DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application is being examined under the pre-AIA first to invent provisions.
Claims 3-5, 14, 16-20 have been canceled. Claims 1-15 are pending.
Applicant's arguments filed 2-5-25 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.
The interview summary sent 4-25-25 was an error and belongs in another case.
Election/Restrictions
Applicants elected Group I, claims 1-15, without traverse in the reply filed on 8-29-24.
Claims 1, 2, 6-13, 15 remain under consideration.
Claim objections
The phrase “comprising coding region(s) for one or more reprogramming factor(s)… …wherein the one or more reprogramming factors [sic] comprise one or more selected from the group consisting of an Oct family gene,… …the Nanog gene” in claim 1 is inaccurate for this application for reasons of record. Applicants’ disclosure is limited to using coding sequences for reprogramming factors (Fig. 2). Applicants do not teach using a reprogramming factor promoter (pg 25, para 48) or non-coding regions which are found in “genes” as claimed. The phrase also uses improper Markush group language because it is missing the genus that describes the items in the group, i.e. “gene” in this case, but that’s inaccurate.
It may be simpler and clearer to say ---a) introducing at least one non-viral vector encoding one or more Oct family protein,… or Nanog into an isolated mammalian somatic cell--- (Fig. 2). Or --- a) introducing at least one non-viral vector comprising one or more nucleic acid sequence encoding one or more reprogramming factor selected from the group consisting of an Oct family protein,… and Nanog into an isolated mammalian somatic cell---. Or ---at least one non-viral vector comprising a nucleic acid sequence encoding an Oct family protein,… …or Nanog---. Because of the way claims 6 and 8-12 are currently written, the best course of action unclear. These suggestions for claim 1 are not intended to indicate allowability. They are made to point out the inaccuracies, redundancies, and discrepancies in the claim set.
One example for clarifying claim 6 is ---wherein the one or more non-viral vector encodes:
i) Oct4 and Sox2 and optionally TERT or SV40 Large T antigen;
ii) Oct4, Klf4, and Sox2 and optionally TERT or SV40 Large T antigen;
iii)… …
v) Oct4, Sox2, Klf4, cMyc, Lin28, and Nanog and optionally TERT or SV40 Large T antigen---.
One example for clarifying claim 8 is ---wherein the one or more non-viral vector encodes:
i) three proteins consisting of an Oct family protein, a Klf family protein, and a Sox family protein; or
ii) four proteins consisting of an Oct family protein, a Klf family protein, a Sox family protein, and a Myc family protein---.
But again, changes to claims 6 and 8-11 will depend on how claim 1 is amended.
Claim Rejections - 35 USC § 112
Enablement
Claims 1, 2, 6-13, 15 remain 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
a) transfecting an isolated human fibroblast with a plasmid encoding iPS reprogramming factors comprising Oct4, Sox2, and Klf4; and
b) culturing the fibroblasts obtained in step a) in pluripotent cell medium [inferred by “ES cell culture medium” on pg 35, lines 28-29] such that iPS cells are obtained;
c) culturing the iPS cells obtained in step b) such that iPS cells that are free of the plasmid are obtained
does not reasonably provide enablement for
introducing a non-viral vector encoding one or more Oct, Klf, Sox, Myc, or Lin family protein or Nanog into isolated mammalian somatic cells such that iPS cells are obtained as broadly claimed.
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.
Withdrawn rejections
The rejection regarding performing the method in any species in vitro or in vivo as broadly claimed other than in isolated mammalian somatic cells has been withdrawn because the claim has been limited to performing the method in isolated mammalian somatic cells.
The rejection regarding introducing the vector(s) encoding the reprogramming factor(s) cells to “produce an induced pluripotent stem cell” in claim 1 without a clear positive step of culturing the cells obtained after introducing the vector(s) and obtaining pluripotent cells has been withdrawn in view of the amendment which adds a step of culturing.
Pending rejections
State of the art
The art at the time of filing is unpredictable regarding how to reprogram somatic cells into iPS cells.
Takahashi (Cell, Aug. 25, 2006, Vol. 126, pg 663-676) introduced four retroviral vectors encoding Oct3/4, Sox2, c-Myc and Klf4 into mouse ES cells and mouse adult fibroblasts cultured under conditions suitable for mouse ES cell culture to obtain induced pluripotent stem (iPS) cells that exhibited mouse ES cell morphology and growth properties and expressed mouse ES cell marker genes. Notably, exogenous Oct-4 introduced into the mouse fibroblasts resulted in only marginal Oct-4 expression. Subcutaneous transplantation of iPS cells into nude mice resulted in tumors containing a variety of tissues from all three germ layers. Following injection into blastocysts, iPS cells contributed to mouse embryonic development. However, c-Myc, which was necessary for pluripotent induction, is an oncogene. Likewise, Klf4 is an oncogene. These data demonstrate that pluripotent cells can be directly generated from mouse fibroblast cultures by adding only a few defined factors using a retroviral transduction. However, as described infra, the set of factors used to produce iPS cells from differentiated mouse cells was insufficient to reprogram human somatic cells to pluripotency using lentiviral vectors without introducing additional changes to the cells.
Maherali (Cell Stem Cell, July 2007, Vol. 1, pg 55-70) discussed the art of reprogramming fibroblasts into iPS using retroviral vectors encoding Oct4, Sox2, c-Myc, and Klf4 and taught Oct4 expression was required for reprogramming of fibroblasts into iPS but not for maintenance of iPS (pg 66, col. 2, paragraph 3).
Blelloch (Cell Stem Cell, Sept. 2007, Vol. 1, pg 245-247) established iPS cells using N-myc instead of c-myc.
Yu (Science, Dec. 2007, Vol. 318, pg 1917-1920) developed new iPS cell lines derived from human fibroblasts, using a similar approach but with retroviral vectors encoding a different set of transcription factors including Oct4, Sox2, Nanog and LIN28. Nevertheless, the Yu's method was much less efficient than the Takahashi's method. As of 2007, there were two problems unsolved; first is the use of retroviral transgenes, and secondly the use of oncogenes (e.g. c-Myc and Klf4). Retroviral infection was the only effective means capable of transgenically delivering four large transcription factor genes into a targeted host cell; however, the random or non-random insertion of multiple retroviral vectors into the targeted cell genome may also affect other non-target genes and may not adequately express all the genes in the targeting construct. Yu taught additional work was required to avoid vectors that integrate into the genome, potentially introducing mutations at the insertion site.
Nakagawa (Nat Biotechnol, Jan. 2008 (published online Nov. 30, 2007), Vol. 26: 101-106) established iPS cells in mice as well as in humans by introducing the three genes of Oct3/4, Sox2 and Klf4, without using c-myc gene.
Since the time of filing, Duinsbergen (Experimental Cell Res. July 9, 2008, Vol. 314, pg 3255-3263) taught making iPS using mouse neural stem cells endogenously expressing SoxB1 transfected with retroviruses encoding Oct4, Klf4, and c-myc (or MYCER).
Eminli (Stem Cells, July 17, 2008, Vol. 26, pg 2467-2474) taught making iPS using mouse neural stem cells transfected with retroviruses encoding Oct4, Sox2, Klf4, and c-myc or retroviruses encoding Oct4, Klf4, and c-myc; Eminli taught the neural stem cells endogenously expressed Sox2.
Okita (Science, Nov. 7, 2008, Vol. 322, pg 949-953) taught making iPS using plasmids instead of viral vectors encoding Oct4 and Sox2 and either Klf4 and c-myc or nanog and lin28.
Li (Cell Stem Cell, Jan. 2009, Pg 16) stated:
“while rat ESClike cells have been established based on certain traits (Demers et al., 2007; Ruhnke et al., 2003; Schulze et al., 2006; Ueda et al., 2008), to date, these lines fall short of exhibiting true pluripotency (e.g., fail to form teratoma or no/little contribution to chimerism) and thus cannot be considered authentic rat ESCs.”
Feng (Cell Stem Cell, April 3, 2009, Vol. 4, pg 301-312) taught Oct4 and Sox2 were required for all methods of reprogramming somatic cells known in the art at the time of filing (pg 302 Table 1) in combination with KIf4 and optionally cMyc (Nakagawa) using a series of assays to establish pluripotency including teratoma formation and germline transmission (pg 308, Table 3). Thus, merely obtaining “GFP+ colonies" using Sox1, Sox3, Sox7, Sox15, and Sox17 or Klf1, Klf2, or Klf5 as described by Nakagawa is inadequate to establish true iPS cells capable of germline transmission were obtained. Feng also summarized 5 examples of making iPS cells known in the art, all of which used Oct4 and Sox2 (pg 302 Table 1). Table 3 (pg 308) also shows iPS cells are determined by teratoma formation, chimeria formation (C), germline transmission (T) or tetraploid complementation (TE). Feng does not teach colony formation on its own was a marker of pluripotency.
Kaji (Nature, April 9, 2009, Vol. 458, pg 771-776) taught transfecting human fibroblasts with a single vector encoding Oct4, Sox2, Klf4, and c-Myc optionally with piggyback transposon.
Hochedlinger (US Patent 8,298,825) transfected somatic cell with Oct4 and Klf4 and optionally Sox2 and/or c-Myc (claim 1) wherein the sequence is introduced by viral vector or plasmid (claim 4).
Teachings in the specification
Pg 12, lines 1-3, incorporates WO 2007/69666 (US Patent Application Publication 2009/0068742). The amendment filed 5-22-14 incorporated two paragraphs and two Tables from WO 2007/69666 (paragraphs 49 and 51, and Tables 1 and 2 on pg 4 and 5 of US Patent Application Publication 2009/0068742). The new paragraphs teach: “The nuclear reprogramming factor provided by the present invention comprises at least a combination of gene products of an Oct family gene, a Klf family gene, and a Myc family gene, for example, a combination of gene products of Oct3/4, Klf4, and c- Myc. Examples of the Oct family gene include, for example, Oct3/4, OctlA, Oct6, and the like. Oct3/4 is a transcription factor belonging to the POU family, and is reported as a marker of undifferentiated cells (K. Okamoto et al., Cell, 60, pp 461-72, 1990). Oct3/4 is also reported to participate in the maintenance of pluripotency (J. Nichols et al., Cell, 95, pp 379-91, 1998). Examples of the Klf family gene include Klf1, Klf2, Klf4, Klf5 and the like. Klf4 (Kruppel like factor-4) is reported as a tumor repressing factor (A. M. Ghaleb et al., Cell Res., 15, pp 92-6, 2005). Examples of the Myc family gene include c-Myc, N-Myc, L-Myc and the like. c-Myc is a transcription control factor involved in differentiation and proliferation of cells (S. Adhikary, M. Eilers, Nat. Rev. Mol. Cell Biol., 6, pp. 635-45, 2005), and is also reported to be involved in the maintenance of pluripotency (P. Cartwright et al., Development, 132, pp. 885-96, 2005). The NCBI accession numbers of the genes of the families other than Oct3/4, Klf4 and c-Myc are as follows:…” and goes on to Table 1 which describes the Klf1, Klf2, Klf5, cMyc, nMyc, lMyc, Oct1A, and Oct6 gene sequences.
The second paragraph teaches: “The nuclear reprogramming factor of the present invention may comprise a gene product other than the aforementioned three kinds of gene products. An example of such gene product includes a gene product of a Sox family gene. Examples of the Sox family gene include, for example, Sox1, Sox3, Sox7, Soxl5, Sox17 and Sox18, and a preferred example includes Sox2. A nuclear reprogramming factor comprising at least a combination of the gene products of four kinds of genes, an Oct family gene (for example, Oct3/4), a Klf family gene (for example, Klf4), a Myc family gene (for example, c-Myc), and a Sox family gene (for example, Sox2) is a preferred embodiment of the present invention from a viewpoint of reprogramming efficiency, and in particular, a combination of a gene product of a Sox family gene is sometimes preferred to obtain pluripotency. Sox2, expressed in an early development process, is a gene encoding a transcription factor (A. A. Avilion et al., Genes Dev., 17, pp. 126- 40, 2003). The NCBI accession numbers of Sox family genes other than Sox2 are as follows…” and goes on to Table 2 which describes the Sox1, 3, 7, 15, 17 and 18 gene sequences.
Example 1 teaches making a plasmid encoding mouse Oct4, Sox2, Klf4 ligated together and a plasmid encoding c-myc (fig. 2, paragraph bridging pg 33-34).
Mouse fibroblasts were transfected with the plasmid (¶ bridging pg 34-35), and the cells were cultured in ES cell medium (pg 35, line 28) such that iPS cells were obtained (pg 36, lines 15-23). The plasmid was integrated into the host genome (pg 36, lines 23-25), or, with modification of the protocol, the plasmid was episomal (pg 36, line 28, through pg 37, line 11).
Example 3: Pluripotency was confirmed in iPS cells without integration by testing for teratoma formation after injection into a mouse (pg 37, lines 24-29) and by injection into a recipient embryo (pg 37, lines 29, through pg 38, line 7; pg 38, lines 16-19).
Example 4: Dental pulp stem cells and fetal stem cells were transfected with separate plasmids encoding Oct4, Sox2, and Klf4 (pg 38-pg 40, line 7; pg 40, lines 8-14). Some cells “exhibited a typical ES cell-like morphology, confirming the establishment of human iPS cells” (pg 40, line 5). Expression of exogenous Oct4, Klf4, and cMyc was detected (pg 40, lines 8-14; Fig. 18). Applicants conclude the cells were iPS cells because of morphology alone on pg 40, line 5; however, the morphology and expression of exogenous reprogramming factors is inadequate to confirm pluripotency for reasons established in the art at the time of filing. Specifically, Feng (2009) taught pluripotency is established by not only detecting expression of the reprogramming factors; it must also include testing the cells for the ability to form teratomas (as described by applicants in Example 1) and germline transmission (pg 308, Table 3, of Feng). Example 4 does not confirm pluripotency using the means known in the art at the time of filing, described by applicants in Example 1, or later described by Feng required to establish pluripotency, i.e. the ability to form teratomas or the ability to contribute to the germline upon being transplanted into a recipient embryo. Accordingly, Example 4 fails to provide adequate guidance that the method of claim 1 results in iPS cells.
Example 5: Dental pulp stem cells were transfected with one plasmid encoding Oct4, Sox2, and Klf4, such that integration of the viruses were obtained (pg 40, lines 17-24; pg 40, line 25). These cells are also described by applicants as iPS cells, but applicants did not confirm pluripotency using the means known in the art or later shown to be required to establish pluripotency, i.e. teratoma formation or the ability to contribute to the germline upon transplantation into a recipient embryo.
Example 6: Fetal stem cells were transfected with separate plasmids encoding Oct4, Sox2, and Klf4, such that integration was detected (pg 40, lines 2-14; pg 40, lines 14-15). These cells are described by applicants as iPS cells, but applicants did not confirm pluripotency using the means known in the art or later shown to be required to establish pluripotency, i.e. teratoma formation or the ability to contribute to the germline upon transplantation into a recipient embryo.
Example 7: fibroblasts were transfected with separate plasmids encoding Oct4, Sox2, and Klf4, such that iPS cells were obtained (pg 41, line 19), and integration was NOT detected (pg 42, lines 1-2).
Claim and breadth
Claim 1 is drawn to a method of producing an iPS cell, comprising the step of introducing at least one non-viral vector encoding one or more Oct, Klf, Sox, Myc, or Lin family protein or Nanog into isolated mammalian somatic cells such that iPS cells are obtained.
The breadth will be discussed in each rejection below.
Rejections
A) The specification does not enable introducing a non-viral vector encoding any one or more Oct, Klf, Sox, Myc, or Lin family protein or Nanog into isolated mammalian somatic cells such that iPS cells are obtained as broadly encompassed by claim 1 other than Oct4/Sox2/KLF4 and optionally cMyc or Oct4/Sox2/Nanog/LIN28.
Claim 1 encompasses using any one or more Oct, Klf, Sox, Myc, or Lin family protein or Nanog for making iPS cells.
Oct family proteins include Oct1, Oct2, Oct3/4, Oct6, Oct7, Oct8, Oct9, Oct11 (Wikipedia description of Octamer transcription factor, 2024). Sox family proteins include Sox1, Sox2, Sox3, Sox15, or Sox17, or Sox18 (Wikipedia description of Sox gene family transcription factor, 2024). Klf family genes include Klf1, Klf2, Klf4, or Klf5 (Wikipedia description of Kruppel-like factors, 2024). Myc family genes include c-myc, l-myc, n-myc (Wikipedia description of Myc, 2024).
The breadth of factors is contemplated on pg 3, lines 10-15. Pg 14, paragraph 28, teaches “in addition to the aforementioned genes, one or more kind of genes selected from the group consisting of 15 Fbxl5, ERas, ECAT15-2, Tell, and (J-catenin may be combined, and/or one or more kind of genes selected from the group consisting of ECAT1, Esgl, Dnmt3L, ECAT8, Gdf3, Soxl5, ECAT15-1, Fthll7, Sall4, Rexl, UTF1, Stella, Stat3, and Grb2 may also be combined… …[as] described in WO2007/69666.”
iPS cells must be capable of becoming all three germ tissues – endoderm, mesoderm, and ectoderm. Neural stem cells are multipotent stem cells that can be likened to a family - although they do give rise to different cells, the cells themselves are within a certain family and therefore, are closely related.
Claim 1 explicitly requires obtaining pluripotent cells. Claim 1 encompasses using Oct4 alone or in combination with Oct3, SALL4, SOX1,2,3,7,15,17,18 KLF4, C-MYC, N-MYC, and/or LIN28.
The specification teaches the factors may be any one or more genes selected from the consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Lin family gene, and Nanog gene to produce an iPS cell.
However, the art at the time of filing is limited to using Oct4, Sox2, Klf4 and optionally C-myc or Oct4/Sox2/Nanog/LIN28:
Okita (Nature, 2007, Vol. 448, pg 313-317) taught “selection for Nanog expression resulted in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells” (abstract). While a “Nanog-reporter-positive colony” may potentially be an iPS cell, Okita (2007) tested the clones for teratoma formation and germline transmission and found “germline competence was variable among Nanog iPS clones (pg 316, col. 2, line 6 of “Discussion"). Therefore, Okita provides evidence that the ability to provide germline transmission and true pluripotency was unpredictable.
Nakagawa (Nat Biotechnol, Jan. 2008, Vol. 26: 101-106) established iPS cells in mice as well as in humans by introducing the three genes of Oct3/4, Sox2 and KIf4, without using c-myc gene. Nakagawa also used Sox1, Sox3, Sox7, Sox15, Sox17, and Sox18 (pg 101, col. 2, line 4) and obtained “GFP+ colonies” using Sox1 and fewer with Sox3, Sox15, and Sox18 (pg 101, col. 2, lines 5-6; Fig. 1A); Sox17 as claimed did not work, and Sox18 “failed to expand the cells” (pg 101, col. 2, line 7). Nakagawa used Klf1, Klf2, and Klf5 instead of Klf4, and obtained “GFP+ colonies” using Klf2 and fewer with Klf1 and Klf5 (pg 101, col. 2, lines 10-12).
See also Yamanaka (US Patent 8,058,065), Yamanaka (US Patent 8,129,187) and Yamanaka (US Patent 8,278,104), and Yu (Science, Dec. 2007, Vol. 318, pg 1917-1920) – none of them established pluripotent cells simply by colony shape.
Since the time of filing, Feng (Cell Stem Cell, April 3, 2009, Vol. 4, pg 301-312) taught Oct4 and Sox2 were required for all methods of reprogramming somatic cells known in the art at the time of filing (pg 302 Table 1) in combination with KIf4 and optionally cMyc (Nakagawa) using a series of assays to establish pluripotency including teratoma formation and germline transmission (pg 308, Table 3). Thus, merely obtaining “GFP+ colonies" using Sox1, Sox3, Sox7, Sox15, and Sox17 or Klf1, Klf2, or Klf5 as described by Nakagawa is inadequate to establish true iPS cells capable of germline transmission were obtained. Feng summarized 5 examples of making iPS cells known in the art, all of which used Oct4 and Sox2 (pg 302 Table 1). Table 1 also shows iPS cells can be determined by teratoma formation or germline transmission (T or G). Colony formation on its own is not necessarily a marker of pluripotency.
In fact, Sox17 was known as a marker for endodermal cells (Kanai-Azuma (Development, 2002, Vol. 129, pg 2367-2379), and Sox18 was associated with skeletal muscle regeneration (Lee, Molecular and Cell. Biol., Oct. 2004, Vol. 24, No. 19, pg 8428-8436); therefore, Sox family proteins are not necessarily interchangeable, do not have the same function as Sox2, and are not associated with the function of reprogramming to the state of pluripotency.
Likewise, Klf1 has been associated with proper maturation of erythroid cells, chromatin remodeling, modulation of the gamma to beta globin switch, and transcription activation (Klf1 description, Wikipedia, 2024) and Klf2 has been associated with lung development, embryonic erythropoiesis, epithelial integrity, T-cell viability, and adipogenesis (Klf2 description, Wikipedia, 2024). Therefore, Klf family proteins are not necessarily interchangeable, do not have the same function as Klf4, and are not associated with the function of reprogramming to the state of pluripotency.
Given the teachings in the specification taken with the art at the time of filing, it would have required those of skill undue experimentation to exclude Oct4, to exclude Sox2, to replace Sox2 with Sox1, Sox3, Sox7, Sox15, or Sox17 (specifically Sox17 didn’t work), to exclude Klf4, or to replace it with Klf1, Klf2 or Klf5. The specification and the art at the time of filing are limited to using plasmid(s) encoding Oct4/Sox2/Klf4 and optionally c-myc or Oct4/Sox2/Nanog/LIN28.
The breadth and specific combinations in claims 6, 8-11 are not enabled for reasons set forth above other than plasmid(s) encoding Oct4/Sox2/Klf4 and optionally c-myc or Oct4/Sox2/Nanog/LIN28.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
B) The specification does not enable using a non-viral vector as broadly encompassed by claim 1 other than a plasmid. Example 1 teaches the plasmid is integrated into the host genome (pg 36, lines 23-25), or, with modification of the protocol, maintaining the plasmid episomally (pg 36, line 28, through pg 37, line 11). Pg 25, para 49, suggests incorporating a DNA sequence, e.g. an SV40 replication origin, that allows autonomous replication of a non-viral vector. However, the specification does not teach the structure/type of any non-viral vectors other than a plasmid. The specification does not correlate plasmids to any other non-viral vectors. Given the teachings in the specification taken with the art at the time of filing, it would have required those of skill undue experimentation to make/use any non-viral vector as broadly encompassed by claim 1 other than a plasmid.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
Written Description
Claims 1, 2, 6-13, 15 remain 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.
Withdrawn rejections
The rejection regarding performing the method in any species in vitro or in vivo as broadly claimed other than in isolated mammalian somatic cells has been withdrawn because the claim has been limited to performing the method in isolated mammalian somatic cells.
The rejection regarding introducing the vector(s) encoding the reprogramming factor(s) cells to “produce an induced pluripotent stem cell” in claim 1 without a clear positive step of culturing the cells obtained after introducing the vector(s) and obtaining pluripotent cells has been withdrawn in view of the amendment which adds a step of culturing.
Pending rejections
Claim 1, its breadth, the teachings in the art, the teachings specification, and Examples are discussed above.
A) The specification lacks written description for introducing a non-viral vector encoding any one or more Oct, Klf, Sox, Myc, or Lin family protein or Nanog into isolated mammalian somatic cells such that iPS cells are obtained as broadly encompassed by claim 1 other than Oct4/Sox2/KLF4 and optionally cMyc or Oct4/Sox2/Nanog/LIN28.
Claim 1 encompasses using any one or more Oct, Klf, Sox, Myc, or Lin family protein or Nanog for making iPS cells.
Oct family proteins include Oct1, Oct2, Oct3/4, Oct6, Oct7, Oct8, Oct9, Oct11 (Wikipedia description of Octamer transcription factor, 2024). Sox family proteins include Sox1, Sox2, Sox3, Sox15, or Sox17, or Sox18 (Wikipedia description of Sox gene family transcription factor, 2024). Klf family genes include Klf1, Klf2, Klf4, or Klf5 (Wikipedia description of Kruppel-like factors, 2024). Myc family genes include c-myc, l-myc, n-myc (Wikipedia description of Myc, 2024).
The breadth of factors is contemplated on pg 3, lines 10-15. Pg 14, paragraph 28, teaches “in addition to the aforementioned genes, one or more kind of genes selected from the group consisting of 15 Fbxl5, ERas, ECAT15-2, Tell, and (J-catenin may be combined, and/or one or more kind of genes selected from the group consisting of ECAT1, Esgl, Dnmt3L, ECAT8, Gdf3, Soxl5, ECAT15-1, Fthll7, Sall4, Rexl, UTF1, Stella, Stat3, and Grb2 may also be combined… …[as] described in WO2007/69666.”
iPS cells must be capable of becoming all three germ tissues – endoderm, mesoderm, and ectoderm. Neural stem cells are multipotent stem cells that can be likened to a family - although they do give rise to different cells, the cells themselves are within a certain family and therefore, are closely related.
Claim 1 explicitly requires obtaining pluripotent cells. Claim 1 encompasses using Oct4 alone or in combination with Oct3, SALL4, SOX1,2,3,7,15,17,18 KLF4, C-MYC, N-MYC, and/or LIN28.
The specification teaches the factors may be any one or more genes selected from the consisting of an Oct family gene, a Klf family gene, a Sox family gene, a Lin family gene, and Nanog gene to produce an iPS cell.
However, the art at the time of filing is limited to using Oct4, Sox2, Klf4 and optionally C-myc or Oct4/Sox2/Nanog/LIN28:
Okita (Nature, 2007, Vol. 448, pg 313-317) taught “selection for Nanog expression resulted in germline-competent iPS cells with increased ES-cell-like gene expression and DNA methylation patterns compared with Fbx15 iPS cells” (abstract). While a “Nanog-reporter-positive colony” may potentially be an iPS cell, Okita (2007) tested the clones for teratoma formation and germline transmission and found “germline competence was variable among Nanog iPS clones (pg 316, col. 2, line 6 of “Discussion"). Therefore, Okita provides evidence that the ability to provide germline transmission and true pluripotency was unpredictable.
Nakagawa (Nat Biotechnol, Jan. 2008, Vol. 26: 101-106) established iPS cells in mice as well as in humans by introducing the three genes of Oct3/4, Sox2 and KIf4, without using c-myc gene. Nakagawa also used Sox1, Sox3, Sox7, Sox15, Sox17, and Sox18 (pg 101, col. 2, line 4) and obtained “GFP+ colonies” using Sox1 and fewer with Sox3, Sox15, and Sox18 (pg 101, col. 2, lines 5-6; Fig. 1A); Sox17 as claimed did not work, and Sox18 “failed to expand the cells” (pg 101, col. 2, line 7). Nakagawa used Klf1, Klf2, and Klf5 instead of Klf4, and obtained “GFP+ colonies” using Klf2 and fewer with Klf1 and Klf5 (pg 101, col. 2, lines 10-12).
See also Yamanaka (US Patent 8,058,065), Yamanaka (US Patent 8,129,187) and Yamanaka (US Patent 8,278,104), and Yu (Science, Dec. 2007, Vol. 318, pg 1917-1920) – none of them established pluripotent cells simply by colony shape.
Since the time of filing, Feng (Cell Stem Cell, April 3, 2009, Vol. 4, pg 301-312) taught Oct4 and Sox2 were required for all methods of reprogramming somatic cells known in the art at the time of filing (pg 302 Table 1) in combination with KIf4 and optionally cMyc (Nakagawa) using a series of assays to establish pluripotency including teratoma formation and germline transmission (pg 308, Table 3). Thus, merely obtaining “GFP+ colonies" using Sox1, Sox3, Sox7, Sox15, and Sox17 or Klf1, Klf2, or Klf5 as described by Nakagawa is inadequate to establish true iPS cells capable of germline transmission were obtained. Feng summarized 5 examples of making iPS cells known in the art, all of which used Oct4 and Sox2 (pg 302 Table 1). Table 1 also shows iPS cells can be determined by teratoma formation or germline transmission (T or G). Colony formation on its own is not necessarily a marker of pluripotency.
In fact, Sox17 was known as a marker for endodermal cells (Kanai-Azuma (Development, 2002, Vol. 129, pg 2367-2379), and Sox18 was associated with skeletal muscle regeneration (Lee, Molecular and Cell. Biol., Oct. 2004, Vol. 24, No. 19, pg 8428-8436); therefore, Sox family proteins are not necessarily interchangeable, do not have the same function as Sox2, and are not associated with the function of reprogramming to the state of pluripotency.
Likewise, Klf1 has been associated with proper maturation of erythroid cells, chromatin remodeling, modulation of the gamma to beta globin switch, and transcription activation (Klf1 description, Wikipedia, 2024) and Klf2 has been associated with lung development, embryonic erythropoiesis, epithelial integrity, T-cell viability, and adipogenesis (Klf2 description, Wikipedia, 2024). Therefore, Klf family proteins are not necessarily interchangeable, do not have the same function as Klf4, and are not associated with the function of reprogramming to the state of pluripotency.
Accordingly, the specification lacks written description for excluding Oct4, excluding Sox2, replacing Sox2 with Sox1, Sox3, Sox7, Sox15, or Sox17 (specifically Sox17 didn’t work), excluding Klf4, or replacing it with Klf1, Klf2 or Klf5 as broadly encompassed by claim 1. The specification and the art at the time of filing are limited to using plasmid(s) encoding Oct4/Sox2/Klf4 and optionally c-myc or Oct4/Sox2/Nanog/LIN28.
The specific combinations in claims 6, 8-11 lack written description for reasons set forth above other than plasmid(s) encoding Oct4/Sox2/Klf4 and optionally c-myc or Oct4/Sox2/Nanog/LIN28.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
B) The specification lacks written description for a non-viral vector as broadly encompassed by claim 1 other than a plasmid. Example 1 teaches the plasmid is integrated into the host genome (pg 36, lines 23-25), or, with modification of the protocol, maintaining the plasmid episomally (pg 36, line 28, through pg 37, line 11). Pg 25, para 49, suggests incorporating a DNA sequence, e.g. an SV40 replication origin, that allows autonomous replication of a non-viral vector. However, the specification does not teach the structure/type of any non-viral vectors other than a plasmid. The specification does not correlate plasmids to any other non-viral vectors. Accordingly, the specification lacks written description for any non-viral vector as broadly encompassed by claim 1 other than a plasmid.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
Indefiniteness
Withdrawn rejections
The rejection of claims 1-15 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being incomplete for omitting essential steps/elements, has been withdrawn because applicants added a culture step that clearly results in obtaining iPS cells.
The rejection of claim 2 regarding the metes and bounds of what applicants consider a non-viral vector “autonomously replicable outside a chromosome” has been withdrawn in view of the amendment.
Pending rejections
Claims 1, 2, 6-13, 15 remain 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.
A) The phrase “comprising coding region(s) for one or more reprogramming factor(s)… …wherein the one or more reprogramming factors [sic] comprise one or more selected from the group consisting of an Oct family gene,… …the Nanog gene” in claim 1 makes the claim indefinite. It is unclear whether the vectors contain just the coding sequence from the gene or the gene itself. Applicants’ disclosure is limited to using coding sequences for reprogramming factors (Fig. 2). Applicants do not teach using a reprogramming factor promoter (pg 25, para 48) or non-coding regions which are found in “genes” as claimed. The phrase also uses improper Markush group language because it is missing the genus that describes the items in the group, i.e. “gene” in this case, which makes it inaccurate and indefinite.
B) Claim 6 is indefinite as newly amended because it does not have a proper nexus with the “coding sequence(s)” or the “genes” in claim 1. Claim 6 refers to reprogramming factors that are proteins, but claim 1 is limited to “coding sequence(s)” or “genes”; therefore, claim 6 does not properly further limit the “coding sequence(s)” or “genes” in claim 1. The phrase “any one combinations (i)-(vi) further comprising TERT and/or the SV40 large T antigen” in item (vi) is grammatically incorrect and inaccurate. The phrase “one combinations” uses singular and plural forms at the same time which does not make sense. The phrase “(i)-(vi)” does not make sense in item (vi) because item (vi) cannot further limit itself. The term “comprising” is grammatically incorrect in conjunction with plural “combinations”. The phrase “and/or” is redundant and can be written more simply as “or” which is open claim language.
C) Claims 8-11 are indefinite as newly amended because they do not have a proper nexus with the “coding sequence(s)” or the “genes” in claim 1. Claims 8-11 refer to reprogramming factors that are proteins, but claim 1 is limited to “coding sequence(s)” or “genes”; therefore, claims 8-11 do not properly further limit the “coding sequence(s)” or “genes” in claim 1.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
Claim Rejections - 35 USC § 102
Claims 1, 2, 6-11, 14, 15 are rejected under 35 U.S.C. 102a1 as being anticipated by Thomson (US Patent Application Publication 2014/0057355) or Thomson (8440461) as evidenced by Boyle (2006/0246064) and Schultz (2004/0235031).
The effective filing date of ‘355 and ‘461 is Mar 23, 2007.
The effective filing date of the instant application is May 2, 2008.
Thomson taught transfecting human somatic cells with “as few as two potency determining factors, e.g., Oct-4 and Sox2, can be sufficient. Efficiency in obtaining reprogrammed cells, however, can be improved by including additional potency-determining factor, such as Lin28, Nanog or both.” (pg 4, last 2 sentences of para 16). “Genetic material encoding a potency-determining factor can be introduced by transfection or transduction into the somatic cells using a vector, such as an integrating- or nonintegrating vector. Of particular interest herein are retroviral vectors. Retroviral vectors, particularly lentiviral vectors, are transduced by packaging the vectors into virions prior to contact with a cell. After introduction, the DNA segment(s) encoding the potency-determining factor(s) can be located extra-chromosomally (e.g., on an episomal plasmid) or stably integrated into cellular chromosome(s)” (pg 11, para 42). “the inventors recognize the desirability of introducing the potency-determining factors into the somatic cells using non-integrating, episomal vectors and obtaining cells from which the episomal vectors are lost (e.g., at a rate of about 5% per generation) by subsequently withdrawing the drug selection used to maintain the vectors during the reprogramming step.” (para 46). Thomson used a human fibroblast which is a mammalian somatic cell (para 107) as required in claim 1.
Thomson taught non-integrating vectors can be a plasmid as required in claim 2 (para 42).
Thompson taught the vectors encoded Oct4 and Sox2 but not cMyc or Klf4 as encompassed by claim 1 (claim 1).
Thompson taught the vectors encoded Oct4 and Sox2 but not cMyc or Klf4 as encompassed by claims 1 and 6 (claim 1).
Thomson used the large T antigen as encompassed by claim and 6 (pg 15, para 53).
Thomson taught introducing the factors using separate vectors (para 37) as required in claim 7.
Thomson taught introducing multiple factors, e.g. Oct4 and Sox2, (claim 1) using separate vectors (para 37) as required in claim 8.
Thomson taught a single vector encoding Oct4 and Sox2 but not cMyc or Klf4 as encompassed by claim 9 (para 37).
Thomson taught a single vector encoding Oct4 and Sox2 with intervening sequences as encompassed by claim 10 (para 37).
Thomson taught a vector encoding Oct4 and Sox2 as encompassed by claim 11 (claim 1).
Thomson taught the somatic cell was human (para 107) as required in claim 15.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
Claim Rejections - 35 USC § 103
Claims 1, 2, 6-13, 15 remain rejected under 35 U.S.C. 103 as being unpatentable over Thomson (US Patent Application Publication 2014/0057355) or Thomson (8440461) as evidenced by Boyle (2006/0246064) and Schultz (2004/0235031).
The effective filing date of ‘355 or ‘461 is Mar 23, 2007.
The effective filing date of the instant application is May 2, 2008.
Thomson taught transfecting human somatic cells with “as few as two potency determining factors, e.g., Oct-4 and Sox2, can be sufficient. Efficiency in obtaining reprogrammed cells, however, can be improved by including additional potency-determining factor, such as Lin28, Nanog or both.” (pg 4, last 2 sentences of para 16). “Genetic material encoding a potency-determining factor can be introduced by transfection or transduction into the somatic cells using a vector, such as an integrating- or nonintegrating vector. Of particular interest herein are retroviral vectors. Retroviral vectors, particularly lentiviral vectors, are transduced by packaging the vectors into virions prior to contact with a cell. After introduction, the DNA segment(s) encoding the potency-determining factor(s) can be located extra-chromosomally (e.g., on an episomal plasmid) or stably integrated into cellular chromosome(s)” (pg 11, para 42). “the inventors recognize the desirability of introducing the potency-determining factors into the somatic cells using non-integrating, episomal vectors and obtaining cells from which the episomal vectors are lost (e.g., at a rate of about 5% per generation) by subsequently withdrawing the drug selection used to maintain the vectors during the reprogramming step.” (para 46). Thomson used a human fibroblast which is a mammalian somatic cell (para 107) as required in claim 1.
Thomson taught non-integrating vectors can be a plasmid as required in claim 2 (para 42). If it is found that Thomson did not adequately teach using non-viral vectors, then it would have been obvious to those of ordinary skill in the art at the time the invention was made to transfect somatic cells with reprogramming factors as described by Thomson using plasmids known in the art at the time of filing. Examples of transfecting human somatic cells with plasmids are provided by Boyle (pg 12, para 119) and Schultz (pg 11, para 109). Those of ordinary skill in the art at the time the invention was made would have been motivated to replace the retroviral vectors of Thomson with plasmids to prevent incorporation into the genome and decrease the risk of risks associated with retroviral vectors.
Thompson taught the vectors encoded Oct4 and Sox2 but not cMyc or Klf4 as encompassed by claim 1 (claim 1).
Thompson taught the vectors encoded Oct4 and Sox2 but not cMyc or Klf4 as encompassed by claim 6 (claim 1).
Thomson used the large T antigen as encompassed by claim 6 (pg 15, para 53).
Thomson taught introducing the factors using separate vectors (para 37) as required in claim 7.
Thomson taught introducing multiple factors, e.g. Oct4 and Sox2, (claim 1) using separate vectors (para 37) as required in claim 8.
Thomson taught a single vector encoding Oct4 and Sox2 but not cMyc or Klf4 as encompassed by claim 9 (para 37).
Thomson taught a single vector encoding Oct4 and Sox2 with intervening sequences as encompassed by claim 10 (para 37).
Thomson taught a vector encoding Oct4 and Sox2 as encompassed by claim 11 (claim 1).
Thomson did not teach administering different vectors at the same time as required in claim 12; however, it would have been obvious to do so. Those of ordinary skill in the art at the time of filing would have been motivated to do so in order to introduce the reprogramming factors at the same time.
Thomson did not teach repeatedly administering different vectors at the same time as required in claim 13; however, it would have been obvious to do so. Those of ordinary skill in the art at the time of filing would have been motivated to do so in order to introduce the reprogramming factors at the same time to sustain a high level of expression of the reprogramming factors over time.
Thomson taught the somatic cell was human (para 107) as required in claim 15.
Response to arguments
Applicants argue the amendment overcomes the rejection. Applicants’ argument is not persuasive for reasons set forth above.
Yamanaka (US Patent 8,058,065), Yamanaka (US Patent 8,129,187) and Yamanaka (US Patent 8,278,104) have been considered but the only non-viral vectors used by Yamanaka are those used to make retroviral particles and not to reprogram somatic cells.
Yu (Science, Dec. 2007, Vol. 318, pg 1917-1920) taught additional work was required to avoid vectors that integrate into the genome, potentially introducing mutations at the insertion site (pg 1919, col. 2, lines 12-17) but does not discount the enablement of Thomson (US Patent Application Publication 2014/0057355) or Thomson (8440461).
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