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 .
Claim Status
Claims 1-2, 4-18, and 20-25 are currently pending and under examination.
Priority
The instant application is the National Stage entry of PCT/US2019/051827, International Filing Date: 09/19/2019, which claims priority to US Provisional Application 62/734,063, filed 09/20/2018.
Accordingly, the effective filing date of the claimed invention is 09/20/2018.
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.
The factual inquiries for establishing a background for determining obviousness under 35 U.S.C. 103 are summarized as follows:
1. Determining the scope and contents of the prior art.
2. Ascertaining the differences between the prior art and the claims at issue.
3. Resolving the level of ordinary skill in the pertinent art.
4. Considering objective evidence present in the application indicating obviousness or nonobviousness.
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.
Claim(s) 1-2, 4-14, 17-18, 20-22, and 24-25 are rejected under 35 U.S.C. 103 as being unpatentable over Donnelly et al. (WO 2018165293 A1), in view of Xuemei Zhang et al. (RNA stores tau reversibly in complex coacervates, PLOS Biology, July/05/2017, pages 1-28), and further in view of Shin et al. (Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets, 2017 January 12, published in: Cell; 168(1-2): 159–171.e14) previously cites on the IDS dated 09/08/2021, and further in view of Young et al. (US20220120736A1).
Regarding claim 1, Donnelly discloses a high-throughput method for mapping or screening intracellular interactions (is a method of inducing a neurodegenerative disease pathology in a cell, comprising the steps: introducing into the cell an expression vector encoding a chimeric polypeptide, comprising: a first nucleotide sequence encoding a light induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein, wherein the first nucleotide sequence is operably linked to a promoter; expressing the chimeric polypeptide; and inducing oligomerization of the chimeric polypeptide by stimulation with blue light (abstract); aggregation is measured by simultaneous chronic blue light exposure and high-throughput automated confocal microscopy (pg. 5, Ls. 18-19)).
Donnelly further discloses providing a plurality of cells (All constructs were fused to a fluorescent protein called mCherry (mCh) to visualize the proteins in live cells (pg. 5, Ls. 23-24)), each cell expressing a phase separation or aggregation system capable of being controlled by at least one wavelength of light (light-induced aggregation of low-complexity domain proteins. Optogenetic TDP-43 fragments containing the low-complexity domain (LCD) undergo 5 progressive aggregations with light stimulation (pg. 5, Ls. 3-20; FIGS. 4A-4E, 5A-5K)) the phase separation or aggregation system comprising a target protein and a fluorescent protein or attached fluorophore introducing into the cell an expression vector encoding a chimeric polypeptide, comprising: a first nucleotide sequence encoding a light induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein, wherein the first nucleotide sequence is operably linked to a promoter; expressing the chimeric polypeptide; and inducing oligomerization of the chimeric polypeptide by stimulation with blue light (abstract).
Donnelly further discloses developing various protein arrangements and blue light exposure paradigms to induce protein aggregation of proteins and protein fragments containing LCD/IDR/prion-like domains (for example, cognate partner), promote the mislocalization of nuclear proteins and recapitulate the neuropathology of neurodegenerative diseases. TDP-43 is a predominantly nuclear protein that contains an IDR/IDD/prion-like domain, and is mislocalized and aggregated in ALS, FTD, and some AD patients, as an example. DNA sequences were engineered to generate amino acid sequences that encode for a the CRY20LIG protein (for example, light-sensitive receptor protein) that clusters when exposed to blue light generated a fusion protein either the entire TDP-43 protein (for example, equivalent of enzymatically dead Cas9 )( (cry2-TDP43-mCh) or just the low complexity domain (LCD/IDD/prion-like domain) (for example, cognate partner), which makes TDP-43 aggregation prone (Cry2-274-mCh). As a control, CRY20LIG alone was used. All constructs were fused to a fluorescent protein called mCherry (mCh) (for example, reporter) to visualize the proteins in live cells. CRY20LIG-mCh reversibly clusters with blue light stimulation but the CRY20LIG-TDP-43-mCh or the truncated CRY20LIG-274-mCh forms irreversible aggregates with specific blue light stimulation paradigms. (pg. 4, Ls. 14-26, FIG. 2). Donnelly further discloses blue light induced oligomerization and aggregation of proteins employing the NcVVD, NcVVD Y50W or NcLOV photoreceptor (for example, light sensitive receptor protein). Donnelly further discloses that the NcVVD or LOV domain have only been shown to homodimerize with blue light stimulation (for example, the cognate partner). A light exposure paradigm to induce oligomerization and aggregation was developed. The top panel shows a schematic of how a single acute stimulation with blue light (405-499 nm) induces the homodimerization of the LOV protein when fused to a protein of interest. The bottom panel shows that chronic stimulation with blue light promotes the homooligomerization of NcVVD (for example, cognate partner equivalent) or LOV fusion proteins that contain a prion-like domain/LCD/IDD (for example, equivalent of enzymatically dead Cas9) (pg. 4, last para., FIG. 3). Donnelly further discloses light-induced aggregation of low-complexity domain proteins. Optogenetic TDP-43 fragments containing the low-complexity domain (LCD) undergos progressive aggregation with light stimulation (for example, equivalent of enzymatically dead Cas9) (pg. 4, last para., FIG. 3). (FIGS. 4A-4B) C-terminal fragments of TDP-43 (optoRRM2+LCD/optoLCD) rapidly oligomerize after a brief pulse (8 sec, 10% laser power, 488nm) of blue light (pg. 5, first para., FIG 4).
Donnelly further discloses introducing at least one chemical or biological agent to the well at a first concentration, the well is maintained at a predetermined temperature (a method of screening for an agent that modulates protein aggregation, … introducing the agent into a culture media comprising the cell (pg. 27, Ls. 5-14)).
Donnelly further discloses irradiating the well with the at least one wavelength of light, in a constant or pulsed fashion, and allowing the phase separation or aggregation system ((FIGS. 4A-4B) fragments of TDP-43 rapidly oligomerize after a brief pulse of blue light when monitored by live confocal microscopy. (FIGS. 4C-4D) Optogenetic LCD fragments also continue to aggregate following brief light pulses, growing in size over time. (FIG. 4E) Persistent blue light stimulation of the TDP-43 LCD forms intracellular aggregates in the cells (pg. 5, Ls. 3-9); The methods can include various degrees of blue light stimulation. In some embodiments, the stimulation is acute or, optionally, chronic. Acute stimulation refers stimulation with pulses of blue light from about 0.2 to about 60 seconds (pg. 29, Ls. 18-20)).
Donnelly further discloses irradiating at least one of the plurality of cells with an additional wavelength of light to cause the fluorescent protein or fluorophore to fluoresce ((FIG. 4E) Persistent blue light stimulation of the TDP-43 LCD forms intracellular aggregates in the cells (pg. 5, Ls. 3-9); (FIG. 5E) Fluorescence recovery after photobleaching (FRAP) imaging was performed to assess dynamicity of optoTDP43 structures (pg. 5, Ls. 3-9)).
Donnelly further discloses quantifying phase separation or aggregation based on an amount of fluorescence within a first region and a second region of the plurality of cells, thereby generating a quantified phase separation or aggregation, the first region containing and the second region not containing ((FIG. 5E) Fluorescence recovery after photobleaching (FRAP) imaging was performed to assess dynamicity of optoTDP43 structures (pg. 5, Ls. 3-9); (FIG. 5G) To confirm direct recruitment of non-optogenetic TDP-43 species to exogenous light-induced optoTDP43 inclusions, EGFP-tagged TDP-43 was co-expressed with optoTDP43 or the Cry2 photoreceptor-only control (pg. 5, Ls. 30-35); Figure 6A shows the chronic stimulation paradigm and Figure 6B shows representative images of a-synuclein clustering with light over time along with quantification of clustering in Figure 6C (pg. 6, Ls. 18-20)). Donnelly further discloses quantification of aggregation using light induced oligomerization leading to insoluble intracellular aggregates that do not recover in FRAP ((FIG. 4A-4E) pg. 5, para. 1).
Donnelly further discloses generating a phase diagram utilizing the quantified phase separation or aggregation (FIGS. 5A-5K show chronic blue light stimulation induces optoTDP43 mislocalization and aggregation that recapitulates pathological hallmarks seen in patient CNS tissue. HEK293 cells expressing optoTDP43-mCh were exposed to 488 nm LED stimulation or darkness for up to 36 hours. (FIG. 5A-5C) Representative images showing optoTDP43 that (FIG. 5B) first experiences gradual cytoplasmic mislocalization, (FIG. 5C) which was confirmed by nuclear/cytoplasmic fractionation. (FIG. 5D) Mislocalization is followed by optoTDP43 aggregation, as measured by simultaneous chronic blue light exposure and high-throughput automated confocal microscopy, that increases in propensity with increasing light exposure (pg. 7, ls. 13-20)).
Further regarding claim 1, Donnelly discloses clustering quantification in FIG 6C and FIG. 6D shows that α-synuclein fused to the LOV (dimerizing photoreceptor) forms intracellular clusters of alpha synuclein compared to unstimulated image (for example a control). FIG. 6E shows that light induced α-synuclein LOV aggregates exhibit pathological hallmarks of synucleinopathies including phosphorylation at serine 129 and p62 positivity. These tau tangles are pathological hallmarks of AD, FTD and CTE in HEK cells. These also exhibit pathological hallmarks of Tauopathy observed in patients including hyperphosphorylation using specific antibodies developed against pathological tau inclusions (FIGS. 7-8) (pg. 37, Lns. 10-15, pg. 6, ln. 16-25). Also, see using CRY2OLIG as a control (pg. 4, ln. 23) (for example, comparing the quantified phase separation or aggregation to a control).
Further regarding claim 1, Donnelly discloses equivalents of enzymatically dead Cas 9. Young discloses methods of detecting, visualizing, condensates, e.g., transcriptional condensates (abstract). Young further discloses that a catalytically inactive site-specific nuclease and an effector domain capable of attaching a DNA, RNA, or protein to the nucleotide sequence is used. In some embodiments, the catalytically inactive site-specific nuclease dCas (e.g., dCas9 or Cpfl) homing endonuclease is used [0103] [0241-0246].
Further regarding claim 1, Donnelly does not expressly disclose placing at least one of the plurality of cells in a well at a first temperature and maintaining the well at a predetermined temperature. Further Donnelly does not expressly disclose determining a binodal or spinodal boundaries and identifying a difference between phase diagrams.
Xuemei Zhang discloses a method of demonstrating physicochemical properties of tau that may predispose it to undergo changes associated with neurodegenerative disease (Authors Summary, pg. 2 first para.), where Human embryonic kidney (HEK) 293Tcells expressing wild-type, full-length human tau (4R2N), mutant tau (P301L-4R2N), or mutant tau (for example, target protein) in a different isoform fused to cyan fluorescent protein (for example, fluorescence protein)(CFP; P301L-4R1N) were crosslinked.
Xuemei Zhang further discloses using eight samples, culturing and well plating them (pg. 15, Material and Methods). Xuemei Zhang further discloses that PAR-iCLIP was applied to detect specific RNAs bound to tau in living cells [30, 32]. Cells were treated with 4-thiouridine (4SU) (Sigma-Aldrich, St. Louis, MO) for 1 hour at a final concentration of 500 MM at 37°C (for example, first temperature), rinsed with ice-cold 1x PBS and irradiated one time with 400 mJ/cm2 of 365 nm UV light on ice (pg. 16, PAR-iCLIP).
Xuemei Zhang further discloses that continuous wave electron paramagnetic resonance (cw EPR) spectra obtained at room temperature (temperature was maintained at room temperature) of 500 μM Δtau187-SL in droplets formed with 1.5 mg/ml poly (A) RNA (blue in A) and 1.5 mg/ml tRNA (green in B) is unaltered from solution before adding RNA (red in A and B). Cw EPR line shape upon adding 125 μM heparin (for example, a chemical or biological agent) show dramatic line broadening (Fig. 6).
Shin discloses optoDroplets method which utilizes an optogenetic platform to achieve precise spatiotemporal control over phase transitions within living cells by fusing intrinsically disordered protein regions (IDRs) to the light-sensitive protein Cryptochrome 2 (Cry2). Shin further discloses condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1, where above a concentration threshold, these constructs undergo light-activated phase separation, forming spatiotemporally definable liquid optoDroplets.
Shin further discloses quantifying phase separation or aggregation and generating a phase diagram and determining the binodal and spinodal phase boundaries (pg. 29; FIG. 3 A-G).
Shin further discloses quantification and statistical analysis, where total concentrations of molecules as well as steady-state background concentrations (for example, subtracting any signal that does not originate from the specifically labeled molecule of interest/identifying a difference between phase diagram in step g) and a phase diagram without a biological or chemical agent) outside clusters were measured from fluorescence images of cells using ImageJ (NIH), and corrected by subtracting background noises measured with areas absent from any cells.
Regarding claim 2, Donnelly discloses monitoring optogenetic TDP-43 fragments containing the low-complexity domain (LCD) undergoing progressive aggregation with light stimulation by live confocal microscopy (pg. 7, para.1; (FIGS. 4A-4B)).
Donnelly does not expressly disclose staining and fixing of cells. Xuemei Zhang discloses that After gel separation, tRNA was stained with SYBR Gold II (Thermo Fisher, Waltham, MA). For quantitative analysis, the fraction of free and bound tRNA was quantified in ImageJ2 (pg. 16, RNA gel mobility shift assay). Xuemei Zhang further discloses UV Cross-Linking ImmunoPrecipitation (for example, fixing cells) (pg. 3, Results).
Regarding claim 4, Donnelly discloses a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein, wherein the first nucleotide sequence is operably linked to a promoter; expressing the chimeric polypeptide; and inducing oligomerization of the chimeric polypeptide by stimulation with blue light (abstract). Donnelly further discloses that the target protein comprises at least one gene regulatory protein (pg. 17, para. 3: In one embodiment, the low complexity domain from a neurodegenerative disease target protein is selected from the group consisting of TDP-43, Alpha synuclein, Tau, Fus, TIAl, SODl, Huntingtin, Ataxin 2, and hnRNP A2B 1, TDP-43 …); reading on limitations of the cell is configured to express a phase separation or aggregation system comprising: a first construct comprising enzymatically dead Cas9 fused or attached to two or more repeating sequences, each repeating sequence including a receptor protein sensitive to at least one wavelength of light, and a second construct comprising a cognate partner of the light-sensitive receptor protein fused to at least one fluorescent protein and at least one gene regulatory protein having a full length or truncated low complexity or intrinsically-disordered protein region, or other folded proteins known to promote at least one of self-interactions, a network of heterotypic (non-self) interactions, or phase separation, and wherein the target protein comprises the at least one gene regulatory protein.
Donnelly discloses DNA sequences were engineered to generate amino acid sequences that encode for a the CRY2OLIG protein that clusters when exposed to blue light generated a fusion protein either the entire TDP-43 protein (cry2-TDP43-mCh) or just the low complexity domain (LCD/IDD/prion-like domain), which makes TDP-43 aggregation prone (Cry2-274-mCh). As a control, CRY2OLIG alone was used. All constructs were fused to a fluorescent protein called mCherry (mCh) to visualize the proteins in live cells. CRY2OLIG-mCh reversibly clusters with blue light stimulation but the CRY2OLIG-TDP-43-mCh or the truncated CRY2OLIG-274-mCh forms irreversible aggregates with specific blue light stimulation paradigms (pg. 6, Ls. 19-26; FIGS. 2A-2B); (FIG. 7D) Summary showing the various optogenetic Tau constructs that colocalize with pathological Tau antibodies after light-induction of neurofibrillary tangle formation (pg.8, Ls. 31-32).
Donnelly further discloses the expression cassettes (constructs) comprising promoter (repeating sequences) operably linked to a second nucleic acid (e.g., polynucleotide) may include a promoter that is heterologous to the second nucleic acid (e.g. polynucleotide) as the result of human manipulation. The expression cassette may comprise an endogenous promoter (pg. 13, Ls.13-16). See also the chimeric constructs: In one aspect, disclosed herein is a nucleotide sequence encoding a chimeric polypeptide, comprising: a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein (pg. 16). See also, developing various protein arrangements and blue light exposure paradigms in pages 3-4 and FIGs 2-4.
Further regarding claim 4, Donnelly and Xuemei Zhang do not expressly disclose that a first construct comprising enzymatically dead Cas9 fused or attached to two or more repeating sequences.
However, Young discloses methods of detecting, visualizing, condensates, e.g., transcriptional condensates (abstract).
Young further discloses that a catalytically inactive site-specific nuclease and an effector domain capable of attaching a DNA, RNA, or protein to the nucleotide sequence is used. In some embodiments, the catalytically inactive site-specific nuclease dCas (e.g., dCas9 or Cpfl) homing endonuclease is used [0103] [0241-0246].
Regarding claims 5 and 6, Donnelly discloses that the well is irradiated with the at least one wavelength of light, in continuous or pulsed fashion, before at least one chemical or biological agent is introduced to the well (The methods can include various degrees of blue light stimulation. In some embodiments, the stimulation is acute or, optionally, chronic. Acute stimulation refers stimulation with pulses of blue light from about 0.2 to about 60 seconds, wherein the wavelength of the blue light can be any herein disclosed blue light wavelength (pg. 32, Ls. 18-21)).
Donnelly further discloses introducing at least one chemical or biological agent to the well at a first concentration (synthetic oligonucleotide adaptors or linkers (pg. 15, L. 27)).
Regarding claims 7 and 8, Xuemei Zhang discloses using neuronal induction media containing Knockout DMEM F12 with N2 (pg. 15. Materials and methods).
Donnelly and Xuemei Zhang do not expressly disclose that at least one chemical or biological agent is a component of a genetic knockout or knockdown screening system from the group consisting of TALEN, shRNA, siRNA and CRISPR-KO.
However, Young discloses Nucleic acid modifications, non-standard nucleotides or nucleosides, etc., such as those known in the art as being useful in the context of RNA interference (RNAi), aptamer, CRISPR technology, polypeptide production, reprogramming, or antisense-based molecules for research or therapeutic purposes may be incorporated in various embodiments … Various non-limiting examples of nucleic acid modifications include Chemical modification of siRNA [0195].
Regarding claim 9, Donnelly discloses capturing an image of the at least one of the plurality of cells while the fluorescent protein or attached fluorophore is fluorescing ((FIG. 5E) Fluorescence recovery after photobleaching (FRAP) imaging was performed to assess dynamicity of optoTDP43 structures).
Regarding claim 10, Xuemei Zhang discloses utilizing image processing software (NanoAnalyze v3.6 software) to analyze particle tracking data (pg. 18, ITC experiments).
Regarding claim 11, Xuemei Zhang discloses With illumination and focus optimized, droplets settling on the cover slide have lower intensity than their surrounding on the images. An image of the 20 mM ammonium acetate buffer was taken to calculate the average intensity to set as threshold in order to classify different parts of the image into droplets and buffer. For each image, the area of the droplets was divided by the total area of the image, generating a percent droplet coverage value on the cover slide. Droplets with eccentricity above 0.9 or equivalent diameter below 1μm were filtered out in order to reduce false reading (pg. 19, last para.).
Regarding claim 12, Xuemei Zhang discloses data about tau and RNA forms droplet in vitro and discloses using standard deviation from n = 3 in a-b, e-f (pg. 23, S6 Fig., Fig. 5).
Regarding claim 13, Xuemei Zhang discloses an image of the 20 mM ammonium acetate buffer was taken to calculate the average intensity to set as threshold in order to classify different parts of the image into droplets and buffer. For each image, the area of the droplets was divided by the total area of the image, generating a percent droplet coverage value on the cover slide. Droplets with eccentricity above 0.9 or equivalent diameter below 1μm were filtered out in order to reduce false reading (pg. 19 last para.).
Regarding claim 14, Donnelly discloses toxicity associated with assay conditions is determined by examining a metric comprised of the number of viable cells detected within each well, by image analysis algorithms ((FIG. SK) Automated high-throughput confocal microscopy was performed to assess the neurotoxicity of light-induced optoTDP43 inclusions. Human ReN neurons expressing TDP43- mCh or optoTDP43 were exposed to chronic blue light stimulation and viability was simultaneously monitored by longitudinal imaging (pg. 10, Ls. 7-11; FIG. 10B)).
Regarding claim 17, Donnelly discloses that steps d-f are repeated under different conditions selected from the group consisting of different light conditions, and different temperature conditions (Chronic stimulation is defined by exposure to blue light having a wavelength from about 400 nm to about 500 nm for a duration of about 1 minute or longer (for example, at least 1 minute, at least 5 minutes, at least 10 minutes, at least 30 minutes, at least 60 minutes, at least 2 hours, at least 4 hours, at least 8 hours, at least 12 hours, at least 24 hours, at least 36 hours, or more) from about 0.1 mW/cm2 to 8 mW/cm2 (within 400 nm - 500 nm wavelength) (pg. 32, LS. 5-10); Light exposure then forces these unique fusion protein arrangements into close proximity and employing chronic or repeated light stimulation, neurodegenerative disease aggregate pathologies of full-length proteins or the LCDs alone can be obtained (pg. 38, Ls. 17-19)).
Regarding claim 18, Donnelly disclose a time-dependent transition into an irreversible aggregation state is monitored by changing the time that the cells are subject to constant or pulsed activating light (All constructs were fused to a fluorescent protein called mCherry (mCh) to visualize the proteins in live cells. CRY2OLIG-mCh reversibly clusters with blue light stimulation but the CRY2OLIG-TDP-43-mCh or the truncated CRY2OLIG-274-mCh forms irreversible aggregates with specific blue light stimulation paradigms. FIG. 2A shows examples of the CRY2OLIG-TDP-43 arrangements and truncated CRY2OLIG-274 arrangements used in this work (pg. 3, Ls. 23-28); (FIGS. 4C-4D) Optogenetic LCD fragments also continue to aggregate following brief light pulses, growing in size over time (FIGs 4A-4E; pg.7, 3-9).
Regarding claims 20 and 21, Donnelly disclose further comprising calculation a difference between the quantified phase separation or aggregation and a baseline phase separation or aggregation (To confirm direct recruitment of non-optogenetic TDP-43 species to exogenous light-induced optoTDP43 inclusions, EGFP-tagged TDP-43 was co-expressed with optoTDP43 or the Cry2 photoreceptor-only control (a control is used for comparison purposes). In cells exposed to chronic blue light stimulation, strong co- localization is observed between optoTDP43 inclusions and EGFP-TDP43, confirming the ability of optoTDP43 inclusions to directly recruit other TDP-43 species (pg. 5, Ls.32-35, pg. 6, Ls. 1-2)).
Additionally, Xuemei Zhang discloses tRNA abundance in CLIP samples vs total small RNA controls in HEK cells (a) and hiPSCderived neurons (b) indicate that tRNA distributions differ between the total tRNA pool and the CLIP tRNA pool. Specifically, the population above the diagonal is much greater, indicating that the tRNA abundance from PAR-iCLIP samples from tau-bound RNA is significantly greater compared to the total small RNA present in HEK or hiPSC neuron cells. (c) Top 10 ranked tRNAs bound by tau in HEK cell and hiPSC-derived neurons (pg. 22, S3 Fig.).
Regarding claim 22, Xuemei Zhang discloses Control experiments with GFP or CDK5 antibody for the lysate expressing those proteins, or with HJ 8.5 antibody for lysates without expressing tau were always done in parallel to rule out false-positive binding caused by the beads (pg. 16, PAR-iCLIP). Xuemei Zhang further discloses a method of droplet quantification from image analysis where they filter out droplets of specific size in order to reduce false readings (pg. 19, last para.).
Regarding claims 24 and 25, Donnelly discloses a high-throughput method for mapping or screening intracellular interactions (is a method of inducing a neurodegenerative disease pathology in a cell, comprising the steps: introducing into the cell an expression vector encoding a chimeric polypeptide, comprising: a first nucleotide sequence encoding a light induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein, wherein the first nucleotide sequence is operably linked to a promoter; expressing the chimeric polypeptide; and inducing oligomerization of the chimeric polypeptide by stimulation with blue light (abstract); aggregation is measured by simultaneous chronic blue light exposure and high-throughput automated confocal microscopy (pg. 5, Ls. 18-19)).
Donnelly further discloses providing a plurality of cells (All constructs were fused to a fluorescent protein called mCherry (mCh) to visualize the proteins in live cells (pg. 5, Ls. 23-24)), each cell expressing a phase separation or aggregation system capable of being controlled by at least one wavelength of light (light-induced aggregation of low-complexity domain proteins. Optogenetic TDP-43 fragments containing the low-complexity domain (LCD) undergo 5 progressive aggregations with light stimulation (pg. 5, Ls. 3-20; FIGS. 4A-4E, 5A-5K)) the phase separation or aggregation system comprising a target protein and a fluorescent protein or attached fluorophore introducing into the cell an expression vector encoding a chimeric polypeptide, comprising: a first nucleotide sequence encoding a light induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein, wherein the first nucleotide sequence is operably linked to a promoter; expressing the chimeric polypeptide; and inducing oligomerization of the chimeric polypeptide by stimulation with blue light (abstract).
Donnelly further discloses developing various protein arrangements and blue light exposure paradigms to induce protein aggregation of proteins and protein fragments containing LCD/IDR/prion-like domains (for example, cognate partner), promote the mislocalization of nuclear proteins and recapitulate the neuropathology of neurodegenerative diseases. TDP-43 is a predominantly nuclear protein that contains an IDR/IDD/prion-like domain, and is mislocalized and aggregated in ALS, FTD, and some AD patients, as an example. DNA sequences were engineered to generate amino acid sequences that encode for a the CRY20LIG protein (for example, light-sensitive receptor protein) that clusters when exposed to blue light generated a fusion protein either the entire TDP-43 protein (for example, equivalent of enzymatically dead Cas9 )( (cry2-TDP43-mCh) or just the low complexity domain (LCD/IDD/prion-like domain) (for example, cognate partner), which makes TDP-43 aggregation prone (Cry2-274-mCh). As a control, CRY20LIG alone was used. All constructs were fused to a fluorescent protein called mCherry (mCh) (for example, reporter) to visualize the proteins in live cells. CRY20LIG-mCh reversibly clusters with blue light stimulation but the CRY20LIG-TDP-43-mCh or the truncated CRY20LIG-274-mCh forms irreversible aggregates with specific blue light stimulation paradigms. (pg. 4, Ls. 14-26, FIG. 2). Donnelly further discloses blue light induced oligomerization and aggregation of proteins employing the NcVVD, NcVVD Y50W or NcLOV photoreceptor (for example, light sensitive receptor protein). Donnelly further discloses that the NcVVD or LOV domain have only been shown to homodimerize with blue light stimulation (for example, the cognate partner). A light exposure paradigm to induce oligomerization and aggregation was developed. The top panel shows a schematic of how a single acute stimulation with blue light (405-499 nm) induces the homodimerization of the LOV protein when fused to a protein of interest. The bottom panel shows that chronic stimulation with blue light promotes the homooligomerization of NcVVD (for example, cognate partner equivalent) or LOV fusion proteins that contain a prion-like domain/LCD/IDD (for example, equivalent of enzymatically dead Cas9) (pg. 4, last para., FIG. 3). Donnelly further discloses light-induced aggregation of low-complexity domain proteins. Optogenetic TDP-43 fragments containing the low-complexity domain (LCD) undergos progressive aggregation with light stimulation (for example, equivalent of enzymatically dead Cas9) (pg. 4, last para., FIG. 3). (FIGS. 4A-4B) C-terminal fragments of TDP-43 (optoRRM2+LCD/optoLCD) rapidly oligomerize after a brief pulse (8 sec, 10% laser power, 488nm) of blue light (pg. 5, first para., FIG 4).
Donnelly further discloses introducing at least one chemical or biological agent to the well at a first concentration, the well is maintained at a predetermined temperature (a method of screening for an agent that modulates protein aggregation, … introducing the agent into a culture media comprising the cell (pg. 27, Ls. 5-14)).
Donnelly further discloses irradiating the well with the at least one wavelength of light, in a constant or pulsed fashion, and allowing the phase separation or aggregation system ((FIGS. 4A-4B) fragments of TDP-43 rapidly oligomerize after a brief pulse of blue light when monitored by live confocal microscopy. (FIGS. 4C-4D) Optogenetic LCD fragments also continue to aggregate following brief light pulses, growing in size over time. (FIG. 4E) Persistent blue light stimulation of the TDP-43 LCD forms intracellular aggregates in the cells (pg. 5, Ls. 3-9); The methods can include various degrees of blue light stimulation. In some embodiments, the stimulation is acute or, optionally, chronic. Acute stimulation refers stimulation with pulses of blue light from about 0.2 to about 60 seconds (pg. 29, Ls. 18-20)).
Donnelly further discloses irradiating at least one of the plurality of cells with an additional wavelength of light to cause the fluorescent protein or an attached fluorophore to fluoresce ((FIG. 4E) Persistent blue light stimulation of the TDP-43 LCD forms intracellular aggregates in the cells (pg. 5, Ls. 3-9); (FIG. 5E) Fluorescence recovery after photobleaching (FRAP) imaging was performed to assess dynamicity of optoTDP43 structures (pg. 5, Ls. 3-9)).
Donnelly further discloses quantifying phase separation or aggregation based on an amount of fluorescence within a first region and a second region of the plurality of cells, the first region containing and the second region not containing (FIG. 5E) Fluorescence recovery after photobleaching (FRAP) imaging was performed to assess dynamicity of optoTDP43 structures (pg. 5, Ls. 3-9); (FIG. 5G) To confirm direct recruitment of non-optogenetic TDP-43 species to exogenous light-induced optoTDP43 inclusions, EGFP-tagged TDP-43 was co-expressed with optoTDP43 or the Cry2 photoreceptor-only control (pg. 5, Ls. 30-35); Figure 6A shows the chronic stimulation paradigm and Figure 6B shows representative images of a-synuclein clustering with light over time along with quantification of clustering in Figure 6C (pg. 6, Ls. 18-20)).
Xuemei Zhang discloses a method of demonstrating physicochemical properties of tau that may predispose it to undergo changes associated with neurodegenerative disease (Authors Summary, pg. 2 first para.), where Human embryonic kidney (HEK) 293Tcells expressing wild-type, full-length human tau (4R2N), mutant tau (P301L-4R2N), or mutant tau (for example, target protein) in a different isoform fused to cyan fluorescent protein (for example, fluorescence protein) (CFP; P301L-4R1N) were crosslinked.
Xuemei Zhang further discloses using eight samples, culturing and well plating them (pg. 15, Material and Methods). Xuemei Zhang further discloses that PAR-iCLIP was applied to detect specific RNAs bound to tau in living cells [30, 32]. Cells were treated with 4-thiouridine (4SU) (Sigma-Aldrich, St. Louis, MO) for 1 hour at a final concentration of 500 MM at 37°C (for example, first temperature), rinsed with ice-cold 1x PBS and irradiated one time with 400 mJ/cm2 of 365 nm UV light on ice (pg. 16, PAR-iCLIP).
Xuemei Zhang further discloses that continuous wave electron paramagnetic resonance (cw EPR) spectra obtained at room temperature (temperature was maintained at room temperature) of 500 μM Δtau187-SL in droplets formed with 1.5 mg/ml poly (A) RNA (blue in A) and 1.5 mg/ml tRNA (green in B) is unaltered from solution before adding RNA (red in A and B). Cw EPR line shape upon adding 125 μM heparin (for example, a chemical or biological agent) show dramatic line broadening (Fig. 6).
Donnelly further discloses that C-terminal fragments of TDP-43 (optoRRM2+LCD/optoLCD) rapidly oligomerize after a brief pulse (8 sec, 10% laser power, 488 nm) of blue light when monitored by live confocal microscopy (for example, determining phase boundary for phase separation/aggregation). These oligomers persist much longer than the Cry2 photoreceptor alone (˜10 min disassembly). (FIGS. 4C-4D) Optogenetic LCD fragments also continue to aggregate following brief light pulses, growing in size over time.
Additionally, Xuemei Zhang discloses Mixing of 4R2N or Δtau187 (similar to K18, see S1 Fig) with tRNA (25 kDa), poly(A) RNA (66~660 kDa), or poly(U) RNA (800~1000 kDa) reliably produced a turbid solution under a wide range of tau:RNA mass ratios and salt concentrations and that according to bright-field microscopy (Fig 4A), droplets formed and phase separated from the bulk aqueous phase with a clearly visible and highly spherical boundary. Tau droplets were capable of merging into a single droplet with the complete and nearly instantaneous loss of any boundary at the fusion interface, indicating that the droplets are fluidic with a relatively low interfacial tension (a series of snapshots capturing the fusion of 2 droplets are shown in Fig 4A). Confocal microscopy images of fluorescence-labeled tau verified that tau was predominantly contained within the droplet (Fig 4B, pg. 8, para. 2); reading on limitations of determining the phase boundary for the phase separation or aggregation system, and monitoring the location of these boundaries, in full or in part, under the action of at least one chemical or biological agent.
Donnelly further discloses that (FIG. 5D) mislocalization is followed by optoTDP43 aggregation, as measured by simultaneous chronic blue light exposure and high-throughput automated confocal microscopy, that increases in propensity with increasing light exposure (pg. 5, ln. 18-20).
Xuemei Zhang further discloses plotting the fraction of bound tRNA to 4R2N tau (from the low and high bands) as a function of tau: tRNA molar ratios (Fig 3E), and compared to theoretical binding saturation curves representing (see Fig 3F, pg. 6, last para.).
Xuemei Zhang further discloses that with a dissociation constant of 0.3 pM, HJ 8.5 pulled down tau under the high-stringency purification conditions of CLIP and remained bound to tau throughout the procedure (pg. 16, PAR-iCLIP). Xuemei Zhang further discloses methods for expression and purification of all recombinant tau variants that are detailed in S1 Text.
Shin discloses optoDroplets method which utilizes an optogenetic platform to achieve precise spatiotemporal control over phase transitions within living cells by fusing intrinsically disordered protein regions (IDRs) to the light-sensitive protein Cryptochrome 2 (Cry2). Shin further discloses condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1, where above a concentration threshold, these constructs undergo light-activated phase separation, forming spatiotemporally definable liquid optoDroplets.
Shin further discloses quantifying phase separation or aggregation and generating a phase diagram and determining the binodal and spinodal phase boundaries (pg. 29; FIG. 3 A-G).
Shin further discloses quantification and statistical analysis, where total concentrations of molecules as well as steady-state background concentrations (for example, subtracting any signal that does not originate from the specifically labeled molecule of interest/identifying a difference between phase diagram in step g) and a phase diagram without a biological or chemical agent) outside clusters were measured from fluorescence images of cells using ImageJ (NIH), and corrected by subtracting background noises measured with areas absent from any cells.
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Donnelly to have used temperature controls, as shown by Xuemei Zhang to better control phase behavior and protein formation. Further, it would have been obvious to one ordinary skilled in the art to have used the quantification and downstream statistical analysis as shown by Shin to better understand the different mechanisms by which a system separates into different phases. There would be a reasonable expectation of success in combining the technique of Xuemei Zhang and Shin to the method of Donnelly because they are all quantifying proteins from image analysis.
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Donnelly, Xuemei Zhang and Shin to have used enzymatically dead Cas9, as shown by Young to localize specific components to an existing condensate and precisely modulate gene expression without altering DNA sequence itself. There would be a reasonable expectation of success in combining the technique of Young to the method of Donnelly and Xuemei Zhang because they are all in the same field of quantifying proteins by modulating condensates from image analysis.
Response to Applicant Remarks
Applicant's arguments filed 12/24/2025 have been fully considered but they are not persuasive.
Applicant states:
claims 1, 24, and 25 are not obvious over the combination of Donnelly, Xuemei Zhang, and Shin, as none of the references alone or in combination teach or disclose, every element of the claim, at least because none of the references disclose a phase separation or aggregation system comprising (i) a target protein; and (ii) a fluorescent protein or fluorophore; (iii) an enzymatically dead Cas9; (iv) a receptor protein sensitive to the at least one wavelength of light; and (v) a cognate partner of the receptor protein..
It is respectfully submitted that this is not persuasive. Applicants’ amendments necessitated a new round of art rejection. As such, the combination of Donnelly, Xuemei Zhang, Shin and Young disclose all limitations of instant claims 1-2, 4-14, 17-18, 20-22, and 24-25.
Claim(s) 15-16 and 23 is/are rejected under 35 U.S.C. 103 as being unpatentable over Donnelly et al. (WO 2018165293 A1), in view of Xuemei Zhang et al. (RNA stores tau reversibly in complex coacervates, PLOS Biology, July/05/2017, pages 1-28), and further in view of Shin et al. (Spatiotemporal Control of Intracellular Phase Transitions Using Light-Activated optoDroplets, 2017 January 12, published in: Cell; 168(1-2): 159–171.e14) previously cites on the IDS dated 09/08/2021, and further in view of Young et al. (US20220120736A1), as applied to claims 1-2, 4-14, 17-18, 20-22, and 24-25 above, and further in view of Greef et al. (US 20180264464 A1).
Claims 15-16 depend from claim 9. The limitations of claim 9 has been taught in the above rejections.
Regarding claims 15 and 16, Donnelly and Xuemei Zhang do not expressly disclose that the image is sent to a trained machine learning algorithm to estimate a first and second concentration.
However, Greef discloses a method of protein microarray for quantifying protein levels [0163]. Greef further discloses biomolecule microarray assembly 1010 may be configured to image and analyze biomolecules other than proteins, such as DNA, cDNA, mRNA, siRNA, peptides, carbohydrates, lipids, whole cells, etc.
Greef further discloses the logic subsystem 1083 may include one or more algorithms for processing and analyzing data received from the camera 1030. Thus, in order to compare the intensity of different wavelengths of light emitted from the labeled analytes in the analyte capture locations 1016, the logic subsystem 1083 may include one or more algorithms or software for image analysis. One or more of a combination of different algorithms for data normalization and statistical techniques, such as artificial neural networks, multivariate statistics, machine learning such as, e.g., pixel feature classification, and tree algorithms may be stored in the logic subsystem 1083 for analyzing the images captured by the camera 1030 to detect for and/or quantify biomarkers in the sample based on relative intensities of different wavelengths of light [0224].
Greef further discloses the duration may be based on the configuration of the assembly, such as the wavelength of light produced by the laser, intensity of the light beam produced by the laser, a distance between the laser and the array, type or excitation wavelength of the fluorophore(s) used in the probe molecules to fluorescently tag the target biomolecules, resolution and/or sensitivity of the camera, concentration or amount of biomarkers bound and/or fluorescently tagged on the microarray [0262].
Regarding claim 23, Donnelly discloses a system for high throughput mapping or screening of intracellular interactions, comprising: at least one light source configured to irradiating under constant or pulsed light conditions at least one well with at least one wavelength of light (The methods can include various degrees of blue light stimulation. In some embodiments, the stimulation is acute or, optionally, chronic. Acute stimulation refers stimulation with pulses of blue light from about 0.2 to about 60 seconds, wherein the wavelength of the blue light can be any herein disclosed blue light wavelength. In some embodiments, the acute stimulation includes pulses of blue light from about 0.5 second to about 30 seconds, from about 1 second to about 20 seconds, or about 5 seconds. The blue light can be provided by a blue light source or a broad- spectrum light source filtered for the disclosed wavelengths (pg. 31, Ls. 18-24)) a receptor protein within a cell is sensitive to, and configured to irradiate a plurality of fluorescent proteins within a cell with at least one wavelength of light the fluorescent proteins are capable of absorbing (All constructs were fused to a fluorescent protein called mCherry (mCh) (pg. 6, Ls. 23-24); a light-induced oligomerization domain comprising a LOV photoreceptor domain (pg. 23, Ls. 24-25)) to visualize the proteins in live cells (pg. 6, Ls. 23-24), wherein the cell expresses a phase separation or aggregation system capable of being controlled by the at least one wavelength of light, the phase separation or aggregation system comprising a target protein or a target protein and a fluorescent protein or attached fluorophore (a nucleotide sequence encoding a chimeric polypeptide, comprising: a first nucleotide sequence encoding a light-induced oligomerization domain and a second nucleotide sequence encoding a low complexity domain from a neurodegenerative disease target protein. (pg. 16, Ls. 5-9)); a detector configured to capture an image of the plurality of fluorescent proteins within the cell (Automated high-throughput confocal microscopy);; causes components of a microscope to change the position of wells such that different wells come into focus; detects a first region in the image with a first intensity, and a second region of the image with a second intensity, the first intensity being higher than the second intensity (Automated high-throughput confocal microscopy was performed to assess the neurotoxicity of light-induced optoTDP43 inclusions. Human ReN neurons expressing TDP43- mCh or optoTDP43 were exposed to chronic blue light stimulation and viability was simultaneously monitored by longitudinal imaging); and determining a concentration of the first region based on the first intensity and a concentration of the second region based on the second intensity.
Donnelly further discloses developing various protein arrangements and blue light exposure paradigms to induce protein aggregation of proteins and protein fragments containing LCD/IDR/prion-like domains (for example, cognate partner), promote the mislocalization of nuclear proteins and recapitulate the neuropathology of neurodegenerative diseases. TDP-43 is a predominantly nuclear protein that contains an IDR/IDD/prion-like domain, and is mislocalized and aggregated in ALS, FTD, and some AD patients, as an example. DNA sequences were engineered to generate amino acid sequences that encode for a the CRY20LIG protein (for example, light-sensitive receptor protein) that clusters when exposed to blue light generated a fusion protein either the entire TDP-43 protein (for example, equivalent of enzymatically dead Cas9 )( (cry2-TDP43-mCh) or just the low complexity domain (LCD/IDD/prion-like domain) (for example, cognate partner), which makes TDP-43 aggregation prone (Cry2-274-mCh). As a control, CRY20LIG alone was used. All constructs were fused to a fluorescent protein called mCherry (mCh) (for example, reporter) to visualize the proteins in live cells. CRY20LIG-mCh reversibly clusters with blue light stimulation but the CRY20LIG-TDP-43-mCh or the truncated CRY20LIG-274-mCh forms irreversible aggregates with specific blue light stimulation paradigms. (pg. 4, Ls. 14-26, FIG. 2). Donnelly further discloses blue light induced oligomerization and aggregation of proteins employing the NcVVD, NcVVD Y50W or NcLOV photoreceptor (for example, light sensitive receptor protein). Donnelly further discloses that the NcVVD or LOV domain have only been shown to homodimerize with blue light stimulation (for example, the cognate partner). A light exposure paradigm to induce oligomerization and aggregation was developed. The top panel shows a schematic of how a single acute stimulation with blue light (405-499 nm) induces the homodimerization of the LOV protein when fused to a protein of interest. The bottom panel shows that chronic stimulation with blue light promotes the homooligomerization of NcVVD (for example, cognate partner equivalent) or LOV fusion proteins that contain a prion-like domain/LCD/IDD (for example, equivalent of enzymatically dead Cas9) (pg. 4, last para., FIG. 3). Donnelly further discloses light-induced aggregation of low-complexity domain proteins. Optogenetic TDP-43 fragments containing the low-complexity domain (LCD) undergos progressive aggregation with light stimulation (for example, equivalent of enzymatically dead Cas9) (pg. 4, last para., FIG. 3). (FIGS. 4A-4B) C-terminal fragments of TDP-43 (optoRRM2+LCD/optoLCD) rapidly oligomerize after a brief pulse (8 sec, 10% laser power, 488nm) of blue light (pg. 5, first para., FIG 4).
Shin discloses optoDroplets method which utilizes an optogenetic platform to achieve precise spatiotemporal control over phase transitions within living cells by fusing intrinsically disordered protein regions (IDRs) to the light-sensitive protein Cryptochrome 2 (Cry2). Shin further discloses condensed phases driven by the IDRs of various RNP body proteins, including FUS, DDX4, and HNRNPA1, where above a concentration threshold, these constructs undergo light-activated phase separation, forming spatiotemporally definable liquid optoDroplets.
Shin further discloses quantifying phase separation or aggregation and generating a phase diagram and determining the binodal and spinodal phase boundaries (pg. 29; FIG. 3 A-G).
Shin further discloses quantification and statistical analysis, where total concentrations of molecules as well as steady-state background concentrations (for example, subtracting any signal that does not originate from the specifically labeled molecule of interest/identifying a difference between phase diagram in step g) and a phase diagram without a biological or chemical agent) outside clusters were measured from fluorescence images of cells using ImageJ (NIH), and corrected by subtracting background noises measured with areas absent from any cells.
Further regarding claim 23 Donnelly and Xuemei Zhang do not disclose memory containing instructions that, when executed by at least one processor: causes the at least one light source to irradiate the at least one well with the at least one wavelength of light the receptor protein within the cell is sensitive to; causes the at least one light source to irradiate the at least one well with the at least one wavelength of light the fluorescent proteins are capable of absorbing; receive an image from the detector.
However, Greef discloses a biomolecule analysis system 1010 [0162] that includes a laser 1018 that may be a violet diode laser and may emit a light beam in a range of wavelengths between 400 and 450 nanometers, and a camera 1030; the biomolecule microarray assembly 1010 may include a memory chip 1033, which may be removably coupled to biomolecule microarray assembly 1010 for storing images captured by the camera 1030; Based on signals received from the user, the controller 1034 may send signals to the laser 1018 to emit light and/or to the camera 1030 for capturing an image [0199]; Logic subsystem 1083 may include one or more processors that are configured to execute software instructions. Additionally, or alternatively, the logic subsystem 1083 may include one or more hardware or firmware logic machines configured to execute hardware or firmware instructions [0191] ([0162-0202], FIG. 10).
It would have been prima facie obvious to one of ordinary skill in the art, before the effective filing date of the claimed invention, to have modified the method of Donnelly, Xuemei Zhang, and Shin to have used the biomolecule microarray assembly, as shown by Greef ([0162-0202], FIG. 10) to automate protein quantification. There would be a reasonable expectation of success in combining the technique of Greef to the method of Donnelly and Xuemei Zhang because they are all quantifying proteins from image analysis.
Response to Applicant Remarks
Applicant's arguments filed 12/24/2025 have been fully considered but they are not persuasive.
Applicant states:
Greef does not cure the deficiencies identified in Donnelly, Xuemei Zhang, and Shin as discussed above, since it also fails to teach or disclose a phase separation or aggregation system comprising (i) a target protein; and (ii) a fluorescent protein or fluorophore; (iii) an enzymatically dead Cas9; (iv) a receptor protein sensitive to the at least one wavelength of light; and (v) a cognate partner of the receptor protein as in the amended independent claim 1.
It is respectfully submitted that this is not persuasive. Applicants’ amendments necessitated a new round of art rejection. As such, the combination of Donnelly, Xuemei Zhang, Shin and young disclose all limitations of instant claims 1-2, 4-14, 17-18, 20-22, and 24-25.
Conclusion
No claims are allowed.
Applicant's amendment necessitated the new ground(s) of rejection presented in this Office action. Accordingly, THIS ACTION IS MADE FINAL. See MPEP § 706.07(a). 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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/G.S./ Examiner, Art Unit 1686
/LARRY D RIGGS II/ Supervisory Patent Examiner, Art Unit 1686