DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Applicant’s response to restriction requirement and amendments to the claims filed on November 9, 2025 have been received and entered. Claims 1-5, 7--8, 10, 12-13, 15, 17-18 have been amended, while claims 19-23 are newly added. Claims 1-5, 7-8, 10, 12-13, 15-22 and 23 are pending in the instant application.
Election/Restrictions
Applicant's election with traverse of claims 1-5 and 18 (group I) in the reply filed on November 9, 2025 is acknowledged. The traversal is on the ground(s) that shared feature that the infusion solution comprises 0.12-2 mM Mg2+ and 0.12-2 mM Ca2+ is inventive over the disclosure of prior art. This is not found persuasive because as stated in previous office action the invention of group I and II-VII lack unity of invention because even though the inventions of these groups require the technical feature of a chimeric embryo comprising aggregating a tetraploid embryo with embryonic stem cells to form a new reconstructed embryo or a chimeric embryo. This special technical feature does not contribute over prior art of record. Further, Applicant has amended the claims to incorporate the limitation of other non-elected independent claims, however, as stated in the previous office action these groups do not share the same or corresponding technical feature for the reasons discussed on page 4 of the restriction requirement mailed on September 11, 2025. Therefore, instant applicant would be examined to the extent claims are drawn to the elected invention of a method for preparing a chimeric embryo or a nonhuman animal (group 1). Please note any claim drawn to method of preparing targeting vector, preparing cell line or preparing a nonhuman animal model using targeting vector and using primer would be withdrawn. A telephone call was made to applicant’s representative on two separate occasions on February 21, 2026 to clarify the election, however no response was returned. The requirement is still deemed proper and is therefore made FINAL.
Priority
This application is a 371 of PCT/CN2020/101681 filed on 07/13/2020, which claims foreign priority from CN202010151592.2 filed on 03/16/2020 and CN202010375045.2 filed on 04/30/2020.
Should applicant desire to obtain the benefit of foreign priority under 35 U.S.C. 119(a)-(d) prior to declaration of an interference, a certified English translation of the foreign application must be submitted in reply to this action. 37 CFR 41.154(b) and 41.202(e).
Failure to provide a certified translation may result in no benefit being accorded for the non-English application.
Information Disclosure Statement
The information disclosure statements (IDS) submitted on 01/20/2023, 02/10/2023 and 11/09.2025 are in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
Claims 1-5, 7-8, 10, 12-13, 15-22 and 23 are under consideration.
Claim Objections
Claims 1-2 and 22 are objected to because of the following informalities: In the instant cases, the method steps of each of these claims recite whole numbers (1, 2, 3), which is same as claim numbering. The whole numbering for method step should be replaced with roman numeral (i, ii, iii, iv etc). Appropriate correction is required.
Claim Rejections - 35 USC § 112-scope of enablement
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 1-5, 7-8, 10, 12-13, 15-22 and 23 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for
A method for preparing a chimeric embryo and a mouse, comprising aggregating a tetraploid embryo with embryonic stem cells to form a new reconstructed embryo or a chimeric embryo, wherein the tetraploid embryo is a tetraploid embryo said method comprising
:(i) obtaining a mouse 2-cell embryo;
(ii) placing the mouse 2-cell embryo in a fusion solution and performing electrofusion in the fusion solution to obtain a tetraploid embryo, wherein the fusion solution comprising 0.12-2mM of Mg2+ and 0.12-2mM of Ca2+;
(iii) culturing the tetraploid embryo obtained in step (iii) in a culture medium, and
(iv) aggregating the tetraploid embryo, with a mouse embryonic stem cells to form a chimeric mouse embryo; wherein the fusion solution comprises 0.10-0.2mM ofMg2+ and 0.10-0.2mM ofCa2+ ; and further comprising
(v) implanting the chimeric mouse embryo into the uterus of a pseudo pregnant mouse to obtain a chimeric mouse,
does not reasonably provide enablement for preparing chimeric human embryo, or any other chimeric nonhuman embryo that involves interspecies tetraploid complementation and chimera formation or using ES cells from any other species for making chimeric embryo, making any other nonhuman animal or injecting the chimeric embryo into any animal as broadly claimed. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
In determining whether Applicant’s claims are enabled, it must be found that one of skill in the art at the time of invention by applicant would not have had to perform “undue experimentation” to make and/or use the invention claimed. Such a determination is not a simple factual consideration, but is a conclusion reached by weighing at least eight factors as set forth in In re Wands, 858 F.2d at 737, 8 USPQ 1400, 2d at 1404. Such factors are: (1) The breadth of the claims; (2) The nature of the invention; (3) The state of the art; (4) The level of one of ordinary skill in the art; (5) The level of predictability in the art; (6) The amount of direction and guidance provided by Applicant; (7) The existence of working examples; and (8) The quantity of experimentation needed to make and/or use the invention.
The office has analyzed the specification in direct accordance to the factors outlines in In re Wands. MPEP 2164.04 states: “[W]hile the analysis and conclusion of a lack of enablement are based on factors discussed in MPEP 2164.01(a) and the evidence as whole, it is not necessary to discuss each factor in written enablement rejection.” These factors will be analyzed, in turn, to demonstrate that one of ordinary skill in the art would have had to perform “undue experimentation” to make and/or use the invention and therefore, applicant’s claims are not enabled.
Nature of the Invention:
The claims are directed to method for preparing a chimeric embryo or a nonhuman animal, comprising aggregating a tetraploid embryo with embryonic stem cells to form a new reconstructed embryo or a chimeric embryo, wherein the tetraploid embryo is a tetraploid embryo at 2-cell stage; the method comprises the following steps:(1) obtaining an animal 2-cell embryo; (2) placing the 2-cell embryo obtained in step (1) in a fusion solution and performing fusion to obtain a tetraploid embryo; (3) culturing the tetraploid embryo obtained in step (2) in a culture medium, (4) aggregating the tetraploid embryo in step (3) with embryonic stem cells to form a chimeric embryo. Thus, the nature of Applicant's invention is the production of inter or intra-mammalian interspecific tetraploid complementation.
Breadth of the claims:
The claims are broadly directed to a method for producing any chimeric embryo of any species or a nonhuman animal by tetraploid complementation using a 2-cell embryo derived from any animal in a fusion solution comprising 0.12-2mM ofMg2+ and 0.12-2mM ofCa2+ and performing fusion under any condition to obtain a tetraploid embryo. Subsequently aggregating the tetraploid embryo with embryonic stem cells derived from same species as 2-cell embryo or genus of other specie to form a chimeric embryo.
The breadth of the claim encompasses embryos that may be obtained from any animal including from a pig, a rat, a mouse, a hamster, a rabbit, a pig, a bovine, a deer, a sheep, a goat, a chicken, a cat, a horse, a dog, an orangutan, and a monkey (see page 3) . The term "embryonic stem cells" or "ES cells" as used herein include embryo-derived totipotent or pluripotent cells capable of promoting the development of any tissue of an embryo after being introduced into the embryo (see page 22 of the specification). In the instant case, claims encompass embryonic stem cells derived from any species of animal that may be allogenic or xenogeneic to the donor 2-cell embryo. Claim1 broadly encompasses chimeric human or interspecies embryo that is not enabling because of the absence of an enabling disclosure: (i) to address the issues of genetic and epigenetic incompatibles between tetraploid embryo derived from any animal to ESC from same or another species; (ii) developmental incompatibility between ESC and tetraploid embryo, (iii) immunological barrier between cross species aggregation of tetraploid embryo with ES cells derived from plurality of different species, and (iv) interspecies tetraploid complementation The disclosure provided by the applicant, in view of prior art, must encompass a wide area of knowledge to a reasonably comprehensive extent. In other word each of these, aspect must be shown to a reasonable extent so that one of the ordinary skills in the art would be able to practice the invention without any undue burden being on such Artisan.
Guidance of the Specification and The Existence of Working Examples:
The specification teaches use of sgRNA1 for ACE2 gene and construction of pX330-sgRNA plasmid (examples 1-2). Example 3 describes evaluation of pX330-sgRNA cleavage efficiency. Example 6 teaches obtaining of mouse embryonic stem cells having humanized ACE2. The specification teaches genotype identification of mouse ES having humanized ACE2 (example 7). The specification teaches placing mouse embryos in 0.3 M mannitol containing 0.1 mM MgSO4, 0.1 mM CaCl2, and 0.3% bovine serum albumin, and subjected to direct current fusion at 60 V for 50 microseconds by using electrofusion device to obtain tetraploid embryos. The tetraploid embryos were then put into KSOM medium of mouse ova in a simplex optimized medium supplemented with amino acids. Then the embryos were aggregated with the embryonic stem cells (the mouse embryonic stem cells having humanized ACE2 obtained in Example 6) to form chimeric embryo (see example 9). It is further noted that the specification discloses the use embryonic stem cells within passage 15 (p15) and more specifically exemplified embryonic stem cells were at passage 12 (example 10). Example 12 shows the use of 4-cell stage in electrofusion solution with increased Mg2+ and Ca2+ concentrations in presence of ES cells of passage 12. The results that the birth rate of the mice and the survival rate of the mice is better in fusion solution containing higher Mg2+ and Ca2+ concentrations as compared to use of traditional fusion solation (see fig. 19 and example 12-13).
State of the Art and Predictability of the Art:
The state of the art teaches summarized by the reference of Yamaguchi (Scientific Reports, 2018, 15289, 1-10) teaches contribution of donor PSC-derived cells is lower in interspecies chimeras than in intraspecies chimeras, and high donor chimerism is associated with anomalies or embryonic (abstract). Yamaguchi states “limitations of interspecies chimeras, including cell contribution and tissue distribution, have not been thoroughly explored. Identifying these limits will not only highlight the temporal and spatial occurrence of a xeno- genic barrier, but may also provide clues regarding which cell types and organs are most amenable to interspecies chimerism and organogenesis” (see page 2, para. 2). Yamaguchi shows that interspecies aggregation often fails at a later stage especially during placenta formation and organogenesis due to species mismatch (see page 8, para. 2-5). This is further supported by Simerly et al (Stem Cell Research (2011) 7,28–40) who reported “cell cycle differences between primate and rodent pluripotent stem cells preclude primate ESCs from participation in development after implantation. The time course of development also differs significantly between rodents and primates. Blastocysts develop in mice within 3.5–4 days, whereas human blastocysts require 5–6 days and nhp primates a week or more” (see page 37, col. 1, last para.). Further, Li et al (Reproduction(2005)13053–59) teach genetic heterozygosity, fitness of tetraploid embryos and fitness of ES cells are crucial parameters influencing survival of mice derived from ES cells by tetraploid embryo aggregation (see abstract). Li reported that the developmental potential of the tetraploid aggregates is strain dependent (see page 52, col. 2, para. 1). In view of foregoing, it is apparent that even if embryonic stem cell enters the embryo the development fails at a later stage due to xenogeneic incompatibility. In the instant case, guidance provided in the specification is limited to placing mouse embryos in 0.3 M mannitol containing 0.1 mM MgSO4, 0.1 mM CaCl2, and 0.3% bovine serum albumin, and subjecting said embryo to electrofusion to obtain tetraploid mouse embryos that is aggregated with the mouse embryonic stem cells to obtain a chimeric mouse embryo (see example 9). The specification fails to provide any guidance to overcome the art recognized unpredictability pertaining to genetic and epigenetic incompatibles between tetraploid embryo derived from any animal to ESC from another species and interspecies tetraploid complementation. . One of skill in the art would have to perform undue experiment to make and use the invention, without reasonable expectation of success.
The claimed method broadly embraces making any chimeric embryo or nonhuman animal, by obtaining a tetraploid embryo from any animal and aggregating said embryo with embryonic stem cell derived from any species to produce chimeric embryo followed by producing a nonhuman animal. . The claim encompasses an extremely large genus of tetraploid embryo derived animal species with allogenic or xenogeneic ES cell species. In this regard, the art teaches that use of tetraploid complementation with any species of ES cells to produce a chimeric embryo or a non-human animal from any species of animal is unpredictable in any species, except for mice. The specification exemplified placing mouse embryos in 0.3 M mannitol containing 0.1 mM MgSO4, 0.1 mM CaCl2, and 0.3% bovine serum albumin, and subjecting said embryo to electrofusion to obtain tetraploid mouse embryos that is aggregated with the mouse embryonic stem cells to produce chimeric mouse embryo and mouse. The specification fails to provide any guidance as to other species of embryonic stem cells capable of populating the tetraploid. At the time of filing, the skilled artisan did not consider the generation of a non-human animals other than the mouse as routine or predictable using tetraploid competition. As noted above, the specification only provides specific guidance for using mouse embryonic stem cells to generate a mouse. The specification provides no additional guidance for making other species of nonhuman animals using interspecies or intraspecies chimera. Before the effective filing date of instant invention, art teaches there is significant unpredictability in isolating, characterizing and using ES cells isolated from genus of nonhuman animal species, and that pluripotency has not been validated or verified in any species of animal other than the mouse. Regarding ES cells, the art teaches that while mouse ES cells have been established, no validated porcine ES cells are available (Brevini et al., 2010, Theriogenology, Vol. 74, pgs. 544-550, see Abstract). Brevini continues to teach that conflicting data regarding the expression of pluripotency markers in porcine ESCs further complicates the understanding and establishment of a porcine ESC cell line (pg. 548 col. 2 para. 4). Brevini concludes by teaching that “many factors, some of which are briefly discussed in the present manuscript, make the establishment of ESC lines in the pig, and in animal species other than mouse and human, a very slow process.” (pg. 548 col. 2 para. 5 lines 1-4). Brevini continues to teach that “Compared with the large number of studies exploring the appropriate culture conditions for mouse and human ESCs, there is a minimal amount of data available for domestic species ESC. That limited information is mainly based on mouse ESC culture systems. As a result, such conditions did not appear to be effective for maintaining stable undifferentiated ESC lines in domestic animals. We are convinced that a major goal at present is to develop better culture formulations in order to obtain homogenous pluripotent outgrowths from pig embryos and identify the best in vitro environment that would facilitate derivation of stable pESC culture” (pg. 546 col. 1 parag. 2 bridge col. 2 parag. 1). Ezashi et al (Annu. Rev. Anim. Biosci. 2016. 4:223–53) reviewed the state of the art and states “papers reporting ESC derivation from swine, cow, and dog significantly outnumber those for sheep, goat, and cat (Figure 2, orange bars), but authentic ESC homologous to those described for rodent have not been established conclusively in any of these species (see page 227, para.1). Ezashi et al continue to teach that “the persistent failure in generating ESC from these same species may stem from a shared problem, namely, instability of the gene networks necessary to maintain pluripotency under the culture conditions employed” (see page 231, para. 1). Tachibana et al (Cell 148, 285–295) show that injected primate ESC fail to integrate into 4n-embryo (see pgae 290, col. 1). In fact, art reported that tetraploid complementation is considered to be the most stringent but extremely inefficient pluripotency test for mouse ESCs. Although the majority of mouse ESC lines do contribute to conventional chimeras, only selected lines are able to produce whole ESC-derived live offspring after aggregation with tetraploid host embryos (see page 291, col. 2, para. 2). It is suggested that use of human ESCs or nonhuman ES (monkey) cell to clone humans or create chimeras seem to be unattainable or more challenging, (see page 292, col. 1, para.1). In view of foregoing, it is apparent that the art teaches that use of ES cells in many mammals has several significant issues of unpredictability and the claims encompass any species of animal, mammal or rodent to be made. Thus, the art of record clearly establishes that before the effective filing date of instant invention the only mouse ES cells were known to colonize the tetraploid embryo, and therefore, the art only enables the use of mouse ES cells to produce chimeric embryo or mouse as embraced by the breadth of the claims. Therefore, in view of the state of the art at the time of filing, the lack of specific guidance in the specification making chimeric embryo or nonhuman animal using tetraploid embryo other than mice, and the breadth of the claims, it would have required under experimentation, without reasonable expectation of success. In view of foregoing, instantly claims invention had many scientific hurdles to overcome to be enabled. The specification fails to provide any type of guidance to overcome these obstacles described by art of record cited above. As such, the art teaches that the claimed method is highly unpredictable and also the art fails to supplement the shortcomings of the specification’s guidance.
In conclusion, in view of breadth of the claims and absence of a strong showing by Applicant, in the way of specific guidance and direction, and/or working examples demonstrating the same, such invention as claimed by Applicant is not enabled for the claimed inventions. An artisan of skill would have required undue experimentation to practice the invention because the art of producing chimeric embryo or a nonhuman animal using tetraploid complementation was unpredictable at the time of filing of this application as supported by the observations in the art record.
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.
Claims 5 and 16 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a natural product without significantly more. The claim(s) recite(s) a tissue, a body fluid, a cell, or debris or extract thereof from a non-human animal or its offspring, wherein the non-human animal is prepared by the method, comprising aggregating a tetraploid embryo with embryonic stem cells to form a new reconstructed embryo or a chimeric embryo, wherein the tetraploid embryo is a tetraploid embryo at 2-cell stage; the method comprises the following steps:(1) obtaining an animal 2-cell embryo; (2) placing the 2-cell embryo obtained in step (1) in a fusion solution and performing fusion to obtain a tetraploid embryo; (3) culturing the tetraploid embryo obtained in step (2) in a culture medium, (4) aggregating the tetraploid embryo in step (3) with embryonic stem cells to form a chimeric embryo and injecting chimeric embryo into an animal as set forth in claim 2 and 15 respectively.
Claim interpretation: Under the broadest reasonable interpretation, the terms of the claim are presumed to have their plain meaning consistent with the specification as it would be interpreted by one of ordinary skill in the art See MPEP 2111. Claims are directed to a tissue, a body fluid, a cell, or debris or extract thereof from a non-human animal or its offspring, wherein the non-human animal is prepared by the method of tetraploid complementation. Claims are written as "Product-by-process" claim language that only limits the scope of a product claim to the extent that the process affects the structure of the claimed product. See In re Thorpe, 777 F.2d 695, 697 (Fed. Cir. 1985) ("The patentability of a product does not depend on its method of production. If the product in a product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process."). The specification refers term chimeric embryo to embryo comprising embryonic stem cells and in a chimeric state. The chimeric embryo can be produced using, for example, the so-called "aggregation method", in addition to the injection method. In the "aggregation method", embryo + embryo, or embryo + cells are enabled to closely adhere to each other in a culture dish to produce a chimeric embryo. The specification teaches animal cloned by this technology are completely developed from ES cells and are called ES animal (see page 2, lines 11-12). This is further evidenced by the teaching of Nagy (Proc. Natl. Acad. Sci. USA, 1991, 90, 8424-8428, IDS) who reported isolating brain, kidney, liver, heart and blood from an ES cell derived mouse (see page 8425, col. 1, para. 1) produced by aggregating the tetraploid embryo with mouse embryonic stem cells (see page 8424, col. 2, last para. to page 8425, col. 1, para. 1, fig. 4). Nagy teaches mouse produced by the method showed no evidence of tetraploid cells in the blood suggesting majority of the resulting mouse are ES-derived mouse. There is nothing in the claims that distinguishes the claimed cell from naturally occurring cell from the mouse. Further, there is no indication and/or evidence in the specification that the claimed process imparts any structural, functional, or otherwise markedly different characteristics to the claimed tissue, a body fluid, a cell, or debris or extract thereof derived from a non-human animal that makes them different from naturally occurring tissue, a body fluid, a cell, or debris or extract thereof from any other wild type mouse (non-human animal).
Step 1: The claim recites a tissue, a body fluid, a cell, or debris or extract thereof from a non-human animal or its offspring, wherein the non-human animal is prepared by the method of tetraploid complementation. Because a tissue, a body fluid, a cell, or debris or extract thereof from a non-human animal are composition of matter, which is a statutory category of invention. Thus, the claim is to at least one statutory category of invention (Step 1: YES).
Step 2A prong 1: An examination of in the revised 101 guidance, with respect to the claimed invention, the answer is yes since the claimed invention is directed to a tissue, a body fluid, a cell, or debris or extract thereof from a naturally occurring product (non-human animal or its offspring therefrom) (judicial exceptions). There is no evidence, in the specification or elsewhere in the claim, showing that the claimed tissue, body fluid, cell, debris or extract thereof from a nonhuman animal produced by a method of tetraploid complementation recited in the claims are markedly different from a naturally occurring mouse. This is further evidenced by the teaching of Nagy as discussed above. Thus, the evidence supports a finding that the claims recite a product of nature, which is a judicial exception to patentable subject matter. See Association for Molecular Pathology v. Myriad Genetics Inc., 569 U.S. 576, 580 (2013). ("[A] naturally occurring DNA segment is a product of nature and not patent eligible merely because it has been isolated.").
Step 2(A), Prong 2 the answer is no with respect to the claimed invention. There are no other additional elements recited in the claims that would amount to significantly more than the judicial exceptions. This is because the claimed invention is drawn to a tissue, a body fluid, a cell, or debris or extract thereof from a non-human animal or its offspring, there are no additional components which impart any additional element or limitation which amount to more than the judicial exception, since the claimed product only recite the judicial exception.
Step 2B: This part of the eligibility analysis evaluates whether the claim as a whole amount to significantly more than the recited exception. MPEP 2106.05. As discussed above with respect to Step 2A Prong one, the claims recite no additional element to the judicial exception. (Step 2B: NO). Thus, the claims are not patent eligible.
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.
Claims 4,7-8, 10, 12-13, 19-22 and 23 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.
Claim 4 recites the limitation "the electrofusion solution" in lines 2, 4, 6, 8, 10, 12 and 14. There is insufficient antecedent basis for this limitation in the claim. It is unclear if electrofusion solution is same as fusion solution of claim 1. Recitation of electrofusion solution instead of fusion solution in claim 1 would obviate the basis of the rejection. Appropriate correction and/or clarification is required.
Claim 7 is vague and indefinite to the extent it is unclear as to how embryonic cells is prepared by using a set of primers. Likewise, claim 10 is vague and indefinite to the extent it not clear as to how embryonic stem cells is prepared by using targeting vector. There is no nexus between ES cell and a set of primer or targeting vector. None of the claim recites ES cell comprises any genetic construct and or primer. It is unclear as how the primer set or targeting vector is used in the claimed method step to prepare the ES cells. Claims 8, 12-13, 19-21 are included in the rejection because they directly or indirectly depend from the rejected base claim.
Claim 13 is vague and indefinite to the extent it is unclear as to step of introducing a target human derived gene into the ES cell occurs before, at or after step (iv) of claim 1. It is unclear how the claimed method step relates to any of the preceding method step. The metes and bounds of the step of introducing a target human derived gene into ES cells could not be ascertained as step (iv) of claim 1 results in chimeric embryo. Claim 22 is unclear as to how the claimed method steps (1)-(3) relates to any of the preceding method step of claim 1 and 13. Claims 22-23 are included in the rejection because they directly or indirectly depend from the rejected base claim. Appropriate correction is required.
Claim 2 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential steps, such omission amounting to a gap between the steps. See MPEP § 2172.01. Claim 2 recite method step similar to claim 1 directed to method of preparing chimeric embryo. The omitted step is : v) implanting/injecting the chimeric nonhuman embryo into the uterus of a pseudo pregnant nonhuman animal to obtain the chimeric nonhuman animal Appropriate correction is required.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 5 and 16 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Nagy et al (Proc. Natl. Acad. Sci, USA, 1993, 90, 8424-8428, IDS). .
Claims are drawn to a tissue, a body fluid, a cell, and debris or extracts thereof from a non-human animal or its offspring, wherein the non-human animal is prepared by the method according to claim 2 and 15.
Claim interpretation: Claims 5, and 16 are product by process claims. MPEP 2113 states” "[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985) (citations omitted).
With respect to claim 5 and 16, Nagy teaches isolated brain, kidney, liver, heart and blood from an ES cell derived mouse (see page 8425, col. 1, para. 1) produced by aggregating the tetraploid embryo with mouse embryonic stem cells to form a chimeric embryo and then transferring the embryo into the uterus of 2.5-day pseudo pregnant recipients (see page 8424, col. 2, last para. to page 8425, col. 1, para. 1, fig. 4). The tissues and blood of the ES cell derived mouse as disclosed by Nagy and those embraced by the instant claims appear to be structurally same. Where the claimed and prior art products are identical or substantially identical in structure or composition, or are produced by identical or substantially identical processes, a prima facie case of either anticipation or obviousness has been established. In re Best, 562 F.2d 1252, 1255, 195 USPQ 430, 433 (CCPA 1977). ''When the PTO shows a sound basis for believing that the products of the applicant and the prior art are the same, the applicant has the burden of showing that they are not.'' In re Spada, 911 F.2d 705, 709, 15 USPQ2d 1655, 1658 (Fed. Cir. 1990). Therefore, the prima facie case can be rebutted by evidence showing that the prior ad products do not necessarily possess the characteristics of the claimed product. In re Best, 562 F.2d at 1255, 195 USPQ at 433.Accordingly, Nagy anticipates claims 5 and 16.
.
Claim Rejections - 35 USC § 103
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.
Claims 1-5, 15-17 and 18 are rejected under 35 U.S.C. 103 as being unpatentable over Ohta (US20090178150, dated 07/09/2009) and McLaughlin (Method of Enzymology, 1991, 55, vol 275, 919-930)/ Darbandi (Int J Reprod BioMed, 2017, Vol. 15. No. 10. 601-612) in view of Suo et al (Journal of Reproduction and Development, 2009, 55, No. 4, 383-385).
Claims are directed to a method for preparing a chimeric embryo, the method comprises the following steps:(1) obtaining an animal 2-cell embryo; (2) placing the 2-cell embryo obtained in step (1) in a fusion solution and performing fusion to obtain a tetraploid embryo; (3) culturing the tetraploid embryo obtained in step (2) in a culture medium, (4) aggregating the tetraploid embryo in step (3) with embryonic stem cells to form a chimeric embryo; wherein the fusion solution comprises 0.12-2mM ofMg2+ and 0.12-2mM ofCa2+. Dependent claims limit the animal is selected from a group consisting of pig, rat, mouse, hamster, rabbit, pig, bovine, deer, sheep, goat, chicken, cat, horse, dog, orangutan, and monkey.
Claim interpretation: Claims 5, and 16 are product by process claims. MPEP 2113 states” "[E]ven though product-by-process claims are limited by and defined by the process, determination of patentability is based on the product itself. The patentability of a product does not depend on its method of production. If the product in the product-by-process claim is the same as or obvious from a product of the prior art, the claim is unpatentable even though the prior product was made by a different process." In re Thorpe, 777 F.2d 695, 698, 227 USPQ 964, 966 (Fed. Cir. 1985) (citations omitted).
With respect to claims 1 and 3, Ohta teaches a method of preparing a chimeric embryo, said method comprising:
obtaining an animal 2-cell embryo
placing a number of two-cell stage embryos that linearly-arranged and then both sides of the embryos are sandwiched with a parallel-double electrode in a medium for electrofusion to select and obtain tetraploid embryos
culturing the tetraploid embryo in culture for overnight (see para 24)
transferring the ES cells to the tetraploid embryos and cultured to produce chimeric embryos (see para. 27), wherein cells may be transferred as cell aggregation (see para. 44), wherein the ES cell and the animal is a mouse ES cells and mouse respectively to produce chimeric mouse embryo (See example 3-4).
Regarding claims 2, 15,18, Ohta teaches implanting the chimeric embryos to a pseudo pregnant mouse to produce chimeric mouse (see claim 1 of ‘150, para. 35 example 3 and 4).
With respect to claim 17, it is noted that claim recites an intended use of the mouse medical research. To the extent, the mouse disclosed in Ohta has same genotype it must necessarily be capable for the use model animals for pathological study and development of new therapy and research of new medicine (see para. 59).
With respect to claims 5 and 16, Ohta teaches cells derived liver and brain from ES mice produced by tetraploid embryo (see fig. 3). The cells derived from the ES mice produced by tetraploid embryo appears to be structurally and functionally similar to one claimed in the instant application.
Ohta differs from claimed invention by not disclosing that the electrofusion solution comprises 0.12-2mM ofMg2+ and 0.12-2mM ofCa2+. (limitation of claims 1 and 4).
However, before the effective filing date of instant application, McLaughlin teaches in order to create an ac field in aqueous solution, a nonelectrolyte is required as a salt to obtain a normal medium osmolarity. The most commonly used nonelectrolyte is mannitol. Small amounts of calcium and magnesium help in membrane healing. BSA acts as a lubricant to minimize adhesion of the embryos to the walls of the chamber and the pipette and to facilitate rotation of the embryos. The contents of the fusion medium are as follows: 0.3 M mannitol, 0. I mM MgSO4, 50uM CaC12, and 3% BSA (see page 926, last para.). Darbandi reported electrolyte medium usually is combination of 0.27-0.3M mannitol, 0.05-0.1 mM Calcium chloride, and 0.05-0.1 mM Magnesium Sulfate in medium, with or without 0.3% BSA (see page 604, col. 2, last para. and page 605, col. 1, para. 1). It is further disclosed that the electrolyte medium works much better than the non-electrolyte (see page 605, col. 1, para. 1). The combination of references differs from claimed invention by not disclosing electrofusion solution containing comprises 0.12-2mM ofMg2+ and 0.12-2mM ofCa2+.
However, before the effective filing date of instant application, it was routine in prior art to equilibrate the fusion in solution at varying condition and voltage to get optimal electrofusion. comprises 0.12-2mM ofMg2+ and 0.12-2mM ofCa2+. Suo teaches identifying different concentration ranging from 0.1mM to 1.4mM at varying electric field ranging from 0.6 to 1.4KV/cm to determine the optimal concentration and electric field for the fusion solution (see page fig. 1, 2 and 3, page 384, col. 1, para. 1).
Therefore, it would have been prima facie obvious for a person of ordinary skill in the art to combine the teachings of prior art to modify the method of Ohta by optimizing the medium for electrofusion as suggested by McLaughlin/ Darbandi in view of Suo, to successfully produce tetraploid embryo, as instantly claimed, with a reasonable expectation of success, before the effective filing date of the instant invention. Said modification amounting to combining prior art elements according to known methods to yield predictable results. Generally, differences in concentration or temperature will not support the patentability of subject matter encompassed by the prior art unless there is evidence indicating such concentration or temperature is critical. It would have been customary for an artisan of ordinary skill to determine the optimal concentration of Mg2+, Ca2+ and electric field to achieve the desired results of obtaining tetraploid embryo. Thus, absent some demonstration of unexpected results from the claimed parameter, the optimization of concentration of Mg2+, Ca2+ and electric field would have been obvious before the effective time of filing of instant invention. "Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation." In re Aller, 220 F.2d 454, 456, 105 USPQ 233, 235 (CCPA 1955). See MPEP 2144.05 One of ordinary skill in the art would be motivated to do so as use of small amounts of calcium and magnesium help in membrane healing of embryo during electrofusion, while BSA acts as a lubricant to minimize adhesion of the embryos to the walls of the chamber and the pipette that also facilitate rotation of the embryos (see McLaughlin). Further, prior art also suggested that such an electrolyte medium works much better than the non-electrolyte for electro fusion. One of skill in the art would have been expected to have a reasonable expectation of success in using the electrofusion solution for preparing tetraploid embryo because prior art successfully reputed optimizing the concentration of divalent ions ranging from 0.1mM to 1.4mM at varying electric field ranging from 0.6 to 1.4KV/cm to determine the optimal concentration and electric field for the fusion solution to prepare tetraploids embryo as evident from the teaching of Suo. It should be noted that the KSR case forecloses the argument that a specific teaching, suggestion, or motivation is required to support a finding of obviousness See the recent Board decision Ex parte Smith, --USPQ2d--, slip op. at 20, (Bd. Pat. App. & Interf. June 25, 2007) (citing KSR, 82 USPQ2d at 1396) (available at http: www. uspto.gov/web/offices/dcom/bpai/prec/fd071925.pdf).
Conclusion
No claims allowed.
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure. Liu et al.,( Physiologia, vol. 71, no. 4, pp. 588-596, 18 June 2019) specifically teaches constructing an ACE2 knockout mouse model using CRISPR/Cas9 technology, and breeding, identifying and validating Ace2 knockout mice. By constructing vectors targeting knockout of Ace2 gene, microinjection of Cas9 mRNA and guide RNA (gRNA) into mouse zygotes in vitro, detection and identification of exon deletions from 3 to 18 of mouse Ace2 gene by PCR and TA clone sequencing, breeding of Ace2 knockout mice and verification of Ace2 mRNA and protein expression in the major organs of Ace2-/Y mice obtained using qRT-PCR and Western blot methods. The results showed successful construction of expressing gRNA vectors and in vitro transcription, successful direct injection of active gRNA and Cas9 mRNA into zygotes to obtain 6 positive F0 generation naïve mice, PCR and gene sequencing identified successful deletion of exons 3 to 18 of the mouse Ace2 gene; an F0 mouse was backcrossed to a wild type mouse to give 3 positive F1 mice, which were then crossed to a wild type mouse to
give an F2 generation in which an Ace2-/+ female heterozygote mouse was selected to be crossed to a wild type mouse to give an F3 generation Ace2-/Y male homozygote mouse.
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/ANOOP K SINGH/ Primary Examiner, Art Unit 1632