DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claims status
Applicants reply filed 10/14/2025 is acknowledged.
Claims 28, 29, 45, 47 is/are cancelled and claims 54 is/are newly added. Claims 25, 27, 30-33, 46, 48-54 is/are currently pending and is/are under examination.
Claim Objections
Claim 25 is objected to because of the following informalities: Claim recites “a promoter specifically expressed in Sertoli cells”. Since promoters are not the expressed elements but rather drive the expression of downstream elements, it is understood that the claimed promoter is one that drives the expression of the fluorescent protein encoded by the reporter system. Following language is recommended to overcome this objection: “a Sertoli cell-specific promoter . Appropriate correction is required.
Claim Interpretation
Claim 25 recites “adult Sertoli cells which are capable of”, claim 33 recites “cells capable of” and claim 54 recites “adult Sertoli cells are also capable of” (emphasis added) having the claimed properties. The phrase ‘capable of’ is broadly interpreted to allow the cells produced to have or be able to develop the claimed properties, for example by further modification such as inclusion of other culture conditions.
Claim Rejections - 35 USC § 112(a) – New, necessitated by claim amendments
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 25, 27, 30-33, 46, 48-54 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification,
while being enabling for
A method for preparing Sertoli-like cells, comprising a step a1): introducing a fluorescent protein reporter system into human fibroblasts; wherein the fluorescent protein reporter system is specifically expressed in Sertoli cells, and includes a Sertoli cell-specific promoter which promotes expression of a fluorescent protein, wherein the promoter is a promoter of an Anti-Mullerian hormone (AMH) gene; and a step a2): increasing the expression level of a protein or protein combination in the human fibroblasts,
wherein the protein or protein combination is selected from one of the following groups:
B2) NR5A1 and GATA binding protein 4 (GATA4),
B6) NR5A1, GATA4 and WT1,
B7) NR5A1, GATA4 and SOX9,
B8) NR5A1, GATA4 and DMRT1,
B12) NR5A1, GATA4, WT1 and SOX9,
B13) NR5A1, GATA4, WT1 and DMRT1, and
B14) NR5A1, GATA4, SOX9 and DMRT1,
wherein the step al) is carried out before the step a2) is carried out;
wherein the method does not comprise increasing the expression level of a combination of NR5A1, GATA4, SOX9, WT1, and DMRT1 in the human fibroblasts; and
wherein the method reprograms the human fibroblasts into Sertoli-like cells which are capable of interacting with germ cells and have immune privilege,
wherein the Sertoli-like cells express the Krt18 and exhibit at least one of the following functions germ cell interaction, endothelial cell attraction and immune privilege.
does not reasonably provide enablement for a method wherein the cells differentiated from the human fibroblasts are “adult Sertoli cells”.
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 and use the invention commensurate in scope with these claims.
There are many factors to be considered when determining whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any necessary experimentation is “undue.” See MPEP § 2164. These factors include, but are not limited to: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, the quantity of experimentation needed to make or use the invention based on the content of the disclosure.
The office has analyzed the specification in direct accordance to the factors outlines in In re Wands. MPEP 2164.04 states: “[W]hile the analysis and conclusion of a lack of enablement are based on factors discussed in MPEP 2164.01(a) and the evidence as whole, it is not necessary to discuss each factor in written enablement rejection.” These factors will be analyzed, in turn, to demonstrate that one of ordinary skill in the art would have had to perform “undue experimentation” to make and/or use the invention and therefore, applicant’s claims are not enabled.
(A) With respect to the breadth of the claims: the claims as currently drafted encompass a method that differentiates human fibroblasts into adult Sertoli cells by expressing a combination of transcription factors recited in claims 25, 46, 48-53. The specification does not provide any specific definition for “adult Sertoli cells”. The term “adult Sertoli cells” is interpreted to mean cells that are the same as Sertoli cells of an adult animal; structurally and phenotypically – wherein structurally same cells are cells have the same genomic, transcriptional and/or at least translational profile. For differentiation of the human fibroblasts, the claims are limited only by the recitation of the specific combination of transcription factors, without any additional method steps. The claims exclude other combinations, specifically excluding a combination of all the 5 transcription factors recited in the other combinations. Consequently, the breadth of the claims is broad.
(B) The nature of the invention: The invention is in the field of dedifferentiation of fibroblasts into other cells by expressing transcription factors known to be involved in maturation in vivo. In the instant case, the human fibroblasts are dedifferentiated into Sertoli-like cells by expressing a combination of transcription factors that were known in the art for involvement in Sertoli cell development.
(C) With respect to the state of the prior art: Transcriptional regulation of Sertoli cells development is well known in the art and each of the five transcription factors recited in the claims were known to be active in immature and mature Sertoli cells (see Figure 1 in Rotgers et al (Endocrine Reviews, October 2018, 39(5):739–759, Published online May 15, 2018; ref of record). Rotgers teaches the relevance of mouse gonadal development to human gonadal development and the overlap between transcriptional regulation of mouse and human gonadal development (page 739, right column, lines 10-15). For examples, mutations in NR5A1 cause human gonadal agenesis and Nr5a1 is also known regulator for mouse gonadal development (page 740, right column, last para).
Furthermore, various combinations of the claimed transcription factors were known to produce Sertoli-like cells in vitro when expressed in fibroblasts (Buganim et al, Cell Stem Cell, 2012; ref of record). Cells produced by Buganim were Krt18 positive (Figure S4) and exhibited functions similar to Sertoli cells in vivo, specifically functions of germ cell interaction, endothelial cell attraction (Figure 5E-F, 6, 7, S5, S7). Buganim started with screening nine transcription factors, identifying five out of these nine transcription factors for their ability to reprogram fibroblasts (Nr5a1, Wt1, Dmrt1, Gata4, and Sox9; Figure 1A, 2A; Results, para 1 and 2), then testing three combinations of these five key transcription factors: two combinations comprising three out of the five transcription factors (Nr5a1, Wt1, Dmrt1 in Figure 1 and, Nr5a1, Wt1 and Sox9 in Figure 2) that induce fibroblast reprogramming into cells that are distinct from fibroblast and similar to immature and mature Sertoli cells and, a combination of all five transcription factors (Nr5a1, Wt1, Dmrt1, Gata4, and Sox9) that convert fibroblasts into ieSCs that are transcriptionally and morphologically similar to embryonic Sertoli cells and successfully incorporate in mouse testis in vivo (Figure 3; page 376; Figure 5; Figure 7). As can be seen in Buganim, the Sertoli-like cells produced in Buganim also share similarities with adult Sertoli cells. See Figure 1E that shows transcriptional profile of fibroblasts expressing Nr5a1, Wt1, Dmrt1 is distinct from fibroblasts but similar to immature and mature Sertoli cells. Figure 3E shoes “ieSCs” that are fibroblasts expressing all five transcription factors have marker expression similar to immature and mature Sertoli cells. Whole genome profiling clusters ieSCs with immature and mature Sertoli cells, Figure 4A. Finally, Buganim shows the phenotypic characteristics of ieSCs which include capability of interacting with germ cells and attracting endothelial cells (Figure 5E-F, 6, 7, S5, S7). The instant claims recite these phenotypes for the “adult” Sertoli cells produced however, as taught by Buganim these phenotype exist in Sertoli-like cells that although share similarities with both immature and mature Sertoli cells, are still distinguishable from mature Sertoli cells such that they could not be identified as “adult Sertoli cells”.
Thus, the prior art taught transcription factors known for Sertoli cell development in vivo and taught methods of reprogramming fibroblasts into Sertoli-like cells by using combinations of the 5 key transcription factors. The prior art taught Krt18 as a marker for Sertoli-like cells and that fibroblasts reprogrammed into Krt18 Sertoli-like cells, although not the same as adult Sertoli cells, exhibited some functions similar to adult Sertoli cells.
(D) With respect to the level of one of ordinary skill: As can be seen from the analysis of the prior art presented above, the level of ordinary skill in this art is high because method for dedifferentiation of fibroblasts in Sertoli-like cells were known, role of key transcription factors in Sertoli cell development were known for both mouse and human and methods specifically using expression of combinations of these key transcription factors to convert fibroblasts in Sertoli-like cells were known.
(E) With respect to the predictability of the art: Even with the high level of knowledge and skill in the art, the art remained unpredictable for reprogramming fibroblasts into adult Sertoli cells. In vitro differentiation methods in the prior art were limited to producing cells that were “Sertoli-like” at best and did not result in conversion of fibroblasts into cells that had the same structural and/or phenotypic identity as Sertoli cells of an adult animal. Critically, it was unpredictable that the combinations of transcription factors that were known to work in vivo to generate adult Sertoli cells from embryonic progenitors (as taught by Rotgers) would convert fibroblasts into adult Sertoli cells in vitro. This is because embryonic progenitors found in vivo in the embryonic gonadal primordium are a distinct cell type from fibroblasts and in vitro environment does not recapitulate an in vivo embryonic environment. However, transcriptions factors identified in vivo as upregulated in various stages of development could be utilized to investigate transcriptions factors that could be used for in vitro methods. This is evident from Buganim. They short-listed nine transcription factors that were highly expressed in the immature Sertoli cells, yet not each of these nine supported differentiation of fibroblasts in vitro (Figure 1). Even for the specific combinations taught by Buganim that successfully converted fibroblasts into cells similar to mature Sertoli cells, it was unpredictable that these cells would acquire full mature Sertoli cells characteristics, especially without further experimentation to identify additional culture parameters that would allow for such a feat. Buganim showed that combination of at least 5 TFs (excluded from the claim) was required to reprogram fibroblasts into cells with features that were more similar to immature Sertoli cells rather than mature Sertoli cells (Figure 3E, 4C) while other combinations comprising 3TFs reprogrammed fibroblasts but these cells were less similar to even immature Sertoli cells than the 5TFs even after 3 weeks in culture (Figure 1F vs 3E).
(F), (G) With respect to the amount of direction and working examples provided by the applicant: The applicants have provided multiple working examples directed to production of Sertoli-like cells from fibroblasts, that similar to Buganim result in cells with Krt18 marker expression and share some functional characteristics with adult Sertoli cells. The applicants have not provided working examples wherein any combination of transcription factor recited in claim 25 produced adult Sertoli cells from fibroblasts.
Embodiment 2 provided in the specification is directed towards a method of differentiating human fibroblasts cells comprising AMH-EGFP reporter by expressing various combinations of the same 5 transcription factors that were taught by Buganim (Figure 3). Similar to Buganim, it shows the necessity of expressing Nr5a1 for producing AMH-EGFP +ve cells from the fibroblasts. The cells produced express EGFP under the AMH promoter and AMH is a known marker of immature Sertoli cells in vitro (see Table 1 in Wang et al (Reproduction (2016) 152 R31–R40; ref of record)) and a marker for mesenchymal cells that differentiate into embryonic Sertoli cells and embryonic Sertoli cells in vivo (see Figure 2a in Xu et al. (Stem Cell Research & Therapy (2019) 10:81; ref of record)). Post-filing art of Wang et al (Cell Stem Cell, 23, 599-814, October 2018; presented by Applicant as Exhibit B with the remarks filed 10/14/2025) shows that adult Sertoli cells also express AMH (Figure 1D), however this does not mean that the cells produced in this embodiment 2 are adult Sertoli cells since AMH is also expressed in immature/embryonic Sertoli cells. Critically, Embodiment 3 clearly shows that the cells produced in Embodiment 2 are not the same as adult/mature Sertoli cells.
In embodiment 3, the specification only tests one of the combination of transcription factors claims i.e. B2 =NR5A1+GATA4 (claim 25; claim 46). No other combination is further tested in the entire application to establish a comparison with adult Sertoli cells or their phenotypic properties. The only other combination of transcription factors tested in this embodiment is the one that comprises all five transcription factors (NR5A1+GATA4+WT1+DMRT1+Sox9 = explicitly excluded from the claimed method). Both combinations tested in this embodiment (i.e. B2 and the excluded combination) resulted in differentiation of fibroblasts into cells that share similarities with adult Sertoli cells. Figure 5 shows that 2F-hiSCs (with B2 combination) tested were distinct from 5F-hiSCs (with all 5 TFs) and also from aSCs (primary adult Sertoli cells). See Figure 3D in Liang et al (eLife 2019;8:e48767. Applicant NPL; ref of record for a better version of this figure from the specification. It can be seen in this heatmap that mere expression of 2 transcription factors (B2) was less similar to aSC then all 5 TFs thus suggesting that expression of only two transcription factors do not result in adult Sertoli cells and most likely result in cells even less mature than the cells expressing all five transcription factors, as taught by Buganim. The inventors themselves identify the cells as Sertoli-like cells in the corresponding NPL: See Liang where they state that “This study reported that human fibroblasts can be reprogrammed to Sertoli-like cells using 5TFs (NR5A1, GATA4, WT1, DMRT1 and SOX9) or 2TFs (NR5A1 and GATA4)” (emphasis added; Discussion, line 1).
The specification does not provide any guidance regarding additional manipulations that could be performed to further differentiate the Sertoli-like cells produced into adult Sertoli cells. Thus, lacking such guidance there is known manipulation of these Sertoli-like cells that would predictably yield adult Sertoli cells. It is, however, clear that neither the prior art not the Applicants produced adult Sertoli cells.
(H) Undue experimentation would be required to practice the invention as claimed due to the amount of experimentation necessary because of the state of the prior art and its unpredictability, and the lack amount of guidance in the form of varied working examples in the specification that would allow an artisan generate adult Sertoli cells from fibroblasts by expressing the combination of transcription factors claimed.
MPEP §2164.01(a), 4th paragraph, provides that, “A conclusion of lack of enablement means that, based on the evidence regarding each of the above factors, the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention without undue experimentation. In re Wright, 999 F.2d 1157, 1562; 27 USPQ2d 1510, 1513 (Fed. Cir. 1993).
After applying the Wands factors and analysis to claims 25, 27, 30-33, 46, 48-54, in view of the applicant’s entire disclosure, and considering the In re Wright, In re Fisher and Genentech decisions discussed above, it is concluded that the practice of the invention as claimed in claims 25, 27, 30-33, 46, 48-54 would not be enabled by the written disclosure. Therefore, claims 25, 27, 30-33, 46, 48-54 are rejected under 35 U.S.C. §112(a) for failing to disclose sufficient information to enable a person of skill in the art to make and use the claimed invention to its full scope.
Claim Rejections - 35 USC § 103 – New, necessitated by claim amendments
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Rejection of Claim(s) 25, 27-28, 31, 45-53 under 35 U.S.C. 103 as being unpatentable over Buganim et al (Cell Stem Cell 11, 373–386, September 7, 2012; ref of record) in view of Rotgers et al (Endocrine Reviews, October 2018, 39(5):739–759, Published online May 15, 2018; ref of record) is withdrawn because claim 25 is now amended to recite limitations from now cancelled claim 29 which was previously rejected in a separate U.S.C. 103 rejection.
Rejection of Claim(s) 29 and 30 under 35 U.S.C. 103 as being unpatentable over Buganim et al (Cell Stem Cell 11, 373–386, September 7, 2012; ref of record) in view of Rotgers et al (Endocrine Reviews, October 2018, 39(5):739–759, Published online May 15, 2018; ref of record) as applied to claims 25 and 28, further in view of Guerrier et al (J Reprod. Fert. Volume 88, 1990; ref of record) as evidenced by AMH gene promoter sequence alignment with SEQ ID No.1 (ref of record) is withdrawn because previous rejections of claims 25 and 28 that this rejection depends from is withdrawn, as noted above.
Rejection Claim(s) 32 and 33 under 35 U.S.C. 103 as being unpatentable over Buganim et al (Cell Stem Cell 11, 373–386, September 7, 2012; ref of record) in view of Rotgers et al (Endocrine Reviews, October 2018, 39(5):739–759, Published online May 15, 2018; ref of record) as applied to claims 25 and 28 as evidenced by FUW-tetO-MCS vector from Addgene (catalog #84008; ref of record), Freshney et al (Culture of Animal cells; 6th edition, Published 2010; pages 541-542; ref of record), Selection Antibiotics (ThermoFischer scientific; ref of record) is withdrawn because previous rejections of claims 25 and 28 that this rejection depends from is withdrawn, as noted above.
Claim(s) 25, 27, 30, 31, 46, 48-54 is/are rejected under 35 U.S.C. 103 as being unpatentable over Buganim et al (Cell Stem Cell 11, 373–386, September 7, 2012; ref of record) in view of Rotgers et al (Endocrine Reviews, October 2018, 39(5):739–759, Published online May 15, 2018; ref of record) and Guerrier et al (J Reprod. Fert. Volume 88, 1990; ref of record) as evidenced by AMH gene promoter sequence alignment with SEQ ID No.1 (ref of record).
Regarding claims 25, 27, 46, 48-53, Buganim teaches a method for preparing induced embryonic Sertoli cells (= Sertoli-like cells) from fibroblasts (Abstract). Buganim teaches deriving fibroblasts from H2B-GFP mice wherein the H2B-GFP reporter system has the H2B promoter that is specifically expressed in the Sertoli-like cells in comparison with the fibroblasts and green fluorescent protein (GFP as required for claim 31; Figure 5A; Experimental Procedures: Cell culture and Mice). Buganim shows that GFP expression is significantly higher in the Sertoli-like cells derived from the fibroblasts (ieSCs) in comparison to the undifferentiated fibroblasts thus displaying specificity to Sertoli-like cells (Figure 5A). Therefore, Buganim’s method comprises a first step of introducing a fluorescent reporter system in the fibroblasts that is specifically expressed in the Sertoli-like cells due to use of a promoter specifically expressed in Sertoli cells such that differentiation of fibroblasts could be confirmed (= claimed step a1 in part).
Next, Buganim’s method comprises transducing fibroblasts with lentiviral vectors (= nucleic acids, as required for claim 27) encoding various combinations of transcription factors (Nr5a1, Gata4, Sox9, Wt1, and Dmrt1) resulting in increasing the expression of the transcription factors in the fibroblasts (Figures 1A, 1C, 2A, 3A; Supplemental Experimental Procedures: Molecular cloning and lentiviral infection). Specifically taught combinations are: (i) Nr5a1, Gata4, Sox9, Wt1, and Dmrt1 (Figure 3-7), (ii) Nr5a1, Wt1, and Dmrt1 (Figure 1C-I), (iii) Nr5a1, Sox9, Wt1, (Figure 2).
Regarding the various combinations of transcription factors recited in claims 25, 46, 48-53, Buganim started with screening nine transcription factors, identifying five out of these nine transcription factors for their ability to reprogram fibroblasts (Nr5a1, Wt1, Dmrt1, Gata4, and Sox9; Figure 1A, 2A; Results, para 1 and 2), then testing three combinations of these five key transcription factors: two combinations comprising three out of the five transcription factors (Nr5a1, Wt1, Dmrt1 in Figure 1 and, Nr5a1, Wt1 and Sox9 in Figure 2) that induce fibroblast reprogramming into cells that are distinct from fibroblast and similar to immature and mature Sertoli cells and, a combination of all five transcription factors (Nr5a1, Wt1, Dmrt1, Gata4, and Sox9) that convert fibroblasts into ieSCs that are transcriptionally and morphologically similar to embryonic Sertoli cells and successfully incorporate in mouse testis in vivo (Figure 3; page 376; Figure 5; Figure 7).
Regarding the phenotype of Sertoli-like cells produced, Buganim teaches that fibroblasts expressing Nr5a1, Gata4, Sox9, Wt1, and Dmrt1 are capable of interacting with germ cells and attract endothelial cells (Figure 7, S5, S7).
Although Buganim teaches H2B-GFP reporter, three combinations of the five transcription factors (Nr5a1, Wt1, Dmrt1, Gata4, and Sox9) and the phenotype of the Sertoli-like cells produced by the combination comprising all 5 transcription factors, Buganim does not teach an AMH-GFP reporter system, other combinations of these same transcription factors as recited in claims 25, 46, 48-53. Additionally, Buganim’s method uses mouse fibroblasts and does not teach human fibroblasts as recited in claims 25.
Guerrier teaches the AMH gene promoter which is a 3.6kb sequence 5’ of AMH gene start site that comprises SEQ ID No. 1 that is specific to immature Sertoli cells (as required by claim 30; see Introduction, para 2 and Figure 2, 3; alignment ref of record).
Regarding the various combinations of transcription factors recited in claims 25, 46, 48-53 not taught by Buganim, transcriptional regulation of Sertoli cells development is well known in the art. Rotgers teaches that WT1, SF1 (=NR5A1) and GATA4 are the key regulators for differentiation of somatic progenitor cells into fetal Sertoli cells (see para 1 and 2 of section ‘Formation of the Gonadal Primordium’; para 1 and 2 of section ‘Molecular pathways that govern Sertoli cell development’ and Figure 1 and 2). Rotgers also teaches that WT1 increases the expression of NR5A1 and that expression of NR5A1 is critical for Sertoli cell development (page 740, right column, para 2, lines 1-10). Rotgers also teaches the criticality of GATA4 in Sertoli cell development, stating that without GATA4 “agenesis of the gonads” occurs (page 740, right column, para 1, lines 13-15). Finally, Rotgers teaches the relevance of mouse gonadal development to human gonadal development and the overlap between transcriptional regulation of mouse and human gonadal development (page 739, right column, lines 10-15). For examples, mutations in NR5A1 cause human gonadal agenesis and Nr5a1 is also known regulator for mouse gonadal development (page 740, right column, last para).
The combination of prior art cited above under 35 U.S.C. 103 satisfies the factual inquiries as set forth in Graham v. John Deere Co., 383 U.S. 1, 148 USPQ 459 (1966). Once this has been accomplished the holdings in KSR can be applied (KSR International Co. v. Teleflex Inc. (KSR), 550 U.S., 82 USPQ2d 1385 (2007)). Guidance pertaining to exemplary rationales that may support a conclusion of obviousness are in MPEP2143.
In the present situation, rationale G (Some Teaching, Suggestion, or Motivation in the Prior Art That Would Have Led One of Ordinary Skill To Modify the Prior Art Reference or To Combine Prior Art Reference Teachings To Arrive at the Claimed Invention) is applicable. MPEP 2143 guides that for rationale G “Office personnel must articulate the following: (1) a finding that there was some teaching, suggestion, or motivation, either in the references themselves or in the knowledge generally available to one of ordinary skill in the art, to modify the reference or to combine reference teachings; (2) a finding that there was reasonable expectation of success; and (3) whatever additional findings based on the Graham factual inquiries may be necessary, in view of the facts of the case under consideration, to explain a conclusion of obviousness
(1) The prior art of Buganim teaches a H2B-GFP reporter system to identify fibroblasts that have differentiated into Sertoli-like cells. Due to this teaching an ordinary artisan recognizes that reporter systems are useful in identifying the differentiation state in culture due to the use of a promoter that allows for cell specific expression and that other reporter systems with promoters that would specifically express in cells produced in comparison to fibroblasts could be substituted in place of H2B-GFP. Guerrier teaches AMH promoter as such a promoter that is specifically expressed in immature Sertoli cells that are Sertoli-like cells. An ordinary artisan would be motivated to use Guerrier promoter to more specifically identify and isolate immature Sertoli cells from Buganim’s culture that can be used in supporting in vivo embryonic testicular development
Further, Buganim teaches three combinations of the five key transcriptional factors for reprogramming of fibroblasts into immature Sertoli cells. Buganim screens nine transcriptional factors to identify the five key transcriptional factors and tests three combinations of these five transcription factors. Due to this teaching an ordinary artisan recognizes that other combinations of the same five transcription factors could also produce Sertoli-like cells and thus motivates an ordinary artisan to identify other combinations of the same five transcription factors. Considering that Buganim was able to reduce the number of transcription factors that can be combined to reprogram fibroblasts into Sertoli-like cells from nine to five and combinations comprising only 3 out those 5 transcription factors are sufficient to reprogram fibroblasts into cells more similar to Sertoli-like cells, this also suggests to and motivates an ordinary artisan to find other combinations of the same five transcription factors comprising fewer than the five to produce immature Sertoli-like cells. Furthermore, although Buganim uses mouse-derived fibroblasts in their method, an ordinary artisan is motivated to apply mouse methods to human cells to allow for clinical applications.
(2) When substituting Buganim’s H2B promoter with Guerrier’s AMH promoter, an ordinary artisan reasonably expects that a fluorescent reporter system comprising AMH gene promoter in place of H2B promoter would identify immature Sertoli cells because Guerrier teaches that the AMH gene expression is specific to immature Sertoli cells and also teaches the sequence of the AMH gene promoter.
Further, since Buganim was able to reduce the number of transcription factors that can be combined to reprogram fibroblasts into Sertoli-like cells from nine to five and combinations comprising only 3 out those 5 transcription factors are sufficient to reprogram fibroblasts into cells more similar to Sertoli-like cells, an ordinary artisan has a reasonable expectation that other combinations of the same 5 transcription factors would also reprogram fibroblasts into cells similar to Sertoli cells i.e. Sertoli-like cells. Teachings from Rotgers about the function of each of the five transcription factors taught by Buganim in Sertoli cell development, specifically the critical role of NR5A1 and GATA4, also enhance the reasonable expectation of an ordinary artisan that other combinations comprising at least NR5A1 and GATA4 could reprogram fibroblasts into Sertoli-like cells. Rotgers also provides reasonable expectation to an ordinary artisan that teachings and method of Buganim that uses mouse fibroblasts could be applied to human fibroblasts due to the overlap between transcriptional regulation of mouse and human gonadal development.
Finally, an ordinary artisan reasonably expects that the cells produced by other combinations of the same 5 transcription factors already taught by Buganim would result in Sertoli-like cells with the ‘capability’ of interacting with germ cells, immune privilege, endothelial cell attraction and lipid droplet formation (as required for claims 25 and 54; see claim interpretation above).
(3) Findings pertaining to the Graham factual inquiries (1-3) are detailed above. Findings pertaining to the Graham factual inquiries 4 are discussed here: The evidence in the specification supporting that each of the claimed transcription factors combinations results in Sertoli-like cells is limited. Figure 3 shows that the claimed transcription factor combinations result in AMH:EGFP+ cells. Although these cells express GFP, cells expressing most of the claimed combination of transcription factors (B6, B7, B8, B12, B13, B14) are not compared with either immature or mature Sertoli cells. Prior art teaches that AMH is a marker for precursors to the immature Sertoli cells (See Guerrier; See analysis of prior art in 112a enablement rejection above). Only one of the claimed combinations (=B2 in claim 25, claim 46) was compared to adult Sertoli cells in Embodiment 3 wherein they were shown to be distinct from both the control fibroblasts and adult Sertoli cells while the combination of all five transcription factors resulted in cells that were more similar to adult Sertoli cells (Figure 5). This suggests that expression of all 5 transcription factors (excluded from the claims; taught by Buganim) results in a more mature phenotype than expression of only NR5A1+GATA4 transcription factors. This is obvious to an ordinary artisan because Buganim shows that fibroblasts transduced with only 3 of the 5 transcription factors result in immature Sertoli-like cells that are less mature than fibroblasts transduced with all 5 transcription factors. Thus, objective evidence in support of non-obviousness is not found in the specification. The data presented in the specification is obvious in light of teachings from Buganim alone and further in light of teachings from Rotgers.
Therefore, the teachings of the cited prior art in the obviousness rejection above provide the requisite teachings with a clear, reasonable expectation. The cited prior art meets the criteria set forth in both Graham and KSR. Therefore, it would be obvious to a person of ordinary skill in the art to modify Buganim’s reporter system to comprise a AMH gene promoter comprising SEQ ID No. 1, as taught by Guerrier, in place of the H2B promoter taught by Buganim and, further to modify the method of Buganim to comprise other combinations (such as recited in claims 25, 46, 48-53) of the same five transcription factors to produce immature Sertoli-like cells. Furthermore, considering the teachings of Rotgers pertaining to human and mouse gonadal development, it would be obvious to a person of ordinary skill in the art to modify the method of Buganim to comprise human fibroblasts.
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in
the art at the effective time of filing of the invention, especially in the absence of evidence to the
contrary.
Claim(s) 32 and 33 are rejected under 35 U.S.C. 103 as being unpatentable over Buganim et al (Cell Stem Cell 11, 373–386, September 7, 2012; ref of record) in view of Rotgers et al (Endocrine Reviews, October 2018, 39(5):739–759, Published online May 15, 2018; ref of record) and Guerrier et al (J Reprod. Fert. Volume 88, 1990; ref of record) as evidenced by AMH gene promoter sequence alignment with SEQ ID No.1 (ref of record) as applied to claim 25 and further in view by FUW-tetO-MCS vector from Addgene (catalog #84008; ref of record), Freshney et al (Culture of Animal cells; 6th edition, Published 2010; ref of record), Selection Antibiotics (ThermoFischer scientific; ref of record).
The teachings of Buganim, Guerrier and Rotgers as applied to claim 25 above are relied upon for the instant rejection.
Buganim, Guerrier and Rotgers render obvious the method of claim 25. Buganim teaches a medium for culturing fibroblasts wherein the culture medium comprises antibiotics (Experimental Procedures: Cell culture and Mice). Since Buganim teaches use of the FUW-tetO-MCS vector (comprising BleoR gene to confer bleomycin resistance to transduced cells; available from Addgene, catalog #84008; see Supplemental Experimental Procedures: Molecular cloning and lentiviral infection) for the generation of their lentivirus, Buganim implicitly teaches bleomycin-based antibiotic selection. Buganim also teaches that differentiation of fibroblasts into embryonic Sertoli cells takes 2 weeks from transduction with the transcription factors (page 376, left column, para2). Although, Buganim does not explicitly state to refresh the culture medium every 3-7 days during the 14 day culture period such a practice is routine and an ordinary artisan would discard exhausted culture media (liquid phase) with fresh culture media as often as required by the cells (See teachings from Freshney in Exercise 9 in Chapter 28). Buganim also teaches isolation of embryonic Sertoli cells expressing green fluorescence from the culture system (Figure 7a; step a6). Therefore, in teachings a method that cultures the cells for the same duration as claimed, Buganim renders obvious the method wherein culture medium is refreshed every 3-7 days, as recited in steps a4, a5.
Taken together, Buganim teaches a method of generating Sertoli-like cells from fibroblasts by transducing fibroblasts with lentivirus encoding transcription factors (step a2), culturing the fibroblasts in a culture medium comprising antibiotics (i.e. adding antibiotics to the culture system; step a3) for 14 days in a medium for culturing fibroblast (step a4 and a5 recite 7 days each) and isolating embryonic Sertoli cells from the culture system based on their fluorescence (step a6, claim 33).
Although Buganim teaches the use of antibiotics (bleomycin) as selection agents, Buganim does not teach the specific antibiotic selection agent recited in the claim step a3, G418. However, both bleomycin and G418 are well-known in the art as antibiotic selection agents in eukaryotic cells (see teachings in Selection Antibiotics listed by ThermoFischer scientific). An ordinary artisan would select the antibiotic to be added to culture medium based on the antibiotic-resistance gene present in the cells to be selected. According to MPEP 2144.06, substituting equivalents known for the same purpose provides strong evidence of obviousness. Thus, disclosure of an antibiotic selection agent such as bleomycin by Buganim in their method renders the use of an equivalent antibiotic selection agent such as G418 prima facie obvious.
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in
the art at the effective time of filing of the invention, especially in the absence of evidence to the
contrary.
Response to Arguments
Applicant’s arguments with respect to the U.S.C. 103 rejection of claim(s) 25, 27-28, 31, 45-53 and, U.S.C. 103 rejection of claim(s) 32 and 33 have been considered but are moot because the new ground of rejection necessitated by amendment to claim 25.
Arguments pertinent to the instant rejection are discussed below.
First, Applicant argue that “the reprogrammed Sertoli cells exhibit gene expression profiles similar to those of adult Sertoli cells (see, e.g., paragraphs [0134]-[0135]1), which can interact with germ cells (see, e.g., paragraphs [0161]-[0167]), and have immune-privileged characteristics (see, e.g., [0168]-[0198]).” (page 7, last para). Applicant point to a review from Cheng and Murk that they claim teaches that “these attributed are exclusively associated with adult Sertoli cells” (page 7, last para). Applicants also point to Liang et al (the post-filing art by the Applicants) which they allege “further confirms that the Sertoli cells in this application are adult Sertoli Cells” based on the transcriptional map of the 2F or 5F hiSCs in Figures 2 and 3 (page 8, para 1). Finally, Applicants point to Wang et al that shows that AMH “can also be detected in adult Sertoli cells” (page 8, para 2) and Krt18 is upregulated in ‘Sertoli cells’ produced by their method in comparison to fibroblast (page 8-9, bridging para). Thus, Applicant conclude that “it is clear from this application that the Sertoli cells recited in the claims refer to adult Sertoli cells, not any Sertoli cells” (page 8, para 2).
In response, reprogramming of fibroblasts “similar to those of adult Sertoli cells” and having some functions similar to adult Sertoli cells does not make the produced cells “adult Sertoli cells”. This has issue has been in addressed in detail in the instant U.S.C. 112a- Scope of Enablement rejection above wherein analysis that addresses each of these arguments in addressed. Briefly, the review of Cheng and Murk discusses the functional characteristics of adult Sertoli cells however they do not teach that these functions are only present in adult Sertoli cells such that immature Sertoli cells do not have some of the functions that are the same as their mature counterparts i.e. adult Sertoli cells. To this end, Buganim compared their Sertoli-like cells with both mature and immature Sertoli cells, showing that they have some similarities with both. Buganim did not consider their Sertoli-like cells as “adult” (a view Applicant agrees with based on remarks on page 9, para 5). Yet, Buganim shows that their Sertoli-like cells interact with germ cells and attract endothelial cells in vitro and in vivo resulting vascularization of gonads (Figures 7, S5, S7). Thus, contrary to Applicants allegations, presence of the claimed functional characteristics does not support the conclusion that the cells produced are “adult”. This conclusion is further strengthened by Applicant’s own disclosure in Liang et al wherein the Applicants identifies the cells produced as “Sertoli-like cells” (Title). The data presented in Applicants NPL, shows that the “Sertoli-like cells” produced expressed Krt18 and explicitly teaches that this was also shown by Buganim (page 8, para 1). The data in Figure 2 in Liang is directed to the “5F-hiSC” which are fibroblasts with all 5 transcriptions factors, a condition the claims explicitly exclude. Thus, this data is not relevant to the argument. The data in Figure 3 in Liang is compares the 2F-hiSC (= claimed B2, NR5A1+GATA4) with 5F-hiSC and adult Sertoli cells. It can be seen in this heatmap that mere expression of 2 transcription factors (B2) was less similar to aSC then all 5 TFs thus suggesting that expression of only two transcription factors do not result in adult Sertoli cells and most likely result in cells even less mature than the cells expressing all five transcription factors, as taught by Buganim. Thus, teachings in Applicant’s own disclosure in the associated NPL do not support the arguments Applicants present here.
Finaly, regarding the AMH and Krt18 reporters. Expression of AMH and even Krt18 in adult Sertoli cells does not preclude their expression in immature Sertoli cells. Although Wang shows that adult Sertoli cells express AMH, Wang (2016), Xu and Guerrier each support that AMH is also expressed in immature Sertoli cells (see discussion of Wang 2016 and Xu in scope of enablement rejection). Thus, expression of AMH does not guarantee that the cells produced are indeed “adult”. Although Krt18 is a marker for Sertoli-like cells, used by both Buganim and Applicant’s NPL disclosure, this does not indicate that the cells are “adult Sertoli cells”.
Taken together, it is concluded that the Applicants disclosure does not enable a method wherein fibroblasts are converted to “adult Sertoli cells”.
Next, Applicants argue that “Buganim specifically concerns preparing induced embryonic Sertoli-like cells (ieSCs), which have different characteristics from adult Sertoli cells” and “Buganim does not teach or suggest that preparing adult Sertoli cells would be desired or that the methods described therein can be modified for preparing adult Sertoli cells” (page 9, para 5 and 6). Thus, Applicant conclude that “a person of ordinary skill in the art seeking to prepare adult Sertoli cells would not have had reason to start with Buganim and then to delete transcription factors from the only successful combination for ieSCs to arrive at the claimed method with a reasonable expectation of success.” (page 10, para 2). Applicant also add that “The cited secondary references do not cure the deficiencies of Buganim.” (page 10, para 4). Specifically, “Rotgers does not show specifically that the claimed combination of transcription factors could be successful in reprogramming human fibroblasts into adult Sertoli cells.” (page 10-11, bridging para).
In response, these arguments rely on the unpersuasive arguments above which contend that the instant Application discloses making adult Sertoli cells. As detailed above in the response to the argument and the instant U.S.C. 112a- Scope of Enablement rejection, the Applicants disclosure does not enable a method wherein fibroblasts are converted to “adult Sertoli cells”. Only Sertoli-like cells are enabled and thus, in this regard, Buganim is not deficient and secondary references are not required to overcome the alleged deficiency.
Finally, Applicant argue that “fluorescent protein reporter system is not merely a reporting system for marking cells. As described according to paragraph [0128] of this application, according to steps 2 to 9, when the dH1*AMH: GFP is replaced with the dH1 fibroblast strain, and other steps are all unchanged, flow cytometry shows that hiSCs (dH1) are nearly not obtained. Thus, this fluorescent protein reporting system helps reprogramming fibroblasts into Sertoli Cells: this is not taught or suggested in the cited references.” (page 13, para 5).
In response, although this para [0128] refers to a flow cytometry data, it does not refer to any figure that shows hiSC were “nearly not obtained”. The FACS analysis described, and in general, measures cells such as hiSC based on reporter expression. Thus it is unsurprising that without the reporter few hiSC could be detected, any that could be detected were because of auto-fluorescence common in the GFP fluorescent spectra. Such control experiments are performed in FACS analysis to identify proper gating such that auto/background fluorescence can be subtracted.
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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to MATASHA DHAR whose telephone number is (571)272-1680. The examiner can normally be reached M-F 8am-4pm (EST).
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Peter Paras Jr. can be reached at (571)272-4517. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/MATASHA DHAR/Examiner, Art Unit 1632
/PETER PARAS JR/Supervisory Patent Examiner, Art Unit 1632