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 .
Claims 1-20 have been canceled. Claims 21-36 have been added.
Election/Restrictions
Applicant’s election without traverse of Group I, claims 21-27, 34-36, in the reply filed on 6-9-26 is acknowledged.
Upon reconsideration, Groups I-III have been recombined.
Claims 21-36 are under consideration.
Claim Objections
The term gRNA in claim 21 should be spelled out before being abbreviated.
The phrase “in a manner to result in impaired or absent Fel d1 protein expression” in claim 21 is an intended use and does not necessarily have to occur.
Claims 21-25 should clearly refer to ---the nucleic acid sequence of SEQ ID NO: 1244-2468 [or 1-1225]---
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.
Enablement
Claims 21-36 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 inactivating a Fel d1 gene in an isolated Felis catus somatic cell using a homology vector (Avner US Patent 10626417), does not reasonably provide enablement for
i) any “edited” Fel d1 gene made using one or more gRNA to inactivate a Fel d1 gene (claims 21, 34) or a cat cell “lacking at least a portion of the Fel d1 gene” (claim 25), any “CRISPR system” (claims 34), or any Felis catus cell lacking any sequence 60% identical to SEQ ID NO: 1-1225 (claim 25) other than an isolated Felis catus cell whose genome comprises an inactivated Fel d1 gene obtained using Cas9 and a gRNA pair that is a) SEQ ID NO: 1234, 1235, 1238 or 1239; and b) SEQ ID NO: 1236, 1237, 1240, or 1241.
ii) gRNAs that are 60%, 70%, or 90% identical to SEQ ID NO: 1244-2468 other than a gRNA pair that is a) SEQ ID NO: 1234, 1235, 1238 or 1239; and b) SEQ ID NO: 1236, 1237, 1240, or 1241 (claims 21-23);
iii) any Felis catus cell line (claim 25) other than a somatic cell line;
iv) any genetically modified cat comprising the Felis catus cell line (claim 28);
v) genetically modifying any Felis catus cell in vivo as encompassed by claim 34;
vi) any “conserved region identified from genomic sequencing data” “from feline tissue samples” (claims 35, 36).
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/or use the invention commensurate in scope with these claims.
Claim 21 is drawn to a method of editing a Fel d 1 gene in an isolated Felis catus cell, the method comprising: introducing i) one or more gRNAs and ii) Cas9 into an isolated Felis catus cell and editing an endogenous Fel d 1 gene of the Felis catus cell in a manner to result in impaired or absent Fel d 1 protein expression; wherein the gRNA are at least 60% identical to gRNA of SEQ ID NOS: 1244 to 2468.
Claim 34 is drawn to a method of deleting a genomic sequence from the genome of a Felis catus cell, comprising: introducing guide RNA into a Felis catus cell and using a CRISPR system directing a double stranded break in the genome of the Felis catus cell, wherein at least a portion of a Fel d 1 gene is removed resulting in impaired or an absence of Fel d 1 protein expression.
Claims 21 and 34 encompass performing the method using Cas9 and one or more gRNAs in the absence of a homology template that causes inactivation of the Fel d1 gene. This concept relies on non-homologous end joining (NHEJ) to cause various random deletions and insertions with the hopes of one of them inactivating the Fel d1 gene.
Claims 21, 34 encompass performing the method using Cas9, one or more gRNAs, and a homology template that complements the target sequences of the gRNAs. This concept relies on homologous recombination wherein the template is incorporated into the Fel d1 gene and causes inactivation of the Fel d1 gene.
Claims 21, 34 also encompass using gRNA that targeting the Fel d1 gene anywhere, i.e. the 5’ end or 3’ end, in a coding region upstream or downstream of a coding region essential for function.
Claims 25-33 encompass any naturally occurring or genetically modified cat cell or cat obtained by any means.
Fig. 1 and 2 show the structure of chains 1 and 2 of the felis catus Fel d1 gene.
Fig. 3 shows a Fel d1 gene map with possible crRNA target sequences 1A, 1B, 2A, 2B, 3A, 3B, 4A and 4B. See pg 26, para 127.
Fig. 4A describes the design strategy for crRNA and target sequences. In particular, applicants suggest “aim[ing] for targeting the more conserved portion of genome sequences” (echoed on pg 25, para 124). One pair of crRNAs (#1/#2) targets flanking regions of Fel d1 (one upstream of chain 2, one downstream of chain 1) (SEQ ID NOs: 680 and 254). The other pair (#3/#4) targets internal regions of Fel d1 (one internal to chain 2, one internal to chain 1) (SEQ ID NOs: 851 and 266). The crRNAs can be mixed “but not 1/3 and 2/4 probably because the distances between those only cover ~one exon”, i.e. #1/#4 and #2/#3. Table 3 (pg 27-28) shows the combinations of crRNA target sites:
PNG
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511
346
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Greyscale
If Cas9 “nickase” is used, then 4 crRNAs are used (Fig. 4B; pg 28, para 131) as shown in Table 4 (pg 28).
Examples are not numbered in the specification.
Examples of cell lines (pg 22) are limited to felis catus cell lines.
Examples for design of guide sequences (pg 24-30) are discussed above.
Examples for “In silico identification of guide RNA target sequences” (pg 30-34) is based on the Broad institute website and full length Fel d1 chain 1 (SEQ ID NO: 1226) and chain 2 (SEQ ID NO: 1227).
Examples of oligo synthesis are discussed on pg 34.
Pg 34-38, Fig. 5-8, discusses how to clone sequences into vectors.
Examples for testing gRNA are discussed on pg 38-40.
Pg 40-41 suggests creating transgenic cats and merely refers to “general procedures for gene targeting and animal cloning” (Denning 2003; Wang 2003).
Pg 41-43 suggests making a knockout cat by transfecting a donor cat cell with vectors encoding Cas9 and a pair of gRNAs followed by nuclear transfer based on Shin (2002) is described on pg 41-43. This discussion is prophetic.
Pg 41-43 suggests modifying the method of Wongsrikeao (2011) for “cloning a genetically engineered Fel d1 knockout cat” by transfecting cat “oocytes” with lentiviral vectors encoding a pair of gRNAs and Cas9 10-12 hours before or after fertilization. Embryos are then cultured and selected for implantation. Contrary to the title of this section, this is not cloning because the no nuclear transfer occurs. This discussion is also prophetic.
Pg 44-45 suggest “cloning a genetically engineered Fel d1 knockout cat” by ES cell manipulation. However, the art at the time of filing was limited to ES-like cat cells; the art did not teach true cat ES cells, i.e. capable of becoming all three germ layers, forming a teratoma, and germline transmission. The art did not teach cat ES cells capable of being cultured long enough to select transfected cells while still maintaining their ES cell phenotype or the conditions required to do so. This discussion is prophetic.
Pg 45 discusses transfecting cat salivary glands with feline immunodeficiency virus (FIV) encoding a pair of gRNAs and Cas9 using a salivary gland-specific promoter. This example is prophetic.
Pg 46 discusses transfecting cat skin with feline immunodeficiency virus (FIV) encoding a pair of gRNAs and Cas9 using a skin-specific promoter. This example is prophetic.
Pg 46-47 discusses systemically administering a feline immunodeficiency virus (FIV) encoding a pair of gRNAs and Cas9 using a skin-specific promoter. This example is prophetic.
Pg 47 suggests introducing a homology vector encoding GFP into a cat zygote such that the sequence encoding GFP is integrated into the Fel d1 gene and the Fel d1 gene is inactivated. While Avner (10626417) described inactivating a Fel d1 gene in a cat cell in vitro using a homology vector, Avner did not obtain a cat zygote or transgenic cat using the homology vector. The art at the time of filing did not teach using a homology vector that targets the Fel d1 gene to make a cat zygote or a transgenic cat. Accordingly, this suggestion is also prophetic.
Pg 47-49 describes how to confirm hypoallergenicity.
The specification does not enable making/using any “edited” Fel d1 gene made using one or more gRNA to inactivate a Fel d1 gene (claims 21, 34) or a cat cell “lacking at least a portion of the Fel d1 gene” (claim 25), any “CRISPR system” (claims 34), or any Felis catus cell lacking any sequence 60% identical to SEQ ID NO: 1-1225 (claim 25) other than an isolated Felis catus cell whose genome comprises an inactivated Fel d1 gene obtained using Cas9 and a gRNA pair that is a) SEQ ID NO: 1234, 1235, 1238 or 1239; and b) SEQ ID NO: 1236, 1237, 1240, or 1241. Claim 21, 34 encompass making any “edit” or deletion in a Fel d1 gene by targeting one or more locations selected from any of at least SEQ ID NO: 1-1225 (claim 39) and claim 25 encompasses a cat cell “lacking at least a portion of the Fel d1 gene”. The specification does not provide an enabled use for making any deletion of any portion of a Fel d1 gene other than one that inactivates the gene. Furthermore, the specification is limited to making a deletion that causes hypoallergenicity which is limited to deleting any portion of Fel d1 that acts as an allergen and decreases the allergenic effect of Fel d1. The specification does not teach which parts of Fel d1 are/are not allergens. Specifically, applicants do not teach a deletion between 3A/3B and 4A/4B would cause hypoallergenicity because the specification does not teach what remains is hypoallergenic, i.e. exon 4 in chain 1 may still be expressed and act equally as allergenic. At minimum, the specification is limited to deletions at the 1st and 2nd sites shown in Table 3, specifically those that delete the entire Fel d1 gene (crRNA 1A and 2A or 2B, or 1B and 2A or 2B). The specification fails to correlate the sites that remove large portions of the initial coding regions of chain 1 and 2 to any sites as broadly encompassed by claim 34. Without such guidance, it would have required those of skill undue experimentation to determine how to make/use any “edited” Fel d1 gene made using one or more gRNA to inactivate a Fel d1 gene (claims 21, 34) or a cat cell “lacking at least a portion of the Fel d1 gene” (claim 25), any “CRISPR system” (claims 34), or any Felis catus cell lacking any sequence 60% identical to SEQ ID NO: 1-1225 (claim 25) other than an isolated Felis catus cell whose genome comprises an inactivated Fel d1 gene obtained using Cas9 and a gRNA pair that is a) SEQ ID NO: 1234, 1235, 1238 or 1239; and b) SEQ ID NO: 1236, 1237, 1240, or 1241.
The specification does not enable making/using gRNAs that are 60%, 70%, or 90% identical to SEQ ID NO: 1244-2468 other than a gRNA pair that is a) SEQ ID NO: 1234, 1235, 1238 or 1239; and b) SEQ ID NO: 1236, 1237, 1240, or 1241 (claims 21-23). The specification does not teach any gRNAs that share 60%, 70%, or 90% identity with SEQ ID NO: 1244-2468. The specification does not teach any such gRNAs would bind adequately to a Fel d1 gene such that inactivation would occur. The specification does not teach any such gRNAs would cause deletion between the areas of interest that cause hypoallergenicity. Given the lack of guidance in the specification taken with the art at the time of filing, it would have required those of skill undue experimentation to determine how to make/use gRNAs other than a gRNA pair that is a) SEQ ID NO: 1234, 1235, 1238 or 1239; and b) SEQ ID NO: 1236, 1237, 1240, or 1241.
Cell lines in claim 25 encompass those described on pg 22, para 110, which are all from felis catus. Claim 25 encompasses an embryonic stem (ES) cell line – ES cells are contemplated on pg 21, para 108; pg 44, para 197-190; pg 47, para 209 for the production of transgenic cats. However, Gomez (Theriogenology, 2010, Vol. 74, pg 498-515) described felis catus ES-like cells incapable of retaining their ability to self-renew, specifically while carrying a transgene, “in long term culture [which] is essential for producing knockout cells and, consequently knockout cats” (pg 513, col. 2, last 6 lines). Since applicants’ disclosure of cat ES cells requires long term culture of transfected ES cells capable of maintaining their phenotype, the ES-like cells described by Gomez would not suffice. Likewise, Ezashi (Annual Rev. Animal Biosci., 2016, Vol. 4, pg 223-253) summarized the art of obtaining pluripotent cells from domesticated mammals. Attempts to obtain feline ES cells were partly successful, the ES-like cells were only able to differentiate into mesoderm and ectoderm in vitro; they were not able to form teratomas (pg 230, last paragraph). Since the time of filing, Dutton (Stem Cells and Develop., 2019, Vol. 28, No. 19, pg 1299-1309) obtained feline ES-like cells that were partly successful as indicated by markers that indicate the ability to differentiate into mesoderm, endoderm, and ectoderm in vitro. Dutton did not teach the ES-like cells were able to form teratomas, differentiate into all cell types, or provide germline transmission. Accordingly, the specification fails to enable making any cell line as broadly encompassed by claim 25 other than a somatic cell line as described on pg 22.
The specification does not enable a cat comprising the Felis catus cell line lacking a portion of Fel d1 gene resulting in impaired/absent Fel d1 expression as required in claim 28. The state of the art of making cats with inactivated genes was unpredictable. Yin (Biol. Reproduction, 2008, Vol. 78, pg 425-431) transfected a cat fibroblast with a retroviral vector encoding red fluorescence protein (RFP) and used it for nuclear transfer to obtain a cloned cat; Yin did not inactivate or target a cat gene. Gomez (Cloning and Stem Cells, 2009, Vol. 11, No. 1, pg 167-175) transfected a cat fetal fibroblast with a retroviral vector encoding green fluorescence protein (GFP) and used it for nuclear transfer to obtain a cloned cat; Gomez did not inactivate or target a cat gene. Avner (US Patent 10626417) disrupted the Fel D1 gene in a felis catus cell in vitro using a homology vector that inserts a marker gene into exon 2 of the gene (Fig. 5). The homology vector has homology arms that target a specific sequence in exon 2 of the felis catus Fel d1 gene. However, Avner and the rest of the art at the time of filing did not teach making a transgenic cat with an inactivated gene in a feline using a homology vector (e.g. the vector described by Avner), using a nuclease (ZFNs, TALENs, or CRISPR/gRNA) via NHEJ (claim 76), or using a nuclease and donor sequence (ZFNs, TALENs, or CRISPR/gRNA in combination with a donor sequence) via homologous recombination (claim 34). Specifically, Avner (10626417) described inactivating a Fel d1 gene in a cat cell in vitro using a homology vector, but Avner did not obtain a cat zygote or transgenic cat using the homology vector. The art at the time of filing did not teach using a homology vector that targets the Fel d1 gene to make a cat zygote or a transgenic cat. Pg 40-41 suggests creating transgenic cats and merely refers to “general procedures for gene targeting and animal cloning” (Denning 2003; Wang 2003). Pg 41-43 suggests making a knockout cat by transfecting a donor cat cell with vectors encoding Cas9 and a pair of gRNAs followed by nuclear transfer based on Shin (2002) is described on pg 41-43. This discussion is prophetic and does not provide reasonable guidance that a cat with an inactivated Fel d1 gene will occur, specifically that it is hypoallergenic (the sole disclosed use of making a genetically modified cat). Pg 41-43 suggests modifying the method of Wongsrikeao (2011) for “cloning a genetically engineered Fel d1 knockout cat” by transfecting cat “oocytes” with lentiviral vectors encoding a pair of gRNAs and Cas9 10-12 hours before or after fertilization. Embryos are then cultured and selected for implantation. Contrary to the title of this section, this is not cloning because the no nuclear transfer occurs. This discussion is also prophetic and does not provide reasonable guidance that a cat with an inactivated Fel d1 gene will occur, specifically that it is hypoallergenic (the sole disclosed use of making a genetically modified cat). Pg 44-45 suggest “cloning a genetically engineered Fel d1 knockout cat” by ES cell manipulation. However, the art at the time of filing was limited to ES-like cat cells; the art did not teach true cat ES cells, i.e. capable of becoming all three germ layers, forming a teratoma, and germline transmission. The art did not teach cat ES cells capable of being cultured long enough to select transfected cells while still maintaining their ES cell phenotype or the conditions required to do so. This discussion is prophetic. The specification does not teach how to make/use a cat with only one or some cells from the cell line. The specification does not provide adequate guidance that one cell or a group of cells with impaired Fel d1 expression would make the cat hypoallergenic. Pg 45 discusses transfecting cat salivary glands with feline immunodeficiency virus (FIV) encoding a pair of gRNAs and Cas9 using a salivary gland-specific promoter. This example is prophetic and fails to provide adequate guidance that the cells of salivary gland WILL have impaired Fel d1 production or that the cat WILL be hypoallergenic. Pg 46 discusses transfecting cat skin with feline immunodeficiency virus (FIV) encoding a pair of gRNAs and Cas9 using a skin-specific promoter. This example is prophetic and fails to provide adequate guidance that the cells of skin WILL have impaired Fel d1 production or that the cat WILL be hypoallergenic. Pg 46-47 discusses systemically administering a feline immunodeficiency virus (FIV) encoding a pair of gRNAs and Cas9 using a skin-specific promoter. This example is prophetic and fails to provide adequate guidance that the cells of the cat WILL have impaired Fel d1 production or that the cat WILL be hypoallergenic. The art also did not teach a genetically modified cat with an inactivated gene, specifically a gene encoding an allergen. The guidance in the specification regarding making a genetically modified cat with an inactivated Fel d1 gene is inadequate for reasons cited above. Accordingly, it would have required those of skill undue experimentation to determine how to make a cat as required in claim 28, specifically a cat with reduced Fel d1 expression that was hypoallergenic (the sole disclosed use of making a genetically modified cat).
The specification does not enable using any cat cell in vivo as encompassed by claims 25 and 34. Claims 25 and 34 encompass editing a cell in vivo using a CRISPR system; however, the specification and the art at the time of filing are limited to administering Cas9 and gRNA to isolated cells. The specification and the art at the time of filing do not correlate CRISPR technology in vitro to in vivo embodiments. Given the lack of guidance in the specification taken with the art at the time of filing, it would have required those of skill undue experimentation to determine how to do so.
The specification does not enable making/using any “conserved region identified from genomic sequencing data” “from feline tissue samples” (claims 35, 36) other than the gene segment that is removed when a gRNA pair is used that is a) SEQ ID NO: 1234, 1235, 1238 or 1239; and b) SEQ ID NO: 1236, 1237, 1240, or 1241. The specification does not correlate deleting the entire Fel d1 gene to any “portion” that is “removed” and “represents a “conserved region identified from genomic sequencing data”. Given the lack of guidance in the specification taken with the art at the time of filing, it would have required those of skill undue experimentation to determine how to do so.
Written Description
Claims 21-36 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention.
The specification lacks written description for any “edited” Fel d1 gene made using one or more gRNA to inactivate a Fel d1 gene (claims 21, 34) or a cat cell “lacking at least a portion of the Fel d1 gene” (claim 25), any “CRISPR system” (claims 34), or any Felis catus cell lacking any sequence 60% identical to SEQ ID NO: 1-1225 (claim 25) other than an isolated Felis catus cell whose genome comprises an inactivated Fel d1 gene obtained using Cas9 and a gRNA pair that is a) SEQ ID NO: 1234, 1235, 1238 or 1239; and b) SEQ ID NO: 1236, 1237, 1240, or 1241. Claim 21, 34 encompass making any “edit” or deletion in a Fel d1 gene by targeting one or more locations selected from any of at least SEQ ID NO: 1-1225 (claim 39) and claim 25 encompasses a cat cell “lacking at least a portion of the Fel d1 gene”. The specification does not provide an enabled use for making any deletion of any portion of a Fel d1 gene other than one that inactivates the gene. Furthermore, the specification is limited to making a deletion that causes hypoallergenicity which is limited to deleting any portion of Fel d1 that acts as an allergen and decreases the allergenic effect of Fel d1. The specification does not teach which parts of Fel d1 are/are not allergens. Specifically, applicants do not teach a deletion between 3A/3B and 4A/4B would cause hypoallergenicity because the specification does not teach what remains is hypoallergenic, i.e. exon 4 in chain 1 may still be expressed and act equally as allergenic. At minimum, the specification is limited to deletions at the 1st and 2nd sites shown in Table 3, specifically those that delete the entire Fel d1 gene (crRNA 1A and 2A or 2B, or 1B and 2A or 2B). The specification fails to correlate the sites that remove large portions of the initial coding regions of chain 1 and 2 to any sites as broadly encompassed by claim 34. Accordingly, the specification lacks written description for any “edited” Fel d1 gene made using one or more gRNA to inactivate a Fel d1 gene (claims 21, 34) or a cat cell “lacking at least a portion of the Fel d1 gene” (claim 25), any “CRISPR system” (claims 34), or any Felis catus cell lacking any sequence 60% identical to SEQ ID NO: 1-1225 (claim 25) other than an isolated Felis catus cell whose genome comprises an inactivated Fel d1 gene obtained using Cas9 and a gRNA pair that is a) SEQ ID NO: 1234, 1235, 1238 or 1239; and b) SEQ ID NO: 1236, 1237, 1240, or 1241.
The specification lacks written description for any gRNAs that are 60%, 70%, or 90% identical to SEQ ID NO: 1244-2468 other than a gRNA pair that is a) SEQ ID NO: 1234, 1235, 1238 or 1239; and b) SEQ ID NO: 1236, 1237, 1240, or 1241 (claims 21-23). The specification does not teach any gRNAs that share 60%, 70%, or 90% identity with SEQ ID NO: 1244-2468. The specification does not teach any such gRNAs would bind adequately to a Fel d1 gene such that inactivation would occur. The specification does not teach any such gRNAs would cause deletion between the areas of interest that cause hypoallergenicity. Accordingly, the specification lacks written description for any gRNAs other than a gRNA pair that is a) SEQ ID NO: 1234, 1235, 1238 or 1239; and b) SEQ ID NO: 1236, 1237, 1240, or 1241.
The specification lacks written description for any cell line as required in claim 25 other than an isolated Felis catus somatic cell whose genome comprises an inactivated Fel d1 gene made using the gRNA pairs described in the paragraph above. Cell lines in claim 25 encompass those described on pg 22, para 110, which are all from felis catus. Claim 25 encompasses an embryonic stem (ES) cell line – ES cells are contemplated on pg 21, para 108; pg 44, para 197-190; pg 47, para 209 for the production of transgenic cats. However, Gomez (Theriogenology, 2010, Vol. 74, pg 498-515) described felis catus ES-like cells incapable of retaining their ability to self-renew, specifically while carrying a transgene, “in long term culture [which] is essential for producing knockout cells and, consequently knockout cats” (pg 513, col. 2, last 6 lines). Since applicants’ disclosure of cat ES cells requires long term culture of transfected ES cells capable of maintaining their phenotype, the ES-like cells described by Gomez would not suffice. Likewise, Ezashi (Annual Rev. Animal Biosci., 2016, Vol. 4, pg 223-253) summarized the art of obtaining pluripotent cells from domesticated mammals. Attempts to obtain feline ES cells were partly successful, the ES-like cells were only able to differentiate into mesoderm and ectoderm in vitro; they were not able to form teratomas (pg 230, last paragraph). Since the time of filing, Dutton (Stem Cells and Develop., 2019, Vol. 28, No. 19, pg 1299-1309) obtained feline ES-like cells that were partly successful as indicated by markers that indicate the ability to differentiate into mesoderm, endoderm, and ectoderm in vitro. Dutton did not teach the ES-like cells were able to form teratomas, differentiate into all cell types, or provide germline transmission. Accordingly, the specification lacks written description for any cell line as broadly encompassed by claim 25 other than a somatic cell line as described on pg 22.
The specification lacks written description for a cat comprising the Felis catus cell line lacking a portion of Fel d1 gene resulting in impaired/absent Fel d1 expression as required in claim 28. The claim encompasses any cat comprising one or more cell from the cell line or a cat whose genome comprises an inactivated Fel d1 gene. The state of the art of making cats with inactivated genes was unpredictable. Yin (Biol. Reproduction, 2008, Vol. 78, pg 425-431) transfected a cat fibroblast with a retroviral vector encoding red fluorescence protein (RFP) and used it for nuclear transfer to obtain a cloned cat; Yin did not inactivate or target a cat gene. Gomez (Cloning and Stem Cells, 2009, Vol. 11, No. 1, pg 167-175) transfected a cat fetal fibroblast with a retroviral vector encoding green fluorescence protein (GFP) and used it for nuclear transfer to obtain a cloned cat; Gomez did not inactivate or target a cat gene. Avner (US Patent 10626417) disrupted the Fel D1 gene in a felis catus cell in vitro using a homology vector that inserts a marker gene into exon 2 of the gene (Fig. 5). The homology vector has homology arms that target a specific sequence in exon 2 of the felis catus Fel d1 gene. However, Avner and the rest of the art at the time of filing did not teach making a transgenic cat with an inactivated gene in a feline using a homology vector (e.g. the vector described by Avner), using a nuclease (ZFNs, TALENs, or CRISPR/gRNA) via NHEJ (claim 76), or using a nuclease and donor sequence (ZFNs, TALENs, or CRISPR/gRNA in combination with a donor sequence) via homologous recombination (claim 34). Specifically, Avner (10626417) described inactivating a Fel d1 gene in a cat cell in vitro using a homology vector, but Avner did not obtain a cat zygote or transgenic cat using the homology vector. The art at the time of filing did not teach using a homology vector that targets the Fel d1 gene to make a cat zygote or a transgenic cat. Pg 40-41 suggests creating transgenic cats and merely refers to “general procedures for gene targeting and animal cloning” (Denning 2003; Wang 2003). Pg 41-43 suggests making a knockout cat by transfecting a donor cat cell with vectors encoding Cas9 and a pair of gRNAs followed by nuclear transfer based on Shin (2002) is described on pg 41-43. This discussion is prophetic and does not provide reasonable guidance that a cat with an inactivated Fel d1 gene will occur, specifically that it is hypoallergenic (the sole disclosed use of making a genetically modified cat). Pg 41-43 suggests modifying the method of Wongsrikeao (2011) for “cloning a genetically engineered Fel d1 knockout cat” by transfecting cat “oocytes” with lentiviral vectors encoding a pair of gRNAs and Cas9 10-12 hours before or after fertilization. Embryos are then cultured and selected for implantation. Contrary to the title of this section, this is not cloning because the no nuclear transfer occurs. This discussion is also prophetic and does not provide reasonable guidance that a cat with an inactivated Fel d1 gene will occur, specifically that it is hypoallergenic (the sole disclosed use of making a genetically modified cat). Pg 44-45 suggest “cloning a genetically engineered Fel d1 knockout cat” by ES cell manipulation. However, the art at the time of filing was limited to ES-like cat cells; the art did not teach true cat ES cells, i.e. capable of becoming all three germ layers, forming a teratoma, and germline transmission. The art did not teach cat ES cells capable of being cultured long enough to select transfected cells while still maintaining their ES cell phenotype or the conditions required to do so. This discussion is prophetic. The specification does not teach how to make/use a cat with only one or some cells from the cell line. The specification does not provide adequate guidance that one cell or a group of cells with impaired Fel d1 expression would make the cat hypoallergenic. Pg 45 discusses transfecting cat salivary glands with feline immunodeficiency virus (FIV) encoding a pair of gRNAs and Cas9 using a salivary gland-specific promoter. This example is prophetic and fails to provide adequate guidance that the cells of salivary gland WILL have impaired Fel d1 production or that the cat WILL be hypoallergenic. Pg 46 discusses transfecting cat skin with feline immunodeficiency virus (FIV) encoding a pair of gRNAs and Cas9 using a skin-specific promoter. This example is prophetic and fails to provide adequate guidance that the cells of skin WILL have impaired Fel d1 production or that the cat WILL be hypoallergenic. Pg 46-47 discusses systemically administering a feline immunodeficiency virus (FIV) encoding a pair of gRNAs and Cas9 using a skin-specific promoter. This example is prophetic and fails to provide adequate guidance that the cells of the cat WILL have impaired Fel d1 production or that the cat WILL be hypoallergenic. The art also did not teach a genetically modified cat with an inactivated gene, specifically a gene encoding an allergen. The guidance in the specification regarding making a genetically modified cat with an inactivated Fel d1 gene is inadequate for reasons cited above. Accordingly, the specification lacks written description for any cat as required in claim 28, specifically a cat with reduced Fel d1 expression that was hypoallergenic (the sole disclosed use of making a genetically modified cat).
The specification lacks written description for any cat cell in vivo as encompassed by claims 25 and 34. Claims 25 and 34 encompass editing a cell in vivo using a CRISPR system; however, the specification and the art at the time of filing are limited to administering Cas9 and gRNA to isolated cells. The specification and the art at the time of filing do not correlate CRISPR technology in vitro to in vivo embodiments. Accordingly, the specification lacks written description for in vivo embodiments.
The specification lacks written description for any “conserved region identified from genomic sequencing data” “from feline tissue samples” (claims 35, 36) other than the gene segment that is removed when a gRNA pair is used that is a) SEQ ID NO: 1234, 1235, 1238 or 1239; and b) SEQ ID NO: 1236, 1237, 1240, or 1241. The specification does not correlate deleting the entire Fel d1 gene to any “portion” that is “removed” and “represents a “conserved region identified from genomic sequencing data”. Accordingly, the concept lacks written description.
Indefiniteness
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 21-36 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 pre-AIA the applicant regards as the invention.
The concept of “editing a Fel d1” gene in the preamble of claim 21 does not have a nexus with “impaired or absent Fel d1 protein expression” in the body of claim 21. Therefore, it is unclear whether any “editing” will do or whether the Fel d1 gene must be “impaired or absent”. It would be clearer to say the method inactivates the Fel d1 gene, i.e. ---A method of inactivating a Fel d1 gene, the method comprising: introducing Cas9 and gRNA into an isolated Felis catus cell……such that the Fel d1 gene is inactivated in the cell---.
The phrases “gRNA of SEQ ID NO: 1244-2468” and “gRNA chosen from SEQ ID NO: 1-1225” in claims 21-25 do not make sense. SEQ ID NOs 1-1225, 1244-2468 are nucleic acids. While gRNA may comprise the nucleic acids, the SEQ ID NOs are not gRNAs as claimed.
The phrase “comprising a Fel d 1 genomic sequence lacking at least a portion of the Fel d 1 genomic sequence of a cell line from which the engineered cell line is derived” in claim 25 makes the claim indefinite. It is unclear when a Fel d1 genomic sequence is “of a cell line from which the engineered cell line is derived” or when/how the cell line is “derived” from an “engineered cell line”. The claim fails to clearly set for the structure of the genetic modification, the genome of the cell line, or the function of the genetic modification.
Claim 28 is indefinite because it does not clearly set forth the structure/function of the cat. The claim fails to clearly set forth the structure of the genetic modification in the cat, the genome of the cat, the genome of one or more cells within the cat, or the function of the genetic modification in the cat. Therefore, claim 28 is missing essential elements.
Claim 33 is indefinite because it does not clearly set forth the structure/function of the progeny. The claim fails to clearly set forth the structure of the genetic modification in the cat, the genome of the cat, the genome of one or more cells within the cat, or the function of the genetic modification in the cat. Therefore, claim 33 is missing essential elements. If the structure of the cat in claim 33 is identical to the cat in claim 28, then claim 33 is a substantial duplicate of claim 28. If the structure of the cat in claim 33 is different than the claim in claim 28, much clarification is required. If the cat in claim 33 does not contain genetically modified cells, much clarification is required.
The phrase “using a CRISPR system directing a double stranded break in the genome of the Felis catus cell” in claim 34 makes the claim indefinite. The metes and bounds of “CRISPR system” cannot be determined because the structures/functions associated with are missing. It is unclear when a CRISPR system “directs” a double stranded break.
The term “represents” phrase “portion of the Fel d1 gene removed represents” in claim 35 makes the claim indefinite. It is unclear when a “portion” of a gene “represents” a “conserved region identified from genomic sequencing data”. It is unclear whether the phrase “conserved region identified from genomic sequencing data” “from feline tissue samples” is attempting to invoke product-by-process language or not. It is unclear how the phrase further limits the “portion” of the “removed” Fel d1 gene.
Claim Rejections - 35 USC § 102
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 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 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 28-33 are rejected under 35 U.S.C. 102a1 as being anticipated by Sartore (Veterinary Sci., 2017, Vol. 4, pg 1-5).
Sartore taught a siberian cat whose genome comprises a deletion of 6 nucleotides causing the loss of two serines in the Fel d1 gene (pg 3, 1st paragraph) that cause decreased concentration of Fel d1 in the saliva (pg 3, 3rd full paragraph). This is equivalent to a cat whose genome comprises an inactivated Fel d1 gene as encompassed by claims 28-33.
Claims 25-33 are rejected under 35 U.S.C. 102a1 as being anticipated by Avner (20200008405, EFD=1-13-16).
Avner taught an isolated Felis catus somatic cell with an inactivated Fel d1 gene engineered using Cas9 and crRNAs (Example 2; Fig. 35, 36). The lack of Fel d1 is hypoallergenic. This is equivalent to claims 25-27. Avner suggested using the cell to clone a cat which has the same structure the cat in claims 28-33 (Fig. 1).
Claims 25-33 are rejected under 35 U.S.C. 102a1 as being anticipated by Avner (10626417).
Avner taught an isolated Felis catus somatic cell with an inactivated Fel d1 gene engineered using a homology vector (claim 1, col. 37, example 2). The lack of Fel d1 is hypoallergenic. This is equivalent to claims 25-27. Avner suggested using the cell to clone a cat which has the same structure the cat in claims 28-33 (Fig. 1).
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 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 set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966), that are applied for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claim 21-36 are rejected under 35 U.S.C. 103 as being unpatentable over Avner (10626417) in view of Cong (Sci., 2013, Vol. 339, No. 6121, pg 819-823) and Gronlund (Int Arch Allergy Immunol, 2010, Vol. 151, pg 265-274).
Avner taught an isolated Felis catus somatic cell with an inactivated Fel d1 gene engineered using Cas9 and crRNAs (Example 2; Fig. 35, 36). The lack of Fel d1 is hypoallergenic. Avner suggested using the cell to clone a cat which has the same structure the cat in claims 47, 48, 69, 70 (Fig. 1). Avner did not teach using Cas9 and two chimeric guide RNAs comprising crRNA and tracrRNA as encompassed by claims 21 and 34.
However, Cong taught genetically modifying an isolated somatic cell using Cas9 and two chimeric gRNAs made of crRNA and tracrRNA that target a gene of interest such that a deletion occurs that cause the gene to be inactivated. The sequence for the Felis catus Fel d1 gene was known in the art at the time of filing.
Thus it would have been obvious to those of ordinary skill in the art at the time of filing to genetically modify an isolated Felis catus somatic cell such that the Fel d1 gene is inactivated as described Avner using Cas9 and gRNA as described by Cong. Those of ordinary skill in the art at the time of filing would have been motivated to replace the homology vector with the CRISPR system to improve specificity. Those of ordinary skill in the art at the time of filing would have had a reasonable expectation of designing gRNA using the teachings of Cong and the sequence of the Fel d1.
SEQ ID NO: 1244-2468 in claims 21-24 are readily apparent from Gronlund.
SEQ ID NO: 1-1225 in claim 25 are readily apparent from Gronlund.
Thus, Applicants' claimed invention as a whole is prima facie obvious in the absence of evidence to the contrary.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure:
Pereyra-Bonnet (Reprod., Fertility and Develop., 2008, Vol. 20, pg 741-749) taught a method of introducing exogenous DNA into feline embryos.
Cho (Cellular Reprogramming, 2010, Vol. 12, No. 9, pg 739-747) confirmed the cat cloned by Yin (see above) passed the RFP transgene on to its offspring via the germline.
Conclusion
No claim is allowed.
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Michael C. Wilson
/MICHAEL C WILSON/
Primary Examiner, Art Unit 1638
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