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 .
DETAILED ACTION
RESPONSE TO AMENDMENT
Status of Application/Amendments/claims
2. Applicant’s amendment filed January 02, 2026 is acknowledged. Claims 2-13, 15, 17-28 and 32-35 are cancelled. Claims 1, 14, 16 and 29-31 are amended. Claims 36-39 are newly added. Claims 1,14, 16, 29-31 and new claims 36-39 are pending in this application. Applicant timely traversed the restriction (election) requirement in the reply filed on September 29, 2023.
3. Claims 1,14, 16, 29-31 and 36-39 are under examination with respect to gene markers of Hypoxia or response to hypoxia in dysregulated biological pathways; genes of negative regulation of apoptotic pathway; a combination of GDNF, FGF2, Testosterone, Retinoic Acid, WNT3A, NRTN, BMP-2, rh beta-NGF, rh Midkine Protein, rh HB-EGF, rh MIF and rh LIF for ligands; a combination of HIF inhibitor, a gonadocorticoid and FGFR ligand for factors in second culture medium; Echinomycin for HIF inhibitor; Notoginsenoside R1 for anti-apoptosis agent; Plerixafor for anti-inflammation agent; Dimethyl-bisphenol A for anti-angiogenesis agent; DL-a-tocopheral acetate (VE) for RO inhibitor; and healthy subject for testicular tissue source in this office action.
4. Applicant’s arguments filed on January 02, 2026 have been fully considered but they are not deemed to be persuasive for the reasons set forth below.
Priority
5. The priority for the claimed subject matter of a process for identifying proliferation factors for increasing human spermatogonial stem cell (SSC) proliferation in vitro comprising steps recited in instant claims in the instant application is July 3, 2023.
Claim Rejections/Objections Withdrawn
6. The rejection of claims 2 and 32-35 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite is moot because the claims are canceled.
The rejection of claims 1-2,14, 16 and 29-35 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112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement is withdrawn in response to Applicant’s amendment to the claims and cancelation of claims 2 and 32-35.
Claim Rejections/Objections Maintained
In view of the amendment filed on January 02, 2026, the following rejections are maintained.
Claim Rejections - 35 USC § 112
7. The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 1,14, 16, 29-31 and 36-39 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor, or for pre-AIA the applicant regards as the invention. The rejection is maintained for the reasons of record and the reasons set forth below.
Response to Arguments
On p. 9 of the response, Applicant argues that the rejection has been overcome in view of amendment to claim 1.
Applicant's arguments have been fully considered but they are not found persuasive. Contrary to Applicant' s arguments, the examiner asserts that based on MPEP§2171-MPEP§2173, claims 1,14, 16, 29-31 and 36-39 are indefinite because:
i. The limitations “one or more factors of Tables 1, 2 and 7-11” in a)-1) of claim 1 and “(A) factors listed in Tables 5 and 6” in item b)-i) of claim 1, the limitation “one or more factors of Tables 7-11” in claim 37, the limitation “additional factors selected from Tables 1, 2 and 7-11” in claim 38, and the limitation “….from (A) factors listed in Tables 5 and 6…” in claim 39 are not concise to define the invention. Note that "Where possible, claims are to be complete in themselves. Incorporation by reference to a specific figure or table “is permitted only in exceptional circumstances where there is no practical way to define the invention in words and where it is more concise to incorporate by reference than duplicating a drawing or table into the claim. Incorporation by reference is a necessity doctrine, not for applicant’s convenience.” Ex parte Fressola, 27 USPQ2d 1608, 1609 (Bd. Pat. App. & Inter. 1993)". See MPEP 2173.05(s). Note that the rejection can be obviated by copying the table contents of the factors listed in Tables 1, 2, 5-6 and 7-11 into the claim itself.
ii. The rest of the claims are indefinite as depending from an indefinite claim.
Accordingly, the rejection of claims 1,14, 16, 29-31 and 36-39 under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite is maintained.
New Grounds of Rejection Necessitated by the Amendment
The following rejections are new grounds of rejections necessitated by the amendment filed on January 02, 2026.
Claim Rejections - 35 USC § 112
8. 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 1,14, 16, 29-31 and 36-39 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a new matter rejection.
Claims 1,14, 16, 29-31 and 36-39 as amended are drawn to a process for identifying proliferation factors for increasing human spermatogonial stem cells (SSC) proliferation in testicular tissued cultured in vitro, comprising:
a) identifying structure-maintaining media (SMM) that maintain somatic tissue structure by:
i) culturing testicular tissue obtained from a subject in a somatic screen medium (SSM), wherein the SSM comprises a base medium (BM) supplemented with one or more factors of Tables 1, 2 and 7-11, wherein the BM is aMEM and 10% Knockout serum replacement (KSR), and wherein, when the testicular tissue is cultured in unsupplemented BM, the cultured testicular tissue exhibits an altered somatic structure by day 7;
ii) identifying among factors in step (a)(i) one or more structure-maintaining factors that are capable of maintaining somatic tissue structure in the cultured testicular tissue at 7 days or longer after start of culture, thereby identifying an SMM, wherein the SMM comprises the BM supplemented with the one or more structure-maintaining factors; and
b) identifying SSC proliferation factors that promote SSC proliferation om the cultured testicular tissue by:
i) culturing testicular tissue in a proliferation screen medium (PSM), wherein the PSM comprises an SMM supplemented with one or more test factors selected from (A) factors listed in Tables 5 and 6 and (B) one or more of the structure-maintaining factors identified in step (a)(ii); and
ii) identifying among test factors in (b)(i) one or more SSC proliferation factors that increase SSC proliferation in testicular tissue cultured in the PSM when compared to SSC proliferation in testicular tissue cultured in the PSM lacking the one or more test factors, thereby identifying the SSC proliferation factors;
c) culturing testicular tissue in a final culture medium (FCM) comprising the SMM and one or more SSC proliferation factors identified in step (b)(ii), wherein SSC proliferation is measured by an increase in the number of EdU/DDX4 double-positive cells in cultured testicular tissue, and wherein the SSCs exhibit increased proliferation for 14 days or longer in the FCM;
wherein altered somatic tissue structure is determined by histological staining showing loss of seminiferous tubular architecture, by EdU labeling identifying replicating cells outside the seminiferous tubules or both.
The instant claims now recite new limitations “a) identifying structure-maintaining media (SMM) that maintain somatic tissue structure… a somatic screen medium (SSM)…….. SMM comprises the BM supplemented with the one or more structure-maintaining factors; b) identifying SSC proliferation factors that promote SSC proliferation om the cultured testicular tissue ….in a proliferation screen medium (PSM)…..c) culturing testicular tissue in a final culture medium (FCM) comprising the SMM and one or more SSC proliferation factors identified in step (b)(ii)…. wherein the SSCs exhibit increased proliferation for 14 days or longer in the FCM;…. histological staining showing loss of seminiferous tubular architecture, by EdU labeling identifying replicating cells outside the seminiferous tubules or both”, which were not clearly disclosed in the specification and claims as filed, and now change the scope of the instant disclosure as filed. Such new limitations recited in the present claims, which did not appear in the specification or original claims, as filed, introduce new concepts and violate the description requirement of the first paragraph of 35 U.S.C. 112.
The specification fails to disclose the new limitations “a) identifying structure-maintaining media (SMM) that maintain somatic tissue structure… a somatic screen medium (SSM)…….. SMM comprises the BM supplemented with the one or more structure-maintaining factors; b) identifying SSC proliferation factors that promote SSC proliferation om the cultured testicular tissue ….in a proliferation screen medium (PSM)…..c) culturing testicular tissue in a final culture medium (FCM) comprising the SMM and one or more SSC proliferation factors identified in step (b)(ii)…. wherein the SSCs exhibit increased proliferation for 14 days or longer in the FCM;…. histological staining showing loss of seminiferous tubular architecture, by EdU labeling identifying replicating cells outside the seminiferous tubules or both” recited in claims 1, 37-39.
The specification only discloses using scRNA-Seq and comparing the RNA transcripts in each cell type in cultured cells dissociated from cultured testicular tissue to the level of RNA transcripts in cells dissociated from tissue directly from a subject without culture based on the scRNA Seq transcriptome profile of the adult human testis atlas to characterize cell types and then based on the scRNA Seq transcriptome profile of the adult human testis atlas, identifying that the expression of genes associated with extracellular exosome, negative regulation of apoptotic process, cytokine, response to hypoxia, actin cytoskeleton, extracellular matrix, and muscle contraction that affect the HIF pathway are changed in cultured Leydig and myoid cells within the cultured testicular tissue, and identifying expression of genes associated with ribosome, focal adhesion, extracellular matrix, and angiogenesis are changed in cultured endothelial cells within the cultured testicular tissue (p. 69-71, paragraphs [00220]-[00223], Example 2, figures 19-20, tables 12-13). The specification teaches screening and testing different small molecules and ligands based on the genes affecting the HIF pathway and different concentrations on germ cell proliferation, and identifying that the HIF-1 inhibitor echinomycin helps maintain testicular tissue structure and germ cell survival (Figures 22-24), and adding a combination of echinomycin, testosterone, FSH, and RA to the medium resulted in better germ cell proliferation when compared to echinomycin alone or ligands alone (Figure 24); iii) culturing and increasing proliferation of spermatogonia using a C2 medium (MEM, 10%KSR, 1% penicillin-streptomycin, 20ng/ml GDNF, 20ng/ml FGF2, 10ug/ml insulin, 20ng/ml EGF and 10uM Testosterone), a C2 medium with 80uM Testosterone or using a combination of the testicular tissue system using the control 2 culture medium (MEM, 10%KSR, 1% penicillin-streptomycin, 5nM echinomycin, 10uM Testosterone, 20ng/ml GDNF, 20ng/ml FGF2, 10ug/ml insulin, 20ng/ml EGF) followed by the SPG culture system using the C2 medium (p. 84-85; p. 88-96, Examples 4, 6-8, tables 12-13 and 15).
Applicant provides no guidance as to what is encompassed in the new limitations recited in instant claims. Accordingly, in the absence of sufficient recitation for the new limitations “a) identifying structure-maintaining media (SMM) that maintain somatic tissue structure… a somatic screen medium (SSM)…..or both”, the specification does not provide adequate written description to support the new limitations recited in claim 1. Support is not found for the new limitations ““a) identifying structure-maintaining media (SMM) that maintain somatic tissue structure… or both” as disclosed in the original specification and thus the recitations constitute new matter absent evidence for their support. Applicant is required to cancel the new matter in the reply to this office action. Alternatively, Applicant is invited to clearly point out the written support for the instant limitations.
Conclusion
9. NO CLAIM IS ALLOWED.
10. The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
De Oliveira et al. (Front. Cell Dev. Biol., 2022;10:782996. Doi:10.3389/fcell.2022.782996) teach using scRNA-seq Approach to identify changes in Spermatogonial stem cell gene expression following in vitro culture and based on gene ontology analysis, upregulated gene expression of genes involved in oxidative phosphorylation in cultured spermatogonia along with downregulation of integral processes including DNA repair and ubiquitin-mediated proteolysis, wherein the cultured spermatogonia is obtained from adult mouse testis and has been cultured for 10 weeks; and the data obtained from the cultured spermatogonia provides a valuable platform for improving SSC culture approaches and therapeutic use (see abstract).
Sohni et al. (Cell Reports, 2019; 26:1501-1517) teach use of scRNA-seq analysis to define cell subsets and corresponding gene markers in the human testis and identifying receptor-ligand pairs in adult cells
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Ramilowski et al. (Nat. Commun. 2015; 6:7866. DOI:10.1038/ncomms8866) teach a map of cell-to-cell communication between 144 human primary cell types and a highly connected signaling network through multiple ligand–receptor paths based on ligands and receptor expressed in the cells (see abstract; Figure 5).
Huleihel et al. (US2020/0199527) teaches a method of culturing and inducing differentiation of human spermatogonia or spermatogonial stem cells, comprising culturing the spermatogonia or spermatogonial stem cells in a three-dimensional methylcellulose culture system (MCS) under conditions capable of proliferating and differentiating the human spermatogonia or spermatogonial stem cells into an elongated spermatid, thereby in vitro maturing the spermatogonia or spermatogonial stem cells (see paragraph [0008]). The 3D culture conditions provided by the MCS and growth factors, in addition to the somatic cells present; the number of examined markers was dependent on the amount of RNA extracted from the samples (see paragraphs [0045] [0057]; [0101]-[0152]; [0338]-[0339]). Huleihel teaches comparing markers, which were positive before culture to after culture (see paragraph [0331]), the process comprising: a) identifying differentially expressed RNA transcripts in single testicular cells grown in vitro under a first set of conditions when compared to expression of the RNA transcripts in single testicular cells directly isolated from the testis of male subjects (expression level of the RNA in the cells of some embodiments of the invention can be determined including various pre-meiotic (e.g., VASA, SALL4, OCT4, PLZF, CD9, A-6-INTEGRIN, GFR-A1, and C-KIT), meiotic (e.g., CREM-1, LDH, ACROSIN, and BOULE) and post-meiotic (e.g., ACROSIN, and PROTAMINE) markers (see paragraphs [0167]; [0217]); method involves the detection of a particular RNA in a mixture of RNAs (see paragraph [0219]); divide the biopsy (according to its size; for each part at least 3mm3 tissue was used) to be used for tissue culture (first priority), histology and RNA extraction (see paragraphs [0045] [0057]; [0101]-[0152]; [0338]-[0339]). Huleihel also teaches that the method is “the first study that shows the expression of different pre-meiotic markers in testicular biopsies from prepubertal cancer patients after chemotherapy….. this study shows for the first time a proof of concept for an in vitro culture system that induces human spermatogonial cells to meiotic and post-meiotic stages including the generation of sperm-like cells” (see paragraph [0064]). Huleihel teaches differentiation of spermatogonial stem cells in a medium comprising TNF-Alpha(TNFaQ) (1-200pg/ml…..50pg/ml), GDNF (10ng/ml or 1-50, 1-40…5, 10,15, 20ng/ml), LIF (10ng/ml or 1-50….20ng/ml), bFGF (10ng/ml; 1-50….5, 15, or 20ng/ml), EGF (20ng/ml, 0.1-200ng/ml….20ng/ml), testosterone (10-8M..10-6M), FSH (25u/ml or 1-100u/ml…25U/ml), retinoic acid (10-8M..10-6M), insulin (25ug/ml), hormone or combinations thereof as recited in claims 14, 16 and 30-31, and wherein the medium comprises aMEM and KSR as in claim 2 (see paragraphs [0057]; [0101]-[0152]; [0284]-[0285]; [0290];[0338]-[0339]).
Kumar et al. (Oncotarget, 2016; 7:85709-85727, as in IDS) teach that over activation of Wnt signaling in germ cells causes defects in proliferation and differentiation leading to premature loss of germ cells and a) performing ScRNA sequencing analysis of control and mutant testes to identify 20 genes (see p.85721,left-col.,1st paragraph) including proliferation and differentiation of germ cells in mammalian testis, which was also confirmed using an in vitro cell culture system (see p. 85721,right col., 1st paragraph); b) growing testicular cells under a second set of culture conditions, based on comparisons in (a), that alleviate dysregulation of the identified pathways by testing for improved growth, survival, physiology, or development i.e. proliferation and differentiation of germ cells in mammalian testis, which was also confirmed using an in vitro cell culture system (see P.85721,right-col., 1st paragraph). Kumar teaches culturing GC1 cells in Wnt3a conditioned media or control media for 5 days, and proteins were isolated from these cells and that Western blot analysis revealed 1.7 fold increase in B catenin expression in GC1 cells cultured with Wnt3a conditioned media relative to controls (see p.85718,right-col.,3rd paragraph, and P..85720,left-col., 1st paragraph); wherein cells grown under the second set of culture conditions exhibit proper identity, growth, and survival when compared to the cells directly isolated from the testis of males (i.e. proliferation and differentiation of germ cells in mammalian testis, which was also confirmed using an in vitro cell culture system, p.85721, right-col., 1st paragraph).
Lee et al. (J. Endocrinol. 2017; 234:R53-R65, as in IDS) teach that hypoxia and Intermittent hypoxia result in changes in testicular morphology and loss of spermatogenic cells in all stages of the spermatogenic cycle (p. R57, 1st paragraph) and echinomycin and its use for inhibition of HIF activity as recited in claims 14 and 16 (see p. R58, left col., bottom of 1st paragraph).
Vlaminck et al. (FEBS J, 2007; 274:5533-5542. doi:10.111/j.1742-4658. 2007.06072.x) teach that the dose of echinomycin in inhibiting HIF activity is at 2nM-10nM (see p. 5534, 2nd col., p. 5535, figures 1).
JP7399406-English translated version (published Sept 10, 2020, priority March 3, 2019) teaches a medium comprising agents for in vitro spermatogenesis induction in a testicular organ culture or a testicular tissue culture comprising spermatogonia or testicular germ cells, wherein the agent includes glutathione (GSH), L-ascorbic acid 2-glucoside (VC) (i.e. vitamin C) and DL-a-toxopheal acetate (VE) (i.e. vitamin E) as in claim 29 (see p.1-5).
11. 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.
12. Any inquiry concerning this communication or earlier communications from the examiner should be directed to CHANG-YU WANG whose telephone number is (571)272-4521. The examiner can normally be reached Monday-Thursday, 7:00am-5:00pm 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, Jeffrey Stucker can be reached at 571-272-0911. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
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Chang-Yu Wang
April 21, 2026
/CHANG-YU WANG/Primary Examiner, Art Unit 1675
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