Development of new protein nanoconjugation strategies to improve enzyme replacement therapy

Abstract

Enzyme replacement therapy (ERT) has revolutionized the management of patients with lysosomal storage disorders (LSDs) over the last few decades. Gaucher disease (GD), a disorder caused by mutations in the GBA1 gene encoding the enzyme ß-glucocerebrosidase (GBA), is the most common LSD. Intravenous infusion of functional GBA into GD patients reverses most of the visceral manifestations of the disease. However, there are important drawbacks associated with ERT, such as poor stability and enzyme inactivation during transport throughout the bloodstream. This limits the biodistribution and therapeutic potential of ERT, which has been shown to be ineffective in the treatment of bone disease. Furthermore, the inability of GBA to cross the blood-brain barrier (BBB) makes this therapy ineffective for treating the neurological manifestations of GD. For that reason, there's a need for the development of enhanced ERT strategies and brain-targeted ERT with GBA. In fact, brain penetration in the specific case of GBA, not only opens the possibility to treat neuropathic GD, but also allows the possible treatment of Parkinson's disease (PD). The clear genetic association of GBA1 gene with PD as a risk factor affecting age of onset and disease pathogenicity represents an opportunity for PD treatment. Furthermore, the inverse proportionality between GBA enzymatic activity and Syn levels and aggregation provide a mechanistic disease modifying treatment opportunity. Nanotechnology offers powerful tools to overcome the limitations of protein therapeutics, particularly through drug delivery systems (DDS) that protect enzymes from degradation and improve their bioavailability. Among these, protein-polymer conjugation has emerged as a leading strategy, enhancing protein stability and pharmacokinetics. While PEGylation has dominated the field, concerns over PEG's non-biodegradability and immunogenicity have prompted interest in alternatives. Poly(Alpha-L-glutamic acid) (PGA) is a biodegradable, synthetically tunable polymer with low immunogenicity, making it an attractive and adaptable candidate for next-generation protein-polymer conjugates. In this thesis, we propose a method for recombinant GBA conjugation to PGA polymer using different conjugation techniques. GBA-PGA conjugates are effectively synthetized, physico-chemically characterized and in vitro evaluated in GBA Knock-Out cellular models. Its efficacy in vivo as an enhanced ERT for visceral GD treatment has been proven in this thesis by comparative intravenous administration of GBA and GBA-PGA conjugates in GD mouse model animals. Intravenously administered GBA-PGA conjugates confer a novel treatment for GD with the capacity to address bone manifestations of the disease. However, intravenously administered GBA-PGA conjugates does not cross the BBB. Nose-to-brain drug delivery has been thoroughly assessed in this thesis as an alternative route of administration by-passing the BBB. Minimal modifications on PGA polymer conferred to GBA-PGA conjugates mucoadhesion properties and brain targeting capacity through the olfactory pathway, potentially following intraneuronal transport to the olfactory bulb. Despite our efforts, minimal amounts of GBA-PGA conjugates reached the brain compartment following this strategy due to several defence mechanisms of the treated organisms, including GBA-PGA uncoupling, PGA trimming and immune-mediated events. Further optimization is on-going to address these multiple issues for effective brain delivery of GBA-PGA conjugates.