DETAILED ACTION
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and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114.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 3-2-26 has been entered.
Claims 1, 3-25, 38, 40 have been canceled. Claims 43-45 have been added. Claims 2, 26-37, 39, 41-45 are pending.
Election/Restrictions
Applicants elected claims 2-6, i.e. Group I, without traverse in the reply filed on 2-26-24. Groups II (claims 7-10) and Group III (claims 11-13) were combined with Group I. Claims 14-16 were regrouped into Group IV because they require the “isolated cell” of claim 2 is “in a human subject” or “administering the mammalian cell”.
Claims 2, 26-37, 39, 41-45 are under consideration.
Claim interpretation
The office action sent 3-7-24 assumed the invention was related to the “ALINK” system which is limited to using the inducer to kill the cell. This was confirmed in applicants’ response filed 9-9-24 which pointed to pg 28, para 158-159, which shows the insertion of “ALINK at the 3’ of the CDL [(cell division locus), i.e. the] CDK1 DNA coding sequence”. It is assumed the invention does not relate to the “EARC” system which is limited to withdrawal of an inducer.
The structures of the “dox-bridge” in the “ALINK” and “EARC” systems in Fig. 1-3 cannot be determined, were not clarified in parent application 15556146, and have not been clarified in this application.
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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. 2A and 2B are discussed on pg 28, paragraphs 158-159, and show the CDK1 gene and the targeting vector “for insertion ALINK at the 3’ of the CDL” (pg 28, line 1 of paragraph 159). However, it is unclear how the targeting vector in Fig. 2B correlates to the ALINK system discussed in Fig. 1 or in the description of Figure 1 on pg 7, para 40, or on pg 14, paragraphs 73, 74, 77, 78; pg 22, para 117-121. It is unclear whether the ALINK system discussed throughout the specification is limited to the co-expression system claimed (in any gene) or if there are broader interpretations.
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 2 requires a mixed population of isolated mammalian cells, i.e. stem cells and cells differentiated from the stem cells, that co-expresses CDK1 and HSV-TK (a negative selection marker) both under the control of an endogenous CDK1 promoter; an inducer of HSV-TK is added to kill the cell. Therefore, the invention appears to relate to the “ALINK” system which is limited to using the inducer to kill the cell. The invention does not appear to relate to 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 2 does not invoke any language about any “dox-bridge”.
Examiner’s response to applicants’ response filed 3-2-26
If, after all this time, pg 8-10 of applicants’ arguments filed 3-2-26 is attempting to say claim 2 IS invoking ALINK and “dox-bridge” language, then much clarification is required. There is so much jargon in the passages of the specification cited by applicants that the structures/functions that define an ALINK system or a “dox-bridge” cannot be determined. Claim 2 as written is clear. Applicants description of an ALINK system or a “dox-bridge” in the specification and in the response filed 3-2-26 is not clear.
Claim objection
The population of mixed cells in claim 2 as newly amended is confusion and contains numerous redundancies. The phrase “the subset of” in claim 2 is redundant. Use of “exogenous” to describe a sequence encoding HSV-TK in a mammalian cell is redundant because HSV-TK is a viral gene; it is not a mammalian gene.
The phrases “gene expressed in all dividing cells of an organism wherein the gene expressed in all dividing cells of an organism comprises an” and “encoding CDK1” in the phrase “a gene expressed in all dividing cells of an organism wherein the gene expressed in all dividing cells of an organism comprises an endogenous cyclin dependent kinase 1 (CDK1) gene encoding CDK1” in claim 2 are extraneous. The phrase can be written more simply as ---an endogenous [CDK1] gene---.
The two cell types in the population in claim 2 are mammalian stem cells and cells differentiated from the stem cells, but that can be written more simply.
The two cell types have the same genetic modification, but that can be written more simply.
The structure of the genetic modification has become even more confusing and burdensome by using numerous “wherein” phrases. The linkage of the HSV-TK coding sequence, CDK1 coding sequence and CDK1 promoter can be written more simply.
Claim 2 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 [HSV-TK] inserted at the 3’ end of an endogenous [CDK1] coding sequence and operably linked to an endogenous CDK1 promoter, wherein the stem cells and cells differentiated from the stem cells are capable of expressing HSV-TK and CDK1 under the control of the endogenous CDK1 promoter and are capable of being killed by ganciclovir when dividing but not when quiescent---.
Claim 26 can be written more simply as ---wherein the stem cells are pluripotent cells---.
Claim 27 can be written more simply as ---wherein the stem cells or cells differentiated from the stem cells are cancer cells---.
Claim 30 should clearly refer to the stem cells or the cells differentiated from the stem cells in claim 2, e.g. wherein the stem cells or cells differentiated from the stem cells are multipotent ---. If applicants are attempting to say a 3rd population of cells is present in claim 30, then that is wholly unclear from the claim as newly amended.
Claim 31 can be written more clearly as ---wherein the cells differentiated from the stem cells are neural epithelial cells---.
Claim 32 can be written more clearly as ---wherein the cells differentiated from the stem cells are terminally differentiated---.
Claim 33 should be reverted back to its previous format.
Claim 34 can be written more clearly as ---wherein the nucleic acid sequence encoding HSV-TK is…---.
Claim 35 should follow the format of genetic modification in claim 2.
Claim 36 can be written more clearly as ---A method of killing diving cells, the method comprising: contacting the population of cells in claim 2 with ganciclovir such that stem cells or cells differentiated from the stem cells that are dividing within the population are killed---.
Claim 37 can be written more clearly by describing the cells that are dividing, i.e. ---wherein the stem cells or cells differentiated from the stem cells that are dividing are cancer cells---.
Claim 39 can be written more clearly by describing the cells that are dividing, i.e. ---wherein the stem cells or cells differentiated from the stem cells that are dividing are human, mouse, rat, or non-human primate cells---.
Claim 41 can be written more clearly using the suggestions for claim 2 above.
Claim 42 should be incorporated into claim 41.
Claim 43 can be written more simply as ---wherein the stem cells are pluripotent and the cells differentiated from the stem cells are multipotent, monopotent, or terminally differentiated---.
Claim 44 can be written more clearly using the suggestions for claim 2 above. But it is a substantial duplicate of claim 2.
Claim 45 does not further limit claim 44 and should be canceled.
Claim Rejections - 35 USC § 112
Written Description
It was well-within the ability of the ordinary artisan to operably link an HSV-TK coding sequence to a CDK1 promoter in any isolated mammalian cell such that co-expression of CDK1 and HSV-TK 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.
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 as required in claim 5. 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 an HSV-TK coding sequence to a TOP2A promoter in any isolated mammalian cell such that co-expression of TOP2A and HSV-TK occur.
Claims 27-29, 37 remain and claims 2, 30-36, 39, 41-45 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.
Pending rejection
A) The specification lacks written description for obtaining the cell population of claim 2 wherein the stem cells or cells differentiated from the stem cells are cancer cells as required in claim 27-29, 37. The scope of cancer stem cells cannot be determined. The specification and the art do not discuss cancer stem cells. Cancer cells encompass melanoma, blastoma, carcinoma, neuroma, etc.
The specification points out that negative selectable markers for cancer therapy exist (pg 19, par 101, 105).
However, the specification does not reasonably teach incorporating the system claimed in any cancer stem cell or any cancer cell as required in claim 27-29, 37. The specification does not teach incorporating an exogenous sequence encoding HSV-TK operably linked to an endogenous CDK1 promoter into a cancer stem cell or a cancer cell. The specification teaches doing so in pluripotent cells but does not correlate those pluripotent cells to cancer stem cells or cancer cells as required in claims 27-29, 37. Accordingly, the specification lacks written description for a population of mixed cells in which the stem cells or cells differentiated from the stem cells are cancer cells as required in claims 27-29, 37.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above. The specification does not teach a mixed population of cells with the genetic modification in claim 2 that contain cancer stem cells or cancer cells differentiated from stem cells as required in claims 27 and 37.
B) Claim 28 requires the cancer cell is a subcutaneous teratoma. Pg 9, para 46, discusses cells comprising an “ALINK” system and treatment of subcutaneous teratomas with ganciclovir. However, claims 2 and 28 do not correlate to the ALINK system for reasons set forth above under “Claim Interpretation”. Pg 31, para 180, discusses a teratoma assay, but does not teach pluripotent cells comprising the system claimed turn into teratomas that comprise the system claimed.
Response to arguments
Applicants argue the specification teaches the structure of a Dox-bridge and the ALINK system. Applicants’ argument is not persuasive for reasons set forth above under “Claim interpretation”. Applicants’ citations provide no specific structure or function of a “dox-bridge”. If applicants are attempting to say claim 2 requires a “dox-bridge” then much clarification is required. If applicants are attempting to say the genetic modification in claim 2 IS the ALINK system, then much clarification is required. More to the point, the specification does not teach a mixed population of cells with the genetic modification in claim 2 that contain subcutaneous teratoma cells or subcutaneous teratoma cells differentiated from stem cells as required in claim 28.
C) Claim 29 requires the cancer cell is a mammary gland cancer cell. Pg 31, para 181-182 taught mammary gland tumor assays but does not teach mammary gland cancer cells comprising the system claimed. Pg 33, para 195 describes Fig. 7C and “ALINK” modified “B6N” cells; however, claims 2 and 29 do not correlate to the ALINK system for reasons set forth above under “Claim Interpretation”.
Response to arguments
Applicants do not specifically address this rejection.
New rejection
D) The specification lacks written description for a mixed population of isolated cells comprising any mammalian stem cell and any cell differentiated from the stem cell as broadly encompassed by claims 2, 41, 44 other than pluripotent cells and teratomas.
Claim 2 is drawn to isolated, genetically modified population of cells comprising mammalian stem cells comprising a genetic modification and cells differentiated from a subset of the mammalian stem cells, the differentiated cells retaining the genetic modification of the mammalian stem cells, wherein the genetic modification comprises comprising an exogenous polynucleotide encoding herpes simplex virus-thymidine kinase (HSV-TK) operably linked to a promoter of a gene expressed in all dividing cells of an organism wherein the gene expressed in all dividing cells of an organism comprises an endogenous cyclin dependent kinase 1 (CDK1) gene encoding CDK1,wherein the polynucleotide encoding HSV-TK is inserted at a 3' end of the CDK1 gene, and wherein the mammalian stem cells comprising the genetic modification and the [[or a]] cells differentiated from the subset of the mammalian stem cells therefrom that retain the genetic modification co-express the HSV-TK and the CDK1 under control of the CDK1 gene promoter and wherein the mammalian stem cells comprising the genetic modification and the cells differentiated from the subset of the mammalian stem cells that retain the genetic modification are capable of being killed by administration of ganciclovir when dividing but not when quiescent.
Claim 41 is drawn to a similar composition except the target gene/promoter is a TOP2A, CENPA, BIRC5, or EEF2 gene/promoter.
Claim is 44 substantially a duplicate of claim 2.
Claim 45 does not further limit claim 44.
Claims 2, 41, 44 encompass 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.
Claims 2, 41, 44 encompass 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 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 2, 41, 44. 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 2, 41, 44. Accordingly, the concept lacks written description other than pluripotent cells and teratomas.
Enablement
Claims 27-29, 37 remain and claims 2, 30-36, 39, 41-45 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.
Pending rejection
A) The specification does not enable making/using a mixed population of cells as required in claim 2 wherein the stem cells or cells differentiated from the stem cells are cancer cells as required in claim 27-29, 37. The scope of cancer stem cells cannot be determined. The specification and the art do not discuss cancer stem cells. Cancer cells encompass melanoma, blastoma, carcinoma, neuroma, etc.
The specification points out that negative selectable markers for cancer therapy exist (pg 19, par 101, 105).
However, the specification does not reasonably teach incorporating the system claimed in any cancer stem cell or any cancer cell as required in claim 27-29, 37. The specification does not teach incorporating an exogenous sequence encoding HSV-TK operably linked to an endogenous CDK1 promoter into a cancer stem cell or a cancer cell. The specification teaches doing so in pluripotent cells but does not correlate those pluripotent cells to cancer stem cells or cancer cells as required in claims 27-29, 37. 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 a population of mixed cells in which the stem cells or cells differentiated from the stem cells are cancer cells as required in claims 27 and 37.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above. The specification does not teach a mixed population of cells with the genetic modification in claim 2 that contain cancer stem cells or cancer cells differentiated from stem cells as required in claims 27-29, 37.
B) Claim 28 requires the cancer cell is a subcutaneous teratoma. Pg 9, para 46, discusses cells comprising an “ALINK” system and treatment of subcutaneous teratomas with ganciclovir. However, claims 2 and 28 lack written description for the ALINK system for reasons set forth above under “Claim Interpretation”. Pg 31, para 180, discusses a teratoma assay, but does not teach pluripotent cells comprising the system claimed turn into teratomas that comprise the system claimed.
Response to arguments
Applicants argue the specification teaches the structure of a Dox-bridge and the ALINK system. Applicants’ argument is not persuasive for reasons set forth above under “Claim interpretation”. Applicants’ citations provide no specific structure or function of a “dox-bridge”. If applicants are attempting to say claim 2 requires a “dox-bridge” then much clarification is required. If applicants are attempting to say the genetic modification in claim 2 IS the ALINK system, then much clarification is required. More to the point, the specification does not teach a mixed population of cells with the genetic modification in claim 2 that contain subcutaneous teratoma cells or subcutaneous teratoma cells differentiated from stem cells as required in claim 28.
C) Claim 29 requires the cancer cell is a mammary gland cancer cell. Pg 31, para 181-182 taught mammary gland tumor assays but does not teach mammary gland cancer cells comprising the system claimed. Pg 33, para 195 describes Fig. 7C and “ALINK” modified “B6N” cells; however, claims 2 and 29 do not correlate to the ALINK system for reasons set forth above under “Claim Interpretation”.
Response to arguments
Applicants do not specifically address this rejection.
New rejection
D) The specification does not enable making/using any mixed population of isolated cells comprising any mammalian stem cell and any cell differentiated from the stem cell as broadly encompassed by claims 2, 41, 44 other than pluripotent cells and teratomas.
Claims 2, 41, and 44 are described above.
Claims 2, 41, 44 encompass 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.
Claims 2, 41, 44 encompass 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 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 2, 41, 44. 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 2, 41, 44.
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 claims 2, 41, 44 other than pluripotent cells and teratomas.
Indefiniteness
The rejection of claim 2 conflict between the preamble and the final lines of the claim has been withdrawn.
The rejection of claim 25 has been withdrawn because the claim has been canceled.
The rejection of claim 38 has been withdrawn because the claim has been canceled.
Claims 28 and 45 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.
The term “subcutaneous” in the phrase “subcutaneous teratoma” in claim 28 does not make sense. Subcutaneous refers to a location in vivo, i.e. under the upper layer of skin, i.e. in the subcutaneous layer of the skin. The cells cannot be “isolated” as require in claim 2 and “subcutaneous” at the same time as required in claim 28. The term “subcutaneous” refers to a place in vivo and cannot further limit an isolated teratoma as required in claim 28. Teratomas located subcutaneously or placed in a culture dish have the same structure/function, so it is unclear how/whether the term “subcutaneous” further limits the teratoma. This makes the claim indefinite.
Claim 45 requires the cell population in vitro of claim 44 is a “mixed in vitro cell culture”; however, this does not further limit claim 44 because it already requires a mixture of two cells in vitro which infers they are in cell culture. Therefore, claim 45 does not further limit claim 44.
Claim Rejections - 35 USC § 103
A) Claims 2, 24, 26, 30-32, 36, 39, 40 remain and claims 27, 28, 37, 43-45 are 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). The HSV-TK gene was inserted into the 3’ end of the Nanog gene (pg 32340, Fig. 1B). This is also evidenced by Rong because Nanog and HSV-TK were co-expressed under the control of the endogenous promoter. This is equivalent to “wherein the polynucleotide encoding HSV-TK is inserted at the 3’ end of the [ ] gene” as required in claim 2.
Rong taught pluripotent cells (abstract, materials and methods) which are the “stem” cells in claim 2. 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 2. Rong also taught pluripotent cells “remaining with their differentiated derivatives” (abstract and throughout) which are the “stem” and “differentiated” cells in claim 2.
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 using a housekeeping promoter, a tissue-specific promoter or a pluripotent-specific promoter were 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).
Suicide strategies for pluripotent cells using an EF1α or Nanog promoter, or any of a number of other promoters were described by Chen and Cheng or in Table 1 of Malecki.
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.
The combined teachings arrive at inserting the HSV-TK gene into the 3’ end of a CDK1 gene, but under the control of the endogenous CDK1 promoter. This is equivalent to “wherein the polynucleotide encoding HSV-TK is inserted at the 3’ end of the CDK1 gene” as newly required in claim 2.
Claim 24 has been included because the cells are capable of being killed upon administration of ganciclovir because expression of HSV-TK is under the control of the CDK1 promoter which is express “when dividing but not when quiescent”.
Claim 26 has been included because an iPS has the exact same structure as the ES cell described by Rong.
Rong taught pluripotent cells (abstract, materials and methods) which are the “stem” cells in claim 27. 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 27. Rong also taught pluripotent cells “remaining with their differentiated derivatives” (abstract and throughout) which are the “stem” and “differentiated” cells in claim 27.
Claim 28 has been included because “subcutaneous” refers to a place in vivo and cannot further limit an isolated teratoma as required in claim 28. Teratomas located subcutaneously or placed in a culture dish have the same structure/function, so the term “subcutaneous” bears no patentable weight.
Claim 30 has been included because the cell of Rong was multipotent.
Claims 31-32 have been included because they further limit a “cell differentiated therefrom” which does not necessarily have to occur.
Claim 34 has been included because Rong inserted the HSV-TK coding sequence into a 3’ UTR of the gene (Fig. 1) and because doing so was obvious. Those of ordinary skill in the art at the time of filing would have been motivated to do so to express the 3’ end of the CDK1 gene.
Claim 36 has been included because administering ganciclovir as described by Rong would result in killing cells with they were dividing when the promoter is the CDK1 promoter as described by the combined teachings. In other words, the combined teachings would result in killing the cells when they were dividing because of the choice of attaching the HSV-TK to the CDK1 promoter and adding ganciclovir.
Rong taught pluripotent cells (abstract, materials and methods) which are the “stem” cells in claim 37. 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 37. Rong also taught pluripotent cells “remaining with their differentiated derivatives” (abstract and throughout) which are the “stem” and “differentiated” cells in claim 37.
Claim 39 has been included because Rong used human, mouse, rat or non-human primate stem cells.
Claim 40 has been included because Rong and the combined teachings as a whole taught a plurality of cells.
Claims 43-45 have been included for reasons set forth above.
Response to arguments
Applicants argue Rong teaches away from using a gene that is found in both differentiated and undifferentiated cells. Applicants’ argument is not persuasive. The claims encompass using all undifferentiated cells, e.g. pluripotent and multipotent cells. The claims are not limited to using differentiated and undifferentiated cells as argued.
While Rong does not teach or suggest using the CDK1 gene, this is the basis of the rejection, and it is not the same thing as “teaching away” from “using a gene that is found in both differentiated and undifferentiated cells”.
Rong does not teach away from “using a gene that is found in both differentiated and undifferentiated cells” because the NANOG gene is found in all cell types and because NANOG is expressed in both differentiated (e.g. cancer) cells and undifferentiated (e.g. pluripotent) cells. Gubelnik (“NANOG expression in human development and cancerogenesis”, Exp. Biol. Med., 2020, Vol. 245, No. 5, pg 456-464) cites numerous references prior to applicants’ filing date of 2015 that clearly establish it was well-known that NANOG was expressed in pluripotent and differentiated cancer cells on pg 456 throughout the first paragraph. Any assertion that Rong teaches away from “using a gene that is found in both differentiated and undifferentiated cells” is completely false.
If applicants are attempting to assert the teachings of Rong have a different mechanism of action, then the rejection provides motivation to make the change to a CDK1 gene and why those of skill would have had a reasonable expectation of doing so. Applicants’ mechanism of action was obvious.
Contrary to applicants argument, applying the suicide gene strategy of Rong to the CDK1 gene WOULD have the same purpose as Rong, i.e. killing cells that escape pluripotency.
The prior art teaches the claim limitations, provides a reason for operably linking HSV-TK and CDK1 coding sequences because the prior art teaches CDK1 is essential for cell division, and for expressing HSV-TK using the CDK1 promoter because it would kill dividing cells (e.g., tumorigenic cells) in the presence of ganciclovir.
Diril has been relied upon for targeting the CDK1 gene for negative selection using HSV-TK. It was obvious to apply the co-expression strategy of Rong to the CDK1 gene. 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.
Applicants response ignores the motivation established by the examiner to specifically target HSV-TK into a CDK1 gene for co-expression.
Applicants have not argued unexpected results. Such an argument should begin with what was expected from the teachings of Rong, i.e. cell killing in pluripotent cells, take into consideration any secondary factors, i.e. the teachings of Diril and the art at the time of filing, compare it with applicants’ results, or provide any indication that applicants’ results were synergistic or unexpected. In the reverse, the discussion does not begin by acknowledging what was expected when genetically modifying a CDK1 gene as described by Diril, take into account secondary considerations, compare what was expected to applicants results, or show that applicants’ results were synergistic or unexpected. The proper analysis for “unexpected results” are missing from applicants’ arguments.
Either Rong or Diril can be used as the primary reference.
Therefore, obviousness rejection A) is apt.
B) Claims 27, 37 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) as applied to claims 2, 24, 26, 27, 28, 30-32, 36, 37, 39, 40, 43-45 and further in view of Hedley (Nature Reviews Cancer, 2007, Vol. 7, No. 11, pg 870-879), Nawa (Anti-Cancer Agents in Medicinal Chem., 2008, Vol. 8, No. 2, pg 232-239), Danks (Cancer Res., 2007, Vol. 67, No. 1, pg 22-25), Neschadim (Mol. Ther., 2012, Vol. 20, No. 5, pg 1002-1013).
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 HSV-TK operably linked to an endogenous CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 for reasons set forth above.
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach performing the method in cancer cells as required in claims 27 and 37.
However, negative selectable markers in cancer cells were well-known as described by Hedley, Nawa, Danks, Shah, Neschadim (see abstract of each).
Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to isolated human cell whose genome comprises an exogenous sequence encoding HSV-TK operably linked to an endogenous CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 as described by Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide using cancer cells described by Hedley, Nawa, Danks and Neschadim. Those of ordinary skill in the art at the time of filing would have been motivated to replace the ES cells of Rong with tumor cells to kill tumor cells and prevent death.
Response to arguments
Applicants argue the combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach claim 2, so the rejection is deficient. Applicants’ argument is not persuasive for reasons set forth above under rejection A).
C) Claims 27, 28, 37 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) as applied to claims 2, 24, 26, 27, 28, 30-32, 36, 37, 39, 40, 43-45 and further in view of Wesselschmidt (2011, “The Teratoma Assay: An In Vivo Assessment of Pluripotency”. In: Schwartz, P., Wesselschmidt, R. (eds) Human Pluripotent Stem Cells. Methods in Molecular Biology, vol 767. Humana Press. https://doi.org/10.1007/978-1-61779-201-4_17).
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 HSV-TK operably linked to an endogenous CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 for reasons set forth above.
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach performing the method in subcutaneous teratomas as required in claim 28.
However, injecting pluripotent cells subcutaneously to determine the amount of teratomas they produce was well known as described by Wesselschmidt (see protocol).
Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to isolated human cell whose genome comprises an exogenous sequence encoding HSV-TK operably linked to an endogenous CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 as described by Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide and inject them subcutaneously to obtain teratomas described by Wesselschmidt. Those of ordinary skill in the art at the time of filing would have been motivated to inject the ES cells of Rong into mice subcutaneously to determine if they were safe for use in humans or whether they would cause teratomas.
Response to arguments
Applicants argue the combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach claim 2, so the rejection is deficient. Applicants’ argument is not persuasive for reasons set forth above under rejection A).
D) Claims 27, 29, 37 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) as applied to claims 2, 24, 26, 27, 28, 30-32, 36, 37, 39, 40, 43-45 and further in view of Guy (Mol. And Cell. Biol., 1992, Vol. 12, No. 3, pg 954-961).
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 HSV-TK operably linked to an endogenous CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 for reasons set forth above.
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach performing the method in mammary tumors as required in claim 29.
However, mammary tumors were well known as described by Guy (see protocol).
Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to isolated human cell whose genome comprises an exogenous sequence encoding HSV-TK operably linked to an endogenous CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 as described by Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide using mammary tumors described by Guy. Those of ordinary skill in the art at the time of filing would have been motivated to use mammary tumor cells instead of ES cells to determine if they could be killed in vivo.
Response to arguments
Applicants argue the combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach claim 2, so the rejection is deficient. Applicants’ argument is not persuasive for reasons set forth above under rejection A).
E) Claim 33 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, 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) as applied to claims 2, 24, 26, 27, 28, 30-32, 36, 37, 39, 40, 43-45 and further in view of Minskaia (BioMed Res. International, 2013, Vol. 2013, ID 291730, pg 1-12) and Szymczak (Nature Biotech., 2004, Vol. 22, No. 5, pg 589-594).
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 HSV-TK operably linked to an endogenous CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 for reasons set forth above.
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach using an F2A sequence between the CDK1 and HSV-TK coding sequences.
However, using an F2A spacer sequence between two coding sequences for co-expression was well-known in the art as evidenced by Minskaia (“Protein co-expression using FMDV 2A: Effect of ‘linker’ residues”) and Szymczak (abstract; paragraph bridging pg 591-592).
Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to isolated human cell whose genome comprises an exogenous sequence encoding HSV-TK operably linked to an endogenous CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 as described by Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide using an F2A spacer described by Minskaia and Szymczak. Those of ordinary skill in the art at the time of filing would have been motivated to replace the IRES of Rong with the F2A spacer of Minskaia and Szymczak for stoichiometric co-expression of multiple proteins to avoid difficulties reported with IRES elements as discussed by Szymczak on pg 592 in the paragraph bridging columns 1 and 2.
Response to arguments
Applicants argue the combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach claim 2, so the rejection is deficient. Applicants’ argument is not persuasive for reasons set forth above under rejection A).
F) Claim 34 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, 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) as applied to claims 2, 24, 26, 27, 28, 30-32, 36, 37, 39, 40, 43-45 and further in view of Yan (Cancer Letters, 2014, Vol. 345, pg 39-47).
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 HSV-TK operably linked to an endogenous CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 for reasons set forth above.
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach inserting the HSV-TK coding sequence into a 3’ UTR of the CDK1 gene.
However, the 3’ UTR of the CDK1 gene was well-known in the art as evidenced by Yan who designed primers using sequences for “CDK1-3’UTR available of NCBI” (pg 40, col. 2, last paragraph).
Thus, it would have been obvious to those of ordinary skill in the art at the time of filing to isolated human cell whose genome comprises an exogenous sequence encoding HSV-TK operably linked to an endogenous CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 as described by Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide and inserting the coding sequence into the CDK1 3’ UTR described by Yan. Those of ordinary skill in the art at the time of filing would have been motivated to insert the HSV-TK coding sequence into the CDK1 3’ UTR to ensure expression of the entire CDK1 coding sequence, thereby allowing co-expression of a functional, complete CDK1 protein along with HSV-TK.
Response to arguments
Applicants argue the combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach claim 2, so the rejection is deficient. Applicants’ argument is not persuasive for reasons set forth above under rejection A).
G) Claims 35, 41, 42 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 2, 24, 26, 27, 28, 30-32, 36, 37, 39, 40, 43-45 and further in view of Hickson (WO 98/37207), 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).
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 HSV-TK operably linked to an endogenous CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 for reasons set forth above.
The combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach operably linking an HSV-TK coding sequence to an endogenous TOP2A promoter and co-expressing HSV-TK and TOP2A as required in claims 35 and 41.
However, Hickson taught operably linking an HSV-TK coding sequence to an endogenous TOP2A promoter (pg 5, lines 10-16; pg 5, line 30 – pg 6, line 1; Example 3, pg 71; claims 4 and 6). Hochhauser, cited by Hickson on pg 19 and throughout, 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”). Therefore, it was well-within the ability of the ordinary artisan to operably link a HSV-TK coding sequence to a TOP2A promoter in any isolated mammalian cell such that co-expression of HSV-TK and TOP2A occurs as required in claims 5 and 10.
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 HSV-TK operably linked to a CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 as described by Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide and operably linking an HSV-TK coding sequence to an endogenous TOP2A promoter such that HSV-TK and TOP2A are co-expressed as described by Hickson, Hochhauser, Morgan, Fraser, and Adachi. Those of ordinary skill in the art at the time of filing would have been motivated to do so because Hickson taught the TOP2A gene could be used to express a cytotoxic protein.
Response to arguments
Applicants argue the combined teachings of Rong, Diril, Ivics, Chen, Cheng, Malecki, Dahler, Liu, Wang, Shao, and Minamide did not teach claim 2, so the rejection is deficient. Applicants’ argument is not persuasive for reasons set forth above under rejection A).
H) Claims 41, 42 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 Hickson (WO 98/37207), 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), 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).
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). The HSV-TK gene was inserted into the 3’ end of the Nanog gene (pg 32340, Fig. 1B). This is also evidenced by Rong because Nanog and HSV-TK were co-expressed under the control of the endogenous promoter. This is equivalent to “wherein the polynucleotide encoding HSV-TK is inserted at the 3’ end of the [ ] gene” as required in claim 2.
Rong taught pluripotent cells (abstract, materials and methods) which are the “stem” cells in claim 2. 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 2. Rong also taught pluripotent cells “remaining with their differentiated derivatives” (abstract and throughout) which are the “stem” and “differentiated” cells in claim 2.
Rong did not teach targeting the TOP2A gene as required in claim 41.
However, Hickson taught operably linking an HSV-TK coding sequence to an endogenous TOP2A promoter (pg 5, lines 10-16; pg 5, line 30 – pg 6, line 1; Example 3, pg 71; claims 4 and 6). Hochhauser, cited by Hickson on pg 19 and throughout, 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”). Therefore, it was well-within the ability of the ordinary artisan to operably link a HSV-TK coding sequence to a TOP2A promoter in any isolated mammalian cell such that co-expression of HSV-TK and TOP2A occurs as required in claim 41.
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 HSV-TK operably linked to a CDK1 promoter, wherein the cell functionally co-expresses HSV-TK and CDK1 as described by Rong and operably linking an HSV-TK coding sequence to an endogenous TOP2A promoter such that HSV-TK and TOP2A are co-expressed as described by Hickson, Hochhauser, Morgan, Fraser, and Adachi. Those of ordinary skill in the art at the time of filing would have been motivated to do so because Hickson taught the TOP2A gene could be used to express a cytotoxic protein.
Suicide strategies for pluripotent cells using a housekeeping promoter, a tissue-specific promoter or a pluripotent-specific promoter were 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).
Suicide strategies for pluripotent cells using an EF1α or Nanog promoter, or any of a number of other promoters were described by Chen and Cheng or in Table 1 of Malecki.
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.
Claim 42 has been included because the cells are capable of being killed upon administration of ganciclovir.
Response to arguments
Applicants argue Rong teaches away from using a gene that is found in both differentiated and undifferentiated cells. Applicants’ argument is not persuasive. The claims encompass using all undifferentiated cells, e.g. pluripotent and multipotent cells. The claims are not limited to using differentiated and undifferentiated cells as argued.
While Rong does not teach or suggest using the TOP2A gene, this is the basis of the rejection, and it is not the same thing as “teaching away” from “using a gene that is found in both differentiated and undifferentiated cells”.
Rong does not teach away from “using a gene that is found in both differentiated and undifferentiated cells” because the NANOG gene is found in all cell types and because NANOG is expressed in both differentiated (e.g. cancer) cells and undifferentiated (e.g. pluripotent) cells. Gubelnik (“NANOG expression in human development and cancerogenesis”, Exp. Biol. Med., 2020, Vol. 245, No. 5, pg 456-464) cites numerous references prior to applicants’ filing date of 2015 that clearly establish it was well-known that NANOG was expressed in pluripotent and differentiated cancer cells on pg 456 throughout the first paragraph. Any assertion that Rong teaches away from “using a gene that is found in both differentiated and undifferentiated cells” is completely false.
If applicants are attempting to assert the teachings of Rong have a different mechanism of action, then the rejection provides motivation to make the change to a TOP2A gene and why those of skill would have had a reasonable expectation of doing so. Applicants’ mechanism of action was obvious.
Contrary to applicants argument, applying the suicide gene strategy of Rong to the TOP2A gene WOULD have the same purpose as Rong, i.e. killing cells that escape pluripotency.
The prior art teaches the claim limitations, provides a reason for operably linking HSV-TK and TOP2A coding sequences because the prior art teaches TOP2A is essential for cell division, and for expressing HSV-TK using the TOP2A promoter because it would kill dividing cells (e.g., tumorigenic cells) in the presence of ganciclovir.
Hickson has been relied upon for targeting the TOP2A gene for negative selection using HSV-TK. It was obvious to apply the co-expression strategy of Rong to the TOP2A gene. The ordinary artisan would have recognized that TOP2A is essential for cell division 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.
Applicants response ignores the motivation established by the examiner to specifically target HSV-TK into a TOP2A gene for co-expression.
Applicants have not argued unexpected results. Such an argument should begin with what was expected from the teachings of Rong, i.e. cell killing in pluripotent cells, take into consideration any secondary factors, i.e. the teachings of Hickson and the art at the time of filing, compare it with applicants’ results, or provide any indication that applicants’ results were synergistic or unexpected. In the reverse, the discussion does not begin by acknowledging what was expected when genetically modifying a TOP2A gene as described by Diril, take into account secondary considerations, compare what was expected to applicants results, or show that applicants’ results were synergistic or unexpected. The proper analysis for “unexpected results” are missing from applicants’ arguments.
Either Rong or Hickson can be used as the primary reference.
Therefore, obviousness rejection H) is apt.
Double Patenting
Claim 44 is objected to under 37 CFR 1.75 as being a substantial duplicate of claim 2. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m). In this case, there are no word in claim 44 that alter the scope of claim 2.
The nonstatutory double patenting rejection is based on a judicially created doctrine grounded in public policy (a policy reflected in the statute) so as to prevent the unjustified or improper timewise extension of the “right to exclude” granted by a patent and to prevent possible harassment by multiple assignees. A nonstatutory double patenting rejection is appropriate where the conflicting claims are not identical, but at least one examined application claim is not patentably distinct from the reference claim(s) because the examined application claim is either anticipated by, or would have been obvious over, the reference claim(s). See, e.g., In re Berg, 140 F.3d 1428, 46 USPQ2d 1226 (Fed. Cir. 1998); In re Goodman, 11 F.3d 1046, 29 USPQ2d 2010 (Fed. Cir. 1993); In re Longi, 759 F.2d 887, 225 USPQ 645 (Fed. Cir. 1985); In re Van Ornum, 686 F.2d 937, 214 USPQ 761 (CCPA 1982); In re Vogel, 422 F.2d 438, 164 USPQ 619 (CCPA 1970); In re Thorington, 418 F.2d 528, 163 USPQ 644 (CCPA 1969).
A timely filed terminal disclaimer in compliance with 37 CFR 1.321(c) or 1.321(d) may be used to overcome an actual or provisional rejection based on nonstatutory double patenting provided the reference application or patent either is shown to be commonly owned with the examined application, or claims an invention made as a result of activities undertaken within the scope of a joint research agreement. See MPEP § 717.02 for applications subject to examination under the first inventor to file provisions of the AIA as explained in MPEP § 2159. See MPEP § 2146 et seq. for applications not subject to examination under the first inventor to file provisions of the AIA . A terminal disclaimer must be signed in compliance with 37 CFR 1.321(b).
The filing of a terminal disclaimer by itself is not a complete reply to a nonstatutory double patenting (NSDP) rejection. A complete reply requires that the terminal disclaimer be accompanied by a reply requesting reconsideration of the prior Office action. Even where the NSDP rejection is provisional the reply must be complete. See MPEP § 804, subsection I.B.1. For a reply to a non-final Office action, see 37 CFR 1.111(a). For a reply to final Office action, see 37 CFR 1.113(c). A request for reconsideration while not provided for in 37 CFR 1.113(c) may be filed after final for consideration. See MPEP §§ 706.07(e) and 714.13.
The USPTO Internet website contains terminal disclaimer forms which may be used. Please visit www.uspto.gov/patent/patents-forms. The actual filing date of the application in which the form is filed determines what form (e.g., PTO/SB/25, PTO/SB/26, PTO/AIA /25, or PTO/AIA /26) should be used. A web-based eTerminal Disclaimer may be filled out completely online using web-screens. An eTerminal Disclaimer that meets all requirements is auto-processed and approved immediately upon submission. For more information about eTerminal Disclaimers, refer to www.uspto.gov/patents/apply/applying-online/eterminal-disclaimer.
Claims 2, 26-37, 39, 41-45 are provisionally rejected on the ground of nonstatutory double patenting as being unpatentable over the claims of U.S. Patent Application 15556146. Although the claims at issue are not identical, they are not patentably distinct from each other because the both require a population of isolated mammalian stem cells and cells differentiated from the stem cells comprising a nucleic acid encoding HSV-TK operably linked to an endogenous CDK1 or TOP2A promoter, wherein the cells are capable of expressing HSV-TK and CDK1 or TOP2A under the control of an endogenous CDK1 or TOP2A promoter.
Conclusion
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure:
Benvenisty (WO 2011/030329; US 20120171213) taught contacting pluripotent cells with an agent that downregulates an activity and/or expression of a polypeptide selected from the group consisting of AW262311 (aka SerpinB6), COX6A1, ZIC2, SOX2, M14087, OTX2, TUBB2B, IL17RD, TMSL8, CRABP1, ZIC3, CDC20, SBK1, TOP2A , DLG7, PTPRZ1, NUF2, NEFL, SPAG5, LOC146909 and survivin (BIRC5), thereby reducing the potential of the pluripotent stem cells to generate teratomas. Methods of treating tumors and compns. capable of same are also provided. . . . from the group consisting of AW262311 (aka SerpinB6), COX6A1, ZIC2, SOX2, M14087, OTX2, TUBB2B, IL17RD, TMSL8, CRABP1, ZIC3, CDC20, SBK1, TOP2A , DLG7, PTPRZ1, NUF2, NEFL, SPAG5, LOC146909 and survivin (BIRC5), thereby reducing the potential of the pluripotent stem cells to generate teratomas. Methods of treating tumors and compns. capable of same are also provided.
Maillet (Scientific Reports, May 2016, 25333, pg 1-13) taught “Doxorubicin-Induced Cardiotoxicity in Human Pluripotent Stem Cell Derived Cardiomyocytes” (title). “Top2B has been shown to be essential for doxorubicin-induced cell death in mouse cells and rat CMs” (bottom of pg 5 which cites Yi, L. L. et al. Topoisomerase IIβ-mediated DNA double-strand breaks: Implications in doxorubicin cardiotoxicity and prevention by dexrazoxane. Cancer Res. 67, 8839–8846 (2007))
Yagyu (Mol. Therapy, May 2015, Vol. 23, No. 9, pg 1475-1485) “The stable genetic introduction of a suicide gene is one of the most appealing safety strategies for hiPSC, 10,11 but to be effective such a safety switch for hiPSC should meet several criteria. The mechanism should have little spontaneous activity to ensure desired survival of hiPSC and their progeny, but should induce essentially complete killing once activated. Killing should be swift in order to regulate acute as well as more chronic toxicities, and the suicide strategy should kill both rapidly dividing undifferentiated hiPSC and their more slowly dividing or postmitotic differentiated progeny. The activating prodrug should be nontoxic and ideally otherwise bio-inert, and finally the system should be non-immunogenic, so that immune responses against the safety switch do not needlessly destroy the cell product. Herpes simplex virus thymidine kinase (HSV-TK) and yeast cytosine deaminase (yCD) have been applied as a safety switches for iPSC, 12–15 but neither possess all of the desired features”
Zhong (Mol. Therapy, 2011, Safeguarding nonhuman primate iPS cells with suicide genes. Vol. 19: pg 1667–1675.
Knoepfler (Stem Cells, 2009, “Deconstructing stem cell tumorigenicity: a roadmap to safe regenerative medicine”, Vol. 27, pg 1050–1056; see “Introduction of a stem cell specific suicide gene”).
Lim (PLoS, July 2013, “Lentiviral vector mediated thymidine kinase expression in pluripotent stem cells enables removal of tumorigenic cells”, Vol. 8: e70543, pg 1-16)
No claim is allowed.
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Michael C. Wilson
/MICHAEL C WILSON/
Primary Examiner, Art Unit 1638
Prosecution Insights