Date: 26th November 2019
Lysosomal storage disorders (LSDs) are a large group of more than 50 clinically recognised inherited metabolic diseases. Lysosomes are sacs of enzymes that normally degrade complex cellular components such as proteins and lipids within the body’s cells. If deficient, as is the case in LSDs, there is an abnormal build-up of various toxic material typically as a consequence of a single enzyme deficiency.
Most LSDs show widespread tissue and organ involvement and brain disease is prevalent in two-thirds of all morbidity as a result. They are also progressive diseases, often resulting in early, permanent damage.
Currently, whilst there is no cure for LSDs, several treatments have been used to alleviate the progression of disease. Bone marrow transplants (BMT) and enzyme replacement therapy (ERT) have shown some degree of success however many LSDs are diagnosed early during infancy and show a high degree of complexity.
With the progressive nature of the disease comes an urgency in time-to-treatment. These time constraints and infantile diagnosis are not conducive with BMT. For example finding suitable donors can be a lengthy process and, if successful, immunosuppressing a young and vulnerable child is not ideal.
Furthermore the transplanted cells need time to engraft and create the therapeutic effects. Also, whilst systemic ERT has proven effective for some LSDs, it cannot be used for aspects of the disease with neurological components as the presence of the blood-brain barrier (BBB) prohibits the translocation of many replacement enzymes into the central nervous system (CNS).
Over the recent months we have reported several methods of targeting, and encapsulating ‘cargo’ in the pursuit of disease treatment. Nanoparticles, nanocapsules, nanomachines and shuttle peptides to name but a few. These methods of protecting and/or targeting cargo offer many advantages over systemic treatment, including reduced immunologic reactions and degradation, enhanced pharmacological responses and controlled released of the payload. The addition of targeting molecules may also allow safe passage through some of the biological barrier such as the BBB.
Now scientists based in Pisa, Italy, have designed an enzyme delivery system, which encapsulates cargo in a nanoparticle, and targets it to the brain.
To support in the treatment of Krabbe Disease (KD), a fatal peadiatric neurodegenerative LSD caused by deficient activity of the enzyme galactosylceramidase (GALC), the authors focused their work on a nanoparticle designed to deliver GALC in vivo into the brain.
Previous work performed by the team, showed the successful in vitro delivery of different enzymes in primary fibroblasts. The enzyme delivery platform, was based on cross-linked enzyme aggregates (CLEAs), encased in a nanoparticle (NP) made of poly-(lactide-co-glycolide) (PLGA); a biocompatible, nontoxic, polymer, which is FDA-approved for use in humans. The work showed encapsulation of four different enzymes which retained excellent activity after delivery.
Using the same method the team designed and synthesised three versions of GALC CLEA–loaded NPs. In order to add functionalisation to the NP, they covalently linked targeting peptides Ang2, g7 or Tf2 to the PLGA.
Initial experiments on dual fluorescently labelled versions of the GALC CLEA NPs in vitro showed successful endocytosis and delivery of the NP into the lysosomes of primary fibroblasts cells.
The next step was to test whether the GALC CLEA NPs could restore enzymatic activity in deficient cells. To achieve this the group used murine cells from a KD mouse model called Twitcher (TWI) and fibroblasts from patients with KD, together with the appropriate control cells in each case.
Dose-response curves and time –response experiments were performed for the three targeted GALC CLEA NPs. In all cases the NPs were able to restore GALC activity to wild type levels in TWI cells. Furthermore, they were more efficient at this rescue than an equivalent exposure of the cells with free recombinant murine GALC. Non-targeted NPs could also rescue GALC activity in the TWI cells. Cells ‘rescued’ for enzymatic activity by the NPs reached wild type levels by 24 hours. Overall, a similar result was observed in KD patient fibroblasts, although subtle differences were observed, suggesting the GALC CLEA NPs could restore GALC activity in murine and human GALC-deficient cells.
Whilst the initial work showed great promise for the nano-delivery system the real test was to assess the method in vivo. Could enzymatic activity recovery be achieved in the brain of TWI mice?
Once the stability of the enzyme in the NPs was confirmed in a biological fluid mimic (free recombinant GALC was not stable), the TWI mice were systemically injected with GALC CLEA NPs and labelled with a fluorophore to enable tracking. Young pre-symptomatic mice were used and examined 4 hours post treatment. Confocal microscopy was used to determine location of NPs and enzyme activity was measured in the keys organs of the central and peripheral nervous system.
GALC activity was up approximately 42% compared to the activity measured for wild type mice in the brains of mice treated with targeted NPs. This was a similar level of activity seen in mice heterozygous for TWI (~45%), a level at which the disease does not manifest. Untreated TWI mice had near undetectable levels of GALC activity, as did TWI mice treated with free recombinant murine GALC or non-targeted NPs.
The liver saw increased levels of GALC activity in all cases of treatment, although this was higher in free recombinant murine GALC or non-targeted NPs TWI mice; likely due accumulation of the non-targeted enzyme. A similar result was seen in the kidneys. In contrast, sciatic nerve and spinal cord appeared to be unchanged following treatment.
The study concluded by assessing the presence of NPs in the brain by confocal microscopy. Fluorescence was only detected in the brain for the three targeted NPs, whilst liver analysis showed fluorescent NPs were present for both targeted and non-targeted NPs.
The authors therefore concluded that targeted GALC CLEAs NPs could induce GALC enzymatic activity via systemic administration in TWI brains. This ‘rescue’ was up to the level required for the disease not to manifest in mice. The presence of NPs was detected in accumulation organs such as the liver and kidney but the spinal cord and sciatic nerves were unaffected.
The study, published in Science Advances, offers great hope for sufferers of KD, and those with LSDs with a neurological component. The fact that targeted NPs could specifically cross the BBB highlights the crucial element of functionalisation via specific targeting peptides. Indeed, the lack of rescued activity in the spinal cord and sciatic nerve, indicates that either these regions require a longer time post treatment for the enzyme activity to accumulate or perhaps other targeting peptides for these areas would be more suitable. Certainly peptide selection is important, and combinatorial therapy may offer advantages in such complex diseases. These are likely areas of future investigation.
This targeted approach offers many advantages over current systemic ERTs using free recombinant enzymes, which are currently the most widely applied method to treat symptoms of non-CNS LSDs but they require long-term, frequent treatment, with often severe side-effects. In the work described here, encapsulating the enzyme protected the cargo and further optimisation may see tuneable release mechanisms to enable more control in the discharge from the NP, and so negate some of the disadvantages of traditional ERT.
As with mice, human heterozygous carriers of the GALC mutation also exhibit a completely healthy phenotype. Thus, the results here that show the ability of this approach to restore enzyme activity to near heterozygote levels in mice are very promising. Combined with the data showing that NPs are able to carry enzymatically active GALC efficiently in human primary fibroblasts, this work looks promising and the findings will hopefully translate into clinical research where longer treatment times, long-term effects and toxicities can be more thoroughly explored.
This is however, an exciting breakthrough and it opens-up the opportunity of a new, exciting approach, not only for CNS-involved LSDs, but for other deficiencies and treatments that would benefit from advances in crossing the BBB to reach the CNS.
With respect to this work and LSDs, the authors initially see this technique as a short term, ‘buffering’ therapy. Applied immediately after birth, halting the progressive destruction of the disease, and ‘buying’ the patient time until a longer term therapy can be used. This would in theory allow gene replacement or editing therapy to be started, or in a more traditional approach offer more time to find a bone marrow donor.
Del Grosso, A., M. Galliani, L. Angella, M. Santi, I. Tonazzini, G. Parlanti, G. Signore and M. Cecchini (2019). “Brain-targeted enzyme-loaded nanoparticles: A breach through the blood-brain barrier for enzyme replacement therapy in Krabbe disease.” Science Advances 5(11): eaax7462.