DETAILED ACTION
Applicant’s response of 12/11/2025 has been received and entered into the application file.
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 .
Priority
The present application is a National Stage application field under 35 U.S.C. 371. The certified copy has been filed in application PCT/JP2021/007669, filed on 03/01/2021.
Also, acknowledgment is made of applicant’s claim for foreign priority under 35 U.S.C. 119 (a)-(d). The certified copy has been filed in parent Application No. JP2020-034255, filed on 02/28/2020.
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.
Election/Restrictions
Applicants have previously elected without traverse Group I, drawn to a production method for a genetically modified megakaryocyte, the method comprising introducing a CRISPR-associated (Cas) family protein and guide RNA (gRNA) into a megakaryocyte to modify a subject gene, whereby a genetically modified megakaryocyte is obtained, wherein the Cas family protein and the gRNA to be introduced into the megakaryocyte in the introducing form a complex beforehand and wherein the subject gene is (i) Human Leukocyte Antigen (HLA)-A, HLA-B, and HLA-C genes, or (ii)32-Microglobulin (Β2M) gene in the reply filed on 08/15/2025.
The restriction requirement between Groups 1-3, as set forth in the Office action mailed on 06/17/2025, had been reconsidered, such that the restriction requirement between groups 1-2 was withdrawn (currently cancelled claim 6 was previously rejoined), whereas the restriction requirement between groups (1 -2) and 3 was maintained.
Claims 1-5, 7-13 are pending. Claim 6 is cancelled. Claim 13 remains withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 08/15/2025.Therefore, claims 1-5 and 7-12 are under examination in the instant application.
Status of Prior Rejections/Response to Arguments
RE: Rejection of claim(s) 12 under 35 U.S.C. 102(a)(1) as being anticipated by Suzuki et al.:
Applicants have traversed the rejection asserting that MK-iPSCs described in Suzuki are not megakaryocytes, but iPSCs obtained by reprogramming megakaryocytes. This is not persuasive. Although Suzuki et al. teaches MK-iPSCs, Suzuki et al. still teaches indirectly genetically modifying megakaryocyte by knocking out β2M from human induced pluripotent cell-derived HLA-I deficient immortalized megakaryocyte progenitor cells (β2M-KO imMKCL). The starting cell population of megakaryocyte was reprogrammed into iPSCs to obtain MK-iPSCs after which an sgRNA expression vector and a CRISPR/Cas9 expression vector were used for transfecting these cells to obtain the genetically modified megakaryocyte (see Suzuki et al. teachings on page 56, column 1, paragraph 5, lines 11-14).
Applicant's arguments filed 12/11/2025 have been fully considered but they are not persuasive. The rejection is therefore maintained.
RE: Rejection of claim(s) 1-11 are rejected under 35 U.S.C. 103 as being unpatentable over Suzuki et al., in view of Zuris et al.:
Applicants have traversed the rejection asserting that in Suzuki et al., genome editing of imMKCLs was not successful, whereas in Example 5 of the instant Application, it was demonstrated that genome editing of megakaryocytes is possible by forming a complex of a Cas family protein and gRNA beforehand and then introducing the complex into megakaryocytes. Applicants further argue that Zuris et al. does not cure the deficiencies of Suzuki at least because it does not teach producing genetically modified megakaryocytes.
Regarding applicants’ argument that genome editing of imMKCLs of Suzuki et al., was not successful, although direct modification of Suzuki’s megakaryocytes into genetically modified megakaryocyte did not work, Suzuki et al. teaches indirectly genetically modifying megakaryocyte by knocking out β2M from human induced pluripotent cell-derived HLA-I deficient immortalized megakaryocyte progenitor cells (β2M-KO imMKCL) by first reprogramming the starting cell population of megakaryocyte into iPSCs to obtain MK-iPSCs after which an sgRNA expression vector and a CRISPR/Cas9 expression vector were used for transfecting these cells to obtain the genetically modified megakaryocyte (see Suzuki et al. teachings on page 56, column 1, paragraph 5, lines 11-14).
Regarding applicants’ argument that Zuris et al. does not cure the deficiencies of Suzuki at least because it does not teach producing genetically modified megakaryocytes, Zuris et al. was only used to cure the deficiency of Suzuki et al. that the Cas family protein and the gRNA to be introduced into the megakaryocyte form a complex before they’re introduced into the cells.
Applicant's arguments filed 12/11/2025 have been fully considered but they are not persuasive. The rejection over claim 6 is moot in view of applicants’ cancellation of claim 6 and maintained over claims 1-5 and 7-11.
New/Maintained Grounds of Rejection
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.
Claim 12 is rejected under 35 U.S.C. 102(a)(1) as being anticipated by Suzuki et al. (Suzuki et al. “iPSC-Derived Platelets Depleted of HLA Class I Are Inert to Anti-HLA Class I and Natural Killer Cell Immunity”. Stem Cell Reports. 2020 Jan 14;14(1):49-59).
Regrading claim 12, Suzuki et al. teaches establishing a β2M knockout human induced pluripotent cell-derived HLA-I deficient immortalized megakaryocyte progenitor cells (β2M-KO imMKCL), by transfecting an sgRNA expression vector and a CRISPR/Cas9 expression vector into 0.8 × 106 megakaryocyte progenitor cell obtained from human pluripotent stem cells (MK-iPSCs) by electroporation (page 56, column 1, paragraph 5, lines 11-14). This reads on a production method for a genetically modified megakaryocyte comprising introducing a CRISPR-associated (Cas) family protein and guide RNA (gRNA) into a megakaryocyte to modify a subject gene and whereby a genetically modified megakaryocyte is obtained, wherein the introduction of the Cas family protein and the gRNA in the introducing is performed by an electroporation method.
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.
Claim(s) 1-5, and 7-11 are rejected under 35 U.S.C. 103 as being unpatentable over Suzuki et al. (Suzuki et al. “iPSC-Derived Platelets Depleted of HLA Class I Are Inert to Anti-HLA Class I and Natural Killer Cell Immunity”. Stem Cell Reports. 2020 Jan 14;14(1):49-59), in view of Zuris et al. (Zuris et al., “Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo”, nature biotechnology, 2015, vol. 33, pp. 73-80).
Suzuki et al. teaches that ex vivo production of platelets depleted of human leukocyte antigen class I (HLA-I) could be useful as means to overcome platelet transfusion refractoriness caused by HLA-I incompatibility
(Summary). β2M knockout human induced pluripotent cell-derived HLA-I deficient platelets (HLA-KOiPLAT)
were developed in a clinically applicable immortalized megakaryocyte progenitor cell (imMGCLs) system by
genetic manipulation (Summary); and the sgRNA expression vector and the CRISPR/Cas9 expression vector were
introduced by electroporation into immortalized megakaryocyte progenitor cell (imMGCLs) obtained from human
pluripotent stem cells, and genetically modified megakaryocytes were obtained (Summary and Experimental
Procedures).
Regrading claim 1, Suzuki et al. teaches establishing a β2M knockout human induced pluripotent cell-derived HLA-I deficient immortalized megakaryocyte progenitor cells (β2M-KO imMKCL), by transfecting an sgRNA expression vector and a CRISPR/Cas9 expression vector into 0.8 × 106 megakaryocyte progenitor cell obtained from human pluripotent stem cells (MK-iPSCs) by electroporation (page 56, column 1, paragraph 5, lines 11-14). This reads on a production method for a genetically modified megakaryocyte comprising introducing a CRISPR-associated (Cas) family protein and guide RNA (gRNA) into a megakaryocyte to modify a subject gene and whereby a genetically modified megakaryocyte is obtained.
However, Suzuki et al. fails to teach that the Cas family protein and the gRNA to be introduced into the megakaryocyte form a complex before they’re introduced.
However, Zuris et al. teaches that transient delivery of functional Cas9:sgRNA protein:RNA complexes circumvents risks associated with other gene delivery methods and has the potential to improve the specificity of genome editing by minimizing the opportunity of agents to modify off-target substrates after the target locus is modified, or to reverse on-target modification (page 78, column 1, paragraph 2, lines 1-6).
Therefore, it would have been prima facie obvious to one of the ordinary skills in the art before the effective filing date of the claimed invention to have introduced the Cas9 family protein and the gRNA into the megakaryocyte of Suzuki et al. as a complex to genetically manipulate the HLA-I complex molecule β2-microglobulin (B2M) in the megakaryocyte with a reasonable expectation of success. One would have been motivated to have done so in order to reduce the risk of off-target compared with introducing by means of plasmids as taught by Zuris et al.
Regrading claim 2: Following discussion of claim 1 above, Suzuki et al. teaches establishing a β2M-KO imMKCL by transfecting an sgRNA expression vector and a CRISPR/Cas9 expression vector into the MK-iPSCs by electroporation (page 56, column 1, paragraph 5, lines 11-14).
Regrading claim 3: Following discussion of claim 1 above, Suzuki et al. teaches establishing a β2M-KO imMKCL by transfecting an sgRNA expression vector and a CRISPR/Cas9 expression vector into the MK-iPSCs by electroporation (page 56, column 1, paragraph 5, lines 11-14).
Regrading claim 4: Following discussion of claim 1 above, Suzuki et al. teaches that for depletion of HLA-I of the megakaryocytes, genetic manipulation of the HLA-I complex molecule β2-microglobulin (β2M) has been shown to be effective. The knockout procedure can completely deplete HLA-I expression for all A, B, and C antigens (“HLA-KO”) (page 50, column 1, paragraph 2, lines 1-8). Knocking out β2M reads on the subject gene to be modified by disruption.
Regrading claim 5: Following discussion of claim 1 above, Suzuki et al. teaches that imMKCLs were created from the megakaryocyte (MK)-lineage differentiation from iPSCs (page 49, column 2, paragraph 1, lines 11-13).
Regrading claim 7, Suzuki et al. teaches that ex vivo production of platelets depleted of human leukocyte antigen class I (HLA-I) could be useful as means to overcome platelet transfusion refractoriness caused by HLA-I incompatibility (Summary). Suzuki et al. further teaches that induced pluripotent cell-derived HLA-I deficient platelets (HLA-KOiPLAT) were developed in a clinically applicable immortalized megakaryocyte progenitor cell (imMGCLs) system by genetic manipulation (Summary), such that an sgRNA expression vector and a CRISPR/Cas9 expression vector were transfected into 0.8 × 106 megakaryocyte progenitor cell obtained from human pluripotent stem cells (MK-iPSCs) by electroporation (page 56, column 1, paragraph 5, lines 11-14). This reads on a production method for a modified platelet comprising introducing a Cas family protein and guide RNA (gRNA) into a megakaryocyte to modify a subject gene and obtaining a modified platelet by producing a platelet from the genetically modified megakaryocyte.
However, Suzuki et al. fails to teach that the Cas family protein and the gRNA to be introduced into the megakaryocyte form a complex before they’re introduced.
However, Zuris et al. teaches that transient delivery of functional Cas9:sgRNA protein:RNA complexes circumvents risks associated with other gene delivery methods and has the potential to improve the specificity of genome editing by minimizing the opportunity of agents to modify off-target substrates after the target locus is modified, or to reverse on-target modification (page 78, column 1, paragraph 2, lines 1-6).
Therefore, it would have been prima facie obvious to one of the ordinary skills in the art before the effective filing date of the claimed invention to have introduced the Cas9 family protein and the gRNA into the megakaryocyte of Suzuki et al. as a complex to genetically manipulate the HLA-I complex molecule β2-microglobulin (B2M) in the megakaryocyte with a reasonable expectation of success. One would have been motivated to have done so in order to reduce the risk of off-target compared with introducing by means of plasmids as taught by Zuris et al.
Regrading claim 8: Following discussion of claim 7 above, Suzuki et al. teaches that transfecting an sgRNA expression vector and a CRISPR/Cas9 expression vector into the MK-iPSCs is done by electroporation (page 56, column 1, paragraph 5, lines 11-14).
Regrading claim 9: Following discussion of claim 7 above, Suzuki et al. teaches that transfecting an sgRNA expression vector and a CRISPR/Cas9 expression vector into the MK-iPSCs is done by electroporation (page 56, column 1, paragraph 5, lines 11-14).
Regrading claim 10: Following discussion of claim 7 above, Suzuki et al. teaches that for depletion of HLA-I of the megakaryocytes, genetic manipulation of the HLA-I complex molecule β2-microglobulin (β2M) has been shown to be effective. The knockout procedure can completely deplete HLA-I expression for all A, B, and C antigens (“HLA-KO”) (page 50, column 1, paragraph 2, lines 1-8). Knocking out β2M reads on the subject gene to be modified by disruption.
Regrading claim 11: Following discussion of claim 7 above, Suzuki et al. teaches that imMKCLs were created from the megakaryocyte (MK)-lineage differentiation from iPSCs (page 49, column 2, paragraph 1, lines 11-13).
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to HANAN ISAM ABUZEINEH whose telephone number is (571)272-9596. The examiner can normally be reached Mon- Fri 8:30-5:00.
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Hanan Isam Abuzeineh
/H.I.A./Examiner, Art Unit 1633
/CHRISTOPHER M BABIC/Supervisory Patent Examiner, Art Unit 1633