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 .
Information Disclosure Statement
The information disclosure statement (IDS) submitted on June 7th, 2024 is acknowledged. The submission is in compliance with the provisions of 37 CFR 1.97. Accordingly, the information disclosure statement has been considered by the examiner.
Priority
Receipt is acknowledged of certified copies of papers required by 37 CFR 1.55.
Claim Summary
Claims 1-9 are pending. Claims 1-9 are under examination and discussed in this Office action.
Specification
Applicant is reminded of the proper content of an abstract of the disclosure.
A patent abstract is a concise statement of the technical disclosure of the patent and should include that which is new in the art to which the invention pertains. The abstract should not refer to purported merits or speculative applications of the invention and should not compare the invention with the prior art.
If the patent is of a basic nature, the entire technical disclosure may be new in the art, and the abstract should be directed to the entire disclosure. If the patent is in the nature of an improvement in an old apparatus, process, product, or composition, the abstract should include the technical disclosure of the improvement. The abstract should also mention by way of example any preferred modifications or alternatives.
Where applicable, the abstract should include the following: (1) if a machine or apparatus, its organization and operation; (2) if an article, its method of making; (3) if a chemical compound, its identity and use; (4) if a mixture, its ingredients; (5) if a process, the steps.
Extensive mechanical and design details of an apparatus should not be included in the abstract. The abstract should be in narrative form and generally limited to a single paragraph within the range of 50 to 150 words in length.
See MPEP § 608.01(b) for guidelines for the preparation of patent abstracts.
The abstract of the disclosure is objected to because it is longer than the allowed word limit. It is suggested that the Applicant provide a more concise summary of the invention. A corrected abstract of the disclosure is required and must be presented on a separate sheet, apart from any other text. See MPEP § 608.01(b).
The disclosure is objected to because of the following informalities: the Brief Description of the Drawings appears to reference incorrect figures in paragraphs [0021] and [0022]. Figures 4 and 5 show experimental data, and Figure 5 shows an example of sequence analysis. Appropriate correction is required.
Claim Interpretation
The generic abbreviation RCA is claimed as a part of the method in step f. of claim 1, and the rest of the claims given their dependence from claim 1. As provided in paragraph [0003] of the specification, RCA is the abbreviation for Rolling Circle Amplified, and will therefore be interpreted as such for examination.
Claim Objections
Claims 1 and 6-9 are objected to because of the following informalities:
Claim 1 has several objections, which are noted in the following list:
Step a. reads "…one m-RNA strand and b) an anchor sequence…”. Since "b)" isn't the final component in the "comprising" list, there should not be an "and" between the a) and b) components making up the linear probe. It is suggested that the phrase read "one m-RNA strand [[and]], b) an anchor sequence".
Step b. reads “…hybridizing the linear probe with its binding region to the m-RNA strand”. This wording is awkward and could be interpreted as the binding region belonging to another sequence entity. It is suggested that the phrase read “hybridizing the binding region of the linear probe to the m-RNA strand”.
Step c. reads “…using the m-RNA strand as template…”. The word “a” should be inserted between “as” and “template”.
Step d. reads “…hybridizing a locator molecule with its 3’ and 5’ ends to the first and second locator regions…”. This wording is awkward and could be interpreted as the 3’ and 5’ ends are hybridizing to the locator molecule itself instead of the linear probe as presumably intended. It is suggested that the phrase read “hybridizing 3’ and 5’ ends of a location molecule to the first and second locator regions”.
Step g. reads “Sequencing at least the UMI portion…” The “S” in “Sequencing” should be lower case.
Step i. reads “…providing the single stranded cDNA oligomer with a first and a second adaptor primer at the 3' and 5' ends obtaining a primed single stranded oligomer; amplification of the primed single stranded oligomer by PCR…”. The word “amplification” in the above phrase should instead read “amplifying” for consistency with the rest of the steps of the claim.
There are several instances of the phrase “reversed transcribed”. In every instance, “reversed” should instead read “reverse”.
Claims 6, 8 and 9 are all missing a period at the end of the claim. These should be inserted.
Claim 7 recites “…first hybridizing the linear locator to the to at least one m-RNA strand…”. The second instance of the word “to” in this phrase should be deleted. Claim 7 also recites “…reversed transcribed…”, which should instead read “reverse transcribed”.
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 1-9 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 1 recites the limitations “…a target sequence of at least one m-RNA strand on a tissue sample…” in the preamble, and later “thereby obtaining the spatial location of the m-RNA on the tissue…”. It is unclear from this recitation if the m-RNA strand originated from the tissue sample or if the m-RNA has been added onto the tissue sample from an outside source. This is due to the language m-RNA “on” the tissue. It is further unclear from this recitation where specifically the m-RNA is located on the tissue. For instance, the m-RNA could be in the extracellular space of the tissue, or it could be in a cell within the tissue sample. Clarification is required.
Claim 1 also recites the limitation “…Sequencing at least the UMI portion of the rolonies thereby obtaining the spatial location of the m-RNA on the tissue…” It is unclear from this recitation how generically sequencing the UMI of the rolonies will result in a spatial location for the m-RNA because the mRNA is “on” the tissue, not “in” the tissue.
Claim 1 also recites the limitation “…multiplying the circular template molecule by RCA on the tissue sample, starting from a primer region thereby creating a rolony; Sequencing at least the UMI portion of the rolonies…”. There is insufficient antecedent basis for the plural term “rolonies” based on the initial introduction of the singular term “rolony”. It is noted that claim 9 also recites plural “rolonies”. Claims 2-9 are also rejected here given their dependence on claim 1 and not further clarifying the identified issues.
Claim 4 contains the trademark/trade name Phusion. Where a trademark or trade name is used in a claim as a limitation to identify or describe a particular material or product, the claim does not comply with the requirements of 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph. See Ex parte Simpson, 218 USPQ 1020 (Bd. App. 1982). The claim scope is uncertain since the trademark or trade name cannot be used properly to identify any particular material or product. A trademark or trade name is used to identify a source of goods, and not the goods themselves. Thus, a trademark or trade name does not identify or describe the goods associated with the trademark or trade name. In the present case, the trademark/trade name is used to identify/describe a DNA polymerase and, accordingly, the identification/description is indefinite. Claim 4 also recites the limitation "the circular locator". There is insufficient antecedent basis for this limitation in the claim. Claim 1, from which claim 4 depends, does not introduce a circular locator, but instead a “circular template” formed from a “locator molecule”. It is therefore unclear if the circular locator is referring to this circular template formed from the locator molecule, or some other locator that has not been previously introduced.
Claim 4 also recites the limitation “Phusion DNA polymerase or non-strand displacement polymerase”. From this recitation, it could be interpreted that Phusion DNA polymerase is not a non-strand displacement polymerase given that the polymerases are presented as separate options. However, as described in the specification, Phusion DNA polymerase is a type of non-strand displacement enzyme (see Page 6, paragraph [0028]). Therefore, it is unclear why Phusion DNA polymerase is being distinguished from non-strand displacement polymerase as currently claimed.
Claim 5 recites the limitation “…the 3’ and 5’ ends of the gap-filled DNA molecule...”. There is insufficient antecedent basis for this limitation in the claim. Claim 1, from which claim 5 depends, does not introduce a gap-filled DNA molecule, but instead a “locator molecule” that has a gap that is filled. It is therefore unclear if the gap-filled DNA molecule is referring to this locator molecule, or some other DNA molecule that has not been previously introduced.
Claim 5 also recites the limitation “…wherein the UMI and the first region of the linear locator…”. There is also insufficient antecedent basis for this limitation in the claim. Claim 1, from which claim 5 depends, does not introduce either a “first region” or a “linear locator”, but instead a “linear probe” containing a “first locator region”. It is therefore unclear if the first region of the linear locator is referring to this linear probe, or some other linear locator that has not been previously introduced. Claims 6 and 7 are also rejected here given their dependence on claim 5 and not further clarifying the identified issues.
Claim 7 recites the limitation “…the locator probe is provided by first hybridizing the linear locator to the to at least one m-RNA strand, complementing the RNA anchor region of the locator probe into a reversed transcribed c-DNA strand and then hybridizing the circular locator to the linear locator.” There is insufficient antecedent basis for a “locator probe”, an “RNA anchor region” of the locator probe, and a “circular locator,” which have not been previously introduced in claims 1, 5, or 6, from which claim 7 depends. A “locator molecule” is introduced in claim 1, but not a “locator probe”. A “linear locator” is introduced in claim 5, but the clarity of this recitation has already been brought into question above. An “anchor sequence comprising a UMI region located between a first and a second locator region” is introduced in claim 1, but not an “RNA anchor region”. A “locator molecule” is introduced in claim 1, but not a “circular locator”. Given all of these issues, it is unclear if any of these recitations are referring to previously claimed probes or molecules, or some other probes or molecules that have not been previously introduced.
Claim 8 recites the limitation "the single stranded oligomer". There is insufficient antecedent basis for this limitation in the claim. Claim 1, from which claim 8 depends, does not introduce a “single stranded oligomer”, but instead a “single stranded cDNA oligomer”. It is therefore unclear if the single stranded oligomer is referring to this single stranded cDNA oligomer, or some other oligomer that has not been previously introduced. Assuming they are the same oligomer, it is also unclear how an oligomer that is sheared into smaller fragments can be amplified with what are presumably the same first and second adaptor primers to the 3’ and 5’ ends. The ends of a fragmented oligomer will change given the shearing process, and therefore it is not clear how the method claimed in claim 8 can be successfully performed.
Claim 9 recites the limitation "the single stranded adapted DNA oligomer". There is insufficient antecedent basis for this limitation in the claim. Claim 1, from which claim 9 depends, does not introduce a “single stranded adapted DNA oligomer”, but instead a “single stranded cDNA oligomer” and a “primed single stranded oligomer”. It is therefore unclear if the single stranded adapted DNA oligomer is referring to either of these previously identified oligomers, or some other oligomer that has not been previously introduced.
Prior Art
The prior art made of record and not relied upon is considered pertinent to applicant's disclosure.
Bava (US 20230287478 A1, effectively filed March 11th, 2022) teaches on in situ detection methods using a probe that hybridizes to a target sequence, the probe containing a barcode within a concatemeric region amplified by RCA (Page 1, paragraphs [0004] and [0009]). The concatemeric region amplified by RCA is primed using a circular or circularized oligonucleotide (Page 1, paragraph [0008]). The concatemeric region is amplified using detectably labeled nucleotides (Page 1, paragraph [0007]). Bava does not teach on the specifically claimed combination of probes, namely that a linear probe with a binding region, anchor sequence comprising a UMI and locator regions, and a primer region hybridizes to an RNA molecule in addition to a locator molecule (e.g. padlock probe) hybridizing to the linear probe. Bava also does not teach that the locator molecule is ligated and amplified with RCA, that a spatial location can be determined by sequencing the locator molecule after it has been ligated and amplified into a rolony, or that spatial location is linked to the specific sequence of the RNA molecule.
Bava (US 20220186300 A1; cited on the IDS filed June 7th, 2024) teaches on in situ analysis methods using a padlock probe to directly hybridize to an RNA transcript for detection of a nucleic acid (Page 28, paragraph [0271]). The padlock probe contains one or more barcode regions (Page 28, paragraph [0271]). A splint primer may be used adjacent to the padlock probe to facilitate DNA-templated padlock ligation and form a circular probe (Page 28, paragraph [0271]). The circular probe is amplified with RCA such that the barcodes are also amplified, and the barcodes are detected with fluorescently labeled detection oligos (Page 28, paragraph [0271]). Bava does not teach on the specifically claimed combination of probes, namely that a linear probe with a binding region, anchor sequence comprising a UMI and locator regions, and a primer region hybridizes to an RNA molecule, in addition to a locator molecule (e.g. padlock probe) hybridizing to the linear probe. Bava also does not teach that detection is done by sequencing, that a spatial location can be determined by sequencing the locator molecule after it has been ligated and amplified into a rolony, or that spatial location is linked to the specific sequence of the RNA molecule.
Glezer (US 20220042083 A1; cited on the IDS filed June 7th, 2024) teaches on detecting a plurality of nucleic acid sequences within a cell in situ (Page 16, paragraph [0087]). This detecting involves direct hybridization of a padlock probe with a known barcode form a set of barcodes to a target in the plurality of nucleic acid sequences (Page 16, paragraph [0087]). The padlock probe also includes a primer binding sequence (Page 16, paragraph [0087]). The barcodes are sequenced to obtain a multiplexed signal in the cell in situ, demultiplexed by comparing known barcodes, and targets are detected based on identification of the known barcodes in the cell (Page 16, paragraph [0087]). Glezer does not teach on the specifically claimed combination of probes, namely that a linear probe with a binding region, anchor sequence comprising a UMI and locator regions, and a primer region hybridizes to an RNA molecule in addition to a locator molecule (e.g. padlock probe) hybridizing to the linear probe. Glezer also does not teach that the locator molecule is ligated and amplified with RCA, that a spatial location can be determined by sequencing the locator molecule after it has been ligated and amplified into a rolony, or that spatial location is linked to the specific sequence of the RNA molecule.
Costa (US 20220235403 A1; cited on the IDS filed June 7th, 2024) teaches on detecting a DNA concatemer that is generated in situ (Page 2, paragraph [0015]). This concatemer can be an RCA product of a circular or circularizable probe (e.g. padlock) probe that hybridizes directly to a nucleic acid molecule in a sample (Page 2, paragraph [0017]). These probes can comprise a barcode sequence that uniquely corresponds to particular nucleic acid molecules (Page 2, paragraph [0017]). The concatemer as taught is detected by detection probes that directly or indirectly hybridize to the concatemer (Page 1, paragraph [0004]). Costa does not teach on the specifically claimed combination of probes, namely that a linear probe with a binding region, anchor sequence comprising a UMI and locator regions, and a primer region hybridizes to an RNA molecule, in addition to a locator molecule (e.g. padlock probe) hybridizing to the linear probe. Costa also does not teach that detection is done by sequencing, that a spatial location can be determined by sequencing the locator molecule after it has been ligated and amplified into a rolony, or that spatial location is linked to the specific sequence of the RNA molecule.
Conclusion
All claims stand rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Allison E Schloop whose telephone number is (703)756-4597. The examiner can normally be reached Monday-Friday 8:30-5 ET.
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/ALLISON E SCHLOOP/Examiner, Art Unit 1683
/Robert T. Crow/Primary Examiner, Art Unit 1683