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 .
1. Claims 1 – 15 and 17 – 28 are pending.
Election/Restrictions
2. Applicant's election with traverse of Group I (claims 1 – 15, 17 – 24, and 26 – 27) in the reply filed on 03/13/2026 is acknowledged. The traversal is on the ground(s) that the claims share a special technical feature over Gao that combines HiUGE with RMCE to enable rapid, iterative, isogenic engineering of a cell at a defined locus; in contrast Gao focuses only on HiUGE for genome modification and Gao does not suggest combining HiUGE with RMCE. This is not found persuasive because the technical feature of Groups I – III is a cell comprising an inserted nucleic acid sequence into its genome and Gao teaches cells and animals made by a method using HiUGE donor vectors for inserting nucleic acids into the genome of cells and interchanging the payload of the donor vectors and that the method relies on additional Cas9 expression strategies. Therefore, the technical feature is not a special technical feature because it does not make a contribution over Gao. Clam 1 recites HiUGE but Applicant’s specification defines HiUGE as vector system and not a method at para. 0038.
The requirement is still deemed proper and is therefore made FINAL.
3. Claims 25 and 28 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 03/13/2026.
Priority
4. This application claims domestic benefit to U.S. Provisional Application 63/179,585 filed on 04/26/2021.
Drawings
5. The drawings filed on 10/26/2023 are acknowledged.
Specification
6. The use of the term Tet-On/TetOff, HisTag, StrepTag, which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Claim Objections
7. Claim 1 is objected to because of the following informalities: in line 11, “a nucleic acid sequence” should read “the nucleic acid sequence” to clarify that the vector of (i) encodes the nucleic acid sequence encoding the second donor polypeptide. Appropriate correction is required.
8. Claim 1 is objected to because of the following informalities: in line 20, “the cells” should read “the selected cells” to clarify “the cells” refers to the cells of step (b). Appropriate correction is required.
9. Claim 12 is objected to because of the following informalities: in line 2, “a slice acceptor” should read “a splice acceptor” to correct the misspelling of “splice”. Appropriate correction is required.
10. Claim 17 is objected to because of the following informalities: in line 1, “the first donor polypeptide” should read “the nucleic acid encoding the first donor polypeptide comprises a sequence encoding a first peptide tag” to clarify that the nucleic acid sequence of step (a) encodes the first peptide tag. Appropriate correction is required.
11. Claim 18 is objected to because of the following informalities: in line 1, “the second donor polypeptide” should read “the nucleic acid encoding the second donor polypeptide comprises a sequence encoding a second peptide tag” to clarify that the nucleic acid sequence of step (c) encodes the first peptide tag. Appropriate correction is required.
12. Claim 21 is objected to because of the following informalities: in line 1, “self-cleaving peptides are” should read “self-cleaving peptide is” because claim 20 recites a singular self-cleaving peptide. Appropriate correction is required.
13. Claim 24 is objected to because of the following informalities: in line 1, “the cell” should read “the population of cells” to clarify that “the cell” refers to the cells of step (a). Appropriate correction is required.
14. Claim 26 is objected to because of the following informalities: in line 1, “the cell” should read “the population of cells” to clarify that “the cell” refers to the cells of step (a). Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
15. Claims 1 – 15, 17 – 24, and 26 – 27 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.
16. 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. Further, it is unclear if recitation of “HiUGE” is meant to impart structure to the nucleic acid sequence and what that structure is, if any. Claims 2 – 15, 17 – 24, and 26 – 27 are also rejected as they depend from claim 1 and do not clarify the grounds of rejection.
17. Regarding claim 2, recitation of “a coding sequence for a gene” lacks antecedent basis because step (a) of claim 1 does not recite “a coding sequence for a gene”. Claim 3 is also rejected as it depends from claim 2 and does not clarify the grounds of rejection.
18. Regarding claim 3, the claim lacks antecedent basis because claim 1 does not recite “fusion polypeptide” or “the gene” in step (c).
19. Claim 4 recites the limitation “the fusion polypeptide” and "the endogenous promoter for the gene" in lines 2 – 3. There is insufficient antecedent basis for this limitation in the claim.
Claim Interpretation
20. For the purpose of applying prior art, claim 1 is interpreted as (a) inserting a nucleic acid sequence encoding a first donor polypeptide into the genome of a population of cells using HiUGE vectors, wherein 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 of step (b) with a nucleic acid encoding a second donor polypeptide by transfecting the selected cells from step (b) with a (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 based on Applicant’s specification at para. 0033 defining RMCE and para. 0038 defining HiUGE.
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.
21. Claim(s) 1 –11, 13 – 15, 17, 18, 22 – 24, 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.), hereinafter Gao in view of Du (Du, Zhong-Wei, et al. Stem cells 27.5 (2009): 1032-1041.), hereinafter Du in view of Ordovas (Ordovás, Laura, et al. Journal of visualized experiments: JoVE 117 (2016): 54718.), hereinafter Ordovas.
Regarding claim 1, Gao teaches a method of inserting a nucleic acid sequence encoding a hemagglutinin (HA) epitope tag (“a nucleic acid sequence encoding a first donor polypeptide”) at the mouse Tubb3 gene in primary neurons (“into the genome of a population of cells”) using HiUGE vectors (“via Homology-independent Genome Engineering”) (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). 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).
Regarding claim 2, Gao teaches inserting the nucleic acid encoding the first donor polypeptide into the Tubb3 locus (page 585, right col. para. 2) but does not teach this is the nucleic acid encoding the second donor polypeptide.
Regarding claim 3, Gao teaches expression of the βIII-tubulin-HA fusion but does not teach “comprising the second donor polypeptide”. Gao teaches an important consideration is that HiUGE donor vectors should be suitable to target diverse coding sequences without introducing premature stop codons that prevent proper expression of the insert (page 584, right col. last para.).
Regarding claim 4, Gao teaches genomic insertion of the HA tag was verified by sequencing the Tubb3 locus confirming the successful HA-epitope integration (page 584, right col. para. 2; Figure 1F). Therefore, the expression of the βIII-tubulin-HA fusion is under the control of the endogenous promoter for Tubb3. Gao does not teach “second donor polypeptide”.
Regarding claim 8, Gao teaches the HA tag is inserted into a noncoding sequence in the genome of the cells (page 587, left col. para. 1; Figure S3F – H; page 595, left col. para. 3). Gao does not teach “the second donor polypeptide”.
Regarding claims 9 – 11, Gao teaches the HA tag is inserted into an intron (“regulatory sequence” of claim 9; “intron” of claims 10 and 11) (page 587, left col. para. 1; Figure S3D).
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 14, Gao teaches inserting mCherry into the Tubb3 locus using HiUGE vectors (page 589, right col. para. 3; Figure 6B – D). Gao does not teach “second donor polypeptide”.
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 claim 18, 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) but does not teach “second donor polypeptide”.
Regarding 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) or an exogenous promoter that is constitutive or inducible or cell-specific of claims 5 – 7 or the one or more recombinase target sites are FRT sites and the recombinase is flippase of claim 22 or the recombinase target sites are loxP sites and the recombinase is Cre recombinase of claim 23 or the cell is a stem cell of claim 26 or the stem cell is an embryonic stem cell of claim 27. However, Gao teaches the donor payloads can be interchangeable for multiplexing and flexible selection of protein modification (page 589, left col. para. 2; page 595, right col. para. 2). Gao teaches the ability to mix and match premade HiUGE donors would simplify the experimental selection of optimal epitopes, diverse fusion proteins, or fusion proteins with variable linkers (page 589, left col. para. 2). Gao teaches creating a mosaic labeling of cells by infecting neurons with a mixture of epitope payloads targeting the Tubb3 locus (page 589, left col. para. 2; Figure 4). Gao teaches after genomic cleavage directed by the GS-gRNA, different payloads can be inserted interchangeably (page 589, left col. para. 2). Gao teaches the data demonstrated the interchangeability of payloads within a single gene locus, thus enabling flexible selection of diverse protein modifications (page 589, left col. para. 2). Gao teaches the HiUGE vectors can be delivered successfully to common cell lines such as HeLa, HEK293T and NIH3T3 to knockin the HA epitope and GFP (page 593, right col. para. 1). Gao teaches one potential drawback to HiUGE is the formation of indels at the targeted loci (page 595, left col. para. 3). 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 but current techniques are often inefficient or resource intensive (page 583, left col. and right col. para. 1).
Regarding step (c) of claim 1 and “second donor polypeptide” of claims 2 – 4, 8, and 14, Du teaches a method of exchanging a first nucleic acid encoding a first donor polypeptide with flanking recombinase target sites with a second nucleic acid encoding a second donor polypeptide with the same flanking recombinase target sites by RMCE by transfecting human embryonic stem cells (hESCs) with a vector comprising the nucleic acid encoding the second donor polypeptide and a vector encoding a recombinase (page 1033, left col. para. 3 – 4 and right col. para. 1; page 1034, right col. last para.; page 1035, left col. and right col.; Figure 1A; page 1036, right col. last para.). Du teaches the nucleic acid encoding the first donor polypeptide (master vector) encodes GFP and a neomycin resistance gene in Figure 1A and was transfected into hESCs to make a master cell line that was selected using neomycin (page 1034, right col. last para.; page 1035, left col. para. 2 and right col. para. 1 – 2). Du teaches the master vector integrated into the genome (page 1036, right col. para. 1). Du teaches the gene encoding GFP could be exchanged for a gene encoding RFP by RCME (Figure 3, page 1036, right col. last para.; page 1037, left col. and right col. para. 1). Du teaches exchanging the GFP gene for Olig2-FLAG (page 1037, right col. para. 2; Figure 4).
Regarding claim 5 – 7, Du teaches vectors encoding an exogenous promoter operably linked to the nucleic acid sequence encoding the second donor polypeptide where the promoter is constitutive (CAG promoter of Figure 4A) or inducible (TetO of Figure 5A) (“exogenous promoter” of claim 5 and “constitutive promoter or an inducible promoter” of claim 6) or neuron-specific (synapsin promoter of Figure 6A) (“cell-specific promoter” of claim 7) (page 1037, right col. para. 2; page 1038, left col. last para. and right col. last para.; page 1039, left col. last para.)
Regarding claim 23, Du teaches the nucleic acids encode loxP sites and the recombinase is Cre recombinase (Figure 1A; page 1036, right col. para. 2).
Regarding claims 26 and 27, Du teaches the cells are hESCs (page 1033, left col. para. 1 – 2; page 1036, right col. last para.).
Du does not teach the recombinase target sites are FRT sites and the recombinase is flippase of claim 22. However, Du teaches building stable transgenic hESC lines remains a challenging and laborious process (page 1032, left col. para. 2). Du teaches the reasons for this include low transfection and cloning efficiency as well as high incidence of transgene silencing caused by the integration site and following cellular differentiation (page 1032, left col. para. 2). Du teaches transgenes are introduced to some unique sites such as the AAV integration site 1 locus (page 1032, right col. para. 2). 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 vector was integrated randomly (page 1036, right col. para. 1). Du teaches stability of transgene expression is highly dependent on the site of integration (page 1039, right col. para. 2). Du teaches the master hESC cell line offers a flexible and simple platform for genetic manipulation of hESCs and versatile transgenic hESC lines may be built upon the master hESC cell line (page 1040, left col. para. 1 and 3). 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. and right col. para. 1). Du teaches replacement of the built-in loxP cassette in the master cell line with any targeting transgene cassette possessing the same loxP sites through RMCE allowed the generation of versatile transgenic hESC lines (page 1033, left col. para. 1). Du teaches the built-in double loxP cassette in the established master hESC lines was specifically replaced by a targeting vector containing the same loxP sites and Cre recombinase (Abstract). One would have been motivated to combine the teachings of Gao and Du wherein the nucleic acid encoding the first donor polypeptide is flanked on each side by one or more recombinase target sites and the first nucleic acid is exchanged for the second nucleic acid by RMCE because both teach methods of inserting transgenes into the genomes of cells where the first transgene inserted can be changed to another transgene where Gao teaches the insertion can be targeted to a gene of interest while Du teaches random insertion and Gao teaches one of the potential drawbacks of HiUGE is the formation of indels at the targeted loci, while RMCE would prevent this because Du teaches the built-in double loxP cassette in the established master hESC lines was specifically replaced by a targeting vector containing the same loxP sites and Cre recombinase.
Regarding FRT and flippase of claim 22, Ordovas teaches a method of RMCE in human stem cells using nucleic acids encoding target gene with flanking FRT sites and flippase at the AAVS1 locus (Abstract; Figure 1A and 2B and 3). Ordovas teaches use of this RMCE system in multiple hESC/iPSC lines requires pre-integration of the described FRT-containing cassette in the AAVS1 locus of each independent line but once generated, its rapidity and simplicity makes it possible to develop semi-high throughput genetic screens in defined isogenic settings for applications that otherwise would be technically very time consuming (page 8, para. 2). Ordovas teaches creation of a master cell line having FRT sites where the cell lines maintained pluripotency and genome integrity and stably expressed GFP in vitro and in vivo (page 4, para. 1). Ordovas teaches genotypic characterization of the new transgenic lines is significantly reduced (no clonal screening is necessary), and characterization associated to off-target nuclease activity is rendered dispensable due to the specificity of the Flippase for FRTs (page 7, last para.). Ordovas teaches characterization can also be reduced in routine RMCE by demonstrating the complete loss of GFP from the master cell line because full cassette exchange and lack of random integration have already been sufficiently demonstrated by PCR (page 7, last para.). Ordovas teaches identification of an appropriate site for ubiquitous stable transgene expression not only in hPSCs but also in their differentiated progenies in vitro and in vivo remains to be solved (page 2, para. 2). Ordovas teaches creating transgenic cell lines could benefit from using site-specific targeted recombinases like Cre of FLPe which are more rapid and less prone to off-target effects (Abstract). Ordovas teaches gene editing ease and flexibility in safe harbor loci is increased through the use of site-specific targeted recombinases like Cre or Flippase which specifically recognize target sequences (loxP or FRT) and catalyze efficient recombination between identical targets and this is a common tool used in mouse transgenesis and allows for RMCE (page 1, para. 2). Ordovas teaches RMCE has been successfully carried out in hPSCs using mostly Cre recombinase even if there are indications that Flippase is more efficient that Cre (page 2, para. 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 a method of inserting and interchanging nucleic acids in the genome of cells using HiUGE vectors with the teachings of Du and Ordovas regarding a method of exchanging inserted nucleic acids in the genome of cells using RMCE where Du teaches loxP/Cre recombinase and Ordovas teaches FRT/Flippase recombinase sites/recombinase to arrive at the claimed method for generating a stable cell line 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 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 a 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 cells via recombination-mediated cassette exchange (RMCE). One would have been motivated to combine the teachings of Gao, Du, and Ordovas in a method of creating transgenic cell lines as 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 but current techniques are often inefficient or resource intensive and Du teaches building stable transgenic hESC lines remains a challenging and laborious process and 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 and Ordovas teaches identification of an appropriate site for ubiquitous stable transgene expression not only in hPSCs but also in their differentiated progenies in vitro and in vivo remains to be solved. One would have a reasonable expectation of success in combining the teachings as Gao teaches the data demonstrated the interchangeability of payloads within a single gene locus, thus enabling flexible selection of diverse protein modifications and Du teaches replacement of the built-in loxP cassette in the master cell line with any targeting transgene cassette possessing the same loxP sites through RMCE allowed the generation of versatile transgenic hESC lines and Ordovas teaches use of this RMCE system in multiple hESC/iPSC lines requires pre-integration of the described FRT-containing cassette but once generated, its rapidity and simplicity makes it possible to develop semi-high throughput genetic screens in defined isogenic settings for applications that otherwise would be technically very time consuming.
22. Claim(s) 12 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gao (Gao, Yudong, et al. Neuron 103.4 (2019): 583-597.), hereinafter Gao in view of Du (Du, Zhong-Wei, et al. Stem cells 27.5 (2009): 1032-1041.), hereinafter Du in view of Ordovas (Ordovás, Laura, et al. Journal of visualized experiments: JoVE 117 (2016): 54718.), hereinafter Ordovas as applied to claims 1 –11, 13 – 15, 17, 18, 22 – 24, 26, and 27 above, and further in view of Zhang (Zhang, Xu, et. al. G3: Genes, Genomes, Genetics 4.12 (2014): 2409-2418.), hereinafter Zhang.
Gao in view of Du and Ordovas make obvious the limitations of claim 1 and 11 as set forth above. Gao teaches the method of knockin with HiUGE vectors relies on NHEJ (page 595, left col. para. 2). Ordovas teaches a second donor polypeptide encoding a splice acceptor site flanking a puromycin resistance gene and a variable experimental cassette X in Figure 1A. Gao, Du, and Ordovas do not teach a splice first donor site.
Zhang teaches a two step method to flexibly engineer the fly genome by combining CRISPR with RMCE (Abstract; page 2409, right col. last para.; page 2410, left col. para. 1 and right col. last para.; page 2411, left col. and right col. para. 1 – 2). Zhang teaches in the second RMCE step, the eye marker introduced in the first step was replaced with DNA sequences of choice including a nucleic acid encoding synthetic exons flanked by a splice acceptor site and a splice donor site (Figure 2, 3B and 4; page 2414, right col.; page 2410, left col. para. 1; page 2415, left col. and right col. para. 1). Zhang teaches the method should generally be versatile for the genetic analysis of muscle in the future (page 2415, right col. para. 1). Zhang teaches RMCE is an established standard technology in Drosophila (page 2410, left col. para. 1). Zhang teaches in Drosophila, CRISPR-NHEJ has mainly been utilized to mutate genes that result in a visible, easily scored phenotype or to mutate GFP transgenes and mutants in genes with no visible phenotype require PCR screening for their identification requiring high mutagenesis rates which may be difficult to achieve at all positions in the fly genome (page 2409, left col. and right col. para. 1). Zhang teaches this bottleneck was addressed by applying CRISPR-induced HDR to insert an attP-site together with a visible marker into the gene of interest which was sometimes flanked by FRT or loxP sites allowing its excision to only leave one attP site (and one loxP or FRT site) within the gene (page 2409, right col. para. 1). Zhang teaches the two-step strategy allows structure-function analysis at the endogenous locus (page 2417, left col. para. 2). Zhang teaches the functionality of the method was verified by the reversion of the lethality for the step 2 alleles in the first intron of salm gene (page 2417, left col. para. 2). Zhang teaches the data suggest that any fly laboratory can engineer their favorite gene for a broad range of applications within approximately 3 months (Abstract; page 2410, left col. para. 1). 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 and multiple fluorescent and affinity tags can be easily inserted or a deleted exon can effectively be replaced by various engineered exon versions (page 2417, para. 2).
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 a method of inserting and interchanging nucleic acids in the genome of cells using HiUGE vectors using NHEJ with the teachings of Du and Ordovas regarding a method of exchanging inserted nucleic acids in the genome of cells using RMCE with the teachings of Zhang regarding a two step method to exchange nucleic acids in the genome of Drosophila comprising CRISPR and RMCE to arrive at the claimed method wherein the nucleic acid encoding the second donor polypeptide is a synthetic exon flanked by a slice acceptor and a splice first donor site. One would have been motivated to combine the teachings of Gao, Du, Ordovas, and Zhang in a method of creating transgenic Drosophila to study muscle function as Zhang teaches the method should generally be versatile for the genetic analysis of muscle in the future and Zhang teaches the two-step strategy allows structure-function analysis at the endogenous locus and 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 and multiple fluorescent and affinity tags can be easily inserted or a deleted exon can effectively be replaced by various engineered exon versions. One would have a reasonable expectation of success in combining the teachings as Zhang teaches the functionality of the method was verified by the reversion of the lethality for the step 2 alleles in the first intron of salm gene.
23. Claim(s) 19 is/are rejected under 35 U.S.C. 103 as being unpatentable over Gao (Gao, Yudong, et al. Neuron 103.4 (2019): 583-597.), hereinafter Gao in view of Du (Du, Zhong-Wei, et al. Stem cells 27.5 (2009): 1032-1041.), hereinafter Du in view of Ordovas (Ordovás, Laura, et al. Journal of visualized experiments: JoVE 117 (2016): 54718.), hereinafter Ordovas as applied to claims 1 –11, 13 – 15, 17, 18, 22 – 24, 26, and 27 above, and further in view of Liu (US-20230141187-A1; Filed 04/21/2021; Published 05/11/2023), hereinafter Liu.
Gao in view of Du and Ordovas make obvious the limitations of claim 1 as set forth above. Gao, Du, and Ordovas do not teach BirA. However, 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 but current techniques are often inefficient or resource intensive (page 583, left col. and right col. para. 1). Gao teaches HiUGE will enable higher-throughput mapping and functional interrogation of proteomes (page 595, left col. para. 1).
Liu teaches a nucleic acid encoding BirA, an HA tag, and an ER retention signal for creating transgenic mice expressing BirA that was microinjected into the pronuclei of fertilized eggs (page 1, para. 0005; page 12, para. 0073; Figure 2; page 13, para. 0080). Liu teaches transgenic mice designed to produce biotinylated polypeptides that are released from a particular cell type or tissue containing that particular cell type (page 1, para. 0004). Liu teaches the identification of well-validated endothelial-specific proteins in the serum of the transgenic mice suggests that the mice represent a robust strategy for the deconvolution of serum to rapidly identify tissue-specific secretion (page 13, para. 0080; page 14, para. 0083). Liu teaches the transgenic secretome mouse provides a genetic platform to identify the in vivo cell or tissue-specific secretome under basal conditions of following a physiological or pathophysiological stress and the model can be used as a discovery platform and aid in the identification of circulating biomarkers such as disease biomarkers (page 14, para. 0083). Liu teaches the tissue origin of certain abundant proteins like albumin are known, but the complete contribution of any given cell type to the plasma proteome currently remains difficult to elucidate (page 1, para. 0003). Liu teaches such information would undoubtedly be useful as there is a growing realization that the abundance of certain proteins might provide unique insight into human health (page 1, para. 0003).
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 a method of inserting and interchanging nucleic acids in the genome of cells using HiUGE vectors using NHEJ with the teachings of Du and Ordovas regarding a method of exchanging inserted nucleic acids in the genome of cells using RMCE with the teachings of Liu regarding a method of preparing BirA transgenic mice to arrive at the claimed method wherein the second donor polypeptide is BirA. One would have been motivated to combine the teachings of Gao, Du, Ordovas, and Liu in a method of creating transgenic mice to identify the tissue origin of proteins as Liu teaches such information would undoubtedly be useful as there is a growing realization that the abundance of certain proteins might provide unique insight into human health and Gao teaches HiUGE will enable higher-throughput mapping and functional interrogation of proteomes. One would have a reasonable expectation of success in combining the teachings as Liu teaches the identification of well-validated endothelial-specific proteins in the serum of the transgenic mice suggests that the mice represent a robust strategy for the deconvolution of serum to rapidly identify tissue-specific secretion.
24. 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.), hereinafter Gao in view of Du (Du, Zhong-Wei, et al. Stem cells 27.5 (2009): 1032-1041.), hereinafter Du in view of Ordovas (Ordovás, Laura, et al. Journal of visualized experiments: JoVE 117 (2016): 54718.), hereinafter Ordovas as applied to claims 1 –11, 13 – 15, 17, 18, 22 – 24, 26, and 27 above, and further in view of Bosch (Bosch JA, et. al. Genetics. 2020 Jan;214(1):75-89), hereinafter Bosch in view of Diao (Diao F, et. al.. Genetics. 2012 Mar;190(3):1139-44), hereinafter Diao.
Gao in view of Du and Ordovas make obvious the limitations of claim 1 as set forth above. Ordovas teaches a 2A self-cleaving peptide in the vector for constructing the master cell line in Figure 1A but does not teach a 2A self-cleaving peptide in the second nucleic acid of claim 20 or T2A of claim 21.
Regarding self-cleaving peptide upstream of the second donor nucleic acid and T2A peptide of claims 20 and 21, Bosch teaches a knockin vector comprising a nucleic acid sequence encoding a T2A peptide upstream from Gal4 in Figure 3A (page 82, left col. para. 1 – 2; page 76, right col. para. 3). Bosch teaches the vector is used in a homology-independent knockin method for Drosophila (Abstract; page 76, left col. para. 3; page 82, left col. para. 1 – 2). Bosch teaches the results indicate that insertion of the vector into 5’ coding sequence can disrupt gene function (page 82, right col. para. 2; page 84, left col. para. 2). Bosch teaches the knockin resulted in lines that expressed Gal4 under the control of the target gene where the nucleic acid was inserted (page 84, left col. para. 3 – 4; Figure 4). Bosch teaches Drosophila is an excellent animal model with which to analyze gene function because of its many genetic tools, fast generation time, and in vivo analysis (page 75, left col.).
Diao teaches in Drosophila, the Gal4-UAS system permits a transgene to be expressed in the same pattern as a gene of interest by placing the Gal4 transcription factor under control of the gene’s DNA regulatory elements (Abstract). Diao teaches if these regulatory elements are not known, expression of Gal4 in the desired pattern may be difficult or impossible and to solve this problem the ribosomal skipping mechanism of the T2A peptide can be used (Abstract). Diao teaches by inserting a construct consisting of the T2A and Gal4-coding sequences in-frame into an exon of an endogenous gene, this property of 2A-like peptides can be used to co-express the Gal4 gene and the endogenous gene in Drosophila (page 1139, right col. para. 2; Figure 1). Diao teaches the T2A-GIFF (T2A-Gal4 in-frame fusion) technique represents a readily adaptable technique for transgene expression in cells expression a gene of interest (page 1140, left col. para. 1). Diao teaches the T2A-GIFF technique can be used to gain genetic access to otherwise inaccessible cells in Drosophila using only the coding sequence of a gene of interest (page 1143, left col. para. 2). Diao teaches T2A-GIFF can be used to selectively mark of manipulate cells expressing individual splice variants of a gene of interest and if desired to mutate the gene to investigate its role in cellular function (page 1143, left col. para. 2). Diao teaches the T2A-GIFF method will be broadly applicable to the investigation of cellular function in Drosophila (page 1143, left col. para. 2).
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 a method of inserting and interchanging nucleic acids in the genome of cells using HiUGE vectors using NHEJ with the teachings of Du and Ordovas regarding a method of exchanging inserted nucleic acids in the genome of cells using RMCE with the teachings of Bosch regarding a homology independent method of knocking in a nucleic acid encoding a T2A peptide upstream of Gal4 with the teachings of Diao regarding inserting a construct consisting of the T2A and Gal4-coding sequences in-frame into an exon of an endogenous gene, this property of 2A-like peptides can be used to co-express the Gal4 gene and the endogenous gene in Drosophila to arrive at the claimed method 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, Du, Ordovas, Bosch, and Diao in a method of creating in a method of creating transgenic Drosophila with disruption in targeted genes to study gene function as Bosch teaches Drosophila is an excellent animal model with which to analyze gene function because of its many genetic tools, fast generation time, and in vivo analysis and Diao teaches the T2A-GIFF technique can be used to gain genetic access to otherwise inaccessible cells in Drosophila using only the coding sequence of a gene of interest and Diao teaches T2A-GIFF can be used to selectively mark of manipulate cells expressing individual splice variants of a gene of interest and if desired to mutate the gene to investigate its role in cellular function. One would have a reasonable expectation of success in combining the teachings Bosch teaches the knockin resulted in lines that expressed Gal4 under the control of the target gene where the nucleic acid was inserted and Bosch teaches the results indicate that insertion of the vector into 5’ coding sequence can disrupt gene function and Diao teaches the T2A-GIFF method will be broadly applicable to the investigation of cellular function in Drosophila.
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.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
25. Claims 1 – 15, 17 – 24, and 26 – 27 are rejected on the ground of nonstatutory double patenting as being unpatentable over claims 1 – 15 of U.S. Patent No. 12325855. Although the claims at issue are not identical, they are not patentably distinct from each other because instant claim 1 is anticipated by patent claim 1.
Patent claim 1 recites a Homology-Independent Universal Genome Engineering (HiUGE) system for gene editing a subject genome, the HiUGE system comprising (a) a Homology-Independent Universal Genome Engineering (HiUGE) vector comprising: (i) a first polynucleotide sequence encoding at least insert; (ii) at least one donor recognition sequence (DRS) flanking each side of the first polynucleotide sequence, the DRS comprising a cleavage site for a CRISPR-based nuclease; based nuclease cleaves the at least one DRS flanking each side of the first polynucleotide and the target gene specific sequence, thereby generating a cleaved first polynucleotide sequence and a cleaved site of the target gene, wherein the (iii) a second polynucleotide sequence encoding a HiUGE vector specific gRNA, wherein the HiUGE vector specific gRNA targets the CRISPR-based nuclease to the DRS and does not target a specific sequence within the subject genome; (iv) a third polynucleotide sequence encoding a first portion of a CRISPR-based nuclease having a first split-intein; and (b) a gene specific vector comprising: (i) a fourth polynucleotide sequence encoding a second portion of a CRIS PR-based nuclease having a second split-intein complementary to the first split-intein, wherein the first portion of a CRISPR-based nuclease and the second portion of a CRISPR-based nuclease can join together to form the CRISPR-based nuclease; and (ii) a fifth polynucleotide sequence that encodes a target gene specific gRNA which targets the CRISPR-based nuclease to a target gene specific sequence within the subject genome. Therefore patent claim 1 is in essence a species of the generic invention of instant claim 1 that recites “via Homology-independent Universal Genome Engineering (HiUGE)” where Applicant’s specification defines HiUGE as “a vector system that allows modification of genomic target loci” at para. 0038. It has been held that a generic invention is “anticipated” by a “species” within the scope of the generic invention. See In re Goodman, 29 USPQ2d 2010 (Fed. Cir. 1993).
Conclusion
No claims allowed.
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/ZANNA MARIA BEHARRY/Examiner, Art Unit 1632
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