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
This action is response to the papers filed on June 07, 2023. Claims 1-20 are currently pending. Claim 1 is an independent claim.
Therefore, claim 1-20 are under examination to which the following grounds of rejection are applicable.
Priority
This application is claiming the benefit under 35 U.S.C. 119(e) of prior-filed provisional application 63/351,330 filed on June 10th 2022.
Thus, the earliest possible priority for this instant application is June 10th 2022.
The disclosure of the prior-filed application, 63/351,330, filed on June 10th 2022, provides adequate support or enablement in the manner provided by the first paragraph of 35 U.S.C. 112 for one or more claims of this application. Specifically, at least claims 1, 2, 8, 11, 15, 18, 26, 27, 29, 30, 40, 53, 56, 57, 59 and 62 of provisional application 63/351,330 filed on June 10, support claims 1-20 of the instant invention.
Accordingly, the effective priority date of claims 1-20 is granted as June 10th 2022.
Information Disclosure Statement
The listing of references in the specification is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Claim Rejections - 35 USC § 112(b): Indefiniteness
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claim 1-20 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 1 is indefinite in its recitation of the term “dedifferentiating said cellular population”. The term " dedifferentiating " is not defined by the claim. ”. The specification does not provide any closed definition as to what is meant by “dedifferentiating”. Although it is acknowledged in the specification some cellular populations are considered by applicant to be " dedifferentiating” (Abstract; para [0083]), these are merely exemplary and non-limiting. The metes and bounds of the claims are unclear particularly since dedifferentiating would vary depending on the nature of a cellular population to be dedifferentiated, and culture conditions. As such, the metes and bounds of the claims cannot be determined.
Claims 2-20 are included in the rejection insofar as they depend directly from claim 1.
Claim 2 is indefinite in its recitation of the phrase “cellular population is an EPC” in line 1. The term EPC refers to a singular endothelial progenitor cell while a cellular population refers to a plurality of endothelial progenitor cells, which lacks antecedent basis. It is recommended to amend the claim to recite “said cellular population is an EPC population”.
Claim 8 is indefinite in its recitation of the phrase “cellular population is a mesenchymal stem cell” in line 1. The term EPC refers to a singular endothelial progenitor cell while a cellular population refers to a plurality of endothelial progenitor cells, which lacks antecedent basis. It is recommended to amend the claim to recite “said cellular population is a mesenchymal stem cell population”.
Claim 9 and 10 are indefinite in its recitation of the phrase “naturally recurring mesenchymal stem cell” in line 1. There is not proper antecedent basis for “naturally occurring mesenchymal stem cell”. Claim 8 recites a “mesenchymal stem cell”. As such the metes and bounds of the claim are indefinite.
Claim 11 is indefinite in its recitation of the phrase “said tissue derived mesenchymal stem cells are derived from umbilical cord tissue.” in line 1. Claim 10, in which it depends on, recites that the mesenchymal stem cell is a bodily fluid. Thus, claim 11 lacks proper antecedent basis.
Claim 12 are indefinite in its recitation of the phrase “tissue derived mesenchymal stem cell” in line 1. There is not proper antecedent basis for “tissue derived mesenchymal stem cell” as claim 10 recites mesenchymal stem cells derived from a bodily fluid.
Claim 13 is indefinite because of its recitation of “capable of” since this phrase refers to a latent ability, and it is unknown whether the ability is expressed or observed in the invention.
Note, it has been held that the recitation that an element is “capable of” performing a function is not a positive limitation, but only requires the ability to so perform. It does not constitute a limitation in any patentable sense. In re Hutchinson, 69 USPQ 138.
Claim 14 is indefinite in its recitation “said pluripotency marker”. There is not proper antecedent basis in the claim for “said pluripotency marker” as claim 13 recites proteins capable of differentiation.
Claim 16 are indefinite in its recitation of the phrase “wherein mRNA” in line 1. There is not proper antecedent basis for “wherein mRNA”.
Claim 15 is indefinite because of its recitation of “capable of” since this phrase refers to a latent ability, and it is unknown whether the ability is expressed or observed in the invention.
Note, it has been held that the recitation that an element is “capable of” performing a function is not a positive limitation, but only requires the ability to so perform. It does not constitute a limitation in any patentable sense. In re Hutchinson, 69 USPQ 138.
Claims 18-20 are indefinite in its recitation of the phrase “redifferentiated cells” in line 1. There is not proper antecedent basis for “redifferentiated cells”.
Claim 18 is indefinite because of its recitation of “capable of” since this phrase refers to a latent ability, and it is unknown whether the ability is expressed or observed in the invention.
Claim Rejections - 35 USC § 112(a)
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 1-20 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 enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
In order to determine compliance with the enablement requirement of 35 U.S.C. 112(a), the Federal Circuit has developed a framework of factors in In re Wands, 858 F.2d 731, 737, 8 USPQ2d 1400, 1404 (Fed. Cir. 1988), referred to as the Wands factors, to assess whether any necessary experimentation required by the specification is "reasonable" or is "undue." The factors considered include (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 in the art; (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.
Breadth of the Claims
The breadth of the claims weighs heavily against enablement. Instant claim 1 is directed to a method for creating an endothelial progenitor cell (EPC) conditioned media by obtaining a cellular population capable of dedifferentiation. The “cellular population” encompasses a cell population able to dedifferentiate in vitro or in vivo, for example in a mammalian subject. Moreover, the claimed genus of cellular population for dedifferentiation encompasses cell populations isolated from insect, fungi, vertebrate, invertebrate, plants, etc. without limitation to any cell type, lineage or tissue origin. Thus, encompassing all cellular populations within and outside of a mammalian cell culture. Although dependent claims recite specific examples of cell types, such examples do not limit the scope of the independent claim. As such, the breadth of the claims is great.
Additionally, Claim 16 is broadly directed to a genus of “mRNA is introduced into said cells” to induce pluripotency-inducing genes, without limiting identity, sequence, order, or function of the mRNA. As written, claim 16 encompasses any mRNA, including mRNAs unrelated to pluripotency of dedifferentiation. The claims therefore an extremely broad genus defined primarily by the functional result rather than by structure or specific methodology.
Nature of Invention
The claimed subject matter involves cellular dedifferentiation, reprogramming, and redifferentiation into endothelial progenitor cells. Such processes are recognized in the art as highly sensitive to cell type, culture conditions and gene expression dynamics. The claimed methods further recite the generation of conditioned media with functional angiogenic properties.
Claim 16 is drawn to the mRNA capable of expression of pluripotency inducing genes for inducing dediffentiation of a cellular population.
State of the Prior Art
The state of prior art does not support enablement for full scope of the claims, as it establishes that cellular dedifferentiation, reprogramming and redifferentiation are highly cell-type specific and differentiating factor-specific processes. As such, there would be no universal method applicable to all cellular populations or all mRNAs.
Takashi and Yamanaka [Published: 2006. Cell, 126(4), 663–676.] demonstrated that pluripotency could be induced in mouse and human fibroblasts using a specific and limited set of transcription factors (Oct3/4, Sox2, c-Myc, and Klf4). More specifically, this work was only limited to fibroblasts and needed specific factor combinations and gene expression conditions. The authors additionally noted that the origin of cells determined the reprogramming efficiency of these dedifferentiated cells, implying that not all cells are capable of dedifferentiation.
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Furthermore, subsequent studies confirmed that reprogramming efficiency depends on the starting cell population. Stadtfeld and Hochendinger [Published: 2010. Genes & development, 24(20) 2239–2263] reported that certain somatic cells are resistant or refractory to reprogramming and that the differentiation state strongly influence reprogramming outcomes [Table 3]. The authors also state that protocols do not encompass all various derivations of iPSCs, and even that one protocol may be more successful than another. Stadtfeld and Hochendinger also demonstrates the hematopoietic stem cells, endothelial cells, and terminally differentiated cells present unique challenges for reprogramming. For instance, the authors recite that “hematopoietic stem and progenitor cells were shown to give rise to iPSCs with significantly higher efficiencies and within a shorter time frame than mature lymphocytes and myeloid cells” [page 2246, col 2 para 2]. As such, the dedifferentiation of cell lineages requires extensive optimization even among related cell populations.
With respect to mRNA-base reprogramming to induce expression of pluripotency inducing genes (instant claim 16), Plews et al. [Published: 2010. PLoS ONE 5(12): e14397] described a highly specific method using modified mRNAs encoding defined pluripotency factors (Oct3/4, Sox2, c-Myc, and Klf4), delivered to human fibroblasts. The work did not disclose that any mRNA could induce pluripotency, nor that mRNA-based approaches would be effective across all cell types. This inherently implies that not all mRNA are capable of expressing pluripotency factors in cells, therefore underscoring that the claims do not enable all types of mRNA.
Quantity of Experimentation Required
The quantity of experimentation required to practice the full scope of the claims would be undue. A skilled artisan would need to independently determine for each call population, whether dedifferentiation is possible, which mRNAs would induce pluripotency, how such mRNA should be delivered, and whether redifferentiation into endothelial progenitor cells can be achieved.
The Amount of Direction Provided by the Inventor
The specification provides insufficient direction and guidance to enable a person of ordinary skill in the art to practice the full scope of the claimed invention. Although claims recite the cellular population to be monocytes, hematopoietic stem cells and mesenchymal stem cells [Claims 6-8], the disclosure fails to exemplify any working examples, levels of gene expression, or culture conditions for any cell type to ensure that they are capable of dedifferentiation. Furthermore, the specification does not provide guidance identifying which cell populations are suitable for dedifferentiation and subsequent redifferentiation into endothelial progenitor cells. As such, a skilled artisan must perform undue experimentation to determine which cells are best suitable for dedifferentiation, and subsequent redifferentiation into endothelial progenitor cells.
With respect to the mRNA limitation in claim 16, the specification provides insufficient guidance regarding the selection and use of mRNA to induce pluripotency. The specification does not identify specific mRNA sequences, combinations of mRNAs, or encoded factors that are effective across different cell populations. Furthermore, it does not provide any guidance on dosage, administration, or control of expression kinetics. Although the specification mentions the use of Sendai virus vectors to deliver mRNA that can induce pluripotency and a plurality of different mRNA genes these are only preferred methods and not working examples, and do not enable the full scope of the claims.
Overall, the specification prophetically provides generalized descriptions and desired results but fails to supply the detailed technical guidance necessary to enable the claimed methods across their full breadth. Instead, the specification requires a skilled artisan to perform undue experimentation to determine which cell populations are workable, which mRNA sequences induce pluripotency, and whether the claimed limitations achieve the functional outcomes that the applicant discloses.
Presence or Absence of Working Examples
The application provides no working examples in the specification demonstrating that dedifferentiation and endothelial progenitor cell redifferentiation can be done in a variety of cell types, nor does it provide examples of using any mRNA strand to achieve the functional outcomes. The specification provides various preferred embodiments of the claimed “cellular population” including endothelial progenitor cells, monocytes, hematopoietic stem cells and mesenchymal stem cells. However, the specification does not provide working examples demonstrating the claimed method using these cellular populations.
Specifically, the disclosure fails to recite any experimental protocols, conditions, or results showing (i) dedifferentiation of such cellular populations (ii) subsequent redifferentiation into endothelial progenitor cells and (iii) the production of conditioned media. Merely listing these cell types as preferred embodiments does not establish full enablement of the claimed invention. Due to the absence of working examples, a person with ordinary skill in the art would not be able to determine, without undue experimentation, whether and how the claimed method could successfully be practiced across the full breadth of the recited cellular population and mRNA.
Conclusion
In light of the unpredictability surrounding the claimed subject matter and the lack of adequate
guidance, one wishing to practice the presently claimed invention would be unable to do so without
engaging in undue experimentation. It is especially noted that applicants provide no data, examples,
figures, etc. demonstrating that any cellular population, or any mRNA can be used to achieve dedifferentiation, and redifferentiation into endothelial progenitor cells. The specification fails to provide sufficient guidance, representative examples, or predictability to allow a person with ordinary skill in the art to practice the invention across its full scope without undue experimentation.
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 (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries 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.
Claim 1 recites a method of creating endothelial progenitor cell (EPC) conditioned media by obtaining a cellular population, dedifferentiating said cellular population, inducing differentiation of said dedifferentiated cell into EPC, and culturing said EPC in a liquid media to obtain a conditioned media.
In the interest of compact prosecution, the term “cellular population” is interpreted as encompassing any population of cells, including progenitor cells, differentiated somatic cells, and stem cells, regardless of tissue source. The term dedifferentiating is reasonably interpreted to include reprogramming differentiated or lineage-restricted cells to a more primitive or pluripotent state.
Claims 1-20 are rejected under 35 U.S.C. 103 as being unpatentable over Kim et al. [Published in 2010. Cell transplantation, 19(12), 1635–1644] over Giorgetti et al. [Published 2009. Cell stem cell, 5(4), 353–357] and in further view of Aoki et al. [Published: 2020. Heliyon, Volume 6, Issue 3, e03493]
Regarding claim 1, Kim discloses a method for developing an endothelial progenitor cell conditioned media (EPC) by obtaining a cellular population and culturing said endothelial progenitor cells to obtain a conditioned media (“For experiments performed with conditioned medium (CM) from EPCs (EPC-CM), the CM was prepared by culturing EPCs in serum-free M199 media”) [page 1636, column 1 para 4 bridging into col 2], falling within the scope of Claim 1, steps d).
However, Kim fails to teach dedifferentiating a cellular population and inducing differentiating of said dedifferentiated cell into EPC or to redifferentiate the said dedifferentiated cells into endothelial progenitor cells.
Giorgetti teaches dedifferentiating a cellular population derived from human umbilical cord blood to pluripotent stem cells [Abstract, page 1, para 1, “…cord blood (CB) represents an alternative and readily-accessible source of stem cells. Here we describe reprogramming of CD133+ CB cells to pluripotency by retroviral transduction of four (OSKM), three (OSK) and as few as two (OS) transcription factors, without the need for additional chemical compounds.”]
Moreover, Aoki teaches re-differentiating induced pluripotent stem cells (iPSCs) into endothelial progenitor cells [Abstract, page 1, “…we report the development of relatively simple differentiation and purification methods for hiPSC-derived EPCs (iEPCs)”; page 1 column 2 paragraph 2, “To address the problems related to a stable supply and consistent quality, hPSC-derived EPCs are considered as a viable alternative to human primary EPCs”]. Furthermore, Aoki discloses that EPCs derived from a cellular source are limited in expandability, have finite proliferative capacity and display variability within primary EPC populations [page 1 col 2 para 1, “However, there are two main problems with the use of ECs and EPCs: (1) human primary EPCs have limited expandability (Igreja et al., 2008) and (2) the properties and characteristics of EPCs are heterogeneous owing to differences in genetic backgrounds and sampling techniques. Especially, the low number and weakened function of EPCs are serious problems for autologous transplantation for patients with lifestyle-related diseases”].
A person with ordinary skill in the art would have been motivated to combine the teachings of Kim, Giogetti and Aoki to arrive at the instant invention because each reference addresses a complementary aspect of producing endothelial progenitor cell-derived conditioned media. Kim teaches the therapeutic utility of condition media obtained by culturing EPCs, but relies on EPCs directly obtained by a cellular source and does not address the limitations associated with dedifferentiating or reprogramming a cell population (claim 1, step b) nor inducing differentiation of said dedifferentiated cell into EPC (claim 1, step c). Giorgetti teaches dedifferentiating a cellular population derived from human umbilical cord blood into CB-derived stem cells and Aoki teaches re-differentiating induced pluripotent stem cells (iPSCs) into endothelial progenitor cells. Moreover, based on Aoki’s teachings on limited expandability of human primary EPCs, a person of ordinary skill in the art would have recognized these limitations as significant obstacles for consistent and scalable production of EPC-conditioned media, and utilized the human cord-blood derived iPSCs of Giorgetti as they hold the ability to differentiate into homogeneous cells and develop more consistent batches of EPC-derived conditioned medium. Furthermore, as both CB-derived stem cells and iPSCs have pluripotency potential, a person with ordinary skill in the art would have recognized that combining the dedifferentiation approach of Giorgetti to result in CB-derived stem cells with the EPC differentiation methods of Aoki to obtain endothelial progenitor cells from iPSCs would allow regenerated EPCs to be produced from pluripotent stem cells in sufficient quality and quantity for use in conditioned media production disclosed by Kim with a reasonable expectation of success. To achieve the claimed conditioned medium, a skilled artisan would have taken the cell population of Giorgetti and dedifferentiate the cells into iPSCs, then re-differentiate the cells into the EPCs discussed in Aoki, then finally culture the cells to develop the EPC conditioned medium discussed in Kim to render the claimed invention obvious.
Regarding claim 2, the combined teachings of Kim, Giorgetti and Aoki render obvious claim 1. Moreover, Aoki teaches epithelial progenitor cells [page 2 col 1 para 2. “Protocols for the efficient generation and differentiation of hPSC derived ECs and EPCs have been recently reported.”).
Regarding claim 3, the combined teachings of Kim, Giorgetti and Aoki render obvious claim 1. Moreover, Kim teaches that the said EPC are derived from cord blood (page 1643 col 2 para 2, “The present study is the first to our knowledge to definitely characterize the therapeutic potential of human cord blood derived EPCs in diabetic wound healing.”].
Regarding claim 4, the combined teachings of Kim, Giorgetti and Aoki render obvious claim 1. Moreover, Kim teaches that the said EPC express VEGF-receptor 2 [page 5 para 4 bridging into page 6, “The RT-qPCR results confirmed that iEPCs derived from three different iPS cell lines exhibited high expression levels of the typical EC markers PECAM1 and cadherin 5 (CDH5, also known as VE-cadherin), and the EPC markers CD34 and kinase insert domain receptor (KDR, also known as VEGF receptor 2), as compared with HUVECs.”].
Regarding claim 7, the combined teachings of Kim, Giorgetti and Aoki render obvious claim 1. Moreover, Giorgetti teaches said cellular population are hematopoietic stem cells [Abstract, page 1 para 2, “Cord Blood (CB) cells are considered an alternative to bone marrow (BM) as a source of haematopoietic stem cells for transplantation”].
Regarding claim 13, the combined teachings of Kim, Giorgetti and Aoki render obvious claim 1. Moreover, Giorgetti teaches the introduction of proteins capable of dedifferentiation [Abstract, page 1, “Here we describe reprogramming of CD133+ CB cells to pluripotency by retroviral transduction of four (OSKM), three (OSK) and as few as two (OS) transcription factors, without the need for additional chemical compounds.”]
Regarding claim 14, the combined teachings of Kim, Giorgetti and Aoki render obvious claim 1. Moreover, Giorgetti teaches the said pluripotency marker to be TRA-1-60 [page 2 para 5, “Immunofluorescence of 6 CBiPS cell lines revealed expression of pluripotency markers such as OCT4, SOX2, TRA-1-81, TRA-1-60, SSEA3, SSEA4, and NANOG”].
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Regarding claim 15, the combined teachings of Kim, Giorgetti and Aoki render obvious claim 1. Moreover, Giorgetti teaches the said introduction of proteins to be OCT4, NANOG, KLF1 and SOX2 [page 2 para 4 “The presence of each retroviral transgene was confirmed by PCR genotyping, demonstrating the insertion of the expected 4, 3 or 2 transcription factors in CBiPS 4F, CBiPS 3F, CBiPS 2F, respectively (Figure 1B).”; page 2 para 5, “Immunofluorescence of 6 CBiPS cell lines revealed expression of pluripotency markers such as OCT4, SOX2, TRA-1-81, TRA-1-60, SSEA3, SSEA4, and NANOG”; Figure 1B attached].
Regarding claim 16, the combined teachings of Kim, Giorgetti and Aoki render obvious claim 1. Moreover, Giorgetti teaches the mRNA is introduced into cells in order to induce pluripotency [page 2 para 2, “As early as 9 days post infection, small colonies started to appear in cells transduced with OSKM, OSK and OS”].
Regarding claim 17, the combined teachings of Kim, Giorgetti and Aoki render obvious claim 1. Moreover, Aoki teaches that the dedifferentiated cells are capable of proliferating for more than 50 passages [page 1 col 2 para 2, “Human pluripotent stem cells (hPSCs), including human induced pluripotent stem cells (hiPSCs) and human embryonic stem cells, proliferate infinitely and have the ability to differentiate into various cell types].
Regarding claim 19, the combined teachings of Kim, Giorgetti and Aoki render obvious claim 1. Moreover, Kim teaches that redifferentiated cells are a direct therapeutic effector [page 1643 col 2 para 2, “The present study is the first to our knowledge to definitely characterize the therapeutic potential of human cord blood derived EPCs in diabetic wound healing.”].
Regarding claim 20, the combined teachings of Kim, Giorgetti and Aoki render obvious claim 1. Moreover, Aoki teaches that redifferentiated cells are more potent than naturally occurring EPCs based on a) VEGF production b) nitric oxide release and c) angiogenesis/capillary formation assay [page 5 para 4 bridging into page 6, “The RT-qPCR results confirmed that iEPCs derived from three different iPS cell lines exhibited high expression levels of the typical EC markers PECAM1 and cadherin 5 (CDH5, also known as VE-cadherin), and the EPC markers CD34 and kinase insert domain receptor (KDR, also known as VEGF receptor 2), as compared with HUVECs.”; Page 8 col 2 para 1 “The mRNA expression levels of vWF and matrix metalloproteinase 1 (MMP1), a marker of angiogenesis, had gradually increased in both groups of iEPCs during expansion culture.” ; Page 9 line 16 “Moreover, the results of western blot analysis confirmed that YAC-treated iEPCs highly expressed nitric oxide synthase 3 (NOS3, also known as endothelial NOS), which prevents EC senescence (Hayashi et al., 2008) (Figures 8C and 8D).”].
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over the combined teachings of Kim, Giorgetti and Aoki as applied to claims 1 and 2, and in further view of Moldenhauer et al. [Published: 2015. Stem cell research, 14(3), 380–395.].
With regard to claims 1 and 2, the combined teachings Kim, Giorgetti, and Aoki render obvious the claimed methodology, as iterated above in the 103 rejection the content of which is incorporated in claims 1 and 2. However, the references fail to teach that the endothelial progenitor cells express an IL-3 receptor.
Prior to the effective filing date, it was known that IL-3 significantly expands endothelial progenitor cells in culture, demonstrating that functional responsiveness of these cells to IL-3. This indicates that EPCs inherently express an IL-3 receptor or a functionally equivalent receptor, as IL-3 requires a receptor-mediated activity [Abstract, page 380, “Circulating endothelial progenitor cells (EPCs) provide revascularisation for cardiovascular disease and the expansion of these cells opens up the possibility of their use as a cell therapy. Herein we show that interleukin-3 (IL3) strongly expands a population of human non-adherent endothelial forming cells (EXnaEFCs) with low immunogenicity as well as pro-angiogenic capabilities in vivo, making their therapeutic utilisation a realistic option”].
Therefore, a person of ordinary skill would have reasonably expected that EPCs would similarly respond to IL-3 through the IL-3 receptor since this receptor is well established in endothelial lineage -related cells.
Claim 6 is rejected under 35 U.S.C. 103 as being unpatentable over the combined teachings of Kim, Giorgetti and Aoki and in further view of Isogai et al. [Published: 2018. Cellular Reprogramming, vol. 20, no. 6, Dec. 2018, pp. 347–355] as evidenced by Yamanaka et al. [Published: 2006. Cell, 126(4), 663–676].
With regard to claim 1, the combined teachings Kim, Giorgetti, and Aoki render obvious the claimed methodology, as iterated above in the 103 rejection the content of which is incorporated in claim 1. However, the references fail to teach a cellular population to be monocytes as described in claim 6.
Isogai teaches the preparation of induced pluripotent stem cells by reprogramming human peripheral blood monocytes [Abstract, page 347, “However, generation of monocyte-derived iPS cells has only been successful when special persistent Sendai virus vectors have been used…Therefore, in this study, as a preculture method for monocytes, a culture method for maintaining activity without using any cytokine was established, and using a commercially available vector without genetic toxicity without damaging the chromosome of the cell, iPS cells derived from monocytes were successfully produced.”].
Prior to the effective filing date, it was well established that terminally differentiated somatic cells such as monocytes can be reprogrammed into iPSCs through the introduction of pluripotency factors as described in Yamanaka et al. The reprogramming process erases lineage-specific differentiation markers and restores pluripotency, thereby constituting the dedifferentiation of a cellular population. Reprogramming monocytes into iPSCs necessarily involves reversal of their differentiated state, and the acquisition of pluripotency markers including OCT4, NANOG, KLF4, and c-MYC [page 350 col 2 para 5-6, “The expression of NANOG (Fig. 3B), OCT3/4 (Fig. 3C), KLF4, and c-MYC increased to 26.4 +/– 0.3, 13.4 +/– 0.2, 1.5 +/– 0.2, and 6789.6 +/ – 0.2, respectively (increased number of expression compared with gene expression in derived cell of monocyte). Expression of SOX2, OCT3/4, and NANOG protein detected…”]. Accordingly, conversion of monocytes to iPSCs is properly characterized as dedifferentiation within the meaning of the claims.
A person with ordinary skill in the art would have been motivated to dedifferentiate monocytes into iPSCs since they provide a renewable and intermediary cell state to allow the subsequent redifferentiation into multiple downstream lineages, including the claimed endothelial progenitor cells with a reasonable expectation of success The use of iPSCs as an intermediate step would have been predictable and routine in the art, as reprogramming techniques were well established and applicable to numerous somatic cell types. Although Giorgetti teaches reprogramming cells derived from cord blood into iPSCS, a person with ordinary skill would have understood that monocytes represent an interchangeable somatic cell source for iPSC generation. Such substitution would have not altered the fundamental reprogramming mechanism or the resulting dedifferentiated state, and still achieve the same downstream EPC differentiation.
Claims 8-12 are rejected under 35 U.S.C. 103 as being unpatentable over the combined teachings of Kim, Giorgetti and Aoki, as applied to claim 1, and in further view of Dupuis et al. [Published: 2021. World journal of stem cells, 13(8), 1094–1111] and Göbel et al. [Scientific reports, 8(1), 11676.]
With regard to claim 1, the combined teachings Kim, Giorgetti, and Aoki render obvious the claimed methodology, as iterated above in the 103 rejection the content of which is incorporated in claim 1. However, the references fail to teach a cellular population using mesenchymal stem cells as recited in claim 8.
Dupuis teaches the differentiation of iPSCs (as described in Giorgetti) into mesenchymal stem cells and various methods thereof [Abstract, page 1094, para 1, “iPSCs represent a new reliable, unlimited source to generate MSCs (MSCs derived from iPSC, iMSCs) from homogeneous and well-characterized cell lines, which would relieve many of the above mentioned technical and biological limitations.”].
Furthermore, through the integration of OSKM factors, it is noted that MSCs are able to dedifferentiate and subsequently differentiated into endothelial progenitor cells. As discussed by Göbel et al., MSCs are capable of being reprogrammed and dedifferentiated into iPSCs through OSKM factors, “Mesenchymal stromal cells of three different donors were transfected at passage six with episomal plasmids coding for OCT3/4, SOX2, KLF4, L-MYC, LIN28, and shRNA for p53, which provides an efficient method for integration-free reprogramming into iPSCs.” [Page 2 para 2]. This further supports that mesenchymal stem cells are amenable to cellular reprogramming. Once dedifferentiated, iPSCs may be predictable redifferentiated into endothelial progenitor cells as taught by Aoki. Thus, whether the derivation of MSCs are directly from tissue or from iPSCs, the claimed pathway of dedifferentiation and redifferentiation into EPCs represents a routine application of known cellular reprogramming techniques with a reasonable expectation of success.
Therefore, it would have been obvious to a person of ordinary skill in the art to select iPSCs as a cellular population to generate mesenchymal stem cells as taught by Dupuis with the combined teachings of Kim, Giorgetti and Aoki for isolating iPSCs from human cord blood since iPSCs hold an inherent property of pluripotency that allow iPSCs to differentiate into various cell types. One of ordinary skill in the art would be able to determine the need to dedifferentiate iPSCs to differentiate into MSCs. The combination represents the predictable use of known techniques according to their established functions. Thus, generating MSCs from iPSCs for use the cellular population in claim 1 would have been obvious to a person of ordinary skill.
Regarding claim 9, the combined teachings of Kim, Aoki, Giorgetti, Dupuis and Göbel render obvious claims 1 and 8. Moreover, Dupuis teaches that mesenchymal stem cells are tissue derived [page 1095, para 2, “In particular, mesenchymal SCs (MSCs) are adult pluripotent SCs that can be found in various tissues at low numbers. They were initially (mid 60s) identified by Friedenstein in the bone marrow of mice[3], but later have been found in many additional human tissues[4] including adipose tissue[5], umbilical cord[6], neural crest cells[7], and dental tissues[8-11]. Basically, all vascularized human tissues seem to harbor MSCs[12])”].
Regarding claim 10, the combined teachings of Kim, Aoki, Giorgetti, Dupuis and Göbel render obvious claims 1 and 8. Moreover, Dupuis teaches that mesenchymal stem cells are derived from a bodily fluid (page 1100 para 1, “Regarding claim 12, the combined teachings of Kim, Aoki, Giorgetti, Dupuis and Göbel render obvious claim 1." As the name suggests, it uses a lysate of platelets obtained from human peripheral blood to supplement iPSC´s growing media, replacing the FBS component, which by its animal origin raised ethical and safety concerns. This method, therefore, could improve the safety of the final iMSCs produced.”).
Regarding claim 11, the combined teachings of Kim, Aoki, Giorgetti, Dupuis and Göbel render obvious claims 1 , 8 and 10. Moreover, Dupuis teaches that mesenchymal stem cells are from the umbilical cord [page 1095, para 2, “In particular, mesenchymal SCs (MSCs) are adult pluripotent SCs that can be found in various tissues at low numbers. They were initially (mid 60s) identified by Friedenstein in the bone marrow of mice[3], but later have been found in many additional human tissues[4] including adipose tissue[5], umbilical cord[6], neural crest cells[7], and dental tissues[8-11]. Basically, all vascularized human tissues seem to harbor MSCs[12].” ].
Regarding claim 12, the combined teachings of Kim, Aoki, Giorgetti, Dupuis and Göbel render obvious claims 1, 8 and 10. Moreover, Göbel teaches stromal cells [page 5 para 3, “Mesenchymal stromal cells were isolated from the femoral bone marrow of different donors after orthopaedic surgery as described before”].
Claim 18 is rejected under 35 U.S.C. 103 as being unpatentable over the combined teachings of Kim, Giorgetti and Aoki, as applied to claim 1, and in further view of Ma et al. [Published: 2007. Journal of Central South University. Medical sciences, 32(3), 466–472.]
With regard to claim 1, the combined teachings Kim, Giorgetti, and Aoki render obvious the claimed methodology, as iterated above in the 103 rejection the content of which is incorporated in claim 1. However, the references fail to teach that the redifferentiated cell population can allow 50 passages while maintaining a stable karyotype.
Ma teaches a safety evaluation of progenitor cells sourced from umbilical cord blood that can be passaged at least 42 times and displayed strong abilities of proliferation and maintained a normal karyotype [Abstract, lines 10-12, “ EPCs were successfully derived from the UCB, and could be passaged to at least 42(nd) generation and had strong abilities of proliferation, acLDL intake and vasoformation, but there was not oncogenicity. They expressed endothelial cell-surface antigens and maintained normal karyotype.”]
Therefore, it was well known before the effective filing date that endothelial progenitor cells could maintain a normal karyotype beyond 40 passages to reach 50 passages, as the inherent proliferative capacity of EPCs was recognized in the art. A person of ordinary skill would have expected that the EPCs could be cultured through many passages without losing their functional characteristics.
Conclusion
Claims 1-20 are rejected.
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/KATRIEL BARCELLANO KASAYAN/ Examiner, Art Unit 1634
/MARIA G LEAVITT/ Supervisory Patent Examiner, Art Unit 1634