DETAILED ACTION
*Note that a new Examiner has been assigned to this application. Please direct all future correspondence to Examiner Katie Pennington, AU 1634. Additional contact information can be found at the end of this paper.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
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. Applicant’s Request for Continued Examination received on 17 June 2025, and Amendment and Arguments/Remarks received on 28 April 2025 have been entered.
Claims 1, 5-7, 12-13, 16-17, 19-20, 30-32, 34-35, 60-62, and 69 were previously pending in the application. Claims 12-13 have been cancelled by Applicant. Claims 1, 60, and 62 are independent claims.
The election of the invention of Group I, drawn to a method for determining the presence and/or level of contamination by pluripotent stem cell (PSC) contaminants in a PSC-derived cell population for further use, remains in effect in the instant application. Claims 60-62 remain withdrawn from consideration as being directed to a nonelected invention.
Claims 1, 5-7, 16-17, 19-20, 30-32, 34-35, and 69 are currently pending and under examination in the instant application. An action on the merits follows.
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Specification
The specification of the disclosure filed 04 December 2020 is newly objected to because the specification lacks a Brief Description of the Drawings, as set forth in 37 CFR 1.74:
BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S): See MPEP § 608.01(f). A reference to and brief description of the drawing(s) as set forth in 37 CFR 1.74.
Additionally, the use of the term “SurePrint G3 Human v16 microRNA 8x60K” on page 29 line 6, “AgiMicroRNA” on page 29 line 21, “Exiqon’s LNA” on page 30 line 14, “Exiqon’s Pick&Mix” on page 30 line 20, “, which is a trade name or a mark used in commerce, has been noted in this application. The term should be accompanied by the generic terminology; furthermore the term should be capitalized wherever it appears or, where appropriate, include a proper symbol indicating use in commerce such as ™, SM , or ® following the term.
Although the use of trade names and marks used in commerce (i.e., trademarks, service marks, certification marks, and collective marks) are permissible in patent applications, the proprietary nature of the marks should be respected and every effort made to prevent their use in any manner which might adversely affect their validity as commercial marks.
Appropriate correction is required.
Withdrawn objections/ Rejections in response to Applicants’ arguments or amendments
Claim Rejections - 35 USC § 103
The rejection of amended, previously presented, and cancelled claims 1, 5-7, 12-13, 16-17, 20, 30, 32 and 34-35 under 35 U.S.C. 103 as being unpatentable over Parr et al., 2016 (Nature Scientific Reports, 6: 32532, p. 1-14, IDS) in view of Asikainen et al., 2015 (PLoS ONE, 10(3): e0116668, p. 1-25), is withdrawn in view of cited art not teaching the level of sensitivity wherein the method is configured to determine whether the level of contamination by PSC contaminants meets the criterion of ten or fewer PSC contaminant cells per one million cells of the sample, as recited in independent claim 1.
A response to Applicant’s arguments with regard to a withdrawn rejection is moot. A response to any argument pertaining to a new or maintained rejection can be found below.
The rejection of amended previously presented claims 1, 17, and 19 under 35 U.S.C. 103 as being unpatentable over Parr et al., 2016 (Nature Scientific Reports, 6: 32532, p. 1-14, IDS) in view of Asikainen et al., 2015 (PLoS ONE, 10(3): e0116668, p. 1-25) as applied to claims 1, 5-7, 12-13, 16-17, 20, 30, 32 and 34-35 above, and further in view of Bours et al., 2016 (WO 2016/150475 A1), is withdrawn in view of cited art not teaching the level of sensitivity wherein the method is configured to determine whether the level of contamination by PSC contaminants meets the criterion of ten or fewer PSC contaminant cells per one million cells of the sample, as recited in independent claim 1.
A response to Applicant’s arguments with regard to a withdrawn rejection is moot. A response to any argument pertaining to a new or maintained rejection can be found below.
The rejection of amended previously presented claims 1 and 31 under 35 U.S.C. 103 as being unpatentable over Parr et al., 2016 (Nature Scientific Reports, 6: 32532, p. 1-14, IDS) in view of Asikainen et al., 2015 (PLoS ONE, 10(3): e0116668, p. 1-25) as applied to claims 1, 5-7, 12-13, 16-17, 20, 30, 32 and 34-35 above, and further in view of Trivedi et al., 2008 (Exp Hematol., 36(3): p. 1-16), is withdrawn in view of cited art not teaching the level of sensitivity wherein the method is configured to determine whether the level of contamination by PSC contaminants meets the criterion of ten or fewer PSC contaminant cells per one million cells of the sample, as recited in independent claim 1.
A response to Applicant’s arguments with regard to a withdrawn rejection is moot. A response to any argument pertaining to a new or maintained rejection can be found below.
The rejection of amended previously presented claims 1 and 69 under 35 U.S.C. 103 as being unpatentable over Parr et al., 2016 (Nature Scientific Reports, 6: 32532, p. 1-14, IDS) in view of Asikainen et al., 2015 (PLoS ONE, 10(3): e0116668, p. 1-25) as applied to claims 1, 5-7, 12-13, 16-17, 20, 30, 32 and 34-35 above, and further in view of Witwer et al., 2013 (RNA Biology, 10:7, p. 1080-1086), is withdrawn in view of cited art not teaching the level of sensitivity wherein the method is configured to determine whether the level of contamination by PSC contaminants meets the criterion of ten or fewer PSC contaminant cells per one million cells of the sample, as recited in independent claim 1.
A response to Applicant’s arguments with regard to a withdrawn rejection is moot. A response to any argument pertaining to a new or maintained rejection can be found below.
New Grounds of Objection/ Rejections in response to Applicants’ arguments or amendments
Claim Objections
Amended independent claim 1 is newly objected to because of the following informalities: claim 1 recites “where” in line 6 which appears to be a typographical misspelling of “wherein”. Appropriate correction is required.
Amended and previously presented claims 1, 16, 19 are newly objected to because of the following informalities: abbreviations such as miRNA should be spelled out at the first encounter in the claims. Appropriate correction is required.
Previously presented claim 7 is newly objected to because of the following informalities: claim 7 recites “a sample PSC-derived cells” in line 2 which appears to be a typographical misspelling of “a sample of PSC0derived cells”. Appropriate correction is required.
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.
Amended and previously presented claims 1, 5-7, 16-17, 19-20, 30-32, 34-35, and 69 are newly 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.
Independent claim 1 has multiple issues of indefiniteness.
Independent claim 1 recites, “the presence” in line 1. There is insufficient antecedent basis for this limitation in the claim.
Independent claim 1 recites, “pluripotent stem cell (PSC) contaminants” or “PSC contaminants” in lines 2, 5, 14, 16-17, and 17, which is indefinite because it is unclear whether the PSCs themselves are the contaminating substance or whether the PSCs are being contaminated by some other substance. Examiner suggests rephrasing “PSC contaminants” as “contaminating PSCs” to clarify that the pluripotent stem cells themselves are contaminating the PSC-derived cell population.
Independent claim 1 recites, “against” in line 4, which is indefinite because it is unclear what Applicant intends to encompass by reciting assaying a cell population “against” a miRNA in that the word “against” implies an antagonism or contradiction between the cell population and the miRNA. Therefore, it is unclear what assaying steps are intended to be encompassed by assaying against the miRNA. Claim 1 recites that the assaying step comprises treating, analyzing, measuring, and determining, none of which incorporate an antagonism or contradiction between the cell population and the miRNA.
Independent claim 1 recites, “known or determined to be” in line 5, which is indefinite because it is unclear if Applicant intends to encompass miRNAs known to be differentially expressed in PSC contaminants at the time of filing or any time prior to implementation of the method, or whether Applicant intends to encompass all differentially expressed miRNAs regardless of when they may be/have been determined to be differentially expressed.
Independent claim 1 recites, “differentially expressed in PSC contaminants” in line 5, which is a relative term which renders the claim indefinite. The term “differentially expressed” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. The claim does not indicate what the miRNA expression is differentially expressed relative to. Additionally, the specification does not provide a limiting definition for “differentially expressed in PSC contaminants” which would define what the differential expression is relative to.
Independent claim 1 also recites, “where one or a panel of miRNAs one or more miRNAs selected from a group consisting of” in lines 6-7, which has multiple issues of indefiniteness. Firstly, the quoted text is indefinite because there is no connection between “one or a panel of miRNAs” and “one or more miRNAs selected from the group consisting of”, such that the two phrases are juxtaposed without any indication of the relationship between the two phrases or other transitions words to indicate context for the two phrases being juxtaposed.
Secondly, to the extent that the “one or a panel of miRNAs” are meant to contain the “one or more miRNAs selected from the group consisting of”, it unclear whether the “one or a panel of miRNAs” comprise, consist of, or consist essentially of the “one or more miRNAs selected from the group consisting of”.
Thirdly, there is no connection between “where one or a panel of miRNAs one or more miRNAs selected from a group consisting of” recited in lines 6-7 and the “one or a panel of two or more pre-determined miRNAs” recited in lines 4-5, such that it is unclear if Applicant intends for the “one or a panel of miRNAs” recited in line 6 and/or the “one or more miRNAs selected from a group consisting of” to be the “one or a panel of two or more pre-determined miRNAs” recited in lines 4-5 or to be alternative/additional miRNAs.
Independent claim 1 recites, “determining therefrom the presence and/or level of contamination” in line 12, which is indefinite because it is unclear what the presence and/or level of contamination is being determined from.
Independent claim 1 recites, “configured to determine” in line 16, which is indefinite because it is unclear whether the “to determine” recited in line 16 is meant to further limit the determining step of line 12-13, or whether the “to determine” is meant to encompass any step of determining.
As such, the metes and bounds of the claim cannot be determined.
Previously presented claims 5, 6, 7, 17, 34, and 35 each recite, “PSC contaminants”, which is indefinite because it is unclear whether the PSCs themselves are the contaminating substance or whether the PSCs are being contaminated by some other substance. As such, the metes and bounds of the claims cannot be determined.
Previously presented claim 5 has multiple additional issues of indefiniteness.
Previously presented claim 5 recites, “wherein the method comprises determining the level of PSC contaminants in a sample of the PSC-derived cell population”, which has multiple issues of indefiniteness in addition to those addressed above. Claim 5 is dependent on claim 1. Firstly, it is unclear whether this step of “determining” is meant to be the same step of determining recited in independent claim 1, or whether it is meant to be an additional step, such that the method is further comprising the determining step recited in claim 5. As such, it is likewise unclear whether “a sample of the PCS-derived cell population” recited in line 2 of claim 5 is the same sample recited in claim 1 line 4.
Further, recitation of “the level of PSC contaminants” lacks antecedent basis in the claim. Independent claim 1 recites “level of contamination by pluripotent stem cell (PSC) contaminants” in lines 1-2, “a level of the one or a panel of two or more pre-determined miRNAs” in lines 9-10, and “level of contamination by PSC contaminants” in lines 12 and 16, but neither claim 5 nor claim 1 have any prior recitation of “a level of PSC contaminants”.
Claim 5 also recites, “a known or pre-determined level of PSC contaminants”, which is indefinite because it is unclear if Applicant intends to encompass levels of PSC contaminants known at the time of filing or any time prior to implementation of the method, or whether Applicant intends to encompass all levels of PSC contaminants regardless of when they may be/have been determined.
As such, the metes and bounds of the claim cannot be determined.
Previously presented claim 6 recites, “in an amount of 10 cell per million”, which is indefinite because it is unclear what “million” is meant to modify. As such, the metes and bounds of the claim cannot be determined.
Previously presented claim 7 recites, “grown out”, which is informal language which contributes to ambiguity of the claim because it is unclear what the cells have been grown out of, in what other way the cells have been “grown out”, or whether “grown out” is meant to claim that the sample of PSC-derived cells has been propagated or cultured in vitro. Therefore, it is unclear what is encompassed by the term “grown out”. As such, the metes and bounds of the claim cannot be determined.
Claim 7 recites, “PSC contaminants are negligible” in line 3, which is a relative term which renders the claim indefinite. The term “PSC contaminants are negligible” is not defined by the claim, the specification does not provide a standard for ascertaining the requisite degree, and one of ordinary skill in the art would not be reasonably apprised of the scope of the invention. It is not clear in reference to what extent the absence of the PSCs is completed.
Previously presented claim 17 recites, “by way of a normalizer or a control” in line 4, which is indefinite because it is unclear what Applicant intends to encompass as “by way of a normalizer or a control”. For example, it is unclear whether “by way of a normalizer or a control” is merely indicating the purpose of the assaying step or whether “by way of a normalizer or a control” is meant to further limit the assaying step. Additionally, it is unclear what is encompassed by the phrase “by way of”, such that it is unclear whether additional method steps are meant to be encompassed. As such, the metes and bounds of the claim cannot be determined.
Previously presented claim 20 recites, “wherein the method comprises assaying”, which is indefinite because it is unclear whether the step of assaying recited in claim 20 is meant to be the same step of assaying recited in independent claim 1, upon which claim 20 depends, or whether the step of assaying recited in claim 20 is meant to be an additional step of assaying, in that the method further comprises the step of assaying as recited in claim 20. As such, it is unclear whether “a sample of the PSC-derived cell population” recited in claim 5 is the same sample of the PSC-derived cell population recited in line 8 of claim 1.
Additionally, claim 20 recites, “assaying a sample of the PSC-derived cell population with a negative control comprising an uncontaminated cell sample”, which is indefinite because it is unclear how a sample of the PSC-derived cell population can be assayed with a negative control comprising another cell sample. For example, it is unclear whether the sample of the PSC-derived cell population is assayed side-by-side with the negative control sample such that they are processed in tandem, but separately, or whether the sample of the PSC-derived cell population is assayed mixed with the negative control sample such that the two samples are combined into a single composition for assaying.
As such, the metes and bounds of the claim cannot be determined.
Previously presented claim 31 recites, “the PSC-derived cells”, which lacks antecedent basis in the claim. Claim 31 is dependent on claim 1. Claim 1 recites “a PSC-derived cell population” in line 2 and “the PSC-derived cell population” in lines 4 and 8, but has no recitation of PSC-derived cells themselves. As such, the metes and bounds of the claim cannot be determined.
Previously presented claim 32 recites, “the PSCs”, which has insufficient antecedent basis in the claim. Claim 32 is dependent on claim 1. Claim 1 recites, “pluripotent stem cell (PSC) contaminants” in line 2; “PSC-derived cell population” in lines 2, 4, and 8; “PSC contaminants” in lines 5, 12, 14, 16-17, and 17; “undifferentiated PSCs” in line 14; and “incompletely differentiated PSCs” in line 15. Therefore, it is unclear to which PSCs “the PSCs” of claim 32 is referring. As such, the metes and bounds of the claim cannot be determined.
Previously presented claim 34 recites, “1 cell and/or 5 cells and/or 10 cells per million”, which is firstly indefinite because it is unclear whether Applicant intends for each of the recited numbers of cells to be “per million”, such that the amount is 1 cell per million and/or 5 cells per million and/or 10 cell per million wherein all amounts are relative, or whether Applicant intends for the amounts to be 1 cell (absolute) and/or 5 cells (absolute) and/or 10 cells per million (relative). Secondly, it is further indefinite because per million is not modifying any noun. As such, it is unclear what the 10 cells are per million of. For example, it unclear whether Applicant intends to encompass an amount of 10 PSC contaminant cells per million cells of the sample, an amount of 10 PSC contaminant cells per million cells of the total sample + PSC contaminants composition, an amount of 10 PSC contaminant cells per million PSC contaminant cells, or a million of some other entity. As such, the metes and bounds of the claim cannot be determined.
Previously presented claims 16, 30, and 69 are included in this rejection due to their dependence on independent claim 1.
Claim Interpretation
Note that independent claim 1 recites “contamination by pluripotent stem cell (PSC) contaminants in a PSC-derived cell population”, and “wherein the PSC contaminants comprise one or more of undifferentiated PSCs and incompletely differentiated PSCs”. The claim does not require that the PSC-derived cell population be a population of cells completely differentiated from PSCs. As such, “PSC-derived cell population” has been interpreted as any cell population which has been derived from PSCs through any method of culturing or modification and is not limited to cells differentiated from PSCs. Also, “PSC contaminants” has been interpreted as any PSC or PSC-derived cell which is unwanted in any PSC-derived cell population. Further, “incompletely differentiated” is interpreted to encompass any differentiation state which is not the desired differentiation state.
Additionally, Independent claim 1 recites, “incompletely differentiated PSCs” in line 15, which is a relative term. However, the specification provides a limiting definition for “incompletely differentiated”, specifically: “By incompletely differentiated PSCs, it is meant PSCs that have not completely differentiated to the cell of the derived cell population, but may be at some intermediate stage, such as a multi-potent stage of differentiation” [page 8 lines 15-16].
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(a) which forms the basis for all obviousness rejections set forth in this Office action:
(a) A patent may not be obtained though the invention is not identically disclosed or described as set forth in section 102 of this title, if the differences between the subject matter sought to be patented and the prior art are such that the subject matter as a whole would have been obvious at the time the invention was made to a person having ordinary skill in the art to which said subject matter pertains. Patentability shall not be negatived 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(a) 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 under 35 U.S.C. 103(a), the examiner presumes that the subject matter of the various claims was commonly owned at the time any inventions covered therein were made absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and invention dates of each claim that was not commonly owned at the time a later invention was made in order for the examiner to consider the applicability of 35 U.S.C. 103(c) and potential 35 U.S.C. 102(e), (f) or (g) prior art under 35 U.S.C. 103(a).
Amended and previously presented claim(s) 1, 5-7, 16-17, 19-20, 30-32, 34-35, and 69 are newly rejected under 35 U.S.C. 103 as being unpatentable over Parr et al. [2016, Nature Scientific Reports, 6: 32532, p. 1-14, IDS, cited in a prior action]; in view of Asikainen et al. [2015, PLoS ONE, 10(3): e0116668, p. 1-25, cited in a prior action]; Zhao et al. [2016, US20160231324A1]; Blumenreich [1990, The White Blood Cell and Differential Count. In: Walker HK, Hall WD, Hurst JW, editors. Clinical Methods: The History, Physical, and Laboratory Examinations. 3rd edition. Boston: Butterworths; Chapter 153]; Bours et al. [2016, WO2016150475A1, cited in a prior action]; and Trivedi et al. [2008, Exp Hematol., 36(3): p. 1-16, cited in a prior action].
Regarding claim 1, Parr teaches microRNA-302a-5p (miR-302a) is highly and specifically expressed in human pluripotent stem cells and gradually decreases to basal levels during differentiation. A new RNA tool, miR-switch as a live-cell-reporter mRNA for miR-302a activity, can specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population and can prevent teratoma formation in an in vivo tumorigenicity assay (e.g., Abstract). MicroRNA switches (miR-switches) were encoded on modified mRNA (modRNA) that post-transcriptionally regulates fluorescent reporters in response to the activity of the human miRNA-302/367 cluster expressed in living cells (Fig. 1a, bottom). Detection of spiked hiPSCs can be down to 0.05% of the total cell number using the miR-302a switch (e.g., p. 2, 2nd full paragraph).
Specific and high level of expression of both hsa-miR-302a-5p and hsa-miR-367-3p was found in three feeder-free human iPSC lines, and expression of miR-302/367 in the iPSC lines is 102-6-fold greater than spontaneously differentiated 201B7 cells, 201B7-derived MDA cells, standard culture lines of NHDF and Hela, and primary hepatocytes and renal cells (e.g., p. 2, 3rd full paragraph). ModRNA encoding miR switches that can sensitively and dynamically respond to the activities of miR-302a and miR-367 in living cells was in vitro transcribed. Translational repression of the reporter protein (human codon optimized azamin green, hmAG) occurs when the mature miRNA binds to an antisense sequence that is optimally placed within the 5’ UTR of the mRNA (Fig. 1a). Tag blue fluorescent protein, tagBFP, was used as an internal fluorescent control to normalize this reporter expression (e.g. p. 2, 4th full paragraph). iPSC technology holds great promise for regenerative medicine and allows for personalized medicine that give targeted therapy without immune complication. There is a need to not only make sure transplanted cells are devoid of residual pluripotent cells but also partially differentiated cells (e.g., p. 1, last two paragraphs).
Parr teaches that miR-302a switch effectively separates hiPSCs and hiPSC-derived differentiated cells. miR-302a switch distinguishes hiPSCs and differentiated cells (e.g., p. 2, last paragraph) (for claim 32).
Further regarding claim 1, Parr does not specifically teach one or a panel of two or more miRNAs selected from hsa-miR-302a-3p, hsa-miR-302b-3p, hsa-miR-302c-3p and hsa-miR-302d-3p.
Asikainen teaches “small RNA molecules, including microRNAs (miRNAs), play critical roles in regulating pluripotency, proliferation and differentiation of embryonic stem cells. miRNA-offset RNAs (moRNAs) are similar in length to miRNAs, align to miRNA precursor (pre-miRNA) loci and are therefore believed to derive from processing of the pre-miRNA hairpin sequence”. Certain moRNA isoforms are notably expressed in hESCs (human embryonic stem cells) from loci coding for stem cell-selective or cancer-related miRNA clusters, and in contrast, only sparse moRNAs in fibroblasts (e.g., Abstract). Table 2 shows 15 most overexpressed miRNAs in hESC lines when compared to HEF-1 foreskin fibroblast by statistical computing using DEseq algorithm. In table 2, the overexpressed miRNA in hESC as compared to foreskin fibroblast include hsa-miR-302a-5p, hsa-miR-302d-3p, hsa-miR-302a-3p, hsa-miR-302b-3p, hsa-miR-302c-3p and hsa-miR-367-3p (e.g., page 4, Table 2). Total RNA was extracted for qRT-PCR with miRVana miRNA isolation kit and reverse transcribed with TaqMan reverse transcription-kit. RNU44 (001094) was used as an endogenous control to quantify pre-miR-103a-2 derivatives (two miRs and two moRs). RNS6B (001093) was used as an endogenous control to quantify hESC/HEF-1 miRNAs (e.g., p. 20, 1st full paragraph).
Therefore, an ordinarily skilled artisan at the time of filing the instant application would have been motivated to use any one of the 15 microRNAs taught by Asikainen, including hsa-miR-302d-3p, hsa-miR-302a-3p, hsa-miR-302b-3p, and/or hsa-miR-302c-3p, as hESC-specific miRNAs for differentiating hESCs from more differentiated cells types.
Further regarding claim 1, the combination of Parr and Asikainen does not specifically teach to determine whether the level of contamination by PSC contaminants meets the criterion of 10 or fewer PSC contaminant cells/million cells, nor that the assay is performed by qRT-PCT, digital PCR, or droplet digital PCR.
However, Zhao teaches a droplet digital detection system (e.g., a droplet digital PCR system), wherein the detection system comprises a nucleic acid-based sensing system for detection of single cells [abstract, 0262]. Zhao teaches that the system can be used to detect rare circulating tumor cells in blood and assess miRNAs using droplet digital RT-PCR [0202, 0262, 0320] with single particle sensitivity from mL volumes [0219], including one circulating tumor cell (CTC) present per 107 leukocytes (e.g., 1 target cell in 10 million total cells) [0256, 0327].
Zhao teaches that the ability to rapidly and selectively detect low abundance targets is crucially important to elucidate new biology, to monitor or detect a disease or disorder, to monitor therapeutic responses, and to develop new therapeutics [0003]. Zhao also teaches that not only is their method highly sensitive at detecting rare cells in a heterogenous population, but their system is high-throughput, multiplexable, and rapid [abstract]. Zhao further teaches that microfluids methods such as their invention have advantages of negligible evaporation of reagents, minimal consumption of expensive biological reagents, low fabrication costs, reduced analysis time, and the ability to integrate various functional components on a single chip [0009].
Therefore, an ordinarily skilled artisan would have been motivated to use a digital droplet PCR system to detect rare cells in a cell population, including one circulating tumor cell (CTC) present per 107 leukocytes (e.g., 1 target cell in 10 million total cells).
Zhao further teaches assaying a negative control cell population comprising uncontaminated cells (e.g., donor blood alone without any spiked in bacterial cells) and positive control samples comprising cell populations spiked with target cells (e.g., donor blood with spiked in bacterial cells) at levels of 1-50 cells/mL of whole blood, including 1 cell, 10 cells, 100 cells, 1000 cells, and 10,000 cells [0144-0148, 0316, Figure 22, 23]. Blumenreich teaches that the blood leukocyte concentration ranges from 4 million to 10 million per mL [column 1 ¶ 1]. Therefore, Zhao teaches spiking in 1 cell per million cells (e.g., 10 cells/mL blood), 5 cells per million (e.g., 5 cells/mL blood) 10 cells per million (e.g., 100 cells/mL blood). Therefore, an ordinarily skilled artisan would have had a reasonable expectation of success in detecting rare cells in a cell population with a sensitivity at least as low as 1 cell in a million cells of the cell sample.
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to use one or a panel of two or more miRNAs selected from hsa-miR-302a-3p, hsa-miR-302b-3p, hsa-miR-302c-3p and hsa-miR-302d-3p for determining the presence of PSC contaminants in a PSC-derived cell population because Parr teaches MicroRNA-302a-5p (miR-302a) is highly and specifically expressed in human pluripotent stem cells and gradually decreases to basal levels during differentiation, and can be used to specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 0.05% of hiPSCs in a heterogeneous population. Further, Asikainen teaches the overexpressed miRNA in hESC as compared to foreskin fibroblast include hsa-miR-302a-5p, hsa-miR-302d-3p, hsa-miR-302a-3p, hsa-miR-302b-3p, and hsa-miR-302c-3p. Both Parr and Asikainen teach hsa-miR-302a-5p are specific and overexpressed in human pluripotent stem cells. Additionally, Zhao teaches the detection of rare cells in a cell population as low as 1 in 10 million cells using droplet digital PCR. Accordingly, it would be obvious for one of ordinary skill in the art to substitute the hsa-miR-302a-5p and/or hsa-miR-367-3p taught by Parr with the hsa-miR-302d-3p, hsa-miR-302a-3p, hsa-miR-302b-3p and/or hsa-miR-302c-3p as taught by Asikainen for the use in a method of detecting pluripotency-specific miRNAs in order to specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of hiPSCs of 1 in 10 million in a heterogeneous population with a reasonable expectation of success.
One having ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to do so in order to specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 1 hiPSC in 10 million cells in a heterogeneous population to prevent teratoma formation in an in vivo tumorigenicity assay as taught by Parr, Asikainen, and Zhao with reasonable expectation of success.
Regarding claims 5-7, 20, and 34-35 as discussed above, Zhao teaches assaying a negative control cell population comprising uncontaminated cells (e.g., donor blood alone without any spiked in bacterial cells) and positive control samples comprising cell populations spiked with target cells (e.g., donor blood with spiked in bacterial cells) at levels of 1-50 cells/mL of whole blood, including 1 cell, 10 cells, 100 cells, 1000 cells, and 10,000 cells [0144-0148, 0316, Figure 22, 23]. Blumenreich teaches that the blood leukocyte concentration ranges from 4 million to 10 million per mL [column 1 ¶ 1]. Therefore, Zhao teaches spiking in 1 cell per million cells (e.g., 10 cells/mL blood), 5 cells per million (e.g., 5 cells/mL blood) 10 cells per million (e.g., 100 cells/mL blood).
Regarding claim 16, as discussed above, Asikainen teaches the motivation to use any one of the 15 microRNAs taught by Asikainen, including hsa-miR-302b-3p, as hESC-specific miRNAs for differentiating hESCs from more differentiated cells types.
Regarding claims 30 and 32, as discussed above, Parr teaches that iPSC technology holds great promise for regenerative medicine and allows for personalized medicine that give targeted therapy without immune complication. Parr also teaches that here is a need to not only make sure transplanted cells are devoid of residual pluripotent cells but also partially differentiated cells (e.g., p. 1, last two paragraphs). Therefore, Parr teaches wherein the PSCs are iPSCs and the intended further use is for cell therapy.
Regarding claim 69, as discussed above, Zhao further teaches wherein the treating and analyzing comprises droplet digital PCR [0202, 0234, 0308].
Regarding claims 17 and 19, the combination of Parr, Asikainen, Zhao, and Blumenreich does not specifically teach using the endogenous non-coding RNA hsa-miR-107 or hsa-miR-130a-3p for normalization or control.
Bours teaches an in vitro method of diagnosing breast cancer by measuring the expression level of microRNAs including miR-16, let-7d and miR-103 in the test biological fluid sample from a subject, comparing the expression level to a reference value of a control biological fluid sample from a healthy subject (e.g., Abstract, claim 1). The expression level in the test or control biological fluid sample is normalized with a mean expression level of the fifty most expressed microRNAs in said test or control biological fluid sample, and the most expressed microRNAs include miR-107 (e.g., claims 12 and 14, Table 3, p. 34-35).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention to use the endogenous non-coding RNA hsa-miR-107 or hsa-miR-130a-3p for normalization or control because Bours teaches miR-107 is one of the fifty most expressed microRNA in the biological sample and teaches using miR-107 as an internal control for normalizing expression levels of other microRNAs in test biological fluid samples. It would be obvious for one of ordinary skill in the art to use hsa-miR-107 as a non-coding and non-specific microRNA to normalize expression level of hsa-miR-302a-3p, hsa-miR-302b-3p, hsa-miR-302c-3p and/or hsa-miR-302d-3p in cells of interest taught by Parr and Asikainen to provide accurate reading of the expression level of hsa-miR-302a-3p, hsa-miR-302b-3p, hsa-miR-302c-3p and/or hsa-miR-302d-3p with reasonable expectation of success.
One having ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to do so in order to specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 1 hiPSC in 10 million cells in a heterogeneous population to prevent teratoma formation in an in vivo tumorigenicity assay as taught by Parr, Asikainen, Zhao, and Blumenreich with reasonable expectation of success.
Regarding claim 31, Parr, Asikainen, Zhao, Blumenreich, and Bours do not specifically teach the PSC-derived cells are mesenchymal stem cells (MSCs).
Trivedi teaches “Derivation and immunological characterization of mesenchymal stromal cells from human embryonic stem cells” (e.g., Title). Undifferentiated ESCs (embryonic stem cells) were cultured on Matrigel-coated plates without feeder cells. The MSCs derived from human ESCs have morphology, cell surface marker characteristics, and differentiation potential similar to adult BM-derived MSCs. MSCs can be derived from human ESCs without feeder cells and the MSCs have cell surface markers, differentiation potentials and immunological properties in vitro that are similar to adult BM-derived MSCs (e.g., Abstract).
It would have been prima facie obvious for one of ordinary skill in the art before the effective filing date of the claimed invention that the PSC-derived cells are mesenchymal stem cells (MSCs) because Parr teaches miR-302a switch effectively separates hiPSCs and hiPSC-derived differentiated cells, and miR-302a switch distinguishes hiPSCs and differentiated cells. Asikainen also teaches that hsa-miR-302a-5p, hsa-miR-302a-3p, hsa-miR-302b-3p, hsa-miR-302c-3p, and hsa-miR-302d-3p are all part of the miR-302/367 hESC-specific cluster and are among the top 15 most overexpressed miRNAs in hESCs compared to HFF-1 foreskin fibroblasts [page 4 ¶ 1, Table 2]. Since Parr teaches PSC-derived cells can be various types of differentiated cells and Trivedi teaches MSCs can be derived from human ESCs without feeder cells, it would be obvious for one of ordinary skill in the art to use MSCs as the PSC-derived differentiated cells in the method of Parr in order to test separation of PSC and PSC-derived MSC by using the method taught by Parr, Asikainen, and Zhao with reasonable expectation of success.
One having ordinary skill in the art before the effective filing date of the claimed invention would have been motivated to do so in order to specifically detect human induced pluripotent stem cells (hiPSCs) down to a spiked level of 1 hiPSCs in 10 million cells in a heterogeneous population to prevent teratoma formation in an in vivo tumorigenicity assay as taught by Parr with reasonable expectation of success.
Insofar as Applicant’s arguments apply to this new rejection, Applicant argues that:
the amendment to independent claim 1 closes the group from which the miRNAs are selected to a group that consists of only those listed in the claim 1, and as such, the combination of Parr and Asikainen fails to teach each and every element recited in claim 1;
in order to arrive at amended claim 1, a PHOSITA would have to both select a different miRNA and apply a different detection technique to that described by Parr to achieve an improvement in sensitivity of fifty times, and there is nothing in Parr that would lead the PHOSITA to adapt the teaching thereof to select a different miRNA than taught by Parr;
Asikainen fails to bolster the deficiencies of Parr such that the combination of Parr and Asikainen fails to teach the instant invention, particularly given the chemical unpredictability in the effects of in vivo expression of various species; and
Examples 3-6 in the present application demonstrate the surprising result in sensitivity of the specific mRNAs recited in claim 1 and, given that these were not expressed in any of the non-pluripotent potential derived cell types, were therefore suitable assay development candidates for detection of residual contaminating undifferentiated PSCs in derived cell products.
However, this is not agreed.
In response to applicant’s arguments against the references individually, it is noted that the test for obviousness is not whether the features of a secondary reference may be bodily incorporated into the structure of the primary reference; nor is it that the claimed invention must be expressly suggested in any one or all of the references. Rather, the test is what the combined teachings of the references would have suggested to those of ordinary skill in the art. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981). One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. See In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Further, the examiner recognizes that obviousness may be established by combining or modifying the teachings of the prior art to produce the claimed invention where there is some teaching, suggestion, or motivation to do so found either in the references themselves or in the knowledge generally available to one of ordinary skill in the art. See In re Fine, 837 F.2d 1071, 5 USPQ2d 1596 (Fed. Cir. 1988), In re Jones, 958 F.2d 347, 21 USPQ2d 1941 (Fed. Cir. 1992), and KSR International Co. v. Teleflex, Inc., 550 U.S. 398, 82 USPQ2d 1385 (2007). In addition, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Specifically, regarding argument 1), note that due to the indefiniteness of amended independent claim 1 has been interpreted to recite an open group wherein the one or a panel of two or more pre-determined miRNAs comprise one or more of the four recited miRNAs. The claim as written does not recite that the one or a panel of two or more pre-determined miRNAs are selected from a group consisting of the four recited miRNAs. Therefore, the amendment to claim 1 as written does not overcome the finding of obviousness over Parr, Asikainen, Zhao, Blumenreich, Bours, and Trivedi for claim 1, nor for the claims depending therefrom.
Regarding argument 2), note that the number of modifications to a base reference is not a factor in determining obviousness.
Additionally, Parr was relied on for teaching a method for specifically detecting human induced pluripotent stem cells (hiPSCs) in a heterogeneous population of cells, wherein the cell population is assayed for the presence of hsa-miR-302a-5p as a marker of hiPSCs due to the high level and high specificity of expression of hsa-miR-302a-5p in hiPSCs relative to non-hiPSC cells, including a loss of hsa-miR-302a-5p expression during differentiation of the hiPSCs into other cell types [abstract]. Parr further teaches that the miR-302/367 cluster is a pluripotency-specific locus [page 2 ¶ 4].
Parr additionally teaches that their method allows sensitive detection of hiPSCs down to a spiked level of 0.05% of hiPSCs in a heterogenous population [abstract, Figure 3], and that it is important to detect and eliminate residual harmful cells with high sensitivity to facilitate the therapeutic application of IPSC technology [page 5 ¶ 3].
Asikainen was cited for teaching that certain moRNA isoforms are notably expressed in hESCs (human embryonic stem cells) from loci coding for stem cell-selective or cancer-related miRNA clusters, and in contrast, only sparse moRNAs in fibroblasts (e.g., Abstract). Table 2 shows 15 most overexpressed miRNAs in hESC lines when compared to HEF-1 foreskin fibroblast by statistical computing using DEseq algorithm. In Table 2, the overexpressed miRNA in hESC as compared to foreskin fibroblast include hsa-miR-302a-5p, hsa-miR-302d-3p, hsa-miR-302a-3p, hsa-miR-302b-3p, hsa-miR-302c-3p and hsa-miR-367-3p (e.g., page 4, Table 2). Asikainen also teaches that hsa-miR-302a-5p, hsa-miR-302a-3p, hsa-miR-302b-3p, hsa-miR-302c-3p, and hsa-miR-302d-3p are all part of the miR-302/367 hESC-specific cluster [page 4 ¶ 1, Table 2]. Therefore, Asikainen teaches both the motivation and the reasonable expectation of success for using and of hsa-miR-302a-5p, hsa-miR-302a-3p, hsa-miR-302b-3p, hsa-miR-302c-3p, and hsa-miR-302d-3p for discrimination of PSCs from non-PSC cell types.
Additionally, Zhao teaches the method of droplet digital PCR for detection of rare cells with a sensitivity at least as low as 1 cell in 10 million cells of a cell sample. Zhao further teaches the motivation to use the droplet digital PCS method such that the ability to rapidly and selectively detect low abundance targets is crucially important to elucidate new biology, to monitor or detect a disease or disorder, to monitor therapeutic responses, and to develop new therapeutics [0003]. Zhao also teaches that not only is their method highly sensitive at detecting rare cells in a heterogenous population, but their system is high-throughput, multiplexable, and rapid [abstract]. Zhao further teaches that microfluids methods such as their invention have advantages of negligible evaporation of reagents, minimal consumption of expensive biological reagents, low fabrication costs, reduced analysis time, and the ability to integrate various functional components on a single chip [0009]. Therefore, Zhao provides the motivation to substitute the method of Parr with a droplet digital PCR method for ultrasensitive detection of rare cells in heterogenous populations.
Regarding argument 3), note firstly that the claims do not recite any particular species for the cells of the invention. Note secondly that both Parr and Asikainen teach human miRNAs which are highly selective for human pluripotent stem cells, and as such a difference in chemical unpredictability in the effects of in vivo expression of various species is not a factor in combining the teachings of Parr and Asikainen. Further, as discussed above, the combination of Parr and Asikainen were not relied on alone to fully teach the instant invention. Zhao teaches the method of droplet digital PCR which provides the high sensitivity for detecting rare cells in a population as recited in the instant claims. Additionally, Bours and Trivedi teach further dependent limitations not encompassed by the teachings of Parr, Asikainen, Zhao, and Blumenreich.
Regarding 4), Asikainen teaches that the four miRNAs recited in claim 1 are highly specifically expressed in pluripotent cells, with all four being in the top 6 most overexpressed miRNAs in hESC lines when compared to HFF-1 foreskin fibroblasts, with p-values ranging from 5E-28 to 2E-30 for differential expression [Table 2]. As such, it would have been obvious to include these four miRNAs as suitable assay development candidates for detection of residual contaminating undifferentiated PSCs in derived cell products.
Example 3 of the instant specification presents microarray analysis to conform cell-specificity of selected iPSC-specific miRNAs [page 33 lines 7-8], which shows that the 4 miRNAs recited in claim 1 are each expressed in pluripotent cells and below the limit of detection of the microarray in a single sample of non-pluripotent cells [Figure 3]. This does not provide any unexpected results in view of Asikainen.
Example 4 of the instant specification presents sensitivity analysis by QRT-PCR of a selected iPSC-specific miRNAs using cell-spiked samples [page 34 lines 13-14], which shows that of the selected subset of 10 miRNAs, the four recited in claim 1 had the highest detection sensitivity potential as assay development candidates for the detection of very low numbers of residual contaminating undifferentiated PCSs in derived cell products due to being able to detect between 100 and 10 iPSCs in 1 million derived MSCs [page 34 line 19-page 35 line 2, Figure 4]. However, this is not unexpected given the teachings of Asikainen that the four miRNAs are among the top 6 most overexpressed in hESCs. Note that of the other 6 miRNAs tested, only one (has-miR-367-3p) was even in the top 15 most overexpressed miRNAs in hESC lines as taught by Asikainen [Table 2], and was less overexpressed as taught by Asikainen than the four recited miRNAs.
Example 5 teaches the development of highly sensitive droplet digital PCR assay with a selected panel of iPSC-specific miRNAs [page 35 lines 11-12], which shows the optimized assay was able to consistently detect less than 10 iPSCs in a background of one million MSCs [page 35 line 23- page 36 line 2, Figure 5, 6]. However, this is not unexpected given the additional teachings of Zhao of detecting single cells as rare as 1 in 10 million by ddPCR, as discussed above.
Example 6 teaches sensitivity analysis by ddPCR of a selected iPSC-specific miRNAs using cell-spiked samples [page 36 lines 21-22], which shows assessment of the sensitivity of the highest sensitivity potential four miRNAs recited in claim 1 for residual contaminating undifferentiated PSCs in derived cell products in cell-spiked samples comprising 1, 5, or 10 iPSCs seeded into 1 million BM-MSCs, wherein the method was able to detect the miRNAs in samples with as few as 5 iPSCs in 1 million BM-MSCs [page 37 liens 1-120, Figure 7]. As discussed above, this level of sensitivity is not unexpected given the teachings of Zhao of ddPCR sensitivities at least as low as 1 in 10 million.
Therefore, Applicant arguments do not overcome a finding of obviousness over Parr, Asikainen, Zhao, Blumenreich, Bours, and Trivedi under 35 USC 103.
Conclusion
No claim is allowed.
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DR. KATIE L. PENNINGTON
Examiner
Art Unit 1634
/KATIE L PENNINGTON/Examiner, Art Unit 1634
/MARIA G LEAVITT/Supervisory Patent Examiner, Art Unit 1634