Genetic deficiency of lysosomal acid maltase or acid alpha-glucosidase (GAA) results in the orphan disease-glycogen storage disease type II (GSDII), acid maltase deficiency or Pompe's disease (PD), encompassing at least five clinical subtypes of varying severity. The current approved enzyme replacement therapy (ERT) for PD is via intravenous infusion of a recombinant human GAA (rhGAA) produced by CHO cells (Myozyme, Genzyme) once every 2 weeks. Although the current ERT has proven to be efficient in rescuing cardiac abnormalities and extending the life span of infants, the response in skeletal muscle is variable. In late-onset patients, only mild improvements in motor and respiratory functions have been achieved and the current ERT is unsatisfactory in the reversal of skeletal muscle pathology. Due to the high cost of production of Myozyme, lifelong treatment can be prohibitively expensive. Hence, our objective is to develop an innovative and affordable approach for ERT via oral administration (Oral-ERT) to maintain a sustained, therapeutic level of enzyme on a daily basis to improve efficacy of treatment and quality of life. We have shown that tobrhGAA expressed in the seeds of transgenic tobacco plants is enzymatically active and can correct the enzyme deficiency in GAA deficient-cells and in tissues in GAA KO mice by IP admin. (Martiniuk et al., Appl Bio Biotech 171:916-926,2013). Additionally, long-term oral treatment showed increased muscle strength, tolerability, negligible antibody titers, decreased glycogen levels, increased GAA in tissues thereby providing support for proof-of-concept for Oral-ERT for Pompe disease. Daily treatment with whole seeds reversed muscle weakness dose dependently. PK showed peak tobrhGAA levels at 8 hrs in serum and 10 hrs in urine. tobrhGAA vs placental GAA enzyme kinetics are very similar.
Aim 1. To characterize tobrhGAA and in vitro in GAA deficient-cells. The tobrhGAA will be evaluated for (1) enzymatic kinetics (2) uptake by AMD cell lines and compared to placental GAA and Myozyme and (3) NGS-genome sequencing/proteomics of the tobrhGAA.
Aim 2. Oral-ERT in GAA KO mice. From preliminary data in young mice where symptoms are still developing, 2x-50mg seeds daily achieved 60% restoration of RW motor activity, thus 150mg should reverse muscle weakness. Additionally, ERT of 5-6 mo. might be required to maintain reduced glycogen accumulation to reverse the secondary affects of autophagic buildup and blockade. We will also investigate reversal of more advanced staged disease in older mice of 7-8 mo. We will administer 50mg or 150mg whole seeds daily in the AM to GAA KO mice (age 4 and 7-8 months) for 1, 3 and 6 months (n=4, per time-point) plus controls-WT and GAA KO mice treated with WT seeds and GAA KO mice treated with Myozyme-IP (50-100 mg/kg/wk-Genzyme for free). Mice will be sacrificed at 1, 3 and 6 mo., tissues, urine/serum are analyzed/monitored for reversal of disease by biochemical, clinical presentation, histology and antibody titers. Expected feasibility outcomes include increased GAA activity/protein in tissues to 10% of normal, reversal of clinical phenotype, and decreased glycogen.
Aim 3. PK and Toxicity (CRL or Sterling Pharm) To address some of the questions by the FDA in our IND, a CRO will perform PK, toxicity and maximum tolerated dose in mice and a second non-rodent animal-rabbit.

Public Health Relevance

The goal of the proposed research is to investigate the feasibility of a novel approach for enzyme replacement (ERT) via oral administration (Oral-ERT) with a recombinant human acid alpha-glucosidase (rhGAA) produced in transgenic tobacco seeds in an animal model for the orphan disease-Pompe's disease. Compared to infusion of the GAA enzyme once every two weeks, the current approved ERT for Pompe's disease, Oral-ERT allows the GAA enzyme to be ingested daily in a pill/gel/capsule form to maintain a sustained, therapeutic level of enzyme to improve the efficacy of treatment and quality of life of Pompe's patients. Lastly, plant-produced GAA enzyme can be produced at a much lower cost, ensuring affordability for lifelong treatment for this devastating disease.

National Institute of Health (NIH)
National Institute of Arthritis and Musculoskeletal and Skin Diseases (NIAMS)
Small Business Innovation Research Grants (SBIR) - Phase I (R43)
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Special Emphasis Panel (ZAR1)
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Wang, Xibin
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Jme Group, Inc.
United States
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