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 .
Claims 1, 3 – 15, and 17 – 28 are pending. Claims 1, 3, 4, 12, 17, 18, 21, 24, and 26 have been amended and claims 2 have been canceled by Applicants’ amendment filed on 7/22/2026. No claims were added.
Claims 25 and 28 were previously withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. The restriction requirement was previously made FINAL. Claims 1, 3 – 15, 17 – 24, 26, and 27 are under consideration to which the following grounds of rejection are applicable.
Withdrawn Specification Objection
The objection to the specification is withdrawn in view of Applicant’s amendment to the specification to include a proper symbol indicating use in commerce.
Withdrawn Claim Objections
The objections to claim 1 are withdrawn in view of Applicant’s amendment to the claim.
The objection to claim 12 is withdrawn in view of Applicant’s amendment to the claim.
The objection to claim 17 is withdrawn in view of Applicant’s amendment to the claim.
The objection to claim 18 is withdrawn in view of Applicant’s amendment to the claim.
The objection to claim 21 is withdrawn in view of Applicant’s amendment to the claim.
The objection to claim 24 is withdrawn in view of Applicant’s amendment to the claim.
The objection to claim 26 is withdrawn in view of Applicant’s amendment to the claim.
Withdrawn Claim Rejections
The rejection of claim 2 under 35 U.S.C. 112(b) is rendered moot in view of Applicant’s cancellation of the claim.
The rejection of claim 2 under 35 U.S.C. 103 is rendered moot in view of Applicant’s cancellation of the claim.
The rejection of claims 1, 3 – 11, 13 – 15, 17, 18, 22 – 24, 26, and 27 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
The rejection of claim 12 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
The rejection of claim 19 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
The rejection of claims 20 and 21 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1.
The rejection of claim 2 on the ground of nonstatutory double patenting as being unpatentable over claims 1 – 15 of U.S. Patent No. 12325855 is rendered moot in view of Applicant’s cancellation of the claim.
Maintained Claim Rejections - 35 USC § 112b
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.
Claims 1, 3 – 15, 17 – 24, and 26 – 27 remain 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.
Regarding claim 1, it is unclear how step (a) of inserting a nucleic acid sequence into the genome of a population of cells is accomplished by “via Homology-independent Universal Genome Engineering” because Applicant’s specification defines HiUGE as “a vector system”, which are nucleic acids. Therefore, the metes and bounds of inserting a nucleic acid sequence into the genome of a population of cells via nucleic acids is unclear. Claims 3 – 15, 17 – 24, and 26 – 27 are also rejected as they depend from claim 1 and do not clarify the grounds of rejection.
Claim 1 is indefinite in its recitation of “wherein the nucleic acid encoding the second donor polypeptide is inserted into a coding sequence for a gene”. Claim 1 recites “inserting a nucleic acid sequence encoding a first donor polypeptide into the genome of a population of cells via HiUGE. The Specification support that the cells were transfected with by a knock-in vector (e.g., a HiUGE StableTag vector) (para [0017] of the published application) where “A second vector (e.g., a DonorTag vector), was then transduced into the puromycin-selected cells, wherein the second vector includes a donor sequence, as well as a hygromycin resistance gene (FIG. 1B).” “with flippase exchanged cassettes between StableTag and DonorTag through the FRT sites via recombinase-mediated cassette exchange (RMCE).” (para [0017] of the published application). Thus the Specification appears to provide support for a knock-in vector into any sequence of a gene and the second nucleic acid sequence encoding the second polypetide flanked by two FRT sites results in their crossover. Thus it is the first nucleic acid sequence and not the second sequence that appears to determine the endogenous location of the incorporated construct at the locus of interest. As such the metes and bounds of the claim are indefinite.
Claim 1 is indefinite in its recitation of “express the second donor
polypeptide and an endogenous polypeptide encoded by the gene” as it is unclear whether the expressed gene is inside or outside the integration site of the extra piece of second donor DNA one or more recombinase target sites in the vector identical to the integrated first donor DNA target site. As such the metes and bounds of the claim are indefinite.
Regarding claim 3, the claim lacks antecedent basis because claim 1 does not recite “fusion polypeptide” in step (c) or the “wherein” clause.
Claim 4 recites the limitation “the fusion polypeptide” in line 2. There is insufficient antecedent basis for this limitation in the claim.
Claim 8 is unclear for the recitation of “the second donor polypeptide is inserted into a noncoding sequence in the genome of the cell.”. Claim 1 has been amended to recite “the second donor polypeptide is inserted into a coding sequence for a gene”. Thus it is unclear how second donor polypeptide can be simultaneously located in a ” coding sequence” and “non-coding sequence”.
Claim 12 is indefinite in its recitation of “flanked by a splice acceptor and a splice first donor site” as it is unclear if this sites are in addition to the one or more recombinase target sites in the vector required in claim 1. As such the metes and bounds of the claim are indefinite.
New Objections/Rejections Necessitated by Amendment
Claim Objections
Claim 3 is objected to because of the following informalities: in line 2 – 3, “and an endogenous polypeptide encoded by the gene” should read “and the endogenous polypeptide encoded by the gene” to clarify that “endogenous polypeptide” refers to the same “endogenous polypeptide” of claim 1. Appropriate correction is required.
Claim 27 is objected to because of the following informalities: in line 1 – 2, “wherein the stem cell is an embryonic stem cell or an induced pluripotent stem cell” should read “wherein the stem cells are embryonic stem cells or induced pluripotent stem cells” because claim 26 has been amended to recite “cells” and not the singular “cell”. Appropriate correction is required.
Claim Rejections - 35 USC § 112
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.
Claims 8 – 12 are 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 1 requires “the second donor polypeptide is inserted into a coding sequence for a gene”. Claims 8 – 11 do not further limit claim 1 and instead broaden claim 1 because they are drawn to inserting the second donor polypeptide into a noncoding sequence in the genome of the cell.
Claim 1 requires “wherein the selected cells express the second donor polypeptide and an endogenous polypeptide encoded by the gene”. Claim 12 does not further limit claim 1 and instead broadens claim 1 to include expression of synthetic polypeptides containing an inserted synthetic exon.
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.
Claim Interpretation
For the purpose of applying prior art, step (a) of claim 1 is interpreted as inserting a nucleic acid sequence encoding a first donor polypeptide into the genome of a population of cells using a HiUGE donor vector based on Applicant’s specification at para. 0033 defining RMCE and para. 0038 defining HiUGE and para. 0038 describing a HiUGE donor vector.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or 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.
Claim(s) 1, 3, 4, 8 – 15, 17, 18, and 24 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gao (Gao, Yudong, et al. Neuron 103.4 (2019): 583-597; previously cited), hereinafter Gao in view of Zhang (Zhang, Xu, et. al. G3: Genes, Genomes, Genetics 4.12 (2014): 2409-2418; previously cited), hereinafter Zhang.
Regarding step (a) of claim 1, Gao teaches a method of inserting a nucleic acid sequence encoding a hemagglutinin (HA) epitope tag or mCherry (“a nucleic acid sequence encoding a first donor polypeptide”) at the C-terminus of the coding sequence of the mouse Tubb3 gene in primary neurons (“into the genome of a population of cells”) using HiUGE vectors including a HiUGE donor vector (“via Homology-independent Genome Engineering” and “wherein the nucleic acid encoding the first donor polypeptide is located in a HiUGE donor vector”) (page 584, right col. para. 2; Figure 1B; page e3, para. 3; page e4, para. 1 – 4; page e5, para. 4 – 5; page e6, para. 1; Figure S1A; page 593, left col. last para. and right col. para. 1; page 590, right col. para. 2; Figure 6A-D). Gao teaches inserting a nucleic acid sequence encoding mCherry into the Tubb3 gene in primary neurons and selecting cells by immunostaining or direct fluorescence (step (b)) (page 589, right col. para. 3; Figure 6B – D). Gao does not teach “wherein the nucleic acid encoding the first donor polypeptide is flanked on each side by one or more recombinase target sites” of step (a) or step (c) or “wherein the nucleic acid encoding the second donor polypeptide is inserted into a coding sequence for a gene, and wherein the selected cells express the second donor polypeptide and an endogenous polypeptide encoded by the gene”.
Regarding claims 13 and 15, Gao teaches inserting mCherry (“selectable marker” of claim 13 and “fluorescent protein” of claim 15) into the Tubb3, Gfap, and Pdha1 locus using HiUGE vectors (page 589, right col. para. 3; Figure 6B – D).
Regarding claim 17, Gao teaches HA tag (page 584, right col. para. 2; Figure 1B; page e3, para. 3; page e4, para. 1 – 4; page e5, para. 4 – 5; page e6, para. 1; Figure S1A).
Regarding “eukaryotic cells” of claim 24, Gao teaches primary neurons (page e3, para. 3; page e5, para. 4 – 5).
Gao does not teach “wherein the nucleic acid encoding the first donor polypeptide is flanked on each side by one or more recombinase target sites” of step (a) or step (c) of claim 1 or any of the limitations of claims 3, 4, 8 – 12, 14, or 18 regarding a nucleic acid encoding a second donor polypeptide. Gao teaches the HiUGE method is CRISPR-mediated method that allows for inserting different nucleic acids (“payload”) into a coding sequence of a gene of interest (GOI) where cells expressing the endogenous GOI fused to the “payload” can be selected for (by fluorescence) as shown in Figure 1A (below). Gao does not teach exchanging one payload at a gene of interest (GOI) for a different payload as required by step (c) of claim 1.
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However, Gao teaches analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, but current approaches are often low throughput and resource intensive (Abstract). Gao teaches selective labeling and manipulation of endogenous proteins are essential to delineating the molecular mechanisms of cell and organismal biology (page 583, left col.). Gao teaches recent advances in exploratory proteomics and gene expression analysis generate sizable datasets that urgently require high-throughput and reliable methods for protein visualization and functional manipulation purposes yet current techniques to enable these strategies are often inefficient or resource intensive (page 583, left col.). Gao teaches single HiUGE donor vectors can be used to modify multiple proteins at their C- or N-termini and that multiple HiUGE payloads of different functional moieties can be inserted into a single protein (page 593, right col. para. 2). Gao teaches HiUGE payloads are universal with respect to gene identity, provided that the modifications are functionally tolerated, and retained during post-translational maturation of the protein products (some proteins may be post-translationally processed at both their C- and N-termini, thus inherently difficult to target) (page 593, right col. para. 2). Thus, a person of ordinary skill reading Gao would understand that when the HiUGE method produces a modification that is not functionally tolerated and/or retained during post-translational maturation, the HiUGE method would need to be used again to modify the gene of interest with a different payload. One of skill would be motivated to look to the art for a simpler way to exchange one payload for another without repeating the HiUGE method.
Regarding “flanked on each side by one or more recombinase sites” of step (a) and step (c) of claim 1, Zhang teaches a two-step CRISPR- and RMCE-based method for genome engineering comprising inserting a nucleic acid sequence encoding dsRed (a fluorescent protein) into a gene by CRISPR where the nucleic acid sequence encoding dsRed is located on a donor vector flanked by recombinase (attP) sites (“flanked on each side by one or more recombinase sites”), followed by exchanging the dsRed for a second nucleic acid sequence by RMCE where cells are contacted with a vector comprising the nucleic acid sequence encoding the second nucleic acid sequence flanked on each side by recombinase (attB) sites and a vector encoding the recombinase (ϕC31)that cleaves the recombinase target sites inserted into the genome and the recombinase sites in the vector (step (c)) (Figure 1; page 2409, right col. last para.; page 2410, left col. para. 1 and 4 and right col. para. 1; page 2411, right col.; page 2412, left col.; Figure 3).
Regarding claim 3, Zhang teaches the cells express Salm fused to V5 or FLAG in Figure 5.
Regarding claim 4, Zhang teaches expression of the Salm fusion protein is under the endogenous salm promoter (page 2416, left col. para. 2; Figure 4 and 5).
Regarding claims 8 – 11, Zhang teaches the second nucleic acid is a synthetic exon (GFP-3xFLAG exon or 2xTY1-V5 exon) that is inserted in intron 1 (“noncoding sequence” of claim 8; “regulatory sequence” of claim 9; “an intron” of claim 10 and 11) of the salm gene (Figure 4 and 5; page 2415, left col. and right col. para. 1).
Regarding claim 12, Zhang teaches in Figure 4B that the nucleic acid sequence encoding V5 or GFP-3xFLAG is flanked by a splice acceptor (SA) site and a splice donor (SD) site.
Regarding claim 14, Zhang teaches in Figure 4B that the vector encoding the second donor polypeptide comprises GFP-3xFLAG and therefore selectable by fluorescence (page 2415, left col. para. 1).
Regarding claim 18, Zhang teaches the peptide tags FLAG and V5 (page 2415, left col. and right col. para. 1; Figure 4 and 5).
Zhang teaches in Figure 5A that the CRISPR step of the two step method resulted in lethality when targeting inserting dsRed at the first intron of the salm gene (salm[1st intron-dsRed]), but this lethality could be overcome by exchanging dsRed for a second nucleic acid encoding TY1-V5 or GFP-3xFLAG by the RMCE step of the two step method as shown in Figure 5B (2415, left col., para. 1; Figure 4). Thus, Zhang teaches combining CRISPR gene editing with RMCE to exchange a first nucleic acid inserted by CRISPR that is not tolerated (lethal) with a second nucleic acid by RMCE that is tolerated. Zhang teaches the functionality of the two-step method was verified by the reversion of the lethality for the step 2 alleles, suggesting that both steps do not generate additional unintended changes on the chromosome (page 2417, left col. para. 2). Zhang teaches the two-step strategy combines the advantages of both CRISPR and RMCE, thus allowing very flexible modifications of a particular gene region with minimal effort (page 2417, left col. para. 2). Zhang teaches multiple fluorescent and affinity tags can be easily inserted or a deleted exon can effectively be replaced by various engineered exon versions (page 2417, left col. para. 2). Zhang teaches the two-step strategy allows structure-function analysis at the endogenous locus without interfering with the regulatory regions included in introns, which cannot be achieved by simply inserting a cDNA at the transcriptional start site (page 2417, left col. para. 2). Zhang teaches the strategy should be generally applicable to most Drosophila genes (page 2416, right col. para. 2). Zhang teaches the two-step CRISPR-RMCE method could be used by any fly laboratory to engineer their favorite gene for a broad range of applications (Abstract; page 2410, left col. para. 1).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Gao regarding inserting a first nucleic acid in the coding sequence of an endogenous gene of interest at the C-terminus by the CRISPR-mediated HiUGE method and selecting cells expressing the polypeptide corresponding to the endogenous gene of interest and the first nucleic acid to delineate the molecular mechanisms of cell and organismal biology through protein visualization and functional manipulation with the teachings of Zhang to generate a two step method that combines CRISPR and RMCE to insert a first nucleic acid into the coding sequence of a gene of interest by CRISPR followed by exchanging the first nucleic acid with a second nucleic acid by RMCE for structure-function analysis at the endogenous locus to arrive at the claimed method comprising: (a) inserting a nucleic acid sequence encoding a first donor polypeptide into the genome of a population of cells via Homology-independent Universal Genome Engineering (HiUGE), wherein the nucleic acid encoding the first donor polypeptide is located in a HiUGE donor vector, and the nucleic acid encoding the first donor polypeptide is flanked on each side by one or more recombinase target sites; (b) selecting cells that express the first donor polypeptide; and (c) exchanging the nucleic acid encoding the first donor polypeptide in the genome of the selected cells with a nucleic acid encoding a second donor polypeptide by contacting the selected cells with: (i) a vector comprising the nucleic acid sequence encoding the second donor polypeptide, wherein the nucleic acid encoding the second donor polypeptide is flanked on each side by the one or more recombinase target sites, and wherein the one or more recombinase target sites are in frame with the coding sequence of the second donor polypeptide; and (ii) a vector encoding a recombinase that cleaves the one or more recombination target sites inserted into the genome of the selected cells and the one or more recombinase target sites in the vector, whereby the nucleic acid encoding the first donor polypeptide is exchanged for the nucleic acid encoding the second donor polypeptide in the genome of the selected cells via recombination-mediated cassette exchange (RMCE); wherein the nucleic acid encoding the second donor polypeptide is inserted into a coding sequence for a gene, and wherein the selected cells express the second donor polypeptide and an endogenous polypeptide encoded by the gene. One would have been motivated to combine the teachings of Gao and Zhang in a two-step method that combines CRISPR-mediated HiUGE with RMCE to allow for exchange of a first nucleic acid inserted into the coding sequence of a gene of interest that is not tolerated and therefore not functional with a second nucleic acid that is tolerated and functional to study endogenous protein function as Gao teaches analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, but current approaches are often low throughput and resource intensive and Gao teaches recent advances in exploratory proteomics and gene expression analysis generate sizable datasets that urgently require high-throughput and reliable methods for protein visualization and functional manipulation purposes yet current techniques to enable these strategies are often inefficient or resource intensive. One would have a reasonable expectation of success in combining the teachings as Gao teaches HiUGE payloads are universal with respect to gene identity, and Gao teaches the HiUGE method results in expression of various payloads when inserted in the coding sequence for the C-terminus of a gene of interest, and Zhang teaches the functionality of the two-step method was verified by the reversion of the lethality for the step 2 alleles, suggesting that both steps do not generate additional unintended changes on the chromosome.
Claim(s) 5 – 7, 23, 26, and 27 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gao (Gao, Yudong, et al. Neuron 103.4 (2019): 583-597; previously cited), hereinafter Gao in view of Zhang (Zhang, Xu, et. al. G3: Genes, Genomes, Genetics 4.12 (2014): 2409-2418; previously cited), hereinafter Zhang as applied to claims 1, 3, 4, 8 – 15, 17, 18, and 24 above, and further in view of Du (Du, Zhong-Wei, et al. Stem cells 27.5 (2009): 1032-1041; previously cited), hereinafter Du.
Gao in view of Zhang make obvious the method of claim 1 as set forth above.
Regarding claims 5 and 6, Gao teaches the HiUGE donor vector comprising the nucleic acid sequence encoding the first donor polypeptide (payload) comprises a U6 promoter operably linked to the payload as shown in Figure 1A. Zhang does not teach the vector comprising the nucleic acid sequence encoding the second donor polypeptide encodes an exogenous promoter operably linked to the nucleic acid sequence (claim 5) that is constitutive or inducible (claim 6) or that is a cell-specific promoter (claim 7).
Regarding claim 5 and “constitutive promoter” of claim 6, Du teaches a method of RMCE for preparing transgenic human embryonic stem cell (hESC) lines by exchanging a GFP-encoding cassette inserted into the genome of hESCs with a RFP-encoding cassette, where the vector encoding the RFP cassette (“nucleic acid sequence encoding the second donor polypeptide” of claim 5) comprises a CAG promoter (“exogenous promoter” of claim 5; “constitutive promoter” of claim 6) operably linked to the sequence encoding RFP (page 1034, left col. para. 3; page 1036, right col. last para.; Figure 3A; page 1037 left col. and right col. para. 1). Du teaches in Figure 3B that the exchange method results in loss of GFP expression and gain of RFP expression. Du teaches exchanging the GFP-encoding cassette with
Regarding “inducible promoter” of claim 6, Du teaches exchanging the GFP-encoding cassette with the constitutive promoter for an inducible promoter to regulate the level of GFP expression (Figure 5A; page 1038, left col. para. 2 and right col. para. 1 – 2).
Regarding claim 7, Du teaches the RMCE for neuron-specific GFP expression with a vector comprising a synapsin (Syn) promoter so that GFP will be turned on only when the hESCs differentiate to synapsin-expressing neurons (page 1038, right col. last para.; Figure 6A). Du teaches it is often necessary to restrict the transgene expression to a particular cell type and this may be achieved by using a cell type-specific promoter (page 1038, right col. last para.; page 1039, left col. para. 1). Du teaches establishing Syn-GFP hESC lines that only showed GFP fluorescence when differentiated into neurons demonstrating that GFP is specifically expressed in the maturing neurons (page 1039, left col. para. 1).
Regarding claim 23, Du teaches the recombinase sites are loxP sites and the recombinase is Cre recombinase in Figure 1A.
Regarding claims 26 and 27, Du teaches hESC undergo RMCE (page 1034, left col. para. 3; page 1036, right col. last para.; Figure 3A; page 1037 left col. and right col. para. 1; page 1038, right col. last para.; Figure 6A).
Du teaches hESCs offer an invaluable tool for revealing human biology and a potential source of functional cells/tissues for regenerative medicine (page 1032, left col. para. 1). Du teaches the utility of hESCs will likely be significantly enhanced and broadened by the ability to build versatile genetically modified hESC lines (page 1032, left col. para. 1). Du teaches building stable transgenic hESC lines remains a challenging and laborious process due in part to high incidence of transgene silencing caused by the integration site and following cellular differentiation (page 1032, left col. para. 2). Du teaches neurons derived from transgenic mice and rats or mouse embryonic stem cells with transgene (GFP or alkaline phosphatase) inserted into the ROSA26 locus often do not exhibit transgene expression (page 1032, right col.). Du teaches identification of an appropriate site for stable transgene expression not only in hESCs but also in their differentiated progenies remains to be solved (page 1032, right col.). Du teaches the master hESC lines with the double loxP exchange cassette and RMCE offer a flexible and simple platform for genetic manipulation of hESCs (page 1040, left col. last para.). Du teaches a transgenic cell line can be easily obtained by Cre recombination-mediated exchange with a target gene of interest, and a series of different genes may be introduced into the same integration site to evaluate gene function without the variation in the level and pattern of gene expression (page 1040, left col. last para.).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Gao regarding inserting a first nucleic acid in the coding sequence of an endogenous gene of interest at the C-terminus by the CRISPR-mediated HiUGE method and selecting cells expressing the polypeptide corresponding to the endogenous gene of interest and the first nucleic acid to delineate the molecular mechanisms of cell and organismal biology through protein visualization and functional manipulation with the teachings of Zhang regarding a two step method that combines CRISPR and RMCE to insert a first nucleic acid into the coding sequence of a gene by CRISPR followed by exchanging the first nucleic acid with a second nucleic acid by RMCE for structure-function analysis at the endogenous locus with the teachings of Du regarding RMCE with a vector comprising an exogenous promoter that may be constitutive or inducible to arrive at the claimed invention wherein the vector comprising the nucleic acid sequence encoding the second donor polypeptide further comprises an exogenous promoter operably linked to the nucleic acid sequence encoding the second donor polypeptide. One would have been motivated to combine the teachings of Gao, Zhang, and Du for controlling the expression of the second donor polypeptide and the endogenous polypeptide to observe endogenous protein localization, translocation, and function in an hESC transgenic cell line as Gao teaches analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, but current approaches are often low throughput and resource intensive and Gao teaches recent advances in exploratory proteomics and gene expression analysis generate sizable datasets that urgently require high-throughput and reliable methods for protein visualization and functional manipulation purposes yet current techniques to enable these strategies are often inefficient or resource intensive. One would have a reasonable expectation of success in combining the teachings as Du teaches a transgenic cell line can be easily obtained by Cre recombination-mediated exchange with a target gene of interest, and a series of different genes may be introduced into the same integration site to evaluate gene function without the variation in the level and pattern of gene expression and Du teaches cell lines produced by RMCE have either constitutive or inducible GFP expression.
Claim(s) 19 and 23 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gao (Gao, Yudong, et al. Neuron 103.4 (2019): 583-597; previously cited), hereinafter Gao in view of Zhang (Zhang, Xu, et. al. G3: Genes, Genomes, Genetics 4.12 (2014): 2409-2418; previously cited), hereinafter Zhang as applied to claims 1, 3, 4, 8 – 15, 17, 18, and 24 above, and further in view of Schopp (Schopp, Isabel M., et. al. JoVE (Journal of Visualized Experiments) 134 (2018): e57479.), hereinafter Schopp.
Gao in view of Zhang make obvious the method of claim 1 as set forth above. Gao and Zhang do not teach BirA of claim 19 or flippase recognition sites of claim 23.
Regarding BirA of claim 19 and FRT sites of claim 22, Schopp teaches when BirA is fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment (Abstract). Schopp teaches a vectors comprising nucleic acids encoding BirA (claim 19) flanked by F3/FRT recombination sites for Flp-mediated recombination (claim 22) for the possibility to rapidly construct stable cell lines by RMCE (page 2, number 3 under Planning of the Cloning Strategy; Figure 2; page 10, para. 1). Schopp teaches RMCE with the vectors in HeLa cells containing a locus for RMCE allows for proximity labeling (page 3, Note under Testing of the Fusion Proteins; page 5, last para.; Figure 1).
Schopp teaches as most cellular functions are performed by proteins that dynamically assemble macromolecular complexes, the identification of protein-protein interactions is a major endeavor in biomedical research (page 1, para. 1 of Introduction). Schopp teaches to complement existing affinity purification approaches for the identification of protein-protein interactions, enzymes have been introduced that allow the proximity-dependent labeling of proteins in living cells and one such enzyme is BirA used in the BioID approach (Abstract). Schopp teaches BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated (Abstract).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Gao regarding inserting a first nucleic acid in the coding sequence of an endogenous gene of interest at the C-terminus by the CRISPR-mediated HiUGE method and selecting cells expressing the polypeptide corresponding to the endogenous gene of interest and the first nucleic acid to delineate the molecular mechanisms of cell and organismal biology through protein visualization and functional manipulation with the teachings of Zhang regarding a two step method that combines CRISPR and RMCE to insert a first nucleic acid into the coding sequence of a gene by CRISPR followed by exchanging the first nucleic acid with a second nucleic acid by RMCE for structure-function analysis at the endogenous locus with the teachings of Schopp regarding RMCE with a vector encoding BirA flanked with flippase sites for proximity labeling to arrive at the claimed invention wherein the second donor polypeptide is BirA and wherein the one or more recombinase target sites are flippase recognition target (FRT) sites, and wherein the recombinase is flippase. One would have been motivated to combine the teachings of Gao, Zhang, and Schopp to identify protein-protein interactions by the BioID method as Gao teaches analysis of endogenous protein localization, function, and dynamics is fundamental to the study of all cells, but current approaches are often low throughput and resource intensive and Gao teaches recent advances in exploratory proteomics and gene expression analysis generate sizable datasets that urgently require high-throughput and reliable methods for protein visualization and functional manipulation purposes yet current techniques to enable these strategies are often inefficient or resource intensive. One would have a reasonable expectation of success in combining the teachings as Schopp teaches when BirA is fused to a protein of interest and expressed in cells, it allows the labeling of proximal proteins in their native environment and Schopp teaches RMCE with the vectors in HeLa cells containing a locus for RMCE allows for proximity labeling and Schopp teaches BioID detects proteins that have been marked within cells no matter whether they are still interacting with the protein of interest when they are isolated.
Claim(s) 20 and 21 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gao (Gao, Yudong, et al. Neuron 103.4 (2019): 583-597; previously cited), hereinafter Gao in view of Zhang (Zhang, Xu, et. al. G3: Genes, Genomes, Genetics 4.12 (2014): 2409-2418; previously cited), hereinafter Zhang as applied to claims 1, 3, 4, 8 – 15, 17, 18, and 24 above, and further in view of Kondo (Kondo, Shu, et al. Cell reports 30.1 (2020): 284-297), hereinafter Kondo.
Gao in view of Zhang make obvious the method of claim 1 as set forth above. Gao and Zhang do not teach a nucleic acid encoding a self-cleaving peptide upstream of the nucleic acid sequence encoding the second donor polypeptide of claim 20 or P2A, E2A, F2A, or T2A of claim 23.
Regarding claims 20 and 21, Kondo teaches a method of exchanging a T2A-GAL4 cassette inserted into the endogenous neurotransmitter receptor genes of Drosophila by CRISPR/Cas9 into a different transcriptional reporter cassettes by RMCE where the sequence encoding T2A (claim 20 and “T2A” of claim 21) is upstream of the sequence encoding the transcriptional reporter (Summary; page 285, left col. last para. and right col. para. 1 – 2; Figure 2A – F; page 286, left col. para. 3; page 291, right col. para. 2; page e2, last para.). Kondo teaches the insertion of T2A-GAL4 at the C-terminus is less likely to disturb the endogenous expression of the target than N-terminal or internal insertion as it does not block communication between the promoter and intronic enhancers (page 291, right col. para. 3). Kondo teaches recombinase sites flanking the T2A-GAL4 cassette allow replacement of T2A-GAL4 with any other reporter genes by RMCE (page 291, right col. para. 3). Kondo teaches unidirectional RMCE offers a more rapid and straightforward way of transgene replacement than other systems (page 291, right col. para. 3). Kondo teaches this versatility allowed for characterization of not only expression patterns but also subcellular localization of dopamine receptor proteins, as well as visualize the dynamic regulation of receptor levels and activity (page 291, right col. para. 3; page 292, left col. para. 1).
Kondo teaches neurotransmitters often have multiple receptors that induce distinct responses in receiving cells (Summary). Kondo teaches expression and localization of neurotransmitter receptors in individual neurons are therefore critical for understanding the operation of neural circuits (Summary). Kondo teaches a major goal in neuroscience is to understand the computations principles of neuronal networks during information processing (page 284, left col.). Kondo teaches using transgenic reporters that recapitulate the expression patterns of endogenous genes is an alternative to visualization of endogenous protein distribution by immunohistochemical techniques (page 284, right col. para. 2 – 3).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to combine the teachings of Gao regarding inserting a first nucleic acid in the coding sequence of an endogenous gene of interest at the C-terminus by the CRISPR-mediated HiUGE method and selecting cells expressing the polypeptide corresponding to the endogenous gene of interest and the first nucleic acid to delineate the molecular mechanisms of cell and organismal biology through protein visualization and functional manipulation with the teachings of Zhang regarding a two step method that combines CRISPR and RMCE to insert a first nucleic acid into the coding sequence of a gene by CRISPR followed by exchanging the first nucleic acid with a second nucleic acid by RMCE for structure-function analysis at the endogenous locus with the teachings of Kondo regarding a two step method that combines CRISPR and RMCE to exchange a T2A-GAL4 cassette with a T2A-lexA cassette as a transcriptional reporter to arrive at the claimed invention wherein the vector comprising the nucleic acid sequence encoding the second donor polypeptide further comprises a nucleic acid sequence encoding a self-cleaving peptide, wherein the nucleic acid encoding the self-cleaving peptide is located upstream of the nucleic acid sequence encoding the second donor polypeptide. One would have been motivated to combine the teachings of Gao, Zhang, and Kondo to identify expression patterns of neurotransmitters as Kondo teaches expression and localization of neurotransmitter receptors in individual neurons are therefore critical for understanding the operation of neural circuits and Kondo teaches using transgenic reporters that recapitulate the expression patterns of endogenous genes is an alternative to visualization of endogenous protein distribution by immunohistochemical techniques. One would have a reasonable expectation of success in combining the teachings as Kondo teaches the method successfully exchanges GAL4 for lexA and the expression pattern can be obtained in Drosophila.
Maintained Double Patenting
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
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Claims 1, 3 – 15, 17 – 24, and 26 – 27 remain rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 – 15 of U.S. Patent No. 12325855 in view of Kondo (Kondo, Shu, et al. Cell reports 30.1 (2020): 284-297), hereinafter Kondo. The rejection has been modified as necessitated by amendment of the claims in the response filed 7/22/2026 Patent claims 1 and 12 are drawn to a HiUGE system for gene editing a subject genome comprising HiUGE vector comprising a first polynucleotide sequence encoding at least one insert, gRNA, and a CRISPR-based nuclease.
Patent claims 1 and 12 lack the vector comprising recombinase target sites and a vector encoding a recombinase and the method steps of instant claim 1.
Kondo teaches a two-step method combining CRISPR/Cas9-mediated insertion of a first nucleic acid contained in a donor vector followed by RMCE with Cre recombinase and a vector comprising a second nucleic acid flanked by recombinase sites (page e2, last para.; Supplemental Methods S1 vector map and sequence; Figure 1A, 2A, 2B; page 285, left col. last para. and right col. para. 1; page 286, left col. para. 3). Kondo teaches recombinase sites flanking the T2A-GAL4 cassette allow replacement of T2A-GAL4 with any other reporter genes by RMCE (page 291, right col. para. 3). Kondo teaches unidirectional RMCE offers a more rapid and straightforward way of transgene replacement than other systems (page 291, right col. para. 3). Kondo teaches this versatility allowed for characterization of not only expression patterns but also subcellular localization of dopamine receptor proteins, as well as visualize the dynamic regulation of receptor levels and activity (page 291, right col. para. 3; page 292, left col. para. 1). Kondo teaches neurotransmitters often have multiple receptors that induce distinct responses in receiving cells (Summary). Kondo teaches expression and localization of neurotransmitter receptors in individual neurons are therefore critical for understanding the operation of neural circuits (Summary). Kondo teaches a major goal in neuroscience is to understand the computations principles of neuronal networks during information processing (page 284, left col.). Kondo teaches using transgenic reporters that recapitulate the expression patterns of endogenous genes is an alternative to visualization of endogenous protein distribution by immunohistochemical techniques (page 284, right col. para. 2 – 3).
It would have been obvious prior to the effective filing date of the invention as claimed for the person of ordinary skill in the art to have modified the system of Patent claims 1 and 12 for HiUGE gene editing a subject genome with a vector comprising a second nucleic acid flanked by one or more recombinase sites that are in frame with the coding sequence of the second donor polypeptide and a vector encoding a recombinase in order to provide a system for exchanging reporters as an alternative to visualization of endogenous protein expression tagged with a reporter encoded by the second nucleic acid distribution by immunohistochemical techniques as taught by Kondo.
Applicant’s Arguments/ Response to Arguments
Applicant Argues: Applicant asserts that the meaning of the term HiUGE is known by one skilled in the art and the method steps for achieving step (a) of inserting a nucleic acid sequence into the genome of cells and the structure of the nucleic acid sequence required for achieving step (a) are clear.
Response to Arguments: The meaning of the term HiUGE is defined in Applicant’s specification as a vector system that allows modification of genomic target loci (para. 0038). While the amendment to claim 1 provides structural limitations to the HiUGE donor vector (based on the specification at para. 0038), the amendment does not clarify the metes and bounds of what claim 1 covers regarding inserting a nucleic acid sequence into the genome of a population of cells via a vector system. Therefore, the rejection under 35 U.S.C. 112(b) is maintained.
Applicant Argues: Applicant asserts that claim 1 has been amended to recite “a fusion polypeptide”.
Response to Arguments: Claim 1 has not been amended to recite “a fusion polypeptide” and therefore, the rejections of claims 3 and 4 under 35 U.S.C. 112 (b) are maintained.
Applicant Argues: Applicant asserts that the Office has not established a persuasive reason to combine the cited references to achieve the claimed invention. Applicant asserts that a person of ordinary skill would have no reason to combine Du and/or Ordovas to arrive at the claimed method. Applicant asserts that the Office has not shown that a person of ordinary skill in the art would have a reasonable expectation of success in using references focused on safe-harbor integration to achieve insertion into any endogenous coding sequence that yields an endogenous polypeptide encoded by that sequence.
Response to Arguments: The previous rejection of claims citing the teachings of Du, Ordovas, and Liu have been withdrawn in view of Applicant’s amendment to claim 1. The new rejections of the claims do not cite the teachings of Liu or Ordovas. Therefore, Applicant’s arguments addressing Liu and Ordovas are moot.
In the new rejections set forth above, Gao in view of Zhang make obvious the limitations of claim 1. A person of ordinary skill in the art would combine Gao and Zhang for a simpler way to exchange one payload for another without repeating the HiUGE method because Gao teaches HiUGE payloads are universal with respect to gene identity, provided that the modifications are functionally tolerated, and retained during post-translational maturation of the protein products (some proteins may be post-translationally processed at both their C- and N-termini, thus inherently difficult to target) (page 593, right col. para. 2). Thus, a person of ordinary skill reading Gao would understand that when the HiUGE method produces a modification that is not functionally tolerated and/or retained during post-translational maturation, the HiUGE method would need to be used again to modify the gene of interest with a different payload. Zhang teaches in Figure 5A that the CRISPR step of the two step method resulted in lethality when targeting inserting dsRed at the first intron of the salm gene (salm[1st intron-dsRed]), but this lethality could be overcome by exchanging dsRed for a second nucleic acid encoding TY1-V5 or GFP-3xFLAG by the RMCE step of the two step method as shown in Figure 5B (2415, left col., para. 1; Figure 4). Thus, Zhang teaches combining CRISPR gene editing with RMCE to exchange a first nucleic acid inserted by CRISPR that is not tolerated (lethal) with a second nucleic acid by RMCE that is tolerated.
Applicant Argues: Applicant asserts that Du is concerned with avoiding transgene silencing and achieving predictable expression by integrating transgenes into carefully selected genomic loci, and therefore does not complement Gao’s approach as they address a different concern. Applicant asserts that based on Du, one of ordinary skill in the art would be skeptical about genome integration outside previously vetted loci. Applicant asserts that Du steers the skilled artisan towards insertion at previously vetted loci and away from broader insertion strategies.
Response to Arguments: This is not found persuasive because claim 1 does not place any limitations of “the genome” of step (a) and (c) or the “coding sequence for a gene” including requiring previously vetted loci and Gao teaches HiUGE payloads are universal with respect to gene identity (page 593, right col. para. 2). Therefore, it is inconsequential which gene is targeted for insertion by the HiUGE method of Gao, followed by RMCE as taught by Zhang. Applicant’s remarks support insertion at any gene because Applicant states that the claimed methods are not limited to preselected “safe sites” (page 15, para. 2).
Applicant Argues: Applicant asserts that Du is at odds with amended claim 1 because Du specifically discloses insertion into noncoding regions.
Response to Arguments: This is not found persuasive Zhang teaches insertion into coding regions by CRISPR/Cas9 followed by RMCE.
Applicant Argues: Applicant requests reconsideration of the double patenting rejection.
Response to Arguments: The double patenting rejection is maintained and has been revised in view of the amendments to the claims.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/Z.M.B./Examiner, Art Unit 1632
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634
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