DETAILED ACTION
Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Election/Restrictions
Applicant’s election without traverse of Group II in the reply filed on 10/20/2025 is acknowledged.
Claims status
Claims 1-5, 16-23 is/are cancelled and claims 24-33 is/are newly added. Claims 6-15, 24-33 is/are currently pending and is/are under examination.
Information Disclosure Statement
The listing of references in the specification; for example para [002-005], is not a proper information disclosure statement. 37 CFR 1.98(b) requires a list of all patents, publications, or other information submitted for consideration by the Office, and MPEP § 609.04(a) states, "the list may not be incorporated into the specification but must be submitted in a separate paper." Therefore, unless the references have been cited by the examiner on form PTO-892, they have not been considered.
Drawings
Figure 1 should be designated by a legend such as --Prior Art-- because only that which is old is illustrated. This figure is from Fig 1d of Protze et al (IDS 3/6/2023). See MPEP § 608.02(g). Corrected drawings in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. The replacement sheet(s) should be labeled “Replacement Sheet” in the page header (as per 37 CFR 1.84(c)) so as not to obstruct any portion of the drawing figures. If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
The drawings are objected to because UMAP plots in Figure 3 and 10 cannot be evaluated due to low resolution and need for color image. Corrected drawing sheets in compliance with 37 CFR 1.121(d) are required in reply to the Office action to avoid abandonment of the application. Any amended replacement drawing sheet should include all of the figures appearing on the immediate prior version of the sheet, even if only one figure is being amended. The figure or figure number of an amended drawing should not be labeled as “amended.” If a drawing figure is to be canceled, the appropriate figure must be removed from the replacement sheet, and where necessary, the remaining figures must be renumbered and appropriate changes made to the brief description of the several views of the drawings for consistency. Additional replacement sheets may be necessary to show the renumbering of the remaining figures. Each drawing sheet submitted after the filing date of an application must be labeled in the top margin as either “Replacement Sheet” or “New Sheet” pursuant to 37 CFR 1.121(d). If the changes are not accepted by the examiner, the applicant will be notified and informed of any required corrective action in the next Office action. The objection to the drawings will not be held in abeyance.
Claim Objections
Claim 10 is objected to because of the following informalities: Claim 10 recites “wherein the starting population of human cardiomyocytes are obtained by culturing human pluripotent stem cells (hPSCs)” (emphasis added). No additional step to the culturing or modification to the hPSC is recited such that hPSC differentiate into cardiomyocytes. Culturing hPSCs is unlikely to differentiate them into cardiomyocytes. Similar to the limitation recited in claim 11, following language is recommended for claim 10 “wherein the starting population of human cardiomyocytes are derived from in vitro”.
Appropriate correction is required.
Claim Rejections - 35 USC § 112(b)
The following is a quotation of 35 U.S.C. 112(b):
(b) CONCLUSION.—The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the inventor or a joint inventor regards as the invention.
The following is a quotation of 35 U.S.C. 112 (pre-AIA ), second paragraph:
The specification shall conclude with one or more claims particularly pointing out and distinctly claiming the subject matter which the applicant regards as his invention.
Claims 6-15, 24-33 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.
Claim 6, step (b) recites “contacting the human cardiomyocytes with an agent”. It is unclear if only the human cardiomyocytes in the starting population of human cardiomyocytes are being contacted with the agent or if the entire population of step (a) is being contacted with the agent. No step of isolation of only human cardiomyocytes from the starting population is recited. This is further unclear since dependent claims that recite the source of or the method for producing the starting population do not necessarily recite a population comprising only cardiomyocytes. For example, starting population derived from human heart would comprise all the cell types present in the human heart. For the purpose of compact prosecution, the claim(s) 1 is/are interpreted as “contacting the starting population of human cardiomyocytes with an agent”.
Claim 12 recites steps for producing the starting population of human cardiomyocytes but produces cardiovascular mesoderm cells which according to the specification do not comprise cardiomyocytes and certainly not CD34+ve cells (Figure 5A). Thus, claim 12 is indefinite because it is unclear how the recited steps produce the starting population of human cardiomyocytes.
Claim 25 recites 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 recites “step (b)” in line 2. Claim depends from claim 13 which recites a step (b) but claim 13 depends from claim 6 which also recites a step (b). It is unclear which step (b) claim 27 refers to.
Claim 31 appears to recite a contingent step (“detecting if”). MPEP 2111.04(II) guides that “The broadest reasonable interpretation of a method (or process) claim having contingent limitations requires only those steps that must be performed and does not include steps that are not required to be performed because the condition(s) precedent are not met”. Additionally, the claim recites that the step is “preferably” perfumed prior to step (b). Recitation of preferences may lead to confusion over the intended scope of a claim. See MPEP 2173.05(d). In the instant case, it is unclear if the step is required to be performed prior to step (b) such that performing step (b) does not occur if the starting population of step (a) in claim 6 is detected to not comprise CD34 expressing cell. Alternatively, if contingent step is performed after step (b) and no CD34 expressing cells are detected, then how does the method generate CD34+ population required to achieve the SANLPC enriched population.
Claims 7-15, 24-33 is/are rejected due their dependence on claim 6 because they do not clarify the 112b issue noted with claim 6.
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 Rejections - 35 USC § 112(a)
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 6-15, 24-33 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 enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention.
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 generating 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 any starting population that comprise human cardiomyocytes, such as directly from human heart (recited in claim 16) or from in vitro culture 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 CD34 binding agent and isolating cells that are functional equivalent of naturally occurring SANPCs. The specification teaches any CD34 binding agent wherein CD34 is not limited to a cell-surface protein [007] such that the claimed CD34 binding agent could bind CD34 gene, mRNA or protein. 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. Thus, similar to claim 7, the breadth of claim 8 remains expansive.
Claim 9 recites additional markers for the isolated SANLPCs but does not teach using these markers for the isolation. Thus, the claim does not limit the active method steps of claim 6 and its breadth remains expansive.
Claim 10-15, 24-28 recite different sources for the starting population of claim 6 but do not include any additional markers or steps for isolating SANLPCs. Consequently, the breadth of these claims 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.
(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 in PTO-892). 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]. Thus, according to Hendrikx CD34 is a marker for stem cells in the adult human heart and not cardiomyocytes, specifically not SAN pacemaker cardiomyocytes.
Mitrofanova et al (J. Cell. Mol. Med. Vol 22, No 1, 2018 pp. 521-532) 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.
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) 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 (hPSC culture and differentiation). 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).
Protze et al (Nature Biotech, Vol. 35, Number 1, Jan 2017; IDS 3/6/2023) 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). 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 is distinct.
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.
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].
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 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 sing 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 NKX2-5- 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.
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 6-15, 24-33, 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 6-15, 24-33 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.
Conclusion
No claim is allowed.
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/MATASHA DHAR/Examiner, Art Unit 1632