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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 11/19/2025 has been entered.
Claim Status
1. The amendment filed 11/19/2025 has been entered. Claims 1, 2, 9, 10, 15, and 20 remain pending and are under consideration. Claims 3 – 8 and 25 – 35 have been cancelled.
Election/Restrictions
2. Applicant's election with traverse of Group I (claims 1, 2, and 9) in the reply filed on 12/20/2024 is acknowledged. The traversal is on the ground(s) that claim 1 and claims dependent therefrom require not any genetically modified fish but a fish whose genome is recited in claim 1. Applicant states that claims 10, 15, and 20 depend from claim 1 and require the fish claimed in claim 1 and requests consideration of claims 1, 2, 9, 10, 15, and 20. Applicant’s arguments are persuasive and the restriction between Group I, V (claim 10), VI (claim 15), and VII (claim 20) is withdrawn.
3. Claims 3 – 8 and 25 – 35 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 12/20/2024.
Priority
4. This application is a National Stage Application of lnternational Application No.
PCT/US2020/041971, filed 07/14/2020, which claims the benefit of U.S. Provisional Patent
Application Serial No. 62/874,075, filed on 07/15/2019.
Withdrawn Claim Rejections
5. The rejection of claims 1, 2, 9, 10, 15, and 20 under 35 U.S.C. 103 is withdrawn in view of Applicant’s amendment to claim 1 requiring a genome deletion in rag2 gene locus and the genetically-modified fish lack mature B cells.
New Claim Rejections
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.
6. Claims 15 and 20 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.
7. Regarding claim 15, it is unclear if the claim requires formation of a tumor from the transplanted cells from a mammalian tumor prior to contacting the fish with a test compound to evaluate the growth of an established tumor in the presence of a test compound or to evaluate growth of the transplanted cells into a tumor in the presence of a test compound. Example 1 and Figure 2 of Applicant’s disclosure teaches engraftment of tumor cells prior to treatment with Olaparib and temozolomide (page 29, lines 30 – 32; page 30, lines 1 – 22).
8. Claim 15 recites “identifying a compound” in line 9. It is unclear if “a compound” is “the test compound” or another compound.
9. Claim 20 recites “for a time sufficient for the stem or progenitor cell to develop” in line 4 – 5. It is unclear what is meant by “to develop” and therefore it is unclear what is meant by “a time sufficient”. For example, it is unclear if “to develop” refers to proliferation, or differentiation, or engraftment, or something else. Claim 20 also recites “its progeny”. As it is unclear what “to develop” means, it is also unclear what “its progeny” means.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
10. Claims 15 and 20 are rejected under 35 U.S.C. 101 because the claimed invention is directed to abstract ideas without significantly more. The claim(s) recite(s) “evaluating the growth of a tumor” (claim 15), “comparing the level of growth of the tumor” (claim 15), “identifying a compound that decreases the level of growth of the tumor” (claim 15), “evaluating the development of the stem or progenitor cell” (claim 20), “comparing the development of the stem or progenitor cell” (claim 20), “identifying an effect of the test compound on development of the stem or progenitor cell” (claim 20).
Claim 15 is drawn to a method of identifying a candidate therapeutic compound for the treatment of a mammalian tumor.
Claim 20 is drawn to a method of detecting an effect of a test compound on development of a cell or tissue.
The Office published Office's new guidance document entitled 2019 Revised Patent Subject Matter Eligibility Guidance, published January 7, 2019. Applicant is directed to the Federal Register, Volume 4, No. 4, pages 50-57 at page 74621.
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Step 1 of the USPTO' s eligibility analysis entails considering whether the claimed subject matter falls within the four statutory categories of patentable subject matter identified by 35 U.S.C. 101: Process, machine, manufacture, or composition of matter. The claims are directed to a process (step 1, Yes).
Step 2A of the 2019 Revised Patent Subject Matter Eligibility Guidance is a two-prong inquiry. In Step 2A Prong One, examiners evaluate whether the claim recites a judicial exception. The limitations of “evaluating”, “comparing”, and “identifying” are processes that under the broadest reasonable interpretation covers performance of these limitations in the mind because nothing in the claims precludes performing “evaluating”, “comparing”, and “identifying” in the mind. For example, “evaluating” tumor growth or development of stem or progenitor cells encompasses visually observing cell growth; “comparing” encompasses mentally comparing visual observations of cell growth; and “identifying” encompasses mentally determining decreased level of tumor growth (claim 15) or any effect on any development of the stem or progenitor cell (claim 20). Accordingly, the claims recite abstract ideas (Step 2A, prong 1: Yes).
In Step 2A Prong Two, examiners evaluate whether the claim recites additional elements that integrate the exception into a practical application of that exception. This evaluation is performed by (a) identifying whether there are any additional elements recited in the claim beyond the judicial exception, and (b) evaluating those additional elements individually and in combination to determine whether the claim as a whole integrates the exception into a practical application. Claim 15 recites the method identifies a candidate therapeutic compound and that the intended use of the candidate therapeutic compound is for the treatment of a mammalian tumor. An evaluation of whether this limitation is insignificant extra-solution activity is then performed. Note that because Step 2A Prong Two analysis excludes consideration of whether a limitation is well-understood, routine, conventional activity, this evaluation does not take into account whether or not the limitation is well-known. When so evaluated, this additional element is insignificant extra-solution activity because of the high-level of generality (treatment of any mammalian tumor, candidate therapeutic compound) such that it amounts to no more than mere instructions to apply the exception to any tumor and any compound. Claim 20 recites the method detects an effect of a test compound on development of stem or progenitor cells. An evaluation of whether this limitation is insignificant extra-solution activity is then performed. When so evaluated, this additional element is insignificant extra-solution activity because of the high-level of generality (any effect, any test compound, any development) such that it amounts to no more than mere instructions to apply the exception to any tumor and any compound. Accordingly, these additional elements do not integrate the abstract ideas into a practical application because they do not impose any meaningful limits on practicing the idea. The claims are directed to abstract ideas (Step 2A, prong 2, No).
In Step 2B, the eligibility analysis evaluates whether the claim as a whole amounts to significantly more than the recited exception, i.e., whether any additional element, or combination of additional elements adds an inventive concept into the claim. As discussed with respect to Step 2A Prong Two, the additional limitations including the intended use recited in claim 15 is at best the equivalent of merely adding the words “apply it” to the judicial exception. Mere instructions to apply an exception cannot provide an inventive concept and does not amount to significantly more (Step 2B: No).
Limitations that were found not to be enough to qualify as ‘‘significantly more” when recited in a claim with a judicial exception include: Adding the words ‘‘apply it” (or an equivalent) with the judicial exception, or mere instructions to implement an abstract idea on a computer; simply appending well-understood, routine and conventional activities previously known to the industry, specified at a high level of generality, to the judicial exception, e.g., a claim to an abstract idea requiring no more than a generic computer to perform generic computer functions that are well-understood, routine and conventional activities previously known to the industry; adding insignificant extrasolution activity to the judicial exception, e.g., mere data gathering in conjunction with a law of nature or abstract idea; or generally linking the use of the judicial exception to a particular technological environment or field of use.
Rejections Necessitated by Amendment
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.
11. Claim(s) 1, 9, 10, 15, and 20 are rejected under 35 U.S.C. 103 as being unpatentable over Langenau (WO2015006455; previously cited), hereinafter Langenau which is cited on the IDS filed 07/29/2022 in view of Nanni (Nanni, Patrizia, et al. PloS one 7.6 (2012): e39626.), hereinafter Nanni, in view of Teng (Teng Y, et. al. BMC Cancer. 2013 Oct 4;13:453), hereinafter Teng in view of Tang (Tang, Qin, et al. Journal of Experimental Medicine 214.10 (2017): 2875-2887; previously cited), hereinafter Tang which is cited on the IDS filed 07/29/2022.
Regarding claim 1, Langenau teaches rag2-deficient (rag2-/-) fish (“a first engineered or induced genetic alteration in recombination-activating gene 2 (rag2) that results in an inactivation of both alleles of rag2”) that are viable, survive to adulthood, can be genotyped by fin clip, and engraft human cells (Figure 8C, 9, 11, 13, 14; page 6, lines 22 – 26; page 26, lines 1 – 3; page 29, lines 3 – 7; page 30, lines 5 – 7; page 31, lines 17 – 20). Langenau teaches these rag2-/- fish have a genomic deletion as shown in Table 1. Langenau does not teach the “genetic alteration in rag2 is amorphic” or “a second engineered or induced genetic alteration in interleukin 2-receptor gamma a (il2rga)” or the genetically modified fish lack mature B cells. However, Langenau teaches homozygous rag2E450fs fish with a reduction in thymic T cells and reduced lymphocytes and reduced transcript expression for the mature B and T cell markers but also teaches these fish are hypomorphic (page 23, lines 18 – 19; page 24, lines 1 – 10). Langenau teaches fish with deletions in il2rga (Table 1) in Example 1 constructed using ZFNs and TALENs (page 21 – 23).
Regarding claim 9, Langenau teaches rag2-/- fish robustly engrafted hematopoietic stem cells after being irradiated with 10 gy and were examined at 10, 20, 30, and 45 days post-injection and were sacrificed after 45-days post-transplantation (page 29, lines 25 – 31; page 30, lines 5 – 14).
Regarding claim 10, Langenau teaches a method for growing a mammalian cell comprising transplanting sterile human and mouse cells into rag2-/- fish (page 2, lines 15 – 18; page 30, lines 28 – 31; page 31, lines 16 – 32). Langenau does not teach rag2-/-; IL2rga-/- fish of claim 1.
Regarding claim 15, Langenau teaches a method for identifying a candidate therapeutic compound for the treatment of a mammalian tumor comprising transplanting cells from a mammalian tumor into a genetically-modified fish; contacting the fish with a test compound; evaluating the growth of the mammalian tumor cells in the presence of the test compound; comparing the level of growth of the mammalian tumor cells in the presence of the test compound to a reference level; and identifying a compound that decreases the level growth of the mammalian tumor cells as a candidate therapeutic compound (page 2, lines 19 – 26). Langenau does not teach rag2-/-, IL2rga-/- fish of claim 1.
Regarding claim 20, Langenau teaches a method for detecting an effect of a test compound on development of a cell or tissue comprising transplanting a stem or progenitor cell into a genetically-modified fish; contacting the fish with a test compound for a time sufficient for the stem or progenitor cell to develop; evaluating the development of the stem or progenitor cell or its progeny in the presence of the test compound; comparing the development of the stem or progenitor cell or its progeny in the presence of the test compound to development of the stem or progenitor cell or its progeny in the absence of the test compound; and identifying an effect of the test compound on development of the stem or progenitor cell or its progeny (page 2, lines 30 – 33; page 3, lines 1 – 7). Langenau does not teach rag2-/-, IL2rga-/- fish of claim 1.
Langenau does not teach the “genetic alteration in rag2 is amorphic” or “a second engineered or induced genetic alteration in interleukin 2-receptor gamma a (il2rga)” or the genetically modified fish lack mature B cells of claim 1. However, Langenau teaches to date, the major hurdle for use of zebrafish as a xenograft transplant model is lack of immune-compromised zebrafish strains (page 8, lines 28 – 30). Langenau teaches blood development and function are highly conserved between zebrafish, mouse, and human (page 8, lines 31 – 32). Langenau teaches the genetic makeup of zebrafish is closely related to other vertebrates, including human and mouse, thus zebrafish serves as an excellent model for the study of vertebrate development and human diseases (page 11, lines 30 – 32; page 12, line 1). Langenau teaches Rag-deficient mice lack fully functional T or B cells but retain largely intact natural killer cell function and to obviate immune rejection mediated by NK cells, investigators have utilized IL2rg-deficient mice (page 7, lines 20 – 32). Langenau teaches zebrafish have an immune system that develops during the first weeks of life including T, B, and NK cells that can detect and kill foreign cells (page 9, lines 1 – 3). Langenau teaches that investigators have capitalized on the short window of immune tolerance in early larval development to implant human cancer cells into 2-day-old-zebrafish but the animals eventually develop immune responses and kill engrafted cells – preventing analysis of animals after 7 days of life (page 9, lines 3 – 7). Langenau teaches the described fish can be used in stem cell biology, regenerative medicine, and cancer research and fish facilitate large-scale transplantation experiments at greatly reduced costs to investigators (page 16, lines 15 – 17). Langenau teaches the immune compromised genetically modified zebrafish can be transplanted with mammalian tumor cells and the fish are useful to screen for therapeutic compounds that modulate tumor formation (page 19, lines 11 – 32). Langenau teaches human and mouse primary cancers and cancer cell lines can be engrafted into immune compromised fish lines (page 20, lines 14 – 15). Langenau teaches zebrafish have many attributes that represent clear advantages over commonly used vertebrate models including 1) fecundity: each female can produce 100-200 eggs per week; 2) small size: thousands of animals can be reared in a relatively small space; 3) reduced cost: mouse per diems range from $0.20-$1.00/day depending on cage size, while fish per diems are <$0.01/day, 4) optical clarity: engraftment of normal and malignant cells can be easily visualized by fluorescent labeling and direct visualization of engrafted cells can be further enhanced by zebrafish lines that lack pigment- aptly called Casper; 5) wide temperature range: zebrafish can be reared at 18°C-35°C, the latter mimicking temperatures seen in mouse and human, 6) high-throughput cell transplantation: 350+ adult syngeneic zebrafish can be transplanted intraperitoneally or retro-orbitally with fluorescently-labeled cancer cells by one investigator in a single day, facilitating large scale experimentation (page 8, lines 16 – 28).
Regarding an amorphic rag2 genetic alteration and a second genetic alteration in il2rga of claim and the genetically modified fish lack mature B cells 1, Nanni teaches Rag2-/-;IL2rg-/- mice that lack T, B, and NK cells (page 2, left col. para. 2; page 7, left col. para. 3; page 3, right col. para. 3). Nanni teaches many human breast cancer cell lines grow poorly in nude mice and usually do not metastasize even when NK activity is blocked (page 2, left col. para. 3). Nanni teaches metastatic dissemination of breast cancer cells in Rag2-/-;Il2rg-/- mice (page 2, left col. para. 4; Table 1). Nanni teaches metastatic dissemination of human breast cancer is a great therapeutic challenge and new treatments are urgently needed (page 2, right col. last para.; page 3, left col. para. 1). Nanni teaches evaluating a drug against metastatic growth in the Rag2-/-;Il2rg-/- mice (page 3, left col. para. 2 – 3). Nanni teaches Rag2-/-;Il2rg-/- mice could unveil the intrinsic tumorigenic and metastatic potential of human cancer cells, which is masked or distorted by the use of poorly permissive hosts (page 5, left col. para. 1). One would have been motivated to combine the teachings of Langenau and Nanni in zebrafish that has an amorphic rag2 genome deletion and an inactivating il2rga mutation where the genetically modified fish lack mature B cells because both teach animal models of cancer, Langenau teaches to date, the major hurdle for use of zebrafish as a xenograft transplant model is lack of immune-compromised zebrafish strains, and Nanni teaches mice that are deficient in both rag2 and il2rg lack T, B, and NK cells allow for studying metastatic cancer and Langenau teaches blood development and function are highly conserved between zebrafish, mouse, and human.
Teng teaches in vivo metastasis assays have traditionally been performed in mice but the process is inefficient and costly (Abstract). Teng teaches injection of metastatic breast cancer cells into zebrafish embryos and 30 hours post injection the spread of cancer cells could be seen throughout the body of the fish (page 1, right col. para. 2). Teng teaches the zebrafish model offers a rapid, robust, and inexpensive means of evaluating the metastatic potential of human cancer cells (Abstract). Teng teaches the well-established systems for drug screening in zebrafish opens up the possibility of identifying therapeutics that can target metastasis (page 8, right col. para. 2). Teng teaches the metastasis phenotype in fish is identical to that in rodents with the advantages that individual cells can be imaged during the metastasis process (page 8, right col. para. 3).
It would have been obvious prior to the effective filing date for the person of ordinary skill in the art to combine the teachings of Langenau regarding a genetically modified, immune compromised fish having either a genome deletion in rag2 or il2rga where the rag2-/- fish have reduced thymic T cells and reduced lymphocytes and reduced transcript expression for the mature B and T cell markers and can engraft human and mouse tumor cells with the teachings of Nanni regarding Rag2-/-;Il2rg-/- mice that lack T, B, and NK cells as a model for human metastatic cancer to arrive at the claimed genetically-modified fish whose genome is homozygous for a first engineered or induced genetic alteration in recombination-activating gene 2 (rag2) that results in an inactivation of both alleles of rag2, wherein the genetic alteration in rag2 is amorphic and comprises genome deletion in rag2 gene locus; and a second engineered or induced genetic alteration in interleukin 2-receptor gamma a (il2rga) wherein the first genetic alteration results in an inactivation of both alleles of rag2; wherein the second genetic alteration that results in an inactivation of both alleles of il2rga, wherein the genetically-modified fish lack mature B cells. One would have been motivated to combine the teachings of Langenau, Nanni, and Teng in an adult immune-compromised zebrafish metastatic cancer model as Langenau teaches the major hurdle for use of zebrafish as a xenograft transplant model is lack of immune-compromised zebrafish strains and Langenau teaches the immune compromised genetically modified zebrafish can be transplanted with mammalian tumor cells and the fish are useful to screen for therapeutic compounds that modulate tumor formation and Nanni teaches metastatic dissemination of human breast cancer is a great therapeutic challenge and new treatments are urgently needed and Teng teaches the zebrafish model offers a rapid, robust, and inexpensive means of evaluating the metastatic potential of human cancer cells. One would have a reasonable expectation of success in combining the teachings as Langenau teaches blood development and function are highly conserved between zebrafish, mouse, and human and Langenau teaches the genetic makeup of zebrafish is closely related to other vertebrates, including human and mouse, thus zebrafish serves as an excellent model for the study of vertebrate development and human diseases and Nanni teaches Rag2-/-;IL2rg-/- mice lack T, B, and NK cells and Nanni teaches metastatic dissemination of breast cancer cells in Rag2-/-;Il2rg-/- mice and Teng teaches the metastasis phenotype in fish is identical to that in rodents with the advantages that individual cells can be imaged during the metastasis process.
12. Claim(s) 2 is/are rejected under 35 U.S.C. 103 as being unpatentable over Langenau (WO2015006455; previously cited), hereinafter Langenau which is cited on the IDS filed 07/29/2022 in view of Nanni (Nanni, Patrizia, et al. PloS one 7.6 (2012): e39626.), hereinafter Nanni, in view of Teng (Teng Y, et. al. BMC Cancer. 2013 Oct 4;13:453), hereinafter Teng as applied to claims 1, 9, 10, 15, and 20 above, and further in view of Tang (Tang, Qin, et al. Journal of Experimental Medicine 214.10 (2017): 2875-2887; previously cited), hereinafter Tang which is cited on the IDS filed 07/29/2022.
Langenau in view of Nanni and Teng make obvious the limitations of claim 1 as set forth above. Regarding claim 2, Langenau teaches rag2-/- fish but does not teach the fish have genotype rag2null/null, il2rgaY91fs-/-. However, Langenau teaches fish with inactivating mutations in il2gra (page 23, lines 6 – 11; Table 1; Figure 5). Langenau teaches to date, the major hurdle for use of zebrafish as a xenograft transplant model is lack of immune-compromised zebrafish strains (page 8, lines 28 – 30). Langenau teaches Rag-deficient mice lack fully functional T or B cells but retain largely intact natural killer cell function and to obviate immune rejection mediated by NK cells, investigators have utilized IL2rg-deficient mice (page 7, lines 20 – 32). Langenau teaches blood development and function are highly conserved between zebrafish, mouse, and human (page 8, lines 31 – 32). Langenau teaches zebrafish have an immune system that develops during the first weeks of life including T, B, and NK cells that can detect and kill foreign cells (page 9, lines 1 – 3). Langenau teaches that investigators have capitalized on the short window of immune tolerance in early larval development to implant human cancer cells into 2-day-old-zebrafish but the animals eventually develop immune responses and kill engrafted cells – preventing analysis of animals after 7 days of life (page 9, lines 3 – 7). Langenau teaches the described fish can be used in stem cell biology, regenerative medicine, and cancer research and fish facilitate large-scale transplantation experiments at greatly reduced costs to investigators (page 16, lines 15 – 17). Langenau teaches the immune compromised genetically modified zebrafish can be transplanted with mammalian tumor cells and the fish are useful to screen for therapeutic compounds that modulate tumor formation (page 19, lines 11 – 32). Teng teaches the zebrafish model offers a rapid, robust, and inexpensive means of evaluating the metastatic potential of human cancer cells (Abstract). Teng teaches the well-established systems for drug screening in zebrafish opens up the possibility of identifying therapeutics that can target metastasis (page 8, right col. para. 2).
Regarding the specific mutation in il2rga of il2rgaY91fs-/- of claim 2, Tang teaches fish with il2rgaY91fs-/- mutation where these mutant fish showed a dramatic loss of T cells and NK cells (page 2880, left col. para. 2; Figure S3; Figure 3A vs 3C). Tang teaches the il2rgaY91fs-/- fish showed an increase in B cells likely resulting from lineage compensation but when combined with another mutation, the resulting fish lacked B cells (Figure 3A, C, and D; page 2880, left col. para. 2). Tang teaches zebrafish share conserved cell types and underlying molecular programs that regulate hematopoiesis similar to those found in mammals (page 2875, left col.).
It would have been obvious prior to the effective filing date for the person of ordinary skill in the art to combine the teachings of Langenau regarding a genetically modified, immune compromised fish having either a genome deletion in rag2 or il2rga where the rag2-/- fish have reduced thymic T cells and reduced lymphocytes and reduced transcript expression for the mature B and T cell markers and can engraft human and mouse tumor cells with the teachings of Nanni regarding Rag2-/-;Il2rg-/- mice that lack T, B, and NK cells as a model for human metastatic cancer with the teachings of Teng regarding zebrafish as models of human metastatic cancer with the teachings of Tang regarding il2rgaY91fs-/- fish show a dramatic loss of T cells and NK cells to arrive at the claimed genetically-modified fish which has a genotype rag2null/null; ilrgaY91fs-/-. One would have been motivated to combine the teachings of Langenau, Nanni, Teng, and Tang in an adult immune-compromised zebrafish lacking NK cells and mature B cells metastatic cancer model as Langenau teaches the major hurdle for use of zebrafish as a xenograft transplant model is lack of immune-compromised zebrafish strains and Langenau teaches Rag-deficient mice lack fully functional T or B cells but retain largely intact natural killer cell function and to obviate immune rejection mediated by NK cells, investigators have utilized IL2rg-deficient mice and Teng teaches the zebrafish model offers a rapid, robust, and inexpensive means of evaluating the metastatic potential of human cancer cells and the well-established systems for drug screening in zebrafish opens up the possibility of identifying therapeutics that can target metastasis. One would have a reasonable expectation of success in combining the teachings as Tang teaches that B cells can be reduced by combining the il2rgaY91fs-/- mutation with another inactivating mutation and Langenau teaches Rag-deficient mice lack fully functional T or B cells and Langenau teaches blood development and function are highly conserved between zebrafish, mouse, and human and Tang teaches zebrafish share conserved cell types and underlying molecular programs that regulate hematopoiesis similar to those found in mammals and Tang teaches il2rgaY91fs-/- fish showed a dramatic loss of T cells and NK cells.
Applicant’s Arguments/ Response to Arguments
13. Applicant Argues: On page 5 – 7, Applicant asserts that Lagenau does teach an amorphic genetic alteration in rag2 that comprises genome deletion in rag2 gene locus as required by amended claim 1.
Response to Arguments: The previous rejection has been withdrawn in view of Applicant’s amendment to claim 1 and arguments regarding the hypomorphic rag2 mutation taught by Langenau. Amended claim 1 does not require a specific genome deletion in the rag2 gene locus. As such, in the new rejection set forth above, Langenau teaches rag2-/- fish comprise a genomic deletion in the rag2 locus (Table 1) and these fish have reduced transcript expression for the mature B and T cell markers but also teaches these fish are hypomorphic (page 23, lines 18 – 19; page 24, lines 1 – 10). Therefore, Langenau does not teach the rag2-/- fish lack mature B cells or that the mutation is amorphic as required by amended claim 1. However, Langenau teaches zebrafish have many attributes that represent clear advantages over commonly used vertebrate models for studying vertebrate development and human diseases but the development of the zebrafish immune system including T, B, and NK cells that can detect and kill foreign cells limits analysis of engrafted human cancer cells beyond 7 days of life (page 9, lines 3 – 7; page 11, lines 30 – 32; page 12, line 1). Thus, Langenau provides motivation for providing an adult immune compromised genetically modified fish allowing for engraftment of mammalian cancer cells as Langenau teaches the immune compromised genetically modified zebrafish can be transplanted with mammalian tumor cells and the fish are useful to screen for therapeutic compounds that modulate tumor formation (page 19, lines 11 – 32). Langenau provides a reasonable expectation of success in such a fish because Langenau teaches blood development and function are highly conserved between zebrafish, mouse, and human and the genetic makeup of zebrafish is closely related to other vertebrates, including human and mouse (page 8, lines 31 – 32; page 11, lines 30 – 32; page 12, line 1). Further, Langenau teaches robust methods to induce targeted gene disruption in zebrafish using ZFNs and TALENs have been developed (page 9, lines 29 – 31) and the rag2-/- fish and il2rga-/- fish were created using ZFNs and TALENs and this technology can be used to effectively create zebrafish gene modifications that mimic mutations found in human and mouse SCID, and engraftment of human and mouse cancer cells into rag2-/- immune compromised zebrafish (page 16, lines 29 – 32; page 17, lines 10 – 13; page 20, lines 14 – 15; page 30, lines 19 – 32; page 31). Nanni teaches Rag2-/-;IL2rg-/- mice that lack T, B, and NK cells as a model for evaluating potential therapeutics against metastatic human breast cancer (page 2, left col. para. 2 and 4; page 7, left col. para. 3; page 3, left col. para. 2 – 3; page 3, right col. para. 3; Table 1). Nanni teaches metastatic dissemination of human breast cancer is a great therapeutic challenge and new treatments are urgently needed (page 2, right col. last para.; page 3, left col. para. 1). Therefore, Nanni provides motivation to combine inactivating mutations in rag2 and il2rga in the fish of Langenau such that the fish lack mature B cells to engraft metastatic mammalian tumor cells to evaluate potential therapeutics. Teng teaches in vivo metastasis assays have traditionally been performed in mice but the process is inefficient and costly (Abstract). Teng teaches injection of metastatic breast cancer cells into zebrafish embryos and 30 hours post injection the spread of cancer cells could be seen throughout the body of the fish (page 1, right col. para. 2). Teng teaches the zebrafish model offers a rapid, robust, and inexpensive means of evaluating the metastatic potential of human cancer cells (Abstract). Teng teaches the well-established systems for drug screening in zebrafish opens up the possibility of identifying therapeutics that can target metastasis (page 8, right col. para. 2). Teng teaches the metastasis phenotype in fish is identical to that in rodents with the advantages that individual cells can be imaged during the metastasis process (page 8, right col. para. 3). Therefore, Teng provides a reasonable expectation of success in studying mammalian cancer cell metastasis in zebrafish.
Conclusion
No claims allowed.
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/ZANNA MARIA BEHARRY/Examiner, Art Unit 1632