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 .
DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 10/09/2025 has been entered.
Applicant' s amendment and response filed on 10/09/2025 has been received and entered into the case.
Amendments
In the reply filed 10/09/2025, Applicant has amended claims 1, 13, 54 and 65.
Claim Status
Claims 1-16 and 54-68 are pending and are considered on the merits. Claims 1 and 54 are independent claims.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 10/09/2025 is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement is being considered by the examiner. The corresponding signed and initialed PTO form 1449 has been mailed with this action.
Withdrawn Claim Objections
The prior objection to claims 1, 13, 54 and 65 is withdrawn in light of Applicant’s amendment.
Withdrawn Claim Rejections - 35 USC § 112(a)
The prior rejection of claims 54-68 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112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement is withdrawn in light of Applicant’s amendment to claim 54 to recite new limitation that the reprogramming transcription factors are selected from a group consisting of: c-myc, Klf4, Sox2, Oct3/4, Klf2, Nanog, Tfcp2L1, and Stat3.
The prior rejection of claims 54-68 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification does not reasonably provide enablement for a method for inducing iPSCs from somatic cells by exclusively using transcription factors selected from Klf2, Nanog, Tfcp2L1 and Stat3, is withdrawn in light of Applicant’s amendment to claim 54 to recite new reprogramming transcription factors c-myc, Klf4, Sox2 and Oct3/4.
New Claim Rejections - 35 USC § 112(a)
NEW MATTER
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 1-16 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.
Amended independent claim 1, and dependent claims 2-16 are now directed to a method for deriving iPSCs comprising two parallel methods using two subsets of PBMCs (herein referred to as Method 1 and Method 2), in which Method 1 comprises a step of culturing the first subset of the PBMCs to expand blood progenitor cells prior to transfection while Method 2 does not comprise this progenitor-expansion step. Claim 1 further encompasses a step of expanding iPSCs for use in tests and results are compared to iPSCs derived from the transfected second subset.
The new scope of amended claim 1 and dependent claims 2-16, directed to Method 2 that does not comprise a progenitor-expansion step (i.e., comprises a direct transfecting), and directed to comparing results of the iPSCs derived from the two subsets of PBMCs, appears to represent new matter. MPEP 2163.06 notes “If new matter is added to the claims, the examiner should reject the claims under 35 U.S.C. 112(a), pre-AIA first paragraph - written description requirement. In re Rasmussen , 650 F.2d 1212, 211 USPQ 323 (CCPA 1981).” Although the basis for this limitation was identified in Applicant’s remarks filed 10/09/2025 as being described in the specification [0097], [0107], [0108], [0140] and [0145]-[0147], a review of the specification by the Examiner did NOT find any specific basis for the recited new Method 2 and a step of comparing results of the iPSCs derived from the two subsets of PBMCs. Specifically, [0097] describes transcription factors, [0107]-[0108] describe culturing PBMCs to expand blood progenitor cells using IL3, IL6, FLT-3, TPO and SCF, [0140] describes within-species genetic variations in NHPs, and [0145]-[0147] describe in vitro testing of the first subset of derived iPSCs, and in vivo testing of the second subset of the derived iPSCs and comparing the results, and the two subsets of derived iPSCs may be during different time periods of iPSC expansion in vitro (underlined by examiner). Therefore, the recited subject matter of new Method 2 that does not comprise a progenitor-expansion step (i.e., comprises a direct transfecting PBMCs) and a step of comparing results of the iPSCs derived from the two subsets of PBMCs, is not supported by the specification and thus represents new matter.
As noted by the MPEP, new matter includes not only the addition of wholly unsupported subject matter, but may also include the introduction of claim changes which involve narrowing the claims by introducing elements or limitations which are not supported by the as-filed disclosure is a violation of the written description requirement of 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph. See, e.g., Fujikawa v. Wattanasin, 93 F.3d 1559, 1571, 39 USPQ2d 1895, 1905 (Fed. Cir. 1996), and Ex parte Ohshiro, 14 USPQ2d 1750 (Bd. Pat. App. & Inter. 1989) (see MPEP 2163.05 (II) Narrowing or Subgeneric Claim).
New Claim Rejections - 35 USC § 112(b)
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 12-13 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 12 and 13 recite the limitation “the transfected cells” in line 1. There is insufficient antecedent basis for this limitation in the claims because the base claim 1 recites “transfecting the first subset of the cultured PBMCs and the second subset of the PBMCs” and recites “transferring each subset of the transfected cells”, thus recites two subsets of transfected cells. It is not clear which subset of transfected cells this limitation “the transfected cells” is referring to. This limitation is examined as being referring to both subsets of transfected cells.
New Claim Rejections - 35 USC § 112(d)
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 8-9 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Claims 8-9 recite the limitation of an additional step of “extracting a quantity of high-molecular weight (HMW) DNA from the second subset of the obtained PBMCs”. However, the amended base claim 1 and claim 7 recite the step of “transfecting … the second subset of the PBMCs” to reprogram the second subset of the obtained PBMCs into iPSCs. Since the steps recited in claims 8-9 require the second subset of the PBMCs to be lysed for DNA extraction, during which the second subset of the PBMCs can no longer be transfected and reprogrammed as recited in claims 1 and 7, claims 8-9 fail to include all the limitations of the claims upon which they depend.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Withdrawn Claim Rejections - 35 USC § 103
The prior rejection of claims 1-8 and 10-16 under 35 U.S.C. 103 as being unpatentable over Roodgar et al., (referred to as “GigaScience”). as evidenced by Roodgar et al., and in view of Rogers et al., is withdrawn in light of Applicant’s amendment to claim 1 to recite new limitations.
The prior rejection of claim 9 under 35 U.S.C. 103 as being unpatentable over Roodgar et al., (referred to as “GigaScience”) as evidenced by Roodgar et al., and in view of Rogers et al., and further in view of Jayakumar et al., is withdrawn in light of Applicant’s amendment to claim 1 to recite new limitations.
New 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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Claims 1-8 and 10-16 are rejected under 35 U.S.C. 103 as being unpatentable over Roodgar et al., (referred to as “GigaScience”. GigaScience, 9, 2020 July 10, p. 1-12. Prior art of record) as evidenced by Roodgar et al., (bioRxiv preprint. doi: https://doi.org/10.1101/635250; posted June 4, 2019. P. 1-22. Prior art of record) and in view of StemSpan (Information sheet of StemSpan Serum-Free Expansion Medium from StemCell Technologies. Published in 2018. P. 1-3), Rogers et al., (Nat Rev Genet. 2014;15(5):347-59. Prior art of record), and Rim et al., (J. Vis. Exp. 2016; (118): e54650, p. 1-7).
With respect to independent claim 1, as stated supra, amended claim 1 is now directed to a method for deriving iPSCs comprising two parallel methods using two subsets of PBMCs (herein referred to as Method 1 and Method 2), in which Method 1 comprises a step of culturing the first subset of the PBMCs to expand blood progenitor cells prior to transfection while Method 2 does not comprise this progenitor-expansion step, with all other steps being the same.
With respect to Method 1, GigaScience teaches a method of chromosome-level genome assembly of a nonhuman primate, a pig-tailed macaque (abstract, related to claim 7). To annotate the assembled genome, GigaScience teaches using RNAseq and proteomics data from the study animal’s PBMCs and iPSCs (generated from the PBMCs) (p. 9, left col, last para “Genome annotation”) and teaches “iPSCs were derived from PBMCs of the study animal [19]” (p. 8, left col, para “Study subject, sample preparation and DNA extraction”. Note that reference [19] is the reference of Roodgar (bioRxiv, 2019)), thus teaches a method for deriving iPSCs from nonhuman primate’s PBMCs using the method of Roodgar (bioRxiv, 2019).
In regard to the preamble and obtaining/recovering PBMCs steps of claim 1, Roodgar teaches a method for deriving induced pluripotent stem cells (iPSCs) from chimpanzees and a pig-tailed macaque (abstract and p. 11, last para). Roodgar teaches “blood samples were obtained from a 12-year old male pig-tailed macaque from the Johns Hopkins University colony. The PBMC (peripheral blood mononuclear cells) were isolated from whole blood in each sample” (p. 11, last para).
In regard to the step of dividing the obtained PBMCs into at least a first subset and a second subset, as stated supra, GigaScience teaches the obtained PBMCs are used for extraction of high molecular weight (HMW) DNA and additionally iPSCs are derived from PBMCs of the study animal accordingly to the method of reference #19 (i.e., Roodgar) (see e.g., p. 8, left col, para “Study subject, sample preparation, and DNA extraction”). Thus, GigaScience teaches dividing the obtained PBMCs into at least a first subset (for deriving iPSCs) and a second subset (e.g., for extracting HMW DNA).
In regard to culturing the first subset of the PBMCs in an HSPC expansion medium for a time period prior to transfection, Roodgar teaches “PBMCs were cultured in medium to expand blood progenitor cells in StemSpan medium for 9 days” (p. 11, last para, note that the StemSpan medium is an HSPC expansion medium).
However, Roodgar is silent on the expansion medium comprising one or more of IL-3, IL-6, FLT-3, TPO and SCF.
StemSpan teaches StemSpan serum-free expansion medium is used for the generation and culture of hematopoietic cell types, “in most applications, addition of specific hematopoietic growth factors, cytokines, and/or other compounds is required for optimal growth” (p. 1, “Product Description” para 2) and teaches to “add desired cytokines, growth factors, and other components to StemSpan SFEM” (p. 1, “Handling/Directions for Use”, step 2). StemSpan teaches the StemSpan expansion supplements are suitable for use with StemSpan expansion medium, such as StemSpan CD34+ Expansion Supplement (10X) (Catalog #02691) for culture and expansion of large numbers of human CD34+ progenitor cells, containing recombinant human (rh) SCF, rh TPO, rh IL-3, rh IL-6, rh Flt3 ligand (p. 2, bullet point #1).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for deriving iPSCs comprising culturing PBMCs to expand blood progenitor cells in HSPC expansion medium StemSpan disclosed by GigaScience as evidenced by Roodgar, by combining one or more of cytokines and growth factors including IL-3, IL-6, FLT-3, TPO and SCF as suggested by StemSpan with a reasonable expectation of success. Since GigaScience as evidenced by Roodgar intends to expand blood progenitor cells in StemSpan expansion medium (Roodgar, p. 11, last para), and since StemSpan suggests addition of specific hematopoietic growth factors and cytokines is required for optimal growth (p. 1, “Product Description” para 2, and see “Handling/Directions for Use”, step 2) and suggests a StemSpan CD34+ Expansion Supplement for culture and expansion of large numbers of human CD34+ progenitor cells containing recombinant human (rh) SCF, rh TPO, rh IL-3, rh IL-6, rh Flt3 ligand (p. 2, bullet point #1), one of ordinary skill in the art would have had a reason to add an expansion supplement containing one or more of IL-3, IL-6, FLT-3, TPO and SCF to the StemSpan HSPC expansion medium as suggested by StemSpan in order to optimally expand blood progenitor cells for iPSC reprogramming.
In regard to transfecting the cultured PBMCs with transcription factors, Roodgar teaches “the cells were transfected with Sendai virus of four transcription factors c-myc, KLf4, Sox2, Oct3/4 … on the day of reprogramming (day 0)” (p. 11, last para).
In regard to no more than one day post-transfection transferring the transfected cells into a container comprising feeder cells, Roodgar teaches “on the day after transfection (day 1), all the cells transfected with Sendai virus were transferred into a 6-well plate (30,000 cells per well) each well containing ~150,000 mouse embryonic fibroblast (MEF) feeder cells” (p. 11, last para).
In regard to no more than one day after transferring, transferring the cells to a second container, Roodgar teaches “on day 2, the floating cells were transferred to new wells of 6-well plate” (p. 11, last para).
In regard to on selected days after transfer to the second container performing adding medium, changing medium and adding cell growth factors until a first cell colony appears, Roodgar teaches “on day 3, 2mL of in Essential-6 media were added into existing 2mL StemSpan medium…. On day 5, all the medium in each well were removed and replaced by E6 medium. … On day 7, all medium was removed and 2 mL of E6 + 100 ng/mL of bFGF were added. On day 8, 9, and 10 media was changed and with E7 (E6 + 100ng/ul of FGF). From day 11, the media was replaced by E8 until days 16 to day 18 when the first colonies of iPSC were observed.” (p. 11, last para).
In regard to passaging the cells by placing each colony in a third container coated with ECM, Roodgar teaches “each colony was manually picked with 1000μL pipet and were cultured on new matrigel-coated plates in E8 media and 2nM of TZV on the first day after passage” (p. 11, last para – p. 12, para 1).
In regard to expanding the first colony, Roodgar teaches the iPSCs are washed and dissociated, and cultured in E8X medium with TZV on MEFs for each passaging during the expansion and maintenance (p. 12, para 1), thus teaches the first cell colonies are expanded. Roodgar teaches the iPSCs are differentiated into cardiomyocytes in vitro (p. 13, last para) and are injected to mouse in vivo for teratoma assay (p, 12, 2nd last para.).
However, GigaScience, Roodgar and StemSpan are silent on the nonhuman primate being a cynomolgus macaque.
Nevertheless, GigaScience compares the phylogenetic relationships among the 3 macaque species (pig-tailed macaque, rhesus macaque and cynomolgus macaque) and human (see e.g., Fig 4). GigaScience acknowledges that different species of nonhuman primates exhibit varying levels of susceptibility to diseases. Studying the variation in disease susceptibility among primate species (including humans) necessitates reliable reference genomes that enable cross-species comparative genomics. However, the qualities of the genome assemblies and annotations for some of the Old World monkeys are inferior to that of the human genome [11] (see p. 2, “Introduction”, para 2). It is noted that the reference #11 is Rogers (2014).
Rogers, as referenced by GigaScience, teaches the two most commonly used nonhuman primates in biomedical research are the rhesus macaque and the cynomolgus macaque, and thus their importance as models for studies of human health and disease justifies extensive analysis of these genomes (e.g., p. 354, last para). Rogers teaches nonhuman primate genomics is expanding the scope of biomedical research with innovative analyses of primate models of human disease, but despite recent progress, both evolutionary and biomedical studies would benefit significantly from additional information (p. 357, right col, para 1). Rogers further teaches macaques generally have higher levels of intra-species genetic variation than humans (see e.g., p. 356, left col, para “Polymorphism within species and disease phenotypes”), which is assessed by individual primate genome projects (p. 349, last para).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of generating individualized genome assembly of a nonhuman primate pig-tailed macaque and annotating the assembly using data from the study animal’s iPSCs derived from PBMCs suggested by GigaScience, Roodgar and StemSpan, by substituting the pig-tailed macaque with a cynomolgus macaque as suggested by Rogers with a reasonable expectation of success. Since GigaScience compares the phylogenetic relationships among the 3 macaque species (pig-tailed macaque, rhesus macaque and cynomolgus macaque) and human (see e.g., Fig 4) and acknowledges the qualities of the genome assemblies and annotations for some of the Old World monkeys are inferior to that of the human genome (see p. 2, “Introduction”, para 2), and since Rogers teaches the cynomolgus macaque is one of the two most commonly used nonhuman primates in biomedical research that justifies extensive analysis of its genome (e.g., p. 354, last para), and teaches both evolutionary and biomedical studies would benefit significantly from additional information (p. 357, right col, para 1), one of ordinary skill in the art would have had a reason to substitute with a cynomolgus macaque as suggested by Rogers in the method of generating and annotating individualized genome assembly suggested by GigaScience, Roodgar and StemSpan in order to perform extensive analysis on the genome of the cynomolgus macaque to benefit the biomedical studies of using cynomolgus macaques as a human disease model.
In summary, GigaScience, as evidenced by Roodgar and in view of StemSpan and Rogers, suggest the Method 1 comprising a step of expanding blood progenitor cells in PBMCs. As stated supra, the only difference between Method 1 and Method 2 is that Method 2 does not comprise the progenitor-expansion step.
However, GigaScience, Roodgar, StemSpan and Rogers are silent on a method for deriving iPSCs from PBMCs that does not comprise a progenitor-expansion step.
With respect to Method 2 that does not comprise a progenitor-expansion step, Rim teaches a method for deriving iPSCs from PBMCs without the difficult expansion process of a specific cell type, such as CD34+ cells (e.g., p. 1, last para). Rim teaches whole blood cells or PBMCs were serially plated onto matrix-coated plates after transduction with Sendai virus containing Yamanaka factors (p. 1, last para), and teaches without any expansion process of a specific cell type, this protocol suggests a relatively simple method to generate iPSCs, even from a small amount of primary blood cells (p. 6, Discussion section, para 3 and p. 3, Result section, para 1).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for deriving iPSCs from PBMCs comprising a step of expanding blood progenitor cells (i.e., Method 1) as suggested by GigaScience, Roodgar, StemSpan and Rogers, by combining a parallel method for deriving iPSCs from PBMCs having the same steps except not having any expansion process of a specific cell type as suggested by Rim with a reasonable expectation of success. Since Rim teaches the method without the difficult expansion process of a specific cell type, such as CD34+ cells, is a relatively simple method to generate iPSCs even from a small amount of primary blood cells (p. 1, last para, p. 6, Discussion section, para 3 and p. 3, Result section, para 1), one of ordinary skill in the art would have had a reason to combine the teaching of Rim in the method of GigaScience, Roodgar, StemSpan and Rogers to test a relatively simple method to generate iPSCs from PBMCs without expanding a specific cell type (Method 2). Since Rim reduces to practice the method of transducing PBMCs with Sendai virus containing Yamanaka factors and then undergoing serially plating onto matrix-coated plates to generate iPSCs (p. 1, last para), and since the method suggested by GigaScience, Roodgar, StemSpan and Rogers comprises the steps of transducing PBMCs with Sendai virus containing Yamanaka factors and then undergoing serially plating onto plates with feeder cells or coated with extracellular matrix (see e.g., Roodgar, p. 11, last para), one of ordinary skill in the art would have had a reasonable expectation of success in using the method of GigaScience, Roodgar, StemSpan and Rogers but omitting the step of expanding a specific cell type as suggested by Rim to generate iPSCs from PBMCs.
In regard to the limitation in the last paragraph directed to expanding iPSCs for use in an in vitro test, an in vivo test or creation of a genome library wherein results are compared to iPSCs derived from the transfected second subset, as stated supra, Roodgar teaches the iPSCs are cultured for expansion (p. 12, para 1) and teaches the iPSCs are differentiated into cardiomyocytes in vitro (p. 13, last para) and are injected to mouse in vivo for teratoma assay (p, 12, 2nd last para.). Rim teaches the iPSCs are tested for in vitro differentiation into three-germ layers (see e.g., Fig 4B).
Although GigaScience, Roodgar, StemSpan, Rogers and Rim are silent on comparing results between the iPSCs derived from the two subsets/methods, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have added a step of comparing results of the tests on the two populations of expanded iPSCs, that are derived from the two subsets of PBMCs with or without a step of expanding blood progenitor cells, in order to validate the characteristics of the iPSCs generated by Method 1 and Method 2.
With respect to claim 2 directed to the expanding comprising washing with PBS and incubating with a cell detachment solution, claim 3 directed to the incubating occurring at about 37°C and claim 4 directed to the expanding comprising aspirating and dissociating the iPSCs into a single cell suspension, Roodgar teaches “for subsequent passaging and expansion, the iPSCs were washed with PBS and were incubated using 1mL of Accumax for one well of a 6-well plate for 5 minutes at 37°C. The cells were then aspirated, and single cell dissociated” (p. 12, para 1, note that Accumax is a cell detachment solution).
With respect to claim 5 directed to differentiating the iPSCs into main lineages including mesoderm and claim 6 directed to differentiating the main lineages into final tissues, Roodgar teaches iPSCs are cultured and treated with CHIR99021 to induce mesodermal differentiation, and from day 7 onwards, cells are placed on RPMI+B27 supplemented with insulin until beating was observed (p. 13, last para “Differentiation of iPSC into cardiomyocytes” and see Supplementary Figure S3).
With respect to claim 7 directed to further comprising generating an individualized genome assembly of the at least one individual, as stated supra, GigaScience teaches a method of generating chromosome-level genome assembly of a NHP (e.g., abstract) and teaches annotating the assembled genome using RNAseq and proteomics data from the study animal’s PBMCs or iPSCs (generated from the PBMCs) (p. 9, left col, last para “Genome annotation”).
With respect to claim 8 directed to extracting HMW DNA from the second subset of the PBMCs, this is examined as extracting HMW DNA from a subset of the PBMCs. As stated supra, GigaScience teaches a subset of PBMCs are used for the extraction of high molecular weight (HMW) DNA using the MagAtrract HMW DNA Kit. The average size of the extracted HMW DNA is ∼53 kb (Supplementary Fig. S1B) (p. 8, left col, para “Study subject, sample preparation and DNA extraction”). Thus, GigaScience teaches extracting HMW DNA from a subset of PBMCs by purification of archival quality DNA.
With respect to claim 10 directed to the transcription factors, as stated supra, Roodgar teaches “the cells were transfected with Sendai virus of four transcription factors c-myc, Klf4, Sox2, Oct3/4” (p. 11, last para).
With respect to claim 11 directed to the feeder cells being MEFs and claim 13 directed to a ratio of the transfected cells to feeder cells being about 1:3 to about 1:7, as stated supra, Roodgar teaches “on the day after transfection (day 1), all the cells transfected with Sendai virus were transferred into a 6-well plate (30,000 cells per well) each well containing ~150,000 mouse embryonic fibroblast (MEF) feeder cells” (p. 11, last para), thus teaches the feeder cells are MEF feeder cells, and the ratio of the transfected cells to the feeder cells is about 1:3 to about 1:7 (30,000:150,000 = 1:5).
With respect to claim 12 directed to the step of transferring the transfected cells into the container having feeder cells further comprising adding a tankyrase 1/2 inhibitor in a range of about 1 µM to about 3 µM, as stated supra, Roodgar teaches all the cells transfected with Sendai virus are transferred into a 6-well plate containing mouse embryonic fibroblast (MEF) feeder cells to reprogram into iPSCs (p. 11, last para).
However, Roodgar is silent on adding a tankyrase 1/2 inhibitor in a range of about 1 µM to about 3 µM in this step.
Nevertheless, Roodgar teaches optimizing nonhuman primate iPSC culture conditions by supplementing with 2μM XAV939 to inhibit Wnt/β-catenin canonical pathway and teaches XAV939 was critical for long-term maintenance of iPSC on MEFs (Figure 1A) (p. 3, last para). Roodgar further teaches the addition of XAV939 reduces spontaneous differentiation of iPSCs and helps maintain pluripotency in NHP iPSCs (p. 5, last para). It is noted that XAV939 is a tankyrase 1/2 inhibitor.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of transferring the transfected cells into a container having feeder cells to reprogram into NHP iPSCs suggested by GigaScience evidenced by Roodgar and in view of StemSpan, Rogers and Rim, by combining adding a tankyrase 1/2 inhibitor, e.g., XAV939 in a range of about 1 µM to about 3 µM as suggested by Roodgar with a reasonable expectation of success. Since Roodgar teaches supplementing with 2μM XAV939 results in long-term maintenance of NHP iPSC on MEFs and reduces spontaneous differentiation of iPSCs and maintains pluripotency in NHP iPSCs (p. 3, last para and p. 5, last para, see Fig 1A), one of ordinary skill in the art would have had a reason to add a tankyrase 1/2 inhibitor XAV939 of 1 µM to 3 µM as suggested by Roodgar in the step of transferring and reprogramming the transfected cells in order to reduce spontaneous differentiation and to maintain pluripotency of the derived iPSCs.
With respect to claim 14 directed to a Sendai virus vector, as stated supra, Roodgar teaches “the cells were transfected with Sendai virus of four transcription factors” (p. 11, last para).
With respect to claim 15 directed to a basic fibroblast growth factor (bFGF), as stated supra, Roodgar teaches “on day 7, all medium was removed and 2 mL of E6 + 100 ng/mL of bFGF were added” (p. 11, last para).
With respect to claim 16 directed to the passaging comprising adding a serine/threonine kinase inhibitor to a medium in the third container, as stated supra, Roodgar teaches “each colony was manually picked with 1000μL pipet and were cultured on new matrigel-coated plates in E8 media and 2nM of TZV” (p. 11, last para – p. 12, para 1, it is noted that TZV is an inhibitor of Rho-associated coiled-coil containing protein kinase (ROCK) which is a serine/threonine kinase, thus TZV is a serine/threonine kinase inhibitor).
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 10/09/2025 are acknowledged.
Applicant first argues that cited references in the prior rejection do not teach or suggested the new limitation of one or more of IL-3, IL-6, FLT-3, TPO or SCF in the expansion medium, or transfecting the second subset of PBMCs (without an expansion step) (Remarks, p. 10-12).
Applicant’s arguments have been fully considered and they are persuasive. Therefore, the prior 103 rejections have been withdrawn. However, as necessitated by amendments, a new ground of rejection has been made over GigaScience as evidenced by Roodgar and in view of Rogers, and in view of StemSpan and Rim. Specifically, StemSpan suggests addition of cytokines and growth factors such as IL-3, IL-6, FLT-3, TPO and SCF in the HSPC expansion medium is required for optimal cell growth (e.g., p. 1, “Product Description” para 2), and Rim teaches without any expansion process of a specific cell type, this protocol suggests a relatively simple method to generate iPSCs, even from a small amount of primary blood cells (p. 6, Discussion section, para 3 and p. 3, Result section, para 1), as discussed above.
Applicant further argues that the disclosed methods provide technical benefits by increasing diversity of precursor cells available for reprogramming by culturing PBMCs under HSPC expansion conditions (Remarks, p.12, para 3).
Applicant’s arguments have been fully considered but they are not persuasive. As discussed above, Roodgar teaches “PBMCs were cultured in medium to expand blood progenitor cells in StemSpan medium for 9 days” (p. 11, last para, note that the StemSpan medium is an HSPC expansion medium), and new prior art StemSpan suggests addition of one or more of IL-3, IL-6, FLT-3, TPO and SCF in the HSPC expansion medium is suitable for expanding a diversity of precursor cells such as CD34+ progenitor cells, hematopoietic cells, erythroid progenitor cells, megakaryocyte progenitor cells and myeloid progenitor cells (see p. 2, bullet points). Accordingly, cited references make obvious the technical benefits of increasing diversity of precursor cells available for reprogramming by culturing PBMCs under HSPC expansion conditions.
Applicant finally argues that the specification provides support and technical rationale for implementing subsets within the claimed method as taught in [0145]-[0147], which demonstrate that the use of subsets facilitates parallel workflows that provide technical benefits including comparison of in vitro and in vivo outcomes and the ability to correlate predictive in vitro assessments with in vivo safety and efficacy data (Remarks, p. 12, last para – p. 13, para 2).
Applicant’s arguments have been fully considered but they are not persuasive. As a first matter, as stated supra, the new limitation of transfecting the second subset of PBMCs that does not undergo a progenitor-expansion step represents new matter. The cited specification [0145]-[0147] describe in vitro testing of the first subset of derived iPSCs, and in vivo testing of the second subset of the derived iPSCs and comparing the results, and the two subsets of derived iPSCs may be during different time periods of iPSC expansion in vitro (underlined by examiner). Thus, the specification does not provide support on a first subset of PBMCs that undergoes a progenitor-expansion step and a second subset of PBMCs that does not undergo a progenitor-expansion step.
Additionally, in response to the argument that the use of subsets facilitates parallel workflows that provide technical benefits including comparison of in vitro and in vivo outcomes and the ability to correlate predictive in vitro assessments with in vivo safety and efficacy data, this purported technical benefit is not commensurate in scope with the claimed method “wherein results are compared to iPSCs derived from the transfected second subset”. In other words, the claimed comparison is between the iPSCs derived from the transfected first subset of the PBMCs and the iPSCs derived from the transfected second subset of the PBMCs. See MPEP 716.02(d).
Claim 9 is rejected under 35 U.S.C. 103 as being unpatentable over Roodgar et al., (referred to as “GigaScience”. GigaScience, 9, 2020 July 10, p. 1-12. Prior art of record) as evidenced by Roodgar et al., (bioRxiv preprint. doi: https://doi.org/10.1101/635250; posted June 4, 2019. P. 1-22. Prior art of record) and in view of StemSpan (Information sheet of StemSpan Serum-Free Expansion Medium from StemCell Technologies. Published in 2018. P. 1-3), Rogers et al., (Nat Rev Genet. 2014;15(5):347-59. Prior art of record), and Rim et al., (J. Vis. Exp. 2016; (118): e54650, p. 1-7), as applied to claims 1 and 7-8 above, and further in view of Jayakumar et al., (Scientific Data. 2021 June; 8: 159. P. 1-9. Prior art of record).
Claim 9 is directed to preparing a genomic library, sequencing long-read fragments and assembling and annotating the long-read fragments to recreate the genome of the individual. As argued by Applicant, the term “long-read” is examined as being exemplified as being at least about 10,000 base pairs but may also be between about 5,000 base pairs to about 50,000 base pairs disclosed in specification [00122].
In regard to a genomic library for sequencing and assembling, GigaScience teaches preparing two different genomic libraries (a linked-read library and a HiC library), sequencing the libraries on Illumina HiSeq 4000, and assembling and scaffolding the data to generate an genome assembly (p. 8, left col, last para – right col). GigaScience further teaches annotating the genome using RNAseq and proteomics data from the study animal’s PBMCs and iPSCs (generated from the PBMCs) (p. 9, left col, last para “Genome annotation”). Thus, GigaScience teaches preparing a genome library from the HMW DNA, sequencing fragments of the HMW DNA in the library, and assembling and annotating to recreate the genome of the individual from which the PBMCs were obtained.
However, GigaScience, Roodgar, StemSpan, Rogers and Rim are silent on long-read fragments that are about 10,000 base pairs.
Nevertheless, Rogers teaches improved assemblies with longer contigs and more complete coverage in high-quality sequence data (that is, comprehensive delineation of segmental duplications, and fewer genes with gaps and errors) are needed and new technologies that provide longer reads will yield better assemblies by filling remaining gaps. The Pacific Biosciences RS II platform is one plausible option for upgrading the quality of primate genomes (Ref# 105) (see p. 356, right col, para 2. It is noted that reference #105 is titled “Mind the gap: upgrading genomes with Pacific Biosciences RS long-read sequencing technology”).
Jayakumar teaches generating a genome assembly of nonhuman primate by sequencing and assembling long reads using Pacific Biosciences (referred to by Rogers) with a read N50 length of 12.05 kbp (i.e., about 12,050 base pairs. See abstract, p. 2, para “Sample preparation, sequencing, and de novo assembly”, also see Fig 1a). Thus, Jayakumar teaches a method of sequencing long-read fragments (N50 length of 12.05 kbp) to assemble a genome of nonhuman primate.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of generating a genome assembly of a cynomolgus macaque by sequencing DNA fragments suggested by GigaScience evidenced by Roodgar and in view of StemSpan, Rogers and Rim, by substituting the DNA fragments with long-read fragments suggested by Rogers and taught by Jayakumar with a reasonable expectation of success. Since Rogers suggests new technologies, e.g., the Pacific Biosciences RS II platform, that provide longer reads will yield better assemblies by filling remaining gaps for upgrading the quality of primate genomes (see p. 356, right col, para 2), and since Jayakumar teaches single-molecule long-read sequencing has drastically increased the contiguity of assemblies (p. 2, para 2) and teaches the resulted genome assemblies have high fidelity and outperform all the available assemblies of this species in terms of contiguity and are valuable resources for non-human primate models and provide an important baseline in human biomedical research (abstract), one of ordinary skill in the art would have had a reason to make this substitution in order to generate a genome assembly with high fidelity and increased contiguity to be used as a resource for non-human primate models and as an important baseline in human biomedical research (Jayakumar, abstract).
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 10/09/2025 are acknowledged and have been discussed above.
Claims 54-59 and 63-68 are rejected under 35 U.S.C. 103 as being unpatentable over Roodgar et al., (referred to as “GigaScience”. GigaScience, 9, 2020 July 10, p. 1-12. Prior art of record) as evidenced by Roodgar et al., (bioRxiv preprint. doi: https://doi.org/10.1101/635250; posted June 4, 2019. P. 1-22. Prior art of record) and in view of Rogers et al., (Nat Rev Genet. 2014;15(5):347-59. Prior art of record).
With respect to independent claim 54, GigaScience teaches a method of chromosome-level genome assembly of a nonhuman primate, a pig-tailed macaque (abstract). To annotate the assembled genome, GigaScience teaches using RNAseq and proteomics data from the study animal’s PBMCs and iPSCs (generated from the PBMCs) (p. 9, left col, last para “Genome annotation”) and teaches “iPSCs were derived from PBMCs of the study animal [19]” (p. 8, left col, para “Study subject, sample preparation and DNA extraction”. Note that reference [19] is the reference of Roodgar (bioRxiv, 2019)), thus teaches a method for deriving iPSCs from nonhuman primate’s PBMCs using the method of Roodgar (bioRxiv, 2019).
In regard to the preamble, Roodgar teaches a method for deriving induced pluripotent stem cells (iPSCs) from chimpanzees and a pig-tailed macaque (abstract and p. 11, last para).
In regard to obtaining PBMCs, Roodgar teaches “blood samples were obtained from a 12-year old male pig-tailed macaque from the Johns Hopkins University colony. The PBMC (peripheral blood mononuclear cells) were isolated from whole blood in each sample” (p. 11, last para).
In regard to the step of dividing the obtained PBMCs into at least a first subset and a second subset, as stated supra, GigaScience teaches the obtained PBMCs are used for extraction of high molecular weight (HMW) DNA and additionally iPSCs are derived from PBMCs of the study animal accordingly to the method of reference #19 (i.e., Roodgar) (see e.g., p. 8, left col, para “Study subject, sample preparation, and DNA extraction”). Thus, GigaScience teaches dividing the obtained PBMCs into at least a first subset (for deriving iPSCs) and a second subset (for extracting HMW DNA).
In regard to culturing the first subset of the PBMCs in an HSPC expansion medium for a time period, Roodgar teaches “PBMCs were cultured in medium to expand blood progenitor cells in StemSpan medium for 9 days” (p. 11, last para, note that the StemSpan medium is an HSPC expansion medium).
In regard to transfecting the cultured PBMCs with a combination of transcription factors selected from a group, Roodgar teaches “the cells were transfected with Sendai virus of four transcription factors c-myc, Klf4, Sox2, Oct3/4 … on the day of reprogramming (day 0)” (p. 11, last para).
In regard to no more than one day post-transfection transferring the transfected cells into a container comprising feeder cells, Roodgar teaches “on the day after transfection (day 1), all the cells transfected with Sendai virus were transferred into a 6-well plate (30,000 cells per well) each well containing ~150,000 mouse embryonic fibroblast (MEF) feeder cells” (p. 11, last para).
In regard to no more than one day after transferring, transferring the cells to a second container, Roodgar teaches “on day 2, the floating cells were transferred to new wells of 6-well plate containing MEF in 2 mL of the Stem Span media.” (p. 11, last para).
In regard to on selected days after transfer to the second container performing adding medium, changing medium and adding cell growth factors until a first cell colony appears, Roodgar teaches “on day 3, 2mL of in Essential-6 media were added into existing 2mL StemSpan medium…. On day 5, all the medium in each well were removed and replaced by E6 medium. … On day 7, all medium was removed and 2 mL of E6 + 100 ng/mL of bFGF were added. On day 8, 9, and 10 media was changed and with E7 (E6 + 100ng/ul of FGF). From day 11, the media was replaced by E8 until days 16 to day 18 when the first colonies of iPSC were observed.” (p. 11, last para).
In regard to passaging the cells by placing each colony in a third container coated with ECM, Roodgar teaches “each colony was manually picked with 1000μL pipet and were cultured on new matrigel-coated plates in E8 media and 2nM of TZV on the first day after passage” (p. 11, last para – p. 12, para 1).
In regard to expanding the first colony, Roodgar teaches the iPSCs are washed and dissociated, and cultured in E8X medium with TZV on MEFs for each passaging during the expansion and maintenance (p. 12, para 1), thus teaches the first cell colonies are expanded. Roodgar teaches the iPSCs are differentiated into cardiomyocytes in vitro (p. 13, last para) and are injected to mouse in vivo for teratoma assay (p, 12, 2nd last para.).
However, GigaScience and Roodgar are silent on the nonhuman primate being a cynomolgus macaque.
Nevertheless, GigaScience compares the phylogenetic relationships among the 3 macaque species (pig-tailed macaque, rhesus macaque and cynomolgus macaque) and human (see e.g., Fig 4). GigaScience acknowledges that different species of nonhuman primates exhibit varying levels of susceptibility to diseases. Studying the variation in disease susceptibility among primate species (including humans) necessitates reliable reference genomes that enable cross-species comparative genomics. However, the qualities of the genome assemblies and annotations for some of the Old World monkeys are inferior to that of the human genome [11] (see p. 2, “Introduction”, para 2). It is noted that the reference #11 is Rogers (2014).
Rogers, as referenced by GigaScience, teaches the two most commonly used nonhuman primates in biomedical research are the rhesus macaque and the cynomolgus macaque, and thus their importance as models for studies of human health and disease justifies extensive analysis of these genomes (e.g., p. 354, last para). Rogers teaches nonhuman primate genomics is expanding the scope of biomedical research with innovative analyses of primate models of human disease, but despite recent progress, both evolutionary and biomedical studies would benefit significantly from additional information (p. 357, right col, para 1). Rogers further teaches macaques generally have higher levels of intra-species genetic variation than humans (see e.g., p. 356, left col, para “Polymorphism within species and disease phenotypes”), which is assessed by individual primate genome projects (p. 349, last para).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of generating individualized genome assembly of a nonhuman primate pig-tailed macaque and annotating the assembly using data from the study animal’s iPSCs derived from PBMCs disclosed by GigaScience and Roodgar, by substituting the pig-tailed macaque with a cynomolgus macaque as suggested by Rogers with a reasonable expectation of success. Since GigaScience compares the phylogenetic relationships among the 3 macaque species (pig-tailed macaque, rhesus macaque and cynomolgus macaque) and human (see e.g., Fig 4) and acknowledges the qualities of the genome assemblies and annotations for some of the Old World monkeys are inferior to that of the human genome (see p. 2, “Introduction”, para 2), and since Rogers teaches the cynomolgus macaque is one of the two most commonly used nonhuman primates in biomedical research that justifies extensive analysis of its genome (e.g., p. 354, last para), and teaches both evolutionary and biomedical studies would benefit significantly from additional information (p. 357, right col, para 1), one of ordinary skill in the art would have had a reason to substitute with a cynomolgus macaque as suggested by Rogers in the method of generating and annotating individualized genome assembly disclosed by GigaScience and Roodgar in order to perform extensive analysis of the genome of the cynomolgus macaque to benefit the biomedical studies of using the cynomolgus macaque as a human disease model.
With respect to claim 55 directed to the expanding comprising washing with PBS and incubating with a cell detachment solution, claim 56 directed to the incubating occurring at about 37°C and claim 57 directed to the expanding comprising aspirating and dissociating the iPSCs into a single cell suspension, Roodgar teaches “for subsequent passaging and expansion, the iPSCs were washed with PBS and were incubated using 1mL of Accumax for one well of a 6-well plate for 5 minutes at 37°C. The cells were then aspirated, and single cell dissociated” (p. 12, para 1, note that Accumax is a cell detachment solution).
With respect to claim 58 directed to differentiating the iPSCs into main lineages including mesoderm and claim 59 directed to differentiating the main lineages into final tissues, Roodgar teaches iPSCs are cultured and treated with CHIR99021 to induce mesodermal differentiation, and from day 7 onwards, cells are placed on RPMI+B27 supplemented with insulin until beating was observed (p. 13, last para “Differentiation of iPSC into cardiomyocytes” and see Supplementary Figure S3).
With respect to claim 63 directed to the feeder cells being MEFs and claim 65 directed to a ration of the transfected cells to feeder cells being about 1:3 to about 1:7, as stated supra, Roodgar teaches “on the day after transfection (day 1), all the cells transfected with Sendai virus were transferred into a 6-well plate (30,000 cells per well) each well containing ~150,000 mouse embryonic fibroblast (MEF) feeder cells” (p. 11, last para), thus teaches the feeder cells are MEF feeder cells, and the ratio of the transfected cells to the feeder cells is about 1:3 to about 1:7 (30,000:150,000 = 1:5).
With respect to claim 64 directed to the step of transferring the transfected cells into the container having feeder cells further comprising adding a tankyrase 1/2 inhibitor in a range of about 1 µM to about 3 µM, as stated supra, Roodgar teaches all the cells transfected with Sendai virus are transferred into a 6-well plate containing mouse embryonic fibroblast (MEF) feeder cells to reprogram into iPSCs (p. 11, last para).
However, Roodgar is silent on adding a tankyrase 1/2 inhibitor in a range of about 1 µM to about 3 µM in this step.
Nevertheless, Roodgar teaches optimizing nonhuman primate iPSC culture conditions by supplementing with 2μM XAV939 to inhibit Wnt/β-catenin canonical pathway and teaches XAV939 was critical for long-term maintenance of iPSC on MEFs (Figure 1A) (p. 3, last para). Roodgar further teaches the addition of XAV939 reduces spontaneous differentiation of iPSCs and helps maintain pluripotency in NHP iPSCs (p. 5, last para). It is noted that XAV939 is a tankyrase 1/2 inhibitor.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of transferring the transfected cells into a container having feeder cells to reprogram into NHP iPSCs suggested by GigaScience evidenced by Roodgar and in view of Rogers, by combining adding a tankyrase 1/2 inhibitor XAV939 in a range of about 1 µM to about 3 µM as suggested by Roodgar with a reasonable expectation of success. Since Roodgar teaches supplementing with 2μM XAV939 results in long-term maintenance of NHP iPSC on MEFs and reduces spontaneous differentiation of iPSCs and maintains pluripotency in NHP iPSCs (p. 3, last para and p. 5, last para, see Fig 1A), one of ordinary skill in the art would have had a reason to add a tankyrase 1/2 inhibitor XAV939 of 1 µM to 3 µM as suggested by Roodgar in the step of transferring and reprogramming the transfected cells in order to reduce spontaneous differentiation and to maintain pluripotency of the derived iPSCs (p. 5, last para).
With respect to claim 66 directed to a Sendai virus vector, as stated supra, Roodgar teaches “the cells were transfected with Sendai virus of four transcription factors” (p. 11, last para).
With respect to claim 67 directed to growth factors comprising a basic fibroblast growth factor (bFGF) and the iPSCs being cultured under feeder-free conditions using culture media and extracellular matrix substrates for maintenance of the iPSCs, as stated supra, Roodgar teaches “on day 7, all medium was removed and 2 mL of E6 + 100 ng/mL of bFGF were added” (p. 11, last para), thus teaches adding a basic fibroblast growth factor (bFGF). Roodgar teaches a step of maintaining iPSCs in an optimized feeder free condition comprising coating the wells with iMatrix-511 (i.e., an extracellular matrix substrate) in E8X medium (p. 11, para 2).
Accordingly, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have chosen a feeder-free condition for maintenance of the iPSCs as taught by Roodgar with a reasonable expectation of success. Since Roodgar has reduced to practice a method of maintaining iPSCs in an optimized feeder free condition using culture media and extracellular matrix substrates (p. 11, para 2), one of ordinary skill in the art would have had a reason to choose this feeder-free condition to maintain the NHP iPSCs in order to reduce the immunogenic effect from xenogeneic feeder cells.
With respect to claim 68 directed to the passaging comprising culturing the cells under feeder-free conditions and adding a serine/threonine kinase inhibitor to the medium in the third container, as stated supra, Roodgar teaches “each colony was manually picked with 1000μL pipet and were cultured on new matrigel-coated plates in E8 media and 2nM of TZV” (p. 11, last para – p. 12, para 1). It is noted that TZV is an inhibitor of Rho-associated coiled-coil containing protein kinase (ROCK) which is a serine/threonine kinase, thus TZV is a serine/threonine kinase inhibitor. It is also noted that the cited step does not have feeder cells, thus the iPSC colonies are cultured under feeder-free conditions with a serine/threonine kinase inhibitor added to the medium.
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 10/09/2025 are acknowledged.
Applicant argues the amendment to claim 54 overcomes the prior 112(a) written description rejection and the prior 112(a) enablement rejection (Remarks, p. 8-9).
Applicant’s arguments have been fully considered and they are persuasive. Therefore, the prior 112(a) rejections have been withdrawn. However, as necessitated by amendments, a new ground of 103 rejection has been made over GigaScience as evidenced by Roodgar and in view of Rogers as discussed above.
Claims 60-62 are rejected under 35 U.S.C. 103 as being unpatentable over Roodgar et al., (referred to as “GigaScience”. GigaScience, 9, 2020 July 10, p. 1-12. Prior art of record) as evidenced by Roodgar et al., (bioRxiv preprint. doi: https://doi.org/10.1101/635250; posted June 4, 2019. P. 1-22. Prior art of record) and in view of Rogers et al., (Nat Rev Genet. 2014;15(5):347-59. Prior art of record), as applied to claim 54 above, and further in view of Madrid et al., (Current Protocols. 2021 March; 1, e88, p. 1-25) and Jayakumar et al., (Scientific Data. 2021 June; 8: 159. P. 1-9. Prior art of record).
With respect to claims 60-62, as stated supra, GigaScience teaches a method of generating chromosome-level genome assembly of a NHP (e.g., abstract) and teaches the study animal’s PBMCs are used to generate iPSCs (p. 9, left col, last para “Genome annotation”). GigaScience teaches a subset of PBMCs are used for the extraction of high molecular weight (HMW) DNA using the MagAtrract HMW DNA Kit. The average size of the extracted HMW DNA is ∼53 kb (Supplementary Fig. S1B) (p. 8, left col, para “Study subject, sample preparation and DNA extraction”). GigaScience teaches preparing two different genomic libraries (a linked-read library and a HiC library), sequencing the libraries on Illumina HiSeq 4000, and assembling and scaffolding the data to generate an genome assembly (p. 8, left col, last para – right col). GigaScience further teaches annotating the genome using RNAseq and proteomics data from the study animal’s PBMCs and iPSCs (generated from the PBMCs) (p. 9, left col, last para “Genome annotation”). Thus, GigaScience teaches the method further comprises generating an individualized genome assembly of the at least one individual wherein the genome assembly is matched to an iPSC line derived from the at least one individual such that a genomic sequence of the iPSC line corresponds to the individualized genome assembly of the at least one individual in claim 60, the method further comprises extracting HMW DNA from the second subset of PBMCs by purification of archival quality DNA in claim 61, and the method further comprises preparing a genome library from the HMW DNA, sequencing fragments of the HMW DNA in the library, and assembling and annotating the fragments to recreate the genome of the individual from which the PBMCs were obtained in claim 62.
However, GigaScience, Roodgar and Rogers are silent on a step of administering the iPSC line autologously in one translational in vivo validation study conducted in the same individual from which the iPSC line was derived in claim 60.
Nevertheless, Rogers teaches nonhuman primates are used as models for human diseases in the scope of biomedical research (p. 357, right col, para 1).
Madrid summarizes progress toward clinical translation of autologous iPSC-based cell therapies (see e.g., Table 1 in p. 4-5 for clinical trials and preclinical stages of development). Madrid teaches autologous iPSC-based cell therapies have promising outcomes and advantages such as a cell therapy product that can engraft without the risk of immune rejection, eliminating the need for immunosuppression and the associated side effects, and enabling autologous iPSC-based therapies to become a more commonplace treatment modality for patients (e.g., abstract).
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method for deriving iPSCs from a non-human primate used as a model for human diseases in biomedical research as suggested by GigaScience, Roodgar and Rogers, by combining a step of administering the iPSC line autologously in a translational in vivo validation study as suggested by Madrid with a reasonable expectation of success. Since Rogers teaches nonhuman primates are used as models for human diseases in the scope of biomedical research (p. 357, right col, para 1), and since Madrid teaches autologous iPSC-based cell therapies have promising outcomes in preclinical and clinical trials and have advantages such as a cell therapy product that can engraft without the risk of immune rejection (e.g., abstract), one of ordinary skill in the art would have had a reason to use the non-human primate as a model for human diseases and to administer the iPSC line autologously to the same study animal in a preclinical translational in vivo validation study as suggested by Rogers and Madrid in order to make progress toward clinical translation of autologous iPSC-based cell therapies to take advantage of the promising outcomes and reduced risk of immune rejection.
In regard to the long-read fragments in claim 62, as argued by Applicant, the term “long-read” is examined as being exemplified as being at least about 10,000 base pairs but may also be between about 5,000 base pairs to about 50,000 base pairs disclosed in specification [00122].
However, GigaScience, Roodgar, Rogers and Madrid are silent on long-read fragments that are about 10,000 base pairs in claim 62.
Nevertheless, Rogers teaches improved assemblies with longer contigs and more complete coverage in high-quality sequence data (that is, comprehensive delineation of segmental duplications, and fewer genes with gaps and errors) are needed and new technologies that provide longer reads will yield better assemblies by filling remaining gaps. The Pacific Biosciences RS II platform is one plausible option for upgrading the quality of primate genomes (Ref# 105) (see p. 356, right col, para 2. It is noted that reference #105 is titled “Mind the gap: upgrading genomes with Pacific Biosciences RS long-read sequencing technology”).
Jayakumar teaches generating a genome assembly of nonhuman primate by sequencing and assembling long reads using Pacific Biosciences (referred to by Rogers) with a read N50 length of 12.05 kbp (i.e., about 12,050 base pairs. See abstract, p. 2, para “Sample preparation, sequencing, and de novo assembly”, also see Fig 1a). Thus, Jayakumar teaches a method of sequencing long-read fragments (N50 length of 12.05 kbp) to assemble a genome of nonhuman primate.
Therefore, it would have been obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the method of generating a genome assembly of a cynomolgus macaque by sequencing DNA fragments disclosed by GigaScience evidenced by Roodgar and in view of Rogers and Madrid, by substituting the DNA fragments with long-read fragments suggested by Rogers and taught by Jayakumar with a reasonable expectation of success. Since Rogers suggests new technologies, e.g., the Pacific Biosciences RS II platform, that provide longer reads will yield better assemblies by filling remaining gaps for upgrading the quality of primate genomes (see p. 356, right col, para 2), and since Jayakumar teaches single-molecule long-read sequencing has drastically increased the contiguity of assemblies (p. 2, para 2) and teaches the resulted genome assemblies have high fidelity and outperform all the available assemblies of this species in terms of contiguity and are valuable resources for non-human primate models and provide an important baseline in human biomedical research (abstract), one of ordinary skill in the art would have had a reason to make this substitution in order to generate a genome assembly with high fidelity and increased contiguity to be used as a resource for non-human primate models and as an important baseline in human biomedical research (Jayakumar, abstract).
Hence, the claimed invention as a whole was prima facie obvious to a person of ordinary skill before the effective filing date of the claimed invention in the absence of evidence to the contrary.
Response to Traversal:
Applicant’s arguments filed on 10/09/2025 are acknowledged and have been discussed above.
Conclusion
No claims are allowed.
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/JIANJIAN ZHU/Examiner, Art Unit 1631