Tchou-Wong, K.-M. | Search bioRxiv

bioRxiv2023

Preclinical studies with ground germinated barley (GGB) for oral enzyme replacement therapy (Oral-ERT) in Pompe disease knockout mice

Genetic deficiency of lysosomal acid maltase or acid -glucosidase (GAA) results in the orphan disease known as glycogen storage disease type II or acid maltase deficiency (AMD) or Pompe disease (PD), encompassing at least four clinical subtypes of varying severity. PD results from mutations in the GAA gene and deficient GAA activity, resulting in the accumulation of glycogen in tissues (primarily muscle) and characterized by progressive skeletal muscle weakness and respiratory insufficiency. The current approved enzyme replacement therapy (ERT) for PD is via intravenous infusion of a recombinant human GAA (rhGAA) secreted by CHO cells (Myozyme, Sanofi-Genzyme) given once every 2 weeks and has shown varying efficacy in patients. Although the current ERT has proven to be very 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. Additional challenges for ERT include insufficient targeting/uptake of enzyme into disease-relevant tissues, poor tolerability due to severe ERT-mediated anaphylactic and immunologic reactions and the prohibitively high cost of lifelong ERT ($250-500K/year adult patient). A consensus at a Nov.-2019 US Acid Maltase Deficiency Association conference suggested that a multi-pronged approach including gene therapy, diet, exercise, etc. must be evaluated for a successful treatment. Our objective is to develop an innovative and affordable approach via barley GAA (bGAA) from ground germinated barley (GGB) or liquid GGB (L-GGB) for Oral-ERT for PD or as a daily supplement to Myozyme. To this end, we hypothesize that a bGAA produced in germinated barley can be ingested daily that allows the maintenance of a therapeutic level of enzyme. We have shown in extensive preliminary data that GGB or L-GGB was (1) enzymatically active, (2) was taken up by GAA KO mice and human WBCs to reverse the enzyme defect that was blocked by mannose-6-phosphate, (3) hydrolyzed glycogen, (4) increased significant changes in the clinical phenotype towards the WT levels in GAA KO mice dose-dependently, (5) taken up by PD myoblasts, lymphoid/fibroblasts cells to reverse the defect, (6) bGAA was [~]70kD, (7) Km, Vmax, pH optima, inhibitors and kinetics was similar to human placental GAA and an rhGAA and (8) was strain specific.

genetics↗

bioRxiv2021

Crotamine as a vehicle for non-viral gene delivery for Pompe disease

Genetic deficiency of lysosomal acid alpha glucosidase or acid maltase (GAA) results in Pompe disease (PD), encompassing at least five clinical subtypes of varying severity. The current approved enzyme replacement therapy (ERT) for PD is via IV infusion every 2 weeks of a recombinant human GAA (rhGAA) secreted by Chinese hamster ovary (CHO) cells (alglucosidase alfa/Myozyme, Sanofi/Genzyme). Although alglucosidase alfa has proven to be efficient in rescuing cardiac abnormalities and extending the life span of the infantile form, the response in skeletal muscle is variable. ERT usually begins when the patients are symptomatic and secondary problems are already present which are compounded by low alglucosidase alfa uptake, transient nature (every 2 weeks with a rapid return to defect levels), variable glycogen reduction, autophagic accumulation, immune response and high cost. A consensus at a recent US Acid Maltase Deficiency (AMD) conference suggested that a multi-pronged approach including gene therapy, diet, exercise, etc. must be evaluated for a successful treatment of PD. Compared to replication defective viruses, non-viral gene transfer offers fewer safety concerns and, if recent studies are validated, has a wider range of cells. In order for gene therapy (GT) to succeed, the gene of interest must be delivered into the affected cell and expressed to overcome the inherited deficiency. Cell penetrating peptides (CPPs) enter eukaryotic cells through an energy-independent mechanism and efficiently carry biologically active and therapeutic molecules into cells and localize in the cytoplasm or nucleus. CPPs are usually covalently linked to the cargo, including peptides and DNA. Crotamine (Cro) from the South American rattlesnake-Crotalus durrissus terrificus venom, can bind electrostatically to plasmid DNA to deliver into cells, including muscle. We have assembled a bacterial expression vector for Cro and purified the recombinant Cro (rCro). Transient transfection in AMD fibroblasts and ex vivo in whole blood from an adult Pompe patient with rCro complexed with the pcDNA3 x hGAA cDNA demonstrated increased GAA activity. In GAA knockout (KO) mice receiving a single injection of rCro complexed to pcDNA3 x hGAA cDNA intraperitoneally (IP), we found increased GAA activity in tissues after 48 hr. After 8 treatments-IP over 55 days, we found increased vertical hang-time activity, reduced glycogen deposition, increased GAA activity/hGAA plasmid in tissues and minimal immune-reaction to rCro. A subsequent study of 5 administrations every 2 to 3 weeks showed reverse of the clinical phenotypes by running wheel activity, Rotarod, grip-strength meter, open field mobility and T-maze. Tissue culture experiments in PD fibroblast, lymphoid and skeletal muscle cell lines showed increased GAA activity after rCro transient gene delivery.

genetics↗