Multiple sulfatase deficiency (MSD) is a devastating, ultra-rare inherited lysosomal storage disorder affecting an estimated 1 in 500,000 individuals (1). Like many other rare diseases, MSD is chronic, degenerative, and debilitating. Individuals with MSD have a significantly shortened life expectancy, averaging around 13 years, and experience poor quality of life in that time. Like many other rare diseases, MSD has historically been understudied and consequently lacks effective treatment options (2,3). Currently, there are no approved therapies to slow or reverse MSD disease progression. Recognizing this unmet need for effective MSD treatment, fifth year CAMB-GTV PhD candidate Vi Pham from Rebecca Ahrens-Nicklas’ lab sought to develop an ex vivo gene therapy approach for MSD (4).
MSD results from pathogenic germline variants in the gene SUMF1, which lead to diminished activity of the SUMF1 protein product formylglycine-generating enzyme (FGE). FGE is required for activation and subsequent activity of all cellular sulfatases, and individuals with MSD are therefore functionally deficient in all cellular sulfatases. In the absence of functioning sulfatases, toxic sulfated molecules like glycosaminoglycans (GAGs) accumulate in lysosomes throughout the body, leading to multiple debilitating symptoms that include bone abnormalities, hepatomegaly, respiratory complications, cardiac dysfunction, hearing loss, and neurologic regression (5).
One attractive therapeutic strategy for monogenic diseases like MSD is gene therapy (6). In vivo adeno-associated virus (AAV)-mediated gene therapy is currently in preclinical development for MSD, and gene therapy in combination with hematopoietic stem cell transplantation (hereafter referred to as HSCT-GT) has been shown to alleviate symptoms and slow disease progression in preclinical models and early phase clinical trials for other lysosomal storage disorders. Vi therefore hypothesized that using HSCT-GT to deliver functional copies of SUMF1 to hematopoietic stem cells derived from individuals with MSD may similarly benefit individuals suffering from MSD.
To begin testing her hypothesis, Vi designed a clinically translatable lentiviral vector expressing a functional copy of human SUMF1. Transducing immortalized patient-derived primary fibroblasts with the SUMF1 lentiviral vector resulted in robust expression of FGE, with increasing vector copy number integrations correlating with higher FGE expression. Importantly, cells transduced with the SUMF1 lentiviral vector exhibited increased activity of three different sulfatases, with one of the three sulfatases exhibiting activity comparable to cells lacking a SUMF1 mutation (wild-type). Cells transduced with the SUMF1 lentiviral vector also exhibited decreased lysosomal accumulation of GAGs. As GAG accumulation is a key factor contributing to the tissue damage and organ dysfunction observed in MSD, this suggests that SUMF1 gene therapy can effectively increase FGE expression and sulfatase activity in FGE-deficient cells.
Given her promising in vitro findings, Vi next assessed the safety, durability, and efficacy of her SUMF1 lentiviral vector in vivo using a mouse model harboring a clinically relevant pathogenic mutation in the Sumf1 gene. Vi performed primary and secondary transplants of hematopoietic stem cells (HSCs) transduced ex vivo with the SUMF1 lentiviral vector. Transplanted SUMF1 HSCs engrafted with high efficiency and differentiated into both erythroid and lymphoid populations, in proportions similar to those observed in untreated wild-type mice. This suggests that exogenous expression of SUMF1 in HSCs does not alter normal hematopoiesis. Importantly, all mice survived to study endpoint without any signs of transplant-related morbidity, indicating that SUMF1 HSCT-GT is well-tolerated and safe. To assess the stability of the SUMF1 lentiviral vector, vector copy number was determined four months post-transplant for both primary and secondary transplant recipient mice and was found to fall within the clinically relevant range for SUMF1. These data lead to the conclusion that the vector stably integrates into the transduced HSCs, resulting in robust FGE expression that is not lost with progressive cell divisions.
To assess the efficacy of her SUMF1 HSCT-GT approach, Vi then investigated the effects of SUMF1 HSCT-GT on sulfatase activity and GAG accumulation in MSD mice. SUMF1 HSCT-GT significantly increased the activity of the sulfatase arylsulfatase A (ARSA) in the spleen, which had a high vector copy number. In contrast, there was no effect in the brain, heart, lung, and liver, which had significantly lower vector copy numbers compared to the spleen. Despite the tissue-specific restoration of sulfatase activity, however, SUMF1 HSCT-GT reduced accumulation of multiple GAG subspecies relative to untreated MSD mice in all five tissues, with the brain, liver, and spleen exhibiting the greatest reductions. Notably, MSD mice receiving non-transduced HSCs exhibited increased GAG levels compared to untreated MSD mice, and the SUMF1 HSCT-GT was able to significantly reduce this transplant-associated GAG accumulation in all five tissues. These data indicate that, while transduced HSCs preferentially localize to some organs, SUMF1 HSCT-GT is effective at reducing the accumulation of some GAG species in multiple organs.
To better understand the efficacy and therapeutic potential of her SUMF1 HSCT-GT approach, Vi next assessed the effects of SUMF1 HSCT-GT on neuroinflammation and neurologic function, as neurological symptoms are a major source of morbidity in individuals with MSD. Importantly, transplanted SUMF1-expressing HSCs can cross the blood-brain barrier and differentiate into microglia-like cells with the potential to secrete activated sulfatases to neighboring cells lacking sulfatase function. SUMF1 HSCT-GT partially reduced neuroinflammation by decreasing the presence of activated microglia, but was unable to improve motor coordination, balance, or muscular strength in MSD mice. SUMF1 HSCT-GT did, however, improve spatial learning and memory, and also reduced neurodegenerative phenotypes in a subset of MSD mice. These data indicate that SUMF1 HSCT-GT can improve spatial learning and reverse memory deficits associated with MSD, though further optimization of the HSCT-GT approach is needed to rescue neuromuscular deficits and slow neurodegeneration.
Collectively, Vi’s data demonstrate that ex vivo lentiviral SUMF1 HSCT-GT is a novel treatment strategy with the potential to improve symptoms and slow disease progression in individuals suffering from MSD. While further optimization is needed to improve the benefits of the SUMF1 HSCT-GT approach in treating the neurological and motor deficits associated with MSD and to improve the efficacy in tissues other than the spleen, Vi’s findings serve as a proof of principle for using HSCT-GT to treat MSD and lay the groundwork for bringing SUMF1 HSCT-GT into clinical trials. Given the lack of therapies to slow or reverse MSD disease progression, Vi’s findings offer hope to individuals living with MSD. Future studies investigating optimized vector constructs, including those expressing functional copies of downstream sulfatases in addition to FGE, as well as studies investigating the combination of SUMF1 HSCT-GT with small molecules therapies currently in development for MSD, may reveal even more effective treatment options to significantly improve quality of life for individuals living with MSD.
Abbreviations
MSD: multiple sulfatase deficiency
FGE: formylglycine-generating enzyme
GAG: glycosaminoglycan
AAV: adeno-associated virus
HSCT-GT: hematopoietic stem cell transplantation - gene therapy
HSC: hematopoietic stem cell
ARSA: arylsulfatase A
Key Terms
Formylglycine-generating enzyme: an enzyme present in the endoplasmic reticulum that catalyzes the conversion of cysteine to formylglycine. Its activity is required for the activation of all sulfatases in humans.
Gene therapy: a technique that seeks to modify the expression of a gene of interest in order to treat disorders resulting from problems in that gene’s expression or function. In this article specifically, we are discussing a type of gene therapy that delivers functional copies of a gene of interest into cells with pathogenic mutations in that gene to promote functional activity of the gene’s protein product.
Hematopoiesis: the process of producing blood cells in the bone marrow.
HSCT-GT: the transplantation of hematopoietic stem cells (HSCs) that have been transduced ex vivo to exogenously express a gene of interest. The process involves 1) deriving HSCs from the patient, 2) transducing patient-derived HSCs ex vivo with a lentiviral vector expressing the gene of interest, 3) irradiating the patient to deplete endogenous untransduced HSCs, and 4) reinfusing the edited HSCs into the patient.
Monogenic disease: a genetic disorder resulting from pathogenic mutations in a single gene.
Neuroinflammation: inflammation of the brain and spinal cord primarily mediated by microglia, which are the resident innate immune cells of the central nervous system.
Sulfatase: an enzyme that catalyzes the hydrolysis of sulfate esters, thereby removing sulfate groups from a range of substrates. Sulfatases play a critical role in lysosomal degradation of sulfated molecules.
SUMF1: the gene encoding formylglycine-generating enzyme.
References
https://www.orpha.net/en/disease/detail/585?name=multiple%20sulfatase%20deficiency&mode=name
Vi Pham et al. Hematopoietic stem cell gene therapy improves outcomes in a clinically relevant mouse model of multiple sulfatase deficiency. Molecular Therapy, 2024. https://doi.org/10.1016/j.ymthe.2024.08.015
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