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 .
Claims 1-15, 18-21, and 42 are pending and under examination.
Priority
Applicant’s claim for the benefit of a prior-filed application provisional application 63/333,573 filed on 04/22/2022 and PCT/US2023/066023 filed on 4/20/2023 under 35 U.S.C. 119(e) or under 35 U.S.C. 120, 121, or 365(c) is acknowledged.
Information Disclosure Statement
The information disclosure statement (IDS) submitted on 07/10/2025 is considered by the examiner.
Drawings
The drawings are accepted.
Specification
The specification is accepted.
Claim Objections
Claim 10 is objected to because of the following informalities: claim 10 recites “a first open reading frame encoding an MEF2C protein, a second open reading frame encoding an CEBPB protein, and a third open reading frame encoding an IRF8 protein.” The examiner recommends amending the claim to recite “a” instead of “an” in front of MEF2C, CEBPB, and IRF8 for consistency across claims.
Appropriate correction is required.
Applicant is advised that should claim 1 be found allowable, claim 20 will be objected to under 37 CFR 1.75 as being a substantial duplicate thereof. When two claims in an application are duplicates or else are so close in content that they both cover the same thing, despite a slight difference in wording, it is proper after allowing one claim to object to the other as being a substantial duplicate of the allowed claim. See MPEP § 608.01(m).
Claim 20 recites “A composition comprising: a population of the PSC of claim 1.” The structure of the PSC of claim 20 is the same as in base claim 1. Thus, despite a slight difference in wording, these claims have substantially the same scope.
Claim Interpretation
Claim 10 recites “PSC of claim 1, wherein the one or more engineered polynucleotide comprises: a first polycistronic polynucleotide that comprises, optionally 5' to 3', a first open reading frame encoding the SPIl protein, a second open reading frame encoding the CEBPA protein, and a third open reading frame encoding the FLI1 protein; and a second polycistronic polynucleotide that comprises, optionally 5' to 3', a first open reading frame encoding an MEF2C protein, a second open reading frame encoding an CEBPB protein, and a third open reading frame encoding an IRF8 protein.”
As written, the order of the open reading frames in the first and second polycistronic polynucleotides is interpreted to be optional (i.e., not a required limitation). However, the claim language requires all three open reading frames in the first and second polycistronic polynucleotides to be present.
Claim Rejections - 35 USC § 112(a) – Written Description
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 21 and 42 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.
Claim 21 recites “A method, comprising:
culturing, in culture media, a population of pluripotent stem cells (PSCs) to produce an expanded population of PSCs; and
expressing in PSCs of the expanded population a SPIl protein, a CEBPA protein, and a FLI1 protein, to produce a population of microglia-like cells.”
Claim 42 recites “A method comprising:
(a) delivering to pluripotent stem cells (PSCs) one or more engineered polynucleotide comprising an inducible promoter operably linked to one or more open reading frame encoding a SPIl protein, a CEBPA protein, and a FLI1 protein, optionally wherein the one or more engineered polynucleotide further comprises an inducible promoter operably linked to one or more open reading frame encoding a MEF2C protein, a CEBPB protein, and/or a IRF8 protein; and
(b) culturing the PSCs in induction media comprising an inducing agent to produce CD11b+, CX3CR1+, ITGAM+, P2RY12+, TMEM119+, TREM2+, TRA-1- 60- and/or POU5F1- microglia-like cells; or seeding the PSCs in feeder-free, serum-free culture media and optionally culturing the PSCs for about 1 to about 24 hours, and culturing the PSCs in induction media comprising an inducing agent to produce CD11b+, CX3CR1+, ITGAM+, P2RY12+, TMEM119+, TREM2+, TRA-1-60- and/or POU5F1- microglia-like cells.”
In analyzing whether the written description requirement is met for genus claims, it is first determined whether a representative number of species have been described by their complete structure. To provide adequate written description and evidence of possession of a claimed genus, the specification must provide sufficient distinguishing identifying characteristics of the genus. The factors to be considered include disclosure of complete or partial structure, physical and/or chemical properties, functional characteristics, structure/function correlation, methods of making the claimed product, or any combination thereof. The disclosure of a single species is rarely, if ever, sufficient to describe a broad genus, particularly when the specification fails to describe the features of that genus, even in passing. (see In re Shokal 113USPQ283(CCPA1957); Purdue Pharma L.P. vs Faulding Inc. 56 USPQ2nd 1481 (CAFC 2000).
The court explained that “reading a claim in light of the specification, to thereby interpret limitations explicitly recited in the claim, is a quite different thing from ‘reading limitations of the specification into a claim,’ to thereby narrow the scope of the claim by implicitly adding disclosed limitations which have no express basis in the claim.” The court found that applicant was advocating the latter, i.e., the impermissible importation of subject matter from the specification into the claim.). See also In re Morris, 127 F.3d 1048, 1054-55, 44 USPQ2d 1023, 1027-28 (Fed. Cir. 1997).
The culturing steps, including the culture media and induction media, of claims 21 and 42 are recited at a high level of generality. For example, claim 42 recites a generic genus of “inducing agent[s]” to be in the induction media. In claims 21 and 42, no other media components are recited, nor any concentrations or culture periods. Claim 21 recites expressing three proteins in the PSCs, but does not comprise a method step of introducing these proteins to the cells, or recite what culture media would result in the expression of these three proteins.
The claim is considered to lack adequate written description for failing to recite the structure that is necessary and sufficient to cause the recited functional language (i.e., producing an expanded population of PSCs; producing microglia-like cells). The specification fails to disclose what structural changes to the method steps of claims 21 and 42 (e.g., culture media composition, culture period) is/are necessary and sufficient to produce and expanded population of PSCs and to produce microglia-like cells, and hHPCs sharing the same functional properties (e.g., what factors in culture media would/would not result in microglia-like cells? How long must the cells be cultured?), and thus the ordinary artisan would not know what modification(s) must be made in order to fulfill the instant recitation.
The specification offers little guidance on culturing the PSCs to differentiate into microglia-like cells. Pg. 23-24 states:
“In some embodiments, PSCs of the expanded population are cultured for no longer than 10 days… In some embodiments, PSCs of the expanded population are cultured in an induction media for about 36 to about 60 hours… Culturing in the second induction media comprises, in some embodiments, several (one or more) media changes. For example, an induction media may be removed and replaced with new (fresh) second indication media every (about) 12 hours, every 24, hours, every 36 hours, or every 48 hours. In some embodiments, an induction media is changed every (about) 24 hours.”
Regarding the culture media, pgs. 25-26 disclose:
“The methods provided herein, in some embodiments, comprise culturing PSCs in a feeder-free, serum-free culture media. Culture media may comprise, for example, a solubilized basement membrane preparation extracted from the Engelbreth-Holm-Swarm (EHS) mouse sarcoma (e.g., Corning® Matrigel® Matrix) (coated at -75 pl/cm2 to ~150 pl/cm2 of lot-based diluted suspension). In some embodiments, the solubilized basement membrane preparation comprises one or more extracellular matrix (ECM) protein and one or more growth factor. For example, the ECM proteins may be selected from Laminin, Collagen IV, heparan sulfate proteoglycans, and entactin/nidogen.
In some embodiments, the culture media further comprises one or more growth factor, for example, selected from recombinant human basic fibroblast growth factor (rh bFGF) (e.g., 80 ng/ml to 120 ng/ml) and recombinant human transforming growth factor pB (rh TGF3) (e.g., 20 pM to 25 pM). In some embodiments, culture media further comprises rh bFGF and rh TGF3. In some embodiments, culture media comprises mTeSRTM Plus medium (STEMCELL Technologies).
In some embodiments, the culture media further comprises a small molecule ROCK inhibitor (e.g., 9 pM to 11 pM), such as Y-27632, to facilitate seeding the of PSCs.
In some embodiments, an inducing agent is added to the culture media to produce an induction media. In some embodiments, the induction media comprises an inducing agent (e.g., doxycycline (e.g., 0.1-1 pg/mL, optionally about 0.5 pg/ml)).”
The specification does not teach a working example where PSCs are differentiated into microglia-like cells expressing SPI1, CEBPA, and FLI1 without transfecting PSCs with expression vectors comprising polynucleotides encoding these proteins.
The art teaches varied methods of culturing PSCs to differentiate into microglia-like cells, with different methods resulting in different expression.
Speicher et al. (Speicher et al., published Dec 2019, cited in IDS filed 07/10/2025) is considered relevant prior art for reviewing different methods of directed differentiation of hPSCs into microglia. Table 1 includes a detailed summary of the media compositions used on specific days of the protocol. The protocol taught by Abud et al. for example, comprises six different media across 38 days to differentiate hPSCs into microglia-like cells. Pandya et al. on the other hand teaches three different culture medias used in their protocol to generate microglia-like cells.
Table 2 of Speicher et al. also teaches that these different protocols result in microglia-like cells with different structures and profiles, as well as different yields and purities.
In summary Speicher et al. demonstrates the importance of the culturing protocol on the structure and function of the resulting microglia-like cells.
Thus, for the reasons outlined above, it is concluded that the claims do not meet the requirements for written description under 35 U.S.C. 112, first paragraph.
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.
Claim 42 is rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention.
Claim 42 recites “or seeding the PSCs in feeder-free, serum-free culture media and optionally culturing the PSCs for about 1 to about 24 hours, and culturing the PSCs in induction media comprising an inducing agent to produce CD11b+, CX3CR1+, ITGAM+, P2RY12+, TMEM119+, TREM2+, TRA-1-60- and/or POU5F1- microglia-like cells.” It is unclear what limitations in the claim are “optional” or not. For example, is seeding the PSCs in feeder-free, serum-free culture media the only required limitation of the alternate step (b)? Is culturing the PSCs in induction media optional, or only culturing the PSCs for about 1 to about 24 hours? It would be remedial to clarify the required limitations of the claimed method. For examination purposes, any recitations following the term “optionally” are interpreted to not be required limitations.
Claim Rejections - 35 USC § 102
The following is a quotation of the appropriate paragraphs of 35 U.S.C. 102 that form the basis for the rejections under this section made in this Office action:
A person shall be entitled to a patent unless –
(a)(1) the claimed invention was patented, described in a printed publication, or in public use, on sale, or otherwise available to the public before the effective filing date of the claimed invention.
(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, 2, 4-6, 18-21, and 42 is/are rejected under 35 U.S.C. 102(a)(2) as being anticipated by Yumoto et al. (US20230323295A1, published 10/12/2023, filed 03/01/2023, foreign priority to JP2022046010A, filed 03/22/2022).
Regarding claims 1 and 20, Yumoto et al. discloses a pluripotent stem cell (PSC) (population- claim 20) comprising: one or more engineered polynucleotide comprising an open reading frame encoding a SPIl protein, a FLI1 protein, and a CEBPA protein (e.g., [0027]).
Regarding claim 2, Yumoto et al. discloses the PSC of claim 1, wherein the one or more engineered polynucleotide comprises a first polycistronic polynucleotide that comprises a first open reading frame encoding the SPIl protein, a second open reading frame encoding the FLI1 protein, and a third open reading frame encoding the CEBP1 protein (e.g., [0024, 0025]; Table 3; Table 4).
Regarding claim 4, Yumoto et al. discloses the PSC of claim 1, wherein the one or more engineered polynucleotide comprises a first polycistronic polynucleotide that comprises the following open reading frames, ordered in the 5' to 3' direction of the first polycistronic polynucleotide: a first open reading frame encoding the SPIl protein, a second open reading frame encoding the CEBPA protein, and a third open reading frame encoding the FLI1 protein (e.g., ABD of Tables) (e.g., [0024, 0025]; Table 3; Table 4).
Regarding claims 5 and 6, Yumoto et al. discloses the one or more engineered polynucleotide comprises an open reading frame encoding one or more or two or more of a MEF2C protein, a CEBPB protein, and a IRF8 protein (e.g., Abstract).
Regarding claims 18 and 19, Yumoto et al. discloses the PSC being induced and/or human (e.g., [0021]).
Regarding claim 21, Yumoto et al. discloses a method, comprising: culturing, in culture media, a population of pluripotent stem cells (PSCs) to produce an expanded population of PSCs; and expressing in PSCs of the expanded population a SPIl protein, a CEBPA protein, and a FLI1 protein, to produce a population of microglia-like cells (e.g., [0024, 0025]; [0073]; Table 3; Table 4).
Regarding claim 42, Yumoto et al. discloses a method comprising:(a) delivering to pluripotent stem cells (PSCs) one or more engineered polynucleotide comprising an inducible promoter operably linked to one or more open reading frame encoding a SPIl protein, a CEBPA protein, and a FLI1 protein, and (b) culturing the PSCs in induction media comprising an inducing agent to produce CD11b+, CX3CR1+, ITGAM+, P2RY12+, TMEM119+, TREM2+, TRA-1- 60- and/or POU5F1- microglia-like cells (e.g., [0073-0075]; Fig. 7).
Claim Rejections - 35 USC § 103
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
Claim(s) 1, 2, 4-7 and 10-15, 18-21, and 42 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yumoto et al. as applied to claims 1, 2, 4-6, 18-21, and 42 above, and further in view of Pawlowski et al. (US20220220441A1, published 07/14/2022, filed 05/27/2020).
As shown above, the base claims are anticipated by the base art, and thus, are also obvious over the base art.
Yumoto et al. does not teach a PSC comprising one or more engineered polynucleotide comprising an ORF encoding IRF8, or an ORF operably linked to a heterologous promoter.
An Artisan, interested in methods of generating microglia from hPSCs, would be aware of Pawlowski et al. for teaching methods of producing microglia from PSCs comprising introducing a nucleotide sequence encoding PU.1 (i.e., SPI1) and IRF8.
Regarding claims 7 and 10, Pawlowski et al. teaches a method for the production of microglia from stem cells, comprising the steps of: a) targeted insertion of a nucleotide sequence encoding a transcriptional regulator protein into a first genomic safe harbour site; and
b) targeted insertion of the coding sequence of the transcription factor PU.1 (SEQ ID NO: 1) into a second genomic safe harbour site, wherein the gene is operably linked to an inducible promoter, which is regulated by the transcriptional regulator protein; expression of PU.1 (SEQ ID NO: 2); and
c) culturing the stem cells received from steps a) and b) with exposure to at least one growth factor or small molecule that recapitulates signaling during at least one stage of embryonic development of microglia or adult microglia proliferation, differentiation or polarization (e.g., claim 1 of Pawlowski et al.).
The method may further comprise insertion of the coding sequence of the gene of the transcription factor IRF8 (SEQ ID NO: 5) and expression thereof (e.g., claim 8 of Pawlowski et al.).
PU.1 and IRF8 (and/or CEBPB) may be transfected in the form of a bi-cistronic expression cassette (e.g., [0174]; Fig. 4e; claims 1, 6, and 8 of Pawlowski et al.).
It would be obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the PSC taught by Yumoto et al. by adding IRF8 to the set of proteins encoded by the one or more engineered polynucleotide comprising an open reading frame as taught by Pawlowski et al. and yield predictable results. Adding IRF8 as taught by Pawlowski et al. would not render the one or more engineered polynucleotide functionless. Adding another protein to an expression cassette is routine in the art. One would be motivated to make this combination because as taught by Pawlowski et al., screening experiments demonstrated rapid induction of myeloid and microglia lineage marker in all three cell lines expressing PU.1 plus any of the other three candidate reprogramming factors (including IRF8), but not in wild-type control hiPSCs or in cells expressing PU.1 alone (e.g., [0174-0175]). Additionally, IRF8 acts as a reprogramming factor for the generation of microglia (e.g., [0010]).
Regarding claims 11-15, Pawlowski et al. teaches stable introduction of an inducible cassette into the genome of a stem cell, whilst being able to control the transcription of that inducible cassette and thereby the inserted transcription factors (e.g., [0010]). The gene may be regulated by an inducible (e.g., heterologous) promoter, such as a Tet Responsive Element (TRE) (e.g., [0012], [0022]). The inducible system may be tetracycline-controlled activation, where transcription is reversibly turned on or off in the presence of the antibiotic tetracycline or one of its derivatives (e.g. doxycycline, which is more stable) (i.e., chemically-inducible) (e.g., [0117-0118]). The promoter may be operably-linked (e.g., [0001]; [0125]).
It would be obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to modify the PSC taught by Yumoto et al. by adding a chemically-inducible promoter to the one or more engineered polynucleotide comprising an open reading frame, with the promoter being operably-linked, as taught by Pawlowski et al. and yield predictable results. Adding the promoter as taught by Pawlowski et al. would not render the one or more engineered polynucleotide functionless. Adding a promoter to an expression cassette in order to express one or more proteins is routine in the art. One would be motivated to make this combination because as taught by Pawlowski et al., an inducible promoter results in the ability to control the expression of a genetic sequence via an analyte, co-factor, regulatory protein, etc. (e.g., [0076]). The Tet-On system taught by Pawlowski et al. is composed of two components; the constitutively expressed tetracycline-responsive transcriptional activator protein (rtTA) and the rtTA sensitive inducible promoter (Tet Responsive Element, TRE). This may be bound by tetracycline or its more stable derivatives, including doxycycline (dox), resulting in activation of rtTA, allowing it to bind to TRE sequences and inducing expression of TRE-controlled genes (e.g., [0118]).
Claim(s) 1-15, 18-21, and 42 is/are rejected under 35 U.S.C. 103 as being unpatentable over Yumoto et al. and Pawlowski et al. as applied to claims 1, 2, 4-7 and 10-15, 18-21, and 42 above, and further in view of Shaimardanova et al. (Shaimardanova, Alisa A et al. “Production and Application of Multicistronic Constructs for Various Human Disease Therapies.” Pharmaceutics vol. 11,11 580. 6 Nov. 2019).
As shown above, the base claims are obvious over the base art.
Regarding claims 3, 8, and 9, Yumoto et al. and Pawlowski et al. do not teach the PSC of claim 1, wherein the one or more engineered polynucleotide comprises a first polycistronic polynucleotide that comprises the following open reading frames, ordered in the 5' to 3' direction of the first polycistronic polynucleotide: a first open reading frame encoding the SPIl protein, a second open reading frame encoding the FLI1 protein, and a third open reading frame encoding the CEBPA protein, or wherein the one or more engineered polynucleotide comprises a second polycistronic polynucleotide that comprises the following open reading frames, ordered in the 5' to 3' direction of the second polycistronic polynucleotide: a first open reading frame encoding the MEF2C protein, a second open reading frame encoding the CEBPB protein, and a third open reading frame encoding the IRF8 protein (or 5’ to 3’: MEF2C, IRF8, CEBPB).
An artisan, interested in the co-expression of genes in multicistronic constructs, would be aware of Shaimardanova et al. for reviewing multicistronic vectors based on IRES nucleotide sequence and self-cleaving 2A peptides.
Shaimardanova et al. teaches self-cleaving 2A peptides as promising candidates for the production of multicistronic vectors due to their small size and self-cleavage ability (e.g., pg. 4, “3. Self-Cleaving 2A Peptides”). When using 2A peptides to express multiple genes, there is better correlation of the expression of genes placed upstream and downstream of the peptide sequence (e.g., Table 1). However, use of 2A peptides can result in incomplete cleavage of
the translated polypeptide (e.g., pg. 4, para 2). Incomplete cleavage of products of 2A-based
multicistronic constructs can lead to a decreased yield of the final product and to the disruption of the protein functions. As such, the unequal expression of proteins located in the different parts of the vector or separated with different 2A peptide sequences forces researchers to pay more attention to the order of genes in multicistronic vectors in order to achieve more significant therapeutic effects (e.g., pg. 10, “10. Conclusions and Future Perspectives”, para 2).
It would be obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to apply the knowledge that the expression of proteins in a multicistronic system depends on the order of the proteins as taught by Shaimardanova et al. to the PSC comprising one or more polynucleotides encoding proteins taught by Yumoto et al. (and Pawlowski et al.) and arrive at the claimed invention. One would be motivated to apply the teachings of Shaimardanova et al. because despite potential issues with incomplete cleavage and protein synthesis, 2A is considered the best way to provide co-expression of several genes compared with other strategies (e.g., pg. 5, para 1). As such, when using this strategy, one would want to apply techniques and previous findings known in the art to optimize gene expression. As taught by Shaimardanova et al., these previous findings include the importance of gene order and 2A peptide sequence order on protein expression levels in multicistronic constructs and vectors (e.g., pg. 5, para 3).
Conclusion
No claims are allowed.
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ALLISON M. JOHNSON
Examiner
Art Unit 1638
/ALLISON MARIE JOHNSON/Examiner, Art Unit 1638
/ROBERT M KELLY/Primary Examiner, Art Unit 1638
Prosecution Insights