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
This application is in response to the papers filed on October 16, 2025. Pursuant to the amendment filed on October 16, 2025, claims 1, 3-5, 7-11 are currently pending of which claims 1, 3-5, and 8-10 have been amended and claims 2, 6, 12, 16, 22-26, and 46-47 have been cancelled. The restriction requirement between Groups I-V was previously made FINAL in the previous Office Action.
Therefore, claims 1, 3-5, 7-11 are currently under examination to which the following grounds of rejection are applicable.
Priority
The present application is a 35 U.S.C. 371 national stage filing of the International Application No. PCT/US2020/034574, filed May 26, 2020. Applicant’s claim for the benefit of a prior-filed parent provisional applications 62/931,126 filed on November 5, 2019 and 62/852,790 filed on May 24, 2019 under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, or 365(c) is acknowledged.
Thus, the earliest possible priority for the instant application is May 24, 2019.
Response to Arguments
Withdrawn Objections/Rejections in response to Applicants' arguments or amendments:
Claim Objections
In view of Applicants' amendment to the claims dated October 16, 2025, wherein claim 8 has been amended, and claim 2 has been cancelled, the objection to claim 8 has been withdrawn and the objection to claim 2 has been rendered moot.
Claim Rejections - 35 USC § 112
In view of Applicants' amendment to the claims dated October 16, 2025, wherein claims 1, 3-5, and 8-10 have been amended and claims 2, 6, 12, 16, 22-26, and 46-47 have been cancelled, the rejection to claims 1, 3-5, and 7-11 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite, have been withdrawn. The rejection to now cancelled claims 2 and 6 have been rendered moot.
Claim Rejections - 35 USC § 103
In view of Applicants' amendment to the claims dated October 16, 2025, wherein claims 1, 3-5, and 8-10 have been amended and claims 2, 6, 12, 16, 22-26, and 46-47 have been cancelled, the rejection to claims 1, 3-5, 7-11 rejected under 35 U.S.C. 103 as being unpatentable over Leeman et al. in view of Cao et al. is withdrawn.
The withdrawn rejections is in view of the amendments to claim 1 wherein the culture medium is serum free, and further specifying the HSC as “mitochondrial membrane potential (MMP)-low, lineage negative (Lin1 stem cell antigen 1 positive (Sea+), cKit+, Signaling Lymphocyte Activation Molecule 1 positive (CD 150+), and Signaling Lymphocyte Activation Molecule 2 negative (CD48-) cells” all of which the prior art references employed do not teach.
Applicants' arguments are moot in view of the withdrawn rejection. A response to any argument pertaining to a new or maintained rejection can be found below.
New Grounds of Rejection:
Claim Rejections - 35 USC § 103
Claims 1, 3-5, 7-11 are newly rejected under 35 U.S.C. 103 as being unpatentable over Leeman et al. (Science 359.6381 (2018): 1277-1283) in view of Cao et al. (Experimental Hematology 43.3 (2015): 229-242) and Grassinger et al. (. This is a new rejection necessitated by Applicants' amendments to the claims in the response filed on October 16, 2025.
Claim 1 is directed to a method of culturing quiescent hematopoietic stem cells, said method comprising:
providing a serum-free culture medium comprising a vacuolar-H+ adenosine triphosphate ATPase (v-ATPase) inhibitor;
introducing into the serum-free culture medium quiescent hematopoietic stem cells, wherein the stem cells are mitochondrial membrane potential (MMP)-low, lineage negative (Lin-) stem cell antigen 1 positive (Sca+), cKit+, Signaling Lymphocyte Activation Molecule 1 positive (CD 150+), and Signaling Lymphocyte Activation Molecule 2 negative (CD48-) cells; and
culturing the quiescent hematopoietic stem cells in the serum-free culture medium to produce cultured quiescent hematopoietic stem cells, wherein said culturing is effective to maintain and promote the quiescence of the cultured quiescent hematopoietic stem cells.
Claim 11 is an isolated population of quiescent hematopoietic stem cells obtained from the method of claim l.
Regarding claims 1 and 11, Leeman discloses a method of culturing quiescent stem cells, said method comprising: providing a culture medium and introducing into the culture medium quiescent stem cells to culture the stem cells and maintain quiescence of the stem cells, wherein the culture medium comprises a vacuolar-H+ adenosine triphosphate ATPase ("v-ATPase") inhibitor (abstract; pg 3, col 3, para 2: “Treatment with bafilomycin A, which blocks lysosomal acidification, led to increased accumulation of protein aggregates (Fig. 3, A and B, and fig. S7A) and to decreased activation in response to growth factors (Fig. 3)”; pg 4, “Fig. 3. Modulation of lysosomal activity in qNSCs affects protein aggregates and the transition from quiescence to activation. (A) Representative immunofluorescence images of primary cultures of qNSCs from 3-month-old mice after 18 hours of treatment with 50 nM bafilomycin A (BafA, a lysosomal V-ATPase inhibitor) or 3 hours of nutrient deprivation in HBSS”).
Leeman teaches the ability of quiescent stem cells to activate declines with age, and that increased lysosomal function helps promote the activation of older quiescent stem cells wherein protein aggregates are cleared (fig. S9H, provided below). The findings revealed an enlarged lysosome within quiescent neural stem cells that contain a large amount of insoluble protein aggregates, wherein the clearance of these aggregates by lysosomal activation may provide a burst of energy for activation (p 6, col 1, par 3). Lastly, Leeman acknowledges that changes in autophagy during aging have been observed in hematopoietic stem cells (p 6, col 2, par 1).
Fig S9H:
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Leeman does not teach the stem cells are hematopoietic stem cells.
Leeman does teach quiescent hematopoietic stem cells have similar mechanisms with quiescent neural stem cells (pg 1, col 3, para 2: “Similar differences in branches of the proteostasis network were observed between the transcriptomes of quiescent and activated stem cells of other tissues (muscle and hematopoietic) (fig. S4F)” (Figure below).
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Secondly, Leeman does not teach culturing quiescent hematopoietic stem cells in serum-free medium, and furthermore wherein the stem cells are mitochondrial membrane potential (MMP)-low, lineage negative (Lin-), stem cell antigen 1 positive (Sca+), cKit+, Signaling Lymphocyte Activation Molecule 1 positive (CD150+), and Signaling Lymphocyte Activation Molecule 2 negative (CD48-) cells.
Cao et al. (Experimental Hematology 43.3 (2015): 229-242) teaches “Autophagy is implicated in hematopoiesis, … Here, we show that autophagy is essential to maintain and regulate the cell cycle of HSPCs in a nutrient-dependent manner. The loss of autophagy via conditional deletion of its essential gene atg7 ablated the cell cycle of HSPCs (abstract); “The quiescent state, described as the G0 phase, is characterized by a diploid DNA content and a low RNA content.” (p 232, col 1, par 2); “Cytometric analysis showed that autophagy inactivation via the conditional knockout of atg7 (atg7−/−) increased the percentage of HPCs at the G0/1 phase and reduced the percentage of HPCs in the S phase, whereas the percentage of HPCs in G2/M phase was not significantly altered (Fig. 1C).” (p 232, col 1, par1).
Cao teaches the culturing of HSC, specifically LSK HSC (lineage negative (Lin-) stem cell antigen 1 positive (Sca+), cKit+) cells in culture media supplemented with a vacuolar-H+ adenosine triphosphate ATPase ("v-ATPase") inhibitor, e.g. bafilomycin A1 (p 230, col 2, par 4- p 231, col 1, par 1; p 229, col 2). The observed outcome was described as, “the inhibition of starvation-induced autophagy at a late stage, namely autolysosome formation, with bafilomycin A1 did not affect the percentage of G0/1 or S phase HSPCs in response to starvation (Fig. 4C). Taken together, these data indicate that the inhibition of starvation induced autophagy at an early, but not late, stage enhances the G1/S transition of HSPCs, suggesting that the early signaling events in autophagy may play a role in limiting the G1/S transition of these cells under conditions of starvation.” (p 239, col 1). Altogether, v-ATPase inhibitor revealed that the HSPC did not become activated and depart from quiescence that would occur with G1/S transition.
The instant disclosure describes “the majority (approximately 75%) of (LSK CD150+CD48-) HSCs contain active mitochondria (primed HSC). In addition, using a combinatorial approach that includes single-cell transcriptomics and high-resolution confocal imaging, it is shown that most, if not all, of HSCs' attributes (including self-renewal) segregate with the minor (< 25%) subpopulation that display relatively low mitochondrial membrane potential (MMP; quiescent HSCs).…MMP-low HSCs, on the other hand, are shown to be enriched in lysosomes that maintain their quiescence.” (par 0017). Based on this understanding, Leeman teaches MMP-low stem cells based on describing qNSCs as having a larger quantity and relative sized lysosomes in comparison to activated stem cell (Fig. 1F and fig. S5B); and therefore are likely MMP low.
Additionally, Cao teaches “The LSK HSC compartment is heterogeneous, consisting of at least two functionally distinct HSC subpopulations, namely long-term and short-term repopulating HSCs (LT-HSCs and ST-HSCs). Long-term repopulating HSCs have extensive self-renewing potential and, upon commitment, give rise to ST-HSCs with more restricted self-renewing capacity that differentiate to lineage-committed hematopoietic progenitor cells (HPCs), primarily in the Lin−c-Kit+ (LK) compartment”. It can be understood that the MMP low HSC described herein are considered LT-HSC wherein the attribute of self-renewal is conserved as supported by the instant disclosure.
Leeman in view of Cao do not teach culturing quiescent hematopoietic stem cells in serum-free medium, and furthermore wherein the stem cells are Signaling Lymphocyte Activation Molecule 1 positive (CD150+), and Signaling Lymphocyte Activation Molecule 2 negative (CD48-) cells.
Grassinger teaches that “analyzed subpopulations of LSK cells isolated from different regions of the BM and showed that CD150+CD48-LSK HSCs within the endosteal BM region have superior proliferative capacity and homing efficiency compared with CD150+CD48-LSK HSCs isolated from the central BM” (abstract). Grassinger further teaches that LSK cells are enriched for HSPCs, and furthermore employed the SLAM markers (CD150+CD48-) to ensure accurate isolation of long-term (LT) HSCs, which have multilineage engraftment potential and unlimited population self-renewal potential, and are quiescent (p 3185, col 1; p 3187, col 1-2). The CD150+CD48-LSK HSCs were cultured in serum-free medium (p 3187, col 1-2).
It would have been prima facie obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have substituted the neural stem cells with hematopoietic stem cells for culturing in serum-free culturing medium with a vacuolar-H+ adenosine triphosphate ATPase ("v-ATPase") inhibitor with the same predictable result of maintaining quiescence.
This reasoning is based on Leeman acknowledging similar proteolytic mechanisms with hematopoietic stem cells, and furthermore, Cao clearly employing a v-ATPase inhibitor with HSC wherein there was no change observed in cell cycle transitions, and lastly the suppression of autophagy via gene knockout revealed an increased percentage of HPCs at the G0/1 phase wherein the G0 phase is characterized as a quiescent state.
In reference to the CD150+CD48-LSK HSCs, it would be obvious to further substitute the HSC of Cao with the CD150+CD48-LSK HSCs taught by Grassinger based on these cells being described as the best representation of quiescent LT-HSC found in the bone marrow, and therefore, are more likely to maintain the quiescent state after treatments with a v-ATPase inhibitor. Moreover, Grassinger provides clear teachings on culturing these cells in serum-free medium, and as such it would be obvious to employ this known technique to obtain the predictable outcome of proliferated HSCs.
Regarding claim 3, dependent on claim 1, Leeman teaches wherein the stem cells are mammalian stem cells (“As lysosomes are central hubs for key cellular trafficking, signaling, and metabolic pathways, targeting of lysosome biogenesis and/or activation to clear aggregates in qNSCs may provide broad strategies to allow activation of old mammalian cells in vivo.” (p 6, col 2, par 1)).
Regarding claim 4, dependent on claim 1, Leeman and Cao employ murine stem cells (Leeman: p 5, col 2; Cao: p 230, col 2, par 4). Furthermore, Cao teaches human HSCs in relation to entering the cell cycle for HSC maintenance. Therefore, it would have been obvious to substitute the mouse HSCs with human HSCs as these are readily known in the art and have been used in cell culturing methods.
Regarding claim 5, dependent on claim 1, Cao teaches wherein the stem cells are peripheral blood cells, cord blood cells, bone marrow cells, amniotic fluid cells, placental blood cells, aorta-gonad mesonephros (AGM), or mixtures thereof (“Hematopoietic repopulation was determined with flow cytometry based on CD45.1+/CD45.2+ population frequency of donor-derived cells in peripheral blood and bone marrow of the recipient mice 4 weeks after transplantation.” (p 231, col 2)).
Regarding claim 7, dependent on claim 1, Leeman teaches wherein the v-ATPase inhibitor is selected from the group consisting of bafilomycin Al, bafilomycin Bl, bafilomycin Cl, bafilomycin D, concanamycin A, concanamycin C, disulfiram, and combinations thereof as seen in the claim 1 rejection above wherein Leeman employs bafilomycin A1.
Regarding claim 8, dependent on claim 1, Cao teaches the culturing of HSCs with Flt-3 and SCF (p 231, col 1, par 1).
Regarding claims 9 and 10, dependent on claim 1, if the quiescent hematopoietic stem cells prior to contact with the ATPase inhibitors are being described, it would have been obvious to select stem cells wherein at least 90% or 99% of the stem cells are in G0 phase. This is based on Cao teaching that the G0 phase is considered the quiescent stage, as seen in the claim 1 rejection, and starting with a high cell population in a quiescent state would be preferable for a method directed to culturing quiescent stem cell.
In the case that these stem cells are produced by the method of claim 1, there is a reasonable expectation that methods of Leeman in view of Cao would lead to at least 90% or 99% of the stem cells being in the G0 phase, based on this method being taught by Cao in which HSCs are introduced to Bafilomycin A1 for which the instant similarly employs for autophagy inhibition.
Response to Applicants' Arguments as they apply to rejection of the claims under 35 USC § 103
Starting on page 4 of the remarks filed on October 16, 2025, Applicants essentially argue the following:
In relation to claim 1, Applicants' state:
“First, Leeman does not acknowledge that neural stem cells and hematopoietic stem cells have similar proteolytic mechanisms. Instead, as noted above, Leeman states that studies observing autophagy during aging of hematopoietic stem cells did not examine the lysosomes and their protein aggregate content during aging. To the extent Leeman describes "[s]imilar differences in branches of the proteostasis network were observed between the transcriptomes of quiescent and activated stem cells of other tissues (muscle and hematopoietic)," Leeman concludes that "quiescent and activated stem cells mobilize different branches of the protein quality-control network." Leeman, p. 1277, first full paragraph in column 3.”
“Second, Cao does not cure the deficiencies of Leeman with respect to claim 1. Specifically, Cao does not examine lysosomes or their protein aggregate content during aging. Instead, Cao is interested in studying the role of autophagy in the regulation of the hematopoietic stem and progenitor cell cycle. See Cao, paragraph spanning pp. 231-232.”
“Neither Leeman, Cao, or the combination of those references teach or suggest culturing a population of hematopoietic stem cells in a serum free culture medium, wherein the quiescent hematopoietic stem cells are MMP-low LSK CD150+/cD4S-, wherein said culturing is effective to maintain and promote the quiescence of the cultured quiescent hematopoietic stem cells, as required by amended claim 1.”
In response to the argument it has been fully considered but is not persuasive due to the following reasons:
Regarding the first presented argument, Leeman describes “qNSCs and aNSCs also expressed different types of genes that encode protein chaperones (Fig. 1D, fig. S4E, and table S6): endoplasmic reticulum (ER) unfolded protein response genes were more highly expressed in qNSCs, whereas the TCP-1 ring complex (TRiC) and prefoldin complex genes were more highly expressed in aNSCs (fig. S4E).” and then concludes that the proteostatis networks differences between those activated versus quiescent stem cells are similar with other tissues, i.e. HSC, as seen in Fig. S4E listed above. Therefore, there is clear motivation to substitute the NSC that have been shown to maintain quiescence with a v-ATPase inhibitor with that of the claimed CD150+CD48-LSK HSCs. Leeman further describes enlarged lysosomes with insoluble aggregates found with quiescent NSC, and that clearance of these proteins leads to activation. Cao further teaches that the inhibition of autophagy by lysosomes via bafilomycin, a v-ATPase inhibitor, does not increase the number of activate HSC. Therefore, it is clear that it would be obvious to use a v-ATPase inhibitor to obtain the predictable outcome of maintaining the quiescent state of CD150+CD48-LSK HSCs.
Regarding the second presented argument, Cao clearly states, “Surprisingly, however, the inhibition of starvation-induced autophagy at a late stage, namely autolysosome formation, with bafilomycin A1 did not affect the percentage of G0/1 or S phase HSPCs in response to starvation (Fig. 4C).”; “We used bafilomycin A1, an inhibitor of autophagic degradation in autolysosomes, (p 239, col 1-2). Therefore, it is clear Cao does acknowledge lysosomes, and in view of autophagy for which it is known in the art that lysosomes participate in. Lastly, Cao not teaching on protein aggregate content during aging, does not render the render it improper to combine with Leeman. The motivation to combine these references is outlines above, and moreover Cao teaches the majority the claim in contacting HSC with a v-ATPase inhibitor for which the claim is directed.
Regarded the third presented argument, the prior rejection was withdrawn in view of the amendments to the claims. The new ground of rejection rely on Leeman in view of Cao and now Grassinger.
The Grassinger reference teaches the claimed CD150+CD48-LSK HSCs, and it is noted that Cao teaches LSK HSCs, but not the CD150+CD48- markers, also known as SLAM markers. In reference to the MMP low marker, the rejection above describes that there is an expectation that the HSC in view of Leeman and Cao are considered MMP low based on the instant Specification describing MMP low cells as having enriched lysosomes for which Leeman teaches for NSC cells. Furthermore, based on the Specification stating, “HSCs' attributes (including self-renewal) segregate with the minor (< 25%) subpopulation that display relatively low mitochondrial membrane potential (MMP; quiescent HSCs (par 0017) there is reasonable expectation that the LT-HSC that comprise the LSK HSC taught by Cao are MMP low based on having the same self-renewal characteristics. Altogether, the MMP low characteristics is understood as being inherent to the LT-HSC described in the prior art references.
Conclusion
Claims 1, 3-5, 7-11 are rejected. No claims are 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 ANGELO RIGA/Examiner, Art Unit 1634
/TERESA E KNIGHT/Primary Examiner, Art Unit 1634