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 .
Response to Amendment/Status of Claims
Receipt of Arguments/Remarks filed on 12/12/2025 is acknowledged. Claim 2 was cancelled. Claims 1,5,6,18 and 19 were amended. Claims 1 and 3-21 are pending and under examination.
Applicant elected the combination of miR-486 and miR-22 for Species A without traverse, and SNORD29 and SNORD68 for Species B in the reply filed on 07/14/2025 and the examiner withdrew the election requirement for Species B in the office action dated 08/20/2025.
The following rejections and/or objections are either reiterated or newly applied and necessitated by amendment. They constitute the complete set presently being applied to the instant application.
Response to Arguments
Applicant’s arguments and amendments, see page 6, filed 12/12/2025, with respect to the objection to the specification containing an embedded hyperlink, and objection to FIG. 9 for referring to colors in the images, have been fully considered and are persuasive due to the amendments to the specification removing the embedded hyperlink and amendments to the description of FIG. 9 removing the recitation of colors. The objection to the specification and drawings has been withdrawn.
Applicant’s arguments and amendments, see pages 6-7, filed 12/12/2025, with respect to the 35 U.S.C. 112(a) Written Description rejection of claims 1-21 have been fully considered and are persuasive, due to the amendments to claims 1 and 18 reciting the HSPCs are CD34+ and the small RNAs comprise miR-486 and miR-22. The 35 U.S.C. 112(a) Written Description rejection of claims 1-21 has been withdrawn.
Applicant’s arguments and amendments, see pages 7-8, filed 12/12/2025, with respect to the 35 U.S.C. 112(a) Scope of Enablement rejection of claims 1-21 have been fully considered and are persuasive, due to the amendments to claims 1 and 18 reciting the HSPCs are CD34+ and the small RNAs comprise miR-486 and miR-22. The 35 U.S.C. 112(a) Scope of Enablement rejection of claims 1-21 has been withdrawn.
Applicant’s arguments and amendments, see page 8, filed 12/12/2025, with respect to the 35 U.S.C. 102(a)(1) rejection(s) of claim(s) 1,2,9 and 13 as anticipated by Qu et al. as evidenced by Mayani et al. have been fully considered and are persuasive due to the amendments to claim 1 to require the HSPCs to be CD34+ and the one or more small RNAs to comprise miR-486 and miR-22 which are not taught by Qu et al. as evidenced by Mayani et al. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of the amendment to claim 1 for a case of obviousness. See the 103 rejection below.
Applicant’s arguments and amendments, see page 9, filed 12/12/2025, with respect to the 35 U.S.C. 102(a)(1) rejection(s) of claim(s) 18 and 20 as anticipated by Papoutsakis et al. have been fully considered and are persuasive due to the amendments to claim 18 to require the HSPCs to be CD34+ and the one or more small RNAs to comprise miR-486 and miR-22 which are not taught by Papoutsakis et al. Therefore, the rejection has been withdrawn. However, upon further consideration, a new ground(s) of rejection is made in view of the amendment to claim 18 for a case of obviousness. See the 103 rejection below.
Applicant’s arguments and amendments, see pages 9-10, filed 12/12/2025, with respect to the 35 U.S.C. 103 rejections of claims 3,11 and 12 as obvious over Qu et al. as evidenced by Mayani et al. and further in view of Papoutsakis et al., claim 4 as obvious over Qu et al. as evidenced by Mayani and further in view of Scadden, have been fully considered and are persuasive due to the amendments to claim 1 to require the HSPCs to be CD34+ and the one or more small RNAs to comprise miR-486 and miR-22 which are not taught by the cited references. Therefore, the above rejections have been withdrawn. However, upon further consideration, new ground(s) of rejections are made in view of the amendment to claim 1 for a case of obviousness. See the 103 rejections below.
Applicant’s arguments and amendments, see pages 11-12, filed 12/12/2025, with respect to the 35 U.S.C. 103 rejection of claims 5 and 6 as obvious over Qu et al. as evidenced by Mayani and further in view of Bianchi and Weiss has been fully considered and is persuasive due to the amendments to claim 5 removing miR-486 and miR-22 from the list of small microRNAs and therefore the cited references do not teach the remaining miRs. Therefore, the 35 U.S.C. 103 rejection of claim 5 has been withdrawn, however a new ground of rejection is made in view of the amendments to claim 5, removing miR-486 and miR-22 and adding “further”, to require the additional miRs recited in addition to miR-486 and miR-22 now required by amended claim 1. See the new 103 rejection for claim 5 below. However, claim 6 remains rejected under 35 U.S.C. 103 as obvious over Qu et al. as evidenced by Mayani et al. further in view of Bianchi et al. and Weiss et al. The response to arguments follows.
Applicant argues on page 12 of response that Bianchi and Weiss do not disclose transferring into CD34+ HSPCs a combination of miR-486 and miR-22 in an effective amount for inducing megakaryocytic differentiation of the HSPCs without MkMPS and therefore Bianchi and Weiss do not cure the deficiencies of Qu. Applicant argues Qu as evidence by Mayani, Bianchi, and Weiss do not teach or suggest each and every limitation of independent claim 1 and dependent claims 5 and 6, for example the references do not disclose transferring into CD34+ HSPCs a combination of miR-486 and miR-22 in an effective amount of inducing megakaryocytic differentiation of the HSPCs without MkMPs, and as discussed above, the method of claim 4 is not obvious in view of the unexpected synergistic effect of the combination of miR-486 and miR-22 on induction of Mk differentiation of the HSPCs.
This is not found persuasive, as while Bianchi and Weiss do not disclose transferring into CD34+ HSPCs a combination of miR-486 and miR-22, the examiner cited Qu et al. as the base reference for transferring into CD34+ HSPCs an miR agent for differentiation into MKs and modified the method of Qu et al. with the teachings of Bianchi and Weiss regarding motivation for transferring miR-486 and miR-22 into the CD34+ cells of Qu et al. See MPEP 2145 IV. One cannot show nonobviousness by attacking references individually where the rejections are based on combinations of references. In re Keller, 642 F.2d 413, 208 USPQ 871 (CCPA 1981); In re Merck & Co., Inc., 800 F.2d 1091, 231 USPQ 375 (Fed. Cir. 1986). Where a rejection of a claim is based on two or more references, a reply that is limited to what a subset of the applied references teaches or fails to teach, or that fails to address the combined teaching of the applied references may be considered to be an argument that attacks the reference(s) individually. Where an applicant’s reply establishes that each of the applied references fails to teach a limitation and addresses the combined teachings and/or suggestions of the applied prior art, the reply as a whole does not attack the references individually as the phrase is used in Keller and reliance on Keller would not be appropriate. This is because "[T]he test for obviousness is what the combined teachings of the references would have suggested to [a PHOSITA]." In re Mouttet, 686 F.3d 1322, 1333, 103 USPQ2d 1219, 1226 (Fed. Cir. 2012).
Regarding the effective amount, the amount of a specific ingredient in a composition is clearly a result effective parameter that a person of ordinary skill in the art would routinely optimize. Optimization of parameters is a routine practice that would be obvious for a person of ordinary skill in the art to employ and reasonably would expect success. It would have been customary for an artisan of ordinary skill to determine the optimal amount of each miR to add in order to best achieve the desired results such as to improve induction of megakaryocytic differentiation. It would have been obvious to one of ordinary skill in the art at the time of the invention to engage in routine experimentation to determine optimal or workable ranges that produce expected results. Where the general conditions of a claim are disclosed in the prior art, it is not inventive to discover the optimum or workable ranges by routine experimentation. In re Aller, 220 F. 2d 454, 105 USPQ 233 (CCPA 1955). NOTE: MPEP 2144.05.
Regarding Applicant’s argument that the method of claim 4 is not obvious in view of the unexpected synergistic effect of the combination of miR-486 and miR-22 on induction of Mk differentiation of the HSPCs, this argument is provided under the response to the rejection of claims 5 and 6, and claim 4 has different limitations than claims 5 and 6, therefore, the examiner is unsure what Applicant is trying to argue for claim 4 regarding claims 5 and 6.
Applicant did argue unexpected results on page 10 of the response under the response to the rejection of claims 3,11 and 12, that the experimental results on pages 17,18 and 22 demonstrate unexpected synergistic effects of miR-486a-5p and miR-22-3p on Megakaryocytic differentiation of CD34+ HSPCs, including Mk maturation (FIGs. 7C,7D), Mk cell expansion (FIG. 7E), total cell expansion (FIG. 7F), and in view of the unexpected synergistic effect of the combination of miR-486 and miR-22 on induction of MK differentiation of the HSPCs the method of claim 1 or any one of the dependent claims 3,11 and 12 is not obvious.
This is not found persuasive, as it does not appear that the results show unexpected synergistic effects. Also, it is noted that page 17, lines 30-31 and page 22, lines 26-29 states that additional enriched miRs are needed for the full effect of TPO, and only claim 5 recites further miRs in addition to miR-486 and miR-22. Any differences between the claimed invention and the prior art may be expected to result in some differences in properties. The issue is whether the properties differ to such an extent that the difference is really unexpected. An unexpected property or result must actually be unexpected and of statistical and practical significance. The burden is on the applicant to establish the results are in fact unexpected, unobvious and of statistical and practical significance. See MPEP 716.02.
Regarding unexpected synergist effects, see MPEP 716.02(a). “Where the combined action of two or more agents is greater than the sum of the action of one of the agents used alone, “synergy” according to the legally accepted definition exists. Regarding the unexpected synergistic results, claims drawn to (unexpectedly) synergistic combinations of known ingredients must be factually supported by data commensurate in scope with the claims. See, In re Kollman, 201 USPQ 193 (C.C.P.A. 1979). (The court affirming a 103 rejection of a claim containing the word “synergistic”, because the claims were not commensurate in scope with the showing of unexpected results, other than at 1:1 ratio for certain specific combinations). Evidence of a greater than expected result may also be shown by demonstrating an effect which is greater than the sum of each of the effects taken separately (i.e., demonstrating "synergism"). Merck & Co. Inc. v. Biocraft Laboratories Inc., 874 F.2d 804, 10 USPQ2d 1843 (Fed. Cir.), cert. denied, 493 U.S. 975 (1989). However, a greater than additive effect is not necessarily sufficient to overcome a prima facie case of obviousness because such an effect can either be expected or unexpected. Applicants must further show that the results were greater than those which would have been expected from the prior art to an unobvious extent, and that the results are of a significant, practical advantage. Ex parte The NutraSweet Co., 19 USPQ2d 1586 (Bd. Pat. App. & Inter. 1991).
In the instant case, the results do not show synergistic effects as defined by MPEP 716.02(a) above (the combined effect is greater than the sum of each of the effects taken separately). FIG. 7A,7B,7C, 7D,7E and 7F do not show an actual synergistic effect of miR-486+22, as the synergistic effect as defined above must show each individual effect and that the combined effect is greater than the additive effect (sum of each of the effects taken separately). While the results in the above figures show that the combined effect of miR-486+22 is greater than each individual effect, it does not show an effect that is greater than the additive effect and therefore does not show unexpected synergistic results.
No response or argument from Applicant was provided for the 35 U.S.C. 103 rejection of claim 10 as unpatentable over Qu et al. as evidenced by Mayani et al. and further in view of Georgantas et al.; claims 14-17 as unpatentable over Qu et al. as evidence by Mayani et al. and further in view of Cornejo et al.; claim 19 as unpatentable over Papoutsakis et al. and further in view of Bianchi et al.; and claim 21 as unpatentable over Papoutsakis et al. and further in view of Qu et al. as evidenced by Mayani et al. The 35 U.S.C. 103 rejections above have been withdrawn for these claims due to the amendments to claims 1 and 18 and new 103 rejections provided based on the amendments to claim 1 and 18 requiring the HSPCs to be CD34+ and the small RNAs to comprise miR-486 and miR-22. See the 103 rejections below.
Priority
This application is a 371 of PCT/US2020/056593, filed 10/21/2021, which claims benefit of 62/923,841, filed 10/21/2019, as reflected on the filing receipt dated 08/19/2022.
Claim Objections
Claim 5 is objected to because of the following informalities: line 3 recites “miR-92” twice. Appropriate correction is required.
Claim Rejections - 35 USC § 112
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 and 3-21 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 1 recites “transferring into the HSPCs an effective amount of one or more small RNAs”….”and wherein the one or more small RNAs comprise miR-486 and miR-22”. Claim 6 recites “wherein the one or more small RNAs consist of miR-486 and miR-22”. It is unclear how the claims could only have one small RNA, as the claims as amended in claim 1 require at least both of miR-486 and miR-22, and claim 6 as amended requires the small RNAs consist of miR-486 and miR-22 and therefore requires only those two small RNAs and no additional small RNAs.
Claims 5 and 7-13 likewise each recite “the one or more small RNAs” and are indefinite as the claims require two small RNAs (miR-486 and miR-22) but could include more. Therefore, these claims are indefinite as it is not clear how there could be only one small RNA.
Claims 3,4 and 14-17 depend from claim 1 and do not correct the issue and are therefore included in the rejection.
Claim 18 recites “an effective amount of one or more exogenous small RNAs”…”and wherein the one or more exogenous small RNAs comprise miR-186 and miR-22”. Claim 19 recites “wherein the one or more exogenous small RNAs consist of miR-486 and miR-22”. It is unclear how the claims could only have one small RNA, as the claims as amended in claim 18 require at least both of miR-486 and miR-22, and claim 19 as amended requires the exogenous small RNAs consist of miR-486 and miR-22 and therefore requires only those two exogenous small RNAs and no additional small RNAs.
Claims 20 and 21 likewise recite “the one or more exogenous small RNAs”, and are indefinite as the claims require two exogenous small RNAs (miR-486 and miR-22) but could include more, and it is unclear how there could be only one exogenous small RNA.
Claim Rejections - 35 USC § 103
The text of those sections of Title 35, U.S. Code not included in this action can be found in a prior Office action.
Claim 1,6,9 and 13 are rejected under 35 U.S.C. 103 as being unpatentable over Qu et al. (Cell Death and Disease (2016)7,e2430), cited on an IDS, as evidenced by Mayani et al. (Stem Cells 1998:16:153-165), cited on an IDS and further in view of Bianchi et al. (Cell Death and Differentiation, 2015; 22, 1906-1921) cited on an IDS, and Weiss et al. (Blood Advances, 8 Jan 2019, Vol. 3, No. 1, pages 33-46).
Regarding claim 1, Qu et al. teach growing CB mononuclear cells in megakaryocytic differentiation medium and transfecting on day 1 with miR-125b mimics, and that transfection of miR-125b mimics increased miR-125b levels ~30 fold compared with negative control mimics (page 2, bottom right column- page 3 left column). Qu et al. teach microRNAs are small non-coding RNA molecules (page 1, left column) and that miR-125b is a small RNA as shown in Fig. 6a. Qu et al. teach that overexpression of miR-125b increases MK differentiation of MNCs (page 5, left column). Regarding the claimed limitation “without megakaryocytic microparticles”, Qu et al. teach the umbilical cord CB MNCs were grown in megakaryocytic differentiation medium (StemSpan Defined Medium in the presence of a cytokine cocktail containing recombinant human TPO, stem cell factor, interleukin-3 rhL-6 and rhL-11) (page 13, left column, 5th paragraph), and therefore teaches the method without MkMPs. As evidenced by Mayani et al., cord blood is a rich source of hematopoietic stem/progenitor cells HSPC (Abstract). Qu et al. teach CD34+ is a positive marker of undifferentiated hematopoietic progenitor stem cells HSCs and also of cord blood CB hematopoietic cells (page 2, left column 2nd paragraph and right column first paragraph). Qu et al. teach compared with CD34+ HSCs, they found an enrichment (10-fold) of miR-125b in CD41hiCD61hi late state MKs, suggesting the miR-125b might be involved in the common process of MK terminal differentiation (page 2, bottom left column, Fig 1d), and that CD34+ hematopoietic cells were differentiated to MKs by culturing in a megakaryocytic differentiation medium (Figure 1(a) page 3).
Qu et al. do not teach the small RNAs are miR-486 and miR-22.
However, before the effective filing date, Bianchi et al. teach miR-486 plays a key role in differentiation of CD34+ hematopoietic progenitor cells HPCs toward erythroid versus megakaryocyte lineage by supporting the erythropoiesis while restraining the megakaryopoiesis and reducing the abnormal expansion of megakaryocytopoiesis (Abstract, page 1906, first column, second paragraph; page 1908, second column, sixth and eighth paragraphs).
Additionally, Weiss et al. teach miR-22 is upregulated in megakaryocytes, and upregulation is a critical step in megakaryocyte differentiation (Abstract). Weiss et al. teach microRNAs are small single-stranded RNAs, and that only a small number of miRNAs have been shown to positively contribute to MK differentiation (page 34, left column). Weiss et al. teach that miR-22 is dramatically upregulated upon megakaryocytic differentiation in adult mice and in cell lines, megakaryocytic differentiation is driven by overexpression of miR-22 and is inhibited by its loss, and the role of miR-22 in the process is mediated through direct targeting of the zinc-finger-transcriptional repressor GFI1 (Page 34, left column).
Regarding claim 9, Qu et al. teach transfecting CB mononuclear cells in megakaryocytic differentiation medium with miR-125b mimics, and therefore teaches the small RNA is synthetic (page 2, bottom right column- page 3 left column).
Regarding claim 13, Qu et al. teach transfecting CB mononuclear cells with miR-125b mimics, and that transfection of miR-125b mimics increased miR-125b levels ~30 fold compared with negative control mimics (page 2, bottom right column- page 3 left column).
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filing date, to have modified the method of Qu et al. as evidenced by Mayani et al, to include transfection of miR-486 and miR-22 into the CD34+ HSPCs based on the teachings of Bianchi et al. and Weiss et al. with a reasonable expectation of success. There would be a reasonable expectation of success because both Qu et al. and Bianchi et al. disclose the differentiation of CD34+ HSPCs into megakaryocytes and erythroids and Bianchi et al. and Weiss et al. pertains to megakaryocytic differentiation. One of ordinary skill in the art would have been motivated to modify the method of Qu et al. as evidence by Mayani et al, and to provide the miR-486 of Bianchi et al. and transfect into the CD34+ HSPCs of Qu et al., in order to redirect differentiation of the CD34+ HSPCs into cells other than megakaryocytes to avoid abnormal expansion of megakaryocytopoiesis, and to provide and transfect the miR-22 of Weiss et al. into the CD34+ HSCPs of Qu et al. in order to drive megakaryocytic differentiation.
Accordingly, claims 1,9 and 13 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
It would have been obvious to a person of ordinary skill in the art before the effective filing date, to have modified the method of Qu et al. as evidenced by Mayani et al, to swap transfection of the miR-125b mimic of Qu et al. and replace with the miR-486 of Bianchi et al. and miR-22 of Weiss et al. into the CD34+ HSPCs of Qu et al. with a reasonable expectation of success. There would be a reasonable expectation of success as this amounts to simple substitution of one known element for another to obtain predictable results and because both Qu et al. and Bianchi et al. disclose the differentiation of CD34+ HSPCs into megakaryocytes and erythroids and Bianchi et al. and Weiss et al. pertain to megakaryocytic differentiation. One of ordinary skill in the art would have been motivated to modify the method of Qu et al. as evidenced by Mayani et al, and to swap the miR-125b mimic with the miR-486 of Bianchi et al. and transfect into the CD34+ HSPCs of Qu et al., in order to redirect differentiation of the CD34+ HSPCs into cells other than megakaryocytes to avoid abnormal expansion of megakaryocytopoiesis, and to provide and transfect the miR-22 of Weiss et al. into the CD34+ HSCPs of Qu et al. in order to drive megakaryocytic differentiation.
Accordingly, claim 6 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claims 3,11 and 12 are rejected under 35 U.S.C. 103 as being unpatentable over Qu et al. as evidenced by Mayani et al. in view of Bianchi et al. and Weiss et al. as applied to claims 1,6,9 and 13 above, and further in view of Papoutsakis et al. (US 20170058262, Published 2 March 2017).
The teachings of Qu et al. as evidenced by Mayani et al., Bianchi et al. and Weiss et al. as applicable to claims 1,6,9 and 13 are described above.
Qu et al., Mayani et al., Bianchi et al. and Weiss et al. do not teach wherein the HSPCs differentiate into megakaryocytes in vitro in the absence of thrombopoietin (TPO) and do not teach transferring the small RNAs into the HSPCs via cellular particles.
However, before the effective filing date, Papoutsakis et al. teach differentiation of HSPCs to megakaryocytes using small RNA (miRNA) (paragraph 0085). Papoutsakis et al. teach hematopoietic stem cells were co-cultured with or without microparticle CMPs and without thrombopoietin to induce Mk differentiation of hematopoietic stem cells (paragraph 0046).
Papoutsakis et al. teach coculture of megakaryocytic microparticles MkMPs with hemoatopoeitic stem and progenitor cells (HSPCs) promoted HSPC differentiation to mature Mks, and that MkMPs can be loaded with desirable molecules for delivery to HSPCs with effectiveness and specificity (paragraph 0017). Papoutsakis et al. teach MkMPs can be used as a means to modify in vitro or in vivo hematopoietic stem and progenitor cells by transferring specific nucleic acids (RNA or DNA molecules) to these cells (paragraphs 0018,0020), and loading MkMPs with exogenous RNA for delivery to target cells (paragraph 0023). Papoutsakis et al. teach MkMPs promote Mk differentiation of CD34+ cells and HPCs, and CD34+ cells were cocultured with our without MkMPs in a medium without TPO (paragraph 0035). Papoutsakis et al. teach MkMPs promote Mk differentiation through transfer of the RNA carried by the MkMPs, and that several signaling molecules carried by the MPs are miRNA (paragraph 0085).
Papoutsakis et al. teach that multiple sources of HSPCs include CD34+ cells from bone marrow, peripheral blood or cord blood are cultured and differentiated to megakaryocytes (paragraph 0107). Papoutsakis et al. recite and teach a method to generate particles comprising culturing cells selected from the group of megakaryocytes and immature megakaryocyte cells, providing said cultured cells to an exposure of biomechanical stress (claims 1-2), isolating the particles by pelleting via a centrifuge (claim 10), and loading of desirable RNAs inside the MkMPs using electroporation (paragraph 0107) and that the RNA includes miRNA (paragraph 0085).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to modify the method of Qu et al. as evidenced by Mayani et al. in view of Bianchi et al. and Weiss et al. by removing TPO from the cytokine cocktail in the culture medium and therefore wherein the cells differentiate in the absence of thrombopoietin as taught by Papoutsakis et al., with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to do so in order to adjust the conditions of the differentiation of the hematopoietic stem cells and to adjust the properties of the produced megakaryocytes, and determine the effect thereof. There would be a reasonable expectation of success because both Qu et al. and Papoutsakis et al. pertain to differentiation of CD34+ HSPCs into megakaryocytes under the influence of small RNA, and Bianchi et al. disclose the differentiation of CD34+ HSPCs into megakaryocytes and erythroids and Weiss et al. also pertains to megakaryocytic differentiation, and would make obvious the limitations of claim 3.
It would have been obvious to one of ordinary skill in the art before the effective filing date, to modify the method of Qu et al. as evidenced by Mayani et al. in view of Bianchi et al. and Weiss et al., based on the teachings of Papoutsakis et al. to transfer the small RNA into the CD34+ HSPCs via cellular particles with a reasonable expectation of success. There would be a reasonable expectation of success because both Qu et al. and Papoutsakis et al. pertain to differentiation of CD34+ HSPCs into megakaryocytes under the influence of small RNA. One of ordinary skill in the art would have been motivated to transfer the small RNA into the HSCPs via cellular particles, because Papoutsakis et al. teach coculture of megakaryocytic microparticles MkMPs with hemoatopoeitic stem and progenitor cells (HSPCs) promoted HSPC differentiation to mature Mks, that MkMPs can be loaded with desirable molecules for delivery to HSPCs with effectiveness and specificity (paragraph 0017) and that MkMPs can be used as a means to modify in vitro or in vivo hematopoietic stem and progenitor cells by transferring specific nucleic acids (RNA) to these cells (paragraphs 0018,0020), and would make obvious the limitations of claims 11 and 12.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claim 4 is rejected under 35 U.S.C. 103 as being unpatentable over Qu et al. as evidenced by Mayani et al., in view of Bianchi et al. and Weiss et al. as applied to claims 1,6,9 and 13 above, and further in view of Scadden et al. (US 8642569, Issued 4 Feb 2014), cited on an IDS.
The teachings of Qu et al. as evidenced by Mayani et al. in view of Bianchi et al. and Weiss et al. as applicable to claims 1,6,9 and 13 are described above.
Qu et al. does not teach wherein the HSPCs are in a subject and differentiate into megakaryocytes in the subject.
However, before the effective filing date, Scadden et al. teach methods and compositions for expanding HSPCs ex vivo and in vivo and for transplanting HSPCs and treatment of anemia in a human subject (Abstract). Scadden et al. teach promoting hematopoietic stem progenitor cell HSPC expansion in a subject by administering an RNAi agent and hematopoietic stem and progenitor cells can differentiate into megakaryocytes in adult body (column 2, lines 30-35; column 24, lines 1-10), and which RNAi agent may be microRNA (column 15 lines 17-20; column 33, lines 41-46). Scadden et al. teach that sources of CD34+ HSPCs are the bone marrow of adults, umbilical cord blood, placenta, mobilized peripheral blood, fetal liver, fetal spleen and aorta-gonad-mesonephros of animals (column 48, lines 63-67), and teach transduction of CD34+ cells from umbilical cord blood with shRNA (column 63, lines 51-59).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to have modified the method of Qu et al. as evidenced by Mayani et al. in view of Bianchi et al. and Weiss et al. to provide the method wherein the CD34+ HSPCs are in a subject and differentiation in the subject as disclosed by Scadden et al, to apply the methodology of stem cell differentiation by RNA transfection for treatment of human diseases such as anemia. There would be a reasonable expectation of success because both Qu et al. and Scadden et al. disclose altering gene expression in HSPCs by treatment with miRNA, and Scadden et al. also discloses and discusses transfection of CD34+ HSPCs. One of ordinary skill in the art would have been motivated to do so because Scadden et al. teach treatment of diseases such as anemia with such expanded HSCPs in vivo.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claim 5 is rejected under 35 U.S.C. 103 as being unpatentable over Qu et al. as evidenced by Mayani et al. in view of Bianchi et al. and Weiss et al. as applied to claims 1,6,9 and 13 above, and further in view of Croce et al. (US 20090209450, Published 20 August 2009).
The teachings of Qu et al. as evidenced by Mayani et al., in view of Bianchi et al. and Weiss et al. as applicable to claims 1,6,9 and 13 are described above.
Qu et al., Mayani et al., Bianchi et al. and Weiss et al. do not teach the small RNAs further comprise one or more microRNAs selected from the group consisting of miR-191, miR-181, miR-378, miR-26, let-7, miR-92, miR-126, miR-21, miR-146, miR-181 or combination thereof.
Before the effective filing date, Croce et al. taught identification of specific miRNAs that are involved in megakaryocytic differentiation and the gene expression in human megakaryocytic cultures from bone marrow CD34+ progenitors was investigated (paragraph 0009). Croce et al. taught identification of microRNA expression profile of CD34+ cell during megakaryocytic differentiation in which 8 microRNAs that predicted megakaryocytic differentiation were identified, including miR-10a, miR-10b, miR-30C, miR-106, miR-126, miR-130a, miR-132 and miR-143 (paragraph 0190). Croce et al. taught after identification of the microRNA expression profile of CD34+ cells during megakaryocytic differentiation, the miRNA expression in AMKL cells lines were investigated, and identified 10 miRNAs upregulated in AMKL cells lines compared with that of CD34 in vitro differentiation megakaryocytes, and included miR-126, and found five miRNAs involved in megakaryocytic differentiation signature (miR-101, miR-126, miR-106, miR-20 and miR-135) that were upregulated in the acute megakaryoblastic leukemic cell lines (paragraph 0201).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to have modified the method of Qu et al. as evidenced by Mayani et al. in view of Bianchi et al. and Weiss et al. by also transferring miR-126 into CD34+ HSPCs based on the teaching of Croce et al. with a reasonable expectation of success. There would be a reasonable expectation of success, because Croce et al. pertains to identification of miRNA expression profiling of CD34+ during megakaryocytic differentiation, and would amount to combining prior art elements according to known methods to yield predictable results. One of ordinary skill in the art would have been motivated to modify the method of Qu et al. as evidence by Mayani et al. in view of Bianchi et al and Weiss et al. and also transfer miR-126 into CD34+ HSPCs because Croce et al. taught identification of miRNA expression profiles of CD34+ cells during megakaryocyte differentiation and that predicted megakaryocyte differentiation which included miR-126, as well as that miR-126 was one of the miRNAs found to be upregulated in the acute megakaryoblastic leukemic cell lines.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claim 10 is rejected under 35 U.S.C. 103 as being unpatentable over Qu et al. as evidenced by Mayani et al. in view of Bianchi et al. and Weiss et al. as applied to claims 1,6,9 and 13 above, and further in view of Georgantas et al. (PNAS, 20 Feb 2007, Vol. 104, No. 8, pages 2750-2755).
The teachings of Qu et al. as evidenced by Mayani et al., Bianchi et al. and Weiss et al. as applicable to claims 1,6,9 and 13 are described above.
Qu et al. as evidenced by Mayani et al., Bianchi et al. and Weiss et al. do not teach wherein the one or more small RNAs are isolated from cells.
However, before the effective filing date, Georgantas et al. teach 33 miRNAs expressed in CD34+ hematopoietic progenitor cells from normal human bone marrow (Abstract) and which might control hematopoietic differentiation (page 2750). Georgantas et al. teach isolation of small RNAs from cells and determining miRNA expression (Page 2755, left column).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to modify the method of Qu et al. as evidenced by Mayani et al. in view of Bianchi et al. and Weiss et al. based on the teachings of Georgantas et al. to arrive at claim 10 with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to do so because Georgantas et al. teach there are many miRNAs expressed in CD34+ hematopoietic progenitor cells and which might control hematopoietic differentiation, and teach methods of isolation of miRNAs from cells to determine miRNA expression thereof, and would make obvious the limitations of claim 10.
Accordingly, claim 10 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claims 14-17 are rejected under 35 U.S.C. 103 as being unpatentable over Qu et al. as evidenced by Mayani et al., in view of Bianchi et al. and Weiss et al. as applied to claims 1,6,9 and 13 above, and further in view of Cornejo et al. (Blood, 4 August 2011, Vol. 118, No. 5, pages 1264-1273).
The teachings of Qu et al. as evidenced by Mayani et al., Bianchi et al. and Weiss et al. as applicable to claims 1,6,9 and 13 are described above.
Qu et al., Mayani et al., Bianchi et al. and Weiss et al. do not teach further administering to the HSPCs a regulator of PI3K signaling pathway, or administering to the HSPCs a regulator of Akt signaling pathway.
However, before the effective filing date, Cornejo et al. teach that the NOTCH signaling pathway as a positive regulator of megakaryocytic lineage specification during hematopoiesis, and the investigation of the role of downstream mediators of NOTCH signaling pathway during megakaryopoiesis and report crosstalk between the NOTCH and PI3K/AKT pathways (Abstract). Cornejo et al. teach the NOTCH pathway specifies megakaryocyte development from hematopoietic stem and progenitor cells, and that in vitro stimulation of human cord blood CD34+ cells with the NOTCH ligand Delt-like-1 promoted the generation of precursors that repopulation the megakaryocytic lineage in vivo (page 1264, right column). Cornejo et al. teach PIK3 and AKT are key intermediates of a signaling pathway activated by several cell-extrinsic signals involved in cell growth and survival (page 1264, right column). Cornejo et al. teach NOTCH signaling activation correlates with low PTEN expression and high levels of phosphorylation of several components of the PI3K/AKT pathway during megakaryocyte development (page 1267, right column). Cornejo et al. teach “Together our data suggest the presence of a positive feedback loop during megakaryopoiesis, involving NOTCH induced activation of the PI3K/AKT pathway, which in turn suppresses the inhibitory role of FOXO factors (Figure 7A). This feed-forward regulatory loop may thereby enable a threshold level of RBPJ-mediated transcription required for megakaryocyte specification from HSCs” (page 1270, right column).
Therefore, it would have been obvious to one of ordinary skill in the art before the effective filing date, to have modified the method of Qu et al. as evidenced by Mayani et al., in view of Bianchi et al. and Weiss et al. with the teachings of Cornejo et al. regarding administering a positive regulatory of the PI3K signaling pathway, or administering a positive regulator of the Akt signaling pathway to enhance megakaryocytic differentiation of HSPCs with a reasonable expectation of success. One of ordinary skill in the art would have been motivated to further administer a positive regulator of the PI3K signaling pathway or a positive regulator of the Akt signaling pathway to the HSPCs, because Cornejo et al. teach the positive correlation between PIK3 and AKT in the signaling pathway with megakaryopoiesis (PIK3 and AKT are key intermediates of a signaling pathway activated by several cell-extrinsic signals involved in cell growth and survival (page 1264, right column), that the NOTCH signaling pathway as a positive regulator of megakaryocytic lineage specification during hematopoiesis, and the presence of a positive feedback loop during megakaryopoiesis, involving NOTCH induced activation of the PI3K/AKT pathway, which in turn suppresses the inhibitory role of FOXO factors (Figure 7A) and this feed-forward regulatory loop may thereby enable a threshold level of RBPJ-mediated transcription required for megakaryocyte specification from HSCs (page 1270, right column)).
Accordingly, claims 14-17 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claim 18-20 are rejected under 35 U.S.C. 103 as being unpatentable over Papoutsakis et al. (US 20170058262, Published 2 March 2017), cited on an IDS, and further in view of Bianchi et al. (Cell Death and Differentiation, 2015; 22, 1906-1921) cited on an IDS, and Weiss et al. (Blood Advances, 8 Jan 2019, Vol. 3, No. 1, pages 33-46).
Regarding claims 18 and 19, Papoutsakis et al. teach coculture of megakaryocytic microparticles MkMPs with hematopoietic stem and progenitor cells (HSPCs) promoted HSPC differentiation to mature Mks, and that MkMPs can be loaded with desirable molecules for delivery to HSPCs with effectiveness and specificity (paragraph 0017). Papoutsakis et al. teach MkMPs can be used as a means to modify in vitro or in vivo hematopoietic stem and progenitor cells by transferring specific nucleic acids (RNA molecules) to these cells (paragraphs 0018,0020), and loading MkMPs with exogenous RNA for delivery to target cells (paragraph 0023). Papoutsakis et al. teach MkMPs promote Mk differentiation through transfer of the RNA carried by the MkMPs, and that several signaling molecules carried by the MPs are miRNA (paragraph 0085).
Papoutsakis et al. teach that multiple sources of HSPCs include CD34+ cells from bone marrow, peripheral blood or cord blood are cultured and differentiated to megakaryocytes (paragraph 0107).
Papoutsakis et al. do not teach the exogenous small RNA comprises or consists of miR-486 and miR-22.
Before the effective filing date, Bianchi et al. teach miR-486 plays a key role in differentiation of CD34+ hematopoietic progenitor cells HPCs toward erythroid versus megakaryocyte lineage by supporting the erythropoiesis while restraining the megakaryopoiesis and reducing the abnormal expansion of megakaryocytopoiesis (Abstract, page 1906, first column, second paragraph; page 1908, second column, sixth and eighth paragraphs).
Additionally, Weiss et al. teach miR-22 is upregulated in megakaryocytes, and upregulation is a critical step in megakaryocyte differentiation (Abstract). Weiss et al. teach microRNAs are small single-stranded RNAs, and that only a small number of miRNAs have been shown to positively contribute to MK differentiation (page 34, left column). Weiss et al. teach that miR-22 is dramatically upregulated upon megakaryocytic differentiation in adult mice and in cell lines, megakaryocytic differentiation is driven by overexpression of miR-22 and is inhibited by its loss, and the role of miR-22 in the process is mediated through direct targeting of the zinc-finger-transcriptional repressor GFI1 (Page 34, left column).
Regarding claim 20, Papoutsakis et al. teach methods for loading MkMPs with exogenous RNA (paragraphs 0023-0024), and wherein miRNA is an RNA carried by the microparticles (paragraph 0085).
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filing date, to have modified the method of Papoutsakis et al, in order to provide the exogenous small RNAs comprise miR-486 as disclosed by Bianchi et al. to redirect differentiation of the HSPCs into cells other than megakaryocytes to avoid abnormal expansion of megakaryocytopoiesis and provide the miR-22 of Weiss et al. There would be a reasonable expectation of success because both Papoutsakis et al. and Bianchi et al. disclose the differentiation of CD34+ HSPCs into megakaryocytes and erythroids and Weiss et al. pertains to megakaryocytic differentiation. One of ordinary skill in the art would have been motivated to modify the method of Papoutsakis et al, and to provide miR-486 of Bianchi et al. and miR-22 of Weiss et al. in order to redirect differentiation of the HSPCs into cells other than megakaryocytes to avoid abnormal expansion of megakaryocytopoiesis, and to provide miR-22 of Weiss et al. in order to drive megakaryocytic differentiation.
Accordingly, claims 18-20 would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Claim 21 is rejected under 35 U.S.C. 103 as being unpatentable over Papoutsakis et al. in view of Bianchi et al. and Weiss et al. as applied to claims 18-20 above, and further in view of Qu et al. (Cell Death and Disease (2016)7,e2430), cited on an IDS, as evidenced by Mayani et al. (Stem Cells 1998:16:153-165), cited on an IDS.
The teachings of Papoutsakis et al., Bianchi et al. and Weiss et al. as applicable to claims 18-20 are described above.
While Papoutsakis et al. teach and recite transfection methods of lipofection, nucleofection, and electroporation for loading MkMps with exogenous molecules or desirable RNAs (Claim 20 and paragraphs 0099,0107), Papoutsakis et al. does not teach further transferring the one or more exogenous small RNAs into the HSPCs by transfection, electroporation, lipofection or nucleofection.
However, before the effective filing date, Qu et al. teach al. teach transfecting CB mononuclear cells with miR-125b mimics, and that transfection of miR-125b mimics increased miR-125b levels ~30 fold compared with negative control mimics (page 2, bottom right column- page 3 left column) and that overexpression of miR-125b increases MK differentiation of MNCs (page 5, left column). As evidenced by Mayani et al., cord blood is a rich source of hematopoietic stem/progenitor cells HSPC (Abstract).
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filing date, to have modified the method of Papoutsakis et al., Bianchi et al. and Weiss et al. to provide a further step of transferring one or more exogenous small RNAs which is miR-125b into HSCPs by transfection as taught by Qu et al. as evidenced by Mayani et al. with a reasonable expectation of success. There would be a reasonable expectation of success because both Qu et al. and Papoutsakis et al. pertain to differentiation of HSPCs into megakaryocytes under the influence of small RNA. One of ordinary skill in the art would have been motivated to do so because Qu et al. teach that transfecting miR-125b mimics into CB mononuclear cells increased MK differentiation, and would make obvious the limitations of claim 21.
Therefore, the invention as a whole would have been prima facie obvious to one of ordinary skill in the art before the effective filing date.
Conclusion
Claims 1 and 3-21 are rejected.
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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/STEPHANIE L SULLIVAN/Examiner, Art Unit 1635
/ABIGAIL VANHORN/ Primary Examiner, Art Unit 1636