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 .
Claims status
Applicants reply filed 6/1/2026 is acknowledged.
Claims 15 is/are cancelled and claims 34 is/are newly added.
Claims 6-14, 24-34 is/are currently pending and is/are under examination.
Withdrawn Objections
The objections presented herein represent the full set of objections currently pending in this application. Any objections not specifically reiterated are hereby withdrawn.
Claim Objections
Claim 25 is objected to because of the following informalities: The preamble of the claim is missing a comma after “claim 12”. Appropriate correction is required.
Claim 34 is objected to because of the following informalities: The claim recites “the starting population of human cardiomyocytes” in line 1. Claim depends from claim 6 that recites “the starting population of human cardiomyocytes comprising CD34+ cardiomyocytes”. For the sake of consistency, claim 34 should be amended to recite “the starting population of human cardiomyocytes comprising CD34+ cardiomyocytes”. Additionally, a semi-colon should be included at the end of line 5 and the extra “and” in line 7 should be deleted. Appropriate correction is required.
Claim Rejections - 35 USC § 112(b) – Maintained in part, New – necessitated by claim amendments
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.
Previous rejection of Claims 6-11, 13-15, 24, 26-33 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or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention is withdrawn in light of claim amendments.
Claims 6-14, 24-34 are rejected and claims 12, 25 remain 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 6 is amended to recite isolating cells that are “(ii) cTnT+, thereby obtaining a CD34+ cardiomyocytes population that is enriched for SANLPCs”. It is unclear how cTNT+ cells are isolated considering in step (b) the starting population is contacted with only one agent that specifically binds CD34. No cTnT-binding agent is recited. Of note, cTnT is an intracellular protein requiring permeabilization of the cell membrane for the agent to enter the cell followed by cell fixation (see [0074]). Such permeabilized and fixed cells cannot be “obtained” for any further use that requires the cells to be viable.
Claim 7-14, 24-34 is/are rejected due their dependence on claim 6 because they do not clarify the 112b issue noted with claim 6.
Claim 8 is amended to recite “wherein isolating step […] comprises removing CD31+ cells”. It is unclear how CD31+ cells are being removed considering that the step only requires isolating cells bound to CD34 binding agent from cells not bound to CD34 binding agent and the specification shows that at least some CD34 bound cells are also CD31+ (Figure 4C).
Claim 9 is amended to recite “isolating CD31- cells”. It is unclear how CD31- cells are being isolated considering that the step only requires isolating cells bound to CD34 binding agent from cells not bound to CD34 binding agent. Although, the specification shows that at least some CD34 bound cells are CD31-, yet some CD34 bound cells are also CD31+ such that use of CD34 alone cannot isolate CD31- cells (Figure 4C).
Claim 12 remains rejected because it continues to recite steps for producing the starting population of human cardiomyocytes but the steps recited produce cardiovascular mesoderm cells which according to the specification do not comprise cardiomyocytes and certainly not CD34+ve cells (Figure 5A). Although the claim is now amended to recite “wherein the starting population of human cardiomyocytes are generated from cardiovascular mesoderm cells by a process comprising” the recited steps, the steps recited do not produce the starting population of human cardiomyocytes, even one’s that are generated from cardiovascular mesoderm cells. For the purpose of compact prosecution, the claim(s) 12 is/are interpreted as recite “wherein the starting population of human cardiomyocytes are generated from cardiovascular mesoderm cells which are produced by a process comprising”
Claim 25 remains rejected because it continues to recite the limitation "the BMP4 component" in line 1. There is insufficient antecedent basis for this limitation in the claim. For the purpose of compact prosecution, the claim(s) 25 is/are interpreted as “the BMP[[4]] component”.
Claim 27 is amended to recite “isolating cells […] that are characterized by SIRPA+ CD90-“. It is unclear if cells that are SIRPA+ and/or CD90- are being isolated or merely cells that could be characterized as “SIRPA+ CD90-“. This is especially unclear because no steps are recited that would perform isolation of a SIPRA+ or CD90- cell. Furthermore, a clear relationship between cells characterized by SIRPA+ CD90- and the starting population of cardiomyocytes that are obtained by this method is not established.
Claim 31 is amended to recite “prior to step (b) measuring the level of one or more cardiomyocyte markers and CD34 in an aliquot of the starting population”. The claim is indefinite because the purpose of this step is unclear. It is unclear if the “aliquot of the starting population” used in this claim is being used in the subsequent steps and/or the information gleaned from measuring the levels of the recited markers alters any of the subsequent steps.
Claims 32 and 33 is/are rejected due their dependence on claim 31 because they do not clarify the 112b issue noted with claim 31.
Claim 34 recites “isolating cardiomyocytes that are characterized by SIRPA+ CD90-, thereby obtaining the starting population of cardiomyocytes“. It is unclear if cells that are SIRPA+ and/or CD90- are being isolated or merely cells that could be characterized as “SIRPA+ CD90-“. This is especially unclear because no steps are recited that would perform isolation of a SIPRA+ or CD90- cell. Furthermore, a clear relationship between cells characterized by SIRPA+ CD90- and the starting population of cardiomyocytes that are obtained by this method is not established.
Claim Rejections - 35 USC § 112(a) – New, necessitated by claim amendments
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.
Rejection of Claims 6-15, 24-33 under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement is withdrawn due to claim amendment. Although claim scope if narrowed, it continues to embrace non-enabled scope as detailed below.
Scope of Enablement
Claims 6-14, 24-34 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for
A method of isolating a cardiomyocyte population enriched for sinoatrial node-like pacemaker cardiomyocytes (SANLPCs), comprising
(a) providing a starting population of human cardiomyocytes comprising CD34+ cardiomyocytes;
(b) contacting the starting population of human cardiomyocytes with an agent that bind specifically to the cell surface CD34 protein; and
(c) isolating cells that are CD34+ thereby obtaining a CD34+ cardiomyocyte population enriched for SANLPCs;
wherein the starting population of human cardiomyocytes comprising CD34+ cardiomyocytes in step (a) is derived from human pluripotent stem cells (hPSC) in vitro by the following steps:
(i) incubating hPSCs in an embryoid body medium to generate embryoid bodies;
(ii) incubating the embryoid bodies in a mesoderm induction medium to generate cardiac mesodermal cells wherein the mesoderm induction medium comprises BMP4 and Activin A;
(iii) incubating the cardiac mesodermal cells in a cardiac induction medium to generate cardiovascular progenitor cells wherein the cardiovascular progenitor cells comprises BMP4, Retinoic Acid, a TGFb inhibitor and a Wnt inhibitor;
(iv) culturing the cardiovascular progenitor cells in a base medium to generate a population comprising cardiomyocytes;
(v) contacting the population comprising cardiomyocytes from step (iv) with agents that bind specifically to the following cell surface proteins: CD90, SIRPA; and
(vi) isolating cells that are SIRPA+ but CD90- which are the starting population of human cardiomyocytes comprising CD34+ cardiomyocytes of step (a).,
does not reasonably provide enablement for a method wherein the starting population of human cardiomyocytes (hCM) comprising CD34+ are produced by any means or derived from any source. Specific method for generating the starting population from hPSC using specific media components is required to generate a starting population that comprises CD34+ SANPLC at sufficient levels and not comprise other CD34+ cells that the claimed enrichment levels could be used. Furthermore, claims are not enabled for using cTnT as a marker that could be used to isolate a hCM population comprising SANLPCs, as recited in the amended claims (see claim interpretation in U.S.C. 112b above). cTnT is not a cell surface marker and labeling with cTnT requires permeabilization and fixation, thus killing of the cell such that cells could not be recovered.
The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
There are many factors to be considered when determining whether there is sufficient evidence to support a determination that a disclosure does not satisfy the enablement requirement and whether any necessary experimentation is “undue.” See MPEP § 2164. These factors include, but are not limited to: the breadth of the claims, the nature of the invention, the state of the prior art, the level of one of ordinary skill, the level of predictability in the art, the amount of direction provided by the inventor, the existence of working examples, the quantity of experimentation needed to make or use the invention based on the content of the disclosure.
The office has analyzed the specification in direct accordance to the factors outlines in In re Wands. MPEP 2164.04 states: “[W]hile the analysis and conclusion of a lack of enablement are based on factors discussed in MPEP 2164.01(a) and the evidence as whole, it is not necessary to discuss each factor in written enablement rejection.” These factors will be analyzed, in turn, to demonstrate that one of ordinary skill in the art would have had to perform “undue experimentation” to make and/or use the invention and therefore, applicant’s claims are not enabled.
(A) With respect to the breadth of the claims:
Claim 6 is directed to methods for isolating a cardiomyocyte population enriched for sinoatrial node-like pacemaker cardiomyocytes (SANLPCs). The specification states “As used herein, the term SANLPCs encompasses sinoatrial node pacemaker cardiomyocytes (SANPCs) obtained (e.g., isolated) from heart tissue (e.g., human heart tissue), as well as cells that have the electrophysiological functions of naturally occurring SANPCs and are generated from tissue cultures, as, for example, further described below” [0035]. Thus, the claim embraces isolating SANLPCs from any starting population of human cardiomyocytes that comprise CD34+ cardiomyocytes, such as directly from human heart (was recited in now cancelled claim 15) or from in vitro culture from of a variety of cell types such as primary heart tissue or, derived from any type of stem cells or somatic cells (recited in claims 10-14, 24-28). Further, the claims embrace methods wherein SANLPCs could be isolated from any of these distinct starting population by contacting the cells in these populations with only cell surface-CD34-binding agent and isolating cells that are functional equivalent of naturally occurring SANPCs. Although the claim is amended to isolation of cells that are also cTnT+, this amendment is indefinite (see U.S.C. 112b rejection above). Furthermore, even if the claim recites a step wherein cells are labelled with a cTnT antibody, such a step would not allow for the intended use of the method i.e. obtention of SANLPCs- enriched population. Consequently, the breadth of the claim is expansive.
Claim 7 limits the binding agent to anti-CD34 antibody but does not limit the starting population, or include any additional markers or steps for isolating SANLPCs. Consequently, the breadth of the claim remains expansive.
Claim 8 limits the method for isolation to FACS or MACS both of which are antibody-based isolation methods. Claim 8 is amended to recite isolation comprising removal of CD31+ cells. This amendment is indefinite (see U.S.C. 112b rejection above). Furthermore, even if the claim recites a step wherein cells are labelled with a CD31 antibody for removal of CD31+ cells, the claim continues to embrace any starting population of human cardiomyocytes that comprise CD34+ produced by any means and does not include any additional markers or steps for isolating SANLPCs. Thus, the breadth of claim 8 remains expansive.
Claim 9 recites isolating CD31- cells. This amendment is indefinite (see U.S.C. 112b rejection above). Furthermore, the claim continues to embrace any starting population of human cardiomyocytes that comprise CD34+ produced by any means and does not include any additional markers or steps for isolating SANLPCs. Thus, the breadth of claim 9 remains expansive.
Claim 10 recites deriving the starting population of claim 6 from various hPSCs but do not recite specific means or include any additional markers or steps for isolating SANLPCs. Consequently, the breadth of this claim is broad.
Claims 11 and 24 recites deriving the starting population of claim 6 from MSCs or even any differentiated cell but do not recite specific means or include any additional markers or steps for isolating SANLPCs. A transdifferentiation method for deriving a starting population of human cardiomyocytes comprising CD34+ cardiomyocytes from MSCs or any differentiated cell is not disclosed in the specification and is not enabled. Although, MSCs and differentiated cells maybe dedifferentiated into iPSCs which could then used in a method to generate the starting population, the claim is not limited to this embodiment. Furthermore, additional markers for isolating SANLPCs are not recited. Consequently, the breadth of this claim is expansive.
Claim 12 and 25 recites steps that generate cardiovascular mesoderm cells from hPSCs which are not CD34+ (see Figure 5A) and not the required starting population. Although steps recited in this claim are essential to generate the starting population from hPSCs because the hPSCs must be differentiated into mesodermal cells, this claim does not recite steps that result in further differentiation of the mesodermal cells into the starting population. Furthermore, additional markers for isolating SANLPCs are not recited. Consequently, the breadth of this claim is broad.
Claims 13 recites steps that generate the starting population from any mesodermal population without reciting specific media components required (recited in claim 14). The claim embraces any mesodermal population, isolated from or derived from any source. Such a method is not enabled. The specification disclosed cardiovascular mesodermal cells derived from hPSCs using the steps recited in claim 12. Furthermore, additional markers for isolating SANLPCs are not recited. Consequently, the breadth of this claim is broad.
Claim 14, 26 and 28 recite media components for claim 13 however there is no enablement for a method wherein these media components could be used to derive the starting population from a mesodermal population, wherein the mesodermal population is isolated from or derived from any source. Furthermore, additional markers for isolating SANLPCs are not recited. Consequently, the breadth of these claims is broad.
Claim 27 limits the mesodermal cells of claim 13 to cardiovascular mesodermal cell but does not require that the cardiovascular mesodermal cells be derived from hPSCs. There is no enablement for a method wherein incubation with a Wnt inhibitor could be used to derive the starting population from a cardiovascular mesodermal population isolated from or derived from any source. Furthermore, additional markers for isolating SANLPCs are not recited. Consequently, the breadth of this claim is broad.
Claim 29, 30 recite additional markers for the isolated SANLPCs but do not teach using these markers for the isolation. Thus, these claims do not limit the active method steps of claims 12 or 14 and its breadth remains broad.
Claim 31-33 recite detection of CD34, additional markers for the isolated SANLPCs and determining number of cells based on other marker expression. Thus, these claim do not limit the starting population of the marker used for isolation and their breadth remains expansive.
Claim 34 limits the starting population of claim 6 to be derived from hPSCs and recites method steps for differentiating hPSCs into the starting population. Although the claim recites isolating cardiomyocytes in step (e) that are characterized by SIRPA+ CD90-, the claim does not positively recite isolating SIRPA+ and CD90- cardiomyocytes. Furthermore, the claim does not recite key media components required. Other media compositions lead to generation of cardiomyocytes from hPSC that do not comprise SANLPC (see discussion from Skelton below). Consequently, the breadth of this claim is broad.
(B) The nature of the invention: The invention is in the field of identifying markers for isolation of a specific type of cardiomyocytes called sinoatrial node pacemaker cardiomyocytes.
(C) With respect to the state of the prior art: CD34 is a well-known marker of hematopoietic stem cells and expressed on various progenitor and mature cell types, including endothelial cells (See CD34 definition from Science Direct; ref of record). In case of heart tissue and cultured cardiomyocytes, use of CD34 expression to identify specific cell types remains unpredictable. Different prior art use CD34 to identify different cell types in a population that comprises cardiomyocytes. None of the prior art teach CD34 as a marker, alone or in combination with other markers, for identifying SANLPCs.
Hendrikx et al (EP 2258833 A2, 2010-12-08) used CD34 expression to identify a unique population of cardiac stem cells (Abstract). The starting population of Hendrikx is from adult human heart, specifically right atrial appendage [0077]. They use ALDH+ and CD34+ as markers for their novel cardiac stem cells wherein CD34 expression is lost upon continued culture [0079-0082]. Critically, they show CD34+ cells in the human right atria by direct staining wherein the cells were surrounded by TnT+ cardiomyocytes but not themselves TnT+ cardiomyocytes [0081]. According to Hendrikx CD34 is a marker for stem cells in the adult human heart and not cardiomyocytes, specifically not SAN pacemaker cardiomyocytes. Thus, it is unpredictable that CD34+ SANLPCs could be isolated from an adult human heart as a starting population.
Mitrofanova et al (J. Cell. Mol. Med. Vol 22, No 1, 2018 pp. 521-532; ref of record) used CD34 expression to identify another unique population in the human heart – the telocytes (Abstract). They teach that “The immunophenotype of telocytes is similar to that of interstitial, endothelial, smooth muscle, mast and haematopoietic stem cells and neurons. They coexpress CD117, vimentin, CD34, SMA, S100 and NSE as well as the gap junction protein Connexin” (page 521-522, bridging para). They specifically focus on the sinoatrial node and identify a CD34+ population that is distinct from the pacemaker cardiomyocytes and the active myocardial cardiomyocytes (Figure 4, 5, 7). Thus, according to Mitrofanova CD34 is a marker for telocytes in the adult human heart and not cardiomyocytes, specifically not SAN pacemaker cardiomyocytes. Thus, it is unpredictable that CD34+ SANLPCs could be isolated from an adult human heart as a starting population.
Similar to the unpredictability in using CD34 as SANLPC marker when the starting population is a primary cell population derived from human heart, use of CD34 as a marker for SANLPCs when the starting population is derived from in vitro differentiation or de-differentiation of stem cells or somatic cells is also unpredictable.
Skelton et al (Stem Cell Research (2014) 13, 172–179; ref of record) used CD34 expression to identify endothelial progenitors in a population of human cardiomyocytes derived from human embryonic stem cells in vitro (Abstract). The differentiation method used comprises a cocktail of small molecules similar to recited in instant claims 12-14, 25, 26, 28; such as BMP4, Activin A, WntA and goes through similar steps i.e. differentiating hESC into cardiac mesoderm which are differentiated into cardiac progenitors (hPSC culture and differentiation; Figure 4). The results show that by day 7 in culture both NKX2-5+ CD34+ cells and NKX2-5- CD34+ cells had an endothelial expression profile and thus deemed vascular progenitors and not cardiomyocyte progenitors (Figure 1). By Day 10, NKX2-5+ SIRPA+ CD34+ cells gave rise to endothelial cells (Supplementary Figure 2C-E) while NKX2-5- SIRPA- cells remained TnT- i.e. not cardiomyocyte precursors (Figure 3). Overall, Skelton identify NKX2-5- SIRPA+ CD34+ cells as endothelial cells derived from hESCs cultured in cardiac induction medium (Figure 4). Thus, it is unpredictable that CD34+ SANLPCs could be isolated from a starting population that is derived from hPSCs using any method and any media components.
Protze et al (Nature Biotech, Vol. 35, Number 1, Jan 2017; ref of record) teaches a specific method for differentiating hPSCs (both hESCs and hiPSCs) into a population of cardiomyocytes enriched for SANLPCs (Abstract). Protze method requires a specific cocktail for differentiation of hPSCs into a mesoderm that has the potential to develop in to SAN like pacemaker cells without which the mesoderm generated lacks a potential to even make SANLPCs (Figure 7). Use of Wnt inhibitor alone produced a population of cardiomyocytes with no pacemaker potential such that inclusion of additional components, specifically BMP4, RA and TGFb inhibitor was required to produce a population comprising sufficient SANLPCs (Figure 7). See also Figure 2, 3 and Supplementary figure 3 for requirement of BMP4, RA and TGFb inhibitor. Without such specific cocktail, it is not even clear that any SANLPCs could be isolated from a population of cardiomyocytes derived from hPSCs. Using their method, Protze identify SANLPCs as NKX2-5- SIRPA+ CD90- (Figure 7). It is notable that Skelton taught NKX2-5- SIRPA+ along with CD34+ as endothelial cells (Figure 4) thus highlighting the unpredictability in using markers to identify specific cell types when the starting population produced from hPSCs via different methods.
Taken together, the prior art taught CD34 as a marker for HSCs or endothelial cells. In the human heart, CD34 marker expression has been used to identify cardiac stem cells or telocytes but not SAN pacemaker cells. Even for in vitro differentiation of human pluripotent stem cells, use of CD34 alone or in combination has not been shown to identify SANLPCs – instead it has been shown to identify endothelial cells.
(D) The level of one of ordinary skill in the art of using markers to identify cells types is high. An ordinary artisan routinely uses a variety of approaches such as FACs and MACs to identify cell specific markers.
(E) With respect to the predictability of the art: Use of CD34 to identify SANLPCs remains unpredictable since prior art does not teach CD34 as a marker for SANLPCs. Critically, it teaches CD34 as a marker for other cell types. It appears based on the starting population, and culture condition for differentiating stem cells, CD34 expression can be used to identify different cell types. Further, based on teaching of Protze it is unpredictable that any method could be used to differentiate hPSCs into SANLPC comprising population since they teach two distinct methods, one of which results in no SANLPCs to even isolate.
(F), (G) With respect to the amount of direction and working examples provided by the applicant: The applicants have provided working examples directed to generation of SANLPC comprising population from hPSCs using Protze’s method (Example 1). Same as Protze, they identify NKX2-5- SIRPA+ CD90- cells as SANLPC [0075].
In Example 2, Applicants probe the specific starting population of NKX2-5- cells at day 20 after differentiation using single cell RNAseq to identify a TNNT2+ NKX2-5- TBX3+ TBX18+ as SANLPCs which were enriched in CD34 mRNA (Figure 3; [0077]). Next, Applicant isolated NKX2-5- SIRPA+ CD90- cells (taught by Protze as SANLPCs) and showed that 30% of these cells expressed CD34 (Figure 4; [0079]). No data is presented to show that this 30% of the population comprising SANLPCs have the electrophysiological function of SAN pacemaker cardiomyocytes. No functional data is provided. Critically, pre-selection with SIRPA and CD90 was required to even achieve 30% SANLPCs.
Furthermore, Applicant acknowledge that CD34 is a known endothelial marker and even in their specific culture condition that is for generating cardiomyocyte population enriched in SANLPCs CD34+ cells co-stained with CD31, another endothelial marker [0080]. A combined expression profile of NKX2-5- SIRPA+ CD90- was required to use CD34 as a SANLPC marker. Furthermore, another differentiation protocol that generates a population of cardiomyocytes from hESCs was found to not express CD34 [0081].
Applicant state that “[0082] Although we also detected a population of CD34+CD31+ endothelial cells in the differentiation cultures, we do not anticipate issues with contamination from endothelial cells because the proportion of these cells was low (~2%) (FIG. 4C).” However, such an anticipation cannot be extended to other methods wherein the method is not specifically modified to enrich SANLPC [0081] or wherein the SANLPC enriched cardiomyocyte population is not first selected using other markers such as NKX2-5- SIRPA+ CD90- or wherein the starting population is the human heart. This is also evident from data in Figure 10 wherein single cell RNA seq of fetal human heart showed CD34 expression in endothelial cell cluster, fibroblast cluster (Example 7; [0093-0094]. Thus, suggesting that starting with fetal human heart, CD34 alone would not isolate SANLPCs, specific microdissection of SAN from the fetal human heart maybe required, however this is not disclosed.
Examples 3 and 4 also use Protze method for generating SANLPC enriched cardiomyocyte population from hPSC and use NKX2-5- SIRPA+ CD90- along with CD34.
Example 5 and 6 also uses Protze method for generating SANLPC enriched cardiomyocyte population from hPSC. Applicant uses MACs and only CD34-based isolation. These example provides support for use of CD34 as a means to enrich for cardiomyocytes and thus also SANLPCs (about 74%) but only when the starting population is the day 20 SANLPC enriched cardiomyocyte population from hPSC produced by Protze’s method (Figure 7, 8).
Example 7 performs single cell RNAseq and CD34 immunostaining in SAN tissue specifically, wherein the SAN tissue was derived from fetal human heart. Of note, this is different from Hendrikx and Mitrofanova that used adult samples. To identify SANLPC cluster in the single cell RNAseq data, Applicant used TNNT2+ NKX2-5- TBX3+ TBX18+ as SAN marker wherein Applicant identify CD34 expression (Figure 10). However, Applicant also identify CD34 expression in endothelial cell cluster, fibroblast cluster [0093-0094], thus underlining the necessity for combination of markers even when the starting specimen is specifically SAN tissue. Immunostaining data indicates the same as the single cell RNAseq data [0095].
The applicants have not provided working examples for a method as broadly claimed. Although CD34 could be used to enrich for cardiomyocytes when the starting population of cardiomyocytes is derived from hPSCs using Protze’s method that already enriches from SANLPC, the data presented here and the prior art does not enable a method wherein any starting population of cardiomyocytes could be used. Due to the teachings in the prior art, it remains unpredictable that CD34 alone is a SANLPC marker or enrich for cardiomyocytes or enrich for SANLPC. The combination of markers taught; such as SIRPA+ CD90- or TNNT2+ NKX2-5- TBX3+ TBX18+ were only tested in limited starting populations. A skilled artisan cannot predict which combination of markers along with CD34 could be used to identify SANLPC from any starting population, as broadly claimed. A combination of SIRPA+ CD90- CD34+ would be required to predicably isolate SANLPC-enriched cardiomyocyte population from a starting population derived from hPSC using Protze’s method that requires specific method steps.
H) Undue experimentation would be required to practice the invention as claimed due to the amount of experimentation necessary because of the expansive breadth of the claims, the state of the prior art and its high unpredictability, and the limited amount of guidance in the form of varied working examples in the specification.
MPEP §2164.01(a), 4th paragraph, provides that, “A conclusion of lack of enablement means that, based on the evidence regarding each of the above factors, the specification, at the time the application was filed, would not have taught one skilled in the art how to make and/or use the full scope of the claimed invention without undue experimentation. In re Wright, 999 F.2d 1157, 1562; 27 USPQ2d 1510, 1513 (Fed. Cir. 1993).
Genentech Inc. v. Novo Nordisk A/S, 42 USPQ2d 1001, 1005 (CA FC), states that, “[p]atent protection is granted in return for an enabling disclosure of an invention, not for vague intimations of general ideas that may or may not be workable,” citing Brenner v. Manson, 383 U.S. 519, 536 (1966) (stating, in the context of the utility requirement, that “a patent is not a hunting license. It is not a reward for search, but compensation for its successful conclusion”). The Genentech decision continued, “tossing out the mere germ of an idea does not constitute enabling disclosure. While every aspect of a generic claim certainly need not have been carried out by an inventor, or exemplified in the specification, reasonable detail must be provided in order to enable members of the public to understand and carry out the invention.” Id. at p. 1005.
After applying the Wands factors and analysis to claims 6-14, 24-34, in view of the applicant’s entire disclosure, and considering the In re Wright, In re Fisher and Genentech decisions discussed above, it is concluded that the practice of the invention as claimed would not be enabled by the written disclosure. Therefore, claims 6-14, 24-34 are rejected under 35 U.S.C. §112(a) for failing to disclose sufficient information to enable a person of skill in the art to make and use the claimed invention.
Response to Arguments
Applicant's arguments filed 6/1/2026 regarding the U.S.C. 112b rejection of claim 12 and 25 have been fully considered but they are not persuasive.
Regarding claims 12 and 25, Applicant argue that amendment to these claims overcome the rejection of record (page 8, para 1-3).
In response, amendment to claim 12 does not overcome the indefiniteness issue with this claim which is that the recited steps do not generate the starting population, not even from mesodermal cells as included in the amendment. Claim 25 is not amended.
Applicant’s arguments with respect to the U.S.C. 112a -Enablement rejection of the claim(s) 6-15, 24-33 have been considered but are moot because the new ground of rejection necessitated by claim amendment.
Arguments pertinent to instant U.S.C. 112a -Scope of Enablement rejection of the claim(s) 6-14, 24-34 are addressed below.
First, Applicant indicate that the claim is narrowed to “a starting population to comprise CD34+ cardiomyocytes” and that isolated cells to be cTnT+ (page 9, para 3). Applicant allege that cTnT is a more definite cardiomyocyte marker than SIRPA+CD90- based on Figure 9B (page 9, para 4).
In response, the issue is the proper identification of what is “a starting population comprising CD34+ cardiomyocytes” embraced by the claims, how it is produced and if the claim is enabled when the starting population comprises any amount of CD34+ cardiomyocytes. Based on the claims, specification and arguments presented, the Applicant envisions “a starting population to comprise CD34+ cardiomyocytes” to be derived from any cell type (see MSCs and differentiated cell claimed in claim 11) and embrace population derived from human heart (see page 11. para 3 about human heart). However, it is unpredictable that a starting population comprising CD34+ cardiomyocytes could be produced from such sources and if the starting population derived from such source would even comprise sufficient CD34+ cardiomyocytes such that an enriched SANLPC-cardiomyocyte population could be isolated using the claimed method (see Hendrikx and Mitrofanova). The starting population used in the instant specification was derived from hPSC using a specific method of Protze that resulted in a starting population comprising at least 35% SANLPCs to begin with (Figure 2) which was further enriched using CD34 in the instant specification. Both Protze and the instant specification teach that use of SANLPC for studying pacemaker function and critically for transplantation was limited due to low enrichment (Figure 3 and 5 in Protze, [005] in instant specification). Thus, properly enabled use of the claimed method requires sufficient enrichment. Yet, there is no predictability that such an enrichment level could be achieved when a starting population is as broadly claimed.
Regarding Figure 9B, it is unclear how this establishes the superiority of TnT to SIRPA and CD90. Compare the similar level of SANLPC enrichment in Figure 9C with Figure 7C, that uses SIRPA and CD90. Critically, use of TnT in the method would fail to reach the intended result of obtention of the SANLPC-enriched cells. See Description of Figure 9 in [0032].
Next, Applicant argue that “the paper of Protze et al does not state that SANLPCs can only be generated from a certain mesoderm” pointing to Supplementary figure 3 that shows that various concentration BMP4 and Activin A could be used with one combination of concentration being superior (page 10, last para; page 11, para 1). Applicant point to teachings from Skeleton identified in the enablement rejection and how they lend to unpredictability however do not address how the issues raised are addressed by amendment or argument (page 10, para 2).
In response, a specific concentration was not required for enablement or scope of enablement. Data in Protze does not enable any combination of small molecules. On the contrary specific combination of small molecules is required. Figure 7 shows that without the specific combination, such as when using only Wnt inhibitor, VLCM are produced not SANLPC. Figure 3 also shows that VLCM do not have SANLPC function. Critically, Skeleton shows that hPSC when exposed to similar small molecules as Protze but in different order does not result in SANLPC. Thus, claims are enabled to a scope wherein the starting population is derived from hPSC using Protze’s method. Of not, specific concentration of small molecules is not required for enablement.
Finally, Applicant point to Protze, Skelton, Hendrikx and Mitrofanova (page 11, para 2) identified in the enablement rejection and how they lend to unpredictability however do not address how the issues raised are addressed by amendment or argument. It appears that Applicant point to Lim (Single-cell transcriptome analysis reveals CD34 as a marker of human sinoatrial node pacemaker cardiomyocytes. Nat Commun 15, 10206 (2024), a post-filing art, that allegedly shows “CD34 is specifically expressed by the pacemaker cardiomyocytes in human SAN tissue and by hPSC-derived SANLPCs, but not by other cardiomyocyte subtypes” to address issues of unpredictability that arise in light of Hendrikx and Mitrofanova (page 11, last para). Applicant point to nothing in Lim that supports their allegation. Applicant acknowledge that even Lim shows that endothelial cells express CD34 and allege that this “most likely explains the detection of some CD34+ cells in the atrial tissue” page 11, last para). A copy of Lim was not provided however for compact prosecution and to properly address Applicant’s argument, Lim is attached in PTO-892.
In response, the issues of unpredictability that arise in light of Hendrikx and Mitrofanova cannot be addressed by another art, such as Lim, that shows a different results. This only lends to further unpredictability in the art. Furthermore, a post-filing art cannot provide enablement support to the instant claims.
In arguendo, data in Lim does not support that CD34, alone or in combination, can be used to isolate SANLPC-enriched population from any starting population as broadly claimed. Lim’s in vitro data uses Protze’s method (page 3, col. 1, last para) and thus could only enable a method wherein the starting population is produced by Protze’ method. Lim’s in vivo data is from fetal human heart wherein for the single cell RNA seq analysis, only clusters that are first preselected from Core SAN had high CD34 expression in about 25% of the cells (see Figure 3C, color of dot indicates avg. expression while size indicates % cells). For immunohistochemistry, human fetal SAN was dissected out thus pre-selecting a SAN population (page 7, col. 1, para 2; Figure 4). Data for adult human heart was from Kanemaru that performed single nuclear RNA seq. Herein again, Core SAN clusters were preselected and within these CD34 expression was found in less than 24% of the cells at about 0.5-1 average fold expression in comparison to other atrial cells (Supplementary figure 3h and i). It is unclear the relevance of RNA seq and especially nuclear RNA seq data to instant claims that are directed to surface expressed CD34 since mRNA, especially only nuclear RNA, does not predict surface expression due to post-transcriptional and post-translation regulation. Taken together, even if Lim was prior art, in vivo data from Lim could not have enabled the full scope of the instant claims.
Conclusion
No claim is allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). Applicant is reminded of the extension of time policy as set forth in 37 CFR 1.136(a).
A shortened statutory period for reply to this final action is set to expire THREE MONTHS from the mailing date of this action. In the event a first reply is filed within TWO MONTHS of the mailing date of this final action and the advisory action is not mailed until after the end of the THREE-MONTH shortened statutory period, then the shortened statutory period will expire on the date the advisory action is mailed, and any nonprovisional extension fee (37 CFR 1.17(a)) pursuant to 37 CFR 1.136(a) will be calculated from the mailing date of the advisory action. In no event, however, will the statutory period for reply expire later than SIX MONTHS from the mailing date of this final action.
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/MATASHA DHAR/Examiner, Art Unit 1632
/ANOOP K SINGH/Primary Examiner, Art Unit 1632
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