DETAILED ACTION
This action is in reply to papers filed 10/15/2025.
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 .
Status of Claims
Claims 1-3, 7-10, 12-20, and 24-26 are pending and examined herein.
Claim 1 is currently amended.
Claim 6 is currently cancelled.
Rejection
The cancellation of claim 6 renders any rejections thereof moot.
The rejection of claim 12 over Garcia-Bennett et al., in view of Cheng et al., Lian et al., Jenova et al., Vivero-Escoto et al., as evidenced by Yakin et al., is withdrawn.
Applicant’s arguments are addressed following the maintained rejections.
Claim Rejections - 35 USC § 112(a)
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 1-3, 7-10, 12-20, 24-26 remain 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 applications subject to pre-AIA 35 U.S.C. 112, the inventor(s), at the time the application was filed, had possession of the claimed invention. This is a New Matter rejection.
Claim 1 is drawn to a method for inducing cardiomyocyte differentiation in iPS cells comprising contacting iPS cells with one or more first porous silica nanoparticles comprising a GSK3 inhibitor, and one or more second porous silica nanoparticles comprising a Wnt signaling inhibitor, wherein the porous silica nanoparticles have a particle size of about 230 nm to about 300 nm. Claim 18 is drawn to a porous silica nanoparticle comprising a GSK3 inhibitor and a Wnt signaling inhibitor wherein the porous silica nanoparticle has a particle size of about 230 nm to about 300 nm. Claim 24 is drawn to a method for inducing cardiomyocyte differentiation in iPS cells comprising contacting iPS cells with a porous silica nanoparticle comprising a GSK3 inhibitor and a Wnt signaling inhibitor wherein the porous silica nanoparticle has a particle size of about 230 nm to about 300 nm.
Therefore, the claims are drawn to porous silica nanoparticles comprising a GSK3 inhibitor and a Wnt signaling inhibitor wherein the porous silica nanoparticles have a particle size of about 230 nm to about 300 nm, and methods of using said porous silica nanoparticles. The specification describes the coated porous silica nanoparticles have an average particle size range between 2 and 20 micrometers in diameter (2,000-20,000 nm) (pg. 3, lines 12-13 and 24-25; pg. 5, lines 22-23). The specification further describes PLGA-porous silica (pSi) (coated) nanoparticles that measured 8.24 ± 3.25 µm in diameter and uncoated pSi particles that measured 265 nm in diameter (Example 1- pg. 12, lines 10-13).
The specification does not describe porous silica nanoparticles having a particle diameter other than 265 nm or a particle size between 2 and 20 micrometers in diameter. While Example 1 in pg. 12 of the specification describes uncoated pSi particles that measured 265 nm in diameter, per dynamic light scattering, the specification and drawings do not provide support for the specific values of 230 nm to about 300 nm in the claimed range. Thus, the concept of porous silica nanoparticles having a particle size of about 230 nm to about 300 nm lacks support other than porous silica nanoparticles having a particle diameter of 265 nm or coated particle size between 2 and 20 micrometers in diameter.
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.
Claims 1-3 remain rejected under 35 U.S.C. 103 as being unpatentable over Garcia-Bennett et al. (Stem Cells Translational Medicine, 2013, 2:906-915) in view of Cheng et al. (Nanoscale, 2016, 8:2011-2021) (ref. of record) in further view of Lian et al. (PNAS, 2012, pp. E1848-E1857) (ref. of record) and Jenova et al. (Proliferation of Adult Human Small Intestinal Epithelial Stem Cells by a GSK-3β Inhibitor Encapsulated in Poly Lactic-co-Glycolic Acid Nanoparticles, Thesis, 2014, University of California, Los Angeles).
With respect to claim 1, Garcia-Bennett teaches a method for the delivery of exogenous trophic factor mimetics of CNTF and GDNF, Cintrofin and Gliafin (differentiation factors), to embryonic stem cells (ESCs) using mesoporous silica nanoparticles (abstract). In an in vitro assay, ESCs were cultured to form embryoid bodies (EBs) and the EBs were cultured in Advanced Dulbecco’s modified Eagle’s medium/F12:Neurobasal with supplements (in vitro cell culture media) and with mesoporous silica nanoparticles loaded with Cintrofin and mesoporous silica nanoparticles loaded with Gliafin to induce differentiation into spinal motor neurons in a single treatment step (pg. 908, right col. par. 2-4). Garcia-Bennett discloses that mesoporous silica nanoparticles are attractive as highly robust and tunable delivery systems for various uses including controlled drug release and controlled release of differentiation factors with the aim of inducing cell differentiation (Introduction right col.). In addition, Garcia-Bennett teaches that altering pore sizes and other physical properties of mesoporous silica may even permit the sequential delivery of trophic factors with defined concentrations locally to the differentiating stem cells, thereby allowing a more direct transfer of in vitro differentiation protocols to in vivo applications (pg. 913, right col., par. 1). Furthermore, Garcia-Bennett adds that an asset of such a vehicle is its applicability in vitro since controlled long-term release of differentiating factors from the mesoporous silica may eliminate the need for repeated administration of these factors in their free form and, thus, save both time and costs, and reduce the risk of culture contamination (pg. 913, right col, par. 1).
While Garcia-Bennett practiced a method of inducing differentiation of pluripotent stem cells by treating the cells simultaneously with mesoporous silica nanoparticles comprising one differentiation factor and other mesoporous silica nanoparticles comprising another differentiation factor, Garcia-Bennett does not teach inducing cardiomyocyte differentiation in iPS cells by treating the cells with a small molecule GSK3 inhibitor and a small molecule Wnt signaling inhibitor loaded in spherical or rod-shaped mesoporous silica nanoparticles, wherein the particles have a size of about 230 nm to about 300 nm.
Cheng discloses the use of spherical FITC-mesoporous silica nanoparticles (FMSNs) as a drug delivery platform to transport 5-azazytidine (small molecule) into P19 embryonic carcinoma stem cells in vitro to induce the stem cells’ differentiation into cardiomyocytes (abstract; pg. 2012, left col., par. 3; pg. 2016, left col., par. 2.4; Fig. 1). P19 cells treated with the mesoporous silica nanoparticles comprising 5-azacytidine showed upregulation of two cardiac marker genes and two differentiation genes, which Cheng teaches it demonstrates the efficient role of the small molecule in inducing differentiation of the cells with the application of the mesoporous silica nanoparticles (pg. 2018, left col., par. 2-3). Cheng’s mesoporous silica nanoparticles possessed a spherical shape and a particle size of 223 ± 6.8 nm with 5-azacytidine loaded, which considering the uncertainty the particle size could be about 230nm (pg. 2012, right col., par. 2.1 to pg. 2013, left col.). Cheng discloses coating the surface of the mesoporous silica nanoparticles with biodegradable polymer PAH (pg. 2013, left col.). It is noted that claim 1 recites a result of the method (achieve delayed-release kinetics of the inhibitors). Since the cited prior art references in combination teach MSNs coated with a polymer as instantly claimed, then the limitation of achieving delayed-release kinetics of the inhibitors is interpreted as an inherent result.
Although Cheng induces the differentiation of pluripotent stem cells into cardiomyocytes by treating the cells with mesoporous silica nanoparticles loaded with a small molecule, Cheng fails to disclose inducing the differentiation of iPS cells with the simultaneous contacting of the cells with mesoporous silica nanoparticles comprising a GSK3 inhibitor and mesoporous silica nanoparticles loaded with Wnt signaling inhibitor, as recited in instant claim 1.
Lian discloses differentiation of iPS cells via small molecule modulation of regulatory elements of Wnt/β-catenin signaling (see abstract; Results in pg. 1). Lian discloses that appropriate modulation of regulatory elements of Wnt signaling alone via small molecule inhibitors is sufficient to drive multiple hPSC lines to differentiate to cardiomyocytes efficiently (pg. E1848, right col., par. 1). Furthermore, by modulating regulatory elements of Wnt signaling, Lian generated populations consisting of up to 98% cardiomyocytes with an extremely high yield from hPSCs without any enrichment and/or purification step (pg. 1, right col.). Lian maintained 19-9-11 iPSCs in fully defined, mTeSR™1 serum-free medium on Matrigel® or Synthemax® for 5 days, then switched to a medium containing CHIR99021 (GSK3-inhibitor), and at day 3 added porcupine inhibitor IWP2 (Wnt signaling inhibitor) (claim 2), which increased the purity of cardiomyocyte cells to 87% (pg. E1853, left col., par. 1).
Lian fails to disclose using porous silica nanoparticles or any other kind of nanoparticle comprising a GSK3 inhibitor and a Wnt signaling inhibitor to induce cardiomyocyte differentiation, as recited in claim 1.
As further support, regarding claims 1 and 3, Jenova teaches the encapsulation of CHIR99021 (small molecule GSK3 inhibitor) using PLGA nanoparticles on small intestinal epithelial stem cells and discusses that the small molecule inhibitor encapsulated in said PLGA nanoparticles was pharmacologically active and efficient (abstract in pg. iii). Jenova encapsulated the small molecule GSK3 inhibitor in PLGA nanoparticles motivated by several of its features including controlled/sustained drug release properties (pg. 10).
Accordingly, it would have been obvious at the effective filing date of the claimed invention to combine the teachings of Garcia-Bennett regarding the delivery of differentiation factors using porous silica nanoparticles, the teachings of Cheng regarding the use of spherical mesoporous silica nanoparticles to transport 5-azazytidine, a small molecule differentiation factor to induce differentiation of pluripotent stem cells into cardiomyocytes, the teachings of Lian regarding the differentiation of iPS cells via small molecule modulation of regulatory elements of Wnt/β-catenin signaling, and the teachings of Jenova regarding the encapsulation of small molecule GSK3 inhibitor CHIR99021, to arrive at the invention as claimed. One of ordinary skill in the art would have been motivated to make the combination since these prior art references teach the simultaneous delivery of differentiation factors in porous silica nanoparticles to induce the differentiation of pluripotent stem cells, and specifically teaches using porous silica nanoparticles to induce differentiation of pluripotent stem cells into cardiomyocytes by delivering small molecules in porous silica nanoparticles. Therefore, it would have been obvious to adapt Lian’s protocol (of inducing differentiation of iPS cells into cardiomyocytes by delivering a GSK3 inhibitor and a Wnt signaling inhibitor) by delivering said small molecules to iPS cells in porous silica nanoparticles simultaneously as taught by Garcia-Bennett since Cheng demonstrated that pluripotent stem cells can be effectively differentiated into cardiomyocytes by delivering small molecules in porous silica nanoparticles and supported by the evidence provided by Jenova of successfully delivering small molecule CHIR99021 encapsulated in nanoparticles.
Furthermore, since the prior art taught that porous silica nanoparticles offer sequential delivery of trophic factors and controlled release of differentiation factors (Garcia-Bennett), one of ordinary skill in the art would have combined the prior art teachings described above with the purpose of efficiently inducing the differentiation of iPS cells into cardiomyocytes while eliminating the need for repeated administration of these factors in their free form, thus saving both time and costs, and reducing the risk of culture contamination, as taught by Garcia-Bennett.
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the time the invention was made, especially in the absence of evidence to the contrary.
Claims 7-10 remain rejected under 35 U.S.C. 103 as being unpatentable over Garcia-Bennett et al. (Stem Cells Translational Medicine, 2013, 2:906-915) in view of Cheng et al. (Nanoscale, 2016, 8:2011-2021) (ref. of record) in further view of Lian et al. (PNAS, 2012, pp. E1848-E1857) (ref. of record) and Jenova et al. (Proliferation of Adult Human Small Intestinal Epithelial Stem Cells by a GSK-3β Inhibitor Encapsulated in Poly Lactic-co-Glycolic Acid Nanoparticles, Thesis, 2014, University of California, Los Angeles) as applied to claims 1-3 above, in further view of Vivero-Escoto et al. (Small, 2010) (ref. of record).
The teachings of Garcia-Bennett, Cheng, Lian and Jenova can be found in the previous rejection above.
With respect to claim 7, neither Garcia-Bennett nor Lian, Cheng or Jenova disclose porous silica nanoparticles coated with PLGA, PEG, PCL or PLLA. While Cheng discloses coating the surface of the mesoporous silica nanoparticles with biodegradable polymer PAH (pg. 2013, left col.) and Jenova teaches PLGA nanoparticles, neither Cheng nor Jenova teach porous silica nanoparticles coated with PLGA or any of the polymers recited in claim 7.
Vivero-Escoto reviews mesoporous silica nanoparticles (MSNs) for intracellular drug delivery (see abstract). Vivero-Escoto discloses surface functionalization of mesoporous-silica-based nanocarriers with polymers for controlled release of various cargos (see abstract). The MSNs can include a protecting polymer, such as PEG (Scheme 1D, pg. 1954).
Accordingly, it would have been obvious to one of ordinary skill in the art at the effective filing date of the claimed invention to coat the porous silica nanoparticles with a biodegradable polymer, such as PEG, since the modification of porous silica nanoparticles with polymers and other molecules to achieve different desired effects was known and described in the prior art, as taught by Cheng and Vivero-Escoto. One of ordinary skill would have been motivated to combine the teachings of Garcia-Bennett with the teachings of Cheng and Lian, as discussed in the previous rejection, and with the Teachings of Vivero-Escoto by coating the porous silica nanoparticles with a biodegradable polymer, such as PEG, with the purpose of modifying the MSNs to achieve controlled release of the differentiation factor, as taught by Vivero-Escoto (pg. 1954, right col., par. 1). Furthermore, Vivero-Escoto taught other benefits of including a polymer, such as shielding the mesoporous silica nanoparticle surface from interacting with opsonizing proteins (Scheme 1D in pg. 1954).
With respect to claims 8 and 9, Garcia-Bennett, Cheng and Jenova do not teach using defined, serum-free medium. Lian teaches maintaining the iPS cells in fully defined, mTeSR™1 serum-free medium on Matrigel® or Synthemax® for 5 days (pg. E1853, left col., par. 1). Lian further teaches that the use of small molecules instead of growth factors ultimately could allow inexpensive and reproducible generation of human cardiomyocytes or multipotent tissue-specific stem cells in completely chemically defined conditions, facilitating translation of these cells to high throughput screening applications or regenerative therapies (pg. E1854, right col, last par.).
Accordingly, a skilled artisan would have been motivated to use a defined, serum-free medium as recited in claims 8 and 9 with the object of developing a robust and scalable method of producing cardiomyocytes that can be translated to highthroughput screening applications or regenerative therapies, as taught by Lian.
Regarding claim 10, Garcia Bennett teaches that the average particle size for mesoporous silica nanoparticles is 12 micrometers (Introduction, right col.), which falls within the claimed range. Furthermore, Cheng teaches that particle size is tunable (pg. 2012, left col., par. 2).
Accordingly, it would have been obvious to prepare and use porous silica nanoparticles coated with a biodegradable polymer having an average particle size range between 2 and 20 micrometers in diameter since the prior art, Garcia-Bennett and Cheng, taught that porous silica nanoparticles have average particle sizes falling in the claimed range and that the particle size is tunable, which would have been determined by a person of ordinary skill in the art depending on the application of the porous silica nanoparticles.
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the time the invention was made, especially in the absence of evidence to the contrary.
Claims 13-16 remain rejected under 35 U.S.C. 103 as being unpatentable over Garcia-Bennett et al. (Stem Cells Translational Medicine, 2013, 2:906-915) in view of Cheng et al. (Nanoscale, 2016, 8:2011-2021) (ref. of record) in further view of Lian et al. (PNAS, 2012, pp. E1848-E1857) (ref. of record) and Jenova et al. (Proliferation of Adult Human Small Intestinal Epithelial Stem Cells by a GSK-3β Inhibitor Encapsulated in Poly Lactic-co-Glycolic Acid Nanoparticles, Thesis, 2014, University of California, Los Angeles) as applied to claims 1-3 above, in further view of Vivero-Escoto et al. (Small, 2010) (ref. of record) as applied to claims 7-10 above ,in further view of Anneren et al (US 20130236970) (ref. of record).
The teachings of Garcia-Bennett, Cheng, Lian and Vivero-Escoto can be found in the previous rejection above.
With respect to claims 13-16, none of Garcia-Bennett, Cheng, Lian and Vivero-Escoto teach performing the differentiation of the iPSCs into cardiomyocytes in a bioreactor. Anneren discloses culture of cells in a bioreactor (abstract). Anneren discloses (abstract and figure 1) the bioreactor comprises a plastic bag container allowing addition of further cell culture medium and culturing the cells under gentle and constant agitation (a vessel formed of a flexible or rigid disposable container coupled to a means for mixing liquid contents in the container, claim 14, part a) (at least one media introduction port in fluid contact with the container, claim 14, part b). Anneren discloses (figure 1) media is pumped out to a waster compartment (at least one media removal port in fluid contact with the container,” claim 14, part c). Anneren discloses [0025] media is pumped out from the bag while carriers remain in the bag by including a filter in the transfer tube (2B).
Anneren discloses [0036] a vessel is divided into a retentate compartment 11;21 and a permeate compartment 12;22 by a screen (which can also be called a strainer or sieve) 13;23. Anneren discloses [0036] when the microcarrier/cell suspension is fed into the vessel via the inlet 16;26, the detached cells pass through the screen 13;23 and are recovered via outlet 19;29, while the microcarriers are retained by the screen and stay in the retentate compartment 11;21.
Anneren discloses [0036] the screen is selected with pores of such a size and structure that detached cells pass though the pores, but the microcarriers do not (wherein the bioreactor further comprises a retention screen formed by pores in and opening through the container, wherein the pores are sized to retain the porous silica nanoparticles in the container while fluid cell culture media is removed from the vessel; claim 15).
Anneren discloses [0041] the microcarrier/cell suspension can be fed into the device via an inlet 36;46 (injecting a fluid through the at least one introduction port;” claim 16, part a) and if a crossflow type separation is desired it can exit the device via an outlet 39;49 (the claimed “removing fluid cell culture media through the at least one media removal port ;” claim16 part b). Anneren discloses [0041] the microcarriers are retained by the screen, while the detached cells can pass through the screen and be recovered via an outlet 39;49. Anneren discloses [0043] a buffer (e.g., PBS) or cultivation medium is flowed through the device to wash the cell- laden microcarriers (the claimed “to maintain a substantially steady stated equilibrium of the fluid volume within the container throughout the differentiation of cardiomyocytes;” claim 16-part c).
Therefore, it would have been obvious to one of ordinary skill to modify Garcia-Bennett’s method in view of Cheng, Lian and Vivero-Escoto by growing the cells in a bioreactor as suggested by Anneren in view of the teachings that the cell culture in a bioreactor allows for clinical scale production [0007]. One of ordinary skill would have had a reasonable expectation of success in growing the cells in a bioreactor since Anneren disclosed that the method is capable of expansion from a small volume with a low number of cells and ending with high numbers of cells suitable for, for example, cell therapy or vaccine antigen production [0008]. Moreover, one of ordinary skill would have been motivated to use the Anneren method of cell culture for expansion of cells in view of the teachings of Anneren that the method is capable of expansion from a small volume with a low number of cells and ending with high numbers of cells suitable for, for example, cell therapy or vaccine antigen production [0008].
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the time the invention was made, especially in the absence of evidence to the contrary.
Claim 17 remains rejected under 35 U.S.C. 103 as being unpatentable over Garcia-Bennett et al. (Stem Cells Translational Medicine, 2013, 2:906-915) in view of Cheng et al. (Nanoscale, 2016, 8:2011-2021) (ref. of record) in further view of Lian et al. (PNAS, 2012, pp. E1848-E1857) (ref. of record) and Jenova et al. (Proliferation of Adult Human Small Intestinal Epithelial Stem Cells by a GSK-3β Inhibitor Encapsulated in Poly Lactic-co-Glycolic Acid Nanoparticles, Thesis, 2014, University of California, Los Angeles) as applied to claims 1-3 above, in further view of Vivero-Escoto et al. (Small, 2010) (ref. of record) as applied to claims 7-10 above, in further view of Anneren et al (US 20130236970) (ref. of record) as applied to claims 13-16 above, in further view of Ma et al (US 2015/0299658) (ref. of record).
The teachings of Garcia-Bennett, Cheng, Lian, Vivero-Escoto and Anneren can be found in the previous rejection above.
With respect to claim 17, none of the previously cited references explicitly teach transplanting induced cardiomyocytes into a mammal.
Ma discloses induced pluripotent stem cells differentiated into myocytes (the claimed cardiomyocytes) in vitro (abstract). Ma discloses (abstract) the human cardiomyocytes obtained via differentiation can be used to treat myocardial infarction (further comprising transplantation of the induced cardiomyocyte into a mammal in need of such treatment; claim 17).
Therefore, it would have been obvious to one of ordinary skill to modify Garcia-Bennett’s method in view of Cheng, Lian, Jenova, Vivero-Escoto and Anneren by implanting the obtained cardiomyocytes into a mammal in need thereof in view of the teachings of Ma that in myocardial infarction, the consequent decline in cardiac function is typically irreversible because adult CMs (cardiomyocytes) have lost their ability to proliferate and are unable to repair necrotic tissue. One of ordinary skill would have had a reasonable expectation of success in implanting iPSC-derived cardiomyocytes as claimed in view of the teachings of Ma that the human cardiomyocytes obtained via differentiation can be used to treat myocardial infarction. Moreover, one of ordinary skill would have been motivated to transplant iPS derived cardiomyocytes in view of the teachings of Ma [0002] that because adult CMs have almost no ability to proliferate, a source of human CMs is apparently needed for regenerative medicine, for example, for treating myocardial infarction.
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the time the invention was made, especially in the absence of evidence to the contrary.
Claims 18-20 and 24-26 are rejected under 35 U.S.C. 103 as being unpatentable over Garcia-Bennett et al. (Stem Cells Translational Medicine, 2013, 2:906-915) in view of Cheng et al. (Nanoscale, 2016, 8:2011-2021) (ref. of record) in further view of Lian et al. (PNAS, 2012, pp. E1848-E1857) (ref. of record) in further view of Bhatia et al. (US 2008/0213377 A1).
With respect to claims 18, 19 and 24, Garcia-Bennett teaches a method for the delivery of exogenous trophic factor mimetics of CNTF and GDNF, Cintrofin and Gliafin (differentiation factors), to embryonic stem cells (ESCs) using mesoporous silica nanoparticles (abstract). In an in vitro assay, ESCs were cultured to form embryoid bodies (EBs) and the EBs were cultured in Advanced Dulbecco’s modified Eagle’s medium/F12:Neurobasal with supplements (in vitro cell culture media) and with mesoporous silica nanoparticles loaded with Cintrofin and mesoporous silica nanoparticles loaded with Gliafin to induce differentiation into spinal motor neurons in a single treatment step (pg. 908, right col. par. 2-4). Garcia-Bennett discloses that mesoporous silica nanoparticles are attractive as highly robust and tunable delivery systems for various uses including controlled drug release and controlled release of differentiation factors with the aim of inducing cell differentiation (Introduction right col.). In addition, Garcia-Bennett teaches that altering pore sizes and other physical properties of mesoporous silica may even permit the sequential delivery of trophic factors with defined concentrations locally to the differentiating stem cells, thereby allowing a more direct transfer of in vitro differentiation protocols to in vivo applications (pg. 913, right col., par. 1). Furthermore, Garcia-Bennett adds that an asset of such a vehicle is its applicability in vitro since controlled long-term release of differentiating factors from the mesoporous silica may eliminate the need for repeated administration of these factors in their free form and, thus, save both time and costs, and reduce the risk of culture contamination (pg. 913, right col, par. 1).
While Garcia-Bennett practiced a method of inducing differentiation of pluripotent stem cells by treating the cells simultaneously with mesoporous silica nanoparticles comprising one differentiation factor and other mesoporous silica nanoparticles comprising another differentiation factor, Garcia-Bennett does not teach a small molecule GSK3 inhibitor and a small molecule Wnt signaling inhibitor loaded in a spherical or rod-shaped mesoporous silica nanoparticle, wherein the particle has a size of about 230 nm to about 300 nm as recited in claim 18, and also fails to teach inducing cardiomyocyte differentiation in iPS cells by treating the cells with a porous silica nanoparticle comprising a small molecule GSK3 inhibitor and a small molecule Wnt signaling inhibitor, wherein the particle has a size of about 230 nm to about 300 nm and wherein the nanoparticle is coated with a biodegradable polymer, as recited in claims 19 and 24.
However, Cheng discloses the use of spherical FITC-mesoporous silica nanoparticles (FMSNs) as a drug delivery platform to transport 5-azazytidine (small molecule) into P19 embryonic carcinoma stem cells in vitro to induce the stem cells’ differentiation into cardiomyocytes (abstract; pg. 2012, left col., par. 3; pg. 2016, left col., par. 2.4; Fig. 1). P19 cells treated with the mesoporous silica nanoparticles comprising 5-azacytidine showed upregulation of two cardiac marker genes and two differentiation genes, which Cheng teaches it demonstrates the efficient role of the small molecule in inducing differentiation of the cells with the application of the mesoporous silica nanoparticles (pg. 2018, left col., par. 2-3). Cheng’s mesoporous silica nanoparticles possessed a spherical shape and a particle size of 223 ± 6.8 nm with 5-azacytidine loaded, which considering the uncertainty the particle size could be about 230nm (pg. 2012, right col., par. 2.1 to pg. 2013, left col.). With respect to claims 19 and 24, Cheng discloses coating the surface of the mesoporous silica nanoparticles with biodegradable polymer PAH (pg. 2013, left col.). It is noted that claims 19 and 24 contain limitations interpreted as expected results of the method (achieve delayed-release kinetics of the inhibitors). Since the cited prior art references in combination teach porous silica nanoparticles coated with a polymer as instantly claimed, then the limitation of achieving delayed-release kinetics of the inhibitors is interpreted as an inherent result.
Although Cheng induces the differentiation of pluripotent stem cells into cardiomyocytes by treating the cells with mesoporous silica nanoparticles loaded with a small molecule, Cheng fails to disclose a mesoporous silica nanoparticle comprising a GSK3 inhibitor and a Wnt signaling inhibitor as recited in claim 18, or inducing the differentiation of iPS cells by contacting the cells with a mesoporous silica nanoparticle comprising a GSK3 inhibitor a Wnt signaling inhibitor, as recited in instant claim 24.
However, Lian discloses differentiation of iPS cells via small molecule modulation of regulatory elements of Wnt/β-catenin signaling (see abstract; Results in pg. 1). Lian discloses that appropriate modulation of regulatory elements of Wnt signaling alone via small molecule inhibitors is sufficient to drive multiple hPSC lines to differentiate to cardiomyocytes efficiently (pg. E1848, right col., par. 1). Furthermore, by modulating regulatory elements of Wnt signaling, Lian was able to generate populations consisting of up to 98% cardiomyocytes with an extremely high yield from hPSCs without any enrichment and/or purification step (pg. 1, right col.). Lian maintained 19-9-11 iPSCs in fully defined, mTeSR™1 serum-free medium on Matrigel® or Synthemax® for 5 days, then switched to a medium containing CHIR99021 (GSK3-inhibitor), and at day 3 added porcupine inhibitor IWP2 (Wnt signaling inhibitor) (claim 2), which increased the purity of cardiomyocyte cells to 87% (pg. E1853, left col., par. 1).
Lian fails to disclose using a porous silica nanoparticle or any other kind of nanoparticle comprising a GSK3 inhibitor and a Wnt signaling inhibitor to induce cardiomyocyte differentiation, as recited in claim 24.
As further support, regarding claims 18-20 and 24-26, Bhatia teaches a nanoparticle for the delivery of one or more agents [0103] [0105], wherein the particle may be a porous silica nanoparticle [0123] that may have a coating layer including coating materials such as PEG [0124] and which can be used to deliver one or more small molecules [0237] [0261].
Accordingly, it would have been obvious at the effective filing date of the claimed invention to combine the teachings of Garcia-Bennett regarding the delivery of differentiation factors using porous silica nanoparticles, the teachings of Cheng regarding the use of spherical mesoporous silica nanoparticles to transport 5-azazytidine, a small molecule differentiation factor to induce differentiation of pluripotent stem cells into cardiomyocytes, the teachings of Lian regarding the differentiation of iPS cells via small molecule modulation of regulatory elements of Wnt/β-catenin signaling, and the teachings of Bhatia regarding PEG-coated porous silica nanoparticles used to deliver multiple small molecules, to arrive at the invention as claimed. One of ordinary skill in the art would have been motivated to make the combination since these prior art references teach in combination the delivery of differentiation factors in porous silica nanoparticles to induce the differentiation of pluripotent stem cells, and specifically teaches using porous silica nanoparticles to induce differentiation of pluripotent stem cells into cardiomyocytes by delivering small molecules in porous silica nanoparticles. Therefore, it would have been obvious to adapt Lian’s protocol (of inducing differentiation of iPS cells into cardiomyocytes by delivering a GSK3 inhibitor and a Wnt signaling inhibitor) by delivering said small molecules to iPS cells in a porous silica nanoparticle as taught by Garcia-Bennett since Cheng demonstrated that pluripotent stem cells can be effectively differentiated into cardiomyocytes by delivering small molecules in porous silica nanoparticles and supported by the disclosure of Bhatia that nanoparticles as claimed can be used to deliver one or more agents, such as small molecules, to the desired cells.
Furthermore, since the prior art taught that porous silica nanoparticles offer sequential delivery of trophic factors and controlled release of differentiation factors (Garcia-Bennett), one of ordinary skill in the art would have combined the prior art teachings described above with the purpose of efficiently inducing the differentiation of iPS cells into cardiomyocytes while eliminating the need for repeated administration of these factors in their free form, thus saving both time and costs, and reducing the risk of culture contamination, as taught by Garcia-Bennett.
Therefore, the invention as a whole was prima facie obvious to one of ordinary skill in the art at the time the invention was made, especially in the absence of evidence to the contrary.
Response to Arguments
Examiner’s note: Any reference cited below is provided solely to rebut Applicant’s arguments.
RE: Declaration submitted under 37 C.F.R. 1.132 and rejection of claims 1-3, 7-10, 12-20, and 24-26 under 35 USC § 112
The declaration under 37 C.F.R. 1.132 filed 10/15/2025 is insufficient to overcome the rejection of claims 1-3, 7-10, 12-20, and 24-26 under 35 U.S.C. 112(a) in the last Office action. The Declaration of Jeffrey Jacot (the "Declaration") states that the uncoated nanoparticles for which the 265 nm dynamic light scattering (DLS) measurement was reported were also measured using scanning electron microscopy (SEM), and that the SEM measurement showed size of 267.32 ± 33.5 nm (p 5 of Remarks; para 5 of Declaration). However, the SEM measurement showing size of 267.32 ± 33.5 nm was not reported in the specification. The specification does not provide explicit or implicit support for the range of about 230 nm to about 300 nm, as claimed. Thus, the assertions set forth in the Declaration are not supported in the specification, and the Declaration is not sufficient to overcome the rejection of the pending claims on the grounds of new matter.
Furthermore, Applicant argues that a person of skill in the art would presume that a large plurality of porous silica nanoparticles would exhibit some variation in particle size and such a person would understand a size measurement reported for such a plurality as representing a distribution of size values. Applicant maintains therefore that reciting a single integer value as a particle size would not reflect the scope of the described subject matter as recognized by those of skill in the art.
In response, a person of skill in the art would not derive a lower range of about 230 nm and an upper range of about 300 nm for porous silica nanoparticles from a single integer value of 265 nm, which is the value recited in the instant specification.
Re: Rejection of claims 1-3 under 35 USC § 103
Applicant argues: The references fail to teach or suggest a method of inducing cell differentiation by modulating Wnt/beta-catenin signaling through a single induction step. The cited combination of references, at best, hint at the claimed method of two-agent delivery as a possibility without really describing or enabling it so as to suggest the features of the claimed method to a skilled person.
Garcia-Bennett describes using mesoporous silica particles (MSPs) to simultaneously two peptide mimetics to transplanted mouse embryonic stem cell-derived motor neuron precursors. The PTO relies on Garcia-Bennett for, inter alia, allegedly teaching that porous silica nanoparticles "offer sequential delivery" of peptide factors. Office Action p. 10. As noted on page 7 of the Office Action, Garcia-Bennett opines that "[a]ltering pore sizes and other physical properties of mesoporous silica "may even permit" sequential delivery. However, it is unclear whether this speculative statement refers to single-step treatment. More importantly, this discussion provides no suggestion of the particular means of single-step sequential delivery resented by the claims. Rather, it suggests a completely different approach, i.e. altering pore size.
In response: Applicant’s arguments have been fully considered, but are not persuasive. As set forth in the body of the rejection, Garcia-Bennett teaches loading mesoporous silica nanoparticles with Cintrofin (“Meso Cintrofin”) and loading a different set of mesoporous silica nanoparticles with Gliafin (“Meso Gliafin”), then contacting the cells with Meso Cintrofin and Meso Gliafin to induce differentiation into spinal motor neurons in a single treatment step (pg. 908, right col. par. 2-4: “For the in vitro differentiation assay and electrophysiological analysis, EBs were dissociated using TrypLE Express on day 5 and plated on poly-L-ornithine-and mouse laminin-precoated coverslips in ADFNB medium supplemented with GDNF (10 ng/ml) and CNTF (10 ng/ml) or with Meso Cintrofin (1 µM) and Meso Gliafin (1 µM) (nonbiotinylated and biotinylated) and cultured for an additional 2–3 days.”).
Furthermore, Garcia-Bennet teaches that “Meso samples(50mg) were added to 0.5ml of Gliafin in a water solution at a concentration of 8.87 mg/ml and stirred at 4°C for 16 hours. Meso samples (25mg) were added to a 0.4-ml Cintrofin water solution at a concentration of 4.4 mg/ml and stirred at 4°C for 16 hours” (p 908, last paragraph), implying that Gliafin and Cintrofin were loaded onto separate mesoporous silica nanoparticles.
Thus, the method taught in Garcia-Bennett amounts to “a single treatment step of simultaneously contacting iPS cells in in vitro cell culture media,” as required by instant claim 1. It is noted that the limitation of “of single-step sequential delivery” (emphasis added), as asserted by Applicant, is not recited in the claims.
Applicant argues: Lian, Cheng and Jenova are each cited to supply various features of claim 1 not taught by Garcia-Bennett. The PTO cites Cheng in particular for teaching the use of a polymer coating and therefore inherently providing delayed release kinetics. Applicant notes that Cheng teaches the
use of a polymer coating (PAH) on MSPs, but not for the purpose of controlling release rate. Rather, Cheng employed the coating specifically to counteract the positive surface charge of 5-azacytidine-loaded particles. Cheng p. 2013. It should be noted that the uncoated particles of the present claims do not exhibit such a charge, and there is no indication that the MSPs in Garcia-Bennett do either. Applicant submits therefore that a skilled person would not be motivated to modify Garcia-Bennett to add a polymer coating in view of Cheng's teaching and consequently would not acquire any inherent property of said coating.
In response: Applicant’s arguments have been fully considered, but are not persuasive. First, Cheng teaches that FMSNs had a negative charge (−37.7 mV) and the FMSNs+5-azacytidine nanocomplex retained the negative charge (−21.9 mV) (p 2013, col 1). Cheng teaches that to make a strong electrostatic interaction with the cell membrane, which itself has a negative charge, the surface of the negatively-charged FMSNs + 5-azacytidine nanocomplex was covered with the positive electrolytic polymer PAH (p 2013, col 1). Thus, Cheng employed the coating specifically to counteract the negative surface charge of 5-azacytidine-loaded particles, and not the positive surface charge of 5-azacytidine-loaded particles, as asserted by Applicant.
Although Garcia-Bennett is silent regarding the charge of the mesoporous silica nanoparticles (MSPs) taught therein, Huang (Nanomaterials and Nanotechnology, 2014, 4(2)) shows that “It is generally accepted that mesoporous silica is negatively charged above the isoelectric point (pH 2–3)” (p 6, col 2, para 1). Thus, the MSP taught in Garcia-Bennett inherently has a negative charge, like the FMSNs + 5-azacytidine nanocomplex taught in Chang.
Because the MSPs taught in Garcia-Bennett are delivered to cells, a person of ordinary skill in the art would have been motivated to coat the surface of the negatively charged MSPs with biodegradable polymer PAH to induce strong electrostatic interaction with the cell membrane, as taught in Cheng.
Finally, because the PAH-coated MSPs, as taught by Garcia-Bennett in view of Cheng, reads on the limitation of instant claim 1 (wherein the one or more second porous silica nanoparticles are coated with a biodegradable polymer to form one or more coated particles), the PAH-coated MSPs taught in the prior art would have the claimed property of achieve delayed-release kinetics of the inhibitor from the one or more coated particles. There is no distinction between the elements which are claimed and the elements taught in the prior art; therefore, any properties that are exhibited by the claimed composition would be the same as those exhibited in the composition taught in the prior art. See MPEP 2112.01(I).
Applicant argues: As a group, Garcia-Bennett, Lian, Cheng and Jenova may be considered to demonstrate the knowledge in the art that nanoparticles can be used to deliver agents of interest to cells. However, the PTO' s combination of various selected aspects of their disclosures to assemble the specific elements of the claimed method is more indicative of hindsight construction rather than the reasoning of one of skill in the art. With particular regard to the claimed approach of sequential administration of differentiation factor in a single-step treatment, at most the cited references suggest that it may be possible. However, none of them-individually or in combination- show or suggest to a skilled person the actual solution devised by the inventors of the present claims. Applicant maintains therefore that the cited references fail to teach or suggest a method having every element set forth in independent claim 1.
In response: In response to applicant's argument that the examiner's conclusion of obviousness is based upon improper hindsight reasoning, it must be recognized that any judgment on obviousness is in a sense necessarily a reconstruction based upon hindsight reasoning. But so long as it takes into account only knowledge which was within the level of ordinary skill at the time the claimed invention was made, and does not include knowledge gleaned only from the applicant's disclosure, such a reconstruction is proper. See In re McLaughlin, 443 F.2d 1392, 170 USPQ 209 (CCPA 1971).
Re: Rejection of claims 7-10 under 35 USC § 103
Applicant argues: Vivero-Escoto is cited in the Office Action for allegedly teaching or suggesting silica nanoparticles coated with polymers. However, Vivero-Escoto fails to remedy the failure of Garcia-Bennett, Cheng, Lian and Jenova to teach or suggest every element of claim 1. Particularly, while Vivero-Escoto may mention assorted options for functionalizing and using MSNs in a variety of contexts, that reference fails to describe or suggest the particular technical solution provided by the claims. The cited references fail to support a finding of obviousness against the present claims.
In response: Applicant’s arguments have been fully considered, but are not persuasive. As set forth in the rejection above, Vivero-Escoto is not relied upon for the rejection of claim 1, which is rendered obvious over Garcia-Bennett, Cheng, Lian, and Jenova. As discussed above, Garcia-Bennett, in view of Cheng, teaches silica nanoparticles coated with polymers.
As set forth in the rejection above, regarding claim 7, Vivero-Escoto teaches the polymer being PEG (Scheme 1D, pg. 1954). The limitations of claims 8-9 are taught in Liang, and the limitations of claim 10 are taught in Garcia-Bennett.
Re: Rejection of claims 13-16 under 35 USC § 103
Applicant argues: Conceding that Garcia-Bennett, Cheng, Lian, Jenova and Vivero-Escoto fail to disclose the use of a bioreactor, the PTO cites Anneren as allegedly curing that deficiency. Upon inspection, however, Anneren fails to cure every deficiency of Garcia-Bennett, Cheng, Lian, Jenova and Vivero-Escoto with respect to the subject matter of claim 1. Specifically, Anneren is silent as to the presently claimed GSK3 and Wnt inhibitors or their sequential administration in a single step.
In response: Applicant’s arguments have been fully considered, but are not persuasive. As asserted by Applicant, Anneren is relied upon for the teachings regarding the use of a bioreactor. Anneren is not relied upon for teachings regarding GSK3 and Wnt inhibitors or their administration in a single step. These limitations are recited in claim 1, which is rendered obvious over Garcia-Bennett, Cheng, Lian, and Jenova, as set forth above. Moreover, is noted that the limitation of “sequential administration in a single step” (emphasis added), as asserted by Applicant, is not recited in the claims.
Re: Rejection of claim 17 under 35 USC § 103
Applicant argues: Conceding that Lian, Cheng, Vivero-Escoto and Anneren fail to disclose the step set forth in claim 17, the Office Action cites Ma as allegedly curing that deficiency. Upon inspection, however, Ma fails to cure every deficiency of Lian, Cheng, Vivero-Escoto, and Anneren with respect to the subject matter of claim 1. Specifically, Ma is silent as to the presently claimed GSK3 and Wnt inhibitors or their sequential administration in a single step.
In response: Applicant’s arguments have been fully considered, but are not persuasive. Ma is not relied upon for teachings regarding GSK3 and Wnt inhibitors or their administration in a single step. These limitations are recited in claim 1, which is rendered obvious over Garcia-Bennett, Cheng, Lian, and Jenova, as set forth above. Moreover, is noted that the limitation of “sequential administration in a single step” (emphasis added), as asserted by Applicant, is not recited in the claims.
Re: Rejection of claims 18-20 and 24-26 under 35 USC § 103
Applicant argues: Applicant maintains that Garcia-Bennett, Lian and Cheng fail to combine to teach or suggest such an approach for forming cardiomyocytes in vitro. Bhatia is cited as allegedly teaching the use of coated particles to deliver a plurality of agents. Bhatia is a large document (103 pages) presenting a broad disclosure listing multiple possible modes and parameters for delivering agents with nanoparticles. However, like Garcia-Bennett, Bhatia does not teach the specific particle and specific delivery method set forth in claims 18 and 24, respectively. Indeed, the description cited in the Office Action (e.g. [0105]) merely states that plural nanoparticles "may be associated with one or more agents to be delivered". However Bhatia does not specifically describe an individual particle configured for sequential delivery of plural agents or methods using such a particle.
In response: Applicant’s arguments have been fully considered, but are not persuasive. The arguments against Garcia-Bennett, Lian and Cheng are addressed above. As set forth in the rejection above, Bhatia is relied upon for its teachings regarding a porous silica nanoparticle [0123] having a PEG coating layer, which can be used to deliver one or more small molecules [0237] [0261]. The limitations regarding inducing cardiomyocyte differentiation in iPS cells (Claims 24-26) are rendered obvious over Garcia-Bennett, Lian and Cheng, as set forth in the rejection.
Conclusion
THIS ACTION IS MADE FINAL. 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.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Risa Takenaka whose telephone number is (571)272-0149. The examiner can normally be reached M-F, 12-7 EST.
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/RISA TAKENAKA/ Examiner, Art Unit 1632
/KARA D JOHNSON/ Primary Examiner, Art Unit 1632
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