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 without traverse of Group I (Claims 1 and 10-11, 13-14, and 29-43; drawn to a method for the production of microglia from stem cells in the reply filed on September 10, 2024, is acknowledged.
Furthermore, Applicant elected the following species of transcription factor, without traverse: CEBPB
Claims 32-33 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected species, there being no allowable generic or linking claim. Election was made without traverse in the reply filed on September 10, 2024.
DETAILED ACTION
The claims filed on June 8, 2026, have been acknowledged. Claims 2-9, 12, 15-28, 45-47, and 51 were cancelled. In light of the Applicant’s elected invention and species, claims 32-33 are withdrawn from further consideration pursuant to 37 CFR 1.142(b) as being drawn to a nonelected invention, there being no allowable generic or linking claim. Claims 1, 10-11, 13-14, 29-31, 34-44, 48-50, and 52-53 are pending and examined on the merits.
Information Disclosure Statement
The information disclosure statement (IDS) filed on June 8, 2026, has been considered.
Maintained 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.
Claims 1, 10-11, 13-14, 31, 34, 37-39, 41-44, 48-50, and 52-53 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No. 20180127714 (Ko; prior art of record), Brownjohn et al. (Stem Cell Reports 10:1294-1307. 2018), World Intellectual Property Organization Patent Application No. 2018/160496 (Blurton-Jones; prior art of record), as evidenced by Boreland et al. (BioRxiv: 1-45. 2023), Garcia-Reitboeck et al. (Cell Reports 24: 2300–2311. 2018), and Pandya et al. (Nature Neuro 20: 753-759. 2017; prior art of record). This rejection is repeated with regards to the rejection in the Non-final Office action mailed on April 29, 2026. Applicant’s traversal is addressed below.
Regarding claims 1 and 52-53, Ko teaches a method of differentiating a human embryonic stem cell (a pluripotent stem cell) into a hematopoietic stem cell, including a step of forcibly expressing at least two and up to seven (out of seven) transcription factors including SPI1 (also known as PU.1) and CEBPB in a pluripotent stem cell. Through addition of doxycycline to human embryonic stem cells having introduced therein those transcription factors as Transgene to be induced by doxycycline, those transcription factors were forcibly expressed in human embryonic stem cells. Figure 1 of Ko shows that the doxycycline inducible system uses a nucleotide sequence encoding the rtTA transcriptional regulator protein as one insertable sequence with the inducible transcription factors under control of the inducible tetO2 (a TRE) promoter as the other insertable nucleotide sequence. The forced expression was performed for 48 hours on from day 3 to day 5 of the culture, followed by culture in a known hematopoietic progenitor cell medium, and cells on day 5 of the culture were harvested. It was found that forced expression (induced with DOX) of transcription factors (such as SPI1 and CEBPB) under known hematopoietic progenitor cell conditions remarkably increased the speed and efficiency of the differentiation into hematopoietic progenitor cells. The expression of CD43 serving as a marker of hematopoietic progenitor cells was confirmed on day 5 of the culture. On day 5, in the control group having introduced therein no transcription factor, the expression of CD43 was hardly recognized. Thus, the method of the present invention has an about 4-fold differentiation inducing ability (differentiation speed) as compared to the related-art method. Irrespective of whether the transcription factors were used alone or in combination thereof, the transcription factors were able to cause the differentiation into hematopoietic progenitor cells. (paragraphs 0069-0076 and Example 6).
Ko teaches that the type of cells into which the cocktail of two or more transcription factors are introduced is not particularly limited, and encompasses pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (Example 2).
Although Ko teaches that they differentiated pluripotent stem cells into HSPCs, Ko does not teach wherein the HSPCs are differentiated into microglia.
Brownjohn teaches a method of generating microglia from pluripotent stem cells comprising: as a starting point for the differentiation of microglia, they followed an established method for the derivation of primitive macrophage precursors (PMPs) from human pluripotent stem cells. Two to three weeks after the initiation of differentiation, PMPs are produced continuously in suspension, and can be harvested for further maturation. For microglia differentiation, they differentiated PMPs over 6–10 days in differentiation media comprising complete RPMI1640 containing a combination of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-34 (IL-34) to produce monocultures that morphologically resemble mature microglia (Figure 1A). Analysis of the proportion of these cells expressing canonical macrophage/microglia markers indicates that this protocol has a high level of efficiency across genetic backgrounds, producing cells positive for Iba1, positive for CD45, and positive for TREM2 (Figure 1B) (Figures 1-2 and page 1295, column 1, paragraph 2-page 1296, column 1, paragraph 2). As can be seen in Figure 1A, the resulting microglia exhibit a ramified phenotype that is similar to what is shown in Figure 5D of the instant application.
Claim 1 does not specifically recite that the stem cells cannot be placed in microglial differentiation media. Furthermore, example 2 of the instant specification recites that cells were cultured in chemically-defined microglia differentiation and maintenance medium.
Boreland evidences that primitive macrophage precursors express markers CD235 and CD43.
As stated above, Ko teaches that their hematopoietic progenitor cells express CD43 but are silent regarding expression of CD235.
However, Blurton-Jones teaches a method of producing human microglial-like (iMGLs) cells from pluripotent stem cells (PSCs). The method comprises the steps: (i) differentiating PSCs using a media supplemented with hematopoietic differentiation factors (to produce induced hematopoietic progenitor cells (CD43+/CD235a+/CD41a+ iHPCs), (ii) isolating CD43+ iHPCs, (iii) differentiating the CD43+ iHPCs into iMGLs using a microglial differentiating media; and (iv) maturing the iMGLs. iPSCs were differentiated into hematopoietic progenitors (iHPCs), which recapitulates microglia ontogeny as iHPCs represent early primitive hematopoietic cells derived from the yolk sac that give rise to rnicroglia during development (paragraph 0005-0029, Example 1, and Figure 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined the in vitro production method of CD43+ HSPCs from pluripotent stem cells of Ko with the CD43+ differentiation media of Blurton-Jones and the CD43+/CD235+ PMP to microglia differentiation method of Brownjohn to arrive at the instantly claimed invention.
One of ordinary skill in the art would have a reason to combine because Ko teaches that their method of making CD43+ HSPCs has about a 4-fold differentiation inducing ability (differentiation speed) as compared to the just using differentiation media. Furthermore, Blurton-Jones teaches that their differentiation media generates CD43+/CD235+ HPCs for further differentiation into microglia after 10 days of culture and Brownjohn teaches that PMPs (CD43+/CD235+ HPCs) can be differentiated to microglia in 6-10 days. As Ko teaches their method results in an increased differentiation speed of CD43+ HSPCs compared to just differentiation media alone, it would have been obvious to use this method to produce and isolate CD43+/CD235+ hematopoietic progenitor cells for the subsequent formation of microglia using the differentiation method of Brownjohn as more CD43+/CD235+ cells could be produced in a shorter period of time. The combined method of generating microglia from iPSCs would result in a rapid and efficient method of generating microglia in 8-17 days of culture depending on how one chooses to set up the differentiation protocol. One would have combined these methods with a reasonable expectation of success because Blurton-Jones has successfully reduced to practice that iPSCs can be differentiated into CD43+/CD235+ HSPCs and Blurton-Jones and Brownjohn have successfully reduced to practice that CD43+/CD235+ HSPCs can be differentiated into microglia using a defined microglia differentiation culture media. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding whether the combined method of Ko, Brownjohn, and Blurton-Jones produces microglia within 20 days of expressing the transcription factors, as stated supra, Ko teaches that the forced expression was performed for 48 hours on from day 3 to day 5 of the culture, followed by culture in a known hematopoietic progenitor cell medium, and cells on day 5 of the culture were harvested. The expression of CD43 serving as a marker of hematopoietic progenitor cells was confirmed on day 5 of the culture (paragraphs 0069-0076 and Example 6). Therefore, the method of Ko generates CD43+ hematopoietic progenitor cells 48 hours after expression of the transcription factors. Regarding the method of Blurton-Jones, they teach that they differentiated iPSCs into CD43+/CD235+ hematopoietic progenitor cells from days 0-10. Then, CD43+/CD235+ hematopoietic progenitor cells were grown in microglial differentiation medium. Therefore, the method of generating CD43+/CD235+ HPCs (i.e. PMPs) of Ko and Blurton-Jones could follow the 5 day culture method of Ko using the differentiation media of Blurton-Jones or continue for an additional 5 days, as done by Blurton-Jones. This would equate to culturing the iPSCs for 2-7 days post-induction of the transcription factors (days 1-3 of culture do not involve transcription factor induction and are, thus, left out of the calculation).
Furthermore, Brownjohn teaches that their method of differentiating CD43+/CD235+ PMPs into microglia occurs after 6-10 days of culture in a combination of granulocyte macrophage colony-stimulating factor (GM-CSF) and interleukin-34 (IL-34) microglia differentiation media.
As such, the combined method of Ko, Brownjohn, and Blurton-Jones would generate a number of microglial cells around 8-17 days after forced expression of the transcription factors, well within the 20 days required by the claims.
Regarding the limitation mature microglia expressing CD68/HEXB/TMEM19 are produced, Brownjohn is silent regarding the expression of CD68 in their microglia. However, Garcia-Reitboeck evidences that IBA1 is coexpressed with CD68 (Supplemental Figure 3). Figure 1 of Brownjohn shows that over 95% of their microglia express IBA1 and would also express CD68. It is worth noting that the Applicant, similarly, identifies IBA1 expression in their microglia but does not examine CD68 expression). Furthermore, Brownjohn teaches that their microglia express TMEM119 (Figure 2) and HEXB (Supplemental Figure 4).
Regarding claims 10-11, as stated supra, Ko teaches that they used doxycycline to induce the expression of the transcription factors (Example 6). Figure 1 of Ko shows that the doxycycline inducible system uses a nucleotide sequence encoding the rtTA transcriptional regulator protein as one insertable sequence with the inducible transcription factors under control of the inducible tetO2 (a TRE) promoter as the other insertable nucleotide sequence.
Regarding claims 13-14, as stated supra, Ko teaches they used human embryonic stem cells (Example 6)
Regarding claim 31, Ko teaches that the CEBPB gene has the nucleotide sequence of SEQ ID NO: 31 which has 100% sequence identity to SEQ ID NO: 3 of the instant application (paragraph 0181).
Regarding claim 34, as stated supra, Ko teaches that as part of their method, SPI1 (i.e. PU.1) and CEBPB can be expressed together to differentiate embryonic stem cells into HSPCs (paragraphs 0069-0076).
Regarding claims 37-39 and 48-49, as an initial matter, the limitation wherein the pluripotent stem cells are cultured with a growth factor or small molecule is broadly considered to include any culture condition used for differentiation of the pluripotent stem cells into microglia regardless of whether the cell type at the time of addition of the culture media is a pluripotent stem cell or a differentiated cell type derived from a pluripotent stem cell. This interpretation is based on examples 1-2 of the instant specification, as these examples do not discuss any media composition for the pluripotent stem cells other than to say the hiPSCs were plated as single cells onto Matrigel in pluripotency maintenance medium. After two days, the media is changed to Dulbecco's modified eagle medium (DMEM)/F12 supplemented with dox for transgene induction plus undefined small molecules and growth factors. Instead, Applicant identifies that initial screening experiments demonstrated rapid induction of myeloid and microglia lineage marker in all three cell lines expressing PU.1 (SEQ ID NO: 2) plus any of the other three candidate reprogramming factors, but not in wild-type control hiPSCs. Furthermore, the growth factors identified in claim 39 and 48 were specifically used in the microglia differentiation media (as identified in Muffat et al. 2016) and would have been cultured with macrophages and not pluripotent stem cells, at that point in the differentiation process.
Brownjohn teaches, as stated supra, that for microglia differentiation, they differentiated PMPs over 6–10 days in differentiation media comprising complete RPMI1640 containing a combination of granulocyte macrophage colony-stimulating factor (GM-CSF; also known as CSF2) and interleukin-34 (IL-34) to produce monocultures that morphologically resemble mature microglia (Figure 1A).
Regarding claim 48, specifically, although Brownjohn does not teach using CSF1 in their microglia differentiation media, Blurton-Jones teaches that they cultured CD43+/CD235+ iHPCs with CSF-1, IL-34, and TGFβ1 to produce microglial cells (Example 1).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined CSF-1, as identified by Blurton-Jones, with the microglia differentiation media of Brownjohn as part of the combined method of differentiating pluripotent stem cells into microglia of Ko, Brownjohn, and Blurton-Jones to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to combine because Brownjohn and Blurton-Jones are focused on differentiating pluripotent stem cells into hematopoietic progenitor cells that are CD43+ and CD235+ and Blurton-Jones and Brownjohn are focused on further differentiating those CD43+/CD235+ hematopoietic cells into microglia. "It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition to be used for the very same purpose.... [T]he idea of combining them flows logically from their having been individually taught in the prior art." In re Kerkhoven, 626 F.2d 846, 850, 205 USPQ 1069, 1072 (CCPA 1980). MPEP 2144.06. As such, it would have been obvious that one could use CSF-1 (M-CSF) as part of the microglia differentiation media of Ko, Brownjohn, and Blurton-Jones. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Regarding claim 41, as stated supra, Ko teaches that the type of cells into which the single transcription factor or the cocktail of two or more transcription factors are introduced is not particularly limited, and encompasses pluripotent stem cells, such as embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) (Example 2).
Regarding claim 42, Brownjohn teaches that their microglia express TREM2 and P2RY12 (Figures 1-2)
Regarding claim 43, as stated supra, Brownjohn teaches that their microglia express, P2RY12, TREM2, and TMEM119. Brownjohn is silent as to whether their induced microglia express CD39. However, Pandya evidences that CD39 is a plasma membrane protein specific to microglia (page 754, column 1, paragraph 1) and is expressed on induced microglia when iPSCs are differentiated into microglia-like cells via a hematopoietic progenitor intermediate (page 755, column 2, paragraph 2-page 756, column 2, paragraph 1). As such, the induced microglia of Brownjohn would also express CD39.
Regarding claim 44, Ko, as stated supra, teaches a step of forcibly expressing at least two and up to seven (out of seven) transcription factors including SPI1 (also known as PU.1) and CEBPB in a pluripotent stem cell. Through addition of doxycycline to human embryonic stem cells having introduced therein those transcription factors as Transgene to be induced by doxycycline, those transcription factors were forcibly expressed in human embryonic stem cells (paragraphs 0069-0076 and Example 6). Ko teaches that viral vectors can be used to introduce the nucleic acids encoding the transcription factors into the pluripotent stem cells (i.e. inserting the coding sequences into the cells) (paragraphs 0160-0161).
Regarding claim 50, Brownjohn teaches that their induced microglia express Iba1 (Figure 1).
Response to Arguments
Applicant's arguments filed June 8, 2026, are acknowledged.
Applicant argues that they respectfully disagrees with the Examiner's characterization of the cited art teachings and application of the same in making the instant rejections. The Examiner's rationale is predicated on the non-evidence-based assumption that the hematopoietic progenitor cells, or HPCs, in Ko are equivalent to the primitive macrophage precursors, or PMPs, in Brownjohn, merely because both cell types express CD43 and CD235. However, this fundamental assumption is incorrect and against the objective evidence that would have been known to one skilled in the art at the time of the invention, including objective evidence in the cited references themselves.
First, CD43 is not a marker that is unique to PMPs or that distinguishes them from other hematopoietic populations. As recognized by one skilled in the art, CD43 is expressed by nearly all hematopoietic cells at various stages of development, with the exception of mature erythrocytes and certain resting B-cell subsets. CD43-positive cells can thus be considered to be almost any cell derivable from an HPC. Accordingly, the mere presence of CD43 does not necessarily mean that two hematopoietic cell populations are biologically identical or developmentally equivalent.
Second, as the Examiner has acknowledged, Ko does not disclose that its HPCs express CD235. This omission is significant because CD235 is associated with primitive hematopoiesis rather than hematopoietic progenitors generally or PMPs definitively. Instead, hematopoiesis occurs in two waves. Embryonic hematopoiesis generates primitive (also known as "yolk-sac") HPCs, including progenitors that give rise to tissue-resident macrophages such as microglia. Permanent definitive hematopoiesis generates definitive HPCs and ultimately hematopoietic stem cells (HSCs). CD235 is a marker of primitive HPCs, and PMPs are cells downstream from primitive HPCs. Because there is no mention in Ko that its HPCs express CD235, a skilled person cannot ascertain whether the methods described therein would generate primitive or definitive HPCs. Simply assuming the presence of CD235 is insufficient evidence to support an obviousness rejection. See MPEP § 2144.03 (requiring examiners to take official notice of facts unsupported by documentary evidence but only where the facts are "capable of such instant and unquestionable demonstration as to defy dispute").
The Examiner has asserted that because the HPCs of Blurton-Jones express CD235, the HPCs disclosed in Ko would express CD235 if substituted with the differentiation media of Blurton-Jones or cultured for 5 additional days in Blurton-Jones's differentiation media. This is also a conclusion without any evidentiary support and an incorrect assumption that would not have been made by a POSA. The Federal Circuit has cautioned that "rejections on obviousness cannot be sustained with mere conclusory statements; instead, there must be some articulated reasoning with some rational underpinning to support the legal conclusion of obviousness." In re Kahn, 441 F.3d 977, 988, (Fed. Cir. 2006); see also MPEP 2142. As set out in paragraph [0141] of Blurton-Jones, the inventors there sought CD235+ ''primitive hematopoietic cells derived from the yolk sac that give rise to microglia during development", not definitive HPCs or HPCs in general. The fact that Blurton-Jones specifically generated CD235+ primitive HPCs does not support the Examiner's conclusion that Ko's HPCs are necessarily or would become CD235+ progenitors, even if cultured in Blurton Jones's differential media, or that Ko's and Blurton-Jones's methods could have been combined to produce CD235+ HPCs in a shortened timeframe.
Third, the overall teachings of Ko would lead a POSA to understand that Ko's progenitors are associated with definitive rather than primitive hematopoiesis. Throughout Ko, substantial emphasis is placed on the ability of the transcription factors to generate progenitors capable of producing HSCs, which as explained earlier arise exclusively from definitive HPCs. See, e.g., Ko at [0069]-[0084], [0147], [0181], [0289], [0290], [0359], [0386], [0411], [0543][0546]. Consequently, a POSA reviewing Ko would reasonably understand that the disclosed progenitor populations are definitive HPCs rather than primitive HPCs, and therefore would not have combined Ko with Brownjohn when the latter's method focuses on the generation of PMPs which, as the name implies, are a form of primitive HPCs.
Because of the incorrect and non-evidence-based assumption on which the present rejection is made, as detailed above, the Examiner has not properly established that Ko' s HPCs are CD43- and CD235-positive, that Ko' s HPCs and Brownjohn' s PMPs are equivalent, or that Ko' s HPCs are capable of following the differentiation pathway and timeline utilized in Brownjohn to arrive at mature microglia. As a result, the Examiner has not established that the combination of Ko, Brownjohn, and Blurton-Jones teach or suggest each and every element of the claimed method Moreover, the Examiner has not established that a POSA would have been motivated to substitute Ko's HPCs into Brownjohn's subsequence differentiation protocol. Nor has the Examiner established that a POSA would have reasonably expected Ko's HPCs to undergo the developmental processes necessary to produce mature microglia expressing CD68, HEXB, or TMEM 119 within the claimed "20 days or less" time frame with a reasonable expectation of success (page 8, paragraph 2-page 10, paragraph 2).
Applicant's arguments and the cited prior art have been fully considered but they are not persuasive.
As an initial matter, the rejection above does not assume the hematopoietic progenitor cells in Ko are equivalent to the primitive macrophage precursors, or PMPs, in Brownjohn, merely because both cell types express CD43 and CD235. As identified in the rejection above, it is acknowledged that Ko does not discuss CD235 expression in their hematopoietic progenitor cells. Instead, the rejection discusses that the CD43+ hematopoietic progenitor cells of Ko could be differentiated in the differentiation media of Blurton-Jones as this media composition is known to produce CD43+, Cd235+, CD41+ positive cells that can then be further differentiated into microglia. As stated in the rejection above, it would have been obvious to combine the in vitro production method of CD43+ HSPCs from pluripotent stem cells of Ko with the CD43+ differentiation media of Blurton-Jones because Ko teaches that their method of making CD43+ HSPCs has about a 4-fold differentiation inducing ability (differentiation speed) as compared to just using differentiation media. As Ko teaches their method results in an increased differentiation speed of CD43+ HSPCs compared to just differentiation media alone, it would have been obvious to use this method to produce and isolate CD43+/CD235+ hematopoietic progenitor cells for the subsequent formation of microglia as more CD43+/CD235+ cells could be produced in a shorter period of time. Therefore, it is clear from the rejection above that it is the combination of the in vitro production method of CD43+ HSPCs from pluripotent stem cells of Ko with the CD43+ differentiation media of Blurton-Jones that is considered to lead to development of CD43+, CD235+ cells rather than just the cells of Ko alone, as argued by the Applicant.
Regarding Applicant’s arguments about CD43 and CD235 marker expression, in order to complete the art of record and rebut Applicant’s arguments, Xu et al. (Nature Communications 11: 1-16. 2020) evidences that microglia originate from yolk sac erythromyeloid progenitors (EMPs) during primitive hematopoiesis. EMPs further develop to primitive macrophage progenitors (PMPs) that migrate into the
developing neural tube and become microglia with ramified processes within the CNS environment. Additionally, they confirmed the identity of their hPSC-derived PMPs by staining with CD235, a marker for YS primitive hematopoietic progenitors, and CD43, a marker for hematopoietic progenitor-like cells (page 2, column 2, paragraph 1). Furthermore, Wurm et al. (Int. J. Mol. Sci. 22:1-13. 2021) evidences that CD235+ hematopoietic stem cells give rise to the yolk sac progenitors in contrast to definitive CD235- hematopoietic stem cells. Additionally Wurm evidences that as part of differentiation of iPSCs into microglia progenitors, the cells start to express CD117 and CD34 followed by CD235, CD41, CD43, and CD14 (page 4, paragraph 6-page 5, paragraph 9). Similarly, Speicher et al. (Molecular Neurodegeneration 14: 1-16. 2019; referenced in IDS) evidences that KDR+/CD235a+ hemogenic endothelial cells subsequently give rise to either primitive CD34+/CD43+ or transient definitive CD34+/CD43− hematopoietic progenitors and KDR+/CD235a− hemogenic endothelial cells are committed towards definitive CD34+/CD43− hematopoietic progenitors from which, eventually, self-renewing hematopoietic stem cells arise (page 12, column 1, paragraph 1).
Furthermore, as stated in the rejection above, Blurton-Jones teaches a method of producing human microglial-like (iMGLs) cells from pluripotent stem cells (PSCs). The method comprises the steps: (i) differentiating PSCs using a media supplemented with hematopoietic differentiation factors (to produce induced hematopoietic progenitor cells (CD43+/CD235a+/CD41a+ iHPCs), (ii) isolating CD43+ iHPCs, (iii) differentiating the CD43+ iHPCs into iMGLs using a microglial differentiating media; and (iv) maturing the iMGLs. iPSCs were differentiated into hematopoietic progenitors (iHPCs), which recapitulates microglia ontogeny as iHPCs represent early primitive hematopoietic cells derived from the yolk sac that give rise to rnicroglia during development (paragraph 0005-0029, Example 1, and Figure 1).
Therefore, the art shows that the combination of CD43+ and CD235+ expression is indicative of primitive hematopoietic cells and is closely associated with PMPs (Boreland, as stated in the rejection above, and Xu both identify PMP cells based on co-expression of CD235 and CD43) and the further differentiation of these cells into microglia. Additionally, as identified by Wurm, CD34 expression occurs then CD43 expression occurs and, as identified by Speicher, CD235+/CD34+/CD43+ cells are associated with primitive hematopoietic progenitors while CD235- or CD34+/CD43- cells are associated with definitive hematopoietic cells. Therefore, the cells of Ko are more likely to be associated with primitive hematopoietic progenitor cells than definitive hematopoietic progenitor cells.
Furthermore, although Ko only discusses expression of CD43 in their cells, Example 6 of Ko, as cited in the rejection above, can be cultured in hematopoietic progenitor cell medium to produce hematopoietic progenitor cells with their transgenic pluripotent stem cells. Ko identifies that culture conditions for differentiating human ES cells into hematopoietic progenitor cells are already known. As the media of Blurton-Jones is known, its clear that this could be used with the cells of Ko as Ko considers any suitable differentiation media acceptable.
Regarding Applicant’s arguments regarding Blurton-Jones being focused on producing CD235+ cells, this is not what is taught by Blurton-Jones. The cited paragraph 0141 specifically teaches that their method comprises the steps: (i) iPSCs were differentiated into hematopoietic progenitors (iHPCs), which recapitulates microglia ontogeny as iHPCs represent early primitive hematopoietic cells derived from the yolk sac that give rise to rnicroglia during development. This protocol (FIG. lBi) yielded primitive iHPCs that are CD43+/CD235a+/CD41+. FACS sorting for CD43+ cells revealed that this approach produced iHPCs with a >90% purity. As such, although the differentiation method produces CD43+/CD235a+/CD41+ hematopoietic progenitors, they are more focused on CD43+ expression as they isolated cells based on CD43+ expression and not CD235+. If Blurton-Jones was specifically interested in CD235 expression, as argued by the Applicant, then they would have isolated cells based on CD235 expression and not CD43 expression.
In order to complete the art of record and rebut Applicant’s arguments, Speicher (Figure 1), Wurm (page 4, paragraph 6-page 5, paragraph 9), and Xu (Figure 1) all identify defined differentiation protocols with specific media compositions and timing for each step for generating microglia from pluripotent stem cells. Therefore, it would have been well understood in the art that the specific differentiation media composition used will lead to certain markers being expressed. As the cells of Ko express CD43 and cells of Blurton-Jones also express CD43, there is no reason to expect that placing the pluripotent stem cells of Ko in the same media as Blurton-Jones would not result in a similar marker expression as the cells of Blurton-Jones as the starting cells are the same (pluripotent stem cells) and both are known to lead to CD43+ expression.
As such, Applicant’s arguments are considered unpersuasive.
Claims 1 and 29-30 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No. 20180127714 (Ko), Brownjohn et al. (Stem Cell Reports10:1294-1307. 2018), World Intellectual Property Organization Patent Application No. 2018/160496 (Blurton-Jones) as applied to claim 1 above, and further in view of NCBI (GenBank: Homo sapiens Spi-1 proto-oncogene (SPI1), transcript variant 2, mRNA; NM_003120.2, 2007). This rejection is repeated with regards to the rejection in the Non-final Office action mailed on April 29, 2026. Applicant’s traversal has been addressed above.
The teachings of Ko, Brownjohn, and Blurton-Jones are as discussed above. Ko teaches SEQ ID NO: 25 as a possible nucleotide sequence for SPI1. SEQ ID NO: 25 has 98.4% sequence identity to SEQ ID NO: 1 of the instant application. Ko teaches that SPI1 may be any of transcription factor PU.1 isoforms 1 and 2 (paragraph 0181)
The combined teachings of Ko and Blurton-Jones do not teach a sequence with 100% sequence identity to SEQ ID NO: 1 (and the resulting amino acid sequence of SEQ ID NO: 2) of the instant application.
NM_003120.2 teaches an SPI1 transcript variant 2 which has 100% sequence identity to SEQ ID NO: 1 (and SEQ ID NO: 2 when translated into an amino acid sequence) of the instant application.
It would have been obvious that this sequence could have been used as the sequence for SPI1 as it had been known since at least 2007, as identified by NM_003120.2. Furthermore, the successful cloning and sequencing of a DNA encoding a known gene and making an amino acid sequence is obvious, and thus unpatentable, if (1) there was some suggestion or motivation in the prior art to clone the DNA, and (2) there was a “reasonable expectation of success,” based on "detailed enabling methodology" in the prior art. Ex parte Kubin, 83 U.S.P.Q.2d (BNA) 1410 (B.P.A.I. 2007), aff'd, 561 F.3d 1351 (Fed. Cir. 2009). Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 1, 10, and 35 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No. 20180127714 (Ko), Brownjohn et al. (Stem Cell Reports10:1294-1307. 2018), World Intellectual Property Organization Patent Application No. 2018/160496 (Blurton-Jones) as applied to claims 1 and 10 above, and further in view of World Intellectual Property Organization Patent Application No. 2017114430 (Qin; English Translation in US Application No. 20190203251). This is a new rejection made in response to the amendments to claim 1. Applicant’s traversal has been fully considered but is considered moot in response to the new rejection of record.
The teachings of Ko, Brownjohn, and Blurton-Jones are as discussed above.
Ko is silent as to the nucleic acid sequence of the rtTA element.
Qin teaches an rtTA element which has 100% sequence identity to SEQ ID NO: 20 of the instant application that is used as part of a Tet on system in human cell lines (abstract).
It would have been obvious that this sequence could have been used as the sequence for the rtTA as it was also used to generate inducible expression of a gene in human cell lines as part of a Tet on system (same as what was used in Ko), as identified by Qin. Furthermore, the successful cloning and sequencing of a DNA encoding a known gene and making an amino acid sequence is obvious, and thus unpatentable, if (1) there was some suggestion or motivation in the prior art to clone the DNA, and (2) there was a “reasonable expectation of success,” based on "detailed enabling methodology" in the prior art. Ex parte Kubin, 83 U.S.P.Q.2d (BNA) 1410 (B.P.A.I. 2007), aff'd, 561 F.3d 1351 (Fed. Cir. 2009). Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 1, 11, and 36 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No. 20180127714 (Ko), Brownjohn et al. (Stem Cell Reports10:1294-1307. 2018), World Intellectual Property Organization Patent Application No. 2018/160496 (Blurton-Jones) as applied to claims 1 and 11 above, and further in view of NCBI (GenBank: Vector pLVX.TRE3G.eGFP, complete sequence; MH325104.1, January 2019). This rejection is repeated with regards to the rejection in the Non-final Office action mailed on April 29, 2026. Applicant’s traversal has been addressed above.
The teachings of Ko, and Brownjohn, and Blurton-Jones are as discussed above.
Ko is silent as to the nucleic acid sequence of the TRE element.
MH325104.1 teaches a TRE element which has 100% sequence identity to SEQ ID NO: 21 of the instant application that is used as part of a Tet on system in human cell lines (page 1, Title). MH325104.1 teaches that their TRE corresponds to a 3rd-generation Tet-responsive promoter that can be activated by binding of Tet-On(R) 3G. Ko teaches that they also used a Tet promoter for inducible expression.
It would have been obvious that this sequence could have been used as the sequence for the TRE as it was also used to generate inducible expression of a gene in human cell lines as part of a Tet on system (same as what was used in Ko), as identified by MH325104.1. Furthermore, the successful cloning and sequencing of a DNA encoding a known gene and making an amino acid sequence is obvious, and thus unpatentable, if (1) there was some suggestion or motivation in the prior art to clone the DNA, and (2) there was a “reasonable expectation of success,” based on "detailed enabling methodology" in the prior art. Ex parte Kubin, 83 U.S.P.Q.2d (BNA) 1410 (B.P.A.I. 2007), aff'd, 561 F.3d 1351 (Fed. Cir. 2009). Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Claims 1, 37, and 40 are rejected under 35 U.S.C. 103 as being unpatentable over United States Patent Application No. 20180127714 (Ko), Brownjohn et al. (Stem Cell Reports10:1294-1307. 2018), World Intellectual Property Organization Patent Application No. 2018/160496 (Blurton-Jones) as applied to claims 1 and 37 above, and further in view of Takata et al. (Immunity 47: 183–198. 2017). This rejection is repeated with regards to the rejection in the Non-final Office action mailed on April 29, 2026. Applicant’s traversal has been addressed above.
The teachings of Ko, Brownjohn, and Blurton-Jones are as discussed above.
The combined teachings of Ko, Brownjohn, and Blurton-Jones do not teach wherein the small molecule CHIR99021 is used as part of the culturing step.
However, Takata teaches a method of differentiating iPSCs into hematopoietic cells that are then differentiated into macrophages and microglia (page 185, column 1, paragraphs 2-3). Takata teaches that they used CHIR99021 as part of their method of differentiating iPSCs into hematopoietic cells (Methods, page e4, paragraph 3).
It would have been obvious to one of ordinary skill in the art before the effective filing date of the claimed invention to have combined the in vitro production method of HSPCs from pluripotent stem cells of the combined teachings of Ko, Brownjohn, and Blurton-Jones with the CHIR99021 differentiation method of Takata to arrive at the instantly claimed invention. One of ordinary skill in the art would have a reason to combine because Ko, Brownjohn, Blurton-Jones, and Takata are all focused on differentiating pluripotent stem cells into hematopoietic progenitor cells and Brownjohn, Blurton-Jones, and Takata are focused on further differentiating those hematopoietic cells into microglia. "It is prima facie obvious to combine two compositions each of which is taught by the prior art to be useful for the same purpose, in order to form a third composition to be used for the very same purpose.... [T]he idea of combining them flows logically from their having been individually taught in the prior art." In re Kerkhoven, 626 F.2d 846, 850, 205 USPQ 1069, 1072 (CCPA 1980). MPEP 2144.06. As such, it would have been obvious that one could use CHIR99021 as part of the hematopoietic differentiation media of Ko, Brownjohn, and Blurton-Jones. Because the prior art teaches all of the elements of the claimed invention, there is a reasonable expectation of success.
Conclusion
THIS ACTION IS MADE FINAL. Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
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/KEENAN A BATES/Examiner, Art Unit 1631
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