DETAILED ACTION
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 .
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 03/17/2026 has been entered.
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.
Claims 1, 2, 6, and 12-15 are rejected under 35 U.S.C. 103 as being unpatentable over Cakir et al. (Development of human brain organoids with functional vascular-like system, Nature Methods, 2019) in view of Peltz et al (US 20160256499A1).
Cakir et al. teaches generation of in vitro organoids which form a vascular-like network differentiated from embryonic stem cells. (Pg 2, Introduction) and use of lentiviral vectors to induce differentiation from stem cells into endothelial cell containing human cortical organoids. (Pg 8, “Generation of human cortical organoids (hCOs) with hETV2 introduction)
Peltz et al. (US 2016O256499A1) teaches methods of production for a population of hepatocyte like cells (iHeps) from a population of adipocyte-derived stem cells (ASCs). (57) This includes utilization of three dimensional culture conditions with a 1st and 2nd culture medium to create a precursor cell population and then forcing hepatocyte differentiation. (0047-0093) Peltz also teaches optional use of vectors to genetically trigger genes of interest to guide differentiation. (0167)
Regarding Claims 1 and 12: Peltz et al. teaches use of adipose derived pluripotent (0049) stem cells which are differentiated into hepatocyte-like cells. (57) Peltz also teaches optional use of genetic alteration by way of vectors with optionally an inducible promotor (0172) to cells in order to provide greater purity for the desired differentiated cells and use of a vector operatively linked to a promoter that is either pan-specific or specifically active in hepatocytes. (0167-0168) In addition to this, Peltz teaches that the cells may be altered to introduce genes which induce repair of genetic defects, enhance survival, or control proliferation. (0167) A person of ordinary skill in the art would know that modification of genes induce production of proteins which induce differentiation within the cell, which meets the known definition of “transcription factor”. This reads on the claimed method of transforming a first population of pluripotent stem cells with a vector encoding one or more transcription factors which induce differentiation of the first population of pluripotent stem cells down a particular lineage path under the control of an inducible promotor to obtain parenchymal-inducible stem cells. Peltz fails to teach induction of a second population of pluripotent stem cells down a vascular committed lineage pathway and creation of a vascularized organoid tissue.
Cakir et al. teaches use of embryonic stem cells previously transfected with BC4 which were additionally transfected with a lentiviral vector for hETV2 (a vascular specific transcription factor) to generate hETV2 expressing human embryonic stem cells. In addition to this, the lentivirus used was combined with a doxycycline inducible promotor. (Pg 8, “Generation of human cortical organoids (hCOs) with hETV2 introduction) Importantly, Cakir teaches that lacking microvasculature within organoids (specifically, hCOs) severely limits the size the organoids can reach in culture; with long-term culture leading to apoptotic cell death within the center of the organoid due to the lack of nutrient distribution within the organoid. (Pg 2, Introduction) Lastly, Cakir teaches that BC4 was differentiated with the induction of hETV2 in differentiation conditions and that when doxycycline is added to the culture media, the vascular lineage hCOs differentiate within the organoid, leading to a vascularized human brain organoid. (Pg 8, “Generation of human cortical organoids (hCOs) with hETV2 introduction) This reads on inducing a second population of pluripotent stem cells down a vascular committed lineage pathway and either before, during, or after inducing vascularization inducing expression of one or more transcription factors to create a vascularized organoid tissue.
Since these two cell populations are cultured individually, this reads on the claimed method of parenchymal-inducible stem cells (cells of Peltz) being expanded in culture prior to combining with the second population of pluripotent stem cells (cells of Cakir) as stated in Claim 12. However, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to add the population of cells taught by Peltz, which lack a vector which induces differentiation towards vascularization, with the population of cells taught by Cakir, which have been transfected with a vector for differentiation into vascularized tissue. One would have been motivated to do so based on the teaching of Cakir, which state the importance of vascularization in organoids to achieve stable long-term culture.
Regarding Claim 2: Cakir teaches that the cells transfected to become organoids are human embryonic stem cells. Regarding Claim 2, it is noted that Claim 1 teaches a first and second population of pluripotent stem cells. For the purposes of examination this is interpreted as reading on either population.
Regarding Claim 6: Peltz et al. teaches methods of production for a population of hepatocyte like cells (a type of parenchymal cell) from a population of adipocyte-derived stem cells. (57)
Regarding Claim 13: Cakir teaches a method for transfecting a population of HES-3 NKX2–1GFP/w endothelial cells already transfected with BC4 to direct differentiation towards cortical organoids with hETV2. Cakir also teaches use of a doxycycline-inducible promotor connected to the hETV2 vector. (Pg 8, “Generation of human cortical organoids (hCOs) with hETV2 introduction)
Regarding Claim 14: Cakir teaches that the vascularized organoids of the claimed invention are derived from human embryonic stem cells. (Pg 2, Introduction)
Regarding Claim 15: Cakir teaches use of a doxycycline-inducible promotor connected to the hETV2 vector which can be activated at any time by the addition of doxycycline to the culture media, thus reading on an inducible promotor operably linked to a transcription factor. (Pg 8, “Generation of human cortical organoids (hCOs) with hETV2 introduction)
Claim 3 is rejected under 35 U.S.C. 103 as being unpatentable over Cakir et al. (Development of human brain organoids with functional vascular-like system, Nature Methods, 2019) in view of Peltz et al (US 2016O256499A1) and Zhao et al. (PiggyBac transposon vectors: the tools of the human gene, Translational Lung Cancer Research, 2016)
The teachings of Cakir and Peltz are described above.
Zhao et al. teaches use of PiggyBac vectors, which are transposons that are movable between vectors and chromosomes through a “cut-and-paste” mechanism. (Pg 120, Abstract)
Regarding Claim 3: Cakir and Peltz fail to teach use of a PiggyBac vector. Zhao et al. teaches that PiggyBac vectors are efficient, safe, and stable and possess the ability to transpose between vectors and chromosomes. (Pg 120, Abstract) It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use the cells taught by Cakir and Peltz with the teaching of Zhao to utilize a PiggyBac vector with the respective transcription factors taught to induce a vascularized organoid tissue construct. One would have been motivated to do so based on the teaching of Zhao which states that PiggyBac vectors are “safe, efficient, and stable”.
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Cakir et al. (Development of human brain organoids with functional vascular-like system, Nature Methods, 2019) in view of Peltz et al (US 2016O256499A1) and Lancaster et al. (Generation of Cerebral Organoids from Human Pluripotent Stem Cells, 2014)
The teachings of Cakir and Peltz are described above.
Lancaster et al. teaches a protocol for the generation of cerebral organoids from human pluripotent stem cells by creation of embryoid bodies (EBs) followed by neural induction and culture in Matrigel. (Pg 5, “Experimental Design”)
Regarding Claim 4: Lancaster et al. teaches a protocol for development of a brain organoid which can be used for developmental and disease studies. (Pg 4, “Applications and limitations of the protocol”) In brief, embryoid bodies are made from either pluripotent or induced pluripotent stem cells. Following EB development, the EBs are placed into neural induction media for 4-5 days. Following this, the EBs are transferred to Matrigel droplets which should allow for formation of expanded neuroepithelium buds within the culture. (Pg 10-16, Procedure) It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use the protocol for the generation of cerebral organoids taught by Lancaster with the two populations of stem cells taught by Peltz and Cakir to produce a vascularized brain organoid. A person would have been motivated to do so and had a reasonable expectation of success due to the protocols taught by Cakir (which details a doxycycline inducible promotor to trigger vascular differentiation (Pg 8, “Generation of human cortical organoids (hCOs) with hETV2 introduction) and the protocol taught by Lancaster detailing generation of brain organoids.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Cakir et al. (Development of human brain organoids with functional vascular-like system, Nature Methods, 2019) in view of Peltz et al (US 2016O256499A1) and Chal et al. (Making muscle: skeletal myogenesis in vivo and in vitro, The Company of Biologists, 2017)
The teachings of Cakir and Peltz are described above.
Chal et al. teaches a detailed overview of skeletal myogenesis and how the knowledge has been applied to differentiate pluripotent stem cells into muscle fibers in vitro. (Pg 2104, Abstract) Chal et al. also teaches a detailed protocol for skeletal myogenesis from pluripotent stem cells (pg 2112, Fig 4) and also direct reprogramming using transcription factors such as Pax3 overexpression. (pg 2113, “Direct reprogramming strategies using transcription factors) It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use the protocol taught by Cakir of developing a vascularized brain organoid in addition to the teaching of the doxycycline inducible promoter with the differentiation protocol of Chal to create a myovascular organoid. One would have had a reasonable expectation of success at doing so due to the differentiation protocol taught by Chal to achieve an in vitro skeletal myogenesis culture and the addition of the doxycycline inducible promotor (hETV2) taught by Cakir to achieve a myovascular organoid.
Claims 7-11 are rejected under 35 U.S.C. 103 as being unpatentable over Cakir et al. (Development of human brain organoids with functional vascular-like system, Nature Methods, 2019) in view of Peltz et al (US 2016O256499A1) and Murohara et al. (US 201403 01988A1)
The teachings of Cakir and Peltz are described above.
Murohara et al. teaches methods of production a vascular progenitor cell sheet derived from induced pluripotent stem cells which forms a multi-celled layer on a gel material. (57) Murohara teaches several different adhesion substances (0065) and that the gel material contains reagents necessary for “the living and maintenance of cells”. (0081-0082) Murohara also teaches that the cell sheets have multiple layers, typically 10 or more, resulting in a three dimensional construct. (0098)
Regarding Claim 7: Neither Cakir nor Peltz teach a layering of the first and second populations on a first and second biocompatible matrix. Murohara teaches that a cell layer is formed by mixing a suitable gel material with cells and allowing the layer to set (0010, 0081-0082) and that multiple cell and gel layers are to be used. (0098) This reads on the claimed method of the first population of pluripotent stem cells being layered in or on a first biocompatible matrix and the second population of pluripotent stem cells being layered in or on a second biocompatible matrix. It would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to use the cell population of Cakir (embryonic stem cells previously transfected with BC4 which were additionally transfected with a lentiviral vector for hETV2 to direct differentiation into vascular like cells) with the cell population of Peltz (hepatocyte like cells (iHeps) from a population of adipocyte-derived stem cells (ASCs)) in the layering method taught by Murohara to create a layered construct of the first and second cell populations on biocompatible matrices. One would have had reason to combine and had a reasonable expectation of success due to the teachings of Murohara, who specifies that their invention can have upwards of 10-20 layers.
Regarding Claims 8 and 9: Murohara states that the gel material contains “the medium components necessary for the living and maintenance of cells” and also goes on to state several variations within the gel composition. (0081-0082) Murohara also teaches that a variety of cell adhesion proteins can be used (0065). This reads on both the claimed method of having the first and second biocompatible matrix comprising the same material (claim 8) and the first and second biocompatible matrix comprising different material compositions. (Claim 9)
Regarding Claim 10: Murohara teaches an example where the gel material contains type 1 collagen as an active ingredient. (0082)
Regarding Claim 11: Peltz teaches use of macroporous microcarriers (0076) to be used in high density three dimensional culture (0067) which provide higher surface area and better mechanical protection to the cells. (0076)
Claim 16 is rejected under 35 U.S.C. 103 as being unpatentable over Cakir et al. (Development of human brain organoids with functional vascular-like system, Nature Methods, 2019) in view of Peltz et al (US 20160256499A1) and Ostertag et al. (US 8399643 B2)
Peltz et al. teaches use of adipose derived pluripotent (0049) stem cells which are differentiated into hepatocyte-like cells. (57) Peltz also teaches optional use of genetic alteration by way of vectors with optionally an inducible promotor (0172) to cells of the claimed method in order to provide greater purity for the desired differentiated cells and use of a vector operatively linked to a promoter that is either pan-specific or specifically active in hepatocytes. (0167-0168) In addition to this, Peltz teaches that the cells of the claimed invention may be altered to introduce genes which induce repair of genetic defects, enhance survival, or control proliferation. (0167) A person of ordinary skill in the art would know that modification of genes induce production of proteins which induce differentiation within the cell, which meets the known definition of “transcription factor”. This reads on the claimed method of transforming a first population of pluripotent stem cells with a vector encoding one or more transcription factors which induce differentiation of the first population of pluripotent stem cells down a particular lineage path under the control of an inducible promotor to obtain parenchymal-inducible stem cells. Peltz fails to teach induction of a second population of pluripotent stem cells down a vascular committed lineage pathway and creation of a vascularized organoid tissue or use of a second hyperactive piggybac transposase.
Ostertag teaches piggybac transposase proteins and nucleic acids encoding said proteins. (57) Specifically, Ostertag teaches that hyperactive transposases are ideal for the mobilization of a transposon from one genomic location to another, making them the ideal choice for yielding a greater number of transposon integrations per cell as opposed to a non-hyperactive transposase. (Col 1, ln 27-35) Ostertag further teaches in an embodiment of the invention the inclusion of a transcriptionally active gene and specifies use of tetracycline, reading on use of a tetracycline inducible expression system. (Col 15, ln 1-16)
Cakir et al. teaches use of embryonic stem cells previously transfected with BC4 which were additionally transfected with a lentiviral vector for hETV2 (a vascular specific transcription factor) to generate hETV2 expressing human embryonic stem cells. In addition to this, the lentivirus used was combined with a doxycycline inducible promotor. (Pg 8, “Generation of human cortical organoids (hCOs) with hETV2 introduction) Importantly, Cakir teaches that lacking microvasculature within organoids (specifically, hCOs) severely limits the size the organoids can reach in culture; with long-term culture leading to apoptotic cell death within the center of the organoid due to the lack of nutrient distribution within the organoid. (Pg 2, Introduction) Lastly, Cakir teaches that BC4 was differentiated with the induction of hETV2 in differentiation conditions and that when doxycycline is added to the culture media, the vascular lineage hCOs differentiate within the organoid, leading to a vascularized human brain organoid. (Pg 8, “Generation of human cortical organoids (hCOs) with hETV2 introduction) This reads on inducing a second population of pluripotent stem cells down a vascular committed lineage pathway and either before, during, or after inducing vascularization inducing expression of one or more transcription factors to create a vascularized organoid tissue.
Since these two cell populations are cultured individually, this reads on the claimed method of parenchymal-inducible stem cells (cells of Peltz) being expanded in culture prior to combining with the second population of pluripotent stem cells (cells of Cakir) as stated in Claim 12. However, it would have been obvious for a person of ordinary skill in the art before the effective filing date of the claimed invention to add the population of cells taught by Peltz, which lack a vector which induces differentiation towards vascularization, with the population of cells taught by Cakir, which have been transfected with a vector for differentiation into vascularized tissue. One would have been motivated to do so based on the teaching of Cakir, which state the importance of vascularization in organoids to achieve stable long-term culture. Furthermore, one skilled in the art would have been motivated to incorporate the teachings of Ostertag regarding a second vector comprising a hyperactive piggybac transposase and first vector comprising a tetracycline inducible expression system as Ostertag teaches that hyperactive piggybac transposons are ideal for a greater number of transposon integrations per cell.
Response to Arguments
Examiner is confused by Applicant’s statement that Peltz is incorrect in referring to the population of stem cells taught as pluripotent. Examiner acknowledges the reference provided by the Applicant to the teachings of Zuk et al, however, Peltz very clearly states in Example 1 of the claimed invention use of ASCs which were reprogrammed into iPS cells with the transfection of Oct4, Sox2, Klf4, and c-Myc. (0236) As one skilled in the art would understand, these four genes are considered to be hallmarks of pluripotency. In addition, Peltz states that the ASCs of Example 1 must be first reprogrammed into and then exit from the pluripotent state prior to differentiation into iPSHeps. (0236) Lastly, the definition Peltz discloses regarding pluripotency states “in the context of cell ontogeny, the adjective “differentiated” or “differentiating” is a relative term… a cell that has progressed further down the developmental pathway than the cell it is being compared with”. As such, pluripotent stem cells can differentiate into lineage-restricted progenitor cells. (0042) Therefore, it is factually accurate to state that Peltz makes use of pluripotent stem cells.
Applicant argues that there is no motive to combine the teachings of Peltz and Cakir to achieve a population of vascularized cortical cells as Cakir teaches the vascularization of a population of cells. However, while Cakir teaches a method of production of a vascularized organoid, Cakir fails to teach use of a parenchymal inducible population of cells, as neither stem cells nor endothelial cells are parenchymal in origin. Therefore, in order to generate a parenchymal-inducible population of stem cells, one skilled in the art must look to Peltz, who teaches use of transcription factors which would induce a population of stem cells down a parenchymal lineage, as described above. Only by virtue of the combination of the teachings of Cakir and Peltz would one skilled in the art arrive at the generation of a vascularized organoid comprising parenchymal-inducible stem cells. In addition, as discussed above, Peltz does definitively teach a population of pluripotent stem cells, so the argument presented by the Applicant regarding combining a vascular differentiation factor with adult (i.e. not pluripotent stem cells) is moot.
Regarding claims 3, 4, 5, and 7-11: Applicant argues that the respective additional references of Zhao (claim 3), Lancaster (claim 4), Chal (claim 5), and Murohara (claims 7-11) fail to overcome the deficiencies of the combination of references of Cakir and Peltz, yet Applicant has neglected to provide any deficiencies outside of the original argument of claim 1 failing to be overcome. As the Applicant recognizes, claims 3, 4, 5, and 7-11 are dependent on claim 1. As the above discussion rendered the arguments of claim 1 unpersuasive, the arguments surrounding claims 3, 4, 5, and 7-11 are found unpersuasive on the same basis.
Conclusion
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/HANNA MARIE THUESON/ Examiner, Art Unit 1638
/Tracy Vivlemore/Supervisory Primary Examiner, Art Unit 1638
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