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 .
Election/Restrictions
Applicant’s election without traverse of Invention I, claim 1, in the reply filed on 8/22/2025 is acknowledged. The restriction indicates that no linking claims exist. It is noted that this application has been transferred to a different examiner than the previous one who was Matasha Dhar.
Status of the Claims
The amended claims 1, 58, 59-76, 78-79, 81-99 were filed 5/23/2022, and claims 2-57, 77, 80 were cancelled.
Claim 1 is under consideration in view of the election. Claims 58, 59-76, 78-79, 81-99 are withdrawn from further consideration pursuant to 37CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim.
Information Disclosure Statement
The information disclosure statements (IDS’s) submitted on 1/05/2023 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statements are being considered by the examiner.
Drawings
The drawings, filed 9/14/2021, are objected to for the following reason: illegible/low quality text identified in the at least Figures: Fig 3d, 4b,c,g,d, Fig 9, Fig 14, Fig 15 , Fig 16, Fig 17 (as well as X axis tick-mark identifiers), Fig 18f, Fig 19c,d, 20d, 21b, and c, e, h (as well for these, the difference in control and experimental icons), f, h (and Further, “Fig 5”, “Fig 7” should be placed at the top of the drawing), 22 (and Fig 22 heading is missing), Fig 23, Fig 24, Fig 26 b, c, Fig 27 b, c, Fig 28d, e, Fig 29 d, e, g (X axis identifiers), Fig 30B (text in graph), Fig 30C-F (text in graph/legends), Fig 31 B, CD, E, G,H, I (text/X-axis), Fig 32 A (Knock in construct type) B (X, Y axis labels), C Construct type, Fig 33 (text), Fig 35 (text). Discerning between gRNA 1 and 2, and CD4 T cells vs CT8 T cells, in Fig 8 or legend icons in Fig14, or Fig 16 is not possible based upon the legends provided.
37 C.F.R. 1.84 states “Character of lines, numbers, and letters. All drawings must be made by a process which will give them satisfactory reproduction characteristics. Every line, number, and letter must be durable, clean, black (except for color drawings), sufficiently dense and dark, and uniformly thick and well-defined.”
Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claim 1 is objected to because of the following informalities: the alphabetical steps recited are a, b, c, f but d and e are not recited. This appears to be a typographical error such that f should be replaced by d. Appropriate correction is required.
Specification
The use of the term Trizol, which is a trade name or a mark used in commerce, has been noted in this application at [501] as well as Quibit and NovaSeq, Illumina found at least at [509] and [510]. These terms, and all other registered marks which are trade names or marks used in commerce, should be accompanied by the generic terminology; furthermore the terms should be capitalized wherever they appear or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 1 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 is indefinite and confusing in the recitation of …1 after bracketed section: [ (a) DNA templates that differ and comprise coding/noncoding sequence, barcode, common primer binding sequence….(ii), iii. a common primer binding sequence, wherein the 5’ and 3’ ends of each template comprise nucleotide sequences that are homologous to genomic sequence flanking target insertion site], “and wherein one or both homologous nucleotide sequences comprise a mismatched nucleotide sequence compared to a homologous sequence in the genomic sequence wherein the mismatched nucleotide sequence is not inserted into the target site during recombination is followed by b) allowing recombination to occur.
The claim reads as though one or both nucleotide sequences do comprise a mismatch sequence relative to homologous genomic sequence and so there is no insertion. This suggests there are no insertions, since one or both flanks mismatch, and mismatches are not inserted. Then there would be no point to recombination or sequencing. Clarification is required.
The wherein clause, after step c) is confusing and discloses, wherein a first primer is complementary to the primer binding sequence and a second primer binds to genomic sequence flanking the insertion site and does not bind to the mismatched nucleotide sequence in the template. However, the prior step indicated that the mismatched sequence in the template was not inserted during recombination. So, it is unclear how this disclosure relates to the amplification step. The second wherein clause, just below this one, also has an equivalent, confusing issue.
Claim interpretation
Regarding the above first rejection, the claim will be given the broadest reasonable interpretation, which suggests some recombination events will occur, and others will not, as a function of mismatch in some instances.
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.
Claims 1 is rejected under 35 U.S.C. 103 as being unpatentable over Gill US2018/0371498A1 filed 2/14/18; cited on IDS) in view of Deyle (cited on IDS).
Re: claim 1, A method for identifying a targeted insertion in the genome of a cell comprising:
introducing into a population of cells (i) a targeted nuclease that cleaves a target region in the genome of the cell to create a target insertion site
Gill disclosed introducing a vector encoding editing cassette, and at least one guide nucleic acid, into cells, producing cells where targeted nuclease cleaves a target region in the genome to create insertion site ([0196][0151]).
(claim cont.) and ii) a plurality of templates that are different by sequence from each other, wherein each DNA template comprises:
a heterologous coding or noncoding nucleic acid sequence
a unique barcode nucleotide sequence that indicates the identity of the heterologous coding or noncoding nucleic acid sequence and
a common primer binding sequence, wherein the 5' and 3' ends of each DNA template comprise nucleotide sequences that are homologous to genomic sequences flanking the target insertion site
Gill disclosed a plurality of templates, or different (distinct) editing sequences, where editing sequences could comprise mutations relative to the target sequence, including silent or non-silent/missense, one or more insertions, deletions, substitutions, exogenous or heterologous [0158].
Gill disclosed generating a library of the unique editing sequences [0167]. The editing cassette comprises primer sites on flanking ends [0250], for amplification [0154]. The cassette comprised barcodes corresponding to the editing sequence ([0160][0165][0169]).
(claim cont.) and wherein one or both homologous nucleotide sequences comprise a mismatched nucleotide sequence compared to a homologous sequence in the genomic sequence, wherein the mismatched nucleotide sequence is not inserted into the target insertion site during recombination
Homology arms were disclosed for sequence adjacent target [0166] added to editing genomic sequence to incorporate the sequence into the desired location via, e.g. homologous recombination [0165].
Homology arms could be on flanking sequence one or both ends of barcode and upstream/downstream of target sequence, including being two distinct homology arms, 5’ and 3’ [0165][0166]. Homology arms may comprise sequence homologous to a different gene than the target [0166]. Gill disclosed that hybridizing nucleic acids could be complementary to target or exhibit mismatches [0181].
Gill also disclosed that nucleic acids of interest could be amplified by PCR [0182].
(claim, cont. b) allowing recombination to occur, creating a population of modified cells
Gill disclosed an editing cassette to edit a site in a target sequence that was coding or non-coding and for which homologous regions within the editing sequence, flank one or more mutations of the cassettes, and are inserted into the target sequence via recombination [0162]. This produces the modified cells.
(claim, cont. c) amplifying DNA from the cells with a pair of primers to generate amplified DNA, wherein a first primer is complementary to the common primer binding sequence, and wherein a second primer binds to the homologous sequence in the genomic sequence flanking the insertion site and does not bind to the mismatched nucleotide sequence in the DNA template; and f) sequencing the amplified DNA to identify a DNA template inserted into the target insertion site for a cell.
Re c) and f), Gill disclosed amplifying DNA from the cells with primers using known amplification techniques, e.g. with PCR …replicating a target sequence. [0132]; PCR to amplify target sequence, and re: f, sequencing nucleic acids of interest [0182].
Gill disclosed the primer sites in the editing cassette flanking other components with primer binding sites generally on each end 5’, 3’ of the editing cassette [0154], thus a first primer complementary to the primer binding sequence and a second primer to homologous flanking sequence were disclosed. Gill did not explicitly disclose primer does not bind to mismatched sequence.
Re claim 1, part a) after I, ii, iii), and c) above, Gill did not explicitly state that mismatch would not be inserted, though one of ordinary skill would recognize that a homologous arm to another gene would be problematic for a subsequent step b) of allowing recombination to occur.
Deyle et al. (hereafter Deyle, Nuc Acids Res, 2014 42: 3119-3124; cited on IDS).
Deyle disclosed important factors that impact successful gene targeting, specifically that natural recurring SNPs along vector arms’ length, between homology arms and target sequence, can reduce gene targeting frequencies. (Abstract). Deyle reasoned that homology arm polymorphism could be used to direct allele-specific targeting, given vector arms can be several kilobases long, thus random mutations (SNPs) every kb or so, would impact the arms’ pairing (Abstract, Pg 1 left col para 3119) and therefore, targeting at an endogenous locus (Pg 3121 right col, final para).
Deyle developed a SNP dependent targeting assay, where two different MLV vectors, (both with 53 bp deletions in neo gene and one also with 6 additional SNPs; delta 53 or delta 53 plus 6 SNPs in the 5’ homology arm) delivered mutated neo gene. This was corrected by an AAV gene-targeting vector with homology with the MLV (Pg 3120 right col final para). Here, human fibrosarcoma cells were transduced with the MLVs with the mutant neo to form polyclonal target provirus. (Pg 3121 left col, para 1). To evaluate fidelity, PCR of the region homologous to AAV vector was sequenced and PCR product analyzed (no mutations). The SNPs in the MLV decreased targeting by five-fold.
A 4bp neo insert could be corrected by AAV, by introducing a deletion. When target had 1 to a few flanking deletions that could be corrected by AAV, targeting frequencies increased with best results when 4bp inserts were present flanking the neo mutation (or when correcting mutations in an endogenous HPRT locus). Additional MLV vectors with homologous arm mutations were tested and even single SNPs affected targeting when near the neo deletion. (Pg 3121 left col para 1). Similarly, SNP mismatches decreased targeting at a chromosomal locus in a human stem cell. Additional examples also provide for the conclusion that polymorphism, even one SNP, impacts gene targeting in cells, can be used to direct allele-specific targeting, and targeting can be inhibited by homologous arm heterology (Pg 3123 right col). Stable integration, was also demonstrated in this work (Pg 3120, right col, final para).
Prior to effective filing date it would have been prima facie obvious to one or ordinary skill in the art to have incorporated the methods of Deyle into the work of Gill and to have used the concept of homologous arm mutations that result in not binding a sequence and not recombining/amplifying, and thus to provide selectively for allele-specific targeting, in Gill’s work. Selective amplification would motivate Gill, since it would improve their technique over non-selective work in incorporating into cells only that which was explicitly desired to be incorporated.
Conclusion
Claim 1 is rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Lisa Horth whose telephone number is (703)756-4557. The examiner can normally be reached Monday-Friday 8-4 EST.
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/LISA HORTH/Examiner, Art Unit 1681
/GARY BENZION/Supervisory Patent Examiner, Art Unit 1681