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 .
Claims 15-16, 18-20, 22, 24-26, 30, and 32-41 are pending.
Status of the Application
Applicant’s response and amendment filed 01 December 2025 are acknowledged and entered.
Applicant has amended Claims 15-16, 18, 20, 22, 24-26, and 30. Applicant has canceled Claims 1-7 (previously withdrawn as being directed to a nonelected invention), 14, 17, 21, 23, 27-29, and 31. Applicant has added Claims 32-41.
Continued Examination Under 37 CFR 1.114
A request for continued examination under 37 CFR 1.114, including the fee set forth in 37 CFR 1.17(e), was filed in this application after final rejection. Since this application is eligible for continued examination under 37 CFR 1.114, and the fee set forth in 37 CFR 1.17(e) has been timely paid, the finality of the previous Office action has been withdrawn pursuant to 37 CFR 1.114. Applicant's submission filed on 01 December 2025 has been entered.
Election/Restrictions
Applicant previously elected the species YTHDF1 so the claims are examined as they pertain to that species.
Response to Amendment
The 112(a) enablement rejection is maintained but updated in response to the claim amendments.
The previous 112(b) rejections are withdrawn in view of the claim amendments.
The 103 rejection is maintained but updated in response to the claim amendments.
Claims 15-16, 18-20, 22, 24-26, 30, and 32-41 are examined.
Arguments applicable to newly applied rejections to amended or newly presented claims are addressed below. Arguments that are no longer relevant are not addressed.
Rejections not reiterated here are withdrawn.
Claim Interpretation
Claims 22 and 32-41 recite the human subject has [FTD] or [AD] (Claims 22 and 32-33) or is determined to have [FTD] or [AD] (Claims 34-41). The broadest reasonable interpretation of those claims encompasses methods of treating FTD or AD.
Claims 34-41 recite a method that comprises a determining step that occurs entirely in the human mind and is considered a judicial exception. These methods were subjected to 101 analysis and the entire method of these claims was determined to be integrated into the specific practical application of increasing YTHDF1 protein expression including to treat a human subject suffering from AD or FTD.
Claim Objections
Claim 20 is objected to because of the following informalities: Claim 20 should recite a semicolon at the end of item (a) before item (b). Right now there is no punctuation there. Appropriate correction is required.
Claim Rejections - 35 USC § 112
The following is a quotation of the first paragraph of 35 U.S.C. 112(a):
(a) IN GENERAL.—The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor or joint inventor of carrying out the invention.
The following is a quotation of the first paragraph of pre-AIA 35 U.S.C. 112:
The specification shall contain a written description of the invention, and of the manner and process of making and using it, in such full, clear, concise, and exact terms as to enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the same, and shall set forth the best mode contemplated by the inventor of carrying out his invention.
Claims 18-19 and 31 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention. This is an enablement rejection. This rejection is new.
The factors to be considered in determining whether a disclosure would require undue experimentation include: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the specification; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. In re Wands, 8 USPQ2d, 1400 (CAFC 1988) and MPEP 2164.01.
The breadth of the claims and the nature of the invention: With respect to claim breadth, the standard under 35 U.S.C. §112(a) entails determining what the claims recite and what the claims mean as a whole. Methods of Claims 18-19 and 30 are drawn to methods of increasing YTHDF1 protein expression in a human subject in need thereof by administering an LNP encapsulating a nucleic acid molecule that encodes YTHDF1 operably linked to a heterologous promoter, wherein the promoter is a neuron-specific promoter, a glial cell-specific promoter, an astrocyte-specific promoter, a microglial cell-specific promoter, or an oligodendrocyte-specific promoter, wherein the nucleic acid molecule encodes a polypeptide consisting of SEQ ID NO: 2 and is administered in an amount sufficient to increase an expression level of the YTHDF1 protein in at least one brain cell of the subject, by at least 10% compared to an endogenous expression level of the YTHDF1 protein in the at least one brain cell prior to administration (Claims 18 and 30); wherein the at least one brain cell is a neuron, a glial cell, an astrocyte, a microglial cell, or an oligodendrocyte (Claim 19); or wherein the promoter is a glial cell (Claim 30).
Note that the claims require measuring expression in a cell in a human subject in vivo before and after treatment. The claims explicitly recite taking two measurements (before and after treatment): from at least one brain cell in a human subject (after treatment) and from the at least one brain cell (before treatment). Accordingly, the enablement rejection raises issues related to whether the claimed invention can predictably be used as claimed in human subjects.
The broadest reasonable interpretation of the methods is that the methods encompass measuring YTHDF1 protein expression in at least one brain cell in a human subject, administering a nucleic acid encoding YTHDF1, and remeasuring YTHDF1 protein expression in the same human brain cell; the second measurement will show that YTHDF1 protein expression increases by at least 10%. The nature of the invention is a method of increasing YTHDF1 expression in a living human subject by administering a nucleic acid encoding YTHDF1. A skilled artisan would not be able to use the method as claimed with a reasonable expectation of success based on guidance provided in the specification and art at the time of the filing of the application.
The state of the art and prior art, the level of one of ordinary skill, and the level of predictability in the art: A review of the art and prior art shows that at time of filing it was not possible to measure protein expression before and after treatment in the same living cell in a human brain in vivo. In addition, nothing in the Spec. demonstrates the invention is enabled for increasing YTHDF1 protein expression in a human brain cell within an organism because Applicant has presented no data demonstrating any 10% increase in YTHDF1 expression in any brain cell, let alone within a living organism, let alone within a human.
Shekaramiz (et al. 2018. Protein fishing from single live cells. J. Nanobiotechnol. 16:67, “Shekaramiz”) teaches (§Abstract):
Intracellular protein and proteomic studies using mass spectrometry, imaging microscopy, flow cytometry, or western blotting techniques require genetic manipulation, cell permeabilization, and/or cell lysis. We present a biophysical method that employs a nanoaspirator to ‘fish’ native cytoplasmic or nuclear proteins from single mammalian cells, without compromising cell viability, followed by ex cellulo quantitative detection.
That indicates that most measurements of protein quantification cannot be used in a living cell or in vivo. Shekaramiz teaches (§Introduction ¶4) they have developed a technique for spatiotemporally-controlled native protein extraction and direct, quantitative detection from a single mammalian cell. However, Shekaramiz’s work did not use cells in a living organism.
The art of Guillaume-Gentil (et al. 2016. Tunable Single-Cell Extraction for Molecular Analyses. Cell 166:506-516, “Guillaume”) teaches (§Abstract) approaches to directly extract the content of living cells remain a challenging but promising alternative to achieving non-destructive sampling and cell-context preservation. Guillaume teaches (§Introduction ¶1-3) measuring endogenous molecules of a single cell requires isolating and lysing the individual cell. Guillaume’s technique is (§Introduction ¶4) a minimally invasive technique that allows sampling a living cell, but Guillaume performs their technique on cultured cells, not a cell in an organism—let alone a cell within an organism’s brain or a human brain.
Furthermore, Liharska (et al. 2026. A study of gene expression in the living human brain. Molec. Psychiatry 31:137–157, “Liharska”) teaches (§Abstract) it is only recently possible to study gene expression in human brain tissue. Liharska teaches (same §) until their study, measuring gene expression in brain tissue required data from two independent cohorts. That indicates that any ability to measure expression in the same cell is unpredictable. Liharska teaches (same §) their study finally enables expand[ing] the scope of medical research to include questions about the molecular basis of human brain health and illness that can only be addressed in living people (e.g., “What happens in the brain at the molecular level as a person experiences an emotion?”). Liharska teaches (§Discussion, final ¶) most expression studies of the human brain use postmortem tissue, which indicates that taking two measurements from the same cell in vivo is unpredictable. Liharska teaches (§Introduction ¶1) [brain cell] samples from living people are largely unavailable and:
… up until now [2025], studies testing [the assumption that gene expression in the postmortem brain is an accurate representation of gene expression in the living brain] in humans have been small in scale, conducted prior to the advent of next-generation sequencing technologies, or limited to comparisons of living and postmortem cohorts not matched for key clinical and technical variables [5–8]. The Living Brain Project (LBP) developed a safe, ethical, and scalable procedure to acquire prefrontal cortex (PFC) tissue from living people for biomedical research purposes…
Liharska teaches (§Discussion ¶3) a limitation of their study is obtaining brain cell sample from living patients suffering from dementia.
Liharska’s paper is about measuring gene—not protein—expression, but the point remains: it is not possible to measure and remeasure biological molecules in the same living cell.
Cools (et al. 2025. In Vivo Visualization and Quantification of Brain Heat Shock Protein 90. J. Nuc. Med. 66:940-947, “Cools”) teaches (§Abstract) a method of quantifying protein in a living brain in vivo, but their method compares rodent models of AD and Parkinson disease vs. age-matched and young controls. Son (et al. 2023. In vivo Protein Footprinting Reveals the Dynamic Conformational Changes of Proteome of Multiple Tissues in Progressing Alzheimer’s Disease. BioRxiv posted 30 May 2023, “Son”) teaches (§Introduction ¶2) visualizing labeled proteins in vivo requires using a transparent worm which was chosen so the laser beam could penetrate the worm. That indicates there are technical difficulties with labeling or visualizing proteins in vivo in nontransparent tissue. Furthermore, Son teaches (§Introduction ¶3-5) methods that require fixing cells with formaldehyde and lysing tissue. A person of ordinary skill would immediately see how any method requiring transparent sample or fixing and lysing a cell wouldn’t translate to a human, let alone an ability to measure protein in the same brain cell in a human subject before and after treatment.
Shekaramiz and Guillaume discuss the difficulties of measuring protein expression in a single isolated living cell ex vivo. Liharska indicates that measuring gene expression in the same brain cell in vivo before and after a treatment is not possible because the most recent techniques allow measuring expression in a cell extracted from a human being. Cools and Son indicate that even as of 2025 or 2023 it was not possible to compare protein expression from the same cell in vivo.
Furthermore, the art teaches that, regarding gene expression and inhibition, specific controls are necessary. Regarding gene expression, the art of CreativeBiolabs (2024. Potency Tests for Gene Therapy Products. Available online at creative-biolabs.com. Accessed on 15 August 2024; “Creative”, of record) teaches (§Preclinical in vivo studies) the development of gene therapy product requires rigorous in vivo studies and quality control to assure drug potency… we have established a most exquisite service platform for the in vivo validation that separately evaluates infection, transcription and resulting protein levels, proper localization… Regarding gene inhibition with RNAi, the art of Han (2018. RNA Interference to Knock Down Gene Expression. Chapter 16 in Disease Gene Identification: Methods and Protocols, Methods in Molecular Biology vol. 1706. Springer; “Han”, of record) teaches:
(§3.4. Assessment of Gene Knockdown Using Reverse Transcription Polymerase Chain Reaction [RT-PCR] and Western Blotting) although reduction in transcript expression usually results in Western Blotting decreased protein abundance, mRNA levels do not always correlate with protein levels. For example, mRNA measurement can overestimate knockdown of genes whose protein products have long half-lives. Therefore, it is necessary to assess protein levels to ensure efficient knockdown of gene expression and to determine the optimal time point for assessing cellular effects of siRNA knockdown. Western blotting is the most widely used technique for detecting proteins (see Note 21)…
As will be discussed below in §The amount of direction provided by the Spec. and the existence of working examples, the instant Spec. does not describe performing those necessary controls to determine that UAS-YTHDF caused gene expression.
What is shown in the Spec. does not demonstrate any claimed outcomes (i.e., an at least 10% increase in YTHDF1 expression in the same human brain cell in vivo vs. before treatment) because Applicant did not measure YTHDF expression in any brain cell of a living organism, let alone in a human.
Altogether, the art indicates that the ability to measure protein expression in a single brain cell in vivo in a human, administer a nucleic acid, and remeasure protein expression in the same brain cell in vivo in a human is not predictable because techniques to measure protein expression require destroying a cell and techniques to measure gene expression are fraught with ethical and safety issues. Even Liharska’s Living Brain Project and Cools’ work, very recent research which discusses what is possible now, years after the filing date of the claimed invention, measured expression in biopsied cells (see Liharska Fig. 1).
Therefore, although the level of an artisan is high, the art of successfully measuring YTHDF1 protein expression in a single brain cell in vivo in a human, administering a nucleic acid encoding YTHDF1, and remeasuring YTHDF1 protein expression in the same brain cell in vivo in a human is unpredictable as evidenced by the state of the art discussed above.
The amount of direction provided by the specification and the existence of working examples: What is enabled by the working examples does not enable the claimed invention.
None of Applicant’s data show measuring YTHDF1 expression in a single brain cell in a living organism, administering a nucleic acid, and remeasuring YTHDF1 expression in the same brain cell in the living organism.
Fig. 9 shows that expression of human TDP-43 plus (presumed) expression of Ythdf operably linked to an upstream activator increases survival by 2 days. However, Applicant didn’t demonstrate that YTHDF1 expression increased by at least 10% in the same brain cell after treatment vs. before treatment because they never measured YTHDF1 expression, let alone in the same cell before and after treatment.
Figs. 11-12 show that inhibiting YTHDF increases the number of brain vacuoles in flies. Those figures don’t measure anything in living fly brains, let alone before and after treatment.
Figs. 13-16 show Ythdf RNAi results in increased baseline protein translation in flies whereas upregulation of Ythdf in neurons reduces total protein translation—not just YTHDF1 protein. Figs. 13-14 show that inhibiting YTHDF leads to increased more global protein translation. Figs. 15-16 show inhibiting YTHDF leads to increased protein translation and (presumptively) expressing YTHDF (i.e., UAS-YTHDF) leads to less overall protein translation of total protein, not just YTHDF1. None of those data show measuring an increase in YTHDF1 after administering a nucleic acid that encodes YTHDF1—let alone a 10% increase, any change in a living fly brain cell or any change in the same brain cell. Furthermore, those data are western blots which Shekaramiz teaches destroys a living cell. Furthermore, Applicant’s data do not measure the amount of specifically YTHDF1 protein after administering a nucleic acid that encodes YTHDF1.
Fig. 22 shows that expression of Ythdf operably linked to an upstream activator increases fly lifespan. No protein expression was measured to demonstrate the construct actually increases YTHDF1 expression, let alone by 10%.
None of these examples provides any evidence whatsoever that Applicant’s nucleic acid molecule resulted in increases—let alone 10% increases—in YTHDF1 protein expression—either in a fly or a human. None of those data show YTHDF1 expression in any living organism or cell before and after treatment with Applicant’s nucleic acid molecule—let alone by using SEQ ID NO 2.
Applicant has four figures of three experiments showing some effects of UAS-YTHDF1 expression. Figs. 9 and 22 demonstrate that flies expressing the UAS-YTHDF construct have longer lifespan. However, Applicant never showed that the UAS-YTHDF construct performs as expected to increase expression of YTHDF1. Those are crucial controls (see discussion of this in the other Enablement rejection below). As discussed, no controls were performed to validate that the UAS-YTHDF construct increases transcription or translation of YTHDF.
Altogether, what Applicant shows does not enable their invention in view of the art and prior art. None of the provided examples provides evidence that they were able to produce any increase—10% or otherwise—in YTHDF1 protein expression in the same human brain cell in a living human subject. Although the level of an artisan is high, the art of measuring protein expression before and after treating a brain cell in vivo—especially a human brain cell which work is fraught with technical difficulties and ethical and safety concerns—is unpredictable. As described above, the art does not teach that measuring protein expression in the same brain cell in vivo in any organism—let alone a human subject—was possible, especially at time of filing the claimed invention.
The art discussed above discusses a crucial control necessary for making a determination of increased expression. The art also teaches that most methods of measuring protein expression require killing a cell but that those that don’t require killing the cell can only be performed on isolated or biopsied cells, not cells within the brain of a living human subject. While a person of ordinary skill would recognize that measuring increased protein expression in a living fly is theoretically possible (because they could quantify fluorescence of a fluorescence-labeled target protein) it is not possible to do that in a human subject. In any case, Applicant has not shown that their method results in any 10% increase in YTHDF1 expression compared to an endogenous YTHDF1 expression level in any living brain cell from any organism, even a fly. That kind of experiment wouldn’t be possible in a human because human tissue is too thick for light of an imaging system to penetrate. (As Son teaches, footprinting an intact cell required using a transparent worm so the laser beam could penetrate.)
The instant Spec. does not provide any evidence that of enablement for the subject matter of Claims 18-19 and 30, and does not provide any guidance on these issues. In addition, the discussion above indicates that Applicant’s data lack crucial experimental controls. The instant Spec. contains research work demonstrating that expressing YTHDF1—presumably expressing YTHDF, because extent of expression wasn’t in fact validated—increases fly survival in the presence of exogenous TDP-43, reduces baseline protein translation in flies, and extends longevity in flies. (Extension of lifespan is also presumed because statistical significance wasn’t measured.) However, it does not support enablement of the claimed method of increasing YTHDF1 expression in a brain cell in a living human subject. In summary, the guidance present in the specification does not provide any guidance in addressing the enablement issues raised in view of the state of art discussion presented above.
The quantity of experimentation needed to make or use the invention: The standard of an enabling disclosure is not the ability to make and test if the invention works but one of the ability to make and use with a reasonable expectation of success. A patent is granted for a completed invention, not the general suggestion of an idea (MPEP 2164.03 and Chiron Corp. v. Genentech Inc., 363 F.3d 1247, 1254, 70 USPQ2d 1321, 1325-26 (Fed. Cir. 2004). The instant specification is not enabling because one cannot follow the guidance presented therein or within the art at the time of filing, and practice the claimed method without first making a substantial inventive contribution. Given the teachings described above, an artisan of ordinary skill would not be able to use the invention as claimed with a reasonable expectation of success. The amount of experimentation required for enabling guidance commensurate in scope with what is claimed goes beyond what is considered “routine” within the art and constitutes undue further experimentation in order to use the method of measuring protein expression in at least one brain cell in a living human subject, administering the claimed nucleic acid molecule, and remeasuring protein expression within the same at least one brain cell (and finding an at least 10% increase in YTHDF1 expression) with any reasonable expectation of success.
Claims 18 and 30 are rejected for those reasons. Claim 19 is rejected because it depends from Claim 18 and doesn’t remedy the issues.
Claims 22 and 32-41 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, as failing to comply with the enablement requirement. The claim(s) contains subject matter which was not described in the specification in such a way as to enable one skilled in the art to which it pertains, or with which it is most nearly connected, to make and/or use the invention. This is an enablement rejection. This rejection is updated in response to the claim amendments.
The factors to be considered in determining whether a disclosure would require undue experimentation include: (A) The breadth of the claims; (B) The nature of the invention; (C) The state of the prior art; (D) The level of one of ordinary skill; (E) The level of predictability in the art; (F) The amount of direction provided by the specification; (G) The existence of working examples; and (H) The quantity of experimentation needed to make or use the invention based on the content of the disclosure. In re Wands, 8 USPQ2d, 1400 (CAFC 1988) and MPEP 2164.01.
The breadth of the claims and the nature of the invention: With respect to claim breadth, the standard under 35 U.S.C. §112(a) entails determining what the claims recite and what the claims mean as a whole. Methods of Claims 22 and 32-41 are drawn to methods of increasing YTHDF1 protein expression in a human subject in need thereof wherein the human subject has either Alzheimer's disease (AD) or frontotemporal dementia (FTD) or is determined to have AD or FTD. The method step comprises a) administering, to the subject in need thereof, a composition comprising a nucleic acid molecule (that can be an mRNA or a DNA) encapsulated by a lipid nanoparticle, wherein the nucleic acid molecule encodes a YTHDF1, YHTDF2, or YTHDF3 protein operably linked to a heterologous promoter, wherein the promoter is a neuron-specific promoter, a glial cell-specific promoter, an astrocyte-specific promoter, a microglial cell-specific promoter, or an oligodendrocyte-specific promoter; and b) thereby causing expression of the YTHDF1, YHTDF2, or YTHDF3 protein encoded by the nucleic acid molecule in at least one cell of the CAN of the subject; and wherein the nucleic acid molecule is administered in a certain amount per dose, wherein the subject has FTD or is determined to have FTD (Claims 24, 32, 35, and 37) or has AD or is determined to have AD (Claims 24, 33-34, 36, and 38-41); wherein the promoter can be any of various types (Claims 36-37) or a cell type specific promoter (Claims 38-41)..
Claims 22 and 32-41 do not explicitly recite treating the FTD or AD in a human subject. However, a person of ordinary skill in the art would see that the claims recite administering to a human subject in need thereof and that the human subject has FTD or AD and would see that the specification discusses treating human subjects. Therefore they would reasonably interpret the claims as methods of treating a human subject using the method. Accordingly, the enablement rejection raises issues related to whether the claimed invention can predictably treat AD and FTD in a human subject.
The broadest reasonable interpretation of the methods is that the methods encompass administering a nucleic acid encoding YTHDF1 and doing so treats AD or FTD. A person of ordinary skill would reasonably interpret that treating means alleviating at least a symptom or improving pathology of the disease. Note also that the Spec. does not contemplate any other use for the claimed methods besides treating a neurodegenerative disease.
MPEP §2164.01(c) teaches, “When a compound or composition claim is limited by a particular use, enablement of that claim should be evaluated based on that limitation. See In re Vaeck, 947 F.2d 488, 495, 20 USPQ2d 1438, 1444 (Fed. Cir. 1991) (claiming a chimeric gene capable of being expressed in any cyanobacterium and thus defining the claimed gene by its use).” The claimed invention recites method claims so they also must be evaluated for enablement of the contemplated use.
The nature of the invention is a method for treating AD or FTD by administering to a human subject a nucleic acid encoding YTHDF1. A skilled artisan would not be able to use the method as claimed with a reasonable expectation of success based on guidance provided in the specification and art at the time of the filing of the application.
The state of the art and prior art, the level of one of ordinary skill, and the level of predictability in the art: A review of the art and prior art shows that the art considers certain animal models appropriate for studying AD and FTD and longevity; the Spec. does not teach use of those animal models. Teachings in the art also indicate that the techniques used in the instant Spec. are not appropriate for modeling AD or FTD (or longevity), and lack crucial controls required for a scientifically-based conclusion that expressing YTHDF1 can treat AD or FTD. The main problems are:
The claims recite a method of administering a nucleic acid encoding YTHDF1 to a patient who has AD or FTD. As discussed, a method of treating those conditions is implied and an artisan would reasonably interpret that as a method of alleviating at least a single symptom or pathology. The art teaches symptoms and pathologies of those diseases, and Applicant does not present evidence the recited methods treat any symptom or pathology of AD or FTD.
The data shown in the Spec. do not use an appropriate model of AD.
Teachings in the art indicate that certain controls are standard when drawing conclusions about the effect of expressing a gene; the Spec. does not describe performing such controls.
Applicant does not present evidence the recited methods treat any symptom of AD or FTD.
The art of Alzheimers.gov (2024. What Is Frontotemporal Dementia? and What is Alzheimer’s disease? Available online at alzheimers.gov. Accessed 14 August 2024; “Alzheimers.gov”, of record) teaches that FTD and AD are characterized by particular symptoms. Here is a list of the symptoms of FTD:
Symptoms of frontotemporal dementia and associated disorders may include:
Decreased energy and motivation
Lack of interest in others
Inappropriate and impulsive behaviors
Not acting considerate of others
Repeating an activity or word over and over again
Changes in food preferences and compulsive eating
Increased interest in sex
Neglect of personal hygiene
Emotional flatness or excessive emotions
Difficulty making or understanding speech
Inability to make common motions, such as using a fork
Problems with balance and walking
Increased clumsiness
Slow movement, falling, body stiffness
Restricted eye movements
Shaky hands
Muscle weakness and loss, fine jerks, wiggling in muscles.
Here is a list of the symptoms of AD:
Memory problems are often one of the first signs of Alzheimer’s. Symptoms vary from person to person, and may include problems with:
Word-finding, or having more trouble coming up with words than other people the same age.
Vision and spatial issues, like awareness of the space around them.
Impaired reasoning or judgment, which can impact decisions.
Other symptoms may be changes in the person’s behavior, including:
Taking longer to complete normal daily tasks.
Repeating questions.
Trouble handling money and paying bills.
Wandering and getting lost.
Losing things or misplacing them in odd places.
Mood and personality changes.
Increased anxiety and/or aggression.
In addition to those symptoms, FTD and AD have specific pathologies. Alzheimers.gov teaches that (§What Causes Frontotemporal Dementia?) people with FTD have abnormal amounts or forms of proteins called tau and TDP-43 inside nerve cells, or neurons, in their brain. The art of Chavan (et al. 2023 Animal models of Alzheimer’s disease: An origin of innovative treatments and insight to the disease’s etiology. Brain Res. 1814:148449; “Chavan”, of record) teaches (§Abstract) the main pathogenic features [of AD] are the development and deposition of senile plaques and neurofibrillary tangles in brain.
Applicant’s data do not demonstrate treatment of any of those symptoms or pathologies for either AD or FTD. Even if symptom is interpreted as pathology, the data shown in the Spec. do not show treatment of any of those AD or FTD pathologies. Applicant has not shown expression of YTHDF1 has any effect on tau or plaques or neurofibrillary tangles or even TDP-43 (which, as described later, is not a model for AD).
The data shown in the Spec. do not use an appropriate model of AD.
The art of Alzforum (2024. Research models: Alzheimer’s disease. Available online at alzforum.org/research-models/. Accessed 14 August 2024; “Alzforum”, of record) teaches a list of research models for AD. Those are all mouse models but even so, not a single one of them teaches any YTHDF protein or mentions eif2α, a protein taught by the Spec. as related to AD and shown in some of Applicant’s data. If either YTHDF or eif2α were any part of an art-recognized model of AD, it stands to reason that at least one of those genes would appear on the Alzforum list.
In addition to Chavan, several references discussing animal models for AD teach those models are based around specific proteins that cause AD so that the model mimics disease pathology. Those proteins are tau, γ-secretase/presenilin, Aβ/APP, and the Spec. does not use any of those models or measure any of those proteins in any experiment, let alone to show treatment of AD by expressing YTHDF.
The art of Jeon (et al. 2020. Genetic Dissection of Alzheimer’s Disease Using Drosophila Models. Int. J. Mol. Sci. 21:884; “Jeon”, of record) teaches (§1.1 Genetics of AD) Aβ…and tau… have been suggested to be important causative molecules in the pathology of AD. Jeon teaches (§1.2. Drosophila Models of Alzheimer’s Disease) drosophila models of AD:
There are three main types of Drosophila AD models according to the transgenes used, and the first type is the γ-secretase-based model… tau-based models have been established and used to study the role of tau in the formation of neurofibrillary tangles and neurotoxicity… most of the Drosophila AD models are based on APP or Aβ expression, since Aβ peptides, the major components of amyloid plaques, are considered to play the most important role in AD [26]. Because there is no conservation of both Aβ peptide sequence in APP and γ-secretase in Drosophila, an essential condition for the generation of Aβ peptides, fly models expressing both human APP and BACE have been used [27–29]. In these models, AD-like phenotypes, such as age-dependent neuronal death, Aβ accumulation, and lethality are observed. [emphasis added.]
Jeon’s Fig. 1 summarizes art-recognized models for AD:
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Those are appropriate models of AD (i.e., “appropriate” for basing conclusions about treating AD) because the proteins tau, γ-secretase, etc. are demonstrated to have a role in AD pathology and to (Jeon) cause AD. Other papers teach animal models of AD are based on the same three proteins.
The art of Chen (and Zhang. 2022. Animal models of Alzheimer’s disease: Applications, evaluation, and perspectives. Zool. Res. 43[6]:1026−1040; “Chen”, of record) teaches (§Introduction ¶1 and 6):
The brains of AD patients are characterized by extracellular senile plaques composed of amyloid β (Aβ), intracellular tau aggregates, and neuronal loss (Price et al., 1995). Previous studies have indicated that AD is typically associated with cytoskeletal alterations, including the formation of neurofibrillary tangles (NFTs), neuropil threads, and axonal pathology… As an essential tool for studying the mechanisms of AD, animal models must recapitulate human pathophysiology. [emphasis added.]
Chen teaches (§INVERTEBRATE AD MODELS-Drosophila melanogaster models):
According to the introduced transgenes, there are generally three types of D. melanogaster AD models, i.e., γ-secretase-based, tau-based, and APP- or Aβ-based models. In the γ-secretase-based models, the presenilin (psn) gene encodes a component of the γ-secretase complex, and overexpression of the FAD-related mutant psn is considered one of the earliest events in AD pathology (Michno, 2009). In this model, psn deficiency causes synaptic abnormalities and defects in associative learning in D. melanogaster larvae (Knight et al., 2007). Thus, γ-secretase-based AD models can help elucidate the role of psn in both development and degeneration and verify the involved pathways and molecular mechanisms. In the tau-based models, studies have shown that D. melanogaster flies present with AD-like phenotypes after human tau expression (Jackson et al., 2002; Wittmann, 2001). Tau can be genetically modified by inducing the expression of wild-type or mutant human tau in D. melanogaster (Shulman & Feany, 2003). In addition, Aβ42 and tau co-expression models have been used to study the relationship between Aβ42 and tau (Folwell et al., 2010). The most common D. melanogaster AD model is the APP- or Aβ-based model, which exhibits some AD-like phenotypes, such as Aβ accumulation and age-dependent neuronal death (Greeve et al., 2004). To better study the role of amyloid plaques in AD pathology, D. melanogaster AD models that directly express Aβ42 in the brain have been developed (Casas-Tinto et al., 2011; Finelli et al., 2004). [emphases added.]
Similarly, the art of Akhtar (et al. 2022. Preclinical Models for Alzheimer’s Disease: Past, Present, and Future Approaches. ACS Omega 7:47504-47517; “Akhtar”, of record) teaches (§Abstract):
Although various preclinical models are available for several diseases, clinical models for AD remain underdeveloped and inaccurate. The pathophysiology of AD mainly includes the presence of amyloid plaques and neurofibrillary tangles (NFT)... To mimic the pathogenesis of human AD, animal models like 3XTg-AD and 5XFAD are the primarily used mice models in AD therapeutics. Animal models for AD include intracerebroventricular-streptozotocin (ICV-STZ), amyloid beta-induced, colchicine-induced, etc., focusing on parameters such as cognitive decline and dementia. Unfortunately, the translational rate of the potential drug candidates in clinical trials is poor due to limitations in imitating human AD pathology in animal models. Therefore, the available preclinical models possess a gap in AD modeling…
Just like the other art teaching relevant AD models, Akhtar teaches (§8.3 Drosophila Model) the drosophila AD models involve the same proteins that cause AD pathology: APP, BACE-1, presenilin and tau orthologs, resulting in amyloid aggregation in the model brain, leading to neurodegeneration and memory loss. Akhtar teaches (§12. Conclusion) the models are useful because they represent pathological features of human AD.
Applicant does not present evidence of using an AD model that recapitulates AD pathophysiology.
The art teaches certain controls are standard in order to draw meaningful conclusions about the effect of gene expression or inhibition.
Research in the art shows certain experimental controls are used to determine that gene therapy results in expression and that lifespan was extended. Details about any of those controls are absent from the instant disclosure.
Specifically, the art of Macip (et al. 2024. Gene Therapy-Mediated Partial Reprogramming Extends Lifespan and Reverses Age-Related Changes in Aged Mice. Cell Reprogram. 26[1]:24-32; “Macip”, of record), Jaijyan (et al. 2022. New intranasal and injectable gene therapy for healthy life extension. PNAS 119[20] e2121499119; “Jaijyan”, of record), and Xue (et al. 2022. dNAGLU Extends Life Span and Promotes Fitness and Stress Resistance in Drosophila. Int. J. Mol. Sci. 23[22]:14433; “Xue”, of record) indicates that certain controls are standard to conclude that expression of a nucleic acid leads to increased lifespan.
Macip teaches (§Abstract) we show that systemically delivered adeno-associated viruses, encoding an inducible OSK system, in 124-week-old male mice extend the median remaining lifespan by 109% over WT controls and enhance several health parameters. Macip expressed the OSK genes from a vector and show increased survival. Macip indicates (Fig. 1BC) statistically significant differences in lifespan among treated vs. control groups and, notably, measured (Fig. 3) expression of the O, S, and K genes to confirm that their system worked as expected to result in higher gene expression in at least some tissues. That is, Macip validated that OSK were significantly overexpressed in their system and that the treatment statistically significantly (i.e., meaningfully) extended lifespan.
Jaijyan teaches (§Significance):
we illustrated that CMV can be used therapeutically as a monthly inhaled or intraperitoneally delivered treatment for aging-associated decline. Exogenous telomerase reverse transcriptase [TERT] or follistatin [FST] genes were safely and effectively delivered in a murine model. This treatment significantly improved biomarkers associated with healthy aging, and the mouse lifespan was increased up to 41% without an increased risk of cancer.
Jaijyan provides ample evidence that (Figs. 1BD, 2ACDE) TERT and FST expression and protein were significantly upregulated vs. control and that they statistically significantly extend lifespan.
Both Macip and Jaijyan used mouse models but the teachings of Xue indicate the same controls (wherein overexpression of the target gene is validated and statistical comparison between lifespan test groups is calculated) are performed in Drosophila. Xue teaches (§Abstract) we found that the overexpression of CG13397 (dNAGLU) ubiquitously, or tissue specifically, in the nervous system or fat body could extend fly life span. It also extended the life span of flies overexpressing human Aβ42, in a Drosophila AD model. To evidence that conclusion, Xue measured (Fig. 2) NAGLU expression in the whole body and different tissues and provided evidence of statistically significant differences (vs. control) in both NAGLU expression and lifespan. Regarding the relevance of NAGLU overexpression to AD, Xue used (Fig. 3) an AD model wherein (§Introduction ¶1, 3; § 2.3. Overexpression of dNAGLU Extends Life Span and Health Span in the Drosophila AD Model, entire §) flies express human β-amyloid (i.e., Aβ42). Notably, Aβ is one of the proteins that has a known correlation to AD and is taught by Chen, Akhtar, and Jeon as a model of AD.
Furthermore, the art teaches that, regarding gene expression and inhibition, specific controls are necessary. Regarding gene expression, the art of CreativeBiolabs (2024. Potency Tests for Gene Therapy Products. Available online at creative-biolabs.com. Accessed on 15 August 2024; “Creative”, of record) teaches (§Preclinical in vivo studies) the development of gene therapy product requires rigorous in vivo studies and quality control to assure drug potency… we have established a most exquisite service platform for the in vivo validation that separately evaluates infection, transcription and resulting protein levels, proper localization… Regarding gene inhibition with RNAi, the art of Han (2018. RNA Interference to Knock Down Gene Expression. Chapter 16 in Disease Gene Identification: Methods and Protocols, Methods in Molecular Biology vol. 1706. Springer; “Han”, of record) teaches:
(§3.4. Assessment of Gene Knockdown Using Reverse Transcription Polymerase Chain Reaction [RT-PCR] and Western Blotting) although reduction in transcript expression usually results in Western Blotting decreased protein abundance, mRNA levels do not always correlate with protein levels. For example, mRNA measurement can overestimate knockdown of genes whose protein products have long half-lives. Therefore, it is necessary to assess protein levels to ensure efficient knockdown of gene expression and to determine the optimal time point for assessing cellular effects of siRNA knockdown. Western blotting is the most widely used technique for detecting proteins (see Note 21)…
As will be discussed below in §The amount of direction provided by the Spec. and the existence of working examples, the instant Spec. does not describe performing those necessary controls to determine that UAS-YTHDF caused gene expression and that RNAi targeting YTHDF inhibited gene expression.
What is shown in the Spec. does not demonstrate any claimed outcomes (i.e., treatment of any symptoms of AD or FTD) because Applicant did not test YTHDF expression in any models of crucial AD pathology and has not provided details of 1) controls necessary to substantiate their findings or 2) statistical comparisons.
Therefore, although the level of an artisan is high, the art of treating AD or FTD by expressing YTHDF1 is unpredictable as evidenced by the state of the art discussed above.
The amount of direction provided by the specification and the existence of working examples: What is enabled by the working examples does not enable the claimed invention. Some version of some of the claims may be enabled if additional evidence is provided, as discussed in this §.
First of all, global suppression of YTHDF (i.e., the result of RNAi targeting YTHDF) isn’t the same as modeling AD (which has a specific pathology). Second, Applicant has not demonstrated that 1) lack of YTHDF produces AD pathology including critical factors or “hallmarks” such as (Jeon) Aβ and/or tau aggregates or hyperphosphorylated tau OR 2) expressing YTHDF reduces any hallmarks of AD pathology or treats any symptoms of AD or FTD. Third, the fact that globally suppressing YTHDF results in a certain outcome doesn’t demonstrate that the opposite outcome occurs when YTHDF is expressed. Furthermore, some of Applicant’s findings are not in line with what would be expected based on what is known about protein translation in AD. Here is what Applicant’s examples show:
Figs. 5-6 show that inhibiting YTHDF in neurons or glial cells reduces fly lifespan.
Figs. 7-8 show that expression of human TDP-43 plus t inhibiting YTHD significantly reduces fly lifespan but that RNAi KD of Ythdc1 does not.
Fig. 9 shows that expression of human TDP-43 plus expression of Ythdf operably linked to an upstream activator increases survival by 2 days.
Figs. 10-12 show that Ythdf depletion reduces fly ability to withstand stress and increases number of brain vacuoles. Fig. 10 shows inhibiting YTHD reduces fly ability to withstand stress. Figs. 11-12 show that inhibiting YTHDF increases the number of brain vacuoles in flies.
Figs. 13-16 show Ythdf RNAi results in increased baseline protein translation in flies whereas upregulation of Ythdf in neurons reduces protein translation. Figs. 13-14 show that inhibiting YTHDF leads to increased more global protein translation. Figs. 15-16 show inhibiting YTHDF leads to increased protein translation and YTHDF expression leads to less protein translation.
Figs. 17-18 show Ythdf RNAi results in increased eif2a-phosphorylation (which is associated with stress granules/cellular stress). Figs. 17-18 show inhibiting YTHDF leads to more eif2α phosphorylation; ¶64 teaches there is an increased eif2a-phosphorylation compared to control flies, indicative of canonical translational repression. Notably, that’s the opposite of what Applicant said in ¶63 which is that YTHDF knockdown indicates an increased amount of protein translation at baseline in KD flies.
Figs. 19-21 show that coexpression of human TDP-43 with Ythdf RNAi increases protein translation and levels of eif2a-phosphorylation in flies. Fig. 19 shows expressing TDP-43 and inhibiting YTHDF leads to increased protein translation. Figs. 20-21 show inhibiting YTHDF leads to increased eif2α phosphorylation.
Fig. 22 shows that expression of Ythdf operably linked to an upstream activator increases fly lifespan.
None of these examples provides evidence that expressing YTHDF1 can treat any symptom of AD or FTD, either in a human subject or in a reasonably representative model for AD or FTD. The examples showing that UAS-YTHDF increases fly lifespan are missing crucial data required for scientifically concluding that expressing YTDHF increases fly lifespan. Even Applicant’s conclusion that lifespan increases when UAS-YTHDF is expressed in the presence of TDP-43 does not equal that expressing UAS-YTHDF can treat a symptom of AD.
Applicant has four figures of three experiments showing some effects of UAS-YTHDF1 expression. Figs. 9 and 22 demonstrate that flies expressing the UAS-YTHDF construct have longer lifespan. However, Applicant never showed that the UAS-YTHDF construct performs as expected to upregulate YTHDF. Those are crucial controls, as shown by the cited papers and discussed by Creative. As discussed, no controls were performed to validate that the UAS-YTHDF construct increases transcription or translation of YTHDF.
Furthermore, Applicant hasn’t shown that the effects on lifespan are statistically significant. Fig. 9 shows a 2 day increase in lifespan and Fig. 22 shows a 7 day increase but it is not known whether the flies in those experiments were actually overexpressing YTHDF because that crucial validation is not shown. Furthermore, whether a 2-day or 7-day increase in fly lifespan is meaningful is not known because a statistical comparison isn’t shown. Then Figs 15-16 show that the UAS-YTHDF construct reduces protein translation. But a link to lifespan and general protein expression is not discussed in the Spec., and teachings in the art of Anisimova (et al. 2018. Protein synthesis and quality control in aging. Aging 10[12]:4269-4288; “Anisimova”, of record) indicate that decreased protein synthesis is an indicator of aging. Fig. 1 shows this in a graphic; an annotated version is shown here:
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Based on that, an artisan would expect that less protein translation means more aging and shorter lifespan. However, Applicant’s data show the opposite: Figs. 15-16 show that UAS-YTHDF decreases protein translation but Fig. 22 shows UAS-YTHDF increases fly lifespan (although no significant differences are shown). Applicant has not provided any explanation for why their result deviates from what would be expected or how their finding reconciles with what is known in the art about less protein synthesis corresponding to greater aging (and therefore, shorter lifespan).
There are further apparent discrepancies between Applicant’s data and their interpretation of it and what is known in the art. For example, Applicant teaches that:
(¶64) In general, an increased level of phosphorylation of eif2a indicates increased levels of stress granules and cellular stress. Increased eif2a phosphorylation is further known to be associated with human neurodegenerative diseases such as AD… there is an increased eif2a-phosphorylation [in YTHDF-RNAi flies] compared to control flies, indicative of canonical translational repression… [emphasis added.]
But Applicant’s previous data clearly shows—and they described one ¶ previous—the opposite, namely that YTHDF-RNAi increased protein translation:
(¶63) as illustrated by these Western blots [Figs. 13-14] , knock-down of Ythdf in fly neurons causes an increased amount of puromycin incorporation. This indicates an increased amount of protein translation at baseline in these flies… Conversely, upregulation of Ythdf in neurons showed the opposite response, with significant decrease in nascent protein translation in dissected brains protein samples FIGs. 15 and 16. [emphasis added.]
At no point does Applicant acknowledge or explain this apparent discrepancy in their data and their interpretation of it. Both assays use the same YTHDF-RNAi flies. One assay (puromycin) sees increases in protein translation and the other sees increases in eif2α phosphorylation. And yet Applicant interprets the puromycin assay as indicat[ing] an increased amount of protein translation at baseline in these flies and the phosphorylation assay as indicate[ing] canonical translational repression. Translation was not measured in the phosphorylation assay but even though previous data indicate that (Figs. 13-16) YTHDF-RNAi probably did increase translation in that assay (since both assays used the same treatment, i.e., YTHDF-RNAi flies), Applicant concludes that the phosphorylation assay is indicative of canonical translational repression. Perhaps there are differences in the experiments that explain the discrepancy. But since little information is provided and the apparent discrepancy is not addressed, any potential scientific explanation is not clear.
Anisimova also teaches (§Signaling pathways controlling longevity via modulation of protein synthesis ¶10) eIF2α is inactive when phosphorylated and when active eIF2α is lacking, synthesis of most protein is suppressed… That means that one would expect to see less global protein translation when they see elevated levels of phosphorylated eif2α, but that is the opposite of what Applicant shows in Figs. 13-21. Figs. 13-16 show that when YTHDF is inhibited with RNAi, there is more protein translation but Figs. 17-18 and 20-21 show that YTHDF-RNAi leads to more eif2α phosphorylation.
Altogether, what is known in the art is not compatible with what Applicant shows regarding the effect of expressing YTHDF on increasing longevity of lifespan. Note that Applicant has not performed standard controls for their experiments. Such standard controls are described in Macip, Jaijyan, and Xue and include validating changes in gene and protein expression as well as statistical comparison of differences in lifespan. Additionally, as discussed above, Bene teaches that lifespan data cannot reasonably be extrapolated between lab animals and humans.
Applicant’s most compelling evidence supporting a link between YTHDF expression and increased longevity are presented in Fig. 22 which shows that UAS-YTHDF extended fly lifespan by a week. However, Applicant has not shown necessary controls, wherein YTHDF expression is validated and wherein lifespan is statistically compared. Therefore the data presented in Fig. 22 cannot be considered to enable the invention. The following claim could possibly be enabled if those data were presented:
Claim #: A method of increasing the longevity of fly lifespan, comprising:
administering a nucleic acid molecule to the fly, under conditions sufficient to cause expression of a YTHDF1 protein encoded by the nucleic acid, in a cell of the central nervous system of the fly,
wherein the nucleic acid molecule encodes a YTHDF1 protein operably linked to a heterologous promoter and/or an upstream activation sequence (UAS); and
wherein the nucleic acid molecule is administered in an amount of 1-5 µg…or 400-500 µg per dose.
Regarding administering the nucleic acid to a human who has AD or FTD (Claims 22 and 32-41)
The instant claims are directed to a gene therapy that is a gene addition wherein YTHDF1 is added by expressing it in a subject. The American Society of Gene and Cell Therapy (2024. Gene and Cell Therapy FAQs. Available online at asgct.org. Accessed 14 August 2024; “Society”, of record) teaches (§Are there different types of gene therapy?):
Gene addition involves inserting a new copy of a gene into the target cells to produce more of a protein. Most often, a modified virus such as adeno-associated virus (AAV) is used to carry the gene into the cells. Therapies based on gene addition are being developed to treat many diseases, including adenosine deaminase severe combined immunodeficiency (ADA- SCID), congenital blindness, hemophilia, Leber’s congenital amaurosis, lysosomal storage diseases, X-linked chronic granulomatous disease, and many others.
That teaches that gene addition inserts a new copy of a gene into target cells to produce more of a protein. In this case, the protein is YTHDF1 and the diseases are AD and FTD. However, there is no documented link in the art between a YTHDF1 and AD or FTD. The art does not document any link between mutated YTHDF1 or lack of YTHDF1 or a reduced amount of YTHDF1 and AD or FTD. Applicant has not presented evidence demonstrating such a link. Therefore one cannot assume that expressing YTHDF will treat AD or FTD.
Besides for Figs. 9, 15, and 22, Applicant’s data are all directed to inhibiting YTHDF1. Applicant has not provided evidence that expressing YTHDF1 can treat any symptom of AD or FTD because nearly all of Applicant’s data are directed to inhibiting YTHDF1 and because when YTHDF is expressed, it is not in a model of AD that has any of the hallmarks of AD (as described by Jeon, Chen, and Akhtar). The Spec. presents few examples that show expressing YTHDF at all: Figs 9, 15-16, and 22. Fig. 9 shows that expressing UAS-YTHDF in a fly who expresses TDP-43—TDP-43 expression is a model for FTD, not a model for AD—extends lifespan. While TDP-43 occurs in AD, TDP-43 expression is not a model for AD and the art of Herman (et al. 2011. β-Amyloid triggers ALS-associated TDP-43 pathology in AD models. Brain Res. 1386: 191-199; “Herman”, of record) demonstrates that TDP-43 occurs only as a result of upstream pathologies. Herman teaches (§Abstract) TDP-43 aggregation is an outcome of AD, not its cause. That TDP-43 is not a pathological hallmark of AD is evidenced by the fact that the art teaches models for AD that center around tau, γ-secretase, and amyloid. Figs. 15-16 show YTHDF expression results in less protein translation. That experiment is not performed in any model of AD or FTD. Fig. 22 shows YTHDF expression increases lifespan; that experiment is not in any model of AD or FTD either. None of those examples demonstrates any ability to treat any symptom or even a pathological hallmark of AD because Applicant did not measure or show any effects related to tau, Aβ, or γ-secretase. Yet, the claims encompass reducing or eliminating at least one symptom.
Applicant does not present evidence demonstrating a link between YTHDF expression and AD because Applicant did not test the effect of YTHDF expression in any AD model. All the references discussing animal models of AD (Akhtar, Chen, Jeon) teach the same pathogenic proteins: γ-secretase/presenilin, tau, and amyloid-β/APP and none of those were examined in Applicant’s experiments. Those proteins are not even discussed in the Spec. Despite concluding that YTHDF can treat AD, Applicant has not discussed YTHDF in relation to any of the AD pathological hallmarks.
The Spec. teaches (¶57) the present disclosure is based in part on the surprising finding that Ythdf (i.e., the D. melanogaster homolog for the human YTHDF proteins), can be used to reduce the pathological effects observed when D. melanogaster is engineered to express human TDP-43. However, the Spec. does not describe reduction in pathological effects that have a known or demonstrated link to AD. The Spec. teaches examples wherein inhibiting YTHDF decreases lifespan and expressing YTHDF slightly increases lifespan. Applicant relies on data showing inhibiting YTHDF results in more brain vacuoles vs. control; inhibiting YTHDF increases protein translation and expressing YTHDF decreases protein translation; and inhibiting YTHDF increases the level of phosphorylated eif2α. None of these experiments demonstrates treatment of any symptom of AD or FTD or even ties YTHDF to any AD hallmark.
In general, Applicant provides data showing that inhibiting YTHDF leads to one outcome and then bases their conclusions on an assumption that expressing YTHDF leads to the opposite outcome. But that conclusion is not scientifically valid, particularly because of a lack of appropriate controls.
The Spec. teaches (¶65; Figs. 19-21):
As explained above, TDP-43 has been identified as a protein implicated in several neurodegenerative diseases, including ALS and FTD, and the expression of TDP-43 in D. melanogaster causes pathological effects similar to those observed in ALS patients, e.g., neuronal degeneration, motor neuron deficits, and shortened lifespan. Furthermore, elevated levels of eif2a phosphorylation are associated with TDP-43 and also with human neurodegenerative diseases generally. These experiments demonstrates that Ythdf is able to reduce the level of eif2a phosphorylation. This set of data further suggests that Ythdf may be administered to a fly to rescue the neurodegeneration phenotype caused by TDP-43.
But these experiments do not demonstrate that Ythdf is able to reduce the level of eif2a phosphorylation or sugges[t] that Ythdf may be administered to a fly to rescue the neurodegeneration phenotype caused by TDP-43 because in the experiments underlying Figs. 19-21, Applicant did not express YTHDF. The experiments show that inhibiting YTHDF increases the level of eif2α phosphorylation because that is what Applicant tested. Furthermore, Applicant’s finding assumes that the YTHDF-RNAi properly inhibited YTHDF, which is not shown by Applicant. It is well-known in the art that (i.e., see discussion of Han) gene and protein expression must be measured in order to properly validate that the RNAi construct works to inhibit gene expression and that protein levels are truly reduced or that (see Creative) a gene expression construct results in gene expression. As discussed in Han, an artisan would not expect that YTHDF-RNAi knocks down levels of YTHDF protein because (Han teaches) mRNA levels do not always correlate with protein levels. That is why (Han teaches) it is necessary to assess protein levels to ensure efficient knockdown of gene expression and to determine the optimal time point for assessing cellular effects of siRNA knockdown. Western blotting is the most widely used technique for detecting proteins. But no experiment in the Spec. Western blots YTHDF1, and the only Western blots (Figs. 13-21) show either total protein or phosphorylated eif2α.
In addition, some of Applicant’s results are the opposite of what an artisan would expect if YTHDF expression can indeed treat AD. Ohno (2014. Roles of eIF2α kinases in the pathogenesis of Alzheimer’s disease. Frontiers Molec. Neurosci. 7:22; “Ohno”; cited in Applicant’s Spec. at ¶64 and of record on an IDS) teaches general protein translation is shut down in AD: (§Abstract) overactivation of regulatory kinases occurs in neurodegenerative diseases such as AD, leading to shutdown of general translation and translational activation of a subset of mRNAs. Therefore, Applicant’s data demonstrating that (Figs. 15-16) the UAS-YTHDF construct causes less protein translation indicate that expressing UAS-YTHDF recapitulates the AD pathology rather than support that expressing YTHDF could treat AD. Even that assumes that proper controls were performed, which they were not in Applicant’s provided data, as discussed. Additionally, Applicant did not measure the effect of YTHDF expression on eif2α phosphorylation, so it is not possible to know whether or not such expression would alleviate eif2α phosphorylation.
Furthermore, the art of Zaccara (and Jaffrey. 2020. A Unified Model for the Function of YTHDF Proteins in Regulating m6A-Modified mRNA. Cell 181:1582–1595; “Zaccara”, of record) teaches that expressing YTHDF leads to experimental artifacts and unpredictable results:
(Fig. S2 caption) Heterologous expression of [YTH]DF proteins causes formation of DF stress granule-like structures… As shown by the absence of these structures in the control-transfected cells (FLAG-3xHA), the granule-like structures are not an artifact of transfection. Thus, expressing DF proteins may lead to uninterpretable results due to the variable formation of protein aggregates that could act to sequester DF proteins and mask their function.
That further demonstrates why it is not possible to draw conclusions about the function of YTHDF based on Applicant’s experiments, especially since crucial controls are not described. We note that stress granules (induced by YTHDF expression) are something that an artisan would want to avoid when attempting to treat AD.
Altogether, Applicant concludes that Ythdf is able to reduce the level of eif2a phosphorylation… this set of data further suggests that Ythdf may be administered to a fly to rescue the neurodegeneration phenotype caused by TDP-43 despite never showing that YTHDF reduces eif2α phosphorylation. The only experiments about eif2α phosphorylation knock down YTHDF. Furthermore, the results demonstrating YTHDF expression leads to reduced protein translation indicate (based on the art of Anisimova and Ohno) that YTHDF expression recapitulates AD pathology. None of that enables expressing YTHDF to treat any symptom of AD.
Regarding FTD, the art of Li (et al. 2010. A Drosophila model for TDP-43 proteinopathy. PNAS 10[7]:3169-3174; “Li”, of record) teaches that (§Abstract) expressing TDP-43 is an appropriate model for FTD. The art of Macip, Jaijyan, Xue, and CreativeBiolabs teaches basic experimental controls that must be present in gene expression studies. Notably Li shows (Fig. S1D) such gene and protein expression data in their TDP-43 flies. Applicant’s Fig. 9 allegedly shows that expressing YTHDF in a model wherein the fly expresses TDP-43 lengthens fly lifespan by two days. However, Applicant has not shown necessary controls wherein TDP-43 and YTHDF expression are each validated and wherein lifespan is statistically compared. Therefore the data presented in Fig. 9 cannot be considered to enable the invention. It is possible that the following claim could be enabled if data validating TDP-43 and YTHDF expression and statistical comparison of lifespans were presented, provided Applicant demonstrates why Zaccara’s findings about YTHDF expression leading to stress granule formation do not apply to Applicant’s study:
Claim #: A method of increasing the longevity of lifespan in a fly with frontotemporal dementia, comprising:
administering a nucleic acid molecule to the fly, to cause expression of a YTHDF1 protein encoded by the nucleic acid, in a cell of the fly’s central nervous system,
wherein the nucleic acid molecule encodes a YTHDF1 protein, operably linked to a heterologous promoter and/or an upstream activation sequence (UAS); and
wherein the nucleic acid molecule is administered in an amount of 1-5 µg…or 400-500 µg per dose;.
Further problems with specific claims are:
Regarding Claims 22 and 32-41, Applicant has not demonstrated that their method results in the YTHDF1 protein encoded by the nucleic acid molecule being expressed, let alone increasing expression of YTHDF1, in at least one cell in the subject’s central nervous system cell because Applicant has not provided any measurements of any levels of YTHDF1 protein—or YTHDF1 gene expression—in any part of any subject, let alone any subject’s CNS.
Regarding delivery to the CNS, Applicant has not demonstrated administering the nucleic acid molecule to the brain because they used genetic constructs, not delivery.
Altogether, what Applicant shows does not enable their invention in view of the art and prior art. None of the provided examples provides evidence that expressing YTHDF1 or administering YTHDF1 protein can treat or eliminate any symptom of AD or FTD. Although the level of an artisan is high, the art of treating AD or FTD is unpredictable. As described above, the art does not teach a model that represents AD pathology or wherein lifespan data may reasonably be extrapolated to other species. The teachings of Anisimova, Ohno, and Zaccara specifically relate to expression of YTHDF and to Applicant’s data. There are unexplained/unaddressed discrepancies in Applicant’s own data and their interpretation of it. The instant Spec. does not provide guidance on these issues. In addition, the discussion above indicates that Applicant’s data lack crucial experimental controls. The instant Spec. contains research work demonstrating that expressing YTHDF1—presumably expressing YTHDF, because extent of expression wasn’t in fact validated—increases fly survival in the presence of exogenous TDP-43, reduces baseline protein translation in flies, and extends longevity in flies. (Extension of lifespan is also presumed because statistical significance wasn’t measured.) However, it does not support enablement of the claimed method that encompasses treating AD or FTD. In summary, the guidance present in the specification does not provide any guidance in addressing the enablement issues raised in view of the state of art discussion presented above.
The quantity of experimentation needed to make or use the invention: The standard of an enabling disclosure is not the ability to make and test if the invention works but one of the ability to make and use with a reasonable expectation of success. A patent is granted for a completed invention, not the general suggestion of an idea (MPEP 2164.03 and Chiron Corp. v. Genentech Inc., 363 F.3d 1247, 1254, 70 USPQ2d 1321, 1325-26 (Fed. Cir. 2004). The instant specification is not enabling because one cannot follow the guidance presented therein or within the art at the time of filing, and practice the claimed method without first making a substantial inventive contribution. Given the teachings described above, an artisan of ordinary skill would not be able to use the invention as claimed with a reasonable expectation of success. The amount of experimentation required for enabling guidance commensurate in scope with what is claimed goes beyond what is considered “routine” within the art and constitutes undue further experimentation in order to successfully use the method that encompasses treating AD or FTD in a human subject with the methods recited by Claims 22 and 32-41with a reasonable expectation of success.
In conclusion, it is possible the claims as written above could be enabled, if Applicant were to provide data showing their expression and RNAi constructs increase/reduce gene and protein expression and statistical analyses of lifespan data, as well as address on the record the discrepancies in their own data/conclusions and teachings of Anisimova, Ohno, and Zaccara discussed above.
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 15 and 24 are rejected under 35 U.S.C. 112(b) or 35 U.S.C. 112 (pre-AIA ), second paragraph, as being indefinite for failing to particularly point out and distinctly claim the subject matter which the inventor or a joint inventor (or for applications subject to pre-AIA 35 U.S.C. 112, the applicant), regards as the invention. This rejection is new.
A claim may be considered indefinite if the resulting claim does not clearly set forth the metes and bounds of the patent protection desired. See MPEP § 2173. In the present instance, Claims 15 and 24 recite …wherein the nucleic acid molecule encodes a YTHDF1 protein operably linked to a promoter… wherein the nucleic acid molecule is an mRNA molecule. The claim(s) are considered indefinite because there is a question or doubt as to what are the metes and bounds of the claim because it is not clear how an artisan would operably link an mRNA to a promoter.
There is no reason why a nucleic acid molecule would comprise both a promoter and an mRNA. An mRNA obviates the need for any promoter. The art of Clancy (et al. 2008. Translation: DNA to mRNA to Protein. Nature Scitable Nature Education 1[1]:1010, “Scitable”) teaches that (Figs. 1 and 3) DNA comprises a promoter that is bound by RNA polymerase which transcribes the DNA[Wingdings font/0xE0]mRNA. mRNA includes no promoter and is itself translated to protein. Therefore, it does not make sense for mRNA to be a nucleic acid that is operably linked to any promoter, and an artisan would not readily understand the claim.
Therefore, in the interest of compact prosecution, the claims are interpreted as requiring mRNA and no promoter.
Claims 20, 15-16, 18-19, 22, 24-26, 30, and 32-37 are rejected on the basis that it contains an improper Markush grouping of alternatives. See In re Harnisch, 631 F.2d 716, 721-22 (CCPA 1980) and Ex parte Hozumi, 3 USPQ2d 1059, 1060 (Bd. Pat. App. & Int. 1984). A Markush grouping is proper if the alternatives defined by the Markush group (i.e., alternatives from which a selection is to be made in the context of a combination or process, or alternative chemical compounds as a whole) share a “single structural similarity” and a common use. A Markush grouping meets these requirements in two situations. First, a Markush grouping is proper if the alternatives are all members of the same recognized physical or chemical class or the same art-recognized class, and are disclosed in the specification or known in the art to be functionally equivalent and have a common use. Second, where a Markush grouping describes alternative chemical compounds, whether by words or chemical formulas, and the alternatives do not belong to a recognized class as set forth above, the members of the Markush grouping may be considered to share a “single structural similarity” and common use where the alternatives share both a substantial structural feature and a common use that flows from the substantial structural feature. See MPEP § 2117.
The Markush groupings of (1) a promoter that is promoter is a neuron-specific promoter, a glial cell-specific promoter, an astrocyte-specific promoter, a microglial cell-specific promoter, or an oligodendrocyte-specific promote (Claims 20, 15-16, 18-19, 22, 24-26, 30, and 32-37) and (2) a promoter that is a synapsin I promoter, a calcium/calmodulin-dependent protein kinase II promoter, a tubulin alpha I promoter, a neuron-specific enolase promoter, and a platelet-derived growth factor beta chain promoter (Claims 16 and 36-37) are improper because the alternatives defined by the Markush grouping do not share both a single structural similarity and a common use for the following reasons:
The alternatives recited in the claims include very different promoters. Although all of the promoters are made out of nucleotides, each recited promoter is a cell-specific promoter or a kind of cell specific promoter and each of those has a different structure of nucleotides. The promoters also lack a common use. For example, each promoter is used to control expression in one cell type. The cell-specific promoters could not be used in place of one another.
Claims 20, 16, and 36-37 are rejected for those reasons. Claims 15-16, 18-19, 22, 24-26, 30, and 32-37 are rejected because they depend from Claim 20 and don’t remedy the issues.
To overcome this rejection, Applicant may set forth each alternative (or grouping of patentably indistinct alternatives) within an improper Markush grouping in a series of independent or dependent claims and/or present convincing arguments that the group members recited in the alternative within a single claim in fact share a single structural similarity as well as a common use.
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.
Claim(s) 20, 15-16, and 25-26 are rejected under 35 U.S.C. 103 as being unpatentable over Shi (et al. 2018. m6A facilitates hippocampus-dependent learning and memory through YTHDF1. Nature 563:249-253, “Shi”, of record on IDS), Pardridge (10 Jan 2020. Blood-Brain Barrier and Delivery of Protein and Gene Therapeutics to Brain. Front. Aging Neurosci. 11:373, “Pardridge”, of record on previous 892), Combs (et al. 2016. Gene Therapy Models of Alzheimer’s Disease and Other Dementias. Chapter 15 in Gene Therapy for Neurological Disorders: Methods and Protocols, Fredric P. Manfredsson (ed.). Methods Molec. Biol. 1382:339-366, “Combs”), and International Publication Number WO 2020/028751 (published 06 February 2020, “WO751”). This rejection is updated in view of the claim amendments.
Shi teaches m6a (m6a) works through YTHDF1 to promote protein translation of target transcripts in response to neuronal stimuli, thereby facilitating learning and memory. Shi teaches (§Main text p. 250, full ¶3; Fig. 3) they re-expressed YTHDF1 in the hippocampus of adult YTHDF1-KO mice. Shi describes (same § and ¶) they delivered via bilateral stereotactic injection an AAV expressing YTHDF1 under (see Fig. 3) the control of a CMV promoter which is a heterologous promoter. Fig. 3C and Extended data Fig. 5 show the mouse hippocampus expressed YTHDF1. The hippocampus comprises neurons. Shi’s experiment demonstrates that their construct was configured to promote expression of the nucleic acid molecule in neurons. Extended data Fig. 5b shows YTHDF1 immunostaining in the same tissue, indicating the construct caused YTHDF1 protein expression in at least one cell of the mouse’s central nervous system.
Shi teaches encapsulating the nucleic acid in an AAV (which can be considered a nanoparticle) but does not teach encapsulating it in a LNP. Shi does not teach administering to a human subject, a neuron, glial cell, astrocyte, microglial cell, or oligodendrocyte-specific promoter, and does not disclose the dose they used.
Shi does not teach administering a DNA molecule or that the nucleic acid molecule at a dose of 5-10 or 10-15 µg per dose.
However, Pardridge, drawn to blood-brain barrier and delivery of protein and gene therapeutics to brain, teaches (§Brain Adeno-Associated Virus Gene Therapy ¶1) AAV gene therapy can cause immune responses. Pardridge teaches (§NON-VIRAL GENE THERAPY OF THE BRAIN WITH TROJAN HORSE LIPOSOMES [THL]-Safety of THL Gene Therapy of Brain ¶1) THL are a kind of liposome comprising plasmid DNA and they elicit no immune reactions and no neuroinflammation.
Regarding the dosing of Claim 20, Pardridge teaches (§NON-VIRAL GENE THERAPY OF THE BRAIN WITH TROJAN HORSE LIPOSOMES [THL]-Brain Delivery of Reporter Genes With THLs ¶1) 12 µg/kg plasmid DNA (pDNA) was successfully delivered to a primate brain. Pardridge teaches (same §, ¶2) whereas AAV has the size limitation of a 2-4 kB transgene, the pDNA can be as large as 22 kB. Pardridge teaches (same § and ¶) using tissue-specific promoters that restrict gene expression to a target tissue.
Pardridge further teaches (§NON-VIRAL GENE THERAPY OF THE BRAIN WITH TROJAN HORSE LIPOSOMES-THL Gene Therapy of Parkinson’s Disease, entire §) administering liposomes comprising pDNA encoding a target gene under the regulation of a brain-specific promoter to rats. Pardridge teaches (same §, ¶3) the rats were treated with 10 µg liposomes carrying the pDNA. Pardridge describes (same §, Fig. 8) the target gene was delivered to neurons.
Those data demonstrate that at time of filing the claimed invention, it was routine and conventional in the art to administer a nucleic acid molecule at a dose of ≈20 µg per dose. They also demonstrate delivery to neurons.
Regarding Claim 25, Pardridge teaches (§NON-VIRAL GENE THERAPY OF THE BRAIN WITH TROJAN HORSE LIPOSOMES-Safety of THL Gene Therapy of Brain) THL are administered weekly. “Weekly” is 4 times per month. Therefore Pardridge teaches the limitations of Claim 25.
Shi and Pardridge do not explicitly teach using a neuron-, glial cell–, astrocyte-, microglial cell–, or oligodendrocyte-specific promoter (Claim 20). Shi and Pardridge do not explicitly teach using a synapsin I promoter.
However, Combs and WO751 teach limitations that Shi and Pardridge do not teach.
Combs, drawn to gene therapy models of Alzheimer’s disease (AD) and other dementias, teaches various cell type specific promoters. Combs teaches (§5 Potential Caveats with Viral Vector Systems ¶2) using cell type-specific promoters to increase specificity to cell type populations or subpopulations. Combs teaches (same § and ¶) the neuron-specific promoter synapsin I as well as glial-specific promoters. Combs teaches (§9 rAAV-Mediated Expression of Other AD- and Dementia-Related Proteins ¶3) promoters that limit expression to astrocytes.
WO751, drawn to compositions for formulating tissue-tropic AAV, teaches (¶157) cell type-specific promoters that restrict expression of a payload to specific cell types. WO751 teaches (same ¶) cell type specific promoters that restrict expression to microglia, astrocytes, oligodendrocytes, and others.
Although Combs and WO751 are largely directed to AAV gene therapy, an artisan of ordinary skill understands that genetic material such as a promoter can be used in AAV gene therapy or in pDNA gene therapy, and will have the same effect(s) on cell type specific expression no matter how it is administered.
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the nucleic acid encoding YTHDF1 mRNA and administration via brain injection of Shi with the THL and pDNA plasmid administered at a weekly dose of 10 µg per dose of Pardridge and the cell-type specific promoters of Combs and WO751 for the benefit of improving memory and learning in a human using a system that doesn’t cause an immune reaction. It would have been obvious to encode the YTHDF1 mRNA of Shi onto the pDNA plasmid of Pardridge under the control of the synapsin I promoter. It would have been obvious to administer the nucleic acid molecule at a weekly dose of 10 µg per dose as taught by Pardridge.
One would have been motivated to do so with a reasonable expectation of success because Shi teaches (§Main text 2-3) Ythdf1 mRNA is preferentially expressed in the hippocampus, a key region in spatial learning and memory and shows (§Figs. 3 and 5) selective YTHDF1 re-expression rescues defects in memory and synaptic plasticity. One would have been motivated to do so because Shi teaches YTHDF1 mediates a process that facilitates learning and memory formation:
(p. 252, left column, full ¶2) m6A methylation of mRNAs facilitates learning and memory formation in the mouse hippocampus, mainly by promoting translation of target transcripts upon neuronal stimulation, and that this effect is mediated through the m6A-binding protein YTHDF1… The presence of YTHDF1 could expedite new protein synthesis that is required for long-lasting changes in synaptic plasticity and thus memory formation… [emphasis added.]
That passage teaches that YTHDF1 protein plays a specific role in memory and learning: promoting translation of target transcripts upon neuronal stimulation. Shi teaches (p. 249, right column, ¶1) long-term memory formation requires protein synthesis.
Shi continues (p. 252, left column, full ¶2): in the hippocampus of Ythdf1-KO mice, stimulus-dependent protein synthesis is attenuated, resulting in less efficient synaptic strengthening and a lower probability of reaching thresholds for memory formation… [emphasis added.] That teaches that memory formation requires a certain threshold of protein synthesis, which in turn requires YTHDF1. Shi teaches that memory formation is at least in part a probability-based outcome that is based on the presence of YTHDF1 functioning in stimulus-dependent protein synthesis. Therefore it would have been obvious for an artisan to try increasing YTHDF1 expression for the benefit of ensuring ample YTHDF1 is present, and thereby reduce the probability of not reaching a threshold of protein synthesis required for memory formation.
One would have been motivated to put the YTFHD1 gene under the control of the neuron-specific promoter—or specifically, the synapsin I promoter—of Combs because Shi describes (p. 250, left column, full ¶1) observing defects in YTHDF1-KO neurons and it would have been obvious to try and remedy those by expressing YTHDF1 in those defective cells. One would have been motivated to use the 10 µg dose and weekly administration of Pardridge because Pardridge’s teachings indicate using that dose and administration scheme was routine and conventional. One would have been motivated to administer the nucleic acid molecule to the brain because Shi teaches (p. 250, left column, full ¶1) defects in the brain. Both Shi and Pardridge indicate administration to the brain was possible: Shi teaches (Fig. 3) injection to the brain and Pardridge teaches (§THL Gene Therapy of Parkinson’s Disease ¶3) that IV administration results in administering the liposome to the brain.
It would have been obvious to use the YTHDF1 encoded on a pDNA of Shi, Pardridge, Combs, and WO751 in a human because Pardridge teaches (§Translation of the THL Technology to Humans ¶1) using THL in human clinical trials.
It would have been obvious to encode the YTHDF1 under the control of any of the promoters taught in the prior art because Combs teaches (§9 rAAV-Mediated Expression of Other AD- and Dementia-Related Proteins, final ¶) the same gene expressed in different cell types can have different effects.
Determining an exact dose and administration schedule would have been a part of routine optimization.
“[C]onducting clinical trials to test for an optimal dose for a drug ‘is generally a routine process[‘].” Eli Lilly and Co. v. Teva Pharmaceuticals USA, Inc., 619 F.3d 1329, 1342 (Fed. Cir. 2010). “In Mayo, the application of the natural law was merely routine optimization of drug dosage to maximize therapeutic effect.” Ariosa Dignostics, Inc. v. Sequenom, Inc., 809 F.3d 1282, 1293 (Fed. Cir. 2015) (Dyk, J., concurring).
Therefore the limitations of Claims 20, 15-16, and 25-26 would have been obvious in view of Shi, Pardridge, Combs, and WO751.
Claim(s) 20, 15-16, and 24-26 are rejected under 35 U.S.C. 103 as being unpatentable over Shi, Pardridge, Combs, and WO751 as applied to claims 20, 15-16, and 25-26 above, and further in view of Tanaka (et al. 2018. In Vivo Introduction of mRNA Encapsulated in Lipid Nanoparticles to Brain Neuronal Cells and Astrocytes via Intracerebroventricular Administration. Molec. Pharmaceut. 15[5]:2060-2067, “Tanaka”, of record). This rejection is updated in view of the claim amendments.
The teachings of Shi, Pardridge, Combs, and WO751 as applicable to Claim(s) 20, 15-16, and 25-26 have been described above.
Shi, Pardridge, Combs, and WO751 teach a method of increasing expression of YTHDF1 protein in a human subject comprising a LNP, wherein the nucleic acid encodes a YTHDF1 operably linked to a neuron-specific promoter, causing the YTHDF1 to be expressed in at least one cell of the subject’s CNS, wherein the nucleic acid molecule is administered in an amount of 10 µg per dose.
Shi, Pardridge, Combs, and WO751 do not teach the nucleic acid molecule is an mRNA.
However, Tanaka, drawn to in vivo introduction of mRNA Encapsulated in lipid nanoparticles to brain neuronal cells and astrocytes via intracerebroventricular administration, teaches (§Abstract) delivery of mRNA encapsulated in LNPs to neurons and astrocytes.
Tanaka teaches (§introduction ¶1) gene therapy wherein genetic material was introduced into the brain putamen was reported to be successful in treating Parkinson’s disease. Tanaka teaches (same §) that outcome indicate[s] the potential feasibility of gene therapy for curing brain disorders which are currently considered to be intractable. Like Pardridge, Tanaka teaches (§Abstract, §Introduction ¶1) virus-based gene vectors (1) raise safety concerns because of immunogenicity and (2) have technological limitations due to size restrictions. Tanaka teaches (§Abstract) mRNA in lipid nanoparticles is a better system for treating brain disorders.
Tanaka teaches (§EXPERIMENTAL SECTION-Preparation of LNPssPalm) LNPs comprising 3 µg mRNA per 45 µL solution. Tanaka teaches (§Conclusion) their successful results demonstrate LNPs represent a potentially useful platform for mRNA-based therapeutics for the treatment of brain disorders. That makes it clear that they envision using their platform and technique for treating conditions that afflict humans and that injection of mRNA to the brain can treat diseases in humans.
Therefore, it would have been obvious to a person of ordinary skill in the art before the effective filing date of the claimed invention to modify the nucleic acid encoding YTHDF1 and brain injection of Shi, Pardridge, Combs, and WO751 with the LNPs encapsulating mRNA at a 3 µg per 45 µL solution dose and brain injection of Tanaka for the benefit of improving memory and learning in a human. One would have been motivated to do so with a reasonable expectation of success because Shi teaches (§Main text 2-3) Ythdf1 mRNA is preferentially expressed in the hippocampus, a key region in spatial learning and memory and shows (§Figs. 3 and 5) selective YTHDF1 re-expression rescues defects in memory and synaptic plasticity. One would have been motivated to encode the YTHDF1 of Shi, Pardridge, Combs, and WO751 as an LNP-encapsulated mRNA or Tanaka for the benefits of administering mRNA which (Tanaka teaches) is less immunogenic. Determining an exact dose and administration schedule would have been a part of routine optimization. One would have had a reasonable expectation of success because both Shi and Tanaka teach expressing a target gene or protein in living animal brains.
“[C]onducting clinical trials to test for an optimal dose for a drug ‘is generally a routine process[‘].” Eli Lilly and Co. v. Teva Pharmaceuticals USA, Inc., 619 F.3d 1329, 1342 (Fed. Cir. 2010). “In Mayo, the application of the natural law was merely routine optimization of drug dosage to maximize therapeutic effect.” Ariosa Dignostics, Inc. v. Sequenom, Inc., 809 F.3d 1282, 1293 (Fed. Cir. 2015) (Dyk, J., concurring).
Therefore modifying the nucleic acid encoding YTHDF1 of Shi, Pardridge, Combs, and WO751 with the LNP-encapsulated mRNA of Tanaka would have produced the limitations of Claim 24 and the mRNA limitation of Claim 15.
Response to Arguments
Applicant's arguments filed 01 December 2025 have been fully considered but they are not persuasive. Arguments that are no longer relevant are not addressed.
Objections
There are minor objections to Claim 20 over punctuation.
112(a) Enablement
Claims 18-19 and 30 are newly rejected under the enablement requirement because Applicant’s Spec. and what was known in the prior art does not enable the invention. The prior and current art indicate that at time of filing the claimed invention it was not possible to measure protein expression in a cell, administer a treatment, and remeasure protein expression in the same cell within the brain of a living human being, and finding a 10% increase in YTHDF1 expression. Applicant’s Spec. does not enable the invention because they have not shown measuring (before treatment) and remeasuring (after treatment) any protein expression in the same brain cell of a living human being, let alone finding a 10% increase. Furthermore, as discussed, those kinds of experiments are not possible for technical, safety, and ethical reasons.
To overcome a prima facie case of lack of enablement, applicant must present argument and/or evidence that the disclosure would have enabled one of ordinary skill in the art to make and use the claimed invention at the time of filing. [emphasis added.]
MPEP §2164.05
Specification Must Be Enabling as of the Filing Date [R-07.2022]
Whether the specification would have been enabling as of the filing date involves consideration of the nature of the invention, the state of the prior art, and the level of skill in the art. The initial inquiry is into the nature of the invention, i.e., the subject matter to which the claimed invention pertains. The nature of the invention becomes the backdrop to determine the state of the art and the level of skill possessed by one skilled in the art.
The state of the prior art is what one skilled in the art would have known, at the time the application was filed, about the subject matter to which the claimed invention pertains. The relative skill of those in the art refers to the skill of those in the art in relation to the subject matter to which the claimed invention pertains at the time the application was filed. See MPEP § 2164.05(b). See Pac. Biosciences of Cal., Inc. v. Oxford Nanopore Techs., Inc., 996 F.3d 1342, 1352, 2021 USPQ2d 519 (Fed. Cir. 2021). [emphasis added.]
MPEP §2164.05(a)
The other enablement rejection of Claims 22 and 32-41 is maintained. Although Applicant has changed the claim dependency, Claims 22 and 32-41 are not enabled because they recite that the human subject has AD or FTD, so the claims are considered methods of treating either of those diseases. Any ability to treat any disease has not been demonstrated in Applicant’s Spec.
Applicant argues (p. 7 ¶2) the amendments to make the claims depend from Claim 20 obviate the rejection and there is no reasonable basis to hold that such methods require more than routine experimentation and that none of these claims require treatment of a symptom of either condition, merely the existence of the condition or an active determination step.
Those arguments are not found persuasive because the claims recite administering the nucleic acid encoding YTHDF1 protein to a human subject who has FTD or AD. As discussed, the Spec. contemplates treating either of those diseases and the broadest reasonable interpretation (BRI) of the claims is a method of treating. After all, why would an artisan administer a nucleic acid to the CNS and obtain YTHDF1 expression in at least one cell in the CNS of a human subject if there were no perceived benefit in doing so? Since the claims recite the human subject has a disease and the Spec. clearly contemplates treating, interpreting the claims that recite a disease as methods of threating that disease is entirely with the BRI of the claims. Therefore Applicant’s arguments cannot be considered to overcome the extensive problems that demonstrate a lack of enablement.
In general, Applicant has not shown that expressing YTHDF1 can treat any symptom of Alzheimer’s disease (AD). Most of Applicant’s data show effects of inhibiting YTHDF. Showing that inhibition of YTHDF causes certain outcomes does not in any way indicate that expressing YTHDF1 can treat a disease, AD, or frontotemporal dementia (FTD), particularly given the lack of crucial experimental controls; the lack of a connection between YTHDF and AD, FTD, or longevity of lifespan; and the discrepancies between what is shown in Applicant’s data and what is known in the art about pathology of AD and longevity of lifespan.
Briefly, an invention directed to a method of treating must demonstrate evidence establishing a nexus (or, in the MPEP, a “correlation”) between the disease and the treatment. In the case of the claimed invention, that nexus has not been demonstrated. Briefly, the method is directed to expressing a nucleic acid encoding a YTHDF1 protein to treat or eliminate any symptom of either FTD or AD.
Applicant has not demonstrated successful expression of YTHDF1 because they have provided no data demonstrating their constructs result in YTHDF1 expression or overall increased YTHDF1 expression. That is why the word “presumed” or “presumably” is used to modify any reported change in expression when discussing Applicant’s data. Applicant has not demonstrated—and there is no demonstrated link known in the art—between any neurodegenerative disease and YTHDF1.
First, Applicant shows only three experiments that could possibly show the effect of expressing a YTHDF protein because only those (presumably) experiments expressed YTHDF:
Figs. 9 and 22 show (presumably) expressing YTHDF in a TDP-43 background increased fly survival. Figs. 15-16 (discussed below) show (presumably) expressing YTHDF reduces puromycin incorporation into nascent proteins.
All other experiments are directed to inhibiting YTHDF:
Applicant demonstrates that (presumably) knocking down YTHDF1 reduces lifespan. Applicant states (¶59; Figs. 7-8) results showing (presumed) KD of YTHDF reduced survival of flies engineered to express TDP-43 compared with flies expressing TDP-43 alone evidences a functional relationship between these two proteins but Applicant has not explained how that finding evidences the functional relationship. Indeed, a finding that YTHDF KD has no further effect on diminishing lifespan compared with TDP-43 alone would be more straightforwardly indicative of a functional relationship. For example, if any gene encoding a protein crucial for metabolism were reduced or eliminated (e.g., ATP synthase), that reduction/elimination would reduce lifespan without necessarily having anything to do a functional relationship with TDP-43. But if both proteins worked in the same pathway, an artisan would expect that the presence of excess TDP-43 would have already inhibited YTHDF so further inhibiting it with RNAi would have no further effect on lifespan.
Applicant’s further experiments (¶62; Figs. 11-12) discuss the effect of (presumably) depleting YTHDF in flies. The Spec. discloses that such depletion results in a reduced ability to withstand stress ( e.g., heat shock) and causes structural anomalies in the brain which resemble anomalies caused by human neurodegenerative diseases and Figs. 11-12 show flies with (presumed) YTHDF KD have more brain vacuoles and says that fly brain vacuoles can be used to quantify neurodegeneration. That indicates that YTHDF1-KD may mimic a phenotype of neurodegenerative disease but it does not demonstrate that expressing YTHDF1 can treat a neurodegenerative disease let (alone FTD or AD).
The Spec. discusses (¶63, Figs. 13-16) (presumed) YTHDF KD increases protein synthesis and that (presumably) expressing YTHDF slightly but statistically significantly reduces protein synthesis vs. control. Notably, the control is not a model for any neurodegenerative disease. Notably, the protein synthesis study did not demonstrate that proteins associated with any neurodegenerative disease are among those elevated by YTHDF KD or decreased by YTHDF expression. Notably, (presumably) expressing YTHDF reduced overall protein synthesis and Applicant didn’t ever measure YTHDF1 protein expression.
The Spec. discusses (¶64-65, Figs. 17-21) experiments showing YTHDF KD has an effect on eif2α phosphorylation levels in the brain. The Spec. discloses that an increased level of phosphorylation of eif2α indicates increased levels of stress granules and cellular stress. Increased eif2α phosphorylation is further known to be associated with human neurodegenerative diseases such as AD. The Spec. discloses (¶65, Fig. 19) YTHDF KD in flies expressing TDP-43 showed more protein expression and (Figs. 20-21) more eif2α protein phosphorylation.
The conclusions that can be drawn from Figs. 15-21 as they pertain to treating neurodegenerative diseases are not clear. The Spec. teaches (¶64, Fig. 18) YTHDF KD increases eif2α phosphorylation compared to control flies (before heat shock, HS), indicative of canonical translational repression. If Fig. 18 shows translation is being repressed in YTHDF KD flies, how does that reconcile with the previous findings (Figs. 13-16) which show that YTHDF KD increases puromycin incorporation into nascent proteins? The Spec. teaches (¶63) puromycin is incorporated into nascent peptides and labels newly synthesized proteins and YTHDF KD causes an increased amount of puromycin incorporation and conversely, upregulation of YTHDF in neurons showed the opposite response, with significant decrease in nascent protein translation. In the Fig. 17-18 experiments, YTHDF KD caused canonical translational repression but in the Fig. 13-16 experiments, YTHDF KD showed more nascent proteins (i.e., more translation). Notably, these issues were mentioned in the Enablement rejection but Applicant’s remarks have not addressed them.
The Spec. discloses (¶65, Figs. 19-21) elevated levels of eif2α phosphorylation are associated with TDP-43 and also with human neurodegenerative diseases generally. These experiments demonstrates that Ythdf is able to reduce the level of eif2α phosphorylation. This set of data further suggests that Ythdf may be administered to a fly to rescue the neurodegeneration phenotype caused by TDP-43.
But the experiments simply do not demonstrate that YTHDF can reduce level of eif2α phosphorylation because no experiment demonstrates that expressing YTHDF has any effect on eif2α phosphorylation. Eif2α phosphorylation was not measured in any experiments wherein YTHDF was expressed. Nor do the data suggest that YTHDF may be administered to rescue the phenotype caused by expressing TDP-43 because the phenotype previously discussed was brain vacuolation and Applicant has shown only that (presumably) expressing YTHDF can (possibly) increase fly lifespan. The only experiments that show (presumed) YTHDF expression have results shown in Figs. 9, 15-16, and 22. All the other experiments look at the effects of decreasing YTHDF using YTHDF KD. Altogether, Applicant has data showing that expressing YTHDF (1) might increase fly life span (if Applicant shows their construct indeed increases YTHDF expression and if the results in Figs. 9 and 22 are statistically meaningful) and (2) reduces puromycin incorporation into nascent proteins, which may indicate reduced translation (based on Figs. 15-16).
None of those experiments demonstrate either a clear link between YTHDF and development of a neurodegenerative disease or treatment—let alone elimination—of a single symptom of any neurodegenerative disease, or the specific diseases FTD or AD. Survival alone is not a symptom of neurodegenerative diseases nor is it specific to neurodegenerative diseases.
The art at time of filing the claimed invention doesn’t discuss a clear nexus between YTHDF1 and FTD or AD. Applicant has not demonstrated a clear correlation between YTHDF1 and any neurodegenerative disease (ND), FTD, and/or AD. Applicant has (presumably) knocked down YTHDF and shows that doing so reduces lifespan and results in brain vacuoles. But that does not mean that YTHDF is related to ND, FTD, and/or AD. Results showing that YTHDF is reduced in individuals suffering from a ND would show some relationship. Furthermore, even in the experiments where they expressed YTHDF, Applicant didn’t investigate how that expression affects TDP-43. That is to say, many things reduce lifespan, reduce protein translation, or produce brain vacuolization. That doesn’t mean that YTHDF is related or that expressing YTHDF is would treat those conditions or that an artisan would deem it beneficial to administer a nucleic acid encoding YTHDF1 to the CNS of a human patient with FTD or AD. Furthermore, Applicant has not demonstrated that depletion of a protein having one result means that expressing the protein would have the opposite result. Then, there is the fact that Applicant’s claims are reasonably interpreted as being directed to treating or eliminating a symptom of FTD or AD but Applicant has not shown remediation of any symptom.
The issue is not exactly with using drosophila as the model organism as it is with the absence of a drosophila model of a neurodegenerative disease. As discussed in the rejection, the art teaches that excess TDP-43 can be a model for FTD and that plaques and neurofibrillary tangles in the brain are pathogenic features of AD. Applicant has used flies that model FTD in only a single experiment wherein they also apply the claimed method, namely expressing YTHDF. Results from that experiment are shown in Fig. 9 but as discussed below, it is not possible to draw conclusions because no statistical evaluation has been made. All other experiments wherein Applicant has expressed YTHDF are simply in a WT background (or at least no discussion of a disease model was made). That is why Applicant’s method is determined to be unpredictable: a nexus between the disease and YTHDF was not known, Applicant doesn’t show such a nexus, and Applicant doesn’t show remediating symptoms of the disease (again, the claimed method of administering to a human subject who has FTD or AD is reasonably interpreted as treating or eliminating a symptom). Applicant is invited to clearly explain the nexus between eif2α and ND/FTD/AD, and then explain how expressing YTHDF affects that.
In short, the issue is that there is no art-recognized nexus between ND/FTD/AD and YTHDF1, and Applicant does not provide data evidencing that nexus and then evidencing how expressing YTHDF1 positively impacts on the disease symptoms. The art does recognize relationships between certain proteins and ND/FTD/AD but those were not used in the Spec., or were used minimally in conjunction with the claimed method, as explained.
Furthermore, determination of statistical significance is necessary to show whether the small differences in lifespan are meaningful. Applicant has only three experiments showing the effect of expressing YTHDF and two of those are related to lifespan. As discussed, there is no known nexus between YTHDF and ND/FTD/AD, so those two experiments are part of what Applicant is asserting establishes that nexus. To evaluate what conclusions may be drawn from the data it is crucial to evaluate their strength. Part of that is asking whether the differences in lifespan/survival are meaningful.
Applicant is invited to present additional data demonstrating the claimed invention is enabled. Such data would include showing, in a TDP-43 expressing fly (thereby producing a ND model), the benefits of expressing YTHDF (thereby applying the claimed method) on reducing TDP-43 levels, reducing brain vacuolization, and/or improving symptoms or pathology of ND/FTD/AD that are recognized as ND/FTD/AD symptoms or pathologies in flies.
Altogether, in the instant case, the evidence supporting expressing YTHDF treats AD or FTD is not credible for the reasons discussed: the fact that Applicant did not use a model relevant to AD; the lack of crucial experimental controls; the absence of a known-in-the-art or demonstrated-by-Applicant link between YTHDF and AD/FTD/lifespan; the specific discrepancies in Applicant’s data, their interpretation of their data and what is known in the art, without any discussion or explanation of those discrepancies.
Therefore the rejection on the basis of lack of enablement must be maintained.
112(b)
The Claims are indefinite because an artisan wouldn’t readily understand the notion of linking mRNA to a promoter. As discussed in the rejection, Scitable teaches that mRNA is itself readily translated to protein and does not comprise any promoter.
The Markush rejection is applied because the various promoters lack a shared structural similarity and a common use. The promoters for cell-specific expression couldn’t be used in place of one another because they are for promoting expression in specific cell types.
103
Claims 20, 15-16, and 24-26 are is rejected under 103 because the claimed method would have been obvious in view of Shi, Pardridge, Combs, WO751, and Tanaka. Applicant argues that the rejection doesn’t comprise the claim elements but that is not found persuasive because Pardridge, Combs, and WO751 teach all of the cell-type specific promoters and it would have been obvious to use them to target YTHDF1 expression specifically to neurons because Shi describes observing defective neurons in their YTHDF1-KO mice.
Regarding Applicant’s arguments that an artisan would not have had any reason to perform steps (a) and (b) in a subject found to have FTD or AD, note that Claim 20 and the other claims rejected under 103 do not recite any FTD or AD.
In response to applicant's argument that the references fail to show certain features of the invention, it is noted that the features upon which applicant relies (i.e., any benefit for FTD or AD) are not recited in the rejected claim(s). Although the claims are interpreted in light of the specification, limitations from the specification are not read into the claims. See In re Van Geuns, 988 F.2d 1181, 26 USPQ2d 1057 (Fed. Cir. 1993).
The reason provided in the rejection for administering a nucleic acid encoding YTHDF1 is to improve memory and learning. Applicant argues that:
reexpression rescuing a decrease or loss of function does not provide a basis for a reasonable expectation that supplementation of YTHDF1 expression would provide a benefit, applying the same logic asserted in the Office Action with respect to Applicant’s own knockdown data. In short, Shi fails to disclose supplementation above endogenous expression levels provides a benefit in a healthy individual.
First, that argument is not persuasive because the motivation to combine falls under an “obvious to try” rationale; see MPEP 2143(I)(E):
To reject a claim based on this rationale, Office personnel must resolve the Graham factual inquiries. Then, Office personnel must articulate the following:
(1) a finding that at the relevant time, there had been a recognized problem or need in the art, which may include a design need or market pressure to solve a problem;
(2) a finding that there had been a finite number of identified, predictable potential solutions to the recognized need or problem;
(3) a finding that one of ordinary skill in the art could have pursued the known potential solutions with a reasonable expectation of success; and
(4) whatever additional findings based on the Graham factual inquiries may be necessary, in view of the facts of the case under consideration, to explain a conclusion of obviousness.
The rationale to support a conclusion that the claim would have been obvious is that "a person of ordinary skill has good reason to pursue the known options within his or her technical grasp. If this leads to the anticipated success, it is likely that product [was] not of innovation but of ordinary skill and common sense.
Regarding (1): Although Shi administered the nucleic acid encoding YTHDF1 to YTHDF1-KO mice, Shi teaches YTHDF1 mediates a process that facilitates learning and memory formation:
(p. 252, left column, full ¶2) m6A methylation of mRNAs facilitates learning and memory formation in the mouse hippocampus, mainly by promoting translation of target transcripts upon neuronal stimulation, and that this effect is mediated through the m6A-binding protein YTHDF1… The presence of YTHDF1 could expedite new protein synthesis that is required for long-lasting changes in synaptic plasticity and thus memory formation… [emphasis added.]
That passage teaches that YTHDF1 protein plays a specific role in memory and learning: promoting translation of target transcripts upon neuronal stimulation. Shi teaches (p. 249, right column, ¶1) long-term memory formation requires protein synthesis.
Shi continues (p. 252, left column, full ¶2): in the hippocampus of Ythdf1-KO mice, stimulus-dependent protein synthesis is attenuated, resulting in less efficient synaptic strengthening and a lower probability of reaching thresholds for memory formation… [emphasis added.] There, Shi teaches that memory formation requires a certain threshold of protein synthesis, which in turn requires YTHDF1. Shi teaches that memory formation is at least in part a probability-based outcome that is based on the presence of YTHDF1 functioning in stimulus-dependent protein synthesis. Since YTHDF1 is upstream of protein translation—and Shi shows it to be a limiting factor in determining the probability of memory formation—an artisan would reasonably conclude that increasing the amount of YTHDF1 would remove it as a limiting factor in protein synthesis, and the outcome of removing that limiting factor would be an increased probability of protein synthesis and, therefore, an increased probability of memory formation.
In short, the “problem to be solved” is the fact that availability of YTHDF1 is a limiting factor in stimulus-dependent protein synthesis and, therefore, a factor that limits the probability of reaching a threshold level of protein synthesis for memory formation to occur.
Regarding (2), there are a finite number of solutions because the single solution to solving the problem of availability of YTHDF1 in protein translation (and the resulting effect on changing the probability of memory formation) is to increase the amount of YTHDF1.
Regarding (3), Shi and the other cited art indicate that one of ordinary skill in the art could have pursued the known potential solution of expressing YTHDF1 with a reasonable expectation of success.
Therefore the combination of prior art fulfills the requirements of an obvious to try rationale.
Furthermore, Applicant’s arguments that there is no reasonable expectation that supplementation of YTHDF1 expression would provide a benefit in a healthy individual are unpersuasive because the claims don’t recite any outcomes in any individual. The claims recite only a method of increasing YTHDF1 protein that requires a single active method step of administering a nucleic acid encapsulated by a LNP. The 103 rejection shows that the active method step would have been obvious in view of the prior art. Shi’s teachings in particular would have motivated an artisan to express a protein (i.e., YTHDF1) that Shi teaches is necessary for memory formation and whose absence reduces the probability of reaching thresholds for memory formation. Note that Shi’s Extended data Fig. 5 demonstrate that their expression construct results in YTHDF1 expression.
In contrast, the Enablement rejections discuss (1) the unpredictability of measuring protein expression in a brain cell in a human subject in vivo and then remeasuring protein expression in the same brain cell and (2) the unpredictability of treating FTD or AD by expressing YTHDF1 because of the lack of any nexus between YTHDF1 and treating FTD or AD, including because crucial controls weren’t performed (including controls that demonstrate that the constructs used in the Spec.’s experiments result in YTHDF1 expression).
Therefore the 103 rejection is maintained.
Conclusion
Claims 15-16,18-20,22,24-26,30 and 32-41 are rejected.
Any inquiry concerning this communication or earlier communications from the examiner should be directed to RUTHIE S ARIETI whose telephone number is (571)272-1293. The examiner can normally be reached M-Th 8:30AM-4PM, alternate Fridays 8:30AM-4PM (ET).
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RUTHIE S ARIETI
Examiner (Ruth.Arieti@uspto.gov)
Art Unit 1635
/RUTH SOPHIA ARIETI/Examiner, Art Unit 1635
/NANCY J LEITH/Primary Examiner, Art Unit 1636