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 .
Status of the Application
Claims 96-121 are pending and under examination.
The following Office Action is in response to Applicant's communication dated 06/10/2024.
Claim Objections
Claim 110(a) is objected to because of the following informalities: typographical omission of “acid” after “target nucleic”.
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.
Claim(s) 96-121 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 96, the phrase “the cell of a live subject” lacks proper antecedent basis.
claim 96, the phrase “the barcode of the mRNA transcript” lacks proper antecedent basis. Earlier limitation recites “a cell barcode”, not “a barcode”.
Claims 97-121 depend from claim 96 and is therefore similarly rejected.
claim 105, the phrase “the intact tissue” lacks proper antecedent basis. Neither claim 104 nor dependency chain leading to claim 105 provides clear antecedent support for “the intact tissue”.
claim 110(a), the phrase “the tissue” lacks proper antecedent basis. Neither claim 107 nor dependency chain leading to claim 110 provides clear antecedent support for “the tissue”.
claim 110(a), recites “a second complementarity region sequence”, then later recites “second complementarity region” or “second complementary region”. It is unclear if these are the same region.
claim 110(g), the phrase “the intact tissue” lacks proper antecedent basis. Neither claim 107 nor dependency chain leading to claim 110 provides clear antecedent support for “the intact tissue”.
Claims 119-121 the phrase “the target nucleic acid” lacks proper antecedent basis. Claims 119-121 all depend from claim 96, but claim 96 does not introduce “target nucleic acid”. Claim 107 introduces that term. It is unclear if claims 119-121 refer to the construct nucleic acid comprising mRNA, 3-UTR region with barcode and poly-adenylation from claim 96, or target nucleic acid of claim 107.
For purposes of examination only, and to facilitate a complete analysis of the claim, the Examiner interprets Claims 119-121 to depend on claim 107 instead of claim 96 because the limitation first introduced in claim 107. This interpretation is adopted solely for examination and does not resolve the lack of clarity in the claim language.
Applicant is reminded to maintain consistent terminology throughout the claim set. Especially where multiple elements have similar properties/all nucleic acid related elements. It is important that the claim clearly distinguish whether an element is newly introduced or refer back to an element previously recited. Proper antecedent basis is one mechanism for maintaining this distinction and ensuring the scope of the claim is clear.
Claim Rejections - 35 USC § 103
In the event the determination of the status of the application as subject to AIA 35 U.S.C. 102 and 103 (or as subject to pre-AIA 35 U.S.C. 102 and 103) is incorrect, any correction of the statutory basis (i.e., changing from AIA to pre-AIA ) for the rejection will not be considered a new ground of rejection if the prior art relied upon, and the rationale supporting the rejection, would be the same under either status.
The following is a quotation of 35 U.S.C. 103 which forms the basis for all obviousness rejections set forth in this Office action:
A patent for a claimed invention may not be obtained, notwithstanding that the claimed invention is not identically disclosed as set forth in section 102, if the differences between the claimed invention and the prior art are such that the claimed invention as a whole would have been obvious before the effective filing date of the claimed invention to a person having ordinary skill in the art to which the claimed invention pertains. Patentability shall not be negated by the manner in which the invention was made.
This application currently names joint inventors. In considering patentability of the claims the examiner presumes that the subject matter of the various claims was commonly owned as of the effective filing date of the claimed invention(s) absent any evidence to the contrary. Applicant is advised of the obligation under 37 CFR 1.56 to point out the inventor and effective filing dates of each claim that was not commonly owned as of the effective filing date of the later invention in order for the examiner to consider the applicability of 35 U.S.C. 102(b)(2)(C) for any potential 35 U.S.C. 102(a)(2) prior art against the later invention.
Zador et al. and Hrvatin et al.
Claim(s) 96-106 and 115-117 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zador et al. (US12031127B2, EFD: April 2017) in view of Hrvatin et al (e.g. US20220025398A1, EFD: December 2019).
Regrading claim 96, Zador discloses a method comprising: (a) introducing into the cell of a live subject a nucleic acid comprising a sequence encoding a messenger RNA (mRNA) transcript comprising a cell barcode, wherein the cell expresses the mRNA transcript; (e.g. MAPseq is a method of brain mapping, where in neurons are uniquely labeled by an expression construct that encodes a barcoded mRNA. The cells express barcoded mRNA, GFP markers [¶0129-0132])
(b) measuring a morphological or functional characteristic of the cell in response to the cell expressing the mRNA transcript; (e.g. MAPseq is used to measure the projection pattern of individual neurons. The reference states MAPseq quantifies projection pattern of large population of neurons in parallel and provide reliable estimate of each neuron’s relative projection strength to each target [¶0141-150].)
(c) identifying the barcode of the mRNA transcript. (e.g. 44 hours post injection, barcode mRNA extracted from dissected cortical target regions, under goes reverse transcription, amplification, and sequencing. Barcode abundance in the target areas was converted to a matrix of single neuron projection patterns. [¶0134]).
However, Zador does not discloses the specific construct design, where barcode is in 3' -untranslated region (3-UTR) and adjacent to the poly-adenylation site.
Hrvatin discloses an AAV/rAAV expression construct comprising a GFP transcript with a barcode and poly-A inserted in the 3-UTR, wherein the barcode is adjacent to poly-A [Fig 2A and ¶0013-0014].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Zador’s barcode expressing construct such that the barcode locates in the 3-UTR adjacent to the poly-A because Hrvatin explains this configuration helps maximize retrieval of the barcode sequence during single-cell RNA sequencing, which primarily captures the 3′ end of transcripts. [¶0321]. Hence Hrvatin provides the construct design and motivation to place barcode in the 3-UTR region and adjacent to poly-A. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Regrading claim 97, Zador and Hrvatin disclose (b) or (c) is performed ex vivo. (e.g. barcode mRNA is obtained from dissected tissue region outside of live subject for sequencing [Zador ¶0134, Hrvatin ¶0256])
Regrading claims 98 and 99, Zador and Hrvatin disclose the nonhuman animal is a mouse.[Zador ¶0008, Hrvatin ¶0009]
Regrading claims 100 and 101, Zador and Hrvatin disclose the nucleic acid encoding the mRNA transcript is introduced into the cell with adeno-associated virus vector. [Zador ¶0047, Hrvatin Fig. 2A]
Regrading claims 102 and 103 Zador and Hrvatin disclose mRNA transcript comprises a coding sequence encoding a fluorescent protein or a bioluminescent protein. (e.g. viral vector comprising GFP [Zador Fig. 3A; Hrvatin Figs. 1A and 2A]
Regrading claim 104, Zador and Hrvatin discloses imaging the fluorescent protein or the bioluminescent protein, wherein a location of the cell expressing the fluorescent protein or the bioluminescent protein is determined from the imaging.[ Zador Fig. 17b,c and ¶0026; Hrvatin Fig. 3a-d and ¶0015].
Regrading claim 105, the limitation “mapping the location of the cell expressing the fluorescent protein or the bioluminescent protein onto a reference image of the intact tissue.” Is interpreted broadly and reasonably to encompass overlaying/merging the image of GFP expressing cells with image of tissue that provide anatomical or cellular reference context, including DAPI-stained image. Under this interpretation, Hrvatin disclose merging GFP expressing cells and reference tissue stained with DAPI [Hrvatin Figs. 3a-d, 10, 17, and 30A]
Regrading claim 106, Zador and Hrvatin disclose cell is a neuron. [Zador abstract; Hrvatin ¶0009]
Regrading claims 115 and 116, Hrvatin discloses the measuring morphological or functional characteristics comprises performing confocal fluorescence microscopy. (e.g. Sections were imaged on a Leica™ SPE confocal microscope using an ACS APO 10×/0.30 CS objective. [¶0265 and 0370])
Regrading claim 117, Hrvatin discloses measuring morphological or functional characteristics comprises performing an electrophysiology measurement. (e.g. FIG. 3A-3M is a series of images and graphs showing hit confirmation and electrophysiology. [¶0015])
Zador et al., Hrvatin et al., and Wang et al.
Claim(s) 107-110, 112-114, and 119-121 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zador et al. (US12031127B2, EFD: April 2017) in view of Hrvatin et al (e.g. US20220025398A1, EFD: December 2019) and Wang et al. (WO2019199579A1, Published: April 2019).
Regrading claim 107, Zador suggested performing in situ gene sequencing of a target nucleic acid [0190], but does not go in details.
Wang discloses methods and systems for in situ gene sequencing of a target nucleic acid in a cell in an intact tissue [abstract].
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to modify Zador and Hrvatin’s method to perform the Wang’s in situ sequencing because Zador already suggested in situ sequencing, Wang discloses in detail a known in situ gene sequencing of target nucleic acids directly in cells in intact tissue. Accordingly, applying Wang’s sequencing method to Hrvatin’s barcoded viral transcript system would have predictably allowed the expressed barcode and/or other target nucleic acids to be sequenced while maintaining spatial context. Thus, providing more detailed nucleic acid profile from same type of mouse brain tissue. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Regrading claim 108, Zador discloses cell barcode is used for measuring morphological or functional characteristics of the cell. (e.g. barcode mRNA is sequenced and barcode abundance in the target areas is converted to a matrix of single neuron projection patterns [¶0134- 0139]. Projection patterns read on morphological characteristics because it describes the physical/anatomical organization of the neuron’s axonal processes and their target regions.[¶0005]).
Regrading claim 109, Hrvatin discloses removing a tissue comprising the cell from the subject prior to performing gene sequencing on the tissue. [¶0153]. In view of Wang, It is obvious to remove tissue from subject prior to performing in situ gene sequencing.
Regrading claim 110 and 112-114, Wang discloses performing in situ gene sequencing comprises: (a) contacting the tissue with at least a pair of oligonucleotide primers under conditions to allow for specific hybridization, wherein the pair of primers comprise a first oligonucleotide and a second oligonucleotide; wherein each of the first oligonucleotide and the second oligonucleotide comprises a first complementarity region, a second complementarity region sequence, and a third complementarity region; wherein the second oligonucleotide further comprises a barcode sequence; wherein the first complementarity region of the first oligonucleotide is complementary to a first portion of the target nucleic acid, wherein the second complementarity region of the first oligonucleotide is complementary to the first complementarity region of the second oligonucleotide, wherein the third complementarity region of the first oligonucleotide is complementary to the third complementarity region of the second oligonucleotide, wherein the second complementary region of the second oligonucleotide is complementary to a second portion of the target nucleic acid, wherein the first portion of the target nucleic is adjacent to the second portion of the target nucleic acid; [¶0004]
(b) adding ligase to ligate the second oligonucleotide and generate a closed nucleic acid circle; [¶0004]
(c) performing rolling circle amplification in the presence of a nucleic acid molecule, wherein the performing comprises using the second oligonucleotide as a template and the first oligonucleotide as a primer for a polymerase to form one or more amplicons; [¶0004]
(d) embedding the one or more amplicons in the presence of hydrogel subunits to form one or more hydrogel- embedded amplicons; [¶0004]
(e) contacting the one or more hydrogel-embedded amplicons having the barcode sequence with a pair of primers under conditions to allow for ligation, wherein the pair of primers comprise a third oligonucleotide and a fourth oligonucleotide, wherein the ligation only occurs when both the third oligonucleotide and the fourth oligonucleotide ligate to the same amplicon; [¶0004]
(f) reiterating step (e); [¶0004]
(g) imaging the one or more hydrogel-embedded amplicons to determine in situ gene sequencing of the target nucleic acid in the cell in the intact tissue. [¶0004]
Regrading claims 119 and 120, Zador and Hrvatin disclose target nucleic acid is viral RNA or viral DNA. (e.g. both references introduced viral barcoded DNA into neurons [Zador ¶0009; ¶Hrvatin 0013]
Regrading claim 121, the limitation “the target nucleic acid is endogenous to the cell.” Is interpreted as native cellular nucleic acids, including nucleic acids in transcriptome of cells being sequences in in situ sequencing, consistent with applicant specification [¶0062]. Wang discloses this limitation (e.g. the disclosed method of in situ transcriptomic sequencing can effectively link imaging-based molecular information with anatomical and activity information, thus elucidating brain function and dysfunction. [¶00131])
Zador et al., Hrvatin et al., Wang et al., and Mir et al.
Claim(s) 111 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zador et al. (US12031127B2, EFD: April 2017) in view of Hrvatin et al (e.g. US20220025398A1, EFD: December 2019), Wang et al. (WO2019199579A1, Published: April 2019) and Mir et al. (US11066701B2, EFD: May 2018).
Regarding claim 111, Zador, Hrvatin, and Wang do not disclose imaging is performed in presence of an antifade buffer comprising an antioxidant.
Mir discloses kit for sequencing comprising anti-fade comprising antioxidants. [¶0319]
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to include Mir’s antioxidants in imaging process of Zador and Hrvatin because Mir explicitly explains that buffers with antioxidants preserve signal stability, hence nucleotide remains detectable for a required period, before it is removed directly or indirectly by the action of light or a voltage applied to an electrode [¶0402]. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Zador et al., Hrvatin et al., and Meyer et al.
Claim(s) 118 is/are rejected under 35 U.S.C. 103 as being unpatentable over Zador et al. (US12031127B2, EFD: April 2017) in view of Hrvatin et al (e.g. US20220025398A1, EFD: December 2019) and Meyer et al. (US10656158B2, EFD: March 2018).
Regarding claim 118, Zador and Hrvatin do not disclose measuring morphological or functional
characteristics comprises performing an enzymatic assay.
Meyer discloses analyzing enzyme activity within a cell by monitoring localization or diffusion state of a tag enzyme using fluorescence imaging. The reference further discloses using a label-free biosensor-based cellular assay to assess enzyme activity.[abstract]
As of the application’ s effective filing date, it would have been prima facie obvious to a person of ordinary skill in the art to include Meyer’s enzymatic assay as the functional characteristic measurement in Zador and Hrvatin method because Hrvatin’s barcoded cell system is directed to associating barcode with cellular characteristics in order to functionally distinguish the populations of neurons that are intermingled within a small region [¶0005], Meyer discloses enzyme activity can be measured at cellular level using fluorescence imaging. Because enzyme activity is a known indicator of cellular biochemical function. Monitoring enzyme activity would have predictably expanded the measurable cellular characteristics from reporter expression to biochemical functional state, while still allow the barcode sequencing to identify assayed cells. See KSR International Co. v. Teleflex Inc., 550 U.S. 398, 415-421, USPQ2d 1385, 1395 — 97 (2007) (MPEP § 2143).
Conclusion
No claims are allowed
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Khai Quynh Tien Pham whose telephone number is (571)272-6998. The examiner can normally be reached M-T, 9-4 ET.
Examiner interviews are available via telephone, in-person, and video conferencing using a USPTO supplied web-based collaboration tool. To schedule an interview, applicant is encouraged to use the USPTO Automated Interview Request (AIR) at http://www.uspto.gov/interviewpractice.
If attempts to reach the examiner by telephone are unsuccessful, the examiner’s supervisor, Heather Calamita can be reached at (571) 272-2876. The fax phone number for the organization where this application or proceeding is assigned is 571-273-8300.
Information regarding the status of published or unpublished applications may be obtained from Patent Center. Unpublished application information in Patent Center is available to registered users. To file and manage patent submissions in Patent Center, visit: https://patentcenter.uspto.gov. Visit https://www.uspto.gov/patents/apply/patent-center for more information about Patent Center and https://www.uspto.gov/patents/docx for information about filing in DOCX format. For additional questions, contact the Electronic Business Center (EBC) at 866-217-9197 (toll-free). If you would like assistance from a USPTO Customer Service Representative, call 800-786-9199 (IN USA OR CANADA) or 571-272-1000.
/KHAI QUYNH TIEN PHAM/ Examiner, Art Unit 1684
/JEREMY C FLINDERS/ Primary Examiner, Art Unit 1684
Prosecution Insights