• enzyme replacement therapy (ERT)
• Krabbe disease
• polymeric nanoparticles

Krabbe disease (KD), also known as globoid cell leukodystrophy, is a rare (one in 100,000 births) autosomal recessive neurodegenerative disorder. KD is classified as a lysosomal storage disease (LSD), a family of over 70 conditions characterized by perturbation in lysosomal homeostasis. KD is caused by mutations in the gene encoding for the lysosomal enzyme β-galactosylceramidase (or galactocerebrosidase; GALC). GALC plays a pivotal role in the physiological turnover of myelin components and the removal of toxic compounds. In detail, GALC deficiency leads to impaired degradation of its substrates, galactosylceramide (GalCer) and galactosylsphingosine (psychosine, PSY). GalCer can be degraded by another lysosomal enzyme, but PSY can be degraded only by GALC. Consequently, PSY accumulation results in the nearly total loss of myelin forming both in the oligodendrocytes and in the Schwann cells. This condition leads to severe demyelination and consequent neurodegeneration of both the central nervous system and peripheral nervous system. Despite this enormous effort, there is no cure for KD, and the current standard of care is mostly supportive only. Nowadays, only the pre-symptomatic transplantation of hematopoietic stem cells (HSCT) finds clinical applications. Over the last few years, the medical and the scientific communities have focused their efforts on alternative therapies: i. the substrate reduction therapy, or the reduction of the PSY accumulation by blocking its synthesis, ii. pharmacological therapies with anti-inflammatory and antioxidant drugs, iii. the gene therapy to replace the afunctional GALC gene, and iv. the enzyme replacement therapy (ERT). The ERT has gained broad interest thanks to the effective results achieved in other diseases. However, applications of such therapy to KD still do not exist due to one major issue to overcome: the blood-brain barrier (BBB). This physiological barrier strictly regulates the passage of molecules, ions, and cells from the blood to the brain, also hampering the penetration of therapeutics.
The aim of this thesis is to investigate an innovative ERT for KD based on brain-targeted polymeric degradable nanoparticles, loaded with GALC aggregates, and functionalized with a brain targeting peptide (Angiopep-2), to restore GALC activity in the brain of a natural murine model called Twitcher (TWI). This thesis is part of the research project nanoERT – Nanoparticle based Enzyme Replacement Therapy for the treatment of Krabbe disease: a pre-clinical study in the Twitcher Mouse (ELA 2019-008I2), funded by the European Leukodystrophy Association (ELA).
This study reports the development of a set of nanoparticles (NPs) formulations, composed of the copolymer poly (D, L-lactide-co-glycolide) (PLGA), and loaded with increasing concentrations of GALC cross-linked enzyme aggregates (CLEAs), from 3 to 300 ug/mg of PLGA. The targeting unit Angiopep-2 is previously conjugated to PLGA, and a small percentage of Angiopep-2-conjugated copolymer is used in NPs preparation. First, CLEAs, NPs, and CLEAs-loaded NPs are formulated to find the best preparation procedures, allowing high repeatability, maximization of enzyme loading, and suitable NPs size. Then, CLEAs-loaded NPs are characterized to study the stability up to 30 days in storage conditions (4°C, diluted in physiological solution) and their shelf life. Specifically, we investigated the enzyme activity and cargo loading, and the morphological stability of formulations with time, identifying the optimal one. Enzyme activity is measured by a specific enzymatic assay based on the cleavage of the substrate 4-methylumbelliferyl β-D-glucopyranoside, while the quantification of the protein cargo is performed by means of the ninhydrin assay. Size changes are evaluated by dynamic light scattering (DLS) analysis. To identify the optimal formulation, we quantified the leakage of CLEAs from NPs in storage conditions by means of enzyme and protein assays.
After the development and the characterization of the system, postnatal day (PND) 20 TWI mice were treated intraperitoneally with CLEAs-loaded NPs and were sacrificed 4 hours (h), 24h, 72h, and 7 days after treatment. Then, GALC enzymatic assay was performed in the brain and sciatic nerve (representing CNS and PNS, respectively) and in two typical accumulation organs, liver and kidneys.
From the obtained results, we identified the best recipe for the synthesis of CLEAs and for the preparation of NPs. We found that the amount of CLEAs used during the preparation affects enzymatic activity (from 0,08 to 12,8 U/mg of NPs), the size of CLEAs-loaded NPs (mean diameter: 150-250 nm), and the protein encapsulation (from 4,5 to 125,9 µg/g of NPs). Thus, we found a formulation suited to our aim in terms of CLEas activity, diameter, encapsulation, and release over time. We found that formulations are stable under storage conditions for up to 30 days (no loss of activity and significant degradation of PLGA); these data contribute to establishing which are the best storage conditions for the formulation and to a better understanding of CLEAs-loaded NPs.
The brain of the TWI mice treated with CLEAs-loaded NPs showed an enzymatic activity (E.A) that is significantly higher than the E.A. of the untreated TWI from 4 to 72 hours after the NPs administration. Overall, these data demonstrate that the formulation gives TWI brain an E.A. that is maintained to a level that could potentially be of clinical interest up to 72 h from the treatment. An increase in E.A.is also observed in the sciatic nerve compared to untreated TWI. Regarding the liver, we found a high E.A. after 24 hours from the treatment, confirming the capability of our formulation to deliver enzymatically active GALC into mice tissues. Regarding the kidneys, we didn’t find an increase in E.A. compared to untreated TWI. In conclusion, our results are really encouraging at this stage, and we believe that the developed formulation could be of potential interest for the treatment of KD. Some additional aspects related to the fate of NPs, such as the potential accumulation of PLGA, and the effectiveness of released GALC in the brain, still need to be clarified, as well as the verification of the impaired functions restoration. All these aspects will be considered in the next experiments, with repeated administrations of CLEAs-loaded NPs and behavioural tests.