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 .
Election/Restrictions
Applicant’s election without traverse of Group III (Claims 18-21; drawn to a method of producing a bovine-induced pluripotent stem cell) in the reply filed on September 4, 2026, is acknowledged.
Claims 1-17 and 22 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention (Groups I-II and IV), there being no allowable generic or linking claim.
DETAILED ACTION
The amended claims filed on June 18, 2024, have been acknowledged. Claims 4-6, 9-12, 14-15, 17, and 21-22 were amended. In light of the Applicant’s elected invention, claims 1-17 and 22 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 18-21 are pending and examined on the merits.
Priority
The applicant claims domestic priority from U.S. provisional application No. 63/291,669, filed on December 20, 2021. Applicant’s claim for the benefit of a prior-filed application under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, 365(c), or 386(c) is acknowledged. Claims 18-21 receive domestic benefit from U.S. provisional application No. 63/291,669, filed on December 20, 2021.
Information Disclosure Statement
The information disclosure statement (IDS) filed on June 18, 2024, has been considered.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claim 18-21 are rejected under 35 U.S.C. 112(a) or pre-AIA 35 U.S.C. 112, first paragraph, as based on a disclosure which is not enabling. The disclosure does not enable one of ordinary skill in the art to practice the invention without identifying how the reprogramming factors are overexpressed and including the culture step and conditions as part of the method of independent claim 18, which is/are critical or essential to the practice of the invention but not included in the claim(s). See In re Mayhew, 527 F.2d 1229, 188 USPQ 356 (CCPA 1976).
The factors to be weighed to evaluate whether a disclosure satisfies the enablement requirement and whether any necessary experimentation is undue are set forth in MPEP 2164.01(a).
(A) The breadth of the claims;
(B) The nature of the invention;
(C) The state of the prior art;
(D) The level of one of ordinary skill;
(E) The level of predictability in the art;
(F) The amount of direction provided by the inventor;
(G) The existence of working examples; and
(H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure.
Although all the factors have been considered, the relevant factors will be addressed below.
Breadth of the claims: Claim 18 recites the following claim language, “A method of producing a bovine-induced pluripotent stem cell, the method comprising:
a. providing a bovine mesenchymal stem cell, and
b. inducing overexpression of a plurality of reprogramming factors in the bovine mesenchymal stem cell, wherein the plurality of reprogramming factors comprise lysine-specific demethylase 4A (KDM4A), OCT4, SOX2,KLF4, cMYC, LIN28, and NANOG.”
The broadest reasonable interpretation is that this method could be done with or without any culturing steps and any media and that inducing can be done by any method, including inducing the endogenous or exogenous genes.
Nature of the invention: The subject matter of the invention relates to a method of generating bovine induced pluripotent stem cells by expressing seven reprogramming factors.
State of the prior art: The prior art teaches that exogenous reprogramming factors are delivered (such as using lentiviruses, plasmids, and piggybac transposons) to bovine cells to reprogram them into pluripotent stem cells (i.e. induced PSCs) after culturing for multiple days, as identified by Zhao et al. (Tissue and Cell 49: 521–527. 2017), Pillai et al. (Anim Sci J. 00: 1–12. 2019), and Heo et al. (Stem Cells and Development 24: 393-402. 2015).
Zhao teaches that ectopic expression of transcription factors like Oct4,Klf4, Sox2, and cMyc can reprogram mouse and human somatic cells to form induced pluripotent stem cells (iPSCs). Using a similar method, iPSCs of domestic animal species, including cattle can be obtained. And many attempts have been made over past years to obtain completely reprogrammed ground state bovine iPSCs. Firstly, different cocktails of transgenes were tried, such as Oct4, Sox2, Klf4and cMyc (OSKM), Oct4, Sox2, Klf4, cMyc and Nanog (OSKMN), Oct4, Sox2, Klf4, cMyc, Nanog and Lin28 (OSKMNL). In general, more than four OSKM factors could improve the efficiency and quality of bovine iPS cells in these reports. Secondly, many efforts were made in the culture system, such as the employing of “2i” small-molecule inhibitors, which comprised a specific inhibitor of the ERK signal pathway known as PD0325901 and a specific inhibitor of GSK3 referred to as CHIRON (CHIR99021). PD0325901 blocks phosphorylation, and hence inhibition, of ERK1/2 and it promotes long-term self-renewal and colony formation in defined media. CHIRON, on the other hand, inhibits GSK3 before effectively stimulating the canonical WNT signaling, which is thought to maintain pluripotency. Growth factors including leukemia inhibitory factor (LIF), basic fibroblast growth factor (bFGF) or bone morphogenetic protein 4 (BMP4) were also added in the culture medium to maintain bovine iPSCs (page 521, column 1, paragraph 1-column 2, paragraph 1).
Zhao teaches that they generated single-cell derived bovine induced pluripotent stem cells (iPSCs) by the introduction of piggyBac transposons with CAG promoting transcription factors (Oct3/4, Sox2, Klf4 and cMyc). After electroporation, bovine fetal fibroblasts (BFFs) were seeded in Knockout DMEM medium supplemented with 15% FBS plus LIF and bFGF on STO feeders at a density of 3–5 × 105 cells per 10 cm plate. The culture medium was changed every other day. Bovine iPS cell colonies were usually picked up at day 15–20. Some defects were observed when using heterogenous transcription factors to derive bovine iPS cells, in previous experiments (Zhao et al., 2013) and (Han et al.,2011). Thus, they constructed bovine transcription factor vectors, PB-CAG-bovine O/C/K/S. During the reprogramming, small aggregates of cells started appearing in the primary plate approximately at day 8, and, over the following few days, some of these cell clumps progressed to form colonies. The RNA transcripts of the endogenous pluripotency markers, including Oct4, Nanog, Sox2,cMyc and Klf4 were detected in bovine single-cell derived iPSCs by RT-PCR (Fig. 4D). The expression of Oct4 was weaker in iPSCs than in blastocyst (Abstract, Figures 1-4, and page 522, column 1, paragraph 2-page 524, column 2, paragraph 3). As can be seen in Figure 3, the transgenes lead to expression of the exogenous genes. In comparison, Figure 4 shows that only some of the endogenous OKSM genes showed induced expression compared to the BFF cells. Specifically, cMyc expression appears to be decreased in comparison to its expression in BFF cells whereas Oct4 and Klf4 may or may not show increased expression.
Pillai teaches that they used retroviral vectors coding for bovine OCT4, SOX2, KLF4, cMYC, and NANOG to produce consistent iPSC‐like cells from both bovine embryonic fibroblasts and cells from the Wharton's jelly. Reprogramming genes were expressed by infecting bovine embryonic fibroblasts (BEFs) or WJCs with lentiviruses or retroviruses according to the experimental plan (Figure 1a), in medium supplemented with 6 μg/ml Polybrene for 24 hr. Seven days after infection, cells were plated on irradiated mouse embryonic fibroblast feeders, and culture medium was changed to reprogramming medium [DMEM/F12 containing 10% fetal bovine serum (FBS) or 15% KnockOutTM serum replacement (KSR) with 1% nonessential amino acids supplement,
1% penicillin–streptomycin, 0.1 mM β‐mercaptoethanol, and growth factors—20 ng/ml of human basic fibroblast growth factor and 10 ng/ml of human leukemia inhibitory factor. Pillai teaches that lentiviral vectors encoding human OSKM and human OSKMN are not effective in reprogramming BEFs to iPSCs while bovine OSKMN genes in retroviral vectors could successfully reprogram BEFs.
Although previous reports have demonstrated that bovine cells could be reprogrammed
using lentiviral or plasmid‐based approaches with human or bovine reprogramming factors, the vectors employed in a majority of these studies were monocistronic. It has been shown that differences in polycistronic vector design can cause significant differences in stoichiometry and expression levels of individual genes, altering reprogramming efficiencies. Although successful reprogramming with a polycistronic OSKM vector in bovine cells has been possible, this method also used small molecule inhibitors to enhance reprogramming efficiency, suggesting that the stoichiometry of OSKM expression levels by itself may not be sufficient. In support, no iPSC‐like colonies appeared when polycistronic MKOS PiggyBac transposon vector was used, but an OKS and c‐Myc PiggyBac transposon vectors could generate bovine iPSC colonies from bovine amnion cells that could generate chimeric fetuses. Adding NANOG to the polycistronic OSKM vectors did not result in ALP‐positive
bovine iPSC colony formation. In contrast, use of monocistronic bovine OSKM reprogramming factors consistently generated ALP‐positive bovine iPSC like
colonies both with and without NANOG (abstract, page 2 column 2, paragraph 3-page 7, column 2, paragraph 2, and Figures 1-4).
Heo teaches that they transduced bovine fibroblasts with the polycistronic lentiviral vector harboring OKSM reprogramming factors (Fig. 1A). At 2 days after infection, fibroblasts were maintained in mouse ES cell medium supplemented with Lif. Dramatic morphological changes in the culture were observed at 7 days after 4-
factor infection (Fig. 1). Two weeks after transduction, we observed primary colony formation in fibroblast cultures. These bovine fibroblast-derived partially reprogrammed cells are self-renewed and stained positively for alkaline phosphatase.
These cells were positive for pluripotent markers such as OCT4, SOX2, and NANOG by immunofluorescence staining. Moreover, pluripotency genes, including bovine OCT4, SOX2, NANOG, KLF4, C-MYC, and REX1, were markedly elevated in these cells compared with bovine fibroblasts by quantitative RTPCR. However, we observed inadequate transgene expression in these cells and no teratoma formation, suggesting incomplete reprogramming of these cells into a pluripotent state. Small molecules such as valproic acid or GSK3b and MEK inhibitor (2i) have been reported to increase the production of naive iPSCs. They found that these agents efficiently upregulated pluripotency 2i treatment in the partially reprogrammed cells increased the expression of REX1, STELLA, NANOG, and LIN 28, markers of naive state in mouse and human system and demonstrate that bovine fibroblasts can be reprogrammed into native like pluripotent cells by the forced expression of four factors with 2i treatment. (page 395, column 2, paragraph 4-page 396, column 2, paragraph 1 and Figure 1).
As such, the prior art shows that there need to be a careful consideration regarding the method of delivering transgenes to the somatic cell (monocistronic vs polycistronic and vector backbone), the transgenes being delivered (human vs bovine), and the media composition for reprogramming the cells for successful induction of pluripotent stem cells.
Level of predictability in the art: The prior art has successfully reduced to practice that the method of delivering transgenes to the somatic cell (monocistronic vs polycistronic and vector backbone), the transgenes being delivered (human vs bovine), and the media composition for reprogramming the cells are all important consideration for successful induction of pluripotent stem cells as the wrong combination of components fails to generate induced pluripotent stem cells. As such, this results in unpredictability about how someone can use this method to generate bovine induced pluripotent stem cells without defining the method of exogenously expressing the reprogramming factors and without including and defining the culture method.
Amount of direction provided by the inventor and existence of working examples: The Applicant discloses that they used defined protocols for generating bovine induced pluripotent stem cells.
The Applicant discloses that for the establishment of bovine iPSCs: An iPSC induction scheme involving combined expression of human reprogramming factors OKMSLN (expressed in three different pMXs-retroviral vectors for 0, KMS, and LN), together with the inhibitor of histone methyltransferase DOT1L (iDOT1L) and the WNT inhibitor (IWR1) was used to reprogram bovine primary mesenchymal stem cell-like cells (bMSCs) derived from bovine placenta (Figure 7A,B). Although obvious cell aggregation was seen in the first two weeks of reprogramming, the development of any PSC-like colonies thereafter was not identified. Other attempts were made to reprogram bovine bMSCs in different PSC culture media, including the knockout serum replacement (KSR)-based ESC medium, mTeSR medium, and a modified epiblast stem cell (EpiSC) medium containing bFGF, Activin A, and WNT agonist CHIR-99021 (FAC), but no PSC-like colonies were secured.
pMXs-KDM4A was incorporated into the OSKMLN reprogramming (termed here KdOSKMLN) (Figure 1B). Z-VAD-FMK, a pan caspase inhibitor, was also added to reduce the apoptosis of bMSC caused by retroviral infection (Figure 1B). With the KdOSKMLN induction, PSC-like colonies were observed from the reprogrammed bMSCs on day 17 (Figure 1C). The colonies were picked from day 19-30 and transferred to mitomycin C-treated mouse embryonic fibroblasts (MEF) feeders. They were cultured in a 1:1 combination of the FAC/KSR media (Figure 1B,C) for the first three passages and thereafter in 1:1 combination of KSR/mTeSR media with the addition of iDOT1L and IWR1 (termed KT medium) (Figure 1B). These bovine cells exhibited strong positive staining of the PSC surface markers alkaline-phosphatase (AP) and the stage-specific embryonic antigen 4 (SSEA4) at different passages (Figure 1D,E). [0106] The silence of exogenous transgenes is one of the key markers for successful reprogramming. Four lines of the established biPSCs (Lines 4-1, 4-6, 3-2, and 04) were tested for the expression of viral transgenes. Since the KdOSKMLN transgenes were expressed either alone (O and Kd) or polycistronically (KMS and LN), specific PCR primers were designed to amplify DNA regions spanning the vector and the cloned human genes. qRT-PCR revealed that for all vectors, the expression of transgenes was inactivated in early passages (P10-P17) and remained silenced in later passages (P25- P43) (Figure 1F) (Example 1).
As can be seen in Applicant’s example, they used a specific combination of polycistronic and monocistronic vectors encoding the reprogramming factors and specific media compositions for producing bovine induced pluripotent stem cells.
As such, the method of generating bovine induced pluripotent stem cells of the instant application requires a defined protocol that is not disclosed in claim 18.
Quantity of experimentation needed: In light of the above factors, the prior art and the Applicant disclose several induction protocols and identify that multiple components of the protocol are important for generating induced pluripotent stem cells as some protocols failed to generate induced pluripotent stem cells. Therefore, a defined protocol is necessary to generate bovine induced pluripotent stem cells, including how the exogenous transgenes are introduced to the cells and the culture step. As such, these essential steps are considered to be missing from claim 18. There would be undue experimentation related to any method of expressing exogenous transgenes or inducing endogenous genes. Furthermore, there would be undue experimentation related to whether there is or is not a culture step and, if there is a culture step, using any culturing media/method to practice the full scope of the claim.
Claims 19-21 are also rejected because of their dependency on claim 18.
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 20-21 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.
Claim 20 recites the following claim limitation “wherein culturing the cell under suitable conditions to promote cell reprogramming comprises culturing the cell in the cell culture medium of any one of the preceding claims.” However, claims 18-19 do not recite any cell culture medium but are still included within the “any one of the preceding claims” language. It is unclear what culture medium would be associated with these claims.
Claim 21 is rejected because of their dependency on claim 20.
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.
Claims 18-19 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No: 20170073643 (Valamehr) and further in view of Arabaci et al. (The FEBS Journal 288: 4798–4811. 2021; Published November 2020).
Valamehr teaches a method of generating bovine induced pluripotent stem cells (iPSCs) comprising: (a) obtaining one or more bovine mesenchymal stem cell; (b) introducing polynucleotides encoding reprogramming factors OCT4, SOX2, NANOG, KLF4, LIN28, and C-MYC into the one or more bovine mesenchymal stem cells; and (c) culturing the bovine mesenchymal stem cells in a cell culture medium to reprogram the one or more bovine mesenchymal stem cells to an induced pluripotent stem cell state (paragraph 0053-0058, 0260-0264, 0435-0440).
Valamehr does not teach wherein KDM4A is also overexpressed.
However, Arabici teaches that somatic cell-specific H3K9me3 is a barrier in reprogramming, where it is associated with reprogramming-resistant genes. Depletion of H3K9me3 via expression of H3K9me3-specific histone demethylases KDM4A improves the derivation of cloned embryos. This establishes H3K9 methylation as a major mechanism to block pluripotency-specific gene expression and that perturbation of this chromatin mark can significantly increase reprogramming.
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have modified the reprogramming method of Valamehr by also introducing reprogramming factor KDM4A, as identified by Arabaci, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to modify with a reasonable expectation of success because Arabici identifies that somatic cell-specific H3K9me3 is a barrier in reprogramming, where it is associated with reprogramming-resistant genes and that KDM4A expression can deplete H3K9me3 to increase reprogramming. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 18-21 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No: 20170073643 (Valamehr) and further in view of Arabaci et al. (The FEBS Journal 288: 4798–4811. 2021; Published November 2020), as applied to claims 18-19 above, and further in view of Wang et al. (Biology Open 8: 1-12. 2019).
The teachings of Valamehr and Arabaci are as discussed above.
The combined teachings of Valamehr and Arabaci do not teach culturing cells in culture media comprising a WNT signaling inhibitor and a histone methyltransferase DOT1L inhibitor.
However, Wang teaches reprogramming human umbilical cord-derived MSCs by overexpressing NANOG and LIN28 (NL) along with OCT4, SOX2, KLF4 and MYC (OSKM) in a culture media comprising an inhibitor of the histone methyltransferase DOT1L (iDOT1L) and WNT antagonist IWR1. OKSM+NL+iDOT1L+IWR1 reprogramming condition exhibited striking increase in the number and ratio of homogeneous TRA-1-60+ colonies (Fig. 4B,C). The positive effects of iDOT1L and IWR1 on NL-enhanced TRA-1-60+ colony formation (one of the best markers for successful iPSC generation) were also correlated with the AP-staining of induced colonies at reprogramming day 18. iDOT1L could enhance reprogramming by suppressing the expression of mesenchymal markers, which can be further enhanced by the addition of IWR1 (abstract, page 2, column 1, paragraph 2-page 6, column 2, paragraph 1, page 9, column 2, paragraph 4-page 10, column 1, paragraph 1, and Figure 4).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have used a culture media comprising IWR1 and iDOT1L for culturing the mesenchymal stem cells during reprogramming, as identified by Wang, to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to use a culture media comprising IWR1 and iDOT1L for culturing the mesenchymal stem cells during reprogramming with a reasonable expectation of success because Wang teaches that the OKSM+NL+iDOT1L+IWR1 reprogramming condition exhibited striking increase in the number and ratio of homogeneous TRA-1-60+ colonies. The positive effects of iDOT1L and IWR1 on NL-enhanced TRA-1-60+ colony formation (one of the best markers for successful iPSC generation) were also correlated with the AP-staining of induced colonies at reprogramming day 18. iDOT1L could enhance reprogramming by suppressing the expression of mesenchymal markers, which can be further enhanced by the addition of IWR1. Therefore, it would have been obvious to use this culture media to improve reprogramming efficiency. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Conclusion
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/KEENAN A BATES/Examiner, Art Unit 1631
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