For over three decades, the LSDs have been at the forefront of drug discovery for the rare diseases. ¹ , ² , ³ , ⁴ This has led to the approval of treatments for 14 of these diseases, including ERTs for 11 (Gaucher, Fabry, Pompe, MPS I, II, IV, VI, and VII, ASMD, alpha‐mannosidosis, and Batten), small molecule therapies for 4 (Gaucher, Fabry, Niemann‐Pick type C, and Cystinosis), and 1 gene therapy for Metachromatic Leukodystrophy. However, despite the remarkable successes in this field, major challenges remain. This includes the limited delivery and impact of many therapies at critical sites of pathology (including the muscle, bones, and brain) and the extremely high cost of treatment. In part, this latter challenge is due to the fact that most of these therapies are useful in only one disease, limiting their commercial potential.

Another emerging challenge is the ability to diagnose LSD patients prior to the onset of symptoms, including as newborns. Early diagnosis leads to improved care and also raises the possibility of early treatment intervention. However, at present it is not possible to reliably predict the clinical course in most LSD patients, particularly prior to clinical presentation. This has led many investigators to focus on identifying biochemical and other surrogate markers that can be easily measured in patients and are predictive of their clinical course. ⁷ , ⁸ , ⁹ , ²⁹ In most cases these molecules are the disease‐specific substrates themselves (e.g., heparan sulfate for MPS III), but recent efforts have also focused on identifying molecules that represent downstream pathologic changes as well. Ideally, these changes might be occurring in more than one LSD. One example of such a molecule that shows promise for several neurologic LSDs is the neurofilament light chain. ³⁰

Similarly, recent efforts have also turned to identifying drugs that can be used in multiple LSDs. For example, Miglustat (marketed as Zavesca®) is a small molecule inhibitor of the enzyme glucosylceramide synthase and is used to treat patients with Niemann‐Pick disease type C and Gaucher disease. ³¹ , ³² Another example is N‐acetyl‐leucine, a small molecule that targets common neurologic mechanisms including neuroinflammation, neuronal membrane potential, and regulation of calcium channels. It is a recently approved treatment for Niemann‐Pick type C and under development for GM₁ and GM₂ gangliosidoses. ³³ , ³⁴ In principle, developing drugs such as these that target more than one disease should drive down the development costs and ultimately the cost of treatment for patients.

In this regard, we and others have focused on inflammation as a common therapeutic target in the LSDs. ³⁵ , ³⁶ , ³⁷ , ³⁸ , ³⁹ , ⁴⁰ This common abnormality, which has been attributed in part to activation of the NF‐kappa beta signaling pathway, has been documented in many LSD animal models, as well as in patients and contributes to pathology in many organs, including the brain and other sites that have proven difficult to treat by traditional therapies. Several years ago, these observations led us to repurpose a known drug, pentosan polysulfate (PPS), for use in the MPS disorders, and its efficacy was demonstrated in mouse and dog models of MPS I, a rat model of MPS VI, and in mice with MPS IIIA, where the impact on neurologic disease was shown. ⁴¹ , ⁴² , ⁴³ This has led to clinical trials in MPS I, II, and VI patients and the ongoing development of this treatment for multiple MPS disorders. ⁴⁴ , ⁴⁵ , ⁴⁶

As part of our effort to study inflammation in the LSDs, we have recently turned to the endocannabinoid system, an evolutionally conserved signaling system that consists of two essential G‐protein coupled receptors, CB1R and CB2R, and several lipid mediators that are carefully regulated by a series of synthetic and degradative enzymes. ¹² , ¹³ , ⁴⁷ CB1R is primarily expressed in neurons and was originally identified as the ligand responsible for the neurotropic effects of the CB, Δ‐9 tetrahydrocannabinol (THC), while CB2R is primarily expressed on immune cells where it plays an important role in modulation of the immune system. ¹² , ¹³ , ⁴⁸ , ⁴⁹ Notably, CB2R expression is upregulated in many disorders associated with inflammation, and CB2R agonists have anti‐inflammatory properties that impact disease progression in many disease models. ¹⁴ , ⁴⁸ , ⁴⁹ Since chronic inflammation is a common feature of many LSDs, independent of the individual enzyme deficiency or type of substrate that is accumulating, we hypothesized that CB2R expression may be elevated in the LSDs and that CB2R agonists may have beneficial effects in one or more of these disorders.

We initiated our work by evaluating the expression of CB2R in plasma from six LSD animal models, chosen randomly among the breeding colonies we maintain in our laboratory. Among these, we found a significant elevation of CB2R in Farber disease and MPS IIIA mice, followed by MPS I and Fabry disease mice. We did not find significant elevation in plasma from ASMD mice or rats with MPS VI. For this analysis, we purposely chose aged LSD animals that exhibited tissue pathology and/or clinical abnormalities, and compared them to age‐matched WT mice derived from within the colonies. To our knowledge, this is one of the only studies in which plasma CB2R levels have been analyzed and shown to correlate with disease. Since CB2R is a G‐coupled receptor generally found in the outer membrane of blood cells, it is not clear why it is elevated in the plasma. We hypothesize that it may be present in small membrane fragments derived from disrupted cells that remain in the plasma after centrifugation, or perhaps is associated with lipoproteins or other plasma components. However, this remains to be proven. Nonetheless, CB2R is clearly elevated in several LSDs and this likely reflects their activated immune state and the proliferation of blood‐derived immune cells that occurs in these disorders.

To confirm the plasma CB2R findings, we next performed IHC analysis on tissues from the two disease models (Farber and MPS IIIA) that exhibited the highest plasma expression. High CB2R levels were also found in the tissues, including the liver, spleen, and brain of Farber mice and the liver and brain of MPS IIIA mice. qPCR analysis of the MPS IIIA animals confirmed these findings and indicated that the IHC observations were due to elevated Cb2r gene expression. We also investigated whether the elevated CB2R expression in these animals was associated with the activated inflammatory state of these animals, as has been shown in other diseases, and would respond to treatments that are known to reduce inflammation. To this end, we treated the Farber mice by ERT using recombinant acid ceramidase. Farber disease is due to the inherited deficiency of acid ceramidase, resulting in the accumulation of the lipid ceramide, which leads to chronic inflammation, cell death, and organ damage. ⁵⁰ ERT has been shown to reduce ceramide levels, leading to the resolution of inflammation and improvement in organ pathology. ²³ Notably, we found that six‐week ERT treatment reduced the levels of CB2R in the Farber mice, consistent with its known impact on inflammation. In the future, it will be of interest to study other treatment modalities that reduce inflammation in this and other LSDs and evaluate their impact on CB2R expression as well.

We then evaluated the therapeutic potential of CB2R modulation in these diseases. CB2R agonists have been shown to reduce inflammation in many disease models, and some have been shown to cross the blood–brain barrier. ¹² , ⁴⁸ , ⁴⁹ We chose two of these agonists and evaluated them using skin fibroblasts obtained from an MPS IIIA patient, and found that both significantly reduced the release of MCP‐1 into the culture media. MCP‐1 is an inflammatory chemokine that is elevated in this and other LSDs. ²⁴ , ²⁵ Based on this finding, we chose one CB2R agonist (JWH133) ²⁶ , ²⁷ and evaluated it in vivo in the MPS IIIA mice. After 7 months of treatment (once weekly, 10 mg/kg), the treated 10‐month‐old MPS IIIA mice exhibited reduced CB2R and MCP‐1 expression in the plasma, reduced GFAP staining in the brain, and improved performance in a hanging test that accessed motor strength and coordination. This latter finding was due to maintenance of stable function in the treated mice, versus declining function in the MPS IIIA mice treated with vehicle alone. To confirm these findings in MPS IIIA, we also treated Farber mice with JWH133 and found reduced MCP‐1 in plasma and liver.

Overall, these findings revealed the potential of CB2R as a novel biomarker and therapeutic target for the LSDs. Importantly, it is elevated in several different LSD models, consistent with the chronic inflammation that is common among them, and at least in the case of MPS IIIA mice, it appears to follow the progression of the disease (e.g., was not elevated at 2 months of age but was highly elevated by 10 months, where the disease pathology is evident). It is not clear why CB2R was not elevated in plasma of ASMD and MPS VI animals, which also exhibit systemic inflammation, but perhaps the age of these animals (5 months for ASMD mice and 8 months for MPS VI rats) was not sufficient to activate this pathway in the blood. Further studies are needed to elucidate these findings and, importantly, determine if the findings in these animal models correlate with those in LSD patients.

The impact of the CB2R agonists in MPS IIIA and Farber disease mice is also worthy of further investigation. Consistent with its impact on other diseases, JWH133 reduced the levels of CB2R and the inflammatory cytokine, MCP‐1, and in the case of MPS IIIA also improved motor function. Notably, GFAP staining in the brain was also reduced, indicating the ability of this molecule to impact neuroinflammation. While these results provide important proof‐of‐concept for using CB2R agonists in these and perhaps other LSDs, studies that evaluate additional clinical and pathological endpoints in the individual models, including survival, are needed. Of note, CB2R activation has also been shown to reduce neuronal loss, protect the blood–brain barrier, elevate beneficial M2 microglia in other disease models, and regulate TFEB‐mediated autophagy, an abnormality that is evident in most LSDs. ⁵⁰ , ⁵¹ It is therefore possible that CB2R treatment may have additional effects in the LSDs, including unexpected outcomes as we observed with PPS, where it was shown to reduce glycosaminoglycan storage in the MPS disorders in addition to reducing inflammation. Indeed, the cross‐talk between inflammation and these other pathways is an important subject for future investigations.

These studies extend our ongoing investigation of the endocannabinoid system in the LSDs. ⁴⁷ Previously, we found that CB1R expression was reduced in the brain of ASMKO mice, and that treatment of these mice with inhibitors of the enzyme fatty acid amide hydrolase, which activates this receptor, led to reduced sphingomyelin levels and improvements in tissue pathology and clinical findings. ⁵² This followed early studies showing that treatment of astrocytes with THC led to activation of CB1R and the enzyme neutral sphingomyelinase, resulting in sphingomyelin degradation. ⁵³ Others have recently reported that aged astrocytes exhibit cholesterol storage in lysosomes that is due, at least in part, to reduced expression of the NPC1 protein that is deficient in Niemann‐Pick type C disease, and that this abnormality could be alleviated by treatment with CBs, including cannabidiol (CBD). ⁵⁴ Thus, the endocannabinoid system remains a potentially important target for the treatment of these and other LSDs and is worthy of further study.

In conclusion, the results reported here are the first investigation of CB2R in the LSDs and the first to reveal its potential as a biomarker and therapeutic target for these diseases. Future studies should extend these findings in the LSD animal models and correlate the animal results to humans.