DETAILED ACTION
Notice of Pre-AIA or AIA Status
1. 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
2. Applicant’s election without traverse of Group II, drawn to system and kit of claims 19-33 in the reply filed on 17 November 2025 is acknowledged.
Claim Status
3. Claims 1, 15 and 19-33 are pending.
Claims 1 and 15 are withdrawn from further consideration pursuant to 37 CFR 1.142(b), as being drawn to a nonelected invention, there being no allowable generic or linking claim.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 19-33 read on the elected invention and have been examined herein.
Examiner’s Comment
4. Regarding Applicant’s comment that claim 1 has been amended to depend from claim 19, it is noted that claim 1 does not clearly require each of the components of the system of claim 19. Rather, claim 1 recites in the preamble of the claim that the method is one for detecting miRNA-21 carried by the EVs “according to the system of claim 19.” It is unclear as to what is intended to be meant by a method or an EV “according to the system of claim 19.” Claim 19 subsequently recites a step of delivering “the specific hairpin probe,” the claim does not clarify that the specific hairpin probe is the probe in the system of claim 19. Claim 19 is not directed to a method that includes clear process steps of using the complete system of claim 19 which system comprises a miRNA-21 standard and a specific hairpin probe. Further, while claim 15, which depends from claim 1, recites “wherein there are 3 specific hairpin probes,” the claim does not set forth a relationship between the “3 specific hairpin probes” and the “specific hairpin probe” comprising probes A, B and C included in the system of claim 19.
Objection to Drawings / Objection to the Specification
5. The drawings are objected to under 37 CFR 1.83(a). The specification describes the figures, for example Figure 14, in terms of particular colors – i.e. red. However, the figures have been filed in black and white. Thus, the description of Figure 14 in the specification is not consistent with the drawing and the specification is also objected to.
If the drawings are intended to be in black and white, the specification should be amended to delete the reference to the recited colors. Alternatively, corrected drawing sheets in compliance with 37 CFR 1.121(d) are required. Note that color drawings are only accepted on rare occasions when they are the only practical medium by which to disclose the subject matter to be patented. See MPEP 608.02(VIII).
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.
If color photographs or color drawings are submitted, it is noted that color photographs and color drawings are not accepted unless a petition filed under 37 CFR 1.84(a)(2) is granted.
A petition for color drawings must be accompanied by the appropriate fee set forth in 37 CFR 1.17(h), one set of color drawings or color photographs, as appropriate, if submitted via EFS-Web or three sets of color drawings or color photographs, as appropriate, if not submitted via EFS-Web, and, unless already present, an amendment to include the following language as the first paragraph of the brief description of the drawings section of the specification:
The patent or application file contains at least one drawing executed in color. Copies of this patent or patent application publication with color drawing(s) will be provided by the Office upon request and payment of the necessary fee.
Color photographs will be accepted if the conditions for accepting color drawings and black and white photographs have been satisfied. See 37 CFR 1.84(b)(2).
Appropriate correction is required.
Claim Objections
6. Claims 23-33 are objected to because of the following informalities:
Claims 23-33 are objected to because the claims recite “RVs” but do not recite the full terminology for “RVs” prior to the first recitation of this acronym. This objection may be obviated by amendment of claim 23 to recite “red blood cell membrane (RBCM)-derived vesicles (RVs).” Appropriate correction is required.
Claim Rejections - 35 USC § 112(b) - Indefiniteness
7. 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 19-33 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.
Claims 19-33 are indefinite over the recitations of “BHQ2 modified on T” and “Cy5 modified on T” because it is not clear as to what is meant by these phrases. For example, it is unclear as to whether it is the BHQ2 moiety that is modified or if it is the T nucleotide that is modified to include a BHQ2 moiety (i.e., a T nucleotide labeled with BHQ2 or a BHQ2-modified T nucleotide).
Claims 19-33 are also indefinite over the recitations of “A-Cy5” and “C-Cy5” because it is unclear as to whether the claims intend to require an (additional) Cy5 at the 3’ end of A and C, or at any other location in A and C, or if A is intended to include only the Cy5 at the T nucleotide at the 11th position of SEQ ID NO: 1 and/or C is intended to include only the Cy5 at the T nucleotide at the 6th position of SEQ ID NO: 3.
Claims 25 and 26 are indefinite over the recitation of “the miRNA-21 standard is diluted with a PBS into different working concentrations during use.” Since the claims are drawn to a kit, it is unclear as to how this limitation is intended to limit the claims because the claims do not include a process step of using the PBS.
Claims 27-30 are indefinite over the recitation of “a reagent required for the preparation of the RVs by extrusion.” This phrase is not clearly defined in the specification or the claims as it relates to a reagent that is necessarily required for the preparation of the RVs by any extrusion process. Note that extrusion encompasses any process of pushing something through a tool or device. It is unclear as to the metes and bounds of a reagent that is necessarily required for preparation of the RVs by any extrusion processes.
Claim Rejections - 35 USC § 112(a) - Enablement
8. 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 19-33 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 the system of claim 19 wherein the system comprises a miRNA-21 standard and comprises three hairpin probes, A, B and C, wherein hairpin probe A has the sequence of SEQ ID NO: 1 and is modified to include BHQ2 at the T at nucleotide position 11 and Cy5 at the T at nucleotide position 53, wherein nucleotide positions are with respect to the 5’ end of SEQ ID NO: 1; hairpin probe B has the nucleotide sequence of SEQ ID NO: 2; and hairpin probe C has the nucleotide sequence of SEQ ID NO: 3 and is modified to include Cy5 at the T at nucleotide position 6 and BHQ2 at the T at nucleotide position 54, wherein the nucleotide positions are with respect to the 5’ end of SEQ ID NO: 3; and in claim 20, the miRNA-21 standard has the nucleotide sequence of SEQ ID NO: 4,
does not reasonably provide enablement for the claimed systems that comprise “a specific hairpin probe” (i.e., a single probe) that comprises each of A, B, and C, and in which A, B, and C have “a nucleotide sequence of” SEQ ID NO: 1, 2 and 3, respectively, and in claim 20, the miRNA-21 standard has “a nucleotide sequence of SEQ ID NO: 4.” 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.
The following factors have been considered in formulating this rejection (In re
Wands, 858F.2 731, 8 USPQ2d 1400 (Fed. Cir. 1988): the breadth of the claims, the
nature of the invention, the state of the prior art, the relative skill of those in the art, the
predictability or unpredictability of the art, the amount of direction or guidance
presented, the presence or absence of working examples of the invention and the quantity of experimentation necessary.
The claims are drawn to a system and a kit comprising a system, wherein the system comprises a miRNA-21 standard and a single hairpin probe that comprises each of A-Cy5, B, and C-Cy5 and wherein A, B, and C have “a nucleotide sequence of” SEQ ID NO: 1, 2 and 3, respectively. Claim 20 is limited to systems in which the miRNA-21 standard has “a nucleotide sequence of SEQ ID NO: 4.”
With respect to the recited “a specific hairpin probe,” in view of the language of “a nucleotide sequence of,” the claims encompass probes that include any one, two or three or more nucleotides of the referenced sequence. In view of the “has” language, the probes may include any number of additional nucleotides of any identity present at the 5’ and/or 3’ ends of the probes. The claims also include a single hairpin probe that includes all three of A, B and C. Thus, the claims encompass a significantly large genus of one or three probes, the probes including any single nucleotide or small fragment of SEQ ID NO: 1-3 and any number of additional nucleotides of any identity. The claims recite that SEQ ID NO: 1 and 3 are modified to include a BHQ2 quencher and a Cy5 fluorescent label at particular nucleotide positions within SEQ ID NO: 1 and 3. Yet, the claims do not require the full length sequence of SEQ ID NO: 1 and 3 (or the full length sequence of SEQ ID NO: 2).
The specification does not provide sufficient guidance for using the broadly claimed specific hairpin probe for the intended purpose of “in situ detection of a micro ribonucleic acid (miRNA)-21 carried by extracellular vesicles (EVs).”
The teachings in the specification are limited to a system that includes at least 3 separate hairpin probes. For instance, the specification (para [0007]) teaches:
“Preferably, there are three specific hairpin probes; sequences of the three specific hairpin probes each include a self-complementary sequence and a complementary palindromic sequence; and two of the specific hairpin probes each are modified with a fluorophore and a quencher.”
There is no guidance provided in the specification as to how probes that include only small fragments of SEQ ID NO: 1-3, and thereby probes which omit the stem structure or all or part of the palindrome sequence, can be used in the disclosed methods for detecting miRNA-21.
There is also no guidance provided as to how the sequences of A, B and C can be linked together into a single probe and then used to detect miRNA-21. Note that the specification teaches that hairpin probe A must first bind to miRNA-21 before the reaction can proceed with hairpin probe B hybridizing to the complex formed between miRNA-21 and probe A, and then with hairpin probe C hybridizing to the complex formed between miRNA-21, probe A and probe B. See, e.g., para [0064]:
“the target detection substance miRNA-21 could only open probe A but not probe B and probe C, and the next DNA self-assembly reaction could occur only after probe A was opened. Specifically, miRNA-21 opened the hairpin probe A-Cy5 to form a 21-A-Cy5 structure. A-Cy5 hybridized to probe B, resulting in a 21-A-Cy5-B triplex structure with more cohesive ends. C-Cy5 continued to be opened and hybridized with the 21-A-Cy5-B to form a four-stranded structure of 21-A-Cy5-B-C-Cy5.”
Although not specifically stated in the specification, the relative location of the BHQ2 quencher and Cy5 fluorophore in the hairpin structure and in the open structure that results from the hybridization reaction with miRNA-21 and with the additional probes (see Figure 1) is critical to detecting a change in the fluorescent signal as indicative of the presence of miR-21. However, again, the specification does not provide sufficient guidance as to how to make and use a representative number of hairpin probes that include only a small portion of SEQ ID NO: 1, 2 and 3, wherein the location of BHQ2 and Cy5 are not defined since the full length sequences of SEQ ID NO: 1 and 3 are not required by the present hairpin probe(s).
Absent specific guidance in the specification, undue experimentation would be required to develop additional probes, including single probes that include only one or two or more of the nucleotides of SEQ ID NO: 1-3 but also have a hairpin structure and can form a complex that can detect miRNA-21, particularly within a extracellular vesicle.
Case law has established that "(t)o be enabling, the specification of a patent
must teach those skilled in the art how to make and use the full scope of the claimed
invention without 'undue experimentation." In re Wright 990 F.2d 1557, 1561. In re
Fisher, 427 F.2d 833, 839, 166 USPQ 18, 24 (CCPA 1970) it was determined that "(t)he
scope of the claims must bear a reasonable correlation to the scope of enablement
provided by the specification to persons of ordinary skill in the art". The amount of
guidance needed to enable the invention is related to the amount of knowledge in the
art as well as the predictability in the art. Furthermore, the Court in Genetech Inc. V
Novo Nordisk 42 USPQ2d 1001 held that "(I)t is the specification, not the knowledge of
one skilled in the art that must supply the novel aspects of the invention in order to
constitute adequate enablement".
In the instant situation, given the lack of specific teachings in the specification
and the lack of guidance provided in the specification and prior art, it would require
undue experimentation for one of skill in the art to make and use the broadly claimed
invention.
9. The prior art made of record and not relied upon is considered pertinent to
applicant's disclosure.
A. Xu et al (CN112877402A; translation included) discloses hairpin probes for detecting miRNA-21 present in a sample (see abstract). Pairs of hairpin probes are disclosed which contain palindromic sequences and include a fluorescent label (e.g. Cy3 or FAM) and a quencher (e.g., BHQ2). In particular, Xu (p. 3 of translation) teaches the following set of hairpin probes (H1 and H2) for detecting miRNA-21, as well as a target miRNA-21 sequence (which sequence is identical to present SEQ ID NO: 4):
The nucleotide sequence of HP1 is: 5'-GATCGCGATCTCAACAT-FAM-CAGTCTGATAAGCTAACATCGTAGCTTATCAGACTG-BHQ-1-3'; wherein GATCGCGATC is a palindrome sequence; TCAACAT (FAM) CAGTCTGATAAGCTA is a miRNA-21 recognition sequence; CAGTCTGATAAGCTA is complementary to TAGCTTATCAGACTG sequence ;
The nucleotide sequence of HP2 is: 5'-AGTTCGAACTT-Cy3-AGCTTATCAGACTGATGTTGATTTTCAGTCTGATAAGCT-BHQ-2-ACGATGT-3', wherein AGTTCGAACT is a palindrome sequence; T-Cy3-AGCTTATCAGACTGATGTTGA is a DNA sequence corresponding to miRNA-21; T -Cy3-AGCTTATCAGACTGA is complementary to TCAGTCTGATAAGCT-BHQ-2-A sequence;
The nucleotide sequence of the MiRNA-21 is: 5'-UAGCUUAUCAGACUGAUGUUGA-3'.
Xu does not teach a hairpin probe or a set of three hairpin probes that comprise present SEQ ID NO: 1, 2 and 3, wherein SEQ ID NO: 1 and 3 have a BHQ2 quencher and a Cy5 fluorophore at the positions in SEQ ID NO 1 and 3 specified in claim 19.
B. Yue et al (Chem. Sci. 2019. 10: 1651 and Supporting Information, p. S1-S7) teaches a system comprising three hairpin probs (H1, H2 and H3). Yue (abstract) characterizes the system as follows:
“For this system, a Y-shaped hairpin trimer tethered with three kinds of hairpins (H1, H2 and H3) is constructed. The introduction of an initiator (I) triggers a cascade of CHA reactions among hairpin trimers, leading to the formation of DNA dendrimers. Through labeling fluorophore/quencher pairs in the hairpin trimers, this catalytic DNA machine is applied as a versatile amplification platform to analyze nucleic acids using microRNA-155 (miR-155) as a model analyte.”
It is further stated at p. 1652:
“The Y-scaffold DNA is Fig. 1 (a) Components and reaction pathways of the bCHA process triggered by an initiator (I) for dynamic self-assembly of DNA dendrimers. Letters marked with * represent the complementary domains to the corresponding unmarked ones. (b) Microscope images and (c) size distribution of hairpin trimers before and after incubation with a 0.1 mM initiator. The concentration of the hairpin trimers is 1.5 mM. Scale bar in (b): 50 mm. composed of three single-stranded DNAs (ssDNAs), Y1, Y2 and Y3, in which each strand contains three segments: along “sticky end” to interact with its complementary sequence on the corresponding hairpin and two parts to hybridize with the other two strands. On the basis of Watson-Crick base pairs, the Y shaped hairpin trimer is formed via the hybridization of the “sticky ends” between the Y-scaffold DNA and hairpins (Fig. 1a, reaction 1).”
See also Figure 3 and Figure S3.
In the Supplementary Information in Table S1, Yue also exemplifies 3 hairpin probes for detecting miRNA-21 and provides the sequence of target miRNA-21, which sequence is identical to present SEQ ID NO: 4. The H1 probe for miRNA-21 includes an internal Dabcyl quencher and a FAM fluorescent label at the 3’ nucleotide. The H3 probe, and the H2 probe, do not include a fluorophore or quencher.
Yue et al does not teach a hairpin probe or a set of three hairpin probes that comprise present SEQ ID NO: 1, 2 and 3, wherein SEQ ID NO: 1 and 3 have a BHQ2 quencher and a Cy5 fluorophore at the positions in SEQ ID NO 1 and 3 specified in claim 19.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to CARLA J MYERS whose telephone number is (571)272-0747. The examiner can normally be reached M-Th 6:30-5:00 EST.
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/CARLA J MYERS/Primary Examiner, Art Unit 1682