MOUSE MODEL FOR POMPE DISEASE AND METHODS OF USE THEREOF

FIELD OF THE INVENTION

The present invention provides mouse models for Pompe disease and methods of using the same to test agents that may be effective in the treatment of Pompe disease.

BACKGROUND OF THE INVENTION Pompe disease (also known as glycogen storage disease type Il (GSD-II), glycogenosis II, acid maltase deficiency (AMD), acid alpha-glucosidase deficiency, and lysosomal alpha-glucosidase deficiency) is an inherited lysosomal storage disorder caused by deficiency of an enzyme called acid α- glucosidase (GAA). The role of GAA within the body is to break down glycogen, the form of sugar stored in living cells for use as energy. Reduced or absent levels of GAA activity leads to the accumulation of glycogen in the affected tissues, including the heart, skeletal muscles (including those involved with breathing), liver, and nervous system. This accumulation of GAA is believed to cause progressive muscle weakness and respiratory insufficiency in individuals with Pompe disease. Pompe disease can occur in infants, toddlers, or adults, and the prognosis varies according to the time of onset and severity of symptoms. It is estimated that Pompe disease affects approximately 5,000 to 10,000 people worldwide.

There is a need for a mouse model to study Pompe disease and to test agents that may be effective in the treatment of Pompe disease.

SUMMARY OF THE INVENTION

The present invention provides mouse models for Pompe disease that are useful to study Pompe disease as well as to test agents that may be effective in the treatment of Pornpe disease. In particular, the present invention provides a knockout transgenic mouse wherein the endogenous alpha-glucosidase gene is disrupted such that the knockout transgenic mouse substantially lacks the capacity to express the gene for mouse alpha-glucosidase and whose genome comprises at least one mutation in a gene encoding human alpha-glucosidase wherein at least one of: the amount of alpha-glucosidase protein and the level of alpha-glucosidase activity in at least one disease-relevant tissue of the knockout transgenic mouse is lower than a wild-type mouse. Likewise, the present invention provides a knockout transgenic mouse wherein the endogenous alpha- glucosidase gene is disrupted such that the knockout transgenic mouse substantially lacks the capacity to express the gene for mouse alpha-glucosidase and whose genome comprises at least one mutation in a gene encoding human alpha-glucosidase wherein the level of glycogen in at least one disease-relevant tissue of the knockout transgenic mouse is higher than a wild-type mouse.

In addition, the present invention provides methods of screening for a therapeutic agent for Pompe disease, comprising administering a test agent to a knockout transgenic mouse provided herein and evaluating the effect of the test agent on at least one of: the amount of alpha-glucosidase protein, the level of alpha-glucosidase activity or the level of glycogen in at least one disease- relevant tissue of the knockout transgenic mouse, wherein at least one of: an increase in the amount of alpha-glucosidase protein, an increase in the level of alpha-glucosidase activity or a reduction in the level of glycogen in at least one disease-relevant tissue relative to a similar knockout transgenic mouse that does not receive the test agent indicates the test agent is therapeutic for Pompe disease. The present invention also provides methods for assessing one or more agents for the treatment of Pompe disease, comprising administering one or more agents to a knockout transgenic mouse provided herein and evaluating the effect of the agents on at least one of: the amount of alpha-glucosidase protein, the level of alpha-glucosidase activity or the level of glycogen in at least one disease-relevant tissue of the knockout transgenic mouse, wherein at least one of: an increase in the amount of alpha-glucosidase protein, an increase in the level of alpha-glucosidase activity or a reduction in the level of glycogen in at least one disease-relevant tissue as compared to a similar knockout transgenic mouse that does not receive one or more agents is therapeutic for Pompe disease. In one embodiment, one or more agents is a pharmacological chaperone, a recombinant alpha glucosidase, an antineoplastic agent, an antirheumatic agent, a reproductive control agent, or any combination of two or more thereof. In one embodiment, one or more agents is DNJ, recombinant alpha glucosidase, Cyclophosphamide, Methotrexate, Rituximab, or any combination of two or more thereof. In one embodiment, one or more agents includes DNJ and recombinant alpha glucosidase. In one embodiment, one or more agents includes DNJ. In one embodiment, at least one mutation is a missense mutation. In one embodiment, at least one mutation is P545L, P285R, E262K, E579K, or a combination of two or more thereof. In one embodiment, at least one mutation is P545L.

In one embodiment, the amount of alpha-glucosidase protein or the level of alpha-glucosidase activity in at least one disease-relevant tissue of the knockout transgenic mouse is in a range of about 3% to about 10% relative to that of a wild-type mouse.

In one embodiment, the level of glycogen in at least one disease-relevant tissue of the knockout transgenic mouse is at least 2-fold higher than that of a wild-type mouse.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1A illustrates the GAA and glycogen levels in a sample from heart tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+, Tg +/- as well as wild-type (WT) mice.

Figure 1B illustrates the GAA and glycogen levels in a sample from gastrocnemius tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+, Tg +/- as well as wild-type (WT) mice. Figure 1C illustrates the GAA and glycogen levels in a sample from heart tissue of hP545L GAA Tg/KO mice expressing "Low" GAA levels having genotype Tg +/+, Tg +/- as well as wild-type (WT) mice.

Figure 1D illustrates the GAA and glycogen levels in a sample from gastrocnemius tissue of hP545L GAA Tg/KO mice expressing "Low" GAA levels having genotype Tg +/+, Tg +/- as well as wild-type (WT) mice.

Figure 2A illustrates a sample of heart tissue from hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ immunohistologically stained for glycogen and magnified 2OX.

Figure 2B illustrates a sample of gastrocnemius tissue from hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ immunohistologically stained for glycogen and magnified 2OX.

Figure 2C illustrates a sample of heart tissue from hP545L GAA Tg/KO mice expressing "Low" GAA levels having genotype Tg +/+ immunohistologically stained for glycogen and magnified 2OX. Figure 2D illustrates a sample of gastrocnemius tissue from hP545L GAA

Tg/KO mice expressing "Low" GAA levels having genotype Tg +/+ immunohistologically stained for glycogen and magnified 2OX.

Figure 2E illustrates a sample of heart tissue from wild-type mice immunohistologically stained for glycogen and magnified 2OX. Figure 2F illustrates a sample of gastrocnemius tissue from wild-type mice immunohistologically stained for glycogen and magnified 2OX.

Figure 3A illustrates a sample of heart tissue from hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ immunohistologically stained with LAMP1 antibody and magnified 2OX. Figure 3B illustrates a sample of gastrocnemius tissue from hP545L GAA

Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ immunohistologically stained with LAMP1 antibody and magnified 2OX.

Figure 3C illustrates a sample of heart tissue from hP545L GAA Tg/KO mice expressing "Low" GAA levels having genotype Tg +/+ immunohistologically stained with LAMP1 antibody and magnified 2OX. Figure 3D illustrates a sample of gastrocnemius tissue from hP545L GAA

Tg/KO mice expressing "Low" GAA levels having genotype Tg +/+ immunohistologically stained with LAMP1 antibody and magnified 2OX.

Figure 3E illustrates a sample of heart tissue from wild-type mice immunohistologically stained with LAMP1 antibody and magnified 2OX. Figure 3F illustrates a sample of gastrocnemius tissue from wild-type mice immunohistologically stained with LAMP1 antibody and magnified 2OX.

Figure 4A illustrates the GAA and glycogen levels in a sample from heart tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ) or orally dosed for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free- base equivalent of DNJ as well as wild-type (WT) mice which were not dosed with DNJ. In addition, the inset illustrates the level of GAA protein detected by Western blotting in a sample from heart tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ) or orally dosed for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free-base equivalent of DNJ.

Figure 4B illustrates the GAA and glycogen levels in a sample from diaphragm tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ) or orally dosed for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free-base equivalent of DNJ as well as wild-type (WT) mice which were not dosed with DNJ. In addition, the inset illustrates the level of GAA protein detected by Western blotting in a sample from diaphragm tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ) or orally dosed for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free-base equivalent of DNJ.

Figure 4C illustrates the GAA and glycogen levels in a sample from gastrocnemius tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ) or orally dosed for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free-base equivalent of DNJ as well as wild-type (WT) mice which were not dosed with DNJ. In addition, the inset illustrates the level of GAA protein detected by Western blotting in a sample from gastrocnemius tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ) or orally dosed for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free-base equivalent of DNJ.

Figure 4D illustrates the GAA and glycogen levels in a sample from soleus tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ) or orally dosed for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free-base equivalent of DNJ as well as wild-type (WT) mice which were not dosed with DNJ. In addition, the inset illustrates the level of GAA protein detected by Western blotting in a sample from soleus tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ) or orally dosed for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free-base equivalent of DNJ.

Figure 4E illustrates the GAA and glycogen levels in a sample from brain tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ) or orally dosed for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free- base equivalent of DNJ as well as wild-type (WT) mice which were not dosed with DNJ. In addition, the inset illustrates the level of GAA protein detected by Western blotting in a sample from brain tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ) or orally dosed for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free-base equivalent of DNJ.

Figure 4F illustrates the GAA and glycogen levels in a sample from skin tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ) or orally dosed for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free- base equivalent of DNJ as well as wild-type (WT) mice which were not dosed with DNJ. In addition, the inset illustrates the level of GAA protein detected by Western blotting in a sample from skin tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ) or orally dosed for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free-base equivalent of DNJ.

Figure 5A illustrates the glycogen levels in a sample from heart tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ), orally dosed (ad libitum in drinking water) with the aim of administering 100 mg/kg/day free-base equivalent of DNJ either daily or using one of three less-frequent on/off regimens (i.e., (i) 3 days on/4 days off; (ii) 5 days on/2 days off; or (iii) 7 days on/7 days off) for 4 weeks as well as wild-type (WT) mice which were not dosed with DNJ. Each bar represents pooled data from two independent studies with the mean±SEM of 14 mice/group analyzed in triplicate. Figure 5B illustrates the glycogen levels in a sample from diaphragm tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ), orally dosed (ad libitum in drinking water) with the aim of administering 100 mg/kg/day free- base equivalent of DNJ either daily or using one of three less-frequent on/off regimens (i.e., (i) 3 days on/4 days off; (ii) 5 days on/2 days off; or (iii) 7 days on/7 days off) for 4 weeks as well as wild-type (WT) mice which were not dosed with DNJ. Each bar represents pooled data from two independent studies with the mean±SEM of 14 mice/group analyzed in triplicate.

Figure 5C illustrates the glycogen levels in a sample from gastrocnemius tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ), orally dosed (ad libitum in drinking water) with the aim of administering 100 mg/kg/day free- base equivalent of DNJ either daily or using one of three less-frequent on/off regimens (i.e., (i) 3 days on/4 days off; (ii) 5 days on/2 days off; or (iii) 7 days on/7 days off) for 4 weeks as well as wild-type (WT) mice which were not dosed with DNJ. Each bar represents pooled data from two independent studies with the mean±SEM of 14 mice/group analyzed in triplicate.

Figure 5D illustrates the glycogen levels in a sample from triceps tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ), orally dosed {ad libitum in drinking water) with the aim of administering 100 mg/kg/day free-base equivalent of DNJ either daily or using one of three less-frequent on/off regimens (i.e., (i) 3 days on/4 days off; (ii) 5 days on/2 days off; or (iii) 7 days on/7 days off) for 4 weeks as well as wild-type (WT) mice which were not dosed with DNJ. Each bar represents pooled data from two independent studies with the mean±SEM of 14 mice/group analyzed in triplicate.

Figure 5E illustrates the glycogen levels in a sample from brain tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ), orally dosed (ad libitum in drinking water) with the aim of administering 100 mg/kg/day free-base equivalent of DNJ either daily or using one of three less-frequent on/off regimens (i.e., (i) 3 days on/4 days off; (ii) 5 days on/2 days off; or (iii) 7 days on/7 days off) for 4 weeks as well as wild-type (WT) mice which were not dosed with DNJ. Each bar represents pooled data from two independent studies with the mean±SEM of 14 mice/group analyzed in triplicate. Figure 5F illustrates the glycogen levels in a sample from spinal cord tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ which were in the control group (0 mg/kg/day DNJ), orally dosed (ad libitum in drinking water) with the aim of administering 100 mg/kg/day free- base equivalent of DNJ either daily or using one of three less-frequent on/off regimens (i.e., (i) 3 days on/4 days off; (ii) 5 days on/2 days off; or (iii) 7 days on/7 days off) for 4 weeks as well as wild-type (WT) mice which were not dosed with DNJ. Each bar represents pooled data from two independent studies with the mean±SEM of 14 mice/group analyzed in triplicate. DETAILED DESCRIPTION OF THE INVENTION

As used herein the following terms shall have the definitions set forth below.

As used herein the term "treatment" means to mitigate or ameliorate one or more symptoms associated with Pompe disease. As used herein the phrase "disease-relevant tissue" means a tissue that displays biochemical or histological pathology associated with Pompe disease, (e.g., an increase in glycogen level relative to tissue that is not pathologic for Pompe disease, or the lysosomal component thereof)- Exemplary disease- relevant tissue includes that from heart, diaphragm, gastrocnemius, soleus, brain and skin.

Two new Pompe transgenic mouse lines were created that express "Moderate" (10%) and "Low" (3%) levels of hP545L GAA, a mutant form of the human enzyme. GAA levels in disease relevant tissues including heart, diaphragm, skeletal muscles, skin and brain of 12-week old 'Low' and 'Moderate' hP545L GAA Tg/KO mouse lines were, on average, 3% and 10% of the levels seen in wild-type littermates, respectively. Importantly, 12-week old 'Low' and 'Moderate' hP545L GAA Tg/KO mice showed accumulation of glycogen, with 6- 10 and 2-3-fold higher levels than wild-type littermates, respectively. Four-week oral administration (ad libitum) of DNJ to 'Moderate' hP545L GAA Tg/KO mice resulted in a significant and dose-dependent increase in tissue GAA activity, protein levels, and processing as well as a concomitant decrease in tissue glycogen levels.

Additionally, both daily and less-frequent dosing regimens (i.e., (i) 3 days on/4 days off; (ii) 5 days on/2 days off; or (iii) 7 days on/7 days off) of DNJ to hP545L GAA Tg/KO mice expressing "Moderate" GAA levels led to a significant reduction in glycogen levels in disease-relevant tissue. In fact, less-frequent dosing regimens resulting in greater glycogen reduction compared to daily dosing.

Though not meant to be bound by theory, it is believed that on/off DNJ dosing regimens exploit the difference in half-life of DNJ (hours) compared to GAA (days). In particular, the "on" period of DNJ provides a period of enhanced protein stabilization and trafficking to lysosomes (maximal chaperone effect) followed by the "off period of DNJ which allows for dissociation and tissue clearance of the chaperone (providing maximal in situ enzyme activity). The "on/off' DNJ dosing regimens thereby result in a larger net gain in cellular GAA activity than daily dosing of DNJ. Importantly, less-frequent dosing regimens of DNJ also utilize less drug than daily dosing which is beneficial to patients as it is more cost-effective.

EXAMPLES The present invention is further described by means of the examples, presented below. The use of such examples is illustrative only and in no way limits the scope and meaning of the invention or of any exemplified term. Likewise, the invention is not limited to any particular preferred embodiments described herein. Indeed, many modifications and variations of the invention will be apparent to those skilled in the art upon reading this specification. The invention is therefore to be limited only by the terms of the appended claims along with the full scope of equivalents to which the claims are entitled.

Mouse Model with Human GAA Mutation To develop mouse models of Pompe disease, a human GAA gene containing a mutation is introduced (i.e., a transgene) into a mouse substantially lacking the capacity to express endogenous GAA (i.e., a knockout). It is understood that any mutation in the human GAA gene that leads to an increase in the level of glycogen accumulation in a cell would provide a transgene useful in the creation of mouse models for Pompe disease.

As many individuals with Pompe disease have missense mutations in the GAA gene, missense mutations in the human GAA gene were screened for their use as a transgene. It is believed that missense mutations alter the structure of GAA, resulting in the accumulation and/or degredation of this enzyme in the endoplasmic reticulum (ER). As a result of the accumulation and/or degredation of GAA in the ER, GAA is unable to reach the lysosome, the part of the cell where GAA breaks down glycogen. It is believed that certain agents, termed "pharmacological chaperones" bind to GAA in the ER and assist the GAA in exiting the ER such that the GAA is able to reduce the level of glycogen present in the cell. For example, an agent currently in Phase Il clinical studies for the treatment of Pompe disease, 1-deoxynojirimycin-HCI (DNJ, also known as

AT2220), is believed to act as a pharmacological chaperone for GAA. Note that the present invention is not intended to be bound by the aforementioned mechanism.

DNJ has been shown to increase GAA activity, GAA protein levels, and GAA processing to the mature form of GAA both in vitro and in vivo. In particular, DNJ has been shown to significantly increase the level of multiple different mutant forms of GAA in Pompe patient-derived fibroblasts and COS-7 cells transiently-transfected with recombinant human GAA (data not shown). Likewise, DNJ has been shown to directly bind to GAA and cause a concentration-dependent increase in the thermostability of recombinant human GAA in vitro (data not shown). In addition, DNJ has been shown to increase the level of GAA activity as well as GAA protein levels and GAA processing to the 95-, 76-, and 70-kDa forms in HEK293 cells transiently-transfected with a mutated form of GAA (including M519V, P545L, G549R, and L552P; data not shown). Furthermore, in vivo, GAA activity has been shown to increase in a dose-dependent manner in both mice and cynologous monkeys administered DNJ orally (data not shown). Likewise, DNJ increased GAA protein levels and processing to the 70-kDa mature form of GAA in both mice and cynologous monkeys (data not shown). Such increase in activity is believed to be specific for GAA, as no increase was observed in two other lysosomal enzymes examined in mice administered DNJ, specifically alpha-galactosidase and beta- glucocerebrosidase (data not shown).

To further study the effects of DNJ on Pompe disease, mouse models were created containing a mutation known to be responsive to DNJ. It is understood that the mouse model of Pompe disease provided herein is useful for the study of Pompe disease as well as for the identification of other agents that may be useful in the treatment of Pompe disease. Likewise, it is understood that the principles taught herein may be applied to the creation of other mouse models that are responsive to other agent(s) as well as the study of Pompe disease and the identification of other agents that may be useful in the treatment of Pompe disease.

Identification of Human GAA Mutation Responsive to DNJ

In vitro studies were conducted to identify a human GAA missense mutation responsive to the agent (DNJ). Out of several mutations initially examined (IVS1AS,T>G,-13, D654E, V8161 , T927I, P545L, M519V, W402R), the P545L (proline 545 leucine substitution) mutation was chosen as it showed a robust, reproducible, dose-dependent and statistically-significant increase in GAA activity after 5-day incubation with DNJ (data not shown). However, other human GAA missense mutations which are even more responsive than P545L to DNJ have subsequently been identified in vitro (including P285R, E262K, E579K; data not shown). Such mutations are also suitable for the generation of transgenic mice for testing DNJ as well as other agents that may be useful in the treatment of Pompe disease.

Generation ofhP545L GAA Transgenic/Knockout

The generation of transgenic (Tg) mice were similar to those described previously for Fabry transgenic mice by Shimmoto et al., "Generation and characterization of transgenic mice expressing a human mutant alpha- galactosidase with an R301Q substitution causing a variant form of Fabry disease," FEBS Lett, 417(1):89-91 (1997) and lshii et al., "Transgenic mouse expressing human mutant alpha-galactosidase A in an endogenous enzyme deficient background: a biochemical animal model for studying active-site specific chaperone therapy for Fabry disease," Biochim Biophys Acta, 1690(3) 250-7 (2004) (both of which are incorporated herein by reference), with the exception that the transgene used was human P545L GAA (SEQ ID NO: 1). Briefly, the P545L mutation was introduced in a human GAA gene (SEQ ID NO: 3) using polymerase chain reaction (PCR) mutagenesis. The resultant mutant human GAA gene was cloned into a pCI mammalian expression vector containing a cytomegalovirus (CMV) promoter at Kpn I and Sal I restriction sites. This transgenic construct was then used for generating founder mice at contract research organization (CRO) Xenogen Corporation (Cranbury, NJ) using routine methodologies described previously by Gordon et al., "Genetic transformation of mouse embryos by microinjection of purified DNA," Proc Natl Acad Sci U S A, 77(12):7380-4 (1980), incorporated herein by reference. Briefly, the vector carrying the transgene was first injected in the pronuclei of mouse oocyte followed by insemination in super-ovulated wild-type C57BL/6 female mice. The pups obtained were screened for the presence of transgene by PCR amplification of tail genomic DNA. Two founder mice were obtained both carrying, a 500 bp fragment amplified using 5'- TACGTATTAGTCATCGCTAT-S' (SEQ ID NO: 5) forward and 5'- ATTAAGTACTCTAGCCTTAA-S' (SEQ ID NO: 6) reverse primers. The PCR amplification consisted of 30 cycles of denaturation at 94 °C for 30 seconds, annealing at 55 ⁰ C for 1 min. and elongation at 72 ⁰ C for 2 min. The founder mice were bred separately with wild-type C57BL/6 mice to obtain two transgenic F1 mouse lines on a wild-type GAA background [Tg (+/-) mGAA (+/+)]. The F1 transgenic lines were then bred with Pompe knockout (KO) mice described in Raben et al., "Targeted disruption of the acid alpha- glucosidase gene in mice causes an illness with critical features of both infantile and adult human glycogen storage disease type II," J Biol Chem, 273(30): 19086- 19092 (1988), incorporated herein by reference (kind gift of Dr. Barry Byrne). The resultant pups were screened for the presence of transgene by PCR amplification using the primers described above as well as the KO alleles as described by Raben et al. (Raben et al., "Targeted disruption of the acid alpha- glucosidase gene in mice causes an illness with critical features of both infantile and adult human glycogen storage disease type II," J Biol Chem, 273(30).19086- 19092 (1988)). Following multiple crossovers, each of the two aforementioned F1 transgenic lines gave mice with 4 different genotypes. These mice were either homozygous for hP545L trangene [Tg (+/+) KO (-/-)], heterozygous [Tg (+/-) KO (-/-)], no transgene [Tg (-/-) KO (-/-)] or carried wild-type [Tg (-/-) KO (+/+)]. The two homozygous transgenic lines expressed either 1 or 10 copies of the transgene designated as 'Low' and 'Moderate,' respectively. The mice were characterized further for their biochemical phenotype (using the assays described below) as well as to test the efficacy of DNJ in vivo.

Preparation of Plasma and Tissue for Assays

Mice were euthanized with CO ₂ and body weights were recorded. Whole blood was drawn into lithium heparin tubes from the inferior vena cava after CO ₂ euthanization. Plasma was collected by spinning blood at 270Og for 10 minutes at 4 ⁰ C. Heart, diaphragm, gastrocnemius, soleus, skin, and brain tissues were removed, washed in cold PBS, blotted dry, and weighed before storing on dry ice. For biochemical assays (GAA activity, glycogen and Western blotting), lysates were prepared by homogenizing ~50 mg tissue for 3-5 seconds on ice with a micro homogenizer in 200 μl_ Lysis Buffer (25mM Bis-Tris pH 6.5, 15OmM NaCI, 1% Triton X-100 pH 6.5). For histology assays, tissues were harvested and fixed immediately in 3.7% Formaldehyde / 90% Ethanol mix. After fixation, the samples were processed in various grades (10, 20, 50, 75, 100%) of alcohol and embedded in paraffin.

Biochemical Assay for Determining GAA Level

Twenty μL of lysate were added to 50 μL Assay buffer containing 3 mM 4- MU-α-D-glucopyranoside (4-MUG) in 50 mM potassium acetate buffer (pH 4.0), and incubated for 1 hour at 37 ⁰ C. Seventy μL of Stop Solution (0.4 M glycine, pH 10.8) were then added and fluorescence read on a Victor ³ plate reader (Perkin Elmer, Waltham, MA) at 355 nm excitation and 460 nm emission and raw fluorescence counts were background subtracted (defined by counts from substrate solution only). A Micro BCA Protein Assay Kit (Pierce, Rockford, IL) was used according to the manufacturer's instructions to determine protein concentration in lysates. A 4-methylumbelliferone (4-MU) standard curve ranging from 1.3 nM to 30 μM was run each day for conversion of fluorescence data to absolute GAA activity. Data are ultimately expressed as nanomoles/milligram of protein/hour (nmol/mg protein/hr). For plasma GAA levels, exactly similar steps as above were followed except that no homogenization was required.

As illustrated in Figures 1A and IB, the GAA level (determined using the aforementioned assay) in samples from heart tissue and gastrocnemius tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ or Tg +/- displayed ~10% of GAA levels as compared to wild-type (WT) mice. Likewise, as illustrated in Figures 1C and 1D, the GAA level (determined using the aforementioned assay) in samples from heart tissue and gastrocnemius tissue of hP545L GAA Tg/KO mice expressing "Low" GAA levels having genotype Tg +/+ or Tg +/- displayed -3% of GAA levels as compared to wild-type (WT) mice.

Biochemical Assay for Determining Glycogen Level Tissue glycogen levels were measured in supernatants of heat denatured

(99°C for 10 min.) lysates as prepared above for GAA assay. Samples were analyzed in duplicates by adding 4 μL of lysate to 36 μL water with and without 10 μL of 800 units/mL of amyloglucosidase enzyme (Sigma) and incubated for 1 hour at 5O ⁰ C. The reaction was stopped by heat inactivation at 100 ⁰ C for 10 min. Finally 200 μL of glucose reagent was added to estimate the amount of glucose released at 340 nm absorbance on Spectramax. A glycogen standard curve ranging from 400 μg/mL to 5 μg/mL was run each day for conversion of absorbance data to absolute glycogen levels. Data are ultimately expressed as μg of glycogen cleaved/milligram of protein. The protein levels were measured in lysates (before denaturing) using Micro BCA Protein Assay Kit according to the manufacturer's instructions.

As illustrated in Figures 1A and 1 B, glycogen accumulation (determined using the aforementioned biochemical assay) in samples from heart tissue and gastrocnemius tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ or Tg +/- were 2-3-fold above the level of glycogen accumulation of wild-type (WT) mice. Likewise, as illustrated in Figures 1C and 1 D ₁ glycogen accumulation (determined using the aforementioned biochemical assay) in samples from heart tissue and gastrocnemius tissue of hP545L GAA Tg/KO mice expressing "Low" GAA levels having genotype Tg +/+ or Tg +/- were 6-10-fold above the level of glycogen accumulation of wild-type (WT) mice.

Histological Assay for Determining Glycogen Level

Paraffin-embedded tissue sections were cut at 5 μm thickness, mounted onto Superfrost Plus, de-paraffinized in Xylene and finally re-hydrated through graded ethanol series to water. Sections were then treated with 0.1% Periodic acid for 5 min, rinsed with tap water (5 sec.) and stained with Schiff's reagent for 15 min. Excess stain was removed by washing in deionized water for 5 sec. followed by tap water for 10 min, and counterstained with Mayer's Hematoxylin solution (Sigma) before mounting on Xylene based media.

As illustrated in Figures 2A-2F, the level of glycogen accumulation (determined using the aforementioned histological assay) in samples from heart tissue and gastrocnemius tissue of hP545L GAA Tg/KO mice expressing "Moderate" or "Low" GAA levels were similar to those levels detected biochemically, as illustrated in Figures 1A-1 D, wherein glycogen accumulation was at a level 2-3-fold and 6-10-fold, respectively, above that of wild-type (WT) mice.

Histological Assay for Determining Lysosomal Proliferation

Tissues were harvested, processed and sectioned as described above for PAS staining. The endogenous HRP activities were blocked in 3% H2O2 / PBS for 60 min followed by washing in PBS for 10 min. Samples were then blocked by Rodent Block M (Biocare Medical) by incubation at room temperature for 30 min, followed by PBS wash (2x 5min). Primary LAMP1 antibody (Abeam, clone 1 D4B) was diluted in background sniper (1:1000) and incubated overnight at 4C. The unbound antibody was washed with PBS (5x 5min) and secondary Rat HRP Polymer (Biocare Medical) was added at room temperature for 10 min. Finally sections were washed in PBS (5x 5min), counterstained with Mayer's

Hematoxylin solution (Sigma) and finally developed with Betazoid DAB Kit (room temperature for 5 min).

As illustrated in Figures 3A-3F, the level of lysosomal proliferation (determined using the aforementioned histological assay) in samples from heart tissue and gastrocnemius tissue of hP545L GAA Tg/KO mice expressing "Moderate" or "Low" GAA levels was significantly increased relative to that of wild-type (WT) mice. However, the level of lysosomal proliferation in samples from heart tissue and gastrocnemius tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels was lower than that observed in samples from hP545L GAA Tg/KO mice expressing "Low" GAA levels.

GAA Protein Level by Western blotting

Tissue lysates (50 μg total protein per lane) were subjected to SDS-PAGE on 12% gels (Bio-Rad, Hercules, CA), transferred to PVDF membranes (Bio- Rad), and immunoblotted with a rabbit anti-human GAA (kind gift of Dr. Barry Byrne) polyclonal antibody (1 :500 dilution). Protein bands were detected using peroxidase-conjugated goat anti-rabbit secondary antibodies (1 :1000) (Jackson lmmunosearch Labs, West Grove, PA) in combination with enhanced chemiluminescence (Pierce). The blots were scanned on an Image Station 4000R using Molecular Imaging Software, version 4.0.

As illustrated in the insets of Figures 4A-F, administration of DNJ to hP545L GAA Tg/KO mice expressing "Moderate" GAA levels led to a dose- dependent increase in GAA protein levels as well as the processing of GAA protein to its 76 kDa mature form in disease-relevant tissues.

Testing Agents Useful for the Treatment of Pompe Disease Daily dosing regimen

Eight-week old male hP545L GAA Tg/KO mice were administered DNJ HCI in drinking water (ad libitum) with at least seven (25 g) mice per group. The mice were dosed for 4 weeks with the aim of administering 30 mg/kg/day; 100 mg/kg/day; and 300 mg/kg/day free-base equivalent of DNJ using dosing solutions prepared at 0.15, 0.5 and 1.5 mg/mL free-base equivalent of DNJ, respectively, based on the daily water consumption of transgenic mice (~5 mL/day per mouse). The dosing solutions were made fresh each week and water consumption was monitored daily. After dosing was completed, mice were euthanized and plasma samples as well as tissue samples harvested as described above. GAA levels and glycogen levels were assayed in samples from disease-relevant tissue of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels which were in the control group (0 mg/kg/day DNJ) or orally dosed with DNJ HCI for 4 weeks with the aim of administering 30, 100, or 300 mg/kg/day free-base equivalent of DNJ as well as wild-type (WT) mice which were not dosed with DNJ. As illustrated in Figures 4A-F, administration of DNJ to hP545L GAA Tg/KO mice expressing "Moderate" GAA levels led to a significant and dose-dependent increase in GAA levels. In addition, as illustrated in Figures 4A-F, administration of DNJ to hP545L GAA Tg/KO mice expressing "Moderate" GAA levels led to a robust reduction in glycogen levels of soleus, skin, and brain, and a moderate reduction in glycogen levels of heart diaphragm, and gastrocnemius as measured biochemically by amyloglucosidase digestion.

Comparison of daily and less-frequent on/off dosing regimens

Eight-week old male hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ were administered DNJ in drinking water (ad libitum) with fourteen mice per group. Mice were orally dosed (ad libitum in drinking water) either daily or using one of three less-frequent on/off regimens (i.e., (i) 3 days on/4 days off; (ii) 5 days on/2 days off; or (iii) 7 days on/7 days off). Each dosing day, the mice received a dosing solution prepared at 0.5 free- base equivalent of DNJ with the aim of administering 100 mg/kg/day free-base equivalent of DNJ based on the daily water consumption of transgenic mice (~5 mL/day per mouse). The dosing solution was made fresh each week and water consumption was monitored daily. Control groups included eight-week old male hP545L GAA Tg/KO mice expressing "Moderate" GAA levels having genotype Tg +/+ as well as wild-type (WT) mice which were not dosed with DNJ.

After dosing was completed, mice were euthanized and tissue samples harvested as described above. Glycogen levels were assayed in samples from disease-relevant tissue (i.e., heart, diaphragm, gastrocnemius, triceps, brain and spinal cord) of hP545L GAA Tg/KO mice expressing "Moderate" GAA levels which were in the control group (0 mg/kg/day DNJ) or orally dosed (ad libitum in drinking water) with the aim of administering 100 mg/kg/day free-base equivalent of DNJ either daily or using one of three less-frequent on/off regimens (i.e., (i) 3 days on/4 days off; (ii) 5 days on/2 days off; or (iii) 7 days on/7 days off) as well as wild-type (WT) mice which were not dosed with DNJ. As illustrated in Figures 5A-F, administration of DNJ to hP545L GAA Tg/KO mice expressing "Moderate" GAA levels led to a significant reduction in glycogen levels (*p<0.05 vs. untreated, Mest) in the majority of tissues examined, with the "on/off regimens resulting in greater glycogen reduction compared to daily administration (#p<0.05 daily vs. "on/off, Mest). Collectively, these results indicate that less-frequent DNJ dosing regimens, with less drug administered per week or dosing cycle, can lead to an even greater reduction in the quantity of accumulated substrate.

Additional agent(s) It is understood that any of the aforementioned assays for measuring GAA levels or glycogen levels may be used to assess agents useful for the treatment of Pompe disease. The effect of such agents may be tested individually or in combination. Exemplary agents include, but are not limited to, "pharmacological chaperones" such as DNJ ₁ recombinant alpha glucosidase (e.g., Myozyme ^® (alglucosidase alfa) available from Genzyme), an antineoplastic agent (e.g., Cyclophosphamide, Methotrexate, Rituximab), an antirheumatic agent (e.g., Rituximab), a reproductive control agent (e.g., Methotrexate), or a combination of two or more thereof. Similarly, different therapeutic regimens may be tested for their efficacy in the mouse models provided. Likewise, different modes of administration may be used to deliver the agent(s) (e.g., DNJ may be administered orally, recombinant α-glucosidase may be introduced via intravenous and/or intrathecal infusion).