Notice of Pre-AIA or AIA Status
The present application, filed on or after March 16, 2013, is being examined under the first inventor to file provisions of the AIA .
Claim Rejections - 35 USC § 101
35 U.S.C. 101 reads as follows:
Whoever invents or discovers any new and useful process, machine, manufacture, or composition of matter, or any new and useful improvement thereof, may obtain a patent therefor, subject to the conditions and requirements of this title.
Claims 1 and 5 are rejected under 35 U.S.C. 101 because the claimed invention is directed to an abstract idea without significantly more.
Claim 1 recites:
a) determining a difference between a first amount of a glycosylated form of alpha-dystroglycan (αDG) in a first sample taken from a subject undergoing treatment for a dystoglycanopahty at a first time and at a second amount of αDG in a second sample taken from the subject at a second time;
b) based at least in part on a),
determining that the treatment should be continued,
determining that the treatment should be discontinued,
determining that the treatment should be adjusted [emphasis added]
Claim 5 recites:
further comprising f) based at least in part on the difference determined in e), i) determining that the treatment should be continued, ii) determining that the treatment should be discontinued, or iii) determining that the treatment should be increased or decreased.
This judicial exception is not integrated into a practical application because the abstract ideas [see the italicized limitations above] are not integrated into a practical application [such as a treatment that is specifically tailored to a disease/disorder].
Examiner notes that “a subject undergoing treatment”, such as recited in claim 1, lines 3, is not a practical application.
Examiner also notes that claim 5 includes an alternative in which there is no treatment [“ii) determining that the treatment should be discontinued”].
The claim(s) does/do not include additional elements that are sufficient to amount to significantly more than the judicial exception because the claims do not recite additional elements that are significantly more than conventional, well-known, and routine in the art. Claim 1 for example recites the step of a) determining a difference at a high level of generality, and thus encompass well-known techniques in the art.
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 16-17 and 20-22 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 written description requirement. The claims contain subject matter which was not described in the specification in such a way as to reasonably convey to one skilled in the relevant art that the inventor or a joint inventor, or for pre-AIA the inventors, at the time the application was filed, had possession of the claimed invention. This is a written description rejection.
The claims require:
“wherein determining the difference between the first amount and the second amount comprises performing a Western blotting analysis”, (see claim 16; and 17, which depends on claim 16), which, as is well-known in the art, implies a required use of antibodies having particular specificities (i.e., function);
"wherein determining the first amount and the second amount comprises contacting the first sample and the second sample with one or more antibodies” (see claim 20), which implies a required use of antibodies having particular specificities (i.e., function);
"wherein an antibody of the one or more antibodies is used to determine an amount of the glycosylated form of αDG having a molecular weight in the range of interest of between about 125 kiloDaltons (kDa) and about 260 kDa” (see claim 21), which implies a required use of an antibody having a particular specificity (i.e., function); and
“wherein the one or more antibodies are selected from AF6868 alpha-dystroglycan, IIH6C4 alpha-dystroglycan, IR800CW Mouse Anti-Sheep, and IR680 Goat Anti-Mouse antibodies” (see claim 22), which requires antibodies of specific types (i.e., functions).
The specification does not describe which amino acid residues, or other molecular components are responsible for the functions claimed. Rather, potential agents must first be screened in an assay to ascertain if the agents have the functions required by the instant claims. The specification fails to disclose the structures common to all members of the genus of peptides encompassed by the broad definition provided by applicant. The specification does not disclose the structure of all of the claimed variant agents and fails to disclose which regions of the agents are responsible for the functions claimed. In the absence of a known or disclosed correlation between structure and function, claims which encompass variants defined by their function are generally not considered described.
Applicant is directed to MPEP § 2163 for guidelines on compliance with the written description requirement. Here, applicant has not described a reasonable number of members of the genus of agents, i.e. the required starting materials for the claims, but rather has presented the public with an idea of how to perform an assay that might identify some peptides that fall within the scope of the claim. Of course, depending on what agents are used in the screening assay, it may well identify none. The Court of Appeals for the Federal Circuit addressed claims of this sort in great detail in University of Rochester v. G.D. Searle and Co. (69 USPQ 2nd 1886, CAFC 2004). In Rochester, the Federal Circuit upheld the district court's ruling that patent claims which recited administration of compounds not disclosed, but rather to be identified in a screening assay, were invalid on their face.
The specification does not describe the structure of the full genus of antibodies responsible for each of the functions claimed. The Federal Circuit has clarified Written Description as it applies to antibodies in the recent decision Amgen v. Sanofi, 872 F.3d 1367 (Fed. Cir. 2017). The Federal Circuit explained in Amgen that when an antibody is claimed, 35 U.S.C. 112(a) (or pre-AIA first paragraph) requires adequate written description of the antibody itself. Amgen, 872 F.3d at 1378-79. The Amgen court expressly stated that the so-called “newly characterized antigen” test, which had been based on an example in USPTO-issued training materials and was noted in dicta in several earlier Federal Circuit decisions, should not be used in determining whether there is adequate written description under 35 U.S.C. 112(a) for a claim drawn to an antibody. Citing its decision in Ariad Pharmaceuticals, Inc. v. Eli Lilly & Co., the court also stressed that the “newly characterized antigen” test could not stand because it contradicted the quid pro quo of the patent system whereby one must describe an invention in order to obtain a patent. Amgen, 872 F.3d at 1378-79, quoting Ariad, 598 F.3d 1336, 1345 (Fed. Cir. 2010). In view of the Amgen decision, adequate written description of an antigen alone is not considered adequate written description of a claimed antibody to that antigen, even when preparation of such an antibody is routine and conventional. Id.
While generically the structure of antibodies is known, the structure of the presently recited antibodies can vary substantially within the above given claimed recitations. As noted in Amgen, knowledge that an antibody binds to a particular epitope on an antigen tells one nothing at all about the structure of the antibody, wherein “instead of analogizing the antibody-antigen relationship to a ‘key in a lock,’ it [is] more apt to analogize it to a lock and ‘a ring with a million keys on it.” (Internal citations omitted). The relevant antibody art confirms this quandary, indicating that “knowledge of an epitope or antigen used to generate a monoclonal antibody is insufficient for making the original antibody available, even if suitable in vitro test systems for screening are used.” See p. 8, lines 3-5 of WO 2009/033743 A1. Therefore, those of skill in the art would not accept that the inventor had been in possession of the full genus of antibodies presently claimed.
Functionally defined genus claims can be inherently vulnerable to invalidity challenge for lack of written description support, especially in technology fields that are highly unpredictable, where it is difficult to establish a correlation between structure and function for the whole genus or to predict what would be covered by the functionally claimed genus. Abbvie Deutschland GMBH & Co. v. Janssen Biotech, Inc. (759 F.3d 1285 (Fed. Cir. 2014). “When a patent claims a genus using functional language to define a desired result, the specification must demonstrate that the applicant has made a generic invention that achieves the claimed result and do so by showing that the applicant has invented species sufficient to support a claim to the functionally-defined genus." Capon v. Eshhar, 418 F.3d 1349 (Fed. Cir. 2005).
Consequently, in the absence of sufficient recitation of distinguishing identifying characteristics, the specification does not provide adequate written description of the claimed genus of binding agents (including antibodies) nor guidance as to which of the myriad of molecules encompassed by the binding agents would meet the limitations of the claims. Further, given the well-known high level of polymorphism of immunoglobulins and antibodies, the skilled artisan would not have recognized that applicant was in possession of the vast repertoire of antibodies encompassed by the claimed invention.
Vas-Cath Inc. v. Mahurkar, 19 USPQ2d 1111 (Fed. Cir. 1991), clearly states that “applicant must convey with reasonable clarity to those skilled in the art that, as of the filing date sought, he or she was in possession of the invention. The invention is, for purposes of the ‘written description’ inquiry, whatever is now claimed.” (See page 1117). The specification does not “clearly allow persons of ordinary skill in the art to recognize that [he or she] invented what is claimed.” (See Vas-Cath at page 1116).
The skilled artisan cannot envision the detailed chemical structure of the genus of binding agents or antibodies, and therefore conception is not achieved until reduction to practice has occurred, regardless of the complexity or simplicity of the method of identification. Adequate written description requires more than a mere statement that it is part of the invention and reference to a potential method of isolating it. The compound itself is required. See Fiers v. Revel, 25 USPQ2d 1601 at 1606 (CAFC 1993) and Amgen Inc. v. Chugai Pharmaceutical Co. Ltd., 18 USPQ2d 1016 (Fed. Cir. 1991). Therefore, the instant claims do not meet the written description provision of 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph.
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 1, 2, 5, 8, 10, 14-17, 20-25, 27-28, 32-35 and 40 are rejected under 35 U.S.C. 112(a) or 35 U.S.C. 112 (pre-AIA ), first paragraph, because the specification, while being enabling for dystroglycanophathy that is LGMD21/R9, does not reasonably provide enablement for other types of dystroglycanopathy. The specification does not enable any person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
While Applicant has support for the claimed invention regarding a subject undergoing treatment for a dystroglycanopathy that is specifically LGMD21/R9, there is no support for the claimed method regarding other types of dystroglycanopathy, despite Applicant’s specification listing other types of dystroglycanopathy.
Examiner directs Applicant’s attention to MPEP §2164.01(a), listing the Wands factors, to guide the assessment of whether a patent specification enables a person skilled in the art to make and use the claimed invention.
Applicant’s claims are broader than what the disclosure supports, and such broader claimed invention requires more experimentation, given the lack of predictability in biochemistry, biological pathways, and the diagnostic art. Applicant’s example and data (such as in example 5, for the claimed invention as it relates to dystroglycanopathy that is specifically LGMD21/R9, such as in paras. 0060, 0061, 0064) does not provide sufficient predictability to ensure that the method works for any and all dystroglycanopathy. While Applicant lists dystroglycanopathy other than LGMD21/R9 (such as in para. 0062), such mentioning does not provide the support necessary to show that the invention is enabled for these other types of dystroglycanopathy.
Thus the amount of experimentation required under these circumstances renders Applicant’s specification as not enabling to a person skilled in the art to which it pertains, or with which it is most nearly connected, to make and use the invention commensurate in scope with these claims.
Claim Rejections - 35 USC § 103
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.
Claim(s) 1, 2, 5, 8, 10, 14-17, 20-25, 27-28, 32-35 and 40 is/are rejected under 35 U.S.C. 103 as being unpatentable over US 20190008881 (hereinafter “Lu”).
Applicant’s claim 1 recites:
A method, comprising:
determining a difference between a first amount of a glycosylated form of alpha- dystroglycan (aDG) in a first sample taken from a subject undergoing treatment for a dystroglycanopathy at a first time and a second amount of aDG in a second sample taken from the subject at a second time;
and b) based at least in part on a), i} determining that the treatment should be continued, ii) determining that the treatment should be discontinued, or iii) determining that the treatment should be adjusted.
Applicant’s claims would have been obvious to one skilled in the art given the following disclosures by Lu, which discloses all the claimed limitations except for determining whether treatment should be continued, discontinued, or adjusted. The differences between Applicant’s claimed invention and the disclosures by Lu are minor differences that would been obvious to one skilled in the art as may be desirable for assessing the disease and its treatment accordingly.
More specifically, Lu discloses the following.
Dystroglycanopathies are a subset of muscular dystrophies characterized by a secondary defect in glycosylation of alpha-dystroglycan (α-DG). The diseases have been linked to autosomal-recessive mutations in at least 18 different genes. They include fukutin-related protein (FKRP), fukutin, like-acetylglucosaminyltransferase (LARGE), POMGnT1, POMT1, POMT2, Isoprenoid Synthase Domain Containing (ISPD), Transmembrane protein 5 (TMEM5), β1,3-N-acetylglucosaminyltransferase1 (B3GNT1), glycosyltransferase-like domain containing 2 (GTDC2), β3-N-acetylgalactosaminyltransferase 2 (B3GALNT2), DOLK, GMPPB, DMP2, DMP3 and SGK196. Biochemical studies have established direct evidence for involvement of a number of the genes in glycosylation modifications of α-DG. Fukutin and Fukutin related protein (FKRP) genes have been recently proposed as Ribitol-5-P transferase that transfers the phosphorated ribitol to the core sugar chain of α-DG. LARGE protein acts as a bifunctional glycosyltransferase, xylosyltransferase and glucuronyltransferase, producing repeating units of [-3-xylose-α1,3-glucuronic acid-β1-] that is the functional glycan chain linking cell membrane protein and extracellular matrix proteins. This LARGE glycan chain is linked to the core O-mannosyl glycans by tandem ribitols. This linkage is critical for muscle health and lack of FKRP function as the result of gene mutations therefore prevents the production of functional glycosylation of α-DG, and disrupts normal interaction between membrane and connective tissues, leading to muscle fiber damage and muscular dystrophy. Para. 0003.
Mutations in the FKRP gene cause a wide spectrum of disease from a milder form of limb-girdle muscular dystrophy (LGMD2I) to severe Walker-Warburg syndrome (WWS), muscle-eye-brain disease (MEB), and congenital muscular dystrophy type 1D (MDC1D). However, little progress has been made for the treatment of the diseases. There is no effective therapy available and only physical therapy and palliative care are being routinely provided as treatment. Para. 0004.
The disclosed invention provides a method of enhancing functional glycosylation of alpha-dystroglycan (α-DG) in a subject without defects in dystroglycan-related genes and in need thereof, comprising administering to the subject an effective amount of ribitol, thereby restoring or enhancing functional glycosylation of α-DG in the subject. Para. 0006.
In a further aspect, the present invention provides a method of treating muscular dystrophy with the levels of ribitol and CDP-ribitol not affected by the diseases, comprising administering to the subject an effective amount of a ribitol, thereby treating the muscular dystrophy in the subject. Para. 0007.
An additional aspect of this invention is a method of treating a disorder in a subject associated with a mutation in a fukutin related protein (FKRP) gene, comprising administering to the subject an effective amount of a ribitol, thereby treating the disorder in the subject. Para. 0008.
Also provided is a method of treating a disorder associated with a mutation in a fukutin related protein (FKRP) gene in a subject, comprising administering to the subject an effective amount of a ribulose, thereby treating the disorder associated with a mutation in a fukutin related protein (FKRP) gene in the subject. Para. 0013.
Disclosed is a method of treating or inhibiting the development of muscle weakness in a subject that is a carrier of a mutated FKRP gene or without defect in glycosylation of α-DG, comprising administering to the subject an effective amount of a ribulose, thereby treating or inhibiting the development of muscle weakness. Para. 0015.
FIG. 1. Discloses fluorescence-activated cell sorting (FACS) for the enhanced expression of glycosylated alpha-DG after ribitol treatment. The breast cancer cell line MCF-7 was seeded in T25 culture flasks and cultured to 75% confluence in DMEM 10% FBS, and then treated with 10 mM ribitol in the same growth medium for 3 days. The cells were then collected by gentle scrapping and washed twice with PBS. The cells were resuspended in 100 microliter PBS and stained with monoclonal antibody IIH6 (Millipore EMD, 1:100 dilution) for 40 minutes and detected with secondary Alexa 594-labeled goat anti-mouse IgM (Invitrogen). The stained cells were washed and then FACS analyzed for the percentage of positive cells and the signal intensity (Alexa594.007). Untreated MCF-7 cells cultured under the same conditions probed with secondary antibody only (Alexa594.005) and with both IIH6 and the secondary antibody (Alexa594.006) are used as controls. Para. 0017.
Fig. 10. Expression of glycosylated α-DG with IIH6 antibody in the epithelial cells after treatment with ribitol (lane 2) and ribulose (lane 3). Lane 1, control untreated cells; Lane 4, cells treated with ribulose-5-phosphate; Lane 5, cells treated with glucose. Functionally glycosylated alpha-DG is barely detectable in the control and the cells treated with ribulose-5-phosphate and glucose. Para. 0026.
The invention is based on the discovery that ribitol, CDP-ribitol, ribose and/or ribulose can restore and/or enhance functional glycosylation of mainly alpha-dystroglycan (α-DG) in cells without defects in the genes related to muscular dyhstrophy and cells with FKRP mutation. Yet, the same functional glycosylated epitope can also modify other proteins. Therefore, restored or enhanced functional glycosylation by ribitol and/or ribulose is not limited to α-DG and the use of the phrase “functional glycosylation of α-DG” represents functional glycosylation of any protein after the use of ribitol and/or ribulose. Para. 0049.
Thus, in one embodiment, the invention provides a method of restoring and/or enhancing functional glycosylation of alpha-dystroglycan (α-DG) in a subject without defects in dystroglycan-related genes and in need thereof, comprising administering to the subject an effective amount of a ribitol, CDP-ribitol, ribose and/or ribulose, thereby restoring and/or enhancing functional glycosylation of α-DG in the subject. Para. 0050.
The invention also provides a method of treating muscular dystrophy without defects in dystroglycan-related genes (e.g., a muscular dystrophy that is not associated with a defect in glycosylation of α-DG) or defects or abnormalities in levels of the ribitol and CDP-ribitol in a subject, comprising administering to the subject an effective amount of a ribitol, CDP-ribitol, ribose and/or ribulose, thereby treating the muscular dystrophy in the subject. Para. 0051.
Furthermore the invention provides a method of treating a disorder associated with (e.g., caused by or resulting from) a mutation in a fukutin related protein (FKRP) gene in a subject, comprising administering to the subject an effective amount of a ribitol, CDP-ribitol, ribose and/or ribulose, thereby treating the disorder associated with a mutation in a fukutin related protein (FKRP) gene disorder associated with a mutation in a fukutin related protein (FKRP) gene in the subject. Para. 0052.
The present invention further provides a method of treating a disorder associated with a defect in glycosylation of alpha-DG, comprising administering to a subject that has or is suspected of having a disorder associated with a defect in glycosylation of alpha-DG an effective amount of an active agent and/or composition of this invention. A subject can be suspected of having a defect in glycosylation of alpha-DG if the subject has muscle weakness even in cases where genetic and biochemical analyses of the subject have failed to identify a causative gene detect. Para. 0069.
Provided is a method of treating a disorder associated with a defect in glycosylation of alpha-DG caused by a mutation in the FKRP gene, comprising administering to a subject that has or is suspected of having a mutation in the FKRP gene an effective amount of an active agent and/or composition of this invention. A mutation in an FKRP gene can be identified by genetic analysis of the nucleic acid of a subject. Para. 0071.
The subject is any animal that can receive a beneficial and/or therapeutic effect from restoration of functional glycosylation of alpha-dystroglycan (α-DG) and/or enhancement of glycosylation of α-DG. Para. 0082.
“Treat,” “treating” or “treatment” as used herein also refers to any type of action or administration that imparts a benefit to a subject that has a disease or disorder, including improvement in the condition of the patient (e.g., reduction or amelioration of one or more symptoms), healing, etc. Para. 0084.
The terms “therapeutically effective amount,” “treatment effective amount” and “effective amount” as used herein are synonymous unless otherwise indicated, and mean an amount of a compound, peptide or composition of the present invention that is sufficient to improve the condition, disease, or disorder being treated and/or achieved the desired benefit or goal (e.g., control of body weight). Those skilled in the art will appreciate that the therapeutic effects need not be complete or curative, as long as some benefit is provided to the subject. Para. 0085.
Determination of a therapeutically effective amount, as well as other factors including dosage forms, routes of administration, and frequency of dosing, may depend upon the particulars of the condition that is encountered, including the subject and condition being treated or addressed, the severity of the condition in a particular subject, the particular compound being employed, the particular route of administration being employed, the frequency of dosing, and the particular formulation being employed. Determination of a therapeutically effective treatment regimen for a subject of this invention is within the level of ordinary skill in the medical or veterinarian arts. The amount of active ingredient that can be combined with the carrier materials to produce a single dosage form varies depending upon the subject being treated and the particular mode of administration. Para. 0086.
Ribitol treatment for 1.5 months improved muscle pathology with a reduction in the number of centrally nucleated fibers (CNF) and inflammation. This can be most clearly demonstrated in the diseased diaphragm, which undergoes more severe and progressive degeneration and fibrosis. Para. 0119.
Fluorescence-activated cell sorting (FACS) was used to show enhanced expression of glycosylated alpha-DG after ribitol treatment. The breast cancer cell line MCF-7 was seeded in T25 culture flasks and then treated with 10 mM ribitol in the same growth medium. The cells were then collected by gentle scrapping and washed twice with PBS. The cells were resuspended in 100 microliter PBS and stained with monoclonal antibody IIH6 (Millipore EMD, 1:100 dilution) for 40 minutes and detected with secondary Alexa 594-labeled goat anti-mouse IgM (Invitrogen). The stained cells were washed and then FACS analyzed for the percentage of positive cells and the signal intensity (Alexa594.007). Untreated MCF-7 cells cultured under the same conditions probed with secondary antibody only (Alexa594.005) and with both IIH6 and the secondary antibody (Alexa594.006) are used as controls. The cells treated with ribitol and detected with the IIH6 antibody showed clearly higher levels of glycosylated alpha-DG with 82% positive cells compared to the untreated controls with only 45% positive cells (FIG. 1). Para. 0123.
Mutations in Paladin Related Protein (FKRP) gene cause dystroglycanopathy characterized by defects in the O-mannosylation of alpha dystroglycan (α-DG). FKRP functions as a ribitol-5-Phosphate (Rbo5P) transferase and is essential for the synthesis of functionally glycosylated α-DG (F-α-DG). We tested the hypothesis that increase in levels of ribitol, a precursor of FKRP substrate, could enhance the transferase efficiency of mutant FKRPs, most of which retain at least partial function. We demonstrate that ribitol supplementation systemically restored therapeutic levels of F-α-DG in both skeletal and cardiac muscles, and importantly improved muscle pathology and function in an FKRP mutant model. Supplementation of ribitol or its derivatives presents a new approach to compensate for the defect in glycosylation of α-DG with potential to treat more than 90% of FKRP dystroglycanopathies. Para. 0125.
Mutations in the FKRP gene cause muscular dystrophies with a wide variation in severity from mild limb girdle muscular dystrophy (LGMD) 2I to severe congenital muscular dystrophy (CMD), Walker-Warburg syndrome, and muscle-eye-brain disease. Mild LGMD2I is presented predominantly as myopathy with progressive degeneration involving both skeletal and cardiac muscles. The continuous loss of muscle fibers and diminishing capacity of regeneration eventually lead to the loss of muscle volume and increase in fibrotic tissues. Consequently, patients gradually lose mobility with impaired and ultimately failure of respiratory and cardiac functions. The severe forms of the disease can affect central nerve and optical systems with developmental delay and mental retardation. Currently no treatment is available although several experimental therapies are being tested pre-clinically. Para. 0126.
FKRP-related muscular dystrophies belong to a subset of the disease characterized by a common secondary biochemical defect in the glycosylation of alpha-dystroglycan (α-DG). Alpha-DG is a peripheral membrane protein extensively glycosylated with both N- and O-linked glycans, the latter acting as a cellular receptor for laminin and other extracellular matrix (ECM) proteins, including agrin, perlecan, neurexin and pikachurin. Importantly, the interaction of α-DG with ECM proteins is critical for maintaining muscle integrity. The structure of the laminin-binding O-mannosylated glycan on the functionally glycosylated α-DG (F-α-DG) has recently been delineated with the following chain: (3GlcA-1-3Xyl-1)n-3GlcA-1-4Xyl-Rbo5P-1Rbo5P-3GalNAc-1-3GlcNAc-1-4(P-6)Man-1-Thr/ser. The entire process of the glycan chain extension pathway is completed by the following proposed transferase activity, sequentially: POMT1 and POMT2 catalyze the initial O-mannosylation of the proteins. Further extension of the sugar chain is carried out by POMGnT2 (GTDC2) and B3GALNT2, FKTN, FKRP, TMEM5 and B4GAT1 successively. Finally, LARGE acts as a bifunctional glycosyltrasferase having both xylosyltransferaase and glucuuronyltransferase activities, producing repeated units of 3GlcA-1-3Xyl-1. Para. 0127.
The disclosed study employed FKRP mutant mice containing a P448L mutation which is associated with a severe dystrophic phenotype in clinic. Our results show that ribitol supplementation can effectively restore therapeutic levels of F-α-DG and significantly improve muscle pathology. Partial improvement in muscle functions is also achieved without obvious side effects. This constitutes a potentially effective and safe treatment to the diseases. Para. 0128.
One month treatment with ribitol increases glycosylation of α-DG in cardiac and skeletal muscles. In the pilot experiment, 4-week-old P448L mice were treated with drinking water supplemented with 5% ribitol for 1 month. Glycosylation of α-DG was analyzed by immunohistochemistry with a monoclonal antibody, IIH6C4, specifically recognizing the laminin-binding epitopes of F-α-DG. Para. 0129.
Prolonged treatment with ribitol maintains enhanced expression of functionally glycosylated α-DG. To assess whether ribitol treatment can maintain a long term effect on glycosylation of α-DG and whether such effect could improve disease pathology or slow progression, we extended the treatment of 5% ribitol in drinking water to the P448L mutant mice to 3 and 6 months and examined the expression of F-α-DG of the treated mice in comparison with age-matched P448L controls. Consistent with the 1 month treatment, all of the muscles from the 3 and 6 month treated mice showed a clear increase in the levels of F-α-DG by immunofluorescence staining with the IIH6C4. Tissue distribution of the enhanced F-α-DG remained similar to that in 1 month-treated muscles. Nearly all fibers in the cardiac muscle and the majority of fibers in both diaphragm and limb muscles of treated mice were clearly positive. The signal intensity was homogenous in the cardiac muscle, but varied considerably in the skeletal muscles, especially in the diaphragm where both strong and weak expression were observed within vicinity. The majority of muscle fibers in the TA muscles of treated mice contained weak signals and thus more homogeneous when compared to the signal in the same muscle after 1 month ribitol treatment. Signal distribution and intensity for F-α-DG were generally similar in the same muscles between 3 and 6 month ribitol-treated cohorts. Western blot analysis with the IIH6C4 antibody confirmed the enhanced expression of F-α-DG in the muscles of ribitol-treated P448L mice (FIG. 2A). Consistent with the signal intensity shown by immunofluorescence detection after 6-month treatment, the levels of F-α-DG semi-quantified from the western blot were distinctly detected in the cardiac muscle and diaphragm of ribitol-treated animals, reaching up to 6 and 8% of normal levels in C57 mice, respectively (FIG. 2B). Only trace or indistinguishable signal was detected in the muscles of the control P448L mice. To further confirm the ribitol-induced modification of α-DG, cardiac muscle samples were also analyzed by western blot with the antibody AF6868, which detects both the functionally glycosylated form of α-DG (150-250 kDa as multiple bands) and the core α-DG representing species of non-functionally glycosylated α-DG (core α-DG, 75-100 kDa). Ribitol-treated mice showed detectable, although limited, increase of the higher molecular weight bands representing F-α-DG when compared to the P448L control (FIG. 2A). Similarly, laminin overlay assay demonstrated specific bands of the enhanced F-α-DG in ribitol-treated muscles, although only weakly, supporting the functionality of the ribitol-induced IIH6C4 positive α-DG. Para. 0130.
Thus, Lu discloses the fundamental steps and limitations of Applicant’s claims [see for example the highlighted terms above]. The differences between Applicant’s claimed inventions and the disclosures by Lu are minor differences, involving familiar steps with predictable outcome to carry out the assay and treatment as taught by Lu, and thus would been obvious to one skilled in the art as may be desirable for assessing the disease and its treatment accordingly.
Conclusion
Any inquiry concerning this communication or earlier communications from the examiner should be directed to Ann Montgomery whose telephone number is (571)272-0894. The examiner can normally be reached Mon-Fri, 9-5:30 PM PST.
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/Ann Montgomery/Primary Examiner, Art Unit 1678
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