rejections does not rely on the same combination of references. Applicant's arguments filed 04/27/2026 have been fully considered but they are not persuasive.
The instant rejections are made with a combination of Collin, Zhang and Raynal as primary references.
Regarding Applicant’s arguments on page 15, “2. Impermissible hindsight”, In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Allowable Subject Matter
SEQ ID NO: 17 appears to be free from prior art.
Claims 17-18 are objected to as being dependent upon a rejected base claim, but would be allowable if rewritten in independent form including all of the limitations of the base claim and any intervening claims.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/A.D./Examiner, Art Unit 1636
/NANCY J LEITH/Primary Examiner, Art Unit 1636
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