DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s amendment filed on 09/30/2025 has been entered.
Amended claims 1, 6-8, 10-13, 20, 28, 35 and new claims 40-41 are pending in the present application, and they are examined on the merits herein.
Response to Amendment
1. The rejection under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, for Lack of Written Description was withdrawn in light of currently amended claims 28 and 35, particularly with the new limitation “wherein expression of the gene is decreased by 40%”.
2. The rejection under 35 U.S.C. 103 as being unpatentable over Hnisz et al (US 2014/0287932) in view of Fanucchi et al (US 2016/0215280), Dixon et al (Nature 485:376-380, 2012; IDS) and Sofueva et al (EMBO J. 32:3119-3129, 2013; IDS) was withdrawn in light of currently amended independent claims 1 and 10, particularly with the new limitation “wherein the CRISPR/Cas9 system comprises Cas9 and a first guide RNA complementary to SEQ ID NO: 8 and a second guide RNA complementary to SEQ ID NO: 9”.
3. The rejection under 35 U.S.C. 103 as being unpatentable over Hnisz et al (US 2014/0287932) in view of Fanucchi et al (US 2016/0215280), Hnisz et al (WO 2017/011710 with the effective filing date of 07/14/2015) and Sofueva et al (EMBO J. 32:3119-3129, 2013; IDS) was withdrawn in light of currently amended independent claims 1 and 10, particularly with the new limitation “wherein the CRISPR/Cas9 system comprises Cas9 and a first guide RNA complementary to SEQ ID NO: 8 and a second guide RNA complementary to SEQ ID NO: 9”.
Claim Rejections - 35 USC § 112 (New Matter)
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.
Amended claims 1, 6-8, 10-13, 20, 28, 35 and 40-41 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. This is a new ground of rejection necessitated by Applicant’s amendment.
Amended independent claims 1 and 10 recite the limitation “wherein the CRISPR/Cas9 system comprises Cas9 and a first guide RNA complementary to SEQ ID NO: 8 and a second guide RNA complementary to SEQ ID NO: 9”. The claims encompass a method of decreasing the expression of a Nanog gene in a mammalian embryonic stem cell (e.g., a murine embryonic stem cell, a human embryonic stem cell, a non-human primate embryonic stem cell, an elephant embryonic stem cell, a dolphin embryonic stem cell, a bat embryonic stem cell, a kangaroo embryonic stem cell) with a CRISPR/Cas9 system comprising Cas9 and a first guide RNA complementary to SEQ ID NO: 8 (20 nts) and a second guide RNA complementary to SEQ ID NO: 9 (20 nts), wherein the Nanog gene is located in an insulated neighborhood (IN) formed by looping of two CTCF interaction sites occupied by cohesin on a chromosome in the cell, and wherein the sequence of at least one of the two CTCF interaction sites is deleted that disrupts binding of CTCF protein. The as-filed specification does not have a written support for the above limitation.
As an initial matter, currently amended 1, 6-8, 10-13, 20, 28, 35 and 40-41 have been introduced in the Amendment filed on 09/30/2025, so the claims themselves are not part of the original disclosure. 37 CFR § 1.115(a)(2). “New or amended claims which introduce elements or limitations that are not supported by the as-filed disclosure violate the written description requirement. . . . While there is no in haec verba requirement, newly added claims or claim limitations must be supported in the specification through express, implicit, or inherent disclosure. . . . The fundamental factual inquiry is whether the specification conveys with reasonable clarity to those skilled in the art that, as of the filing date sought, applicant was in possession of the invention as now claimed.” MPEP § 2163, part (I)(B); see also MPEP § 2163.02. In this case, the original specification does not convey the particular invention recited in currently amended claims 1, 6-8, 10-13, 20, 28, 35 and 40-41 with reasonable clarity to skilled artisans. “The trouble is that there is no such disclosure, easy though it is to imagine it.” MPEP § 2163.05, part (II) (quoting In re Ruschig, 379 F.2d 990, 995, 154 USPQ 118, 123 (CCPA 1967)).
In the Amendment filed on 09/30/2025 (at page 6, first paragraph), Applicant cited paragraphs [0166] and [0279] of the as-filed specification as an alleged written support for the above limitation. However, both cited paragraphs [0166] and [0279] are related to SEQ ID NO: 8 and SEQ ID NO: 9 genomic sequences complementary to guide RNAs in the genome editing experiments for V6.5 murine embryonic stem cells; and not for any other mammalian embryonic stem cells (see also paragraphs [00165], [00167], [[0175], [0176], and Fig. 4B). There is also no evidence of record indicated that both of murine target genomic sequences of SEQ ID NO: 8 and SEQ ID NO: 9 are 100% identical to those or even present in the genomes of other mammalian embryonic stem cells. Please note that the specification teaches that sgRNA targeting regions are around 200 base pairs up- and downstream of a boundary CTCF binding site (paragraph [001670]). Moreover, with a CRISPR-Cas9 system comprising Cas9 and a first guide RNA complementary to SEQ ID NO: 8 and a second guide RNA complementary to SEQ ID NO: 9, only one and not two CTCF interaction sites occupied by cohesion is deleted (please see reproduced Fig. 4B below). Accordingly, the as-filed specification does not have a written support for the limitation recited in currently amended claims 1, 6-8, 10-13, 20, 28, 35 and 40-41.
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The fact that the person of ordinary skill in the art could have carried out the claimed invention without undue experimentation based on applicants’ disclosure is inadequate to meet this requirement. “The Federal Circuit has pointed out that, under United States law, a description that merely renders a claimed invention obvious may not sufficiently describe the invention for the purposes of the written description requirement of 35 U.S.C. 112.” MPEP § 2163, part (I)(A).
Therefore, given the lack of sufficient guidance provided by the originally filed specification, it would appear that Applicants did not contemplate or have possession of invention as now claimed at the time the application was filed.
Claim Rejections - 35 USC § 112 (Scope of Enablement)
Amended claims 1, 6-8, 10-13, 20, 28, 35 and 40-41 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for:
A. A method of decreasing expression of a murine Nanog gene, the method comprising contacting a murine embryonic stem cell with a CRISPR/Cas9 system, wherein the Nanog gene is located in an insulated neighborhood (IN) formed by looping of two CTCF interaction sites occupied by cohesin on a chromosome of the cell, wherein the CRISPR-Cas9 system comprising Cas9 and a first guide RNA complementary to SEQ ID NO: 8 and a second guide RNA complementary to SEQ ID NO: 9 that result in the deletion of one of the two CTCF interaction sites and disrupting binding of CTCF protein, and wherein expression of the gene is decreased by 40%; and
B. A method of decreasing expression of a Nanog gene in a murine embryonic stem cell in a cell culture, the method comprises deleting a DNA sequence comprising one of two CTCF interaction sites that flank the Nanog gene on a chromosome in the cell using a CRISPR/Cas9 system, wherein the Nanog gene is located in an insulated neighborhood (IN) formed by looping of the two CTCF interaction sites occupied by cohesin on the chromosome, wherein the CRISPR/Cas9 system comprises Cas9 and a first guide RNA complementary to SEQ ID NO: 8 and a second guide RNA complementary to SEQ ID NO: 9, and wherein expression of the gene is decreased by 40%;
does not reasonably provide enablement for other methods for decreasing expression of a Nanog gene in any other mammalian embryonic stem cells as claimed broadly. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention commensurate in scope with these claims. This is a new ground of rejection necessitated by Applicant’s amendment.
The factors to be considered in the determination of an enabling disclosure have been summarized as the quantity of experimentation necessary, the amount of direction or guidance presented, the state of the prior art, the relative skill of those in the art, the predictability or unpredictability of the art and the breadth of the claims. Ex parte Forman, (230 USPQ 546 (Bd Pat. Appl & Unt, 1986); In re Wands, 858 F.2d 731, 8 USPQ 2d 1400 (Fed. Cir. 1988)).
The instant specification is not enabled for the instant broadly claimed invention for the reasons discussed below.
1. The breadth of the claims
Amended claims 1, 6-8, 28 and 40 encompass a method of decreasing expression of a Nanog gene in any amount, not necessarily limited to a 40% decrease, in any mammalian embryonic stem cell (e.g., a murine embryonic stem cell, a human embryonic stem cell, a non-human primate embryonic stem cell, an elephant embryonic stem cell, a dolphin embryonic stem cell, a bat embryonic stem cell, a kangaroo embryonic stem cell) with a CRISPR/Cas9 system comprising Cas9 and a first guide RNA complementary to SEQ ID NO: 8 (20 nts) and a second guide RNA complementary to SEQ ID NO: 9 (20 nts), wherein the Nanog gene is located in an insulated neighborhood (IN) formed by looping of two CTCF interaction sites occupied by cohesin on a chromosome in the cell, and wherein the sequence of at least one of the two CTCF interaction sites (including the sequences of both CTCF interaction sites) is deleted that disrupts binding of CTCF protein.
Amended claims 10-13, 20, 35 and 41 encompass a method of decreasing expression of a Nanog gene in any amount, not necessarily limited to a 40% decrease, in any mammalian embryonic stem cell (e.g., a murine embryonic stem cell, a human embryonic stem cell, a non-human primate embryonic stem cell, an elephant embryonic stem cell, a dolphin embryonic stem cell, a bat embryonic stem cell, a kangaroo embryonic stem cell) in a cell culture, the method comprises deleting a DNA sequence comprising at least one of two CTCF interaction sites (including deleting the sequences of both CTCF interaction sites) that flank the Nanog gene on a chromosome in the cell using a CRISPR/Cas9 system, wherein the Nanog gene is located in an insulated neighborhood (IN) formed by looping of the two CTCF interaction sites occupied by cohesin on the chromosome, wherein the CRISPR/Cas9 system comprises Cas9 and a first guide RNA complementary to SEQ ID NO: 8 and a second guide RNA complementary to SEQ ID NO: 9. Dependent claim 20 encompasses expression of a gene located outside the insulated neighborhood in which the Nanog gene is located is also altered (encompassing both increased expression and/or decreased expression).
2. The state and the unpredictability of the prior art
Before the effective filing date of the present application (09/30/2015), virtually nothing was known about the use of a CRISPR/Cas9 system comprising Cas9 and first guide RNA complementary to SEQ ID NO: 8 and a second guide RNA complementary to SEQ ID NO: 9 to delete at least one of the two CTCF interaction sites that flank a Nanog gene on a chromosome in a mammalian embryonic stem cell to decrease expression of the Nanog gene, wherein the Nanog gene is located in an insulated neighborhood (IN) formed by looping of the two CTCF interaction sites occupied by cohesin on the chromosome as evidenced at least by the teachings of Hnisz et al (US 2014/0287932), Hnisz et al (WO 2017/011710 with the effective filing date of 07/14/2015), Fanucchi et al (US 2016/0215280), Dixon et al (Nature 485:376-380, 2012; IDS) and Sofueva et al (EMBO J. 32:3119-3129, 2013; IDS). With respect to the state of the CRISPR/Cas9 system before the effective filing date of the present application (09/30/2015), Zhang et al (Molecular Therapy-Nucleic Acids 4, e264, 2015; doi:10.1038/mtna.2015.37) stated “[t]he high frequency of off-target activity (≥50%)-RGEN (RNA-guided endonuclease)-induced mutations at sites other than the intended on-target site is one major concern, especially for therapeutic and clinical applications” (Abstract); and “Efforts toward modifications of sgRNA sequences to enhance the specificity of sgRNAs without compromising on-target efficiency have not provided consistent results.11,60 In addition, chromatin accessibility has been reported to be one major determinant of in vivo binding. As Zhang and Sharp reported recently, there are hundreds of thousands of “seeds+NGG” sites in the genome, yet <1% are actually bound by dCas9, and most of the matches are in promoters, enhancers and genes.21 Therefore, when we design sgRNA, we should choose sgRNAs in promoters, enhancers, and genes as far as possible to improve the target cleavage efficiency. However, those sites cleaved by CRISPR/Cas9 system can be both on-target and off-targets sites and we need to balance it according to different experimental purposes” (page 5, right column, first full paragraph). Additionally, Wu et al (Quant. Biol. 2:59-70, 2014) discussed numerous factors that could affect target specificity of the CRISPR-Cas9 system include: PAM, seed, Cas9/sgRNA abundance, target or guide sequence, accessibility of seed match genomic sites, abundance of seed match genomic sites, epigenetics, target sequence length, and sgRNA scaffold (section titled “Determinants of Cas9/sgRNA specificity” on page 6). Wu et al also stated clearly “Despite intense study, the rules governing the specificity of Cas9/sgRNA targeting, especially target cutting and mutation remain elusive. At this stage, it is still challenging to predict genome-wide off-targets of Cas9 with any significant confidence….The current rules of Cas9/sgRNA specificity are likely incomplete and biased” (see section title “Perspective” on page 11). The physiological art is also recognized as unpredictable (MPEP 2164.03).
3. The amount of direction or guidance provided
Apart from disclosing using a CRISPR/Cas9 system comprising two plasmids expressing Cas9 and sgRNA targeting regions around 200 base pairs up- and downstream of a boundary CTCF binding site to generate murine ESC lines with CTCF site deletions at five Prdm14, mir-290-295, Pou5fl, Nanog and Tdgf1 super-enhancer domains, including the deletion of a boundary CTCF site at the Nanog SD (super-enhancer domain) using a first guide RNA complementary to SEQ ID NO: 8 and a second guide RNA complementary to SEQ ID NO: 9 that resulted in about 40% reduction in Nanog transcript (Example 1, particularly paragraphs [00165]-[00168], [00183], [00276]-[00279]; genomic sequences complementary to gRNAs listed in the Table at page 26; Figure 4B); the instant specification failed to provide sufficient guidance for an ordinary skilled artisan on how to decrease expression of a Nanog gene in any amount other than a 40% decrease (e.g., 50%, 70%, 80% or more decrease) in a murine embryonic stem cell using a CRISPR/Cas9 system comprising a first guide RNA complementary to SEQ ID NO: 8 and a second guide RNA complementary to SEQ ID NO: 9, let alone in any other mammalian embryonic stem cell as encompassed broadly by currently amended claims. Moreover, there is no evidence of record indicating or suggesting that a CRISPR/Cas9 system comprising Cas9 along with a first guide RNA complementary to SEQ ID NO: 8 and a second guide RNA complementary to SEQ ID NO: 9 is capable of deleting both CTCF interactions that flank a Nanog gene located in an insulated neighborhood formed by looping of the two CTFC interaction sites occupied by cohesin on a chromosome as evidenced at least by Fig. 4B of the present application. Additionally, there is also no evidence of record indicating that both of the murine target genomic sequences of SEQ ID NO: 8 and SEQ ID NO: 9 are 100% identical to those or even present in the genomes of other mammalian embryonic stem cells. Please note that the specification teaches that sgRNA targeting regions are around 200 base pairs up- and downstream of a boundary CTCF binding site (paragraph [001670]).
With respect to dependent claim 20 reciting the limitation “wherein expression of a gene located outside the insulated neighborhood is also altered”; the instant specification stated explicitly “CRISP-mediated deletion of the boundary CTCF site C1 of the Nanog SD led to a ͠͠ 40% drop in Nanog transcript levels (Figure 4B). In this case, there was no significant change in the level of the Dppa3 transcript (Figure 4B). These results indicate that normal expression of the Nanog transcript is dependent on the C1 CTCF site” (top of paragraph [00279]). Thus, the as-filed specification also fails to provide sufficient guidance for an ordinary skill in the art on how to alter (increase or decrease) expression of a gene located outside of the insulated neighborhood in which the Nanog gene is located in a murine embryonic stem cell via the use of a CRIRP/Cas9 comprising Cas9 along with a first guide RNA complementary to SEQ ID NO: 8 and a second guide RNA complementary to SEQ ID NO: 9, let alone in any other mammalian embryonic stem cell as encompassed by the instant claim.
Since the prior art before the effective filing date of the present application failed to provide sufficient guidance regarding to the aforementioned issues, it is incumbent upon the present application to do so. Given the state of the prior art, coupled with the lack of sufficient guidance provided by the present application, it would have required undue experimentation for a skilled artisan to make and use the instant invention as claimed broadly.
As set forth in In re Fisher, 166 USPQ 18 (CCPA 1970), compliance with 35 USC 112, first paragraph requires:
That scope of claims must bear a reasonable correlation to scope of enablement provided by specification to persons of ordinary skill in the art; in cases involving predictable factors, such as mechanical or electrical elements, a single embodiment provides broad enablement in the sense that, once imagined, other embodiments can be made without difficulty and their performance characteristics predicted by resort to known scientific laws; in cases involving unpredictable factors, such as most chemical reactions and physiological activity, scope of enablement varies inversely with degree of unpredictability of factors involved.
Moreover, the courts have also stated that reasonable correlation must exist between scope of exclusive right to patent application and scope of enablement set forth in the patent application (27 USPQ2d 1662 Ex parte Maizel.).
Accordingly, due to the lack of sufficient guidance provided by the specification regarding to the issues set forth above, the state and unpredictability of the relevant art, and the breadth of the instant claims, it would have required undue experimentation for one skilled in the art to make and use the instant broadly claimed invention.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Quang Nguyen, Ph.D., at (571) 272-0776.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s SPE, James Douglas (Doug) Schultz, Ph.D., may be reached at (571) 272-0763.
To aid in correlating any papers for this application, all further correspondence regarding this application should be directed to Group Art Unit 1633; Central Fax No. (571) 273-8300.
Any inquiry of a general nature or relating to the status of this application or proceeding should be directed to (571) 272-0547.
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/QUANG NGUYEN/Primary Examiner, Art Unit 1631