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 .
Detailed Action
This action is in response to the papers filed April 28, 2026.
Amendments
Applicant's response and amendments, filed April 28, 2026, is acknowledged. Applicant has cancelled Claims 1-144, 146-151, 156, 159-162, 164-165, and 167, amended Claims 145, 157-158, and 163, and withdrawn Claims 152-155, 157-158, and 163.
Claims 145, 152-155, 157-158, 163, and 166 are pending.
Election/Restrictions
Applicant has elected without traverse the following species, wherein:
i) the alternative knock-in effect of the essential gene is to express the essential gene, as recited in Claim 145
ii) the alternative target essential gene is GAPDH, as recited in Claim 160;
iii) the alternative additional method step is selecting against a second plurality of cells of the population of starting cells, as recited in Claim 147;
iv) the alternative indel break location is within the last exon of the essential gene, as recited in Claim 149;
v) the alternative 5’ and 3’ homology arm structural homology limitation is a 5' homology arm upstream of the knock-in cassette and a 3' homology arm downstream of the knock-in cassette, as recited in Claim 151; and
vi) the alternative donor template structural limitation is the exogenous partial coding sequence of the essential gene in the knock-in cassette encodes a C-terminal fragment of a protein encoded by the essential gene, as recited in Claim 156.
Claims 145, 152-155, 157-158, 163, and 166 are pending.
Claims 152-155, 157-158, and 163 are pending but withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a non-elected invention, there being no allowable generic or linking claim.
Claims 145 and 166 are under consideration.
Priority
This application is a continuation of application 17/923,358 filed on November 4, 2022, which is a 371 of PCT/US21/30744 filed May 4, 2021. Applicant’s claim for the benefit of a prior-filed application provisional application 63/019,950 filed May 4, 2020 under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, or 365(c) is acknowledged.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
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.
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
1. The prior rejections of Claims 145-146 and 166 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, and under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, are withdrawn in light of Applicant’s cancellation of the negative limitation ‘wherein the donor template does not comprise a reporter gene’.
2. Claims 145 and 166 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement.
The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
Claim 145 has been amended to recite a method of producing a population of genetically modified mammalian cells using a CRISPR/Cas system comprising:
i) a gRNA comprising a targeting domain comprising SEQ ID NO:95; and
ii) a Cas12a/Cpf1 nuclease,
wherein the CRISPR/Cas system introduces a break within the first 50 base pairs of GAPDH exon 9.
Claim 166, dependent upon Claim 145, recites wherein the CRISPR/Cas system introduces a break within the first 21 base pairs of GAPDH exon 9.
The claims suffer from a gap in the structure/function nexus and/or method step/function nexus for the gene editing parameters by which the functional limitations:
i) “wherein the break is located within the first 50 base pairs of exon 9 of GAPDH” (Claim 145); and/or
ii) “wherein the break is located within the first 21 base pairs of exon 9 of GAPDH” (Claim 166),
is/are be achieved.
In analyzing whether the written description requirement is met for genus claims, it is first determined whether a representative number of species have been described by their complete structure. To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, methods of making the claimed product, or any combination thereof. The disclosure of a single species is rarely, if ever, sufficient to describe a broad genus, particularly when the specification fails to describe the features of that genus, even in passing. (see In re Shokal 113USPQ283(CCPA1957); Purdue Pharma L.P. vs Faulding Inc. 56 USPQ2nd 1481 (CAFC 2000).
The court explained that “reading a claim in light of the specification, to thereby interpret limitations explicitly recited in the claim, is a quite different thing from ‘reading limitations of the specification into a claim,’ to thereby narrow the scope of the claim by implicitly adding disclosed limitations which have no express basis in the claim.” The court found that applicant was advocating the latter, i.e., the impermissible importation of subject matter from the specification into the claim.). See also In re Morris, 127 F.3d 1048, 1054-55, 44 USPQ2d 1023, 1027-28 (Fed. Cir. 1997).
Those of ordinary skill in the art recognize that functional property of the CRISPR/Cas system to generate a break in the genome of a target host cell, and corresponding genus of molecular lesions thereof, is dependent upon many different variable parameters, including, but not limited to:
the gRNA target seed nucleotide sequence [parameter 1];
the gRNA concentration [parameter 2];
the type of Cas nuclease [parameter 3];
the concentration of the Cas nuclease [parameter 4];
the means by which the gRNA and/or Cas nuclease is introduced into the target host cell, e.g. electroporation protocol, transfection protocol, viral transduction protocol, nucleofection protocol, etc… [parameter 5]; and
the assay means by which the gene editing lesions are determined or measured [parameter 6].
The claim(s) also denote(s) that there is a genus of gRNAs and/or Cas nucleases and/or gene editing method step conditions that, do not, in fact, yield:
i) a break located within the first 50 base pairs of exon 9 of GAPDH; and/or
ii) a break located within the first 21 base pairs of exon 9 of GAPDH.
Parameter 1
Claim 145 has been amended to recite wherein the gRNA targeting domain (syn. seed sequence) comprises SEQ ID NO:95 (21 nucleotides in length), as shown below:
atcttctaggtatgacaacga
which is at the intron (italicized)/exon 9 boundary of the human GAPDH gene (GenBank AY340484, human GAPDH coding sequence, 2003).
The specification discloses the gRNA targeting domain (syn. seed sequence) may be 10-30 nucleotides in length [0686].
Thus, the GAPDH target region gRNA encompasses targeting domain nucleotide sequences that contain up to 9 additional nucleotides 5’ or 3’ to SEQ ID NO:95, e.g.:
xxxxxxxxxatcttctaggtatgacaacga;
xxxxxxxatcttctaggtatgacaacgaxx;
xxxxxatcttctaggtatgacaacgaxxxx;
xxatcttctaggtatgacaacgaxxxxxxx; or
atcttctaggtatgacaacgaxxxxxxxxx.
Thus, the claims encompass a genus of about 20 different gRNA targeting domains comprising the nucleotide sequence of SEQ ID NO:95.
The specification discloses a small genus of about 37 GAPDH gRNA seed sequences, e.g. Table 7, for which only 5 exhibited measurable editing of GAPDH and resulted in significant cell death at the population level due to expression of a non-functional GAPDH protein or a GAPDH protein with insufficient function (e.g. Example 1, [780]; Figures 1-2). Applicant’s own data clearly evidence that not all GAPDH gRNA seed sequences within the region of 108 nucleotides to which the gRNA target seed sequence is complementary and hybridizes, including 5’ upstream and/or 3’ downstream of the first 50 base pairs of GAPDH exon 9, yield the claimed functional result. For example, RSQ24576, substantially overlapping RSQ22237, did not achieve the editing results of RSQ22337. Similarly, RSQ22336, RSQ24574, and RSQ24575, just a few nucleotides away from RSQ22337, did not achieve the editing results of RSQ22337.
Rather, Applicant discloses a small subgenus of only 6 GAPDH gRNAs with no in-silico off-target effects (Figure 2) and only 5 GAPDH gRNAs apparently able to achieve the recited functional properties.
Thus, it is axiomatic that those of ordinary skill in the art would immediately recognize that the breadth of the gRNA targeting domain (syn. seed sequence) lengths encompassed by the claims, 10-30 nucleotides in length [0686], is vastly greater than the specific 21 nucleotide gRNA targeting domain (syn. seed sequence) lengths, and their specific corresponding nucleotide sequences, actually reduced to practice.
Those of ordinary skill in the art would also immediately recognize that the few specific species of GAPDH gRNA targeting domain (syn. seed sequence) lengths, and their corresponding specific nucleotide sequences, do not adequately represent the claimed genus of gRNA targeting domains (syn. seed sequences) lengths, and their corresponding nucleotide sequences, because the claimed genus is highly variant.
Zhang et al (U.S. Patent 10,494,621; of record) is considered relevant prior art for having disclosed that while truncated guide sequences may reduce off-target activity at some reference target site(s), they increased off-target activity at other target sites (Example 14, col. 464, lines 52-54).
Xie et al (RNA-Guided Genome Editing in Plants Using a CRISPR-Cas System, Molecular Plant 6(6): 1975-1983, 2013; available online August 17, 2013; of record) is considered relevant prior art for having taught that guide sequences of 22 nucleotides in length comprising one or more nucleotide mismatches relative to a first target site (PS3), wherein said guide sequence is now capable of hybridizing to said first target site and to a second, different target site ("off-target"), even though at least 10 nucleotides of the guide sequence seed region remain unchanged (Figure 4).
Cho et al (Analysis of off-target effects of CRISPR/Cas-derived RNA-guided endonucleases and nickases, Genome Research 24: 132-141, 2013; available online November 19, 2013; of record) is considered relevant prior art for having taught guide sequences of 20 nucleotides in length comprising one or more nucleotide mismatches relative to a first target site ("on"), wherein said guide sequence is no longer capable of hybridizing to said first target site but instead to a second, different off-target site ("Off4", "Off5"), even though at least 10 nucleotides of the guide sequence seed region remain unchanged (Figure 1B). Thus, while the single guide polynucleotide comprising a guide sequence of 10 to 16 nucleotides in length appears to have lost the recited functional property relative to a first, arbitrary "target sequence", it has gained the recited functional property relative to a second, arbitrary "target sequence".
Thus, the ordinary artisan would immediately recognize that Applicant’s specification does not support the enormous breadth of GAPDH gRNA target sequences encompassed by the instantly claimed invention.
The claimed genus of GAPDH gRNA seed sequences is highly variant, and the 5 specific GAPDH gRNAs whose target domain (syn. seed sequence) consists of 21 nucleotides do not adequately represent the enormous genus of structurally and functionally undisclosed gRNA seed sequences comprising target domains (syn. seed sequences) of 21-30 nucleotides encompassed by the claims.
Parameter 2
The claims fail to recite the working concentration of the gRNA with which to generate the recited GAPDH gene editing.
The specification discloses transfecting target cells with GAPDH gRNA/Cas12a ribonucleoprotein complexes (RNPs) by nucleofection or electroporation (e.g. Example 2, [0783]) at varying concentrations, e.g. 0.0625, 0.25, 1, or 4uM [0785], for which the 4uM RNP concentration resulted in the highest editing levels.
However, the independent claim, nor any claims, neither recite(s) nor require(s) nucleofection or electroporation of GAPDH gRNA/Cas12a ribonucleoprotein complexes (RNPs).
Li et al (Optimization of Genome Engineering Approaches with the CRISPR/Cas9 System, PLoS One 9(8): 105779; 10 pages, available online August 28, 2014; of record) is considered relevant prior art for having taught optimization of the CRISPR/Cas9 system for genome engineering (Title), wherein said optimization includes comparison of different guide RNA target sequences, individually or in combination, e.g. GFP KO gRNAs (Table 1; pg 3, col. 2), as such is common practice in the art (See also citation #13 to Mali et al, "target specificity screening"). Li et al taught that the cleavage efficiency, ranging from 20% to 49%, "varied based on the transfection efficiency" (pg 3, col. 2).
Liang et al (Rapid and highly efficient mammalian cell engineering via Cas9 protein transfection, J. Biotechnol. 208: 44-53, May 21, 2015; of record) is considered relevant prior art for having taught that the indel efficiency of a Cas9 protein/gRNA RNP complex varies widely, between 25% and 85%, depending upon the electroporation conditions (Figure 4a).
Schumann et al (Generation of knock-in primary human T cells using Cas9 ribonucleoproteins, Proc. Nat'l Acad. Sci. 112(33): 10437-10442, available online July 27, 2015; of record) is considered relevant prior art for having taught that the editing efficiency of a Cas9/gRNA ribonucleoprotein complex delivered in vitro into primary human T cells varies between 4% to 29%, depending upon the dose of the Cas9/gRNA RNP concentration (pg 10438, col. 2; Figure 4d).
Parameter 3
Claim 145 has been amended to recite the nuclease is a Cas12a/Cpf1 nuclease.
The specification discloses the Cas12a/Cpf1 nuclease includes variants thereof (e.g. [0532]).
Terns et al (U.S. 2011/0189776; of record) is considered relevant prior art for having disclosed a multitude of different Cas-type enzymes (e.g. Figure 5F, at least 29 different Cas-type enzymes), not all of which have actually been functionally characterized.
Doudna et al (U.S. 2016/0060653; of record) is considered relevant prior art for having disclosed Cas nucleases, wherein the amino acid sequence of the Cas nuclease may have as little as 75% identity to a reference nuclease (e.g. [0198]). SEQ ID NO:993 is 1688 amino acids in length.
75% identity allows for as many as 422 amino acid changes, 20^422, which is essentially an infinite genus of Cas variants.
(www.calculator.net/exponent-calculator.html; last visited January 5, 2026; of record)
The specification discloses Cas12a proteins of 1274 or 1353 amino acids in length (e.g. pg 254, Table 5), whereby 75% identity allows for as many as 319 to 338 amino acid changes, which is/are essentially an infinite genus of Cas12a/Cpf1 variants.
Those of ordinary skill in the art immediately recognize that Applicant simply does not possess infinity, nor the essentially infinite genus of Cas12a/Cpf1 variants encompassed by the claims.
Briner et al (Guide RNA Functional Modules Direct Cas9 Activity and Orthogonality Molecular Cell 56: 333-339, October 23, 2014; of record) is considered relevant prior art for having taught that changes to the nucleotide sequence(s) and length(s) of the gRNA molecule affects the ability of the gRNA to bind to different Cas9 orthologs, subsequently affecting the ability of the gRNA variants/Cas9 ortholog variants to efficiently cleave the intended target site nucleotide sequence (e.g. Figure 1). Briner et al taught that, "the molecular basis of selective Cas9:guide-RNA interactions is poorly understood" (Abstract). Distinct portions of the sgRNA are predicted to form various features that interact with Cas9 and/or the DNA target. However, the boundaries and respective roles of these portions remain to be determined (pgs 333-334, joining para.).
Parameter 4
The claims fail to recite the working concentration of the Cas12a/Cpf1 nuclease with which to generate the recited GAPDH gene editing.
As discussed above, the specification discloses transfecting target cells with GAPDH gRNA/Cas12a ribonucleoprotein complexes (RNPs) by nucleofection or electroporation (e.g. Example 2, [0783]) at varying concentrations, e.g. 0.0625, 0.25, 1, or 4uM [0785], for which the 4uM RNP concentration resulted in the highest editing levels.
However, the independent claim, nor any claims, neither recite(s) nor require(s) nucleofection or electroporation of GAPDH gRNA/Cas12a ribonucleoprotein complexes (RNPs).
As discussed above, Li et al taught that the cleavage efficiency, ranging from 20% to 49%, "varied based on the transfection efficiency" (pg 3, col. 2).
As discussed above, Liang et al taught that the indel efficiency of a Cas9 protein/gRNA RNP complex varies widely, between 25% and 85%, depending upon the electroporation conditions (Figure 4a).
As discussed above, Schumann et al taught that the editing efficiency of a Cas9/gRNA ribonucleoprotein complex delivered in vitro into primary human T cells varies between 4% to 29%, depending upon the dose of the Cas9/gRNA RNP concentration (pg 10438, col. 2; Figure 4d).
Parameter 5
The claims are broad for the means by which the gRNA and/or Cas12a/Cpf1 nuclease is/are introduced into the target host cell, e.g. electroporation protocol(s), transfection protocol(s), viral transduction protocol(s), nucleofection protocol(s) from which the limitation(s):
i) a break located within the first 50 base pairs of exon 9 of GAPDH; and/or
ii) a break located within the first 21 base pairs of exon 9 of GAPDH,
is/are to be achieved.
As discussed above, the specification discloses transfecting target cells with GAPDH gRNA/Cas12a ribonucleoprotein complexes (RNPs) by nucleofection or electroporation (e.g. Example 2, [0783]) at varying concentrations, e.g. 0.0625, 0.25, 1, or 4uM [0785], for which the 4uM RNP concentration resulted in the highest editing levels.
However, the independent claim, nor any claims, neither recite(s) nor require(s) nucleofection or electroporation of GAPDH gRNA/Cas12a ribonucleoprotein complexes (RNPs).
Parameter 6
The claims are broad for the assay means by which the gene editing lesions are determined or measured, from which the limitation wherein the break is located within the first 50 base pairs of exon 9 of GAPDH [structure] is to be determined.
As discussed in prior Office Actions, Galetto et al (Targeted approaches for gene therapy and the emergence of engineered meganucleases, Expert Opinion Biological Therapy 9(10): 1289-1303, 2009; of record) is considered relevant prior art for having reviewed the general mechanism by which nucleases mediate insertion of the artisan’s exogenous polynucleotide of interest into the artisan’s genomic target site of interest. As illustrated in Figure 2A (provided below), while the nuclease recognizes its target site and cleaves the DNA, the actual molecular mechanism by which the exogenous polynucleotide of interest integrates into the artisan’s genomic target site of interest via homologous recombination occurs through homologous recombination at the 5’ and 3’ homology arms that flank the exogenous polynucleotide that is to be inserted into the genomic target site by homologous recombination.
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As discussed in prior Office Actions, San Filippo et al (Mechanism of Eukaryotic Homologous Recombination, Ann. Rev. Biochem. 77:229-57, 2008; of record) is considered relevant prior art for having taught that a double-stranded break in the host cell’s genome naturally results in end resection of the cleaved DNA strands. The single strand ends perform strand invasion into the template sequence, upon which DNA synthesis repairs the gaps. Homologous recombination to resolve the 5’ and 3’ D-loops may occur anywhere within the D-loops, including much farther away from the actual site of the initial double-stranded break (e.g. Figure 1), as shown below:
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As discussed in prior Office Actions, Nimonkar et al (BLM–DNA2–RPA–MRN and EXO1–BLM–RPA–MRN constitute two DNA end resection machineries for human DNA break repair, Genes & Development 25: 350-362, 2011; of record) is considered relevant prior art for having taught that the end-resection mechanism in human cells can naturally remove a few hundred nucleotides (e.g. pg 350, col. 2) to a few thousands of nucleotides (e.g. pg 357, col. 2).
As discussed in prior Office Actions, Joung et al (U.S. 2014/0295556; of record) disclosed that the CRISPR/Cas9 system naturally generates deletions ranging in size from 2, 6, 14, 22, 56, 78, 94, 115, 181, 202, and 365 nucleotides at the target site where the nuclease causes a break in the endogenous genome (e.g. Figure 3C).
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Koike-Yusa et al (Genome-wide recessive genetic screening in mammalian cells with a lentiviral CRISPR-guide RNA library, Nature Biotechnol. 32(4): 267-273, 2014; of record) is considered relevant prior art for having taught a CRISPR/Cas9 method of editing target cells comprising the use of lentiviral vectors to transduce and deliver the Cas9 nuclease and gRNA into the cells (e.g. Figure 1), wherein the resulting indels ranged from less than 9bp to as many as 45bp (e.g. pg 269, col. 2), when measured about 14 days post infection (Online Methods, libraries and screening; off-target and cleavage analysis).
As discussed in prior Office Actions, Han et al (available online August 29, 2019; of record) taught a method of selecting a population of genetically modified cells, the method comprising the steps of:
A) contacting a population of starting cells with:
(i) a nuclease that causes a break within an endogenous coding sequence of an essential gene in the cells, wherein the essential gene encodes GAPDH (e.g. Title; Abstract, “efficient transgene knock-in in the endogenous GAPDH gene via CRISPR/Cas9 mediated homologous recombination”), and
(ii) a donor template that comprises a knock-in cassette comprising an exogenous coding sequence for a gene product of interest in frame with and downstream (3') of a partial coding sequence of the essential gene (Figure 1a),
wherein the knock-in cassette is integrated into the genome of a first plurality of cells of the population of starting cells by homology-directed repair (HDR) of the break,
wherein the first plurality of cells express:
(a) the gene product of interest, and
(b) the gene product encoded by the essential gene (e.g. pg 4, Section 3.1 Results, “When HR-mediated knock-in events occurred, the 2A-GFP fragment was inserted in frame with the endogenous GAPDH coding sequence, and because the self-cleaving 2A peptide exists, the GAPDH and GFP can be expressed separately (Figure 1a)”); and
B) selecting those cells comprising the knock-in cassette that express the GAPDH gene and the gene product of interest.
While Han et al demonstrated a reduction to practice of their gene-editing method using a reporter gene encoding a fluorescent protein, to wit, GFP (e.g. Figure 1a), Han et al taught that the method may be used instead to insert the artisan’s foreign gene of interest into the GAPDH locus for stable expression (e.g. Abstract; pg 2, para 3, “integrate exogenous genes”), e.g. for biomedical applications (e.g. pg 2, lines 2-3).
GenBank AY340484 (2003; of record) evidences the human GAPDH coding region of 5441-5530, shown below, which includes the intron (italicized), Applicant’s gRNA target sequence (underlined), exon 9, and stop codon (TAA, bold, underlined):
tcttttcatcttctaggtatgacaacgaatttggctacagcaacagggtggtggacctcatggcccacatggcctccaaggagtaa
Han et al taught the GAPDH sgRNA (underlined) targeted a site that is at least 4 nucleotides 5’ to the last coding sequence of the last GAPDH exon (e.g. Figure 1), as shown below:
tcttttcatcttctaggtatgacaacgaatttggctacagcaacagggtggtggacctcatggcccacatggcctccaaggagtaa
Claim 145 recites wherein the edit is within the first 50 nucleotides of exon 9 (bolded), which overlaps with the gRNA target sequence of Han et al, as shown below:
tcttttatcttctaggtatgacaacgaatttggctacagcaacagggtggtggacctcatggcccacatggcctccaaggagtaa
Claim 166 recites wherein the edit is within the first 21 nucleotides of exon 9 (bolded), which is only 21 nucleotides away from the gRNA target sequence of Han et al, as shown below:
tcttttatcttctaggtatgacaacgaatttggctacagcaacagggtggtggacctcatggcccacatggcctccaaggagtaa
Applicant argues that the Examiner’s contention that Han’s method would inherently and naturally result in a break within the first 50 base pairs of GAPDH exon 9 is incorrect because Han et al themselves taught that by TIDE analysis, the CRISPR/Cas9 system primarily generated indels of up to 6 nucleotides, and thus contradicts the assertion that Han’s methods would generate a large deletion that would encompass a region within the first 50 basepairs of GAPDH exon 9.
Applicant’s argument is unpersuasive because:
The TIDE analysis of Han et al only speaks to the results achieved per their specific CRISPR/Cas9 method step parameters, not the overall ability of the gRNA itself when used in other CRISPR/Cas9 method conditions and/or other CRISPR/Cas method conditions encompassed by the breadth of the instantly claimed method recited at a high level of generality.
That Han et al did not observe the same range of deletions of 2-365 nucleotides in length disclosed by Joung et al (Figure 3C) does not mean that they cannot exist and/or will never exist by the broadly claimed instant method, but rather that the single gRNA used by Han et al (e.g. pg 2, Methods, 2.1, “one sgRNA targeting the GAPDH locus” and/or the single sgRNA/Ca9 editing protocol, e.g. respective gRNA and Cas formulation, to wit, PX330 vector expressing Cas9 and sgRNA (e.g. pg 2, Methods, 2.2), donor:editing vector ratio of 1:1, by which the CRISPR/Cas system was introduced into the target mammalian cells to perform the GAPDH gene edits.
Those of ordinary skill in the art have long-recognized that there are different method step parameters by which the CRISPR/Cas gene editing system is performed. The experimental condition of Han et al is but one example, and is not limiting.
Allen et al (Predicting the mutations generated by repair of Cas9-induced double-strand breaks, Nature Biotechnol. 37(1): 64-72, available online November 27, 2018; of record) is considered relevant prior art for having taught a CRISPR/Cas9 method of editing target cells comprising the use of lentiviral vectors to transduce and deliver the gRNA into Cas9-expressing cells (e.g. Figure 1). Allen et al taught that they measured indel formation anywhere from 7 to 20 days post-infection with the CRISPR/Cas9 system to evaluate the effect of time-point choice (e.g. Online Methods, Screening and sequence of repair outcomes).
Allen et al also taught that to analyze indel prevalence by size, they classified indels into deletions of 1–30 nt and insertions of 1–10 nt. In this case, information about deletions larger than 30 and insertions larger than 10 nt was excluded from the analysis because they are not well detected by our method (Suppl Methods).
Thus, the ordinary artisan would immediately recognize that mere discussion of smaller deletions and/or insertions does not mean that larger deletions and insertions do not exist, but rather that such events were excluded from the analyzed data set for reasons of simplifying the analysis. Other detection means may be used to identify larger deletions and insertions.
Deletions of 30 nucleotides, per Allen et al, using the Han et al gRNA (underlined), would substantially overlap with DNA breaks within the first 50 nucleotides (bolded) and/or the first 21 nucleotides of GAPDH exon 9, as shown below:
tcttttatcttctaggtatgacaacgaatttggctacagcaacagggtggtggacctcatggcccacatggcctccaaggagtaa
Kosicki et al (Dynamics of Indel Profiles Induced by Various CRISPR/Cas9 Delivery Methods, Progress in Molecular Biology and Translational Science (52): 49-67, dx.doi.org/10.1016/bs.pmbts.2017.09.003, 2017; of record) is considered relevant prior art for having taught different CRISPR/Cas9 delivery formats, e.g. lentiviral transduction, plasmid lipofection, ribonuclear protein electroporation (e.g. pg 50; Figure 4), whereby different indel results are achieved using different methods and when measured at different days, e.g. Days 2-14, post-introduction of the CRISPR/Cas system into the target host cell (e.g. Figures 6-8).
Methods for detection of indels differ widely in their ability to resolve alleles, sensitivity, requirement for technical expertise, difficulty of results analysis, cost, time and effort required (pg 51), including, but not limited to Enzyme Mismatch Cleavage (EMC)/Surveyor assay, Tracking of Indels by DEcomposition (TIDE), Next Generation Sequencing (NGS), and Indel Detection by Amplicon Analysis (IDAA), each of which varies by their degree of sensitivity and resolution.
Using the gRNA of Han et al per the CRISPR/Cas9 experimental methods of Zhang et al, Xie et al, Cho et al, Joung et al, Church et al, Li et al, Liang et al, Schumann et al, Koike-Yusa et al, Allen et al, and/or Kosicki et al would be reasonably expected by the ordinary artisan to yield different indel results than the single experimental condition of Han et al, as detected by TIDE analysis, whereby depending on the indel detection assay and arbitrary selection of detection parameters, one will arrive at the detection of different indel sizes.
Han et al taught only the use of the TIDE analysis detection assay used to determine the GAPDH CRISPR/Cas9 indels. However, this does not mean that other, larger indels were not present. Rather, it is limited to the selective results of TIDE assay. Those of ordinary skill in the art would reasonably understand that other indel detection methods including, but not limited to Enzyme Mismatch Cleavage (EMC)/Surveyor assay, Next Generation Sequencing (NGS), and Indel Detection by Amplicon Analysis (IDAA), each of which varies by their degree of sensitivity and resolution would be reasonably expected to yield different results than the TIDE analysis of Han et al.
The claims fail to recite, and the specification fails to disclose, the nexus between the method step parameters 1-6, individually and/or in combination(s) and/or subcombination(s) thereof, that are necessary and sufficient to predictably achieve the recited limitation(s) of:
i) a break located within the first 50 base pairs of exon 9 of GAPDH; and/or
ii) a break located within the first 21 base pairs of exon 9 of GAPDH.
The claims fail to recite, and the specification fails to disclose, a first gRNA of the genus of 20 gRNAs [parameter 1], and its working concentration [parameter 2], that is paired with a first Cas12a/Cpf1 nuclease of the essentially infinite genus of structurally and functionally undisclosed Cas12a/Cpf1 nuclease variants [parameter 3], and its working concentration [parameter 4], and the means by which the gRNA and Cas12a/Cpf1 nuclease are introduced into the target host cells [parameter 5], but does not necessarily and predictably yield a break located within the first 21 base pairs of exon 9 of GAPDH (Claim 166), nor within the first 50 base pairs of exon 9 of GAPDH (Claim 145), and the assay means by which the gene editing lesions of 21 and/or 50 nucleotides is/are determined or measured [parameter 6], as opposed to
a second gRNA of the genus of 20 gRNAs [parameter 1], and its working concentration [parameter 2], that is paired with a second Cas12a/Cpf1 nuclease of the essentially infinite genus of structurally and functionally undisclosed Cas12a/Cpf1 nuclease variants [parameter 3], and its working concentration [parameter 4], and the means by which the gRNA and Cas12a/Cpf1 nuclease are introduced into the target host cells [parameter 5], that will necessarily and predictably yield a break located within the first 21 base pairs of exon 9 of GAPDH (Claim 166), but does not necessarily and predictably yield a break located within the first 50 base pairs of exon 9 of GAPDH (Claim 145), and the assay means by which the gene editing lesions of 21 and/or 50 nucleotides is/are determined or measured [parameter 6], for example.
The Federal Circuit has explained that a specification cannot always support expansive claim language and satisfy the requirements of 35 U.S.C. 112 “merely by clearly describing one embodiment of the thing claimed.” LizardTech v. Earth Resource Mapping, Inc., 424 F.3d 1336, 1346, 76 USPQ2d 1731, 1733 (Fed. Cir. 2005).
Without a correlation between structure and function, the claim does little more than define the claimed invention by function. That is not sufficient to satisfy the written description requirement. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406 (“definition by function ... does not suffice to define the genus because it is only an indication of what the gene does, rather than what it is’).
The claims fail to recite, and the specification fails to disclose, the nexus between the method step parameters 1-6, individually and/or in combination(s) and/or subcombination(s) thereof, that are necessary and sufficient to predictably achieve the recited limitation(s) of:
i) a break located within the first 50 base pairs of exon 9 of GAPDH; and/or
ii) a break located within the first 21 base pairs of exon 9 of GAPDH.
Applicant is essentially requiring the ordinary artisans to discover for themselves the method step parameters 1-6, individually and/or in combination(s) and/or subcombination(s) thereof, which Applicant fails to disclose.
A claim may be rendered indefinite by reference to an object that is variable. (MPEP §2173.05(b)).
If there are multiple ways to achieve and/or determine ‘wherein the break is located within the first 50 and/or 21 base pairs of exon 9 of GAPDH, e.g. gRNA target sequence(s), gRNA and/or Cas nuclease concentration(s), experimental means by which the gRNAs and/or Cas nucleases are introduced into the cell, amount of time after introducing the gRNA(s) and/or Cas nuclease(s) into the cell from which indel(s) and/or GAPDH function is/are determined, and/or the assay means of detecting indels, yet each yields a different result, then the claim may be indefinite because it is unclear which method is to be performed to determine infringement.
Thus, for the reasons outlined above, it is concluded that the claims do not meet the requirements for written description under 35 U.S.C. 112, first paragraph.
MPEP 2163 - 35 U.S.C. 112(a) and the first paragraph of pre-AIA 35 U.S.C. 112 require that the “specification shall contain a written description of the invention ....” This requirement is separate and distinct from the enablement requirement. Ariad Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1340, 94 USPQ2d 1161, 1167 (Fed. Cir. 2010) (en banc)
Dependent claims are included in the basis of the rejection because they do not correct the primary deficiencies of the independent claim(s).
Response to Arguments
Applicant argues that the specification provides ample written description support for the claims.
Applicant’s argument(s) has been fully considered, but is not persuasive. The court explained that “reading a claim in light of the specification, to thereby interpret limitations explicitly recited in the claim, is a quite different thing from ‘reading limitations of the specification into a claim,’ to thereby narrow the scope of the claim by implicitly adding disclosed limitations which have no express basis in the claim.” The court found that applicant was advocating the latter, i.e., the impermissible importation of subject matter from the specification into the claim.). See also In re Morris, 127 F.3d 1048, 1054-55, 44 USPQ2d 1023, 1027-28 (Fed. Cir. 1997).
The claims fail to recite, and the specification fails to disclose, the nexus between the method step parameters:
the genus of about 20 gRNAs comprising the nucleotide sequence of SEQ ID NO:95 [parameter 1];
the gRNA working concentrations, respectively [parameter 2];
the essentially infinite genus of structurally and functionally undisclosed Cas12a/Cpf1 nuclease variants [parameter 3];
the essentially infinite genus of structurally and functionally undisclosed Cas12a/Cpf1 nuclease variant working concentrations, respectively [parameter 4];
the means by which the gRNA [parameter 1] and Cas12a/Cpf1 nuclease [parameter 2] are introduced into the target host cells, respectively [parameter 5]; and
the assay means by which the gene editing lesions of 21 and/or 50 nucleotides is/are determined or measured [parameter 6],
individually and/or in combination(s) and/or subcombination(s) thereof, that are necessary and sufficient to predictably achieve the recited limitation(s) of:
i) a break located within the first 50 base pairs of exon 9 of GAPDH; and/or
ii) a break located within the first 21 base pairs of exon 9 of GAPDH.
3. Claims 145 and 166 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
The Examiner incorporates herein the above 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, rejection.
Claim 145 has been amended to recite a method of producing a population of genetically modified mammalian cells using a CRISPR/Cas system comprising:
i) a gRNA comprising a targeting domain comprising SEQ ID NO:95; and
ii) a Cas12a/Cpf1 nuclease,
wherein the CRISPR/Cas system introduces a break within the first 50 base pairs of GAPDH exon 9.
Claim 166, dependent upon Claim 145, recites wherein the CRISPR/Cas system introduces a break within the first 21 base pairs of GAPDH exon 9.
The claims suffer from a gap in the structure/function nexus and/or method step/function nexus for the gene editing parameters by which the functional limitations:
i) “wherein the break is located within the first 50 base pairs of exon 9 of GAPDH” (Claim 145); and/or
ii) “wherein the break is located within the first 21 base pairs of exon 9 of GAPDH” (Claim 166),
is/are be achieved.
As discussed above, the claims fail to recite, and the specification fails to disclose, the nexus between the method step parameters:
the genus of about 20 gRNAs comprising the nucleotide sequence of SEQ ID NO:95 [parameter 1];
the gRNA working concentrations, respectively [parameter 2];
the essentially infinite genus of structurally and functionally undisclosed Cas12a/Cpf1 nuclease variants [parameter 3];
the essentially infinite genus of structurally and functionally undisclosed Cas12a/Cpf1 nuclease variant working concentrations, respectively [parameter 4];
the means by which the gRNA [parameter 1] and Cas12a/Cpf1 nuclease [parameter 2] are introduced into the target host cells, respectively [parameter 5]; and
the assay means by which the gene editing lesions of 21 and/or 50 nucleotides is/are determined or measured [parameter 6],
individually and/or in combination(s) and/or subcombination(s) thereof, that are necessary and sufficient to predictably achieve the recited limitation(s) of:
i) a break located within the first 50 base pairs of exon 9 of GAPDH; and/or
ii) a break located within the first 21 base pairs of exon 9 of GAPDH.
As discussed above, GenBank AY340484 (2003; of record) evidences the human GAPDH coding region of 5441-5530, shown below, which includes the intron (italicized), Applicant’s gRNA target sequence (underlined), exon 9, and stop codon (TAA, bold, underlined):
tcttttcatcttctaggtatgacaacgaatttggctacagcaacagggtggtggacctcatggcccacatggcctccaaggagtaa
Han et al taught the GAPDH sgRNA (underlined) targeted a site that is at least 4 nucleotides 5’ to the last coding sequence of the last GAPDH exon (e.g. Figure 1), as shown below:
tcttttcatcttctaggtatgacaacgaatttggctacagcaacagggtggtggacctcatggcccacatggcctccaaggagtaa
Claim 145 recites wherein the edit is within the first 50 nucleotides of exon 9 (bolded), which overlaps with the gRNA target sequence of Han et al, as shown below:
tcttttatcttctaggtatgacaacgaatttggctacagcaacagggtggtggacctcatggcccacatggcctccaaggagtaa
Claim 166 recites wherein the edit is within the first 21 nucleotides of exon 9 (bolded), which is only 21 nucleotides away from the gRNA target sequence of Han et al, as shown below:
tcttttatcttctaggtatgacaacgaatttggctacagcaacagggtggtggacctcatggcccacatggcctccaaggagtaa
Applicant argues that the Examiner’s contention that Han’s method would inherently and naturally result in a break within the first 50 base pairs of GAPDH exon 9 is incorrect because Han et al themselves taught that by TIDE analysis, the CRISPR/Cas9 system primarily generated indels of up to 6 nucleotides, and thus contradicts the assertion that Han’s methods would generate a large deletion that would encompass a region within the first 50 basepairs of GAPDH exon 9.
Applicant’s argument is unpersuasive because:
The TIDE analysis of Han et al only speaks to the results achieved per their specific CRISPR/Cas9 method step parameters, not the overall ability of the gRNA itself when used in other CRISPR/Cas9 method conditions and/or other CRISPR/Cas method conditions encompassed by the breadth of the instantly claimed method recited at a high level of generality.
Those of ordinary skill in the art have long-recognized that there are different method step parameters by which the CRISPR/Cas gene editing system is performed. The experimental condition of Han et al is but one example, and is not limiting.
As discussed above, those of ordinary skill in the art previously recognized that different CRISPR/Cas9 delivery formats, e.g. lentiviral transduction, plasmid lipofection, or ribonuclear protein electroporation, yield different indel results using different methods and when measured at different days, post-introduction of the CRISPR/Cas system into the target host cell.
Methods for detection of indels differ widely in their ability to resolve alleles, sensitivity, requirement for technical expertise, difficulty of results analysis, cost, time and effort required, including, but not limited to Enzyme Mismatch Cleavage (EMC)/Surveyor assay, Tracking of Indels by DEcomposition (TIDE), Next Generation Sequencing (NGS), and Indel Detection by Amplicon Analysis (IDAA), each of which varies by their degree of sensitivity and resolution.
Han et al taught only the use of the TIDE analysis detection assay used to determine the GAPDH CRISPR/Cas9 indels. However, this does not mean that other, larger indels were not present. Rather, it is limited to the selective results of TIDE assay. Those of ordinary skill in the art would reasonably understand that other indel detection methods including, but not limited to Enzyme Mismatch Cleavage (EMC)/Surveyor assay, Next Generation Sequencing (NGS), and Indel Detection by Amplicon Analysis (IDAA), each of which varies by their degree of sensitivity and resolution would be reasonably expected to yield different results than the TIDE analysis of Han et al.
A claim may be rendered indefinite by reference to an object that is variable. (MPEP §2173.05(b)).
If there are multiple ways to achieve and/or determine ‘wherein the break is located within the first 50 and/or 21 base pairs of exon 9 of GAPDH, e.g. gRNA target sequence(s), gRNA and/or Cas nuclease concentration(s), experimental means by which the gRNAs and/or Cas nucleases are introduced into the cell, amount of time after introducing the gRNA(s) and/or Cas nuclease(s) into the cell from which indel(s) and/or GAPDH function is/are determined, and/or the assay means of detecting indels, yet each yields a different result, then the claim may be indefinite because it is unclear which method is to be performed to determine infringement.
The instant claims as a whole do not apprise one of ordinary skill in the art of its scope and, therefore, does not serve the notice function required by 35 U.S.C. 112, second paragraph, by providing clear warning to others as to what constitutes infringement of the patent.
Response to Arguments
Applicant argues that Han et al targets the 3’ end of GAPDH exon 9, not the 5’ end.
Applicant’s argument(s) has been fully considered, but is not persuasive. GAPDH exon 9 is only 69 nucleotides in length.
Either the ability of the Han et al gRNA to introduce a break within the first 21 and/or 50 nucleotides of GAPDH exon 9 is an inherent property of (that naturally flows from) the gRNA structure(s) and/or GAPDH gene editing method step(s), or it is not, and something structural and/or method step(s) must change.
As discussed above, it is considered natural law of enzymology and cell biology that the gRNA of Han et al would yield a break in within the first 21 and/or 50 nucleotides of GAPDH exon 9.
As discussed above, instantly claimed method step parameters are recited at a high level of generality, including the GAPDH gene editing method parameters of Han et al.
Han et al taught only the use of the TIDE analysis detection assay used to determine the GAPDH CRISPR/Cas9 indels. However, this does not mean that other, larger indels were not present. Rather, it is limited to the selective results of TIDE assay. Those of ordinary skill in the art would reasonably understand that other indel detection methods including, but not limited to Enzyme Mismatch Cleavage (EMC)/Surveyor assay, Next Generation Sequencing (NGS), and Indel Detection by Amplicon Analysis (IDAA), each of which varies by their degree of sensitivity and resolution would be reasonably expected to yield different results than the TIDE analysis of Han et al.
A claim may be rendered indefinite by reference to an object that is variable. (MPEP §2173.05(b)).
As discussed above, the claims fail to recite, and the specification fails to disclose, the nexus between the method step parameters:
the genus of about 20 gRNAs comprising the nucleotide sequence of SEQ ID NO:95 [parameter 1];
the gRNA working concentrations, respectively [parameter 2];
the essentially infinite genus of structurally and functionally undisclosed Cas12a/Cpf1 nuclease variants [parameter 3];
the essentially infinite genus of structurally and functionally undisclosed Cas12a/Cpf1 nuclease variant working concentrations, respectively [parameter 4];
the means by which the gRNA [parameter 1] and Cas12a/Cpf1 nuclease [parameter 2] are introduced into the target host cells, respectively [parameter 5]; and
the assay means by which the gene editing lesions of 21 and/or 50 nucleotides is/are determined or measured [parameter 6],
individually and/or in combination(s) and/or subcombination(s) thereof, that are necessary and sufficient to predictably achieve the recited limitation(s) of:
i) a break located within the first 50 base pairs of exon 9 of GAPDH; and/or
ii) a break located within the first 21 base pairs of exon 9 of GAPDH.
A claim may be rendered indefinite by reference to an object that is variable. (MPEP §2173.05(b)).
If there are multiple ways to achieve and/or determine ‘wherein the break is located within the first 50 and/or 21 base pairs of exon 9 of GAPDH, e.g. gRNA target sequence(s), gRNA and/or Cas nuclease concentration(s), experimental means by which the gRNAs and/or Cas nucleases are introduced into the cell, amount of time after introducing the gRNA(s) and/or Cas nuclease(s) into the cell from which indel(s) and/or GAPDH function is/are determined, and/or the assay means of detecting indels, yet each yields a different result, then the claim may be indefinite because it is unclear which method is to be performed to determine infringement.
4. Claim 166 is rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claim 145 has been amended to recite a method of producing a population of genetically modified mammalian cells using a CRISPR/Cas system comprising:
i) a gRNA comprising a targeting domain comprising SEQ ID NO:95; and
ii) a Cas12a/Cpf1 nuclease,
wherein the CRISPR/Cas system introduces a break within the first 50 base pairs of GAPDH exon 9.
Claim 166, dependent upon Claim 145, recites wherein the CRISPR/Cas system introduces a break within the first 21 base pairs of GAPDH exon 9.
Either this is an inherent property of (that naturally flows from) the structure(s) and/or method step(s) positively recited in the independent claim, or it is not, and something structural and/or method step(s) of independent Claim 145 must change.
To the extent it is an inherent property of (that naturally flows from) the product/method of the independent claim, then the instant claim fails to further limit the independent claim.
Furthermore, in regard to instant claims, it is noted that the “wherein the CRISPR/Cas system introduces a break within the first 21 base pairs…” clause does not recite any additional structure(s) and/or active method step(s), but simply states a characterization or conclusion of the results of structure(s) and/or active method step(s) positively recited in Claim 145. Therefore, the "wherein" clause is not considered to further limit the method defined by the claim and has not been given weight in construing the claims. See Texas Instruments, Inc. v. International Trade Comm., 988 F.2d 1165, 1171,26 USPQ2d 1018, 1023 (Fed Cir. 1993) ("A 'whereby' clause that merely states the result of the limitations in the claim adds nothing to the patentability or substance of the claim."). See also Minton v. National Assoc. of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003) ("A whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.").
Claim 166 fails to recite one or more structural changes to the Claim 145 gRNA comprising a targeting domain comprising SEQ ID NO:95.
Claim 166 fails to recite one or more structural changes to the Claim 145 Cas12a/Cpf1 nuclease.
Claim 166 fails to recite one or more method step changes to the Claim 145 step of contacting the target mammalian cells with the Claim 145 gRNA comprising a targeting domain comprising SEQ ID NO:95 and the Claim 145 Cas12a/Cpf1 nuclease.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
5. Claim 166 is rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement.
The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The Examiner incorporates herein the above 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, and 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, rejections.
Claim 145 has been amended to recite a method of producing a population of genetically modified mammalian cells using a CRISPR/Cas system comprising:
i) a gRNA comprising a targeting domain comprising SEQ ID NO:95; and
ii) a Cas12a/Cpf1 nuclease,
wherein the CRISPR/Cas system introduces a break within the first 50 base pairs of GAPDH exon 9.
Claim 166, dependent upon Claim 145, recites wherein the CRISPR/Cas system introduces a break within the first 21 base pairs of GAPDH exon 9.
Either this is an inherent property of (that naturally flows from) the structure(s) and/or method step(s) positively recited in the independent claim, or it is not, and something structural and/or method step(s) of independent Claim 145 must change.
The claim denotes that not all structures and/or method steps of the independent claim are able to achieve the functional properties recited in the dependent claim.
To the extent it is not an inherent property of (that naturally flows from) the product/method of the independent claim, then the instant claim lack adequate written description for failing to recite the structural and/or method step changes of the Claim 145 method in order to achieve the functional properties recited in the dependent claim.
Furthermore, in regard to instant claims, it is noted that the “wherein the CRISPR/Cas system introduces a break within the first 21 base pairs…” clause does not recite any additional structure(s) and/or active method step(s), but simply states a characterization or conclusion of the results of structure(s) and/or active method step(s) positively recited in Claim 145. Therefore, the "wherein" clause is not considered to further limit the method defined by the claim and has not been given weight in construing the claims. See Texas Instruments, Inc. v. International Trade Comm., 988 F.2d 1165, 1171,26 USPQ2d 1018, 1023 (Fed Cir. 1993) ("A 'whereby' clause that merely states the result of the limitations in the claim adds nothing to the patentability or substance of the claim."). See also Minton v. National Assoc. of Securities Dealers, Inc., 336 F.3d 1373, 1381, 67 USPQ2d 1614, 1620 (Fed. Cir. 2003) ("A whereby clause in a method claim is not given weight when it simply expresses the intended result of a process step positively recited.").
Claim 166 fails to recite one or more structural changes to the Claim 145 gRNA comprising a targeting domain comprising SEQ ID NO:95, thereby transforming or otherwise modifying a first gRNA comprising a targeting domain comprising SEQ ID NO:95 that, while able to introduce a break within the first 50 base pairs of GAPDH exon 9, is unable to introduce a break within the first 21 base pairs of GAPDH exon 9 into a second gRNA comprising a targeting domain comprising SEQ ID NO:95 that is now necessarily and predictably able to introduce a break within the first 21 base pairs of GAPDH exon 9, for example.
Claim 166 fails to recite one or more structural changes to the Claim 145 Cas12a/Cpf1 nuclease, thereby transforming or otherwise modifying a first Cas12a/Cpf1 nuclease that, while able to introduce a break within the first 50 base pairs of GAPDH exon 9, is unable to introduce a break within the first 21 base pairs of GAPDH exon 9 into a second Cas12a/Cpf1 nuclease that is now necessarily and predictably able to introduce a break within the first 21 base pairs of GAPDH exon 9, for example.
Claim 166 fails to recite one or more method step changes to the Claim 145 step of contacting the target mammalian cells with the Claim 145 gRNA comprising a targeting domain comprising SEQ ID NO:95 and the Claim 145 Cas12a/Cpf1 nuclease, thereby transforming or otherwise modifying a first CRISPR/Cas system contacting step that, while able to introduce a break within the first 50 base pairs of GAPDH exon 9, is unable to introduce a break within the first 21 base pairs of GAPDH exon 9 into a second CRISPR/Cas system contacting step that is now necessarily and predictably able to introduce a break within the first 21 base pairs of GAPDH exon 9, for example.
Without a correlation between structure and function, the claim does little more than define the claimed invention by function. That is not sufficient to satisfy the written description requirement. See Eli Lilly, 119 F.3d at 1568, 43 USPQ2d at 1406 (“definition by function ... does not suffice to define the genus because it is only an indication of what the gene does, rather than what it is’).
MPEP 2163 - 35 U.S.C. 112(a) and the first paragraph of pre-AIA 35 U.S.C. 112 require that the “specification shall contain a written description of the invention ....” This requirement is separate and distinct from the enablement requirement. Ariad Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1340, 94 USPQ2d 1161, 1167 (Fed. Cir. 2010) (en banc)
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 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 of this title, 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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103(a) are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
6. The prior rejection of Claims 145, 147, and 166 under AIA 35 U.S.C. 103 as being unpatentable over Han et al (available online August 29, 2019; of record) in view of Joung et al (U.S. 2014/0295556; of record), Zhu et al (2015; of record), Yesbolatova et al (available online April 24, 2019; of record), Li et al (April 7, 2015; of record), Galetto et al (2009; of record), San Filippo et al (2008; of record), and Nimonkar et al (2011; of record) is withdrawn in light of Applicant’s amendments to independent Claim 145 to recite a gRNA comprising SEQ ID NO:95, to recite a Cas12a/Cpf1 nuclease, and to cancel recitation of the negative limitation ‘does not comprise a reporter gene’, necessitating new grounds of rejection.
7. Claims 145 and 166 are rejected under AIA 35 U.S.C. 103 as being unpatentable over Han et al (available online August 29, 2019; of record) in view of Joung et al (U.S. 2014/0295556; of record), Zhu et al (2015; of record), Yesbolatova et al (available online April 24, 2019; of record), Li et al (April 7, 2015; of record), Galetto et al (2009; of record), San Filippo et al (2008; of record), Nimonkar et al (2011; of record), and Kim et al (Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells, Nature Biotechnology 34(8): 863-868, doi.org/10.1038/nbt.3609, available online June 6, 2016).
Determining the scope and contents of the prior art, and Ascertaining the differences between the prior art and the claims at issue.
Instant claims are directed to a CRISPR/Cas gene-editing method of inserting into the GAPDH exon 9 coding region a knock-in expression cassette comprising an exogenous partial coding sequence of GAPDH fused to a gene product of interest, thereby expressing both GAPDH and the gene product of interest from the thus-edited GAPDH locus.
Claim 145 recites the gRNA targeting domain comprising the nucleotides of SEQ ID NO:95.
As discussed above, GenBank AY340484 (2003; of record) evidences the human GAPDH coding region of 5441-5530, shown below, which includes the intron (italicized), Applicant’s gRNA target sequence (underlined), exon 9, and stop codon (TAA, bold, underlined):
tcttttcatcttctaggtatgacaacgaatttggctacagcaacagggtggtggacctcatggcccacatggcctccaaggagtaa
GAPDH exon 9 is only 69 nucleotides in length and encodes only the last 22 amino acids of GAPDH protein, as shown below:
YDNEFGYSNRVVDLMAHMASKE*
With respect to Claim 145, Han et al is considered relevant prior art for having taught a CRISPR/Cas gene-editing method of inserting into the GAPDH exon 9 coding region a knock-in expression cassette comprising a gene product of interest, thereby expressing both GAPDH and the gene product of interest from the thus-edited GAPDH locus (e.g. Title; Abstract, “efficient transgene knock-in in the endogenous GAPDH gene via CRISPR/Cas9 mediated homologous recombination”).
Han et al taught the donor template comprises a knock-in cassette comprising an exogenous coding sequence for a gene product of interest in frame with and downstream (3') of a partial coding sequence of the GAPDH gene (e.g. Figure 1a; pg 4, Section 3.1 Results, “When HR-mediated knock-in events occurred, the 2A-GFP fragment was inserted in frame with the endogenous GAPDH coding sequence, and because the self-cleaving 2A peptide exists, the GAPDH and GFP can be expressed separately (Figure 1a)”).
The donor template and 5’ homology arm of Han et al naturally comprises the coding sequences of the 5’ portion of the GAPDH exon 9, fused in frame with and upstream of, the exogenous GFP coding sequence.
While Han et al demonstrated a reduction to practice of their gene-editing method using a reporter gene encoding a fluorescent protein, to wit, GFP (e.g. Figure 1a), Han et al taught that the method may be used instead to insert the artisan’s foreign gene of interest into the GAPDH locus for stable expression (e.g. Abstract; pg 2, para 3, “integrate exogenous genes”), e.g. for biomedical applications (e.g. pg 2, lines 2-3).
Han et al taught the GAPDH sgRNA (underlined) targeted a site that is at least 4 nucleotides 5’ to the last coding sequence of the last GAPDH exon (e.g. Figure 1), as shown below:
tcttttcatcttctaggtatgacaacgaatttggctacagcaacagggtggtggacctcatggcccacatggcctccaaggagtaa
Joung et al is considered relevant prior art for having disclosed that the CRISPR/Cas9 system naturally generates deletions ranging in size from 2, 6, 14, 22, 56, 78, 94, 115, 181, 202, and 365 nucleotides at the target site where the nuclease causes a break in the endogenous genome (e.g. Figure 3C).
Thus, it is considered natural law of cell biology and ezymology that the break in the GAPDH exon 9 coding sequence caused by the CRISPR/Cas system of Han et al inherently and naturally introduces a break within the first 21 and/or 50 base pairs of GAPDH exon 9.
Claim 145 recites wherein the edit is within the first 50 nucleotides of exon 9 (bolded), which overlaps with the gRNA target sequence of Han et al, as shown below:
tcttttatcttctaggtatgacaacgaatttggctacagcaacagggtggtggacctcatggcccacatggcctccaaggagtaa
Since the Patent Office does not have the facilities for examining and comparing genus of molecular lesions caused by the Han et al guide RNA and DNA breaks of the CRISPR/Cas9 system of the prior art reference, the burden is upon Applicants to show a distinction between the material structural and functional characteristics of the claimed generically recited essential gene target site and corresponding generically recited nuclease and the guide RNA and CRISPR/Cas9 system of the prior art. See In re Best, 562 F.2d 1252, 195 USPQ 430 (CCPA 1977) and In re Fitzgerald et al., 205 USPQ 594.
Han et al do not teach ipsis verbis wherein the exogenous coding sequence or exogenous partial coding sequence of the essential gene corresponds to endogenous coding sequence of the essential gene located downstream (3’) of the break, thereby restoring the coding region of the essential gene and rescuing the loss of function of the gene product.
However, prior to the effective filing date of the instantly claimed invention, Zhu et al is considered relevant prior art for having taught a method of selecting a population of cells comprising a knock-in expression cassette comprising a heterologous gene of interest, e.g. EGFP, using the CRISPR/Cas gene editing system (e.g. Figures 5a and 6a), whereby the donor template comprises an exogenous coding sequence for the target gene in frame with and upstream (5’) to said heterologous gene of interest, e.g. EGFP, and wherein the exogenous coding sequence for the target gene corresponds to endogenous coding sequences downstream (3’) of the double-stranded break (e.g. Figure 6a).
Similar to Han et al, the gRNA of Zhu et al cleaves the DNA just 9 nucleotides (3 amino acids) upstream of the translation stop codon. The 5’ homology arm of the donor template encodes the last 20 amino acids of the target gene and fuses in frame the EGFP coding sequence. The 3’ homology arm encodes the nucleotide sequence downstream of the translation stop codon, and introduces a new stop codon several nucleotides downstream (Figure 6a).
Zhu et al selected for those cells expressing the knock-in cassette (e.g. Figures 5-6).
Similarly, Yesbolatova et al is considered relevant prior art for having taught a method of selecting a population of cells comprising a knock-in expression cassette comprising a heterologous peptide of interest, e.g. peptide tag, using the CRISPR/Cas gene editing system (e.g. Figure 2a; C-terminal tagging donor), whereby the donor template comprises an exogenous coding sequence for the target gene in frame with and upstream (5’) to said heterologous peptide tag, and wherein the exogenous coding sequence for the target gene corresponds to endogenous coding sequences downstream (3’) of the double-stranded break.
Yesbolatova et al taught that they identify CRISPR/Cas9 target sites within 50bp upstream of the stop codon (e.g. pg 74, col. 1), and the donor template encodes the tag cloned in frame with the gene of interest, so as to be able to express a fusion protein (e.g. pg 74, col. 2).
Yesbolatova et al selected for those cells expressing the knock-in cassette (e.g. Figures 1, 4).
Similarly, Li et al is considered relevant prior art for having taught a method of selecting a population of cells comprising a knock-in expression cassette comprising a heterologous protein of interest, e.g. EGFP, fused in frame with and downstream of the last exon of the target gene using the CRISPR/Cas gene editing system (e.g. Figure 1a), whereby the donor template comprises an exogenous coding sequence for the target gene in frame with and upstream (5’) to said heterologous protein, and wherein the exogenous coding sequence for the target gene corresponds to endogenous coding sequences downstream (3’) of the double-stranded break.
The gRNA of Li et al targets the intron upstream of the last exon of the target gene (exon 13), and the 5’ homology arm encodes the intron nucleotides and entire last exon fused in frame with EGFP, all of which are 3’ downstream to the gRNA target site cut by the CRISPR/Cas9 system.
Li et al taught a demonstration to extend the application of the knock-in strategy to other exogenous genes by substituting the first artisan’s gene of interest, GFP gene, for a second artisan’s gene of interest, to wit, GAL4 transactivator (e.g. pg 636, col. 2).
Han et al do not teach wherein the gRNA targeting domain comprises the nucleotide sequence of SEQ ID NO:95.
Galetto et al is considered relevant prior art for having reviewed the general mechanism by which nucleases mediate insertion of the artisan’s exogenous polynucleotide of interest into the artisan’s genomic target site of interest. As illustrated in Figure 2A (provided below), while the nuclease recognizes its target site and cleaves the DNA, the actual molecular mechanism by which the exogenous polynucleotide of interest integrates into the artisan’s genomic target site of interest via homologous recombination occurs through homologous recombination at the 5’ and 3’ homology arms that flank the exogenous polynucleotide that is to be inserted into the genomic target site by homologous recombination.
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San Filippo et al is considered relevant prior art for having taught that a double-stranded break in the host cell’s genome naturally results in end resection of the cleaved DNA strands. The single strand ends perform strand invasion into the template sequence, upon which DNA synthesis repairs the gaps. Homologous recombination to resolve the 5’ and 3’ D-loops may occur anywhere within the D-loops, including much farther away from the actual site of the initial double-stranded break (e.g. Figure 1), as shown below:
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Nimonkar et al is considered relevant prior art for having taught that the end-resection mechanism in human cells can naturally remove a few hundred nucleotides (e.g. pg 350, col. 2) to a few thousands of nucleotides (e.g. pg 357, col. 2).
With respect to the presence of 5’ and 3’ homology arms in the donor construct,
Han et al taught wherein the donor template comprises a 900 nucleotide 5' homology arm upstream of the knock-in cassette and a 900 nucleotide 3' homology arm downstream of the knock-in cassette, said 5’ homology arm comprising intronic sequences 5’ to exon 9 (e.g. Figure 1a).
Thus, those of ordinary skill in the art would have reasonably expected that the integration of the knock-in expression cassette of Han et al using the gRNA of Han et al would be molecularly indistinguishable from the integration of the knock-in expression cassette of Han et al using a gRNA comprising the nucleotide sequence of SEQ ID NO:95 because the gRNA target sequences are so close to each other in the GAPDH exon 9 and the actual 5’ homology arm recombination event takes place 5’ upstream of the cleavage site induced by both gRNAs.
Zhu et al taught wherein the donor template comprises a 5' homology arm upstream of the knock-in cassette and a 3' homology arm downstream of the knock-in cassette (e.g. Figures 5a, 6a).
Yesbolatova et al taught wherein the donor template comprises a 5' homology arm upstream of the knock-in cassette and a 3' homology arm downstream of the knock-in cassette (e.g. Figure 2a).
Li et al taught wherein the donor template comprises a 5' homology arm upstream of the knock-in cassette and a 3' homology arm downstream of the knock-in cassette (e.g. Figure 1a).
Han et al, Joung et al, Zhu et al, Yesbolatova et al, Li et al taught/disclosed wherein the Cas nuclease is a Cas9 nuclease.
Han et al do not teach wherein the Cas nuclease is a Cas12/Cpf1 nuclease.
However, prior to the effective filing date of the instantly claimed invention, Kim et al is considered relevant prior art for having taught that because Cpf1 nucleases recognize a different PAM motif than Cas9 nucleases, the CRISPR/Cpf1 system could provide an alternative to the CRISPR/Cas9 system (e.g. Abstract), and has about the same editing efficiency as the CRISPR/Cas9 system (e.g. Figure 4d-e).
Kim et al taught that using the same gene-editing method parameters, Cpf1 nucleases generate indels of substantially the same sizes as Cas9 nucleases (e.g. Figure 4c, Cas9 indels ranging 1-21 nucleotides, Cpf1 indels ranging 2-16 nucleotides).
As discussed above, Joung et al disclosed that the CRISPR/Cas9 system naturally generates deletions ranging in size from 2, 6, 14, 22, 56, 78, 94, 115, 181, 202, and 365 nucleotides at the target site where the nuclease causes a break in the endogenous genome (e.g. Figure 3C).
Thus, one of ordinary skill in the art would have reasonably inferred that Cpf1 nucleases, being substantially similar to Cas9 nucleases, as demonstrated by Kim et al, would similarly naturally generates deletions ranging in size from 2, 6, 14, 22, 56, 78, 94, 115, 181, 202, and 365 nucleotides at the target site where the nuclease causes a break in the endogenous genome, as per Joung et al, under the same or substantially the same gene-editing method conditions as Cas9. Such deletions would substantially overlap with the same lesions as generated by the gRNA/Cas9 system of Han et al.
Resolving the level of ordinary skill in the pertinent art.
People of the ordinary skill in the art will be highly educated individuals such as medical doctors, scientists, or engineers possessing advanced degrees, including M.D.'s and Ph.D.'s. Thus, these people most likely will be knowledgeable and well-read in the relevant literature and have the practical experience in molecular biology, gene editing technologies, and natural law of cell biology regarding DNA repair. Therefore, the level of ordinary skill in this art is high.
"A person of ordinary skill in the art is also a person of ordinary creativity, not an automaton." KSR International Co. v. Teleflex Inc., 550 U.S. ___, ___, 82 USPQ2d 1385, 1397 (2007). "[I]n many cases a person of ordinary skill will be able to fit the teachings of multiple patents together like pieces of a puzzle." Id. Office personnel may also take into account "the inferences and creative steps that a person of ordinary skill in the art would employ." Id. at ___, 82 USPQ2d at 1396.
Considering objective evidence present in the application indicating obviousness or nonobviousness.
The focus when making a determination of obviousness should be on what a person of ordinary skill in the pertinent art would have known at the time of the invention, and on what such a person would have reasonably expected to have been able to do in view of that knowledge. This is so regardless of whether the source of that knowledge and ability was documentary prior art, general knowledge in the art, or common sense. M.P.E.P. §2141.
The rationale to modify or combine the prior art does not have to be expressly stated in the prior art; the rationale may be expressly or impliedly contained in the prior art or it may be reasoned from knowledge generally available to one of ordinary skill in the art, established scientific principles, or legal precedent established by prior case law. In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988); In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992). See also In re Kotzab, 217 F.3d 1365, 1370, 55 USPQ2d 1313, 1317 (Fed. Cir. 2000) (setting forth test for implicit teachings); In re Eli Lilly & Co., 902 F.2d 943, 14 USPQ2d 1741 (Fed. Cir. 1990) (discussion of reliance on legal precedent); In re Nilssen, 851 F.2d 1401, 1403, 7 USPQ2d 1500, 1502 (Fed. Cir. 1988) (references do not have to explicitly suggest combining teachings); and Ex parte Levengood, 28 USPQ2d 1300 (Bd. Pat. App. & Inter. 1993) (reliance on logic and sound scientific reasoning). See MPEP §2144.
Prior to the effective filing date of the instantly claimed invention, it would have been obvious to one of ordinary skill in the art to modify the 5’ homology arm of Han et al to further comprise exogenous coding sequence or exogenous partial coding sequence of the essential gene corresponds to endogenous coding sequence of the essential gene located downstream (3’) of the break, as taught by Zhu et al, Yesbolatova et al, and Li et al with a reasonable expectation of success because those of ordinary skill in the art previously recognized and successfully reduced to practice such design principles in order to generate their desired fusion protein knock-in construct.
Wherein the break is located within the first 50 or 21 nucleotides of GAPDH exon 9, as discussed above, Joung et al disclosed that the CRISPR/Cas9 system naturally generates deletions ranging in size from 2, 6, 14, 22, 56, 78, 94, 115, 181, 202, and 365 nucleotides at the target site where the nuclease causes a break in the endogenous genome (e.g. Figure 3C).
Nimonkar et al taught that the end-resection mechanism in human cells can naturally remove a few hundred nucleotides (e.g. pg 350, col. 2) to a few thousands of nucleotides (e.g. pg 357, col. 2).
Galetto et al and San Filippo et al taught that homologous recombination to resolve the 5’ and 3’ D-loops may occur anywhere within the D-loops, including much farther away from the actual site of the initial double-stranded break.
In the case where the claimed ranges "overlap or lie inside ranges disclosed by the prior art" a prima facie case of obviousness exists. In re Wertheim, 541 F.2d 257, 191 USPQ 90 (CCPA 1976); In re Woodruff, 919 F.2d 1575, 16 USPQ2d 1934 (Fed. Cir. 1990). It is routine procedure to optimize component amounts to arrive at an optimal product that is superior for its intended use, since it has been held where the general conditions of a claim are disclosed in the prior art, discovering the optimum or workable ranges involves only routine skill in the art. Similarly, a prima facie case of obviousness exists where the claimed ranges or amounts do not overlap with the prior art but are close enough that one skilled in the art would have expected them to have the same properties. See M.P.E.P. §2144.05(I).
Instant application fails to disclose an element of criticality for the break to be within the 1st, 2nd, 5th, 10th, 15th, 20th, 21st, 23rd, 27th, 30th, 35th, 40th, 43rd, 47th, and/or 50th, as opposed to the 52nd, 54th, 58th, 61st, 64th, and/or 66th nucleotide of the GAPDH exon 9.
In light of the natural law of cell biology that the double-stranded break naturally results in end resection lengths of up to hundreds of nucleotides, whereupon after strand invasion and DNA synthesis, the actual resolution between the donor template and the genomic DNA may occur anywhere within the D-loop, including hundreds of nucleotides 5’ and/or 3’ to the actual target site DNA break, it is considered that the gRNA of Han et al would also be capable of generating recombination events within the first 50 and /or 21 nucleotides of the GAPDH exon 9.
The 5’ homology arm of Han et al is 900 nucleotides in length, and encodes the entire last exon of GAPDH (Figure 1a, exon 9).
Zhu et al taught the 5’ homology arm was almost 800 nucleotides in length (pg 6), and encoded the entire last exon (e.g. Figure 6a, exons 9 and 10).
Yesbolatova et al taught the 5’ homology arm is about 1000 nucleotides in length (pg 74, col. 2), and encodes the last coding exon of the target gene (Figure 2a).
Li et al taught the 5’ homology arm is about 1300 nucleotides in length and encodes the last coding exon of the target gene (Figure 1).
Even if one was to argue that the resulting GAPDH knock-in sequence is missing the last amino acid, upstream of the exogenous gene of interest, there is no objective evidence, or element of criticality for, the absence of the last amino acid of GAPDH to render the edited GAPDH gene inactive or loss of function.
The "mere existence of differences between the prior art and an invention does not establish the invention's nonobviousness." Dann v. Johnston, 425 U.S. 219, 230, 189 USPQ 257, 261 (1976). The gap between the prior art and the claimed invention may not be "so great as to render the [claim] nonobvious to one reasonably skilled in the art."Id.
Furthermore, both Zhu et al and Yesbolatova et al taught the 5’ homology arm of the donor template included the C-terminal amino acid coding sequences fused in frame with the peptide or polypeptide of interest, thereby restoring the entire amino acid sequence of the target gene that is cleaved by the gRNA.
Prior to the effective filing date of the instantly claimed invention, it also would have been obvious to one of ordinary skill in the art to substitute a first GAPDH exon 9 gRNA targeting sequence, as per Han et al, with a second GAPDH exon 9 gRNA targeting sequence, to wit, SEQ ID NO:95, in a method of editing and inserting a knock-in expression cassette in the endogenous GAPDH exon 9 in a mammalian cell with a reasonable expectation of success because the simple substitution of one known element for another would have yielded predictable results to one of ordinary skill in the art at the time of the invention. M.P.E.P. §2144.07 states "The selection of a known material based on its suitability for its intended use supported a prima facie obviousness determination in Sinclair & Carroll Co. v. Interchemical Corp., 325 U.S. 327, 65 USPQ 297 (1945).” An artisan would have been motivated to substitute a first GAPDH exon 9 gRNA targeting sequence with a second GAPDH exon 9 gRNA targeting sequence, to wit, SEQ ID NO:95, in a method of editing and inserting a knock-in expression cassette in the endogenous GAPDH exon 9 in a mammalian cell because those of ordinary skill in the art previously recognized the scientific and technical concepts that the GAPDH exon 9 is only 69 nucleotides in length, and “a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipate success, it is likely that product not of innovation but of ordinary skill and common sense.” One of ordinary skill in the art would understand how to design nucleic acid molecules encoding guide RNA target sequences directed to the previously known GAPDH exon 9 nucleotide sequences, the artisan merely using readily available online guide RNA tool resources with which to identify the finite potential options within the 69 nucleotides of GAPDH exon 9, whereby the ordinary artisan could have pursued the known potential options with a reasonable expectation of success, and the number of possible GAPDH exon 9 guide RNA target sequences from which to choose is neither astronomical nor insurmountable. It would be only routine experimentation to determine which guide RNAs, paired with their corresponding Cas nuclease, achieve the desired editing activity in GAPDH exon 9.
There is no disclosed element of criticality for a gRNA targeting sequence comprising the nucleotides of SEQ ID NO:95, as opposed to other gRNA targeting sequences that also target GAPDH exon 9 in a method of inserting a knock-in expression cassette into the GAPDH exon 9.
The "mere existence of differences between the prior art (Han et al gRNA) and an invention (instant gRNA target sequence SEQ ID NO:95) does not establish the invention's nonobviousness." Dann v. Johnston, 425 U.S. 219, 230, 189 USPQ 257, 261 (1976). The gap between the prior art and the claimed invention may not be "so great as to render the [claim] nonobvious to one reasonably skilled in the art."Id.
As shown be Kim et al, those of ordinary skill in the art would have reasonably expected that, under the same gene editing method experimental conditions, the gRNA/Cas9 system of Han et al would generate breaks within the first 21 and/or 50 nucleotides of GAPDH exon 9 as per instantly recited gRNA SEQ ID NO:95/Cpf1.
The "mere existence of differences between the prior art (Han et al gRNA) and an invention (instant gRNA target sequence SEQ ID NO:95) does not establish the invention's nonobviousness." Dann v. Johnston, 425 U.S. 219, 230, 189 USPQ 257, 261 (1976). The gap between the prior art and the claimed invention may not be "so great as to render the [claim] nonobvious to one reasonably skilled in the art."Id.
There is no disclosed element of criticality for the location and/or size of the breaks within GAPDH exon 9, as it pertains to a method of inserting a knock-in expression cassette into the GAPDH exon 9. Again, the GAPDH exon 9 is only 69 nucleotides in length.
Han et al taught wherein the donor template comprises a 900 nucleotide 5' homology arm upstream of the knock-in cassette and a 900 nucleotide 3' homology arm downstream of the knock-in cassette, said 5’ homology arm comprising intronic sequences 5’ to exon 9 (e.g. Figure 1a).
Thus, those of ordinary skill in the art would have reasonably expected that the integration of the knock-in expression cassette of Han et al using the gRNA of Han et al would be molecularly indistinguishable from the integration of the knock-in expression cassette of Han et al using a gRNA comprising the nucleotide sequence of SEQ ID NO:95 because the gRNA target sequences are so close to each other in the GAPDH exon 9 and the actual 5’ homology arm recombination event takes place 5’ upstream of the cleavage site induced by both gRNAs.
Prior to the effective filing date of the instantly claimed invention, it also would have been obvious to one of ordinary skill in the art to substitute a first Cas nuclease, e.g. Cas9, with a second Cas nuclease, to wit, Cpf12a/Cpf1, in a method of editing the endogenous GAPDH locus in a mammalian cell with a reasonable expectation of success because the simple substitution of one known element for another would have yielded predictable results to one of ordinary skill in the art at the time of the invention. M.P.E.P. §2144.07 states "The selection of a known material based on its suitability for its intended use supported a prima facie obviousness determination in Sinclair & Carroll Co. v. Interchemical Corp., 325 U.S. 327, 65 USPQ 297 (1945).” An artisan would have been motivated to substitute Cas9 with Cpf1 in a method of editing the endogenous GAPDH locus in a mammalian cell because those of ordinary skill in the art previously recognized the scientific and technical concepts that they may use Cpf1 instead of Cas9 to edit their gene of interest in mammalian host cells, as successfully demonstrated by Kim et al, whereby Cpf1 was an art-recognized alternative to Cas9 and has substantially the same efficiency as Cas9.
It is proper to "take account of the inferences and creative steps that a person of ordinary skill in the art would employ." KSR Int'l Co. v. Teleflex Inc., 127 S. Ct. 1727, 1741,82 USPQ2d 1385, 1396 (2007). See also Id. At 1742, 82 USPQ2d 1397 ("A person of ordinary skill is also a person of ordinary creativity, not an automaton.").
It should be noted that the KSR case forecloses the argument that a specific teaching, suggestion, or motivation is required to support a finding of obviousness. See the recent Board decision Ex parte Smith, —USPQ2d—, slip op. at 20, (Bd. Pat. App. & Interf. June 25, 2007) (citing KSR, 82 USPQ2d at 1396) (available at http: www. uspto.gov/web/offices/dcom/bpai/prec/fd071925 .pdf).
With respect to Claim 166, the claim is not considered to further limit the independent claim. See the above 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, rejection.
To the extent Applicant argues otherwise, see the above 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, and 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, rejections.
The cited prior art meets the criteria set forth in both Graham and KSR, and the teachings of the cited prior art provide the requisite teachings and motivations with a clear, reasonable expectation of success. Thus, the invention as a whole is prima facie obvious.
Response to Arguments
Applicant argues that the cited references do not teach or fairly suggest a gRNA comprising the nucleotide sequence of SEQ ID NO:95.
Applicant’s argument(s) has been fully considered, but is not persuasive.
The "mere existence of differences between the prior art (Han et al gRNA) and an invention (instant gRNA target sequence SEQ ID NO:95) does not establish the invention's nonobviousness." Dann v. Johnston, 425 U.S. 219, 230, 189 USPQ 257, 261 (1976). The gap between the prior art and the claimed invention may not be "so great as to render the [claim] nonobvious to one reasonably skilled in the art."Id.
Han et al taught wherein the donor template comprises a 900 nucleotide 5' homology arm upstream of the knock-in cassette and a 900 nucleotide 3' homology arm downstream of the knock-in cassette, said 5’ homology arm comprising intronic sequences 5’ to exon 9 (e.g. Figure 1a).
Thus, those of ordinary skill in the art would have reasonably expected that the integration of the knock-in expression cassette of Han et al using the gRNA of Han et al would be molecularly indistinguishable from the integration of the knock-in expression cassette of Han et al using a gRNA comprising the nucleotide sequence of SEQ ID NO:95 because the gRNA target sequences are so close to each other in the GAPDH exon 9 and the actual 5’ homology arm recombination event takes place 5’ upstream of the cleavage site induced by both gRNAs.
Applicant argues secondary consideration that the specification demonstrates a 30-fold increase in knock-in efficiency as compared to the method of Han et al.
Applicant’s argument(s) has been fully considered, but is not persuasive.
As a first matter, instant claims recite no such efficiency requirement.
As a second matter, instant independent claim fails to recite the method step parameters necessary and sufficient to achieve the asserted secondary consideration. See the above 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, and 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, rejections. Thus, instant claims are prima facie not commensurate in scope to the asserted secondary considerations.
Double Patenting
8. The prior provisional rejections of Claims 145, 147 and 166 on the ground of nonstatutory double patenting as being unpatentable over claims 1, 83, 125, 145, 147, 150, 152, 162, 186-187, 190, 192, 199, 205, 227, 229-230 of copending Application No. 17/923,358 (reference application; U.S. 2023/0227856; claim set filed July 13, 2023) are withdrawn in light of Applicant’s amendments to instant independent Claim 145, limitations not recited in the co-pending application’s claims.
Conclusion
9. No claims are 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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KEVIN K. HILL
Examiner
Art Unit 1638
/KEVIN K HILL/Primary Examiner, Art Unit 1638
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