DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 12/2/2025 has been entered.
Withdrawn Rejection/Objection
The pending rejection of claims 1-6, and 10-15, 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or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention, is withdrawn. The scope of the claims have changed substantially by amendment and appear to have addressed the issues of concern. However, the amendments have also introduced new issues of indefiniteness. See the new rejections below.
The objection to claims 10-15 is withdrawn. The amendments to the claims remove the recitation under objection.
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 1-6 and 10-15 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.
Claims 1-3 each recite, “wherein at least one selected from the group consisting of Cas 9 proteins and Cas9 mRNAs accumulate in the at least one fertilized egg cell during production of the at least one unfertilized egg cell by the non-human organism so as to function to a 2 cell-stage from fertilization of the at least one unfertilized egg cell”. The recitation within this wherein clause, “so as to function to a 2 cell-stage from fertilization of the at least one unfertilized egg cell” renders the wherein clause indefinite because it is not apparent to what “so as to function…” refers. Is it referring to the function of the Cas9 protein or mRNA or the unfertilized egg cell”? As such, claim 1 is indefinite because the “so as to function” limitations lack sufficient antecedent basis to clearly define the intended metes and bound of the claim.
Claims 46 and 10-15 depend upon claim 1. As such, these dependent claims also comprise the indefinite subject matter discussed above.
Claim Rejections - 35 USC § 112
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 1-6 and 10-15 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 the following:
A method for modifying at a target gene in an unfertilized egg cell from a genetically modified non-human animal, the method comprising:
(i) isolating an unfertilized egg cell from a genetically modified non-human animal; and
(ii) introducing at least one gRNA comprising a nucleic acid sequence complementary to a target sequence in the target gene in the genome of the unfertilized egg cell isolated of (i),
wherein the isolated unfertilized egg cell of (i) comprising at least 3 copies of an exogenous Cas9 gene in its genome,
wherein the at least three exogenous Cas9 gene are operably linked to a promoter that drives expression of the at least three copies of the exogenous Cas9 gene during oogenesis prior to MII meiosis,
wherein the at least three copies of the Cas9 gene are expressed during oogenesis and prior to egg cell division in the unfertilized egg cell and Cas9 protein accumulates in unfertilized egg the MII stage of meiosis,
wherein the gRNA is introduced into the unfertilized egg cell during oogenesis and prior to MII stage of meiosis and the gRNA complexes with the accumulated Cas9 protein in the nucleus of the unfertilized cell prior to MII stage of meiosis to form a gRNA/Cas9 complex, and
wherein the Cas9 of the gRNA/Cas9 complex cleaves DNA at the target sequence in the genome of the unfertilized egg prior to MII stage of meiosis, thereby modifying at least one target gene in the unfertilized egg
, does not reasonably provide enablement for the following:
1) a unfertilized egg isolated from genetically modified animal of any species that does not comprising the exogenous Cas9 genes in its own genome; and
2) an unfertilized egg that expresses the exogenous Cas9 genes during any type point in the life of the unfertilized egg cell other than during oogenesis and before MII meiosis;
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.
While determining whether a specification is enabling, one considers whether the claimed invention provides sufficient guidance to make and use the claimed invention, if not, whether an artisan would require undue experimentation to make and use the claimed invention and whether working examples have been provided. When determining whether a specification meets the enablement requirements, some of the factors that need to be analyzed are: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, and whether the quantity of any necessary experimentation to make and use the invention based on the content of the disclosure is “undue”.
Nature of Invention: The claims are directed to a method of genetically modifying a target DNA in an unfertilized egg derived from a transgenic organism that has introduced Cas9 genes into its genome, and thus also are introduced in the genome of the unfertilized egg. The method relies upon accumulation of the Cas9 gene in the egg cell and a subsequence introduction if a gRNA specific for a target gene present in the genome of the unfertilized egg. The gRNA is able interact with the Cas9 and target the Cas9 to the target gene in the genome of the unfertilized egg and allow a cleavage of the target gene to occur. The unfertilized egg may or may not have the Cas9 genes upon cell division and thus potentially can arrive at a egg or zygote that has the genetic modification of the target gene but not the exogenous Cas9 gene.
This scope of enablement rejection is being made because the claims as written lack key structural/functional element for the method to function.
Breadth of the claims: The claims recite the use of a genetically modified organism and unfertilized eggs therefrom. The breadth of “non-human organism” which encompasses any and all living organisms, animal and non-animal in nature with the exception of humans. The claims recite unfertilized egg cell is isolated from “a genetically modified organism having a nuclear genome into which at least three copies of genes encoding Cas9 are introduced”. The location of the at least three copies of genes encoding Cas9 are not designated in the claim. As such the breadth of the genomic modification is that it can be anywhere in the genome on any chromosomes (same or different). The genomic modification can be homozygous or hemizygous. Because the claim does not specify that the isolated unfertilized egg comprising the genetic modification. The breadth of the claims encompass isolating a fertilized egg from a genetically modified organism that is heterozygous for the genetic modification. This embodiment means that the breadth of the unfertilized egg is one that has the genetic modification or does not and resembles a wildtype unfertilized egg. The claim does not specify when the Cas9 is to be expressed in the unfertilized egg nor does not is specify when the Cas9 must accumulate. As such, expression of the Cas9 and its accumulation can occur at anytime period. The claims do not specify a particular time point at which the gRNA is introduced into the unfertilized egg. As such it can be introduces before, during, or after accumulation, before during or after cell division starts to occur. The claim do does not specify that the gRNA comprising a target sequence to a target gene present in the genome of the unfertilized egg. As such, the gRNA can comprise any target sequence. As such, the claims are broadly written.
Specification Guidance: The specification provides the following guidance (citations for the pre-grant publication):
[0090] In the present specification, examples of the “non-human organism” include, but are not limited to, E. coli, Bacillus subtilis, yeast, insect, plant (such as monocotyledon or dicotyledon), bird animals such as chickens, mammals such as mice, rats, hamsters, guinea pigs, rabbits, dogs, cats, horses, cows, sheep, pigs, goats, marmosets, and monkeys. Among these, mammals are preferable, and rodents such as mice, rats, guinea pigs, hamsters, and rabbits are preferable.
[0121] <Egg Cell>
[0122] In one embodiment, the present invention provides an egg cell derived from the genetic modification non-human organism having a nuclear genome into which at least 3 copies of the genes that code for Cas9 are introduced.
[0123] In the egg cell of the present embodiment, a plurality of different genes or a plurality of locations in the same gene can be edited at the same time with high efficiency. In addition, according to a fertilized egg obtained by fertilizing the egg cell of the present embodiment and sperm derived from the same species of the organism, it is possible to rapidly produce the genetic modification non-human organism at a low cost.
[0124] The egg cell of the present embodiment is derived from a female of the genetic modification non-human organism described above.
[0125] In the present specification, the term “egg cell” means a female reproductive cell, including an oocyte and an unfertilized egg.
[0126] As the egg cell derived from the genetic modification non-human organism according to the present embodiment, in a case where the genetic modification non-human organism is a heterozygote having the Cas9 gene on one side of a homologous chromosome in the nuclear genome, there is an egg cell having, within the nucleus, the nuclear genome into which at least 3 copies of the Cas9 genes are introduced, and there is an egg cell not having the nuclear genome into which the Cas9 genes are introduced. Any of the egg cells can be utilized to modify a target gene. This is because protein or mRNA of maternally derived Cas9 (hereinafter will be referred to as maCas9) accumulates in the egg cell during the oogenesis and gradually decomposes after functioning to the 2-cell stage from the fertilized egg. Therefore, in a case where it is desired to produce the genetic modification non-human organism that does not have a foreign gene and in which a target gene is modified, it is preferable to use the egg cell not having, within the nucleus, the nuclear genome into which the Cas9 gene is introduced.
[0127] It is sufficient that the genetic modification non-human organism from which the egg cell is derived in the present embodiment have the nuclear genome into which at least 3 copies of the Cas9 genes are introduced, and protein or mRNA of maCas9 can accumulate in an oocyte during the oogenesis. In addition, the larger the copy number of the introduced Cas9 gene becomes, the more the accumulated protein or mRNA of maCas9 increases, which is preferable. The copy number of the introduced Cas9 gene may be 3, 4, 5, 6, 7, 8, 9, 10, 20, 50 or more, for example.
[0147] The method of the present embodiment will be described in detail below.
[0148] [Introduction Step]
[0149] First, guide RNAs are introduced into cells derived from the genetic modification non-human organism, the egg cells described above, or the fertilized eggs described above. As a method for introducing the guide RNAs, known gene introduction methods (for example, calcium phosphate method, electroporation method, lipofection method, coagulation method, microinjection method, particle gun method, DEAE-dextran method, and the like) can be used. Among these, the electroporation method is preferable because the operation is simple. In the genetic modification method utilizing the CRISPR system of the related art, the member of Cas9 mRNA was large and an introduction efficiency was poor. However, in the method of the present embodiment, the cells derived from genetic modification non-human organism in which Cas9 can be expressed, the egg cells, or fertilized eggs are used, and therefore it is possible to easily and efficiently introduce the guide RNAs by the electroporation method and the like.
[0150] In a case where an introduction target is a cell derived from the genetic modification non-human organism, the guide RNA can be introduced into a living genetic modification non-human organism. In this case, the introduction method may be a method known to those skilled in the art, such as intra-arterial injection, intravenous injection, subcutaneous injection, intranasal introduction, transbronchial introduction, intramuscular introduction, percutaneous introduction, or oral introduction, and the intravenous injection is preferable.
[0151] In addition, in a case where a tissue-specific promoter is operably linked to the upstream (5′ side) of the Cas9 gene, by the guide RNA being directly introduced into the entire body of the genetic modification non-human organism or target tissues, the genetic modification is performed only in the target tissues by the guide RNA and Cas9, and therefore the dynamics of diseases can be observed.
[0152] In a case where the introduction target is a fertilized egg fertilized by the above egg cell, or a fertilized egg, it is preferable to use a fertilized egg of 4 hours to 24 hours, preferably 6 hours to 12 hours, and more preferably 6 hours to 8 hours after fertilization, in which male pronucleus is developed. With the time after fertilization being within the above range, an expression level of protein or mRNA of maCas9 becomes high, and an embryo of a so-called mosaic-type in which both the cell in which the target gene is modified and the cell in which the target gene is not modified are present, is prevented from being generated. Therefore, it is possible to modify the target gene with high efficiency.
[0153] In addition, as described in the above section, “Fertilized Eggs”, in a case where it is desired to produce the genetic modification non-human organism which does not have, within in the nucleus, the nuclear genome into which the Cas9 gene is introduced, and in which the target gene is modified, a fertilized egg to be used is preferably a fertilized egg obtained by fertilizing an egg cell not having, within the nucleus, the nuclear genome into which the Cas9 gene is introduced, and sperm derived from an organism not having, within the nucleus, the nuclear genome into which the Cas9 gene is introduced, or a wild-type organism. In the egg cell not having, within the nucleus, the nuclear genome into which the Cas9 gene is introduced, protein or mRNA of maCas9 is accumulated. Therefore, in a case where the fertilized egg to be used is a fertilized egg obtained by fertilizing an egg cell not having, within the nucleus, the nuclear genome into which the Cas9 gene is introduced, and sperm derived from an organism not having, within the nucleus, the nuclear genome into which the Cas9 gene is introduced, or a wild-type organism, protein or mRNA of maCas9 is transiently present excessively in a short period of the 2-cell stage from the fertilized egg. Accordingly, as shown in the examples described later, mosaic mutations become significantly lower than those of the genomic editing method of the related art in which synthetic Cas9 mRNA and guide RNA are microinjected into the fertilized egg.
[0154] In a case of introducing the guide RNA into the fertilized egg, a concentration of the guide RNA is not particularly limited, but as the concentration becomes higher, genetic modification can be performed with higher probability. A concentration of the guide RNA may be, for example, 1 ng/μL to 300 ng/μL.
[0155] In addition, in a case where the fertilized egg is derived from the genetic modification non-human animal (particularly, a non-human mammal), after introduction of the guide RNA, the fertilized egg is implanted to the uterus or the oviduct of the corresponding non-human animal so as to be generated, and therefore it is possible to easily obtain the genetic modification non-human animal.
[0156] In a case where two or more locations of one target gene are modified, or in a case where two or more of genes are used as target genes, two or more types of the guide RNAs containing, in the 5′ end region, a polynucleotide including a base sequence complementary to the base sequence of each gene, may be designed so as to be introduced. Accordingly, it is possible to edit a plurality of different genes or a plurality of locations in the same gene at the same time with high efficiency.
[0157] In the method of the present embodiment, a foreign gene may be introduced together with the guide RNA. By introducing a foreign gene together, it is possible to obtain the genetic modification non-human organism having the nuclear genome in which after cleavage of a target gene, the foreign gene is inserted into the target gene by homologous recombination.
[0158] In addition, it is preferable that the foreign gene not have a PAM sequence. In the foreign gene, for example, in a case where the PAM sequence is changed to a non-PAM sequence by one base substitution not changing the amino acid sequence, and the like, it is possible to insert the foreign gene on the nuclear genome by homologous recombination and it is also possible to prevent the foreign gene from being cleaved again by the endogenous Cas9.
[0159] It is preferable that the foreign gene be introduced using a vector containing the foreign gene. In the vector containing the foreign gene, it is preferable that a DNA having a sequence homologous to a site into which a target gene is insert be linked to the 5′ end and the 3′ end of the foreign gene. The number of bases of the DNA having a sequence homologous to a site into which a target gene is inserted is preferably 100 bases to 300 bases. In the vector containing the foreign gene, the promoter, the polyadenylation signal, the NLS, the fluorescent protein marker gene, which are described in the above section, “Genetic Modification Non-Human Organism”, and the like may be operably linked to the 5′ end or the 3′ end of the foreign gene.
[0160] In a case of use in the present specification, the “vector” is a tool that enables or facilitates the transfer of entities from one environment to another environment. For example, some vectors used in recombinant DNA technology enable entities such as segments of DNA (for example, heterologous DNA segments such as heterologous cDNA segments) to be transferred into non-human organism cells. In the present embodiment, the vector includes viral vectors (for example, lentiviral vectors, baculovirus vectors, adenovirus/adeno-associated viral vectors, and the like), bacterial vectors, protozoal vectors, DNA vectors, or recombinant vectors which may contain recombination thereof.
[0161] [Cleavage Step]
[0162] Cas9 expressed in the cells derived from the genetic modification non-human organism, the egg cell derived from the genetic modification non-human organism, or the fertilized egg derived from the genetic modification non-human organism, and the guide RNA are combined in mild conditions in vitro or in vivo so as to form a Cas 9-guide RNA complex. The mild conditions indicate a temperature and a pH at which a protein does not decompose or denature, and the temperature may be 4° C. to and 40° C. and the pH may be 4 to 10. Accordingly, the Cas9-RNA complex can be formed even in the body of the living genetic modification non-human organism.
[0163] In the Cas9-RNA complex, some of the guide RNA binds to the target gene and Cas9 recognizes the PAM sequence in the target gene. Subsequently, the target gene is cleaved at a cleavage site located 3 bases upstream so as to produce blunt ends. More specifically, Cas9 recognizes the PAM sequence, a double helix structure of the target gene is peeled from the PAM sequence as a starting point, followed by annealing with the base sequence complementary to the target gene in the guide RNA, and therefore the double helix structure of the target gene is partially loosened. At this time, Cas9 cleaves the phosphodiester bond of the target gene at the cleavage site located 3 bases upstream of the PAM sequence so as to create blunt ends.
[0164] [Modification Step]
[0165] Subsequently, in a region determined by complementary binding between the guide RNA and the target gene, it is possible to obtain a target gene subjected to the modification according to the purpose.
[0166] In the present specification, the term “modification” means that the base sequence of the target gene is changed. Examples of thereof include a change in the base sequence of a target double-stranded polynucleotide by cleavage of a target gene and insertion of an exogenous sequence after the cleavage (physical insertion or insertion by replication via homology directed repair), a change in the base sequence of a target gene by non-homologous end joining (NHEJ: DNA ends generated by cleavage are bonded again) after the cleavage, and the like. By the modification of the target gene in the present embodiment, introduction of mutation into the target gene or destruction of a function of the target gene is possible. Therefore, in the present embodiment, in a case of using the fertilized egg, a genetic modification non-human organism in which a function of the target gene is destroyed (knockout) or substituted (knockin) can be easily produced.
[0167] <Application and Utilization Method>
[0168] In one embodiment, the present invention provides a composition and a method for performing genetic modification. In contrast to previously known methods for targeted genetic recombination, the present invention enables efficient and inexpensive implementation and thus is adaptable to any cell or organism. Any segment of the target gene of a cell or an organism can be modified by the method of the present invention. This method utilizes both a homologous recombination process in which all cells are endogenous and a non-homologous recombination process.
[0169] In addition, in one embodiment, the present invention provides a method for performing targeted DNA insertion or targeted DNA deletion. This method includes a step of transforming a cell using a nucleic acid construct having a donor DNA. The scheme concerning the DNA insertion or the DNA deletion after cleavage of a target gene can be determined by those skilled in the art according to known methods.
[0170] In addition, in one embodiment, the present invention is utilized in both somatic cells and reproductive cells and provides genetic manipulation at a specific locus.
[0171] In addition, in one embodiment, the present invention provides a method for breaking genes in somatic cells. The gene overexpresses products harmful to cells or organisms and expresses products harmful to cells or organisms. Such a gene can be overexpressed in one or more cell types that arise in diseases. Disruption of the overexpressed gene according to the method of the present invention may lead an individual suffering from a disease caused by the overexpressed gene to be healthier.
[0172] That is, disruption of only a small proportion of the genes of the cells works and an expression level thereof is reduced, obtaining a therapeutic effect.
[0173] In addition, in one embodiment, the present invention provides a method for breaking a gene in reproductive cells. A cell in which a specific gene is disrupted can be selected so as to produce an organism not having a function of the specific gene. In the cell in which the gene is disrupted, the gene can be completely knocked out. A deficiency in a function of this specific cell may have a therapeutic effect.
[0174] In addition, in one embodiment, the present invention further provides insertion of a donor DNA that codes for a gene product. This gene product has a therapeutic effect in a case of constitutive expression.
[0175] For example, in a population of pancreatic cells, in order to cause the insertion of a donor DNA that codes for an active promoter and an insulin gene, there is a method in which the donor DNA is inserted into an individual suffering from diabetes. The population of pancreatic cells containing exogenous DNA can then produce insulin and thus can treat the individual suffering from diabetes.
[0176] In addition, the donor DNA can be inserted into crops and can cause the production of drug-related gene products. Genes of protein products (such as insulin, lipase, and hemoglobin) can be inserted into plants together with regulatory elements (constitutively active promoter or inducible promoter) so as to produce large quantities of drug medicine in plants. Such protein products can then be isolated from plants. Genetic modification plants or genetic modification organisms can be produced by a method using a nucleic acid transfer technique (McCreath, K. J., et al. (2000) Nature 405: 1066-1069; Polejaeva, I. A., et al., (2000) Nature 407: 86-90). Tissue-specific vectors or cell-specific vectors can be utilized to provide gene expression only in selected cells.
[0177] In addition, in one embodiment of the present invention, it is possible to select cells that produce cells which are utilized in reproductive cells and in which insertion is performed in a designed manner and all subsequent cell divisions have designed genetic modification.
[0178] In addition, in one embodiment, the present invention can be applied to all organisms, cultured cells, cultured tissues, and cultured nuclei (including cells, tissues, or nuclei that can be used to regenerate intact organisms), and gametes (for example, eggs or sperm at various stages of their development).
[0179] In addition, in one embodiment, the present invention can be applied to cells derived from any organism (including, but are not limited to, insects, fungi, rodents, cows, sheep, goats, chickens, and other agronomically important animals, and other mammals (including, but are not limited to, dogs, cats, and humans).
[0180] In addition, in one embodiment, the present invention can be applied in the production of animal disease models (for example pathological model mice).
[0181] In addition, in one embodiment, the present invention can be used in plants. The plants are not particularly limited, and the present invention can be used in any of various plant species (such as monocotyledon or dicotyledon).
EXAMPLES:
[Test Example 2] Ramp2 Genetic Modification Test
[0218] In the fertilized eggs derived from the Cas9 mouse, protein and mRNA of Cas9 (both or any one thereof) (hereinafter will be referred to as maCas9) were accumulated in the cell as a maternal factor, and it was considered that the Cas9 transgene is not required for genomic editing, and therefore the following test was carried out.
[0219] (1) Introduction of Guide RNA
[0220] Only R2-gRNA (SEQ ID NO: 27) for Ramp2 gene modification was microinjected into the pronucleus of the fertilized eggs obtained by artificial fertilization of various crossing pairs. A genotype in the fertilized eggs produced by crossing is shown in FIG. 4A. In addition, a base sequence homologous to the Ramp2 gene in R2-gRNA is shown in FIG. 4C. In FIG. 4A, subsequently, the injected fertilized eggs were cultured to blastocysts in potassium-supplements simplex-optimized medium (KSOM).
221] (2) Determination of Cas9 Genotype
[0222] In regard to one embryo of the blastocysts, Cas9 genotype was determined using the same method as in (3) of Example 1. The results are shown in FIG. 4B.
[0223] (3) T7 endonuclease I (T7EI) Assay
[0224] PCR was carried out from a nuclear extract of one embryo of the blastocysts using a Ramp 2-5S primer and a Ramp 2-6A, and therefore a PCR product was obtained. The PCR product was added to T7 endonuclease I (manufactured by New England BioLabs Japan), and therefore a DNA fragment was obtained. Subsequently, a fragment cleaved by agarose electrophoresis was confirmed. The results are shown in FIG. 4D.
[0225] Based on FIGS. 4B and 4D, in a case where artificial fertilization was carried out between the Tg/+ mice, or between a Tg/+ female mouse and a +/+ male mouse, it was found that an indel mutation (a genetic mutation due to base insertion or deletion) occurred at a high rate of 92% to 94%. Accordingly, it became clear that genomic editing is possible at a high rate by Cas9 in the Tg fertilized egg.
[0226] On the other hand, in a case where artificial fertilization was carried out between a +/+ female mouse and a Tg/+ male mouse, an indel mutation was 23%, which is a low rate. Furthermore, the inventors of the present invention have investigated a relationship between a genotype of a blastocyst and a genomic editing ability, and therefore have found that an indel mutation occurs at a high rate even in a blastocyst in which the Cas9 transgene is not present.
[0227] In addition, based on FIG. 4B, in a case where in vitro fertilization was cared out between Tg/+ mice or between a Tg/+ female mouse and a +/+ male mouse, in all +/+ blastocysts, a Ramp 2 locus (5+7/5+7) had a mutation. It is known that genes on the genome are not yet expressed in cells of a fertilized egg stage. Transcripts of genes on the genome are gradually made until the 2-cell stage.
[0228] Therefore, based on this result, it is considered that protein and mRNA of maCas9 (both or any one thereof) are responsible for genomic editing in the fertilized egg.
[0229] In addition, as shown in FIG. 4B, it was possible to confirm this phenomenon in another line, FCas9-13.
As such, the breadth of the guidance from the specification is much narrower than the claims. The specification specifically teaches the mechanism of the invention relies upon the egg or egg cell comprising the transgene that comprises multiple copies on the Cas9 gene in a heterozygous configuration so as to have the Cas9 on one set of chromosomes. The method also requires that the Cas9 accumulate in the egg cell prior to any cell division and that the gRNA be introduced when the Cas9 has accumulated and cell division has not occur. While the specification contemplates any non-human organism, specification only provides specific guidance to mice.
State of the Art/Scientific Rationale: Regarding the breadth of “non-human genetically modified organism”, the vast majority of organisms, all bacterial and microbes, most plants, invertebrates, and even some animals reproduce asexually, i.e. without the use of an egg. In contrast, about 99% of all animal reproduce sexually (Knight printout from https://geneticliteracyproject.org/2018/09/21/how-did-sex-start. Page 1-9; See pp2-3). Given that the vast majority of organism due not have eggs, the vast majority of the species of the claimed method would fail to be genetically modified as claimed. As such, the breadth of the claims to non-human organism is not enabled because the vast majority of the species of genus organism would fail in the method as claimed.
Regarding the breadth of a “unfertilized egg from a genetically modified non-human organism having…at least 3 copies of gene that code for Cas9”, wherein the non-human organism is heterozygous for the copies of the gene, Greaney et al (Human Reproduction Update 24(4):135-161, 2018) teaches that during meiosis haploid gametes are formed by two sequences rounds of chromosomal segregation (p. 138). Thus the unfertilized egg is haploid and during the process of oogenesis will loose half their chromosome to the production of two gametes. If the genetically modified non-human organisms of the claims where heterozygous or hemizygous Cas9 genes, the breadth of the claimed unfertilized egg cell encompasses one that comprises the Cas9 genes and one that does not have the Cas9 genes. Thus the breadth of the claimed unfertilized egg encompasses a wild-type egg which will fail to function in the claimed method because the unfertilized egg is required to express the Cas9 genes and for Cas9 to accumulate in the unfertilized egg to cause the genetic modification by Cas9. As such, the art suggests that the unfertilized egg requires at least Cas9 genes in its genome. As such defining the genetic modification of the unfertilized egg by referring to the genetically modified organism from which is came will not predictably arrive at a modification of the target gene as the method requires.
Regarding the time of the expression of the three Cas9 genes introduced into the genome, as discussed above, the timing of the expression and accumulation of Cas9 in the unfertilized eggs and introduction of the gRNA is critical in the instantly claimed method. As discussed above, Greaney teaches that two rounds of chromosomal segregation occur to arrive at the unfertilized egg. For the genetic modification of the unfertilized egg to predictably and consistently occur the Cas9 genes must be expressed between during the time between the two meiotic chromosomal segregations because if it is express MI cell one of the oogonia will possibly express the Cas9 and therefore will not predictably arrive at Cas9 accumulation as need for the genetic modification to occur. As such the method requires expression driven by a promoter specific to oogenesis that allows for expression and accumulation of Cas9 in the unfertilized egg and the breadth of Cas9 expression at any type point driven by any promoter is neither predictable, supported by the specification, or enabled by the art.
As such, the breadth of the claims lack enablement While the specification encompasses isolating a unfertilized egg cell from any non-human organism comprising or lacking the Cas9 genes in its genome, expressing the Cas9 gene at any time point to accumulate Cas9 and introducing a gRNA having into the unfertilized egg to modify a target gene, the specification provide much narrower predictable guidance to isolating a fertilized egg cell from a non-human animal, said egg cell comprising the at least 3 Cas9 transgene in its genome and the transgene being operably linked to a promoter that drives expression during oogenesis prior to MII and introducing a gRNA into the unfertilized egg. The art and scientific logic demonstrates that vast number of the embodiments encompassed by the breadth of the claims would fail to predictable modify a target gene as claimed.
Therefore at the time of filing the skilled artisan would need to perform an undue amount of experimentation without a predictable degree of success to implement the invention as claimed.
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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
(1) Claim(s) 1-6, 11, 13, and 15, as amended, previously presented, or newly added, is/are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Chapman (US 2019/0330660 A1 effectively filed:3/4/2016).
Regarding claims 1-3, Chapman discloses he method comprises culturing a cat oocyte, introducing into the cat oocyte a pair of guide RNAs and the Cas9 protein, wherein the guide RNAs are designed to be encoded by a Fel d 1 genomic sequence of a 5′ or 3′ flanking region([0054]). These disclosures encompass the limitations of introducing a gRNA encoding a target sequence in the genome into an unfertilized egg that comprises Cas9. Chapman discloses additional embodiments include transgenic or knock-out cats produced according to any method described herein ([0055]). These disclosures encompass modifying at least one gene because the method requires the knockout to happen in the oocyte genome to arrive at the knockout cat.
Regarding claims 2-3, Chapman discloses the limitations of PAM ([0123]; [0124]; Table 6 and 7; [0142])
Regarding claim 4, as discussed above, a pair of guide RNAs were introduced, meeting the limitations of two or more guide RNAs as claimed.
Regarding claim 5-6, Chapman discloses that the vectors for guide RNA comprise addition foreign gene sequence that is not a PAM ([0068] to [0074]; [0144]; [0156]).
Regarding claims 11, 13, and 15, Chapman does not disclose that plurality of unfertilized egg cells does not have a nuclear genome comprising the at least 3 copies of Cas9. As such, the negative limitations of claims 11, 13, and 15 are met.
In conclusion, the prior art of Chapman anticipates the claims because it discloses all of the limitations of the claims.
(2) Claim(s) 1-6, 11, 13, and 15, as amended, previously presented, or newly added is/are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Li (US2018/0251728 effectively filed 7/2/2015.
Regarding claims 1-3, Li discloses the nuclei of the haploid cells carrying sgRNA were injected into mature oocytes by ICAHCI, followed by intracytoplasmic injection of Cas9 mRNA into the reconstructed oocyte (where this protocol was known as Lenti-sgRNA+Cas9 injection). See [0343]. These disclosures encompass the limitations of introducing a gRNA encoding a target sequence in the genome into an unfertilized egg that comprises Cas9. Li discloses In this experiment, haploid cells carrying sgRNA were injected into mature oocytes by ICAHCI and then Cas9 was injected into the reconstructed oocytes (where this protocol was known as Lenti-sgRNA+Cas9 injection). A total of 45 SC mice carrying one sgRNA were born, of which 22 had genetic mutations (Table 2). Sequencing of the PCR product of the gene of interest revealed that 10 mice had only a monoallelic modification, and other 12 mice were biallelic mutant (which is 23.5% of all born SC mice). Sequencing by TA cloning of the 7 biallelic mutant SC mice revealed that approximately 63% of the clones carried the insertion or deletion mutations (Table S4). See [0347]. As such, at least one target gene was knockout.
Regarding claims 4, Li discloses the use of multiple guide RNAs. See Example 2 [0246]-[0250]; [0292]-[0301].
Regarding claims 5-6, Li discloses introducing a GFP report gene [0195].
Regarding claims 11, 13, and 15, Li does not disclose that plurality of unfertilized egg cells does not have a nuclear genome comprising the at least 3 copies of Cas9. As such, the negative limitations of claims 11, 13, and 15 are met.
In conclusion, the prior art of Li anticipates the claims because it discloses all of the limitations of the claims.
(3) Claim(s) 1-3, 5-6, 11, 13, and 15, as amended, previously presented, or newly added, is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Suzuki (Suzuki et al. Scientific Reports (2014) 4:7621 DOI:10.1038/srep07621.pp.1-7.)
Regarding claims 1-3, Suzuki discloses Our previous work has shown that cRNA injected into mII oocytes takes 3~4 h before it is discernibly expressed21,22. We therefore reasoned that loading mII oocytes with Cas9 cRNA prior to sperm/ gRNA injection might enhance editing by lengthening the time for Cas9 expression. To investigate this, we sequentially injected oocytes first with Cas9 cRNA and after 3,4 h, eGFP gRNA plus sperm from a 129-eGFP hemizygote (Fig. 1A). As in the 1-step method, the Cas9 system efficiently eliminated green fluorescence compared to controls (Fig. 1B–D). These results were confirmed by analogous experiments with a Nanog-eGFP knock-in line (Fig. 1E and Supplementary Fig. S1A). This shows that genome targeting by injecting mII oocytes with sperm, Cas9 cRNA and gRNA can be achieved high efficiencies and is not locus-specific. To evaluate whether the sequential injection enhanced editing, we performed a comparative titration of gRNA (1-step vs sequential methods), holding the concentration of injected Cas9 cRNA constant and non-limiting at 100 ng/ml (Fig. 1B–D). Editing in the 1-step method became inefficient when ,1 ng/ml eGFP gRNA was injected (Fig. 1C). However, a similar editing efficiency in the sequential method was obtained with 0.01 ng/ml eGFP gRNA, corresponding to ,7 3 108 gRNA molecules per injection (Fig. 1C). Therefore, in these experiments, the sequential method of Cas9-mediated paternal genomic editing was ,100-fold more sensitive than the 1-step method (See page 2 and figure 1).
Regarding claims 5-6, Suzuki discloses that the eggs comprising cRNA encoding Cas9 are injected with gRNA and sperm heads as discussed above. Sperm head comprise DNA comprising foreign genes that do not have PAM sequences.
Regarding claims 11, 13, and 15, Suzuki does not disclose that plurality of unfertilized egg cells does not have a nuclear genome comprising the at least 3 copies of Cas9. As such, the negative limitations of claims 11, 13, and 15 are met.
Response to Arguments
Applicant's arguments filed 12/2/2025 have been fully considered but they are not persuasive.
In response to Applicant’s argument is the after-final rejection, the reader is referred to the after-final advisory action. These argument also suggest that the amendment that specify isolating the unfertilized egg from the genetically modified non-human organism means that the unfertilized egg requires the Cas9 transgene in its genome. In response, Applicant is not giving them their broadest reasonable interpretation. One embodiment is where the unfertilized egg is isolated from a non-human organism that is hemizygous for the introduced Cas9 transgenes. Because the non-human organism is hemizygous, she has oocytes that have the transgene and oocytes that do not because of meiosis to form haploid unfertilized egg cells. Since the 102 rejection are made upon this interpretation, the rejection are still applicable. One possible means is to recite the Cas9 transgene are present in the genome of the unfertilized eggs. See suggestion in the scope of enablement rejection above.
No claims are allowed.
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MARCIA S. NOBLE
Primary Examiner
Art Unit 1632
/MARCIA S NOBLE/Primary Examiner, Art Unit 1632