DETAILED ACTION
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
This office action is in response to an amendment filed 1/15/2026.
Claims 1 and 4-23 are pending.
This application is filed as a 371 filing of PCT/US2021/039897 filed 6/30/2021 which claims the benefit of U.S. Provisional Application No. 63/046,024 filed June 30, 2020, and U.S. Provisional Application No. 63/110,405 filed November 6, 2020.
Information Disclosure Statement
An information disclosure statement filed 1/15/2026 has been identified and the documents considered. The corresponding signed and initialed PTO Form 1449 has been mailed with this action. Initials indicate that the document has been considered even if the reference is lined through.
Response to Amendments
Applicants amendment to amend claim 1 is sufficient to overcome the rejection in part under 35 USC 112 second. As well, cancellation of claim 3 is sufficient to overcome the objection to the claim. Similarly, the rejection under 35 USC 112 4th has been overcome by cancellation of this claim. Applicants arguments were sufficient to overcome the rejection under 35 USC 102.
Claim Objections
Claim 5 is objected to because of the following informalities: claim 5 refers to “the strand” which for completeness should refer to –the cityRNA—or –the one of the strands--. This is true of claims 6 and 7. This is a new observation. Appropriate correction is required.
Claim Rejections - 35 USC § 112, second paragraph
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 1 and 4-23 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. This rejection is maintained for reasons of record. The dependent claims are included in the rejection as they fail to overcome the basis of the rejection.
Claim 1 recites that the RISC is loaded with cityRNA, step c). However, the RISC is loaded with the dsRNA which comprises the city RNA. By reciting that only the cityRNA is loaded, it makes unclear if the cityRNA is separated from the dsRNA or if the entire sequences is loaded onto the RISC. It is noted that this language is associated with claims 8 and 9.
Response to arguments
Applicants argue that the specification teaches that the cityRNA can be introduced to AGO3 separately or as part of a double stranded nucleic acid which makes clear that the cityRNA is introduced to AGO3. This argument is not sufficient to overcome the rejection. The claim was not rejected for lack of description. Rather, the claim is unclear. The claim provides for a dsRNA which comprises a cityRNA. However, the claim continues “exposing the AGO3 associated RISC loaded with cityRNA to the target mRNA”. The AGO3 is exposed to the entirety of the dsRNA which allows loading of the dsRNA into RISC. Consistency of language leads to clarity wherein changing the reference mid-claim makes the claim unclear. By reciting just the cityRNA the dsRNA is not referenced in this latter recitation.
Claim Rejections - 35 USC § 112, first paragraph
The following is a quotation of the first paragraph of 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 1 and 4-23 are rejected under 35 U.S.C. 112, first paragraph, because the specification, while being enabling for cleaving a target sequence in a cell comprising AGO3 in vitro comprising preparing a targeting double stranded RNA (dsRNA) that hybridizes to a target sequence wherein the ds RNA is 14-nt miR-20a, 14-nt let-7a, 14-nt miR-92a, 14-nt miR-17, 14-nt miR-18a and introducing the double-stranded RNA into the cell wherein the double-stranded RNA forms RNA induced silencing complexes (RISC) to cleave the target sequence, does not reasonably provide enablement any other embodiment. 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. This rejection is maintained for reasons of record.
The test of enablement is whether one skilled in the art could make and use the claimed invention from the disclosures in the patent coupled with information known in the art without undue experimentation (United States v. Telectronics, Inc., 8 USPQ2d 1217 (Fed. Cir. 1988)). Whether undue experimentation is required is not based on a single factor but is rather a conclusion reached by weighing many factors (See Ex parte Forman, 230 USPQ 546 (Bd. Pat. App. & Inter, 1986) and In re Wands, 8USPQ2d 1400 (Fed. Cir. 1988); these factors include the following:
1) Nature of invention. The instant claims are drawn to a method of silencing a target mRNA with the intention of treating a genetic disorder. Silencing the mRNA appears to mean preventing the translation of the mRNA into protein or other products encoded by the mRNA.
2) Scope of the invention. The scope of the invention is extremely broad in that the target nucleic is any and the means of “exposing” the target nucleic acid to RISC and then “exposing” the AGO3 associated RISC loaded with the double stranded RNA comprising cityRNA is not clear as to how a person can mediate the exposure. Furthermore, applicants claim use of the method to treat any disease or disorder in dependent claims. This combination of large breadth and lack of guidance couples lack of descriptive elements ( nature of the cityRNA and what is necessary to mediate “exposure” coupled with the nascent state of the art leads to a number of inoperable elements detailed below.
3) Number of working examples and guidance. The specification teaches in vitro activation of AGO2 and AGO3 using filter-binding with 14- or 23-nt single-stranded miR-20a called pl4ss and p23sa, respectively. The kinetics of nucleic acid cleavage with pl4ss and p23sa with each are below.
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Those tiny guide RNAs (tyRNA) that lead to cleavage were then labeled cityRNA.
Example 2 demonstrated that AGO2 and AGO3 have different activation mechanisms although both cleave target RNAs at the same position. And furthermore that cleavage required rather specific sequences.
“Hereafter, ~18-nt miRNA derivatives are named tiny RNAs (tyRNA) to distinguish from their intact miRNAs (19~23-nt). In addition, tyRNAs capable of catalytically activating AGOS, such as the 14-nt miR-20a and let-7a, are referred to as cleavage-inducing tyRNAs (cityRNAs). On the other hands, tyRNAs incapable of activating AGO3, such as the 14-nt miR-16 and miR-19b. are called non-cityRNAS.”
4) State of the art. Argonaute protein family AGO3 has slicer function which means it cleaves complementary target RNAs. The function is dependent on target sequences to target the correct site for cleavage and cleavage-inducing tiny guide RNAs (cityRNAs). AGO3 loaded with cityRNA with small RNAs (miRNA or siRNA that act as guide RNA that recognizes the target sequence) to form ribonucleoproteins referred to as the RNA-induced silencing complexes (RISCs). This complex in turn targets and degrades mRNAs. AGO3 is activated by let-7a, miR-27a, and specific miR-17-92 families to make AGO3 an extremely competent slicer by an ~ 82-fold increase in target cleavage. Their use is being proposed as an alternative to CRISPR Cas wherein the improvement in the system is based on the more simplistic mechanism i.e. lack of need of a PAM sequence and the utilization of ty RNA (see Sun et al, abstract). The family of proteins have low sequence homology but are highly conserved in architecture and function across prokaryotic AGO and eukaryotic long, short and PIWI-RE (Sun, figure 1).
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These domains are described in detail by Kropocheva, page 858, col 1-2.
To date, the application of AGO proteins is in detection of target sequences (see Kropocheva, figure 4).
5) Unpredictability of the art. Applicants have claimed a broad and generic method of silencing target mRNA. The steps themselves for in vivo use are not provided and furthermore, exact targets and outcomes are not provided for either. Rather, the disclosure teaches in vitro application.
The use of AGO for clinical applications is a nascent desired pathway (see Kropocheva et al, page 863, col 2) wherein the post filing publication states,
Although no success was yet achieved in the use of Argonautes for genome editing, their diversity, both structural and functional, allows us to hope that they will soon provide complimentary tools for genome manipulations and biotechnology along with Cas nucleases. At present, studies of the prokaryotic Argonaute family proteins already led to their practical applications in new highly sensitive methods for detecting nucleic acids, including SARS-CoV-2 RNA. In the near future, we could expect the appearance of works using Argonautes as effective tools for manipulations with nucleic acids in vitro, and in vivo.
This is more clearly stated by Wu et al,
Taken together, it is becoming clear that Argonaute proteins contain nucleic acid-guided nuclease activity, but there are still no characterized Argonaute proteins that show robust genome editing ability.
Hence, the art has established that even post-filing, the art of gene editing with AGO is nascent leaving it highly unpredictable that one would be able to use the invention as broadly claimed. The claimed method does not provide for AGO administered and appears to rely on cellular provision of the protein. There is no indication that this is even implied in the claim. Secondly, the target sequence is any and the cityRNA any. However, the method demonstrates functionality with a more limited set of targeting dsRNA that hybridizes to a determined target sequence and furthermore requires that the cityRNA be specific. The specification teaches only use of the following. And furthermore, extrapolations of sequences led to no activation. It appears therefore that specific sequences are necessary to activate AGO.
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Hence, even for in vitro use, the method is not enabled as recited.
Considering in vivo use, the art of gene editing has progressed with the application of CRISPR/CAS wherein even this art is hindered from in vivo general applicability as the guide sequences, target genes and modes of delivery have not progressed to overcome general issues with delivery. The method does not provide sufficient guidance to overcome art recognized obstacles for in vivo delivery i.e. organ barriers, failure to persist, side-effects in other organs, virus neutralizing antibodies, humoral immunity, normal tropism of the vector to other organs and more. The challenge is to maintain the efficiency of delivery and expression while minimizing any pathogenicity of the vector or delivery agent (for a review see Lino especially page 1252). The inability to develop an adequate means of overcoming obstacles such as humoral; responses and refractory cells limits the successful means by which the nucleic acid can be administered. The physiological art is recognized as unpredictable. (MPEP 2164.03.) In cases involving predictable factors, such as mechanical or electrical elements, a single embodiment provides broad enablement in the sense that, once imagined, other embodiments can be made without difficulty and their performance characteristics predicted by resort to known scientific laws. In cases involving unpredictable factors, such as most chemical reactions and physiological activity, the scope of enablement obviously varies inversely with the degree of unpredictability of the factors involved. In this case, the delivery is broadly stated as being administered. Furthermore, the method is not applicable to treating any disease or disorder and it is not clear how it is to be used to treat any disease or disorder given the nascent state of the art, the lack of guidance and specifics.
Finally, a number of claims refer to outcomes without provided the how as to the means by which these outcomes are arrived or in some cases that they are not inherent properties. Claims 8-12, 20 and 21 in particular are drawn to outcomes but the steps leading thereto are not provided. Claim 8, for example, requires that the target nucleic acid is cleaved and claim 9 silenced and claim 10 gene-specific silencing. It is not clear what leads to the step outcomes. This is true of claim 11, 20 and 21 wherein these appear to be either desired outcomes (claim 20 and 21) and potentially inherent outcomes (claims 8-11). This is a limited descriptive element wherein the claims broadly and incompletely claim the inventive elements. Structural features that could distinguish the compounds of the claimed genus from others not encompassed by the genus are missing from the disclosure.
6) Undue experimentation. The claims have been evaluated in light of the art at the time of filing and found not to be commensurate in scope with the specification. MPEP 2164.05 teaches, “However, the examiner should carefully compare the steps, materials, and conditions used in the experiments of the declaration with those disclosed in the application to make sure that they are commensurate in scope; i.e., that the experiments used the guidance in the specification as filed and what was well known to one of skill in the art. Such a showing also must be commensurate with the scope of the claimed invention, i.e., must bear a reasonable correlation to the scope of the claimed invention. The invention recites use of a broad group of sequence. Given the unpredictability of the art, the poorly developed state of the art with regard to predicting the structural/ functional characteristics of antagonists, the lack of adequate working examples and the lack of guidance provided by applicants, the skilled artisan would have to have conducted undue, unpredictable experimentation to practice the claimed invention.”
Response to arguments
Applicants arguments reflect that of the Declaration also submitted in response to the rejection under 35 USC 112, first. They are addressed combined with the response by applicants together below. This includes the two references provided with the response. As a first issue, applicants assert but do not provide support that the use of RNA guided Argonaute activity were well characterized and routine. A search of the art demonstrates that this use known and characterized at the time of filing was not extended to in vivo use to treat a disease or disorder.
Applicants argue that the target sequence and cityRNA are limited in scope as claimed. Once the target sequence is defined, the cityRNA must share identity. However, the claims as recited have any size dsRNA that further comprises any cityRNA that is limited only by the length being 12-16 nucleotides. However, applicants indicate in the disclosure that the nature of the cityRNA is critical,
[[0105]] To find more cityRNAs, 14-nt tyRNAs of miR-17, miR-18a, miR-19a, miR-27a, and miR-92a (FIG. 8A) were tested for in vitro target cleavage. Again, AGO2 reduced slicing activity with their 14-nt tyRNAs whereas AGO3 became a remarkably competent slicer when loaded with all except for the 14-nt miR-19a (FIG. 1E and FIG 8C). These results indicate that not all tyRNAs can catalytically activate AGO3 and that some cityRNAs make AGO3 a superior slicer to AGO2.
[[0107]] In summary, AGO2 and AGO3 have distinct guide lengths optimized for their activation. AGO2 cleaves any RNAs including a sequence fully complementary to the guide RNA, which means that any guide RNAs can activate AGO2. This is not the case for the AGO3 activation. Only specific tyRNAs can serve as cityRNAs due to their unique sequences. These multiple requirements extremely limit the opportunities for catalytically activating AGO3.
In essence, applicants have analyzed the mechanism of cityRNA binding and AGO3 cleavage and to contradict applicants statement indicate that specific sequences are functional with only a potential for generic characterization. Size and properties are very limited.
[[0130]] A preliminary study revealed that the 14-nt tyRNAs of miR-20a and let-7a, but not of miR-16 or miR-19b, converted AGO3 to a strong slicer. The tyRNAs of miR-20a and let-7a share five nucleotides, U1, A3, G5, U6, and U9 (FIG. 1 ra). Among these, only A3, G5, and U6 are unique to miR-20a and let-7a but not to miR-16 or miR-19b (FIG. 14 a ), suggesting that these three nucleotides are possible determinants for cityRNA-directed RNA cleavage.
Applicants expand on this teaching by showing in vitro activity with 14-nt miR-20a, 14-nt let-7a, 14-nt miR-92a, 14-nt miR-17, 14-nt miR-18a, and 14-nt miR-27a. However, the results did not suggests miR-27a was very effective at target cleavage (see Figure 1E and 8C). This is reflected in the expanded scope but still does not generate support or enablement for use of any cityRNA that is only characterized by being 12-16 nucleotides. As well, the dsRNA is especially uncharacterized except to comprise the cityRNA. The entire set up is simply insufficient to provide the specificity of hybridization and cleavage as required of the claims.
Applicants argue that the specification provides detailed examples of how to prepare gRNAs and test the activity. However, this activity is in vitro. The claims are clearly directed to therapeutic use. Claims 13 and 14 direct one to use the method of silencing generically on a disease or disorder. This means generic diseases or disorders and related target gene and target sequences are subjected to a method of silencing using AGO3 wherein no details are provided to guide applicant. This is complicated by the lack of knowledge and expertise in the art to use AGO3 for such purposes. In fact, there is no demonstrated use of AGO for therapy at the time of filing. Specific targets and diseases and disorders must be identified and tested. There are issues of nucleic acid access i.e. organ barriers off tissue effects as well as degradation to consider with therapeutic uses. Even CRISPR/Cas system which has been explored for over a decade for such use is limited as to disorder that applicability is successful. Zhang and Parks provided for by applicants analyzes binding properties of cityRNAs and aspects of targeting by these sequences. It does not demonstrate an in vivo ability especially to treat a disease or disorder.
Considering the target sequence for a disorder or disease, the method of cleavage is not particularly specific. The cityRNA functions to target and activate AGO3as a dual function. The identified sequences capable of this function are typically miRNA or other small RNAs that thus bind to genomic miRNA or small RNAs. Thus, it is not clear how target specific silencing occurs.
The sequence fully complementary to a 14 nt cityRNA appears every 67 million nucleotides (= 413 = 67 × 106). Assuming that about 93% of the human genome is transcribed (Consortium et al., 2007), AGO3 loaded with a 14 nt cityRNA could cleave approximately 100 sites. This number decreases because among 14–15 nt tyRNAs, only ones with specific sequences can serve as cityRNAs (i.e., catalytically activate AGO3) (Park et al., 2020). Nevertheless, the synthesis of cityRNAs appears to be a powerful tool for the cell to change gene expression drastically by cleaving many transcripts. Therefore, cells would need to regulate the biogenesis of tyRNAs strictly.
There simply is no direction, guidance or teachings on how to use argonautes. In fact, it is not clear how introduction of AGO proteins to treat disorders related to cancer would impact the disorder as their involvement in tumorigenesis is contraindicative of use (see conclusion of Nowake and Sarshad).
Secondly, this lack of clear direction is exacerbated by claim language which complicates the lack of enablement. Specifically, claim 1 recites “exposing” the dsRNA to RISC and the loaded RISC to the target mRNA. Claim 4 which recites that Dicer cleaves the RNA prior to exposure to RISC. In vivo, there is no ability to control “exposure” and cleavage by DICER prior to exposure as in vivo you cannot control when events happen. In other words, there is no hand of man in these steps when considering the step in vivo.
Third, claims 8-12, 20 and 21 recite what appear to be desired outcomes but no steps. The description of these claims therefore lacks. For example, claim 8 recites “wherein the target nucleic acid is cleaved by AGO3 associated RISC loaded with cityRNA” which has no additional steps but according to the disclosure is the consequence of the method as claimed. This is true of all of claims 8-12, 20 and 21.
As a whole, the nascent state of the art, the lack of guidance in the disclosure and the large breadth of the claims contribute to the established scope of enablement as set forth above.
Conclusion
The art did not teach linkage of cityRNA to a targeting sequence such that the cityRNA is directed to a specific locus until applicants publication of Parks, 2020.
THIS ACTION IS MADE FINAL. 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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/MARIA MARVICH/Primary Examiner, Art Unit 1634