Reliable and Fast Genotyping Protocol for Galactosylceramidase (Galc) in the Twitcher (Twi) Mouse.

Twitcher (Twi) is a neurological Krabbe disease (KD, or globoid cell leukodystrophy) spontaneous mutant line in mice. The genome of the Twi mouse presents a single nucleotide polymorphism (SNP), leading to an enzymatically inactive galactosylceramidase (Galc) protein that causes KD. In this context, mouse Twi genotyping is an essential step in KD research. To date, the genotyping method used is labor-intensive and often has ambiguous results. Here, we evaluated a novel protocol for the genotype determination of Galc mutation status in Twi mice based on the allele-discrimination real-time polymerase chain reaction (PCR). Here, DNA is extracted from Twi mice (n = 20, pilot study; n = 120, verification study) and control group (n = 10, pilot study; n = 30 verification study) and assessed by allele-discrimination real-time PCR to detect SNP c.355G>A. Using the allele-discrimination PCR, all of the samples are identified correctly with the genotype GG (wild-type, WT), GA (heterozygote, HET), or AA (homozygote, HOM) using the first analysis and no animals are not genotyped. We demonstrated that this novel method can be used to distinguish KD timely, accurately, and without ambiguity in HOM, WT, and HET animals. This protocol represents a great opportunity to increase accuracy and speed in KD research.

Current treatment options and novel nanotechnology-driven enzyme replacement strategies for lysosomal storage disorders.

Lysosomal storage disorders (LSDs) are a vast group of more than 50 clinically identified metabolic diseases. They are singly rare, but they affect collectively 1 on 5,000 live births. They result in most of the cases from an enzymatic defect within lysosomes, which causes the subsequent augmentation of unwanted substrates. This accumulation process leads to plenty of clinical signs, determined by the specific substrate and accumulation area. The majority of LSDs present a broad organ and tissue engagement. Brain, connective tissues, viscera and bones are usually afflicted. Among them, brain disease is markedly frequent (two-thirds of LSDs). The most clinically employed approach to treat LSDs is enzyme replacement therapy (ERT), which is practiced by administering systemically the missed or defective enzyme. It represents a healthful strategy for 11 LSDs at the moment, but it solves the pathology only in the case of Gaucher disease. This approach, in fact, is not efficacious in the case of LSDs that have an effect on the central nervous system (CNS) due to the existence of the blood-brain barrier (BBB). Additionally, ERT suffers from several other weak points, such as low penetration of the exogenously administered enzyme to poorly vascularized areas, the development of immunogenicity and infusion-associated reactions (IARs), and, last but not least, the very high cost and lifelong needed. To ameliorate these weaknesses lot of efforts have been recently spent around the development of innovative nanotechnology-driven ERT strategies. They may boost the power of ERT and minimize adverse reactions by loading enzymes into biodegradable nanomaterials. Enzyme encapsulation into biocompatible liposomes, micelles, and polymeric nanoparticles, for example, can protect enzymatic activity, eliminating immunologic reactions and premature enzyme degradation. It can also permit a controlled release of the payload, ameliorating pharmacokinetics and pharmacodynamics of the drug. Additionally, the potential to functionalize the surface of the nanocarrier with targeting agents (antibodies or peptides), could promote the passage through biological barriers. In this review we examined the clinically applied ERTs, highlighting limitations that do not allow to completely cure the specific LSD. Later, we critically consider the nanotechnology-based ERT strategies that have beenin-vitroand/orin-vivotested to improve ERT efficacy.

Chronic lithium administration in a mouse model for Krabbe disease.

Krabbe disease (KD; or globoid cell leukodystrophy) is an autosomal recessive lysosomal storage disorder caused by deficiency of the galactosylceramidase (GALC) enzyme. No cure is currently available for KD. Clinical applied treatments are supportive only. Recently, we demonstrated that two differently acting autophagy inducers (lithium and rapamycin) can improve some KD hallmarks in-vitro, laying the foundation for their in-vivo pre-clinical testing. Here, we test lithium carbonate in-vivo, in the spontaneous mouse model for KD, the Twitcher (TWI) mouse. The drug is administered ad libitum via drinking water (600 mg/L) starting from post natal day 20. We longitudinally monitor the mouse motor performance through the grip strength, the hanging wire and the rotarod tests, and a set of biochemical parameters related to the KD pathogenesis [i.e., GALC enzymatic activity, psychosine (PSY) accumulation and astrogliosis]. Additionally, we investigate the expression of some crucial markers related to the two pathways that could be altered by lithium: the autophagy and the ß-catenin-dependent pathways. Results demonstrate that lithium has not a significant rescue effect on the TWI phenotype, although it can slightly and transiently improves muscle strength. We also show that lithium, with this administration protocol, is unable to stimulate autophagy in the TWI mice central nervous system, whereas results suggest that it can restore the ß-catenin activation status in the TWI sciatic nerve. Overall, these data provide intriguing inputs for further evaluations of lithium treatment in TWI mice.

Brain-targeted enzyme-loaded nanoparticles: A breach through the blood-brain barrier for enzyme replacement therapy in Krabbe disease.

Lysosomal storage disorders (LSDs) result from an enzyme deficiency within lysosomes. The systemic administration of the missing enzyme, however, is not effective in the case of LSDs with central nervous system (CNS)-involvement. Here, an enzyme delivery system based on the encapsulation of cross-linked enzyme aggregates (CLEAs) into poly-(lactide-co-glycolide) (PLGA) nanoparticles (NPs) functionalized with brain targeting peptides (Ang2, g7 or Tf2) is demonstrated for Krabbe disease, a neurodegenerative LSD caused by galactosylceramidase (GALC) deficiency. We first synthesize and characterize Ang2-, g7- and Tf2-targeted GALC CLEA NPs. We study NP cell trafficking and capability to reinstate enzymatic activity in vitro. Then, we successfully test our formulations in the Twitcher mouse. We report enzymatic activity measurements in the nervous system and in accumulation districts upon intraperitoneal injections, demonstrating activity recovery in the brain up to the unaffected mice level. Together, these results open new therapeutic perspectives for all LSDs with major CNS-involvement.