DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant's election with traverse of Group 1 (1, 5, 7, 15, 20-23, 25-27, 29, 32-34) in the reply filed on 12/8/2025 is acknowledged. The traversal is on the ground(s) that no reference is cited by the Action and it is not clear how the Action arrived at the conclusion that no corresponding technical feature exists without doing a comparison to the prior art. In particular, no conclusion could have been made that the prior art includes a "composition comprising a plurality of enteric neurons, wherein at least about 30% of the enteric neurons express nitric oxide synthase (NOS1)," as recited in claim 1, without comparison to at least one prior art reference. Prior to Applicant's disclosure, such a composition was entirely unknown. This is not found persuasive because the examiner indicated that there is not a common special technical feature across the groups of claims and NOT that the technical feature does not make a contribution over the prior art (which would require citation to prior art). Furthermore, for example (as in the previously mailed Office action), Group 2 (claim 5 and its dependents) requires exposing nitrergic agents to enteric neural crest cells, which is a different feature than required by claim 1 (a plurality of enteric neurons).
The requirement is still deemed proper and is therefore made FINAL.
Claims 35, 42, 45-46, 50-51, 53, 77, 79, and 85 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected inventions, there being no allowable generic or linking claim. Applicant timely traversed the restriction (election) requirement in the reply filed on 12/8/2025.
Claims 1, 5, 7, 15, 20-23, 25-27, 29, 32-34, 35, 42, 45-46, 50-51, 53, 77, 79, and 85 are pending and claims 1, 5, 7, 15, 20-23, 25-27, 29, and 32-34 have been examined herein.
Priority
The instant claims herein are examined utilizing the accepted effective filing date of 3/25/2020 for the basis of any prior art rejections.
Specification
The disclosure is objected to because it contains an embedded hyperlink and/or other form of browser-executable code. Applicant is required to delete the embedded hyperlink and/or other form of browser-executable code; references to websites should be limited to the top-level domain name without any prefix such as http:// or other browser-executable code. See MPEP § 608.01.
Claim Objections
Claim 25 is objected to because of the following informalities:
Claim 25 recites “smooth muscle cells proximate to or adjacent to the plurality of enteric neurons”. While it doesn’t rise to the level of 112(b) issue, the claim language is awkward. The examiner suggests removing the “proximate to or adjacent to the plurality of enteric neurons”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 5, 20, 29 and 33 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claims 5 and 29 recites “at least about.” This limitation utilizes an approximation, which renders the claim indefinite because there is no disclosure in the specification or in the prior art that provides any indication as to what range is covered by the term “about”. The lower threshold value is unclear, thus one
of ordinary skill would not be able to ascertain what would meet the limitation of “at least about”.
Claims 20 and 33 recite the limitation "wherein the plurality of cells expressing NOS1 are derived from a combination of two or more of: human inducible pluripotent stem cells that are in culture at least about 12 days; cells that express human CD49 and SOX10 in culture from about 12 to about 15 days; cells that express human TRKC, PHOX2B and EDNRB in culture from about 15 to about 30 days; cells that express human TRKC and TUJ1 in culture from about 30 days to about 45 days". The claim language is prima facie confusing because it is unclear what applicant intends to claim. Does applicant intend to claim that the enteric neurons of the instant invention are derived from a mixed population of cells? The specification does not clarify the issues with claim and there are no embodiments nor working examples in which applicant utilizes a combination of cells (like those claimed) to derive the enteric neurons. The examiner is interpreting that (as in the prior art rejections below) that human pluripotent stem cells as well as derivative cells expressing CD49 and SOX10 are capable of creating enteric neurons. Please note that the examiner also recognizes that this limitation is a product-by-process limitation (see MPEP 2113).
It is noted that any interpretation of the claims set forth above does not relieve Applicant of the responsibility of responding to rejections made based on said interpretations. If the actual interpretation of the claims is different than that posited by the Examiner, additional rejections and art may be readily applied in a subsequent final Office action.
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 5 and 29 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the written description requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for 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.
The written description requirement for a claimed genus may be satisfied through sufficient description of a representative number of species by actual reduction to practice, reduction to drawings, or by disclosure of relevant, identifying characteristics, i.e., structure or other physical and/or chemical properties, by functional characteristics coupled with a known or disclosed correlation between function and structure, or by a combination of such identifying characteristics, sufficient to show the inventor was in possession of the claimed genus. See, e.g., Ariad Pharm., Inc. v. Eli Lilly & Co., 598 F.3d 1336, 1340, 94 USPQ2d 1161, 1167 (Fed. Cir. 2010); University of California v. Eli Lilly & Co., 119 F.3d 1559, 43 USPQ2d 1398 (Fed. Cir. 1997) at 1406; Juno Therapeutics, Inc. v. Kite Pharma, Inc., 10 F.4th 1330, 1337, 2021 USPQ2d 893 (Fed. Cir. 2021) ("[T]he written description must lead a person of ordinary skill in the art to understand that the inventor possessed the entire scope of the claimed invention. Ariad, 598 F.3d at 1353–54 ('[T]he purpose of the written description requirement is to ensure that the scope of the right to exclude, as set forth in the claims, does not overreach the scope of the inventor's contribution to the field of art as described in the patent specification.' (internal quotation marks omitted).").
A "representative number of species" means that the species which are adequately described are representative of the entire genus. Thus, when there is substantial variation within the genus, one must describe a sufficient variety of species to reflect the variation within the genus. See AbbVie Deutschland GmbH & Co., KG v. Janssen Biotech, Inc., 759 F.3d 1285, 1300, 111 USPQ2d 1780, 1790 (Fed. Cir. 2014). The issue is whether the skilled artisan would understand inventor to have invented, and been in possession of, the invention as claimed.
Independent claim 1 recites “A composition comprising a plurality of enteric neurons, wherein at least about 30% of the enteric neurons express nitric oxide synthase (NOS 1).” Dependent claims 5 and 29 respectively recite “wherein the NOS1 comprises at least about 70% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, or a functional fragment thereof.” The instant specification defines “fragment” as “a portion of a polypeptide or nucleic acid molecule . . . contains, preferably, at least about 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or about 90% of the entire length of the reference nucleic acid molecule or polypeptide”. It also defines “functional fragment” as “any portion of a polypeptide or nucleic acid sequence from which the respective full-length polypeptide or nucleic acid relates that is of a sufficient length and has a sufficient structure to confer a biological affect that is at least similar or substantially similar to the full-length polypeptide or nucleic acid upon which the fragment is based . . . said portion encodes a polypeptide of a certain length and/or structure that is less than full-length but encodes a domain that still biologically functional as compared to the full-length or wild-type protein. In some embodiments, the functional fragment may have a reduced biological activity, about equivalent biological activity, or an enhanced biological activity as compared to the wild- type or full-length polypeptide sequence upon which the fragment is based”. The scope of the sequences encompassed by the claims include 1) sequences having at least 70% identity to SEQ ID NO: 10-12 and 2) functional variants of sequences having at least 70% identity to SEQ ID NO: 10-12. All of these as outlined above are extremely problematic as the specification fails to teach and/or provide support for the possession of NOS1 mutants having 70% identity and all “functional fragments” having nebulous biological activity somewhere in the realm of being “functional.” The specification fails to define, and is devoid of, the relevant structures and functional characteristics necessary to reasonably appraise one of ordinary skill of Applicant’s possession of the instantly claimed “functional fragments.”
Issue 1: Possession of NOS1 mutants with 70%+ identity to SEQ ID NO: 10, 11, and 12:
In support of Applicant’s broadly claimed genus, Applicant only discloses SEQ IDs 10-12. However, there are no working examples or sequences from Applicant’s disclosure in which NOS1 mutants having at minimum 70% identity to the sequences recited above and capable of maintaining nebulous “functionality” (ranging from reduced to enhanced functionality as defined in the specification). Thus, the data generated for the disclosed sequences cannot reasonably be extrapolated to and applied to support possession of the entire claimed genus of NOS1 mutants as claimed, because no one species, combination, or variant accounts for the variability amongst the claimed genus. As in Ariad, merely drawing a fence around the outer limits of a purported genus is not an adequate substitute for describing a variety of materials constituting the genus and showing that one has invented a genus and not just a species. “A patent is not a hunting license. It is not a reward for the search, but compensation for its successful conclusion.” Brenner v. Manson, 383 U.S. 519, 536 (1966).
Issue 2: Art recognized correlation between structure-function relationship between NOS1 mutants with 70%+ identity to SEQ ID NO: 10, 11, and 12
Applicant has not provided information as to what parts of SEQ ID NO: 10-12 are important for the desired NOS1 activity or what modifications could be made that would allow each of the mutants to retain activities at 70% identity to the sequence. The specification fails to disclose how SEQ ID NO: 10-12 could be modified to a minimum 70% sequence identity and still possess its desired activity as a NOS1 mutant. Even with knowledge in the art regarding modification of amino acid sequences, one of ordinary skill would not know what sequence features are required (or which 30% of the sequence could be modified) for the outcome of having a NOS1 mutant that still retains enough functional activity as compared to the parent sequence without a recognized correlation between structure and function. As such, those of ordinary skill would not be able to identify, without further testing, which amino acid sequences that have at least 70% sequence identity to SEQ ID NO: 10-12 would be able to have the activities desired.
Issue 3: Functional fragments
The specification contains no disclosures
There is also no disclosure as to what structure(s) must be present or retained on the fragment of NOS1 that would result in a functional fragment. Applicant has not has not provided any information or steps as to how one of ordinary skill would obtain a functional fragment of NOS1, or any sufficient distinguishing structure-function relationship with respect to the broad genus as claimed. This data cannot be extrapolated to any and all possible fragments of NOS1. Even with knowledge in the art regarding modification of genes, one of ordinary skill would not reasonably know, based on the disclosure provided, what structures or functions are required for the outcome of having a functional fragment of NOS1 without a recognized correlation between structure and function.
There is no disclosure as to what structure(s) must be present or retained on the “functional fragment” or functional characteristics that would distinguish a functional fragment of a protein from a non-functional fragment. Applicant has provided no information as to how a fragment would be considered “functional”, distinguishing characteristics, or the amount of biological activity that is required for the fragment to work. There is no sufficient structure-function relationship with respect to the broad genus as claimed. The data in the specification cannot be extrapolated to any and all possible counterparts of NOS1 sequences/fragments. Even with knowledge in the art regarding the modification of proteins, one of ordinary skill would not reasonably know, based on the disclosure provided, what structures or functions are required for the outcome of creating a functional fragment of these proteins without a recognized correlation between structure and function.
The specification, then, is considered devoid of sufficiently detailed, relevant, identifying characteristics demonstrating that Applicant was in possession of the claimed genus of NOS1 sequences, variants, or fragments, i.e., additional complete or partial structures, other physical and/or chemical properties, functional characteristics coupled with a known or disclosed correlation between function and structure, or some combination thereof demonstrating possession of the claimed genus. Therefore, claims 5 and 29 are rejected under 35 U.S.C. 112(a) for lack of written description.
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claim 1, 5, 7, 15, 20-23, 25-27, 29, and 32-34 are rejected under 35 U.S.C. 101 because the claimed invention is directed to a judicial exception (natural product) without significantly more. This judicial exception is not integrated into a practical application and does not include additional elements that are sufficient to amount to significantly more than the judicial exception for the reasons set forth below:
Step 1 (Statutory Category): This part of the eligibility analysis evaluates whether the claim falls within any statutory category. Here, the claims recite (1) a composition comprising a plurality of enteric neurons, wherein at least about 30% of the enteric neurons express nitric oxide synthase (NOS 1), (2) A system comprising a cell culture vessel comprising a plurality of enteric neurons supported in a culture medium, wherein at least about 20% of the enteric neurons express nitric oxide synthase, and (3) A pharmaceutical composition comprising: a) a therapeutically effective amount of one or a plurality of enteric neurons; and b) a pharmaceutically acceptable carrier. These are compositions of matter; therefore, the claims fall within a statutory category. [Step 1: YES]
Step 2A (Judicial Exceptions), Prong 1: This part of the eligibility analysis evaluates whether the claim recites a judicial exception. A claim “recites” a judicial exception when the exception is “set forth” or “described” in the claim (see MPEP 2106.04(II)). Because the claims recite nature-based product limitations, the markedly different characteristics analysis is used to determine if the nature-based product limitations are a product of nature exception (see MPEP 2106.04(c)(I)). This analysis is performed by comparing the nature-based product limitations in the claims to its naturally occurring counterparts to determine if it has markedly different characteristics (see MPEP 2106.04(c)(II).
The appropriate natural counterpart to the claimed enteric neurons is enteric neurons as found in its natural state. The second step in the analysis requires identifying appropriate characteristics to compare. In this case, the appropriate characteristics pertain to the naturally occurring enteric neurons is expressing NOS1 (as instantly claimed).
Sanders et al (Br J Pharmacol. 2019 Jan;176(2):212-227; Epub Sept 2018) discusses nitric oxide and its role as a non‐adrenergic, non‐cholinergic inhibitory neurotransmitter in the gastrointestinal tract (title). The reference teaches that NO is a neurotransmitter released from enteric inhibitory neurons and responsible for modulating gastrointestinal (GI) motor behavior. Enteric neurons express nNOS (NOS1) that associates with membranes of nerve varicosities. NO released from neurons binds to soluble guanylate cyclase in post‐junctional cells to generate cGMP. cGMP‐dependent protein kinase type 1 (PKG1) is a major mediator but perhaps not the only pathway involved in cGMP‐mediated effects in GI muscles based on gene deletion studies. NOS1+ neurons form close contacts with smooth muscle cells (SMCs), interstitial cells of Cajal (ICC) and PDGFRα+ cells, and these cells are electrically coupled (SIP syncytium).
Thus, the embodiments of the claims encompass naturally occurring enteric neurons. Thus, the claims recite a judicial exception, a natural product. [Step 2A, Prong 1: YES]
Therefore, the analysis proceeds to Step 2A Prong 2.
Step 2A (Judicial Exceptions), Prong 2: This part of the eligibility analysis evaluates whether the claims as a whole integrate the recited judicial exception into a practical application of the exception. This evaluation is performed by (a) identifying whether there are any additional elements recited in the claims beyond the judicial exception, and (b) evaluating those additional elements individually and in combination to determine whether the claims as a whole integrate the exception into a practical application. The enteric neurons as claimed is a product of nature, and the additional elements (derivations from pluripotent stem cells, cell culture vessel and medium, pharmaceutically acceptable carrier, etc. as in instant claims) do not impose a practical use or application of the claimed natural products. In this regard, the claims fail to recite additional elements that integrate the judicial exception natural products into a practical application. [Step 2A, Prong 2: NO]
Step 2B (Significantly More): This part of the eligibility analysis evaluates whether the claims as a whole amount to significantly more than the recited exception, i.e., whether any additional element, or combination of additional elements, adds an inventive concept to the claim (MPEP 2106.05). The composition of instant claim 7, 20, and 32-33 are products-by-process. Product-by-process claims are not dependent on the manner in which they were produced (see MPEP 2113). There are no additional limitations in the additional claims that depend from the independent claims that add an inventive concept to the judicial exception. As such, none of these limitations impose a practical use or application of the claimed natural products as stated in Step 2A2, and thus do not add significantly more to the exception. [Step 2B: NO]
The claims fail to recite additional elements that are sufficient to amount to significantly more than the judicial exception. Therefore, the claims do not qualify as eligible subject matter under 35 USC 101.
Claim Rejections - 35 USC § 102
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 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.
(a)(2) the claimed invention was described in a patent issued under section 151, or in an application for patent published or deemed published under section 122(b), in which the patent or application, as the case may be, names another inventor and was effectively filed before the effective filing date of the claimed invention.
Claim(s) 1 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Sanders et al (Br J Pharmacol. 2019 Jan;176(2):212-227; Epub Sept 2018).
Sanders et al discusses nitric oxide and its role as a non‐adrenergic, non‐cholinergic inhibitory neurotransmitter in the gastrointestinal tract (title). The reference teaches that NO is a neurotransmitter released from enteric inhibitory neurons and responsible for modulating gastrointestinal (GI) motor behavior. Enteric neurons express nNOS (NOS1) that associates with membranes of nerve varicosities. NO released from neurons binds to soluble guanylate cyclase in post‐junctional cells to generate cGMP. cGMP‐dependent protein kinase type 1 (PKG1) is a major mediator but perhaps not the only pathway involved in cGMP‐mediated effects in GI muscles based on gene deletion studies. NOS1+ neurons form close contacts with smooth muscle cells (SMCs), interstitial cells of Cajal (ICC) and PDGFRα+ cells, and these cells are electrically coupled (SIP syncytium). This anticipates “A composition comprising a plurality of enteric neurons, wherein at least about 30% of the enteric neurons express nitric oxide synthase (NOS 1)” as in instant claim 1. The non‐purinergic portion of electrophysiological responses to NANC nerve stimulation (inhibitory junction potentials; IJPs) was found to be NO, and responses were mimicked by exogenous NO and NO donors in gastrointestinal (GI) muscles of laboratory animals and humans. It was then found that NO comes from enteric neurons with Dogiel type 1 morphology, and it is synthesized by the cerebellar isoform of NOS [neuronal NOS (nNOS) also known as NOS1].
Thus, absent evidence to the contrary, Sanders anticipates instant claim 1.
Claim(s) 1, 7, 15, 20-22, and 25 is/are rejected under 35 U.S.C. 102(a)(1) as being anticipated by Fattahi et al (Nature. 2016 Mar 3;531(7592):105-9. doi: 10.1038/nature16951. Epub 2016 Feb 10; Ref. 1 of NPL in IDS filed 7/11/24).
Fattahi teaches the efficient derivation and isolation of ENS progenitors from human pluripotent stem cells (hPSCs) and their further differentiation into functional enteric neurons (abstract) (“composition comprising a plurality of enteric neurons wherein at least about 30% of the enteric neurons express nitric oxide synthase (NOS 1)” as in instant claim 1; “wherein the enteric neurons are derived from one or a plurality of pluripotent stem cells” as in instant claim 7). ENC cells from the 11 day induction protocol were aggregated into 3D spheroids (5 million cells/well) in Ultra Low Attachment 6-well culture plates (Fisher Scientific, 3471) and cultured in Neurobasal (NB) medium supplemented with L-Glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3uM, Tocris Bioscience, 4423) and FGF2 (10nM, R&D Systems, 233-FB-001MG/CF) (“A system comprising a cell culture vessel comprising a plurality of enteric neurons supported in a culture medium, wherein at least about 20% of the enteric neurons express nitric oxide synthase.” as in instant claim 21; “wherein from about 20 to about 60% of the enteric neurons express nitric oxide synthase” as in instant claim 22; “wherein the cell culture comprises the composition of claim 1” as in instant claim 23). After 4 days of suspension culture, the spheroids are plated on Poly Ornithine/Laminin/Fibronectin (PO/LM/FN) coated dishes (prepared as described previously26) in Neurobasal (NB) medium supplemented with L-Glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing GDNF (25 ng/ml, Peprotech, 450-10) and Ascorbic acid (100 uM, Sigma, A8960-5G). The ENC precursors migrate out of the plated spheroids and differentiate into neurons in 1-2 weeks. The cells were fixed for immunostaining or harvested for gene expression analysis at Day 25, Day 40 and Day 60 of differentiation. To facilitate the isolation of pure NC populations we performed a candidate surface marker screen and identified CD49D (α4-integrin) as an epitope that reliably marks early SOX10+ NC lineages (Fig. 1b, Extended Data Fig. 1a-c). We next used CD49D to demonstrate robustness of RA-based NC induction across hPSC lines (both human embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells; Extended Data Fig. 1d). Purified CD49D+ NC precursors, derived in the presence of RA, expressed HOXB2-B5 indicative of vagal identity4,5 but not more caudal HOX transcripts such as HOXB9 (Fig. 1c). In further agreement with vagal identity, CD49D+, RA-treated NC precursors expressed markers of early enteric NC (ENC) lineages2 including PAX3, EDNRB and RET (Fig. 1d, Extended Data Fig. 1e,f). To address whether hESC-derived ENC precursors are capable of recreating ENS neuronal diversity we maintained purified CD49D+ ENC precursors in 3D spheroids for 4 days followed by differentiation as adherent cultures in the presence of ascorbic acid and GDNF (Fig. 2a). The 3D spheroid step was required to retain high levels of SOX10::GFP expression (Fig. 2b). Replating of 3D spheroids under differentiation conditions yielded immature neurons expressing Tuj1 and the enteric precursor marker PHOX2A (day 20; Fig. 2b). The majority of PHOX2A+ cells were positive for TRKC (NTRK3), a surface marker expressed in enteric neuron precursors8 suitable for enrichment for PHOX2A+ and ASCL1+ precursors (Extended Data Fig. 2a,b). Temporal expression analyses (Extended Data Fig. 2c-e) showed maintenance of ENC neuronal precursor marker expression by day 40 of differentiation (Fig. 2c,d) followed by an increase in the percentage of mature neurons by day 60 (Fig. 2e,f). In agreement with enteric neuron identity we observed a broad range of neurotransmitter phenotypes including 5HT+, GABA+ and NOS+ neurons. The presence of these neurotransmitters in neurons derived from CD49D+ purified NC precursors indicates ENC origin, since those neurotransmitters are not expressed in other NC lineages. Indeed, no 5HT+ neurons were observed in parallel cultures derived from HOX-negative, CD49D+ cells (Extended Data Fig. 3a,b; “wherein the plurality of cells expressing NOS1 are deficient in expression of any one or combination of: 5HT” as in instant claim 15; “wherein the plurality of cells expressing NOS1 are derived from a combination of two or more of: human inducible pluripotent stem cells that are in culture at least about 12 days; cells that express human CD49 and SOX10 in culture from about 12 to about 15 days” as in instant claim 20). For connectivity studies, enteric neurons were derived from a hESC line expressing channelrhodopsin-2 (ChR2)-EYFP under control of the human Synapsin promoter. An optogenetic reporter line10 was used to allow for light-induced control of neuronal activity (Fig. 2g). GABA+ and 5HT+ neurons in these co-cultures were closely associated with SMCs (Extended Data Fig. 4c). Interestingly, co-culture of day 25 neurons with SMCs triggered accelerated neuronal maturation as illustrated by the increased expression of SYN::EYFP (Extended Data Fig. 4d). Conversely, hESC-derived SMCs also showed signs of accelerated maturation under co-culture conditions as illustrated by the expression of mature markers (MYH11, AchR; Extended Data Fig. 4e) and the ability to contract in response to pharmacological stimulation (,Supplementary videos 1-6, Extended Data Fig. 4f) (“wherein the cell culture vessel further comprises: (i) smooth muscle cells proximate to or adjacent to the plurality of enteric neurons; and (ii) a hydrogel” as in instant claim 25).
Thus, absent evidence to the contrary, Fattahi anticipates the instant invention of claims 1, 7, 20-22, and 25.
Claim(s) 1, 7, 20-22, 25-27, and 32-33 is/are rejected under 35 U.S.C. 102(a)(1) and (a)(2) as being anticipated by Studer et al (US20180291339A1, 6/22/2018; published 10/11/2018; Ref. 1 of US patent application publications in IDS filed 7/11/2024).
Studer discloses in vitro methods of inducing differentiation of stem cells into enteric neural crest lineage cells (abstract). The stem cells are human pluripotent stem cells (para 29). The method further comprises subjecting said population of differentiated cells to conditions favoring maturation of said differentiated cells into a population of enteric neurons (para 30). At least 70% of the cells express markers such as NOS (para 31 and para 39) (“A composition comprising a plurality of enteric neurons, wherein at least about 30% of the enteric neurons express nitric oxide synthase (NOS 1)” as in instant claim 1; “wherein the enteric neurons are derived from one or a plurality of pluripotent stem cells” as in instant claim 7). The composition comprises a population of matured enteric neurons (para 379). The composition can further comprise a biocompatible scaffold or matrix such as a hydrogel (para 380). The composition is a pharmaceutical composition that comprises a pharmaceutically acceptable carrier (para 381) (“A pharmaceutical composition comprising: a) a therapeutically effective amount of one or a plurality of enteric neurons; and b) a pharmaceutically acceptable carrier” as in instant claim 26; “wherein from about 20% to about 100% of the enteric neurons express NOS1” as in instant claim 27; “wherein the one or plurality enteric neurons are derived from human inducible pluripotent stem cells” as in in instant claim 32). The conditions favoring maturation comprises culturing the differentiated ENS precursors in a suitable cell culture medium, where the culture medium refers to a liquid that covers cells in a culture vessel, such as a Petri plate, a multi-well plate, and the like, and contains nutrients to nourish and support the cells (para 285) (“A system comprising a cell culture vessel comprising a plurality of enteric neurons supported in a culture medium, wherein at least about 20% of the enteric neurons express nitric oxide synthase” as in instant claim 21; “wherein from about 20 to about 60% of the enteric neurons express nitric oxide synthase” as in instant claim 22; “wherein the cell culture comprises the composition of claim 1” as in instant claim 23; “wherein the cell culture vessel further comprises: . . . (ii) a hydrogel” as in instant claim 25). The differentiated ENS precursors cells further express one or more SOX10+ neural crest lineage marker. In certain embodiments, the SOX10+ neural crest lineage marker is CD49D (para 363) (“wherein the plurality of cells expressing NOS1 are derived from a combination of two or more of: human inducible pluripotent stem cells that are in culture at least about 12 days; cells that express human CD49 and SOX10 in culture from about 12 to about 15 days” as in instant claim 20 and 33). SMC progenitors were plated on PO/LM/FN coated culture dishes (prepared as described previously38) three days before addition of ENC-derived neurons. The neurons were dissociated (using accutase, Innovative Cell Technologies, AT104) at day 30 of differentiation and plated onto the SMC monolayer cultures (para 443) (“wherein the cell culture vessel further comprises: (i) smooth muscle cells proximate to or adjacent to the plurality of enteric neuron” as in instant claim 25).
Thus, absent evidence to the contrary, Fattahi anticipates the instant invention of claims 1, 7, 20-22, 25-27, and 32-33.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
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) 5 is/are rejected under 35 U.S.C. 103 as being unpatentable over Fattahi et al (Nature. 2016 Mar 3;531(7592):105-9. doi: 10.1038/nature16951. Epub 2016 Feb 10; Ref. 1 of NPL in IDS filed 7/11/24) in view of Bredt et al (US5268465A, 1/18/1991, published 12/7/1993).
The difference between Fattahi and the instant invention is that it does not teach that the NOS1 expressed by the enteric neurons has at least about 70% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, or a functional fragment thereof.
Bredt teaches purifying nitric oxide synthase from human cells (abstract). It teaches that nitric oxide synthase to a number of anatomical sites, including retina, intestine, adrenal gland, and vasculature (abstract). Antisera has detected NOS in autonomic nerve fibers in the retina, in cell bodies and nerve fibers in the myenteric plexus of the intestine, in adrenal medulla, and in vascular endothelial cells (para 2 of Detailed Description). The NOS1 as described in Bredt has 100% identity to SEQ ID NO: 11 as claimed in instant claim 5 (see screenshot below).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to create NOS1 expressing enteric neurons as taught by Fattahi, where the NOS1 has 100% sequence identity to SEQ ID NO: 11 as taught by Bredt, to arrive at the instantly claimed invention. Given that Bredt shows that human intestinal neurons express NOS1, one of ordinary skill would reasonably expect that the enteric neurons of Fattahi to express the same or similar NOS1 proteinas taught by Bredt.
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Claim(s) 29 is/are rejected under 35 U.S.C. 103 as being unpatentable over Studer et al (US20180291339A1, 6/22/2018; published 10/11/2018) in view of Bredt et al (US5268465A, 1/18/1991, published 12/7/1993).
The difference between Studer and the instant invention that it does not teach that the NOS1 comprises at least about 70% sequence identity to SEQ ID NO: 10, SEQ ID NO: 11 or SEQ ID NO: 12, or a functional fragment thereof.
Bredt teaches purifying nictric oxide synthase from human cells (abstract). It teaches that nitric oxide synthase to a number of anatomical sites, including retina, intestine, adrenal gland, and vasculature (abstract). Antisera has detected NOS in autonomic nerve fibers in the retina, in cell bodies and nerve fibers in the myenteric plexus of the intestine, in adrenal medulla, and in vascular endothelial cells (para 2 of Detailed Description). The NOS1 as described in Bredt has 100% identity to SEQ ID NO: 11 as claimed in instant claim 5 (see screenshot below).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to create NOS1 expressing enteric neurons as taught by Studer, where the NOS1 has 100% sequence identity to SEQ ID NO: 11 as taught by Bredt, to arrive at the instantly claimed invention. Given that Bredt shows that human intestinal neurons express NOS1, one of ordinary skill would reasonably expect that the enteric neurons of Studer to express the same or similar NOS1 protein as taught by Bredt.
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Claim(s) 34 is/are rejected under 35 U.S.C. 103 as being unpatentable over Studer et al as applied to claims 1, 7, 20-22, 25-27, and 32-33 above, and further in view of Fattahi et al (Nature. 2016 Mar 3;531(7592):105-9. doi: 10.1038/nature16951. Epub 2016 Feb 10; Ref. 1 of NPL in IDS filed 7/11/24).
The difference between Studer and the instant invention is that it does not teach that the plurality of cells expressing NOS1 are deficient in expression of any one or combination of: ChAT, 5HT, and GABA.
Fattahi teaches the efficient derivation and isolation of ENS progenitors from human pluripotent stem cells (hPSCs) and their further differentiation into functional enteric neurons (abstract). ENC cells from the 11 day induction protocol were aggregated into 3D spheroids (5 million cells/well) in Ultra Low Attachment 6-well culture plates (Fisher Scientific, 3471) and cultured in Neurobasal (NB) medium supplemented with L-Glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing CHIR99021 (3uM, Tocris Bioscience, 4423) and FGF2 (10nM, R&D Systems, 233-FB-001MG/CF). After 4 days of suspension culture, the spheroids are plated on Poly Ornithine/Laminin/Fibronectin (PO/LM/FN) coated dishes (prepared as described previously) in Neurobasal (NB) medium supplemented with L-Glutamine (Gibco, 25030-164), N2 (Stem Cell Technologies, 07156) and B27 (Life Technologies, 17504044) containing GDNF (25 ng/ml, Peprotech, 450-10) and Ascorbic acid (100 uM, Sigma, A8960-5G). The ENC precursors migrate out of the plated spheroids and differentiate into neurons in 1-2 weeks. The cells were fixed for immunostaining or harvested for gene expression analysis at Day 25, Day 40 and Day 60 of differentiation. To facilitate the isolation of pure NC populations we performed a candidate surface marker screen and identified CD49D (α4-integrin) as an epitope that reliably marks early SOX10+ NC lineages (Fig. 1b, Extended Data Fig. 1a-c). We next used CD49D to demonstrate robustness of RA-based NC induction across hPSC lines (both human embryonic (hESCs) and induced pluripotent (hiPSCs) stem cells; Extended Data Fig. 1d). Purified CD49D+ NC precursors, derived in the presence of RA, expressed HOXB2-B5 indicative of vagal identity4,5 but not more caudal HOX transcripts such as HOXB9 (Fig. 1c). In further agreement with vagal identity, CD49D+, RA-treated NC precursors expressed markers of early enteric NC (ENC) lineages2 including PAX3, EDNRB and RET (Fig. 1d, Extended Data Fig. 1e,f). To address whether hESC-derived ENC precursors are capable of recreating ENS neuronal diversity we maintained purified CD49D+ ENC precursors in 3D spheroids for 4 days followed by differentiation as adherent cultures in the presence of ascorbic acid and GDNF (Fig. 2a). The 3D spheroid step was required to retain high levels of SOX10::GFP expression (Fig. 2b). Replating of 3D spheroids under differentiation conditions yielded immature neurons expressing Tuj1 and the enteric precursor marker PHOX2A (day 20; Fig. 2b). The majority of PHOX2A+ cells were positive for TRKC (NTRK3), a surface marker expressed in enteric neuron precursors8 suitable for enrichment for PHOX2A+ and ASCL1+ precursors (Extended Data Fig. 2a,b). Temporal expression analyses (Extended Data Fig. 2c-e) showed maintenance of ENC neuronal precursor marker expression by day 40 of differentiation (Fig. 2c,d) followed by an increase in the percentage of mature neurons by day 60 (Fig. 2e,f). In agreement with enteric neuron identity we observed a broad range of neurotransmitter phenotypes including 5HT+, GABA+ and NOS+ neurons. The presence of these neurotransmitters in neurons derived from CD49D+ purified NC precursors indicates ENC origin, since those neurotransmitters are not expressed in other NC lineages. Indeed, no 5HT+ neurons were observed in parallel cultures derived from HOX-negative, CD49D+ cells (Extended Data Fig. 3a,b “the plurality of cells expressing NOS1 are deficient in expression of any one or combination of: 5HT” as in instant claim 34).
Therefore, it would have been obvious prior to the effective filing date of the instantly claimed invention to create as taught by Studer, where the cells are deficient in 5HT as taught by Fattahi, to arrive at the instantly claimed invention. As Fattahi shows that enteric neurons derived from human pluripotent stem cell can be negative for 5HT, one of ordinary skill would modify the method of Studer to include culturing CD49D+ cells to produce enteric neurons as taught by Fattahi with a reasonable expectation of advantageously being able to still produce functional enteric neurons as taught by the prior art.
Conclusion
No claim is allowed.
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/G.R./Examiner, Art Unit 1632
/KARA D JOHNSON/Primary Examiner, Art Unit 1632