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 .
Priority
This application is a national phase application under 35 U.S.C. § 371 that claims priority to International Application No. PCT/US2021/018160, filed February 16, 2021, which
claims priority to U.S. Provisional Patent Application Serial No. 62/977,604, filed February 17, 2020.
Restriction/Election
Applicant’s election without traverse of election of species I) an effective amount of fibroblasts; II) the hematopoietic cells express c-kit, Sca-1, CD34, and/or CD33 as the species of pluripotent state that allows for de-differentiation of the fibroblasts into hematopoietic cells: and III) idiopathic pulmonary fibrosis as the species of telomere-associated condition, in the reply filed on 07/18/2025 is acknowledged. Applicant notes that claims 1-6, 12-13, 25-26, 32-33, 35-37, and 45 read on the elected species and claims 29-31, 46, 48 and 49 are generic. Therefore, claims 14-15, 18-19, 22, 24 have been withdrawn from consideration at this time.
Claims 7-11, 16-17, 20-21, 23, 27-28, 34, 38-44, 47, and 50-52 are cancelled.
Claims 1, 3, 4, 6, 33, 37 are amended in the claim set filed on 12/12/2025.
Claims 1-6, 12-15, 18-19, 22, 24-26, 29-33, 35-37, 45-46 and 48-49 are pending.
It is noted that claim 29 recites “the hematopoietic cells are leukocytes” does not read on the elected “hematopoietic cells express c-kit, Sca-1, CD34, and/or CD33 as the species of pluripotent state that allows for de-differentiation of the fibroblasts into hematopoietic cells”. In this regard, hematopoietic cells are multipotent stem/progenitor cells as recited in instant claim 5 whereas leukocytes are terminally differentiated cells. Claim 31 depends from claim 30 and recites “wherein the leukocytes are peripheral blood mononuclear cells.”.
Accordingly, claims 14-15, 18-19, 22, 24, and 30-31 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on 07/18/2025.
Claims 1-6, 12-13, 25-26, 29, 32-33, 35-37, 45-46, and 48-49 are currently under examination.
Summary: The rejections documented in the Non-Final Office Action mailed on 08/14/2025 have been withdrawn in light of claim amendments and Applicant’s arguments filed on 12/12/2025. The new grounds of rejections documented in this Final Office Action are necessitated by claim amendments fired on 12/12/2025.
Claim Rejections - 35 USC § 112
The following is a quotation of 35 U.S.C. 112(d):
(d) REFERENCE IN DEPENDENT FORMS.—Subject to subsection (e), a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
The following is a quotation of pre-AIA 35 U.S.C. 112, fourth paragraph:
Subject to the following paragraph [i.e., the fifth paragraph of pre-AIA 35 U.S.C. 112], a claim in dependent form shall contain a reference to a claim previously set forth and then specify a further limitation of the subject matter claimed. A claim in dependent form shall be construed to incorporate by reference all the limitations of the claim to which it refers.
Claims 33 and 37 are rejected under 35 U.S.C. 112(d) or pre-AIA 35 U.S.C. 112, 4th paragraph, as being of improper dependent form for failing to further limit the subject matter of the claim upon which it depends, or for failing to include all the limitations of the claim upon which it depends.
Amended claim 1 filed on 12/12/2025 reads as follows: A method for treating a telomere-associated condition in a subject comprising providing to the subject an effective amount of isolated fibroblasts, conditioned media from fibroblast culture, microvesicles obtained from fibroblasts, exosomes obtained from fibroblasts, apoptotic vesicles derived from fibroblasts, nucleic acids obtained from fibroblasts, proteins obtained from fibroblasts, lipids obtained from fibroblasts, and/or fibroblast-derived products, wherein the fibroblasts comprise cell-surface expression of CD73.
Amended claim 33 filed on 12/12/2025 reads as follows: The method of claim 1, wherein the fibroblasts express CD73, CD90, CD105, CD14, CD45, and/or CD34 on the cell surface.
Amended claim 37 filed on 12/12/2025 recites the limitation: The method of claim 36, wherein the fibroblasts: ---- “(e) express CDl0, CD13, CD44, CD73, CD90, PDGFr-a, PD-L2, HLA-A, HLA-B, and/or HLA-C on the cell surface.
Claims 33 and 37 further broadening the scope of claim 1 because claim 1 requires “the fibroblasts comprise cell-surface expression of CD73”. However, claims 33 and 37 depend from claim 1, and recites CD73 as one of multiple alternative cell surface markers.
Applicant may cancel the claim(s), amend the claim(s) to place the claim(s) in proper dependent form, rewrite the claim(s) in independent form, or present a sufficient showing that the dependent claim(s) complies with the statutory requirements.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
Claims 1-3, 35-37, and 49 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Wilkinson et al. (2017) (Wilkinson et al., Development of a Three-Dimensional Bioengineering Technology to Generate Lung Tissue for Personalized Disease Modeling, Stem Cells Transl Med, 2017 Feb;6(2):622-633. doi: 10.5966/sctm.2016-0192. Epub 2016 Sep 15) as evidence by Sachs et al. (2012) (Sachs et al., Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites, Cell Tissue Res., 2012 Aug;349(2): 505-15.doi: 10.1007/s00441-012-1423-7. Epub 2012 May 25).
Amended claim 1 filed on 12/12/2025 reads as follows: A method for treating a telomere-associated condition in a subject comprising providing to the subject an effective amount of isolated fibroblasts, conditioned media from fibroblast culture, microvesicles obtained from fibroblasts, exosomes obtained from fibroblasts, apoptotic vesicles derived from fibroblasts, nucleic acids obtained from fibroblasts, proteins obtained from fibroblasts, lipids obtained from fibroblasts, and/or fibroblast-derived products, wherein the fibroblasts comprise cell-surface expression of CD73.
Regarding claims 1-3, and 49, Wilkinson et al. teaches that “Here, we present a method for the generation of self-assembled human lung tissue and its potential for disease modeling and drug discovery for lung diseases characterized by progressive and irreversible scarring such as idiopathic pulmonary fibrosis (IPF). Tissue formation occurs because of the overlapping processes of cellular adhesion to multiple alveolar sac templates, bioreactor rotation, and cellular contraction. Addition of transforming growth factor-β1 to single cell-type mesenchymal organoids resulted in morphologic scarring typical of that seen in IPF but not in two-dimensional IPF fibroblast cultures. Furthermore, this lung organoid may be modified to contain multiple lung cell types assembled into the correct anatomical location, thereby allowing cell-cell contact and recapitulating the lung microenvironment.” (See Abstract). Wilkinson et al. teaches that “Adult lung biopsies were procured according to the University of California, Los Angeles (UCLA) institutional review board protocol (no. 08-09-038-01), from the UCLA Medical Center at the time of lung transplantation. Lung biopsies were obtained from five healthy adults. The induced pluripotent stem cells (iPSCs) were generated as per established protocols by Karumbayaram et al. (See right column, page 623). Wilkinson et al. further teaches that “We found that the system can be adapted to include any combination of cell types and that iPSC-derived mesenchymal cells were also amenable to culture in these organoids (Fig. 2). The organoid formation kinetics and cell morphology of the iPSC-derived organoids was indistinguishable from those derived from fetal lung fibroblasts. Furthermore, the mesenchymal iPSCs demonstrated the ability to be differentiated along several lineages, including osteogenic and adipogenic lineages (supplemental online Fig. 2). Therefore, we were able to demonstrate the ability to personalize this approach for disease modeling and drug discovery. (See right column, page 626).
Regarding the limitation “for treating a telomere-associated condition in a subject” recited in the preamble is the intended result of practicing the only active step recited in claim 1, “providing to the subject an effective amount of isolated fibroblasts”. Moreover, “a telomere-associated condition” encompasses any condition that is directly or indirectly associated, in the context of any genetic association and/or any molecular association, with any telomere function. In this regard, as evidenced by Sachs et al. (2012), a condition of telomere shorting indicates induction of senescence (See Fig. 1C, right column of page 509, and left column of page 514).
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Figure 2. Successful integration of iPSC-derived fibroblasts into organoid model. (Ai): Representative organoid generated using fetal lung fibroblasts. (Aii, Aiii): Confocal immunofluorescence micrographs of fetal lung fibroblast organoid sections for vimentin, collagen I, a-SMA, and DAPI. (Bi): Representative organoid generated using iPSC-derived lung fibroblasts. (Bii, Biii): Confocal immunofluorescence micrographs of iPSC-derived lung fibroblast organoid sections for vimentin, collagen I, a-SMA, and DAPI. Abbreviations: DAPI, 49,6-diamidino-2-phenylindole; iPSC, induced pluripotent stem cell; a-SMA, a-smooth muscle actin.
Regarding claims 35-36, Wilkinson et al. teaches Human fetal lung fibroblasts (FLFs) were isolated from 18- to 20- week-old fetal lungs (Advanced Bioscience Resources, Alameda, CA). Tissues were finely minced and dissociated using 1 mg/ml collagenase/dispase (Roche, Basel, Switzerland, http://www. roche.com) and 0.1 mg/ml DNase (Sigma-Aldrich) with rotation for 45 minutes at 37°C. After washing in media containing1% fetal bovine serum (FBS), a single-cell suspension was generated using 100- and 40-mm cell strainers. To remove red blood cells, the suspension was incubated in red blood cell lysis buffer (BD Biosciences, San Jose, CA, http://www.bdbiosciences.com) for 15 minutes at room temperature. Cells were then plated in 6-well tissue culture plates and cultured in Dulbecco’s modified Eagle’s medium (DMEM)/F12 medium containing 10% FBS (Corning). Human umbilical vein endothelial cells (HUVECs) and small airway epithelial cells (SAECs) were maintained according to the manufacturer’s recommendations (Lonza, Basel, Switzerland, http://www.lonza.com) in endothelial growth medium (EGM)-2 (Thermo Fisher) and small airway growth medium (SAGM) (Lonza), respectively. (See right column, page 623).
Regarding claim 37, the Human umbilical vein endothelial cells (HUVECs) taught by Wilkinson et al. reads on “wherein the fibroblasts are capable of differentiating into vascular endothelial cells” recited in (d) of claim 37.
Regarding the limitation “wherein the fibroblasts comprise cell-surface expression of CD73” recited in claim 1, this limitation is as evidence by the teachings of Sachs et al. (2012).
Sachs et al. (2012) teaches “Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites” (See Title), and flow cytometry for detection of cell surface markers “A total of 500,000 cells/sample were centrifuged at 1,000g for 1 min at 4 °C, resuspended in 75 μl of FACS buffer (PBS, 2 % FBS) and incubated on ice for 10 min. Cells were immunolabeled with 13 μg/μ1 of the respective primary antibody (CD14, CD29, CD31, CD34, CD45, CD73, or CD 105) (Millipore) for 30 min at 4 °C with rotation, followed by centrifugation and 3 washes with FACS buffer. Secondary antibody (Alexa 488 anti-mouse; Molecular Probes) was added at a dilution of 1:400 and incubated in the dark at 4 °C with rotation for 30 min. After washing, the resulting pellet was resuspended in 500 μl PBS and filtered through a 35-μm nylon mesh to remove aggregated cells. Cell suspensions were kept on ice until cytometric analysis, which was performed using a Coulter Epics XL-MCL (Beckman Coulter, Brea, CA, USA) (See left column, page 508); and summary presented in Table 1 (See page 511).
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Applicant’s arguments: Wilkinson does not teach expressly or inherently treating any disorder, much less a telomere-associated condition, nor administering fibroblasts to a subject, much less fibroblasts expressing CD73 on their cell surface. Thus, claim 1 is novel over Wilkinson.
Response to Applicant’s arguments: Claim 1 as written recites one active “providing to the subject an effective amount of isolated fibroblasts, ---, wherein the fibroblasts comprise cell-surface expression of CD73”, which is clearly anticipated by the teachings of Wilkinson et al. (2017) as evidence by Sachs et al. (2012). It is noted that the limitation “for treating a telomere-associated condition in a subject” recited in the preamble is the intended result of practicing the only active step recited in claim 1, “providing to the subject an effective amount of isolated fibroblasts”. Moreover, “a telomere-associated condition” encompasses any condition that is directly or indirectly associated, in the context of any genetic association and/or any molecular association, with any telomere function. In this regard, Wilkinson et al. teaches “a method for the generation of self-assembled human lung tissue and its potential for disease modeling and drug discovery for lung diseases characterized by progressive and irreversible scarring such as idiopathic pulmonary fibrosis (IPF)” (See Abstract). It is further as evidenced by Sachs et al. (2012), a condition of telomere shorting at molecular level indicates induction of senescence (See Fig. 1C, right column of page 509, and left column of page 514).
Claims 1-5, 32-33, 45-46 and 49 are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Barczyk et al. (2015) (Barczyk et al., Stem Cell-Based Therapy in Idiopathic Pulmonary Fibrosis, Stem Cell Rev Rep, 2015 Aug;11(4):598-620. doi: 10.1007/s12015-015-9587-7) as evidence by Sachs et al. (2012) (Sachs et al., Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites, Cell Tissue Res., 2012 Aug;349(2): 505-15.doi: 10.1007/s00441-012-1423-7. Epub 2012 May 25).
Regarding claims 1-3 and 49, Barczyk et al. teaches that “Idiopathic pulmonary fibrosis is a progressive fibrosing disorder for which there is no cure and no pharmacological treatment capable of increasing in a meaningful way the survival rate. Lung transplantation remains the only possible treatment for patients with advanced disease, although the increase in 5-year survival is only 45 %. Some preclinical studies have generated promising results about the therapeutic potential of exogenous stem cells. However, two initial clinical trials involving the endobronchial or systemic delivery of autologous adipose tissue-derived or unrelated-donor, placenta-derived mesenchymal stem cells have not convincingly demonstrated that these treatments are acceptably safe. The results of other ongoing clinical trials may help to identify the best source and delivery route of mesenchymal stem cells and to estimate the risk of unwanted effects related to the mesenchymal nature of the transplanted cells. Considering that most of the therapeutic potential of these cells has been ascribed to paracrine signaling, the use of mesenchymal stem cell-derived secretome as an alternative to the transplantation of single cell suspension may circumvent many regulatory and clinical problems. Technical and safety concerns still limit the possibility of clinical applications of other promising interventions that are based on the use of human amnion stem cells, embryonic stem cells or induced pluripotent stem cells to replace or regenerate the dysfunctional alveolar epithelium. We summarize the current status of the field and identify major challenges and opportunities for the possible future integration of stem cell-based treatments into the currently recommended clinical management strategy for idiopathic pulmonary fibrosis.” (See Abstract).
Regarding the limitation “wherein the fibroblasts comprise cell-surface expression of CD73” recited in claim 1, this limitation is as evidence by the teachings of Sachs et al. (2012).
Sachs et al. (2012) teaches “Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites” (See Title), and flow cytometry for detection of cell surface markers “A total of 500,000 cells/sample were centrifuged at 1,000g for 1 min at 4 °C, resuspended in 75 μl of FACS buffer (PBS, 2 % FBS) and incubated on ice for 10 min. Cells were immunolabeled with 13 μg/μ1 of the respective primary antibody (CD14, CD29, CD31, CD34, CD45, CD73, or CD105) (Millipore) for 30 min at 4 °C with rotation, followed by centrifugation and 3 washes with FACS buffer. Secondary antibody (Alexa 488 anti-mouse; Molecular Probes) was added at a dilution of 1:400 and incubated in the dark at 4 °C with rotation for 30 min. After washing, the resulting pellet was resuspended in 500 μl PBS and filtered through a 35-μm nylon mesh to remove aggregated cells. Cell suspensions were kept on ice until cytometric analysis, which was performed using a Coulter Epics XL-MCL (Beckman Coulter, Brea, CA, USA) (See left column, page 508); and summary presented in Table 1 (See page 511).
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Regarding claims 4-5, Barczyk et al. teaches that “A cell therapy based on the systemic administration of allogeneic MSCs with potent immunosuppressive properties may potentially prolong the survival of these patients by reducing the risk of rejections following organ transplantation and by allowing a reduced use of immunosuppressive drugs, which are much less tolerated by lung transplant recipients with IPF and associated telomere defects than by other recipients. The feasibility of this approach is already supported by specific in-vivo studies on organ transplantations and by the clinical benefit demonstrated with a similar treatment in children who develop an acute and life-threatening GVHD following allogeneic transplantation of hematopoietic stem cells” (See left column, page 616). Barczyk et al. further teaches that “additional attractive candidates for lung repair in IPF are HAECs, which have been harvested from term-delivered fetal membranes and display multilineage differentiation potential” (right column, page 610), which cites “Ilancheranet al. (2007). Stem cells derived from human fetal membranes display multi-lineage differentiation potential. Biology of Reproduction, 77, 577–588) (See reference #98, page 619). Barczyk et al. further cites “Mattoli, S., Bellini, A., & Schmidt, M. (2009). The role of a human hematopoietic mesenchymal progenitor in wound healing and fibrotic diseases and implications for therapy. Current Stem Cell Research & Therapy, 4, 266–280.” (See reference #49, page 618).
Regarding claim 32, routes of administration, Barczyk et al. teaches that “Additional attractive candidates for lung repair in IPF are HAECs, which have been harvested from term-delivered fetal membranes and display multilineage differentiation potential. In nude mice, the intravenous injection of these cells 24 h after bleomycin-induced lung injury was followed by their migration to the damaged alveoli and to areas of ongoing fibrogenesis in the lungs. Treatment with HAECs was associated with reduced production of proinflammatory and profibrotic cytokines, diminished collagen deposition, and attenuated fibrosis (Table 2). (See right column, page 610).
Regarding claims 1 and 33, wherein the fibroblasts express CD73, CD90, CD 105, CD 14, CD45, and/or CD34, this limitation is as evidence by the disclosure of Sachs et al. (2012) which teaches that fibroblasts express CD29, CD73, and CD105 based on flow cytometry analysis on cell surface markers. Moreover, Barczyk et al. teaches that “As reported elsewhere, one major limitation of studies employing adipose tissue-derived stromal cells and MSC is the heterogeneity of the administered cells and contamination with non-mesenchymal cells. The adipose tissue derived stromal cells-stromal vascular fraction includes adipose stromal stem and progenitor cells, pericytes, and fibroblasts, as well as contamining hematopoietic stem and progenitor cells, endothelial cells, lymphocytes, and monocytes/macrophages. In the study described above, the cells isolated from the lipoaspirates were not expanded in culture but were activated by treatment with autologous platelet-rich plasma and low-level laser irradiation before administration, on the basis of previously reported experience. No more than 1 % of the activated cells expressed the pan-hematopoietic marker CD45 and over 45 % of them expressed surface markers shared by stromal cells and MSCs (CD73, CD90, CD105) on flow cytometry analysis. (See left column, page 609).
Regarding a cellular graft recited in claims 45-46, Barczyk et al. teaches that “Lentiviral transduction of mouse MSCs (Tulane) and mouse HSCs to express KFG and promote AEC2 repair” and “Reduction in the expression of type I procollagen mRNA, lung collagen contents and fibrotic changes after bone marrow transplantation with transduced HSC.” (See Table 2, page 604).
Applicant’s arguments: Barczyk does not teach expressly or inherently treating a telomere-associated condition, such as idiopathic pulmonary fibrosis, by administering to a subject isolated fibroblasts that express CD73 on their surface.
Response to Applicant’s arguments: Claim 1 as written recites one active step of “providing to the subject an effective amount of isolated fibroblasts, ---, wherein the fibroblasts comprise cell-surface expression of CD73”, which is clearly anticipated by the teachings of Barczyk et al. (2015) as evidence by Sachs et al. (2012). It is noted that the limitation “for treating a telomere-associated condition in a subject” recited in the preamble of claim 1 is the intended result of practicing the only active step. Moreover, “a telomere-associated condition” encompasses any condition that is directly or indirectly associated, in the context of any genetic association and/or any molecular association, with any telomere function”. In this regard, Barczyk et al. teaches that “A cell therapy based on the systemic administration of allogeneic MSCs with potent immunosuppressive properties may potentially prolong the survival of these patients by reducing the risk of rejections following organ transplantation and by allowing a reduced use of immunosuppressive drugs, which are much less tolerated by lung transplant recipients with IPF (Idiopathic pulmonary fibrosis) and associated telomere defects than by other recipients” (See left column, page 616). It is further as evidenced by Sachs et al. (2012), a condition of telomere shorting at molecular level indicates induction of senescence (See Fig. 1C, right column of page 509, and left column of page 514).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1, 3-6, 12-13, 33, and 35-37 are rejected under 35 U.S.C. 103 as being unpatentable over Barczyk et al. (2015) (Barczyk et al., Stem Cell-Based Therapy in Idiopathic Pulmonary Fibrosis, Stem Cell Rev Rep, 2015 Aug;11(4):598-620. doi: 10.1007/s12015-015-9587-7) in view of Sachs et al. (2012) (Sachs et al., Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites, Cell Tissue Res., 2012 Aug;349(2): 505-15.doi: 10.1007/s00441-012-1423-7. Epub 2012 May 25), and Kim et al. (2013) (Kim et al., Human iPS cell-derived hematopoietic progenitor cells induce T-cell anergy in in vitro-generated alloreactive CD8+ T cells, Blood, 2013 Jun 27;121(26):5167-75. doi: 10.1182/blood-2012-11-467753. Epub 2013 May 17)
The teachings of Barczyk et al. (2015) and Sachs et al. (2012) have been documented above in the rejection of claims 1-5, 32-33, 45-46 and 49 under 35 U.S.C. 102(a)(1).
Barczyk et al. (2015) does not explicitly teach the limitations (i) presence and/or absence of marker gene expression recited in claim 6; and (ii) transfection of the fibroblasts with Oct4, Soz-2, Nanog, and/or Lin28 recited in claims 12-13; (iii) wherein the fibroblasts express CD73, CD90, CD 105, CD 14, CD45, and/or CD34 recited in claim 33; (iv) the fibroblasts isolated from umbilical cord tissue recited in claims 35 and 36; and (v) the presence and/or absence of marker gene expression in the fibroblast recited in claim 37.
It is worth noting that Sachs et al. (2012) teaches “Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites” (See Title), and flow cytometry for detection of cell surface markers “A total of 500,000 cells/sample were centrifuged at 1,000g for 1 min at 4 °C, resuspended in 75 μl of FACS buffer (PBS, 2 % FBS) and incubated on ice for 10 min. Cells were immunolabeled with 13 μg/μ1 of the respective primary antibody (CD14, CD29, CD31, CD34, CD45, CD73, or CD105) (Millipore) for 30 min at 4 °C with rotation, followed by centrifugation and 3 washes with FACS buffer. Secondary antibody (Alexa 488 anti-mouse; Molecular Probes) was added at a dilution of 1:400 and incubated in the dark at 4 °C with rotation for 30 min. After washing, the resulting pellet was resuspended in 500 μl PBS and filtered through a 35-μm nylon mesh to remove aggregated cells. Cell suspensions were kept on ice until cytometric analysis, which was performed using a Coulter Epics XL-MCL (Beckman Coulter, Brea, CA, USA) (See left column, page 508); and summary presented in Table 1 (See page 511).
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Furthermore, (i) Regarding claim 6 (a), the hematopoietic cells express c-kit, Sca-1, CD34, and/or CD33, Kim et al. (2013) teaches that “Human induced pluripotent stem cells (iPSCs) have emerged as an alternative source of pluripotent stem cells that can be used for tissue regeneration in place of the controversial human embryonic stem cells. However, immunologic knowledge about iPSC derivatives remains enigmatic. Here, we characterized human iPS-derived CD34+ hematopoietic progenitor cells (HPCs). --- These data indicate for the first time that HPCs induce T-cell anergy, a unique characteristic of iPSC derived cells that confers immunologic advantage for allogenic transplantation. Although iPSCs are ideal for patient-tailored treatments with the anticipation that no immunosuppression will be required, in cases of gene defects, their derivatives could be used to treat diseases in non-histocompatible recipients. (See Abstract).
Regarding claim 6 (b), the hematopoietic cells do not express a lineage marker, Kim et al. (2013) teaches that “In this study, we show that iPSC-derived CD34+ iPS-HPCs poorly express classical MHC antigens, lack CD80 and CD86, and highly express the T-cell inhibitory ligand PD-L1. (See left column, page 5168).
It is noted that both claims 5 and 6 depend from claim 4. Relevant to “an immuno-deficient host” recited in claim 5, Kim et al. (2013) teaches with “Key Points” that Human iPSCs differentiate into CD34+ HPCs (hematopoietic progenitor cells) and iPSC-derived HPCs induce T-cell anergy (i.e. absence of the normal immune response to a particular antigen or allergen).
(ii) Regarding claims 12-13, Kim et al. (2013) teaches that “Yamanaka and colleagues established induced pluripotent stem cells (iPSCs) by reprogramming fibroblasts into a pluripotent state by means of retroviral transduction of 4 factors: Oct 3/4, Sox2, Klf4, and c-Myc. (See right column, page 5167 of Kim et al.).
(iii) Regarding claim 33, wherein the fibroblasts express CD73, CD90, CD105, CD14, CD45, and/or CD34, Kim et al. teaches that “To determine the phenotype of iPS-HPCs, the
differentiated cells were isolated using 0.05% trypsin and stained with various antibodies directed against hematopoietic cell markers (Figure 1A). On day 9 of the differentiation process, HPCs expressed CD34 (17%), the erythroid cell marker CD235a (8.61%), CD34/CD43
(5.9%), and CD34/CD45 (3.3%). These results are summarized in Figure 1B. (See right column, page 5168).
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Figure 1A-1B. Human iPS-HPCs. (A) To determine the phenotype of iPS-HPCs, iPSCs cultivated on OP9 cells were harvested on differentiation day 9 and phenotypically characterized for various hematopoietic cell markers. iPS-HPCs expressed the hematopoietic cell surface markers CD34, CD43, CD45, and CD235a. We used the Tra-1-85 antibody to discriminate human cells. (B) The relative percentages of CD341, CD431, and CD451 cells during the differentiation days 7, 9, and 12.
(iv) Regarding the fibroblasts isolated from umbilical cord tissue recited in claims 35-36, Kim et al. (2013) teaches that Hematopoietic stem cells (HSCs) that are used in clinical transplantation are derived from bone marrow, peripheral blood, or umbilical cord blood (UCB). (See Introduction, left column, page 5167)
(v) Regarding the presence and/or absence of marker gene expression in the fibroblast recited in claim 37, Kim et al. (2013) teaches that “Here, we studied the MHC expression by iPS-HPCs, iPSCs, and the parental fibroblasts. ESCs, ESC-HPCs, and UCB- CD34+ cells were included as controls. Interestingly, MHC expression by iPSCs was less than that by fibroblasts and by UCB-CD34+ cells (Figure 3). This is consistent with our own studies on rat embryoniclike stem cells, which poorly expressed MHC, now a well-established characteristic of ESC-HPCs. Further, iPS-HPCs expressed the nonclassical MHC molecules HLA-G and HLA-E (Figure 3 and supplemental Table 3), albeit at modest levels. Additionally, HLA-G expression varied among the iPS cell lines used (supplemental Table 3). (See bridging paragraph between columns, page 5169, Kim et al., 2013).
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Figure 3. MHC expression profiles of human iPSCs and iPS-HPCs. Parental fibroblasts, iPSCs, iPS-HPCs, hESCs, ESC-HPCs, and UCB-CD341 cells (control) were stained with anti–HLA-ABC, HLA-DR, HLA-G, and HLA-E antibodies. The gray histograms indicate the isotype control. These data show that reprogrammed human iPSCs exhibit low expression of HLA-ABC compared with parental fibroblasts, whereas the expression of other molecules was not significantly different. The expression of the MHC molecules on iPSCs is very similar to that of hESCs. This pattern is also seen in iPS-HPCs and hESC-HPCs. However, the nonclassical MHC molecule HLA-G is upregulated after differentiation. All cell types expressed HLA-E. These data are representative of 7 experiments.
A skilled artisan would have been motivated to incorporate the teachings of Kim et al. (2013) into the teachings of Barczyk et al. (2015) and Sachs et al. (2012) because Kim et al. (2013) specifically teaches the methodology of inducing human pluripotent stem cells (iPSCs) by reprogramming fibroblasts into a pluripotent state by means of retroviral transduction of 4 factors: Oct 3/4, Sox2, Klf4, and c-Myc; and provide the immunologic knowledge about of iPS cell-derived hematopoietic progenitor cells.
There would have been a reasonable expectation of success because the teachings by Kim et al. (2013) clearly demonstrate in Figure 3 the MHC expression profiles of human iPSCs and iPS-HPCs by immunologic staining and characteristics of parental fibroblasts, iPSCs, iPS-HPCs, hESCs, ESC-HPCs, and UCB-CD341 cells (control) using anti–HLA-ABC, HLA-DR, HLA-G, and HLA-E antibodies.
Applicant’s arguments: Barczyk and Kim do not teach or suggest every element of claim 1 as required to support an obviousness rejection.
Response to Applicant’s arguments: Claim 1 as written recites one active “providing to the subject an effective amount of isolated fibroblasts, ---, wherein the fibroblasts comprise cell-surface expression of CD73”, which is clearly taught by Sachs et al. (2012). It is noted that the limitation “for treating a telomere-associated condition in a subject” recited in the preamble is the intended consequence of practicing the only active step recited in claim 1, “providing to the subject an effective amount of isolated fibroblasts. Moreover, “a telomere-associated condition” encompasses any condition that is directly or indirectly associated, in the context of any genetic association and/or any molecular association, with any telomere function. In this regard, Barczyk et al. teaches that “A cell therapy based on the systemic administration of allogeneic MSCs with potent immunosuppressive properties may potentially prolong the survival of these patients by reducing the risk of rejections following organ transplantation and by allowing a reduced use of immunosuppressive drugs, which are much less tolerated by lung transplant recipients with IPF (Idiopathic pulmonary fibrosis) and associated telomere defects than by other recipients”. Additionally, it is further as evidenced by Sachs et al. (2012), a condition of telomere shorting at molecular level indicates induction of senescence (See Fig. 1C, right column of page 509, and left column of page 514).
Claims 25-26 and 29 are rejected under 35 U.S.C. 103 as being unpatentable over Barczyk et al. (2015) (Barczyk et al., Stem Cell-Based Therapy in Idiopathic Pulmonary Fibrosis, Stem Cell Rev Rep, 2015 Aug;11(4):598-620. doi: 10.1007/s12015-015-9587-7) in view of Sachs et al. (2012) (Sachs et al., Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites, Cell Tissue Res., 2012 Aug;349(2): 505-15.doi: 10.1007/s00441-012-1423-7. Epub 2012 May 25), Kim et al. (2013) (Kim et al., Human iPS cell-derived hematopoietic progenitor cells induce T-cell anergy in in vitro-generated alloreactive CD8+ T cells, Blood, 2013 Jun 27; 121(26): 5167-75. doi: 10.1182/blood-2012-11-467753. Epub 2013 May 17), as applied to claims 1, 3-6, 12-13, 33, and 35-37 above, further in view of Faner et al. (2012) (Faner et al., Abnormal lung aging in chronic obstructive pulmonary disease and idiopathic pulmonary fibrosis, Am J Respir Crit Care Med, 2012 Aug 15;186(4):306-13. doi: 10.1164/rccm.201202-0282PP. Epub 2012 May 10.)
The teachings of Barczyk et al. (2015), Sachs et al. (2012), and Kim et al. (2013) have been documented above in the rejection of claims 1, 3-6, 12-13, 33, and 35-37 under 35 U.S.C. 103.
The combined teachings of Barczyk et al. (2015), Sachs et al. (2012), and Kim et al. (2013) do not explicitly teach reducing the rate of shorting of telomere length in a patient with idiopathic pulmonary fibrosis as recited in claim 25-26 and 29.
Faner et al. teaches that “Aging is a natural process characterized by progressive functional impairment and reduced capacity to respond appropriately to environmental stimuli and injury. The incidence of two common chronic respiratory diseases (chronic obstructive pulmonary disease [COPD] and idiopathic pulmonary fibrosis [IPF]) increases with advanced age. It is plausible, therefore, that abnormal regulation of the mechanisms of normal aging may contribute to the pathobiology of both COPD and IPF. This review discusses the available evidence supporting a number of aging mechanisms, including oxidative stress, telomere length regulation, cellular and immune-senescence, as well as changes in a number of antiaging molecules and the extracellular matrix, which are abnormal in COPD and/or IPF. A better understanding of these abnormalities may help in the design of novel and better therapeutic interventions for these patients.” (See Abstract). Faner et al. teaches that “The cellular and molecular mechanisms of physiological aging are still not well understood. Oxidative stress, telomere length regulation, cellular and immune-senescence, as well as changes in a number of antiaging molecules and in the extracellular matrix are thought to be key mechanisms. This
review discusses the available evidence that these mechanisms are abnormal in COPD and/or IPF (Table 1) and can therefore contribute to the pathogenesis of both diseases.” (See right column, page 306).
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Faner et al. teaches that B-MSCs (bone marrow-derived mesenchymal stem cells) are characterized by a quiescent state with low metabolic activity and are primarily in the G0 phase of the cell cycle. This quiescent state is maintained by both extrinsic and intrinsic mechanisms and has been postulated to be a way of preserving their long-term proliferative potential and genomic integrity. Conversely, DNA damage checkpoints and several repair pathways are cell cycle dependent, and the quiescent state of B-MSCs can underlie the propensity of these cells to accumulate DNA damage during aging, ultimately leading to rapid stem cell depletion or exhaustion (Figure 2). (See right column, page 308).
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Figure 2. Schematic representation of the mechanisms by which aged B-MSCs (bone marrow-derived mesenchymal stem cells) increase susceptibility to the development of fibrosis
through senescence and exhaustion of the stem cells. This hypothesis is supported by the observation that aging B-MSCs accumulate damage in their DNA and experience a decrease in their response to soluble factors, resulting in a decrease in their ability to repair damaged organs.
These observations are providing novel mechanisms to account for the higher incidence of
chronic fibrosing lung disorders in subjects exposed to tobacco, asbestos, and other agents
known to stimulate DNA damage.
Faner et al. teaches that “With the current state of knowledge we can only speculate that as the ethiological factors driving these two diseases (mainly epithelial injury and others still unknown for IPF, and smoking for COPD) are different, they result in the mechanism of normal aging being altered differently, in different sites, or with different repair mechanisms/capacity. As a corollary, we propose that the aging process is abnormal rather than accelerated in these patients, because these two diseases appear to be the result of defective and/or exhausted mechanisms of repair rather than their shift (accelerated aging) with early accumulations of these defects. (See Conclusions, page 311).
A skilled artisan would have been motivated to incorporate the teachings of Faner et al. (2012) into the combined teachings of Barczyk et al. (2015), Sachs et al. (2012), and Kim et al. (2013) because Faner et al. (2012) specifically teaches decreased telomere length in peripheral blood leukocytes (PBLs), and telomerase mutations are found in familial pulmonary fibrosis (FPF) and sporadic IPF (See Table 1); and proposed that the aging process is abnormal in IPF patients, because the disease appear to be the result of defective and/or exhausted mechanisms of repair.
Moreover, Faner et al. (2012) specifically teaches that senescent B-MSCs and fibrocytes increase the susceptibility to IPF because of abnormal lung repair in IPF patients (See Table 1); and that “Abnormalities in cellular senescence have also been demonstrated in patients with IPF, particularly in bone marrow-derived stem cells. These cells can be divided in two groups: (1) hematopoietic stem cells (B-HSCs) and (2) mesenchymal stem cells (B-MSCs). Both have been implicated in the pathogenesis of IPF.” (See right column, page 2012).
According to the proposed mechanisms of Faner et al. by which aged B-MSCs (bone marrow-derived mesenchymal stem cells) increase susceptibility to the development of fibrosis through senescence and exhaustion of the stem cells, there would have been a reasonable expectation of success to reduce the rate of shorting of telomere in the cells of a patient with IPF as recited in claims 25-26 and 29, by introducing iPSCs derived cells, including newly differentiated mesenchymal stem cells (B-MSCs), into the IPF patient based on combined teachings of Barczyk et al. (2015), Sachs et al. (2012), and Kim et al. (2013) to overcome defective and/or exhausted mechanisms of repair in the IPF patients.
Applicant’s arguments: Claim 1 is patentable over Barczyk and Kim as described above for failing to teach or suggest every element of the claimed invention as required to support an obviousness rejection. Fan er fails to cure the deficiencies of Barczyk.
Response to Applicant’s arguments: Claim 1 as written recites one active “providing to the subject an effective amount of isolated fibroblasts, ---, wherein the fibroblasts comprise cell-surface expression of CD73”, which is clearly taught by Sachs et al. (2012). It is noted that the limitation “for treating a telomere-associated condition in a subject” recited in the preamble is the intended consequence of practicing the active step, “providing to the subject an effective amount of isolated fibroblasts. Moreover, “a telomere-associated condition” encompasses any condition that is directly or indirectly associated, in the context of any genetic association and/or any molecular association, with any telomere function. In this regard, Barczyk et al. teaches that “A cell therapy based on the systemic administration of allogeneic MSCs with potent immunosuppressive properties may potentially prolong the survival of these patients by reducing the risk of rejections following organ transplantation and by allowing a reduced use of immunosuppressive drugs, which are much less tolerated by lung transplant recipients with IPF (Idiopathic pulmonary fibrosis) and associated telomere defects than by other recipients”. Additionally, it is further as evidenced by Sachs et al. (2012), a condition of telomere shorting at molecular level indicates induction of senescence (See Fig. 1C, right column of page 509, and left column of page 514).
Claims 1, 45 and 48 are rejected under 35 U.S.C. 103 as being unpatentable over Barczyk et al. (2015) (Barczyk et al., Stem Cell-Based Therapy in Idiopathic Pulmonary Fibrosis, Stem Cell Rev Rep, 2015 Aug;11(4):598-620. doi: 10.1007/s12015-015-9587-7) in view of Sachs et al. (2012) (Sachs et al., Defining essential stem cell characteristics in adipose-derived stromal cells extracted from distinct anatomical sites, Cell Tissue Res., 2012 Aug;349(2): 505-15.doi: 10.1007/s00441-012-1423-7. Epub 2012 May 25), and Wang et al. (2011) (Wang et al., Lithium, an anti-psychotic drug, greatly enhances the generation of induced pluripotent stem cells, Cell Res 2011 Oct;21(10):1424-35. doi: 10.1038/cr.2011.108. Epub 2011 Jul 5).
The teachings of Barczyk et al. (2015) and Sachs et al. (2012) have been documented above in the rejection of claims 1-5, 32-33, 45-46 and 49 under 35 U.S.C. 102(a)(1). Claims 48 depends from claims 1, and 45.
Barczyk et al. (2015) and Sachs et al. (2012) do not explicitly teach the limitations further comprising providing a lithum-containing compound recited in claims 45 and 48.
Regarding claim 45, providing a lithum-containing compound, Wang et al. (2011) teaches that “Lithium (Li), a drug used to treat mood disorders, greatly enhances iPSC generation from both mouse embryonic fibroblast and human umbilical vein endothelial cells. Li facilitates iPSC generation with one (Oct4) or two factors (OS or OK). The effect of Li on promoting reprogramming only partially depends on its major target GSK3β. Unlike other GSK3β inhibitors, Li not only increases the expression of Nanog, but also enhances the transcriptional activity of Nanog. We also found that Li exerts its effect by promoting epigenetic modifications via downregulation of LSD1, a H3K4-specific histone demethylase. Knocking down LSD1 partially mimics Li's effect in enhancing reprogramming. Our results not only provide a straightforward method to improve the iPSC generation efficiency, but also identified a histone demethylase as a critical modulator for somatic cell reprogramming” (See Abstract).
Regarding claim 48, providing a lithum-containing compound is lithum chloride, Wang et al. (2011) teaches that “in this study, we discovered that Li can enhance the reprogramming of somatic cells to iPSCs. By adding LiCl to the culture medium for a short period of time (day
3-8), we can obtain high-quality iPSCs with efficiency greater than 10% in both 4F- and 3F induced reprogramming. in MEF. Li also enhanced two factor (OS)- or one factor (Oct4)-mediated reprogramming in HUVEC, indicating that the mechanism of its action might be similar in both mouse and human system. (See Right column, Discussion, page 1430, Wang et al., 2011).
A skilled artisan would have been motivated to incorporate the teachings of Wang et al. (2011) into the teachings of Barczyk et al. (2015) and Sachs et al. (2012) because Wang et al. (2011) specifically teaches that “Lithium, an anti-psychotic drug, greatly enhances the generation of induced pluripotent stem cells” (See Title).
There would have been a reasonable expectation of success because the teachings by Wang et al. (2011) clearly demonstrate the in Figure 3 that Lithium (5 mM LiCl) enhances the generation of mouse and human iPS cells with one or two factors.
Applicant’s arguments: Claim 1 is patentable over Barczyk as described above for failing to teach or suggest every element of the claimed invention as required to support an obviousness rejection. Wang fails to cure the deficiencies of Barczyk.
Response to Applicant’s arguments: Claim 1 as written recites one active “providing to the subject an effective amount of isolated fibroblasts, ---, wherein the fibroblasts comprise cell-surface expression of CD73”, which is clearly taught by Sachs et al. (2012). It is noted that the limitation “for treating a telomere-associated condition in a subject” recited in the preamble is the intended consequence of practicing the active step. Moreover, “a telomere-associated condition” encompasses any condition that is directly or indirectly associated, in the context of any genetic association and/or any molecular association, with any telomere function. In this regard, Barczyk et al. teaches that “A cell therapy based on the systemic administration of allogeneic MSCs with potent immunosuppressive properties may potentially prolong the survival of these patients by reducing the risk of rejections following organ transplantation and by allowing a reduced use of immunosuppressive drugs, which are much less tolerated by lung transplant recipients with IPF (Idiopathic pulmonary fibrosis) and associated telomere defects than by other recipients”. Additionally, it is further as evidenced by Sachs et al. (2012), a condition of telomere shorting at molecular level indicates induction of senescence (See Fig. 1C, right column of page 509, and left column of page 514).
Conclusion
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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/WU CHENG W SHEN/ Supervisory Patent Examiner, Art Unit 1682
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