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 .
Claims 1-15, 17, 18, 20, 21, 23, 25-46, 49-58, 60-72, 74, 78, 81, 83, 88-94 have been canceled. Claims 16, 19, 22, 24, 47, 48, 59, 73, 75-77, 79, 80, 82, 84-87, 95-100 remain pending.
Election/Restrictions
Applicants elected Group I, claims 1, 2, 4-6, 9, 11, 13, 16-19, 22, 24, 26, 28, 29, 32, 35, 43, 44, without traverse in the reply filed on 10-5-18. Group I is drawn to a method of controlling cell proliferation using the “ablation link” (ALINK) system as opposed to the “inducible exogenous activator or regulation of a CDL” (EARC) (pg 4 of the restriction requirement sent 7-5-18; pg 8 of the response filed 10-5-18).
Applicants also elected the species of CDK1 as the genetically modified gene in the cell, but the species requirement was withdrawn in the office action sent 11-2-18.
Claims 84-87 (added 1-31-20) remain directed to an invention that is independent or distinct from the invention originally claimed for reasons of record and remain withdrawn from consideration as being directed to a non-elected invention. See 37 CFR 1.142(b) and MPEP § 821.03.
Claims 16, 19, 22, 24, 47, 48, 59, 73, 75-77, 79, 80, 82, 95-100 remain under consideration.
Applicant's arguments filed 4-27-26 have been fully considered but they are not persuasive.
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim interpretation/objections
The structures of the “dox-bridge” in the “ALINK” and “EARC” systems in Fig. 1-3 cannot be determined and have not been clarified on the record.
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Fig. 1A shows “induced negative effectors of proliferation” (iNEP; pg 8, para 40) equal “EARC” + “ALINK” (bottom right corner of each of the four scenarios); however, the structure of the “EARC”, “ALINK” and the final iNEP cannot be determined. Fig. 1B discloses 5 scenarios for expressing “CDL protein” and “ALINK”; however, the structure of the “dox-bridge”, ALINK, and iNEPs are unclear.
Fig. 3B and C appear to have the structure of some genetic modifications, but the labels in Fig. 3B cannot be discerned, and it is unclear how the structures in Fig. 3B and C correlate to the transcriptional linkage in claim 16 or the ability to inhibit proliferation of a cell as required in claim 16. Accordingly, the structures of the “ALINK” and “EARC” systems, specifically the Dox-bridge and “iNEPs”, cannot be determined in Fig. 1-3; therefore, it cannot be determined how the structure of the “ALINK”, “EARC” or “iNEPs” in Fig. 1-3 correlate to the structure of the vector in claim 16.
The function of the ALINK system is to allow co-expression of the host gene and a negative selectable marker; the presence of an “inducer of the negative selectable marker” causes cell death (pg 20, para 95). “Negative selectable markers” and “inducers of negative selectable markers” were well-known in the art, e.g. ganciclovir (GCV) (para 6, 98, 102), Caspase 9 (iCasp/AP1903) (para 6, 106), dCK.DM (para 100), and gene-direct enzyme/prodrug therapy” (GEPT) (para 101), e.g. deaminase/5-fluorocytosine (CD/5-FC) (para 102, 104), and carboxyl esterase/irinotecan (CE/CPT-11) (para 102, 105). The function of the EARC system allows expression of the host gene only in the presence of the inducer; the absence or withdrawal of the inducer causes cell death (pg 22, par 109). The EARC system does not appear to be part of the claim set because the claim set is limited to co-expression of the negative selectable marker and the gene of interest and to contacting the negative selectable marker with an inducer that is capable of killing the cell. Therefore, it appears that the function of the vector claimed is limited to the “ALINK” system.
Claim 16 simply requires the cells that co-expresses an endogenous gene and a negative selection marker both under the control of the endogenous gene’s promoter; an inducer of the negative marker is added to kill the cell. The invention relates to the “ALINK” system which is limited to using the inducer to kill the cell, but not the “EARC” system which is limited to withdrawal of an inducer. The specification and the art do not teach the structure of any “dox-bridge” used in “ALINK” inducible gene expression system, but claim 16 does not invoke any language about any “dox-bridge”.
The phrase “wherein the genetically modified mammalian differentiated cells within the population being a direct product of differentiating a subset of the genetically modified mammalian stem cells within the population in vitro” in claim 16 makes the claim confusing.
The phrases “within the population” and “within the population in vitro” are extraneous.
The concept of a cell population comprising “genetically modified mammalian stem cells” and “genetically modified mammalian differentiated cells”, wherein the “genetically modified mammalian differentiated cells” are “a direct product of differentiating a subset of the genetically modified mammalian stem cells within the population in vitro” in claim 16 invokes product-by-process without clearly setting forth the process step required to achieve the product, i.e. differentiating isolated genetically modified mammalian stem cells such that stem cells and differentiated cells are obtained.
The product-by-process language in claim 16 is problematic because differentiated cells having the exact same structure from a different culture container can be mixed with stem cells to arrive at the same product. The product-by-process language fails to bear patentable weight because it does not distinguish the “differentiated cells” from any other cells having the same structure and function. The concept is also problematic because it is attempting to say the stem cells and differentiated cells never left the culture container and are still maintained together in that container, but the phrase “direct product of differentiating [the] stem cells” fails to capture this concept.
It is unclear when “genetically modified mammalian differentiated cells” are a “direct product” of stem cells as required in claim 16, i.e. it is unclear whether the phrase is limited to the next cell type within the lineage of differentiation or if it encompasses any cell type within the lineage. For example, if the stem cells are pluripotent cells, it is unclear whether the phrase is limited to “activated pluripotent” cells (the next step in differentiation of pluripotent cells) or if embryoid bodies, mesoderm, endoderm and ectoderm lineages are encompassed by the phrase.
The concept of “differentiating a subset of the” stem cells in claim 16 makes it unclear because there are no subsets of the stem cells – they are all the same.
Claim 16 can be written more simply and concisely as ---A population of cells comprising:
i) isolated mammalian stem cells;
ii) isolated cells differentiated from the stem cells,
each which comprises a nucleic acid sequence encoding a negative selectable marker inserted at the 3’ end of an endogenous [CDK1, TOP2A, CENPA, BIRC5, or EEF2] coding sequence and operably linked to an endogenous [CDK1, TOP2A, CENPA, BIRC5, or EEF2] promoter, wherein the stem cells and cells differentiated from the stem cells are capable of expressing the negative selectable marker and [CDK1, TOP2A, CENPA, BIRC5, or EEF2 under the control of the endogenous CDK1, TOP2A, CENPA, BIRC5, or EEF2 promoter and are capable of being killed by ganciclovir, 5-fluorocytosine, irinotecan, or AP1903 when dividing but not when quiescent---.
Claims 19, 48, 75, 80 should clearly set forth the second genetic modification following the format of genetic modification in claim 16.
Written Description
It was well-within the ability of the ordinary artisan to operably link a negative selectable marker to a CDK1 promoter in any isolated mammalian cell such that expression of CDK1 and the negative selectable marker occur.
It is assumed that sequencing mammalian homologs of human or mouse CDK1 genes was well-within the ability of the ordinary artisan at the time of filing.
It is assumed that using any well-known negative selectable marker was well-within the ability of the ordinary artisan at the time of filing.
The claims do not invoke any specific language about a “dox-bridge”. There is no structure in the claims that is specific to the indecipherable structures in Fig. 1A or 1B.
The ability to make/use any genetic modification homozygous, heterozygous, or compound heterozygous in an isolated mammalian cell was well-within the ability of the ordinary artisan at the time of filing.
Hochhauser (J. Biol. Chem., 1992, Vol. 267, No. 26, pg 18961-18965) characterized the TOP2A promoter. Targeting the TOP2A promoter with a marker gene was well-known (Morgan, Mol. Pharm., 2001, Vol. 59, pg 203-211, Fig 1; Fraser, Mol. Pharm., 1995, Vol. 47, pg 696-706; Fig. 1; Adachi, Biochem. & Biophys. Res. Comm., 1997, Vol. 230, pg 105-109; Plasmid construction; et al.). Hickson (WO 98/37207) operably linked the TOP2A promoter to a sequence encoding a cytotoxic protein (Example 3, pg 71). Therefore, it was well-within the ability of the ordinary artisan to operably link a negative selectable marker to a TOP2A promoter in any isolated mammalian cell such that expression of TOP2A and the negative selectable marker occur.
Withdrawn rejection
The rejection regarding any mammalian stem cells and differentiated cells in vivo or in vitro as broadly encompassed by claim 16 has been withdrawn because the claims have been limited to in vitro cells.
New rejection
Claims 16, 19, 22, 24, 47, 48, 59, 73, 75-77, 79, 80, 82, 95-100 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention.
The concept of a cell population comprising “genetically modified mammalian stem cells” and “genetically modified mammalian differentiated cells”, wherein the “genetically modified mammalian differentiated cells” are “a direct product of differentiating a subset of the genetically modified mammalian stem cells within the population in vitro” in claim 16 lacks written description other than pluripotent cells and teratomas.
Claim 16 is drawn to An in vitro population of genetically modified mammalian cells comprising genetically modified mammalian stem cells and genetically modified mammalian differentiated cells, the genetically modified mammalian differentiated cells within the population being a direct product of differentiating a subset of the genetically modified mammalian stem cells within the population in vitro wherein the genetically modified mammalian stem cells and the genetically modified mammalian differentiated cells comprise an exogenous polynucleotide encoding a negative selectable marker inserted into a locus of at least one endogenous gene selected from the group consisting of cyclin dependent kinase 1 (CDK1), DNA topoisomerase II alpha (TOP2A), centromere protein A (CENPA), baculoviral IAP repeat containing 5 (BIRC5), and
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eukaryotic translation elongation factor 2 (EEF2), such that the polynucleotide encoding the negative selectable marker is operably linked to a promoter of the at least one said endogenous gene, wherein the genetically modified mammalian stem cells and the genetically modified mammalian differentiated cells co-express[[es]] the negative selectable marker and a protein encoded by the at least one said gene, and wherein contacting the genetically modified mammalian stem cells and the genetically modified mammalian differentiated cells with an inducer of the negative selectable marker is capable of killing the genetically modified mammalian stem cells and the genetically modified mammalian differentiated .
Claim 16 encompasses any stem cell including a totipotent, pluripotent, or multipotent cell. Multipotent cells include hair, skin, eye, muscle, bone, cartilage, heart, lung, liver, pancreas, kidney, reproductive organ, sperm, egg, et al. stem cells.
Claim 16 encompasses any cells differentiated from any stem cell including a multipotent or terminally differentiated cell. Multipotent cells include hair, skin, eye, muscle, bone, cartilage, heart, lung, liver, pancreas, kidney, reproductive organ, sperm, egg, et al. stem cells. Terminally differentiated cells can be from any organ in the mammalian body.
The scope of the product-by-process language in claim 16 cannot be determined because differentiated cells having the exact same structure from a different culture container can be mixed with stem cells to arrive at the same product, and such embodiments are not disclosed by applicants.
The specification does not teach when “genetically modified mammalian differentiated cells” are a “direct product” of stem cells as required in claim 16, i.e. it is unclear whether the phrase is limited to the next cell type within the lineage of differentiation or if it encompasses any cell type within the lineage. For example, if the stem cells are pluripotent cells, it is unclear whether the phrase is limited to “activated pluripotent” cells (the next step in differentiation of pluripotent cells) or if embryoid bodies, mesoderm, endoderm and ectoderm lineages are encompassed by the phrase.
The concept of “differentiating a subset of the” stem cells in claim 16 lacks written description because there are no subsets of the stem cells – they are all the same.
Rong (J. Biol. Chem., 2012, Vol. 287, pg 32338-32345) decreased the risk of malignancy of a population of human embryonic stem (ES) cells in transplant therapies by introducing the HSV-TK suicide gene via homologous recombination into the 3’ UTR of the endogenous Nanog gene. Nanog and HSV-TK were co-expressed, and the presence of ganciclovir caused the cells to die (pg 32340, Fig. 1). Rong taught pluripotent cells (abstract, materials and methods) which are the “stem” cells in claim 16. Rong taught teratomas which differentiate from and are together with the pluripotent cells (abstract, materials and methods) – the teratomas are the “differentiated” cells in claim 16. Rong also taught pluripotent cells “remaining with their differentiated derivatives” (abstract and throughout) which are the “stem” and “differentiated” cells in claim 16.
The specification and the art at the time of filing are limited to pluripotent cells as the starting material (Examples 1-7). The specification does not correlate pluripotent cells as the starting material to totipotent cells or to hair, skin, eye, muscle, bone, cartilage, heart, lung, liver, pancreas, kidney, reproductive organ, sperm, egg, et al. stem cells.
The specification is limited to culturing the pluripotent cells with the ALINK system in pluripotent media and adding ganciclovir (Example 1). The specification does not teach culturing the pluripotent cells with the ALINK system in differentiation such that a mixed population of cells is obtained. The specification does not teach differentiating the pluripotent cells into hair, skin, eye, muscle, bone, cartilage, heart, lung, liver, pancreas, kidney, reproductive organ, sperm, egg, et al. stem cells and killing the pluripotent or multipotent cells that are dividing by adding an inducer of a negative selectable marker as broadly encompassed by claim 16. The specification does not teach differentiating the pluripotent cells into fully differentiated hair, skin, eye, muscle, bone, cartilage, heart, lung, liver, pancreas, kidney, reproductive organ, sperm, or egg cells and killing the pluripotent or fully differentiated cells that are dividing by adding ganciclovir as broadly encompassed by claim 16. Accordingly, the concept lacks written description other than pluripotent cells and teratomas.
Accordingly, the concept lacks written description.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth in the rejection.
Enablement
Claims 16, 19, 22, 24, 47, 48, 59, 73, 75-77, 79, 80, 82, 95-100 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for isolated mammalian pluripotent cells and cells that are a direct product of differentiating the pluripotent cells in vitro, does not reasonably provide enablement for stem cells and differentiated cells in vivo or differentiated cells that are not a direct result of differentiating the stem cells in vitro. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make/use the invention commensurate in scope with these claims.
Claim 16 and its scope are discussed above.
The specification and the art at the time of filing are limited to pluripotent cells as the starting material (Examples 1-7). The specification does not correlate pluripotent cells as the starting material to totipotent cells or to hair, skin, eye, muscle, bone, cartilage, heart, lung, liver, pancreas, kidney, reproductive organ, sperm, egg, et al. stem cells.
The specification is limited to culturing the pluripotent cells with the ALINK system in pluripotent media and adding ganciclovir (Example 1). The specification does not teach culturing the pluripotent cells with the ALINK system in differentiation such that a mixed population of cells is obtained. The specification does not teach differentiating the pluripotent cells into hair, skin, eye, muscle, bone, cartilage, heart, lung, liver, pancreas, kidney, reproductive organ, sperm, egg, et al. stem cells and killing the pluripotent or multipotent cells that are dividing by adding ganciclovir as broadly encompassed by claim 16. The specification does not teach differentiating the pluripotent cells into fully differentiated hair, skin, eye, muscle, bone, cartilage, heart, lung, liver, pancreas, kidney, reproductive organ, sperm, or egg cells and killing the pluripotent or fully differentiated cells that are dividing by adding ganciclovir as broadly encompassed by claim 16.
Given the lack of guidance in the specification taken with the art at the time of filing, it would have required those of skill undue experimentation to determine how to make/use any mixed population of isolated cells comprising any mammalian stem cell and any cell differentiated from the stem cell as broadly encompassed by claim 16 other than pluripotent cells and teratomas.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth in the rejection.
The product-by-process language in claim 16 is problematic because differentiated cells having the exact same structure from a different culture container can be mixed with stem cells to arrive at the same product. The product-by-process language fails to bear patentable weight because it does not distinguish the “differentiated cells” from any other cells having the same structure and function. The concept is also problematic because it is attempting to say the stem cells and differentiated cells never left the culture container and are still maintained together in that container, but the phrase “direct product of differentiating [the] stem cells” fails to capture this concept.
It is unclear when “genetically modified mammalian differentiated cells” are a “direct product” of stem cells as required in claim 16, i.e. it is unclear whether the phrase is limited to the next cell type within the lineage of differentiation or if it encompasses any cell type within the lineage. For example, if the stem cells are pluripotent cells, it is unclear whether the phrase is limited to “activated pluripotent” cells (the next step in differentiation of pluripotent cells) or if embryoid bodies, mesoderm, endoderm and ectoderm lineages are encompassed by the phrase.
The concept of “differentiating a subset of the” stem cells in claim 16 makes it unclear because there are no subsets of the stem cells – they are all the same.
Claim Rejections - 35 USC § 103
A) Claims 16, 22, 24, 47, 59, 73, 76, 77, 79, 82, 95, 96 remain rejected under 35 U.S.C. 103 as being unpatentable over Rong (J. Biol. Chem., 2012, Vol. 287, pg 32338-32345) in view of Diril (PNAS, 2012, Vol. 109, No. 10, pg 3826-3831), Ivics (Mol. Therapy, 2015, Vol. 23, No. 9, pg 1417-1420), Chen (Biomaterials, 2013, Vol. 34, pg 1701-1711), Cheng (Biomaterials, 2012, Vol. 33, pg 3105-3204), Malecki (Stem Cell Res. & Therapy, 2014, Vol. 5, No. 73, pg 1-10), Dahler (J. Cell. Physiol., 1998, Vol. 177, pg 474-482), Liu (Biochem. & Biophysical Res. Comm., 1998, Vol. 246, pg 696-702), Wang (Shijie Huarent Xiaohua Zazhi, 2005, Vol. 13, No. 19, pg 2381-2385, abstract only), Shao (Zhongguo Ganzangbring Zazhi, Dianziban, 2013, Vol. 5, No. 2, pg 10-13, abstract only), and Minamide (PLoS One, 2014, Vol. 9, No. 1, e84460, pg 1-15).
Rong decreased the risk of malignancy of a population of human embryonic stem (ES) cells in transplant therapies by introducing the HSV-TK suicide gene via homologous recombination into the 3’ UTR of the endogenous Nanog gene. Nanog and HSV-TK were co-expressed, and the presence of ganciclovir caused the cells to die (pg 32340, Fig. 1). Rong differentiated isolated mammalian pluripotent cells into neural progenitors (pg 32339, col. 2, “Differentiation of hESCs into neural progenitor cells”) which are stem cells and differentiated cells that are a “direct product of differentiating a subset of stem cells” as required in claim 16.
Rong did not teach targeting the CDK1 gene.
However, the ability to target the CDK1 gene for negative selection using the HSV-TK gene in pluripotent cells was well-known in the art as described by Diril. Diril transfected isolated mouse ES cells with a targeting vector comprising a nucleic acid sequence encoding HSV-TK operably linked to the CDK1 gene (Fig. 1A, item II). ES cells containing the vector were selected using ganciclovir for selection (pg 3831, col. 1, last paragraph).
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The CDK1 and TK must be co-expressed in the cells that are killed as claimed as evidenced by the structure of the insertion and the ability to negatively select using ganciclovir. Killing the cell using ganciclovir simply means the crossover event did not occur, CDK1 and TK were co-expressed, and the undesirable cells were negatively selected using ganciclovir.
Suicide strategies for pluripotent cells and cells differentiated therefrom using a variety of promoters were well-known in the art at the time of filing using a housekeeping promoter, a tissue-specific promoter or a pluripotent-specific promoter. This is described by Ivics who taught “Yagyu et al. wisely settled for the promoter of the housekeeping gene human elongation factor-1 alpha (EF1α) in their study, because it provides fairly robust transgene expression in a wide range of cell types. Other promoter elements providing cell type-specific expression may be considered in the future; for example, promoters exclusively active in pluripotent stem cells would allow selective elimination of iPS cells from heterogeneous cell populations, including both iPS cells and their differentiated progeny (Figure 1C)” (pg 1419, col. 2, end of 1st para, beginning of 2nd para).
Chen, Cheng, and Malecki also described a variety of pluripotent cell suicide strategies using a variety of promoters (Chen - EF1α or Nanog; Cheng - EF1α or Nanog; Malecki - Table 1).
Therefore, those of skill would have recognized that changing the pluripotent-specific promoter of Rong to the CDK1 promoter had the advantage of killing a “wide range of cell types” as described by Ivics and was merely a matter of design strategy.
The desire to specifically target the CDK1 promoter for expressing a marker gene was also well-known as evidenced by Dahler (abstract), Liu (pg 697, col. 1, Plasmid DNA constructs), Wang (abstract), Shao (abstract), Minamide (Luciferase-CDK1; pg 6, last full para).
Thus it would have been obvious to those of ordinary skill in the art at the time of filing to introduce an HSV-TK cassette into the 3’ end of a gene in a pluripotent cell to prevent teratoma formation in the presence of ganciclovir as described by Rong, wherein the gene was CDK1 described by Diril. The ordinary artisan would have recognized that CDK1 is essential for cell division (title of Diril article) and that expressing TK using the CDK1 promoter would kill dividing cells, such as tumorigenic cells. That artisan would have been motivated to target the CDK1 promoter instead of the pluripotent-specific promoter described by Rong to express TK in pluripotent tumorigenic cells as well as their dividing offspring.
In the reverse, those of ordinary skill in the art at the time of filing would have been motivated to modify the CDK1-specific expression system of Diril to the co-expression inducible system described by Rong to obtain an inducible kill switch for pluripotent cells.
Those of ordinary skill would have had a reasonable expectation of modifying the CDK1 gene with a targeting vector that causes co-expression of CDK1 and HSV-TK (without the need for the neo cassette or a crossover event) as evidenced by Diril who taught how to target the 3’ end of the CDK1 gene and insert an HSV-TK cassette. The ability to modify the CDK1 gene for marker gene expression was well-within the ability of the artisan as evidenced by Dahler, Liu, Wang, Shao, and Minamide.
Claims 22, 76 have been included because Rong used HSV-TK.
Claims 24, 77, 96 have been included because Rong used human cells.
Claims 47, 79, 95 have been included because the combined teachings require HSV-TK operably linked to a CDK1 promoter that allows co-expression of CDK1 and HSV-TK for reasons set forth above.
Claim 59, 82 has been included because the combined teachings require using HSV-TK and ganciclovir.
Claim 73 has been included because the combined teachings require using ganciclovir to induce HSV-TK expression and cell death.
Response to arguments
Applicants' argue Rong only addresses killing pluripotent cells (which have a risk of forming teratomas) and does not contemplate killing cells differentiated from pluripotent cells. Applicants’ argument is not persuasive. The claims are product claims; they are not method claims and do not require an active step of killing derivatives of pluripotent cells. The mechanism of action of Rong may be different than applicants’, but the mechanism of action of the combined references is exactly the same as applicants’. Finally, the combined references teach a system for killing any dividing cells, i.e. pluripotent cells and their derivatives, which is the same mechanism of action described by applicants.
Applicants argue Diril is limited to transient selection of clones, not safety. Applicants’ argument is not persuasive because Diril used HSV-TK expressed under the control of a CDK1 promoter to kill cells.
Applicants argue Ivics discusses two options and is not limited to using housekeeping promoters for expression in pluripotent cells. Applicants’ argument is not persuasive. Ivics provides evidence that it was well-known to use housekeeping promoters for expression of suicide strategies in pluripotent cells – this is irrefutable.
Applicants’ discussion of Malecki is noted, but is not persuasive. Malecki merely proves that a variety of pluripotent cell suicide strategies using a variety of promoters were well-known and that the promoter used was interchangeable (Malecki - Table 1).
Overall, Rong differentiated isolated mammalian pluripotent cells into neural progenitors (pg 32339, col. 2, “Differentiation of hESCs into neural progenitor cells”) each containing the system claimed under the control of a nanog (a pluripotent-specific) promoter, and killed pluripotent cells using gancyclovir. But killing dividing cells using HSV-TK under the control of a CDK1 promoter (a housekeeping gene associated with cell division) was well-known as described by Diril. Those skilled would have been motivation to change the pluripotent-specific promoter of Rong to the CDK1 promoter associated with cell division because suicide strategies for pluripotent cells and cells differentiated therefrom using housekeeping promoters were well-known (Ivics).
B) Claims 19, 48, 75, 80, 97 remain rejected under 35 U.S.C. 103 as being unpatentable over Rong (J. Biol. Chem., 2012, Vol. 287, pg 32338-32345) in view of Diril (PNAS, Vol. 109, No. 10, pg 3826-3831), Ivics (Mol. Therapy, 2015, Vol. 23, No. 9, pg 1417-1420), Chen (Biomaterials, 2013, Vol. 34, pg 1701-1711), Cheng (Biomaterials, 2012, Vol. 33, pg 3105-3204), Malecki (Stem Cell Res. & Therapy, 2014, Vol. 5, No. 73, pg 1-10), Dahler (J. Cell. Physiol., 1998, Vol. 177, pg 474-482), Liu (Biochem. & Biophysical Res. Comm., 1998, Vol. 246, pg 696-702), Wang (Shijie Huarent Xiaohua Zazhi, 2005, Vol. 13, No. 19, pg 2381-2385, abstract only), Shao (Zhongguo Ganzangbring Zazhi, Dianziban, 2013, Vol. 5, No. 2, pg 10-13, abstract only), and Minamide (PLoS One, 2014, Vol. 9, No. 1, e84460, pg 1-15) as applied to claims 16, 22, 24, 47, 59, 73, 76, 77, 79, 82, 95, 96 and further in view of Lang (Gene, 1998, Vol. 221, pg 255-266), Hochhauser (J. Biol. Chem., 1992, Vol. 267, No. 26, pg 18961-18965), Morgan (Mol. Pharm., 2001, Vol. 59, pg 203-211), Fraser (Mol. Pharm., 1995, Vol. 47, pg 696-706), Adachi (Biochem. & Biophys. Res. Comm., 1997, Vol. 230, pg 105-109), and Hickson (WO 98/37207).
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide taught a composition comprising isolated human stem cells and “differentiated” cells whose genome comprises an exogenous sequence encoding TK operably linked to the promoter of an endogenous gene, wherein the cells functionally expresses TK and protein encoded by the endogenous gene.
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach targeting a second gene that was TOP2A as encompassed by claims 19, 48, 75, 80, 97.
However, Lang taught the structure of the TOP2A gene (“Structural organization of the human TOP2A and TOP2B genes”). Hochhauser characterized the TOP2A promoter (title; Figures). Targeting the TOP2A promoter with a marker gene was well-known as evidenced by Morgan (Fig 1), Fraser (Fig. 1), and Adachi (“Plasmid construction”). Hickson operably linked the TOP2A promoter to a sequence encoding a cytotoxic protein (Example 3, pg 71). Therefore, it was well-within the ability of the ordinary artisan to operably link a negative selectable marker to a TOP2A promoter in any isolated mammalian cell such that expression of TOP2A and the negative selectable marker occur.
Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to make an isolated human cell whose genome comprises an exogenous sequence encoding TK operably linked to the promoter of an endogenous gene, wherein the cell functionally expresses TK and protein encoded by the endogenous gene as described by Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide and targeting a second gene that is TOP2A as described by Lang, Hochhauser, Mogan, Fraser, Adachi, and Hickson. Those of ordinary skill in the art at the time of filing would have been motivated to do so because Hickson taught the gene could be used to express a cytotoxic protein.
Response to arguments
Applicants' argue the addition of Lang, Hochhauser, Morgan, Fraser, Adachi, and Hickson does not remedy the deficiencies above. Applicants’ argument is not persuasive for reasons set forth above.
C) Claims 16, 22, 24, 59, 73, 76, 77, 82, 96, 97 remain rejected under 35 U.S.C. 103 as being unpatentable over Rong (J. Biol. Chem., 2012, Vol. 287, pg 32338-32345) in view of Lang (Gene, 1998, Vol. 221, pg 255-266), Hochhauser (J. Biol. Chem., 1992, Vol. 267, No. 26, pg 18961-18965), Hickson (WO 98/37207), Ivics (Mol. Therapy, 2015, Vol. 23, No. 9, pg 1417-1420), Chen (Biomaterials, 2013, Vol. 34, pg 1701-1711), Cheng (Biomaterials, 2012, Vol. 33, pg 3105-3204), Malecki (Stem Cell Res. & Therapy, 2014, Vol. 5, No. 73, pg 1-10), Morgan (Mol. Pharm., 2001, Vol. 59, pg 203-211), Fraser (Mol. Pharm., 1995, Vol. 47, pg 696-706), Adachi (Biochem. & Biophys. Res. Comm., 1997, Vol. 230, pg 105-109).
Rong decreased the risk of malignancy of a population of human embryonic stem (ES) cells in transplant therapies by introducing the HSV-TK suicide gene via homologous recombination into the 3’ UTR of the endogenous Nanog gene. Nanog and HSV-TK were co-expressed, and the presence of ganciclovir caused the cells to die (pg 32340, Fig. 1). Rong differentiated isolated mammalian pluripotent cells into neural progenitors (pg 32339, col. 2, “Differentiation of hESCs into neural progenitor cells”) which are stem cells and differentiated cells that are a “direct product of differentiating a subset of stem cells” as required in claim 16.
Rong did not teach targeting the TOP2A gene.
However, the ability to target the TOP2A gene for negative selection using the HSV-TK gene in pluripotent cells was well-known in the art as described by Hickson. Hickson transfected isolated mammalian cells with a targeting vector comprising a nucleic acid sequence encoding HSV-TK operably linked to the TOP2A gene (claim 4).
Suicide strategies for pluripotent cells using a variety of promoters were well-known in the art at the time of filing using a housekeeping promoter, a tissue-specific promoter or a pluripotent-specific promoter. This is described by Ivics who taught “Yagyu et al. wisely settled for the promoter of the housekeeping gene human elongation factor-1 alpha (EF1α) in their study, because it provides fairly robust transgene expression in a wide range of cell types. Other promoter elements providing cell type-specific expression may be considered in the future; for example, promoters exclusively active in pluripotent stem cells would allow selective elimination of iPS cells from heterogeneous cell populations, including both iPS cells and their differentiated progeny (Figure 1C)” (pg 1419, col. 2, end of 1st para, beginning of 2nd para).
Chen, Cheng, and Malecki also described a variety of pluripotent cell suicide strategies using a variety of promoters (Chen - EF1α or Nanog; Cheng - EF1α or Nanog; Malecki - Table 1).
Therefore, those of skill would have recognized that changing the pluripotent-specific promoter of Rong to the TOP2A promoter had the advantage of killing a “wide range of cell types” as described by Ivics and was merely a matter of design strategy.
The desire to specifically target the TOP2A promoter for expressing a marker gene was also well-known as evidenced by Hickson, Morgan (Fig 1), Fraser (Fig. 1), and Adachi (“Plasmid construction”).
Thus it would have been obvious to those of ordinary skill in the art at the time of filing to introduce an HSV-TK cassette into the 3’ end of a gene in a pluripotent cell to prevent teratoma formation in the presence of ganciclovir as described by Rong, wherein the gene was TOP2A described by Lang, Hochhauser, Hickson, Morgan, Fraser, and Adachi. The ordinary artisan would have recognized that TOP2A is essential for cell division (Lang, Hochhauser, Hockson) and that expressing TK using the TOP2A promoter would kill dividing cells, such as tumorigenic cells. That artisan would have been motivated to target the TOP2A promoter instead of the pluripotent-specific promoter described by Rong to express TK in pluripotent tumorigenic cells as well as their dividing offspring.
In the reverse, those of ordinary skill in the art at the time of filing would have been motivated to modify the TOP2A-specific expression system of Hickson to the co-expression inducible system described by Rong to obtain an inducible kill switch for pluripotent cells.
Those of ordinary skill would have had a reasonable expectation of modifying the TOP2A gene with a targeting vector that causes co-expression of TOP2A and HSV-TK (without the need for the neo cassette or a crossover event) as evidenced by Hickson who taught how to target the 3’ end of the TOP2A gene and insert an HSV-TK cassette. The ability to modify the TOP2A gene for marker gene expression was well-within the ability of the artisan as evidenced by Hickson, Morgan, Fraser, and Adachi.
Claims 22, 76 have been included because Rong used HSV-TK.
Claims 24, 77, 96 have been included because Rong used human cells.
Claim 59, 82 have been included because the combined teachings require using HSV-TK and ganciclovir.
Claim 73 has been included because the combined teachings require using ganciclovir to induce HSV-TK expression and cell death.
Response to arguments
Applicants' argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
D) Claim 98 remains rejected under 35 U.S.C. 103 as being unpatentable over Rong (J. Biol. Chem., 2012, Vol. 287, pg 32338-32345) in view of Diril (PNAS, Vol. 109, No. 10, pg 3826-3831), Ivics (Mol. Therapy, 2015, Vol. 23, No. 9, pg 1417-1420), Chen (Biomaterials, 2013, Vol. 34, pg 1701-1711), Cheng (Biomaterials, 2012, Vol. 33, pg 3105-3204), Malecki (Stem Cell Res. & Therapy, 2014, Vol. 5, No. 73, pg 1-10), Dahler (J. Cell. Physiol., 1998, Vol. 177, pg 474-482), Liu (Biochem. & Biophysical Res. Comm., 1998, Vol. 246, pg 696-702), Wang (Shijie Huarent Xiaohua Zazhi, 2005, Vol. 13, No. 19, pg 2381-2385, abstract only), Shao (Zhongguo Ganzangbring Zazhi, Dianziban, 2013, Vol. 5, No. 2, pg 10-13, abstract only), and Minamide (PLoS One, 2014, Vol. 9, No. 1, e84460, pg 1-15) as applied to claims 16, 22, 24, 47, 59, 73, 76, 77, 79, 82, 95, 96 and further in view of GenBank Accession #: BC002703 (2002) and Regnier (Mol. & Cell. Biol., 2005, Vol. 25, No. 10, pg 3967-3981).
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide taught an isolated human cell whose genome comprises an exogenous sequence encoding TK operably linked to the promoter of an endogenous CDK1 gene, wherein the cell functionally co-expresses TK and CDK1.
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach targeting the CENPA gene as required in claim 98.
However, the human CENPA gene was well-known in the art at the time of filing as shown by BC002703.
Thus it would have been obvious to those of ordinary skill in the art at the time of filing to make an isolated human cell whose genome comprises an exogenous sequence encoding TK operably linked to the promoter of an endogenous gene, wherein the cell functionally expresses TK and protein encoded by the endogenous gene as described by the combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide using the CENPA gene described by BC002703. Those of ordinary skill in the art at the time of filing would have been motivated to use the CENPA gene because it is essential for chromosome segregation (title of Regnier). Those of ordinary skill in the art at the time of filing would have recognized teratoma cells are tumorigenic and dividing cells, so targeting the expression of TK in conjunction with CENPA would target tumorigenic dividing cells. In the reverse, those of ordinary skill in the art at the time of filing would have been motivated to modify the teachings of Diril and apply it to the CENPA gene of BC002703 and the pluripotent cells of Rong so they had a kill switch if the pluripotent cells formed teratomas.
Response to arguments
Applicants' argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
E) Claim 99 remains rejected under 35 U.S.C. 103 as being unpatentable over Rong (J. Biol. Chem., 2012, Vol. 287, pg 32338-32345) in view of Diril (PNAS, Vol. 109, No. 10, pg 3826-3831), Ivics (Mol. Therapy, 2015, Vol. 23, No. 9, pg 1417-1420), Chen (Biomaterials, 2013, Vol. 34, pg 1701-1711), Cheng (Biomaterials, 2012, Vol. 33, pg 3105-3204), Malecki (Stem Cell Res. & Therapy, 2014, Vol. 5, No. 73, pg 1-10), Dahler (J. Cell. Physiol., 1998, Vol. 177, pg 474-482), Liu (Biochem. & Biophysical Res. Comm., 1998, Vol. 246, pg 696-702), Wang (Shijie Huarent Xiaohua Zazhi, 2005, Vol. 13, No. 19, pg 2381-2385, abstract only), Shao (Zhongguo Ganzangbring Zazhi, Dianziban, 2013, Vol. 5, No. 2, pg 10-13, abstract only), and Minamide (PLoS One, 2014, Vol. 9, No. 1, e84460, pg 1-15) as applied to claims 16, 22, 24, 47, 59, 73, 76, 77, 79, 82, 95, 96 and further in view of Alteri (Oncogene, 2003, Vol. 22, pg 8581-8589).
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide taught an isolated human cell whose genome comprises an exogenous sequence encoding TK operably linked to the promoter of an endogenous gene, wherein the cell functionally expresses TK and protein encoded by the endogenous gene.
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach targeting the BIRC5 (aka survivin) gene as required in claim 99.
However, the human BIRC5 gene was well-known in the art at the time of filing as shown by Alteri who described the structure/function (pg 8581) and its role in cell division (pg 8582).
Thus it would have been obvious to those of ordinary skill in the art at the time of filing to make an isolated human cell whose genome comprises an exogenous sequence encoding TK operably linked to the promoter of an endogenous gene, wherein the cell functionally expresses TK and protein encoded by the endogenous gene as described by the combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide using the BIRC5 gene described by Alteri. Those of ordinary skill in the art at the time of filing would have been motivated to use the BIRC5 gene because it is essential for cell division (pg 8582, col. 2 of Altieri).
Those of ordinary skill in the art at the time of filing would have recognized teratoma cells are tumorigenic and dividing cells, so targeting the expression of TK in conjunction with BIRC5 would target tumorigenic dividing cells. In the reverse, those of ordinary skill in the art at the time of filing would have been motivated to modify the teachings of Diril and apply it to the BIRC5 gene of Alteri and the pluripotent cells of Rong so they had a kill switch if the pluripotent cells formed teratomas.
Response to arguments
Applicants' argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
F) Claim 100 remains rejected under 35 U.S.C. 103 as being unpatentable over Rong (J. Biol. Chem., 2012, Vol. 287, pg 32338-32345) in view of Diril (PNAS, Vol. 109, No. 10, pg 3826-3831), Ivics (Mol. Therapy, 2015, Vol. 23, No. 9, pg 1417-1420), Chen (Biomaterials, 2013, Vol. 34, pg 1701-1711), Cheng (Biomaterials, 2012, Vol. 33, pg 3105-3204), Malecki (Stem Cell Res. & Therapy, 2014, Vol. 5, No. 73, pg 1-10), Dahler (J. Cell. Physiol., 1998, Vol. 177, pg 474-482), Liu (Biochem. & Biophysical Res. Comm., 1998, Vol. 246, pg 696-702), Wang (Shijie Huarent Xiaohua Zazhi, 2005, Vol. 13, No. 19, pg 2381-2385, abstract only), Shao (Zhongguo Ganzangbring Zazhi, Dianziban, 2013, Vol. 5, No. 2, pg 10-13, abstract only), and Minamide (PLoS One, 2014, Vol. 9, No. 1, e84460, pg 1-15) as applied to claims 16, 22, 24, 47, 59, 73, 76, 77, 79, 82, 95, 96 and further in view of Kaneda (PNAS, 1984, Vo. 81, pg 3158-3162) and Nakamura (International journal of oncology, 2009, Vol. 34, No. 5, pg 1181-1189).
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide taught an isolated human cell whose genome comprises an exogenous sequence encoding TK operably linked to the promoter of an endogenous gene, wherein the cell functionally expresses TK and protein encoded by the endogenous gene.
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach targeting the EEF2 gene as required in claim 100.
However, the human EEF2 gene was well-known in the art at the time of filing as shown by Kaneda who described it as “an essential factor for protein synthesis” (first line of abstract) and Nakamura who taught it was essential for progression into the G2 phase of the cell cycle (abstract).
Thus it would have been obvious to those of ordinary skill in the art at the time of filing to make an isolated human cell whose genome comprises an exogenous sequence encoding TK operably linked to the promoter of an endogenous gene, wherein the cell functionally expresses TK and protein encoded by the endogenous gene as described by the combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide using the EEF2 gene described by Kaneda and Nakamura. Those of ordinary skill in the art at the time of filing would have been motivated to use the EEF2 gene because it is essential for protein production and cell division. Those of ordinary skill in the art at the time of filing would have recognized teratoma cells are tumorigenic and dividing cells, so targeting the expression of TK in conjunction with EEF2 would target tumorigenic dividing cells. In the reverse, those of ordinary skill in the art at the time of filing would have been motivated to modify the teachings of Diril and apply it to the EEF2 gene of Kaneda and Nakamura and the pluripotent cells of Rong so they had a kill switch if the pluripotent cells formed teratomas.
Response to arguments
Applicants' argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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Michael C. Wilson
/MICHAEL C WILSON/
Primary Examiner, Art Unit 1638
Prosecution Insights