DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This Office action is in response to the communication filed 4-11-24.
Claims 1-10 are pending in the instant application.
Claim Rejections - 35 USC § 112
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 1-10 are rejected under 35 U.S.C. 112, first paragraph, because the specification, while being enabling for the in vitro transfection of human fibroblasts with three dimensional RNA constructs comprising BRN-2 repeats comprising SEQ ID No.13 in combination with siRNA targeting PTB of SEQ ID Nos.14 and 15, and for the in vitro transfection of human fibroblasts with three dimensional RNA constructs comprising repeating sequences of OCT4 comprising SEQ ID No. 7 in combination with the repeating sequences of CBF-beta comprising SEQ ID No. 8, wherein human fibroblasts were reprogrammed into nerve cells or osteoblasts respectively, does not reasonably provide enablement of making and using compositions for inducing direct reprogramming of any somatic cell comprising a first active ingredient comprising a three dimensional RNA construct comprising one or two types of transcripts, each having a repeating unit sequence for the expression of any transcription factor, and a second active ingredient comprising a three dimensional RNA construct comprising one or two types of transcripts each having a repeating unit sequence for inhibiting the expression of any transcription factor and a second active ingredient comprising a three dimensional RNA construct comprising one or two types of transcripts each having a repeating unit sequence for inhibiting the expression of any transcription factor.
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/or use the invention commensurate in scope with these claims.
The following factors have been considered in determining that the specification does not enable the skilled artisan to make and/or use the invention over the broad scope claimed.
The breadth of the claims:
The claims are broadly drawn to compositions for inducing direct reprogramming comprising a first active ingredient comprising a three dimensional RNA construct comprising one or two types of transcripts, each having a repeating unit sequence for the expression of any transcription factor, and a second active ingredient comprising a three dimensional RNA construct comprising one or two types of transcripts each having a repeating unit sequence for inhibiting the expression of any transcription factor, which transcription occurs by rolling circle transcription, and which expression or inhibition occurs for 30 days or more after a single transfection, which repeating sequence optionally comprises octamer binding transcription factor 4 (OCT4) and a repeating sequence for core binding factor subunit beta (CBF-beta) for direct reprogramming from a somatic cell to an osteoblast, or wherein the repeating sequence optionally comprises BRN2 and which repeating sequence is for inhibition of polypyrimidine tract binding protein (PTB), which direct reprogramming is from a somatic cell to a nerve cell, which cell for transfection optionally comprises fibroblasts, muscle cells, nerve cells, gastric mucosa cells, goblet cells, G cells, pericytes, astrocytes, B cells, blood cells, epithelial cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and umbilical cord blood stem cells.
Teachings in the specification
The specification teaches the following:
Example 1. Preparation of three-dimensional RNA construct expressing DsRed- Express2 and confirmation of effect thereof
Preparation of three-dimensional RNA construct expressing DsRed-Express2
[63] Integrated DNA Technologies, Inc. was contacted to prepare a plasmid (pIDTSmart(Amp) or pUCIDT(Amp) into which the DsRed-Express2 mRNA encoding sequence was inserted and the transcription termination site was removed. The DsRed-Express2 mRNA encoding sequence is operably linked to the T7 promoter and an internal ribosomal entry site (IRES).
[64] The T7 promoter, IRES, and DsRed-Express2 mRNA sequence included in the plasmid are shown in SEQ ID NOS: 1 to 3 in Table 1…
[67] A three-dimensional RNA construct, in which transcripts including a plurality of repeated DsRed-Express2 mRNA sequences are self-assembled, was prepared by transcribing the plasmid at high concentration using the rolling circle transcription (RCT) method. When the rolling circle transcription (RCT) reaction is performed under conditions where the concentration of ribonucleotide triphosphate (rNTP) and the concentration of the plasmid are above a certain level, long transcripts can self-assemble by tangling and twisting to thereby form a three- dimensional construct.
68] The three-dimensional RNA construct prepared above was injected into cells to confirm whether red fluorescent protein (DsRed-Express2) was stably and continuously expressed in the cells. Specifically, 5 nM of plasmid DNA, a 4 mM ribonucleotide solution mix (New England Biolabs), a reaction buffer (80 mM Tris-HCl, 4 mM spermidine, 12 mM MgCl₂, and 2 mM dithiothreitol), and 50 units/mL of T7 RNA polymerase (New England Biolabs) were added to a tube and mixed, and then the mixture was reacted in an incubator at 37°C for 20 hours, and thereby a three-dimensional RNA construct, in which transcripts including the DsRed-Express2 mRNA region are self-assembled, was synthesized.
[73] It was possible to control the size of the three-dimensional RNA construct by changing the concentration of the reactants. The average size of the three-dimensional construct was increased as the concentration of the template plasmid DNA was increased from 1 nM to 25 nM, as the concentration of the ribonucleotide triphosphate mix as a reactant was increased from 4 mM to 8 mM, and as the concentration of T7 RNA polymerase as a reaction enzyme was lowered from 5,000 units/mL to 50 units/mL.
[83] A three-dimensional RNA construct expressing DsRed-Express2 was transfected into primary human dermal fibroblasts (HDFs)
[89] On the 5th, 10th, and 15th day after the start of incubation, the expression level of red fluorescent protein was confirmed using fluorescence microscopic images and flow cytometry, and compared with the untreated control group (indicated as untreated in FIG. 6).
[90] According to FIG. 6, in the cells to which the three-dimensional RNA construct expressing DsRed-Express2 was delivered, the number of red fluorescent protein-expressing cells (M1) and the mean fluorescence intensity (MFI) of the cells continued to increase for 15 days. This confirmed that genes can be expressed stably and efficiently merely by one-time transfection of the three-dimensional RNA construct into cells, and this result is contrary to the fact that several rounds of mRNA delivery are generally required for the expression of a target protein.
[94] A three-dimensional RNA construct expressing octamer-binding transcription factor 4 (OCT4) and core-binding factor subunit beta (CBF-ß), which are transcription factors that cause differentiation into osteoblasts, was prepared in the same manner as in Example 1-1. In the OCT4-expressing plasmid and the CBF-ß-expressing plasmid, as the ribosome binding site, the Kozak sequence was used instead of the IRES sequence.
[95] The Kozak sequence, OCT4 mRNA encoding sequence, and CBF-β encoding sequence included in the above plasmid, and the unit sequence of the three-dimensional RNA construct expressed from the plasmid containing these are shown in Table 2 below. The unit sequence of the OCT4-expressiing RNA construct of SEQ ID NO: 7 includes a T7 promoter, Kozak, and OCT mRNA; the unit sequence of the CBF-ß-expressing RNA construct of SEQ ID NO: 8 includes a T7 promoter, Kozak, and CBF-ß mRNA. These unit sequences refer to sequences unit in which a promoter, a ribosome binding site, and OCT4 or CBF-ß mRNA are operably linked so as to express OCT4, CBF-ß, etc.
[96] The prepared three-dimensional RNA constructs were (1) an OCT4-expressing three-dimensional RNA construct in which a transcript including the OCT4 mRNA sequence was self-assembled (hereinafter referred to as O), (2) a CBF-ß-expressing three-dimensional RNA construct in which a transcript including the CBF-ß mRNA sequence was self-assembled (hereinafter referred to as C), and (3) a three-dimensional RNA construct expressing OCT4 and CBF-B, in which a transcript including the OCT4 mRNA sequence and a transcript including the CBF-β mRNA sequence are mixed in a ratio of 1:1 to 2:1 and self-assembled (hereinafter referred to as OC). In (3), the mixing ratio of the two transcripts can be adjusted depending on the type of cells to be transfected or the ratio of the proteins to be expressed. The RNA constructs (1) to (3) above were prepared such that the average size was 100 nm to 400 nm by adjusting the plasmid concentration.
An experimental group (O) was prepared in the same manner as in
Example 1, in which human fibroblasts were treated with the OCT4-expressing three- dimensional RNA construct at a concentration of 2 µg/mL and incubated for 24 hours.
[104] Additionally, an experimental group (O+C) was prepared, in which human fibroblasts were treated with an OCT4-expressing three-dimensional RNA construct and a CBF-ß- expressing three-dimensional RNA construct at a concentration of 2 µg/mL, respectively, at daily intervals and incubated for 24 hours.
106] According to FIG. 7, the experimental group (O+C) in which both the OCT4-expressing three-dimensional RNA construct and the CBF-ß-expressing three-dimensional RNA construct were transfected, the activity of phosphatase ALP appeared 16 days after the incubation in an osteogenic differentiation medium, thus confirming reprogramming into osteoblasts.
[108] According to FIG. 8, in the experimental group (0) transfected with only OCT4, which is a transcription factor having a role of increasing stemness, there was no significant difference in the expression level of OPN compared to the untreated control group (indicated as Control), and the direct reprogramming into osteoblasts did not occur.
[109] However, in the experimental group (O+C), in which both the OCT4-expressing three-dimensional RNA construct and the CBF-ß-expressing three-dimensional RNA construct were transfected, the RNA expression level of OPN increased significantly at 16 days after incubation in an osteogenic differentiation medium. Therefore, it was confirmed that the combination of the OCT4-expressing three-dimensional RNA construct and the CBF-ß-expressing three- dimensional RNA construct is suitable for direct reprogramming into osteoblasts.
[115] (3) Confirmation of function of three-dimensional RNA construct expressing both OCT4 mRNA and CBF-β mRNA
[116] Human fibroblasts were treated once with the three-dimensional RNA construct of (3) expressing OCT4 and CBF-β prepared in Example 2-1 at a concentration of 4 µg/mL and incubated for 21 days. In order to confirm the functionality of osteoblasts, the production of mineralized calcium deposits was evaluated through ARS staining.
[117] According to FIG. 10, it was confirmed that the ARS staining intensity was higher in the experimental group (mixed, indicated as OC), in which the three-dimensional RNA construct expressing OCT4 and CBF-β was delivered once, compared to the experimental group (individual, indicated as O+C), in which each of the three-dimensional RNA construct expressing OCT4 and the three-dimensional RNA construct expressing CBF-ß were delivered at daily intervals. Therefore, it was confirmed that the three-dimensional RNA construct, in which a transcript including OCT4 mRNA and a transcript including CBF-ß mRNA were mixed and self-assembled, more efficiently induced direct reprogramming into osteoblasts.
[120] The three-dimensional RNA construct (OC), in which a transcript including OCT4 mRNA and a transcript including CBF-β mRNA are mixed and self-assembled, were transfected into human fibroblasts, and Osteo-Image mineralization analysis was performed on the 7th, 14th, and 21st days to cross-confirm whether the cells possessed functionality as osteoblasts.
[126] In summary, in the experimental group (OC) where the cells were treated at a concentration of 4 µg/mL with a three-dimensional RNA construct, which simultaneously includes OCT4-expressing RNA and CBF-ß-expressing RNA, and incubated for 24 hours, on the 7th day after incubation of the cells in an osteogenic medium, the expressions of both Runx2 (i.e., a marker of early osteoblasts) and OPN (i.e., a marker of mature osteoblasts) are significantly increased and the cells are promptly reprogrammed into osteoblasts and are able to express their functions.
[130] A BRN2-expressing three-dimensional RNA construct was prepared using substantially the same method as Example 1-1 using the BRN2 mRNA-expressing plasmid (including the Kozak sequence) shown in Table 3 below. The average size of the synthesized BRN2-expressing three-dimensional RNA construct was 100 nm to 400 nm.
[131] A three-dimensional RNA construct, which expresses siRNA that inhibits the expression of polypyrimidine tract binding protein (PTB) (i.e., siRNA-PTB), was synthesized through the following two steps.
[132] In the first step, circular DNA for expressing sense RNA for part of the PTB gene and circular DNA for expressing antisense RNA for part of the PTB gene were prepared. Long- stranded DNA (10 µM) of SEQ ID NO: 10 or SEQ ID NO: 11, where the 5' end is phosphorylated, was mixed with promoter DNA (10 µM) of SEQ ID NO: 12, denaturation was performed at 95°C for 2 minutes using a thermal cycler, and annealing was performed while gradually lowering the temperature to 25°C over 1 hour. The resultant was mixed with a reaction solution (30 mM Tris-HCl (pH 7.8), 10 mM MgCl₂, 10 mM DTT, 1 mM ATP) and T4 DNA ligase (3 unit/mL) and T4 DNA ligase (3 units/mL) and the mixture was annealed in a hybridization oven at 25°C to prepare PTB antisense RNA expressing circular DNA (SEQ ID NOS: 10 and 12) and PTB antisense RNA expressing circular DNA (SEQ ID NOS: 11 and 12).
[133] Since the promoter DNA of SEQ ID NO: 12 was designed to be complementary to 16 bases at the 5' end and 6 bases at the 3' end of SEQ ID NOS: 10 and 11 DNA, circular DNA can be prepared by ligation with long-stranded DNA. Additionally, the region corresponding to SEQ ID NO: 12 in circular DNA becomes a binding site and a replication start site (a promoter region) for T7 RNA polymerase.
[135] The two types of synthesized circular DNA (PTB sense RNA expression DNA and PTB antisense RNA expression DNA) in an amount of 0.5 µM each, a ribonucleotide solution mix (4 mM), and T7 RNA polymerase (80 units/µL) were mixed with a reaction buffer (80 mM Tris- HCI, 12 mM MgCl₂, 4 mM spermidine, 2 mM dithiothreitol, pH 7.9 (25°C)) and reacted at 37°C for 20 hours to synthesize a three-dimensional RNA construct expressing siRNA-PTB.
[143] After transfecting human fibroblasts with BRN2-expressing three-dimensional RNA construct and siRNA-PTB-expressing three-dimensional RNA construct, the changes in cell phenotype were observed using an optical microscope while incubating for 28 days.
[144] According to FIG. 13, the changes in cell phenotype began on the 3rd day after transfection, and the morphological characteristics of the nerve cells became prominent on the 14th day; therefore, it was confirmed that direct reprogramming to nerve cells was occurring rapidly. In addition, even on the 28th day after transfection, the phenotype of the nerve cells was maintained and the survival rate was high; therefore, it was confirmed that both the BRN2- expressing three-dimensional RNA construct and the siRNA-PTB-expressing three-dimensional RNA constructs were sufficiently safe to be maintained for 28 days.
[Emphases added] [Citations omitted].
The examples provided in the instant specification, of the in vitro transfection of human fibroblasts with three dimensional RNA constructs comprising BRN-2 repeats comprising SEQ ID No.13 in combination with siRNA targeting PTB of SEQ ID Nos.14 and 15, and for the in vitro transfection of human fibroblasts with three dimensional RNA constructs comprising repeating sequences of OCT4 comprising SEQ ID No. 7 in combination with the repeating sequences of CBF-beta comprising SEQ ID No. 8, wherein human fibroblasts were reprogrammed into nerve cells or osteoblasts respectively, are not representative or correlative of the ability to make and use compositions for inducing direct reprogramming of the array of somatic cells claimed comprising a first active ingredient comprising a three dimensional RNA construct comprising one or two types of transcripts, each having a repeating unit sequence for the expression of any transcription factor, and a second active ingredient comprising a three dimensional RNA construct comprising one or two types of transcripts each having a repeating unit sequence for inhibiting the expression of any transcription factor.
In light of the teachings in the specification, one skilled in the art would not accept on its face the examples provided in the instant disclosure as being correlative or representative of the ability to make and use the broad genus of compositions claimed. Since the specification fails to provide the requisite guidance for making and using the broad genus of compositions claimed, and since determination of the factors required for accomplishing reprogramming is highly unpredictable, it would require undue experimentation to practice the invention over the broad scope claimed.
For these reasons, the instant rejection for lacking enablement over the full scope claimed is proper.
Claims 1-6, 8 and 10 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.
The breadth of the claims:
The claims are broadly drawn to compositions for inducing direct reprogramming comprising a first active ingredient comprising a three dimensional RNA construct comprising one or two types of transcripts, each having a repeating unit sequence for the expression of any transcription factor, and a second active ingredient comprising a three dimensional RNA construct comprising one or two types of transcripts each having a repeating unit sequence for inhibiting the expression of any transcription factor, which transcription occurs by rolling circle transcription, and which expression or inhibition occurs for 30 days or more after a single transfection, which repeating sequence optionally comprises octamer binding transcription factor 4 (OCT4) and a repeating sequence for core binding factor subunit beta (CBF-beta) for direct reprogramming from a somatic cell to an osteoblast, or wherein the repeating sequence optionally comprises BRN2 and which repeating sequence is for inhibition of polypyrimidine tract binding protein (PTB), which direct reprogramming is from a somatic cell to a nerve cell, which cell for transfection optionally comprises fibroblasts, muscle cells, nerve cells, gastric mucosa cells, goblet cells, G cells, pericytes, astrocytes, B cells, blood cells, epithelial cells, neural stem cells, hematopoietic stem cells, mesenchymal stem cells, and umbilical cord blood stem cells.
The specification fails to provide the requisite guidance for making and using the large genus of repeat sequences for the expression or inhibition of transcription factors, whereby three dimensional RNA constructs are self assembled and direct reprogramming is successfully induced in the array of cells claimed.
The examples provided in the specification, as set forth in the enablement rejection above, do not provide a representative number of species for the vast genus of self assembling repeat RNA sequences encompassed by the claims.
Since the disclosure fails to describe the common attributes and characteristics concisely identifying members of the proposed genus, and because the claimed genus is highly variant, the description provided is insufficient. One of skill in the art would reasonably conclude that the disclosure fails to provide a representative number of species to describe the broad genus of compositions instantly claimed.
Thus, Applicant was not in possession of the broadly claimed genus.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
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.
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.
Claims 1-6, 8,and 10 is/are rejected under 35 U.S.C. 103 as being obvious over Pub. No. 1020200142537 (12-22-2020), KR 102122336, Lee et al (WO 2020/085554),
Chang et al (Biomaterials, 11.034, pages 1-31 (2018)), and Xue et al (Cell, Vol. 152, pages 82-96 (2013)), the combination in view of Miller et al (Nature Genetics, Vol. 32, pages 645-649 (2002)), Yamamoto et al (PNAS, Vol. 112, No. 19, pages 6152-6157 (2015)) and Zhu et al (Stem Cells and Development, Vol. 27, No. 11, pages 736-744 (2018)).
The applied references have a common inventor with the instant application. Based upon the earlier effectively filed date of the references, they constitute prior art under 35 U.S.C. 102(a)(2).
This rejection under 35 U.S.C. 103 might be overcome by: (1) a showing under 37 CFR 1.130(a) that the subject matter disclosed in the reference was obtained directly or indirectly from the inventor or a joint inventor of this application and is thus not prior art in accordance with 35 U.S.C.102(b)(2)(A); (2) a showing under 37 CFR 1.130(b) of a prior public disclosure under 35 U.S.C. 102(b)(2)(B); or (3) a statement pursuant to 35 U.S.C. 102(b)(2)(C) establishing that, not later than the effective filing date of the claimed invention, the subject matter disclosed and the claimed invention were either owned by the same person or subject to an obligation of assignment to the same person or subject to a joint research agreement. See generally MPEP § 717.02.
The claims are drawn to compositions for inducing direct reprogramming comprising a first active ingredient comprising a three dimensional RNA construct comprising one or two types of transcripts, each having a repeating unit sequence for the expression of any transcription factor, and a second active ingredient comprising a three dimensional RNA construct comprising one or two types of transcripts each having a repeating unit sequence for inhibiting the expression of any transcription factor, which transcription occurs by rolling circle transcription, and which expression or inhibition occurs for 30 days or more after a single transfection, which repeating sequence optionally comprises octamer binding transcription factor 4 (OCT4) and a repeating sequence for core binding factor subunit beta (CBF-beta) for direct reprogramming from a somatic cell to an osteoblast, or wherein the repeating sequence optionally comprises BRN2 and which repeating sequence is for inhibition of polypyrimidine tract binding protein (PTB), which direct reprogramming is from a somatic cell to a nerve cell, which cell for transfection comprises fibroblasts.
KR 102122336 (See IDS filed 4-11-24) teaches compositions for producing induced pluripotent stem cells, which compositions comprise mRNA for expressing transcription factors that induce somatic cells and adult stem cells to be dedifferentiated into induced pluripotent stems. The compositions also comprise self assembled RNA nanoparticles comprising micro RNAs and siRNAs. The RNA nanoparticles have a diameter of 50-200 nm. The nanoparticles also optionally comprise complex RNA nanoparticles including microRNA and siRNA, and mRNA for expressing transcription factors optionally comprising Oct4. These are encoded by a plasmid comprising a T7 RNA polymerase promoter and generates a circular double stranded plasmid (see esp. pages 3-5, Examples 1, 3 and 6, claims 1-7).
Pub. No. 1020200142537 (12-22-2020) (See IDS filed 4-11-24) teach reprogramming of non-neural cells to neurons upon suppression of PTB and expression of the transcription factor Brn2 (abstract and Figure on pages 1 and 54).
Lee et al (WO 2020/085554) teach the production of induced pluripotent stem cells using self assembled RNA nanoparticles for cell transformation, which RNA comprises mRNA expressing transcription factors, miRNA and/or siRNA for inhibiting transcription factors, leading to the dedifferentiation of somatic cells or adult stem cells and producing induced pluripotent stem cells (see the Abstract).
Chang et al (Biomaterials, 11.034, pages 1-31 (2018)) (See IDS filed 4-11-24) teach novel gene delivery systems for reprogramming of induced cardiomyocytes (iCMs) comprising the administration of nanoparticles loaded with Gata4, Mef2c and Tbx5. See abstract. Compositions comprising these nanocomplexes had high reprogramming efficiency in human and mouse somatic cells. Chang teaches the in vivo conversion of fibroblasts into cardiomyocytes after myocardial infarction. Last paragraph, page 5; Bridging paragraph, pages 14-15.
Xue et al (Cell, Vol. 152, pages 82-96 (2013)) (See IDS filed 4-11-24) teach the inhibition of RNA binding polypyrimidine tract binding (PTB) protein by miR-124 to induce transdifferentiation of fibroblasts into functional neurons. Xue teaches that a key event during neuronal induction is the relief of PTB mediated blockage of micro RNA action on multiple components of the REST complex, derepressing a large array of neuronal genes comprising miR-124 and multiple neuronal specific transcription factors in non0neuronal cells, converting a negative feedback loop to a positive one that elicits cellular reprogramming to the neuronal lineage. Abstract. Xue teaches that neuronal differentiation is well studied paradigm as a consequence of transcription reprogramming. Text on pages 82-84, Figure 1 on page 84, Figure 3 on page 86, text in Discussion on page 93).
Miller et al (Nature Genetics, Vol. 32, pages 645-649 (2002)) teaches the requirement of core binding factor beta (CBF-beta) for bone formation. Miller teaches the rescue of fetal liver hematopoiesis in CBF-beta deficient mice embryos (see the first paragraph on page 645, Table 1 on page 647).
Yamamoto et al (PNAS, Vol. 112, No. 19, pages 6152-6157 (2015)) teach the reprogramming of fibroblasts into osteocalcin producing cells upon transduction with runt related transcription factor 2 and OCT4. OCT4 and L-Myc also induced fibroblasts to produce bone matrix (see the abstract on page 6152, Significance on page 6152).
Zhu et al (Stem Cells and Development, Vol. 27, No. 11, pages 736-744 (2018)) teach the conversion of astrocytes into neural progenitors and neurons upon inducible expression of Brn2 (see the abstract on page 736 and the last paragraph on page 738).
It would have been obvious to provide compositions comprising octamer binding transcription factor 4 (OCT4) and a repeating sequence for core binding factor subunit beta (CBF-beta) for direct reprogramming from a somatic cell to an osteoblast because the prior art taught the roles of these transcription factors in bone formation and osteoblast formation. It would also have been obvious to provide compositions comprising BRN2 and which repeating sequence is for inhibition of polypyrimidine tract binding protein (PTB), which direct reprogramming is from a somatic cell to a nerve cell, because the prior art taught the role of suppression of PTB and the role of the transcription factor BRN2 in neuronal development and reprogramming of somatic cells to neural cells. One would have been motivated to provide compositions comprising three dimensional RNA constructs comprising sequences encoding these transcription factors or siRNA specifically targeting PTB for reprogramming because the delivery of self assembling RNA had been taught in the prior art for successful transfection or transduction of somatic cells, including fibroblasts.
For these and the aforementioned reasons, the instant invention would have been obvious to one of ordinary skill in the art prior to the effective filing date of the instant invention.
Allowable Subject Matter
SEQ ID Nos. 7, 8, 13-15 appear free of the prior art searched and of record.
Conclusion
Certain papers related to this application may be submitted to Art Unit 1637 by facsimile transmission. The faxing of such papers must conform with the notices published in the Official Gazette, 1156 OG 61 (November 16, 1993) and 1157 OG 94 (December 28, 1993) (see 37 C.F.R. ' 1.6(d)). The official fax telephone number for the Group is 571-273-8300. NOTE: If Applicant does submit a paper by fax, the original signed copy should be retained by applicant or applicant's representative. NO DUPLICATE COPIES SHOULD BE SUBMITTED so as to avoid the processing of duplicate papers in the Office.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Jane Zara whose telephone number is (571) 272-0765. The examiner’s office hours are generally Monday-Friday, 10:30am - 7pm. If attempts to reach the examiner by telephone are unsuccessful, the examiner's supervisor, Jennifer Dunston, can be reached on (571)-272-2916. Any inquiry of a general nature or relating to the status of this application should be directed to the Group receptionist whose telephone number is (703) 308-0196.
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Jane Zara
8-25-26
/JANE J ZARA/Primary Examiner, Art Unit 1637
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