Efficient engraftment of genetically modified cells is necessary to ameliorate central nervous system involvement of murine model of mucopolysaccharidosis type II by hematopoietic stem cell targeted gene therapy

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Abstract

Mucopolysaccharidosis type II (MPS II) is a lysosomal storage disease (LSD) caused by a deficiency of the iduronate-2-sulfatase (IDS) that catabolizes glycosaminoglycans (GAGs). Abnormal accumulations of GAGs in somatic cells lead to various manifestations including central nervous system (CNS) disease. Enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) are the currently available therapy for MPS II, but both therapies fail to improve CNS manifestations. We previously showed that hematopoietic stem cell targeted gene therapy (HSC-GT) with lethal irradiation improved CNS involvement in a murine model of MPS II which lacks the gene coding for IDS. However, the strong preconditioning, with lethal irradiation, would cause a high rate of morbidity and mortality. Therefore, we tested milder preconditioning procedures with either low dose irradiation or low dose irradiation plus an anti c-kit monoclonal antibody (ACK2) to assess CNS effects in mice with MPS II after HSC-GT. Mice from all the HSC-GT groups displayed super-physiological levels of IDS enzyme activity and robust reduction of abnormally accumulated GAGs to the wild type mice levels in peripheral organs. However, only the mice treated with lethal irradiation showed significant cognitive function improvement as well as IDS elevation and GAG reduction in the brain.

These results suggest that an efficient engraftment of genetically modified cells for HSC-GT requires strong preconditioning to ameliorate CNS involvement in cases with MPS II.

Introduction

Mucopolysaccharidosis type II (MPS II, Hunter syndrome, OMIM 309900) is an X-linked recessive inherited lysosomal storage disease (LSD) caused by deficiency of iduronate-2-sulfate (IDS) that degrades the glycosaminoglycans (GAGs), including heparan sulfate and dermatan sulfate [1,2]. Deficiency of the lysosomal enzyme leads to a progressive accumulation of GAGs in systemic organs and causes various symptoms including hepatosplenomegaly, skeletal deformities, valvular heart disease, upper airway obstruction, and central nervous system (CNS) involvement [3,4]. Among these symptoms, the CNS involvement is most key issue to affect patients' quality of life. Enzyme replacement therapy (ERT) is available for patients with MPS II and about 2000 patients have received clinical benefits from it (personal communication). However, the delivered enzyme does not cross the blood-brain barrier (BBB), and these patients gain few CNS therapeutic effects [5,6]. Hematopoietic stem cell transplantation (HSCT) is another therapeutic option with CNS therapeutic effects for some LSDs. The therapeutic mechanism involves cross correction by brain migrated donor cells that secrete the normal enzyme and effectively correct its deficiency in the surrounding tissues [7]. HSCT has shown therapeutic benefits for peripheral tissues, but its CNS impact in cases with MPS II remains unclear [5,[8], [9], [10], [11]].

Other therapies have been investigated in animal models and human patients to improve the CNS involvement in patients with MPS II [5]. One approach involves the direct infusion of enzyme to the CNS to bypass the BBB. We and others have reported that the MPS II mice treated with intraventricular ERT showed increased IDS activities, decreased GAGs in the brain and behavioral improvements [12], and similar results have been reported for other types of MPS diseases [13,14]. In a clinical trial, intrathecal IDS administration led to robust reduction in cerebrospinal fluid GAGs [15]. Another approach involves engineered enzymes that can penetrate the BBB [5]. Fusion proteins with anti-insulin receptor antibody or anti-transferrin receptor antibody can cross the BBB [16,17]. In the case of MPS II, IDS was fused with the anti-transferrin receptor antibody. The animal study and phase I/II human trial using this mechanism produced promising results [18,19].

Gene therapies (GT) have also been considered promising strategies for treating CNS involvement of LSDs including MPS II [20]. Three gene therapy approaches have been considered. The first one is in vivo gene therapy using adeno-associated virus (AAV) vectors. Local or systemic administration of AAVs carrying the normal IDS gene improves CNS manifestations of MPS II mice [[21], [22], [23], [24]]. An early phase human trial of direct administration of AAV to the brain in patients with MPS III also showed positive effects [25,26]. As the second, the gene editing strategy has also been tested in mice [27,28] and humans [29]. The IDS gene was precisely inserted into the albumin locus in the liver of mice using gene editing technology. As a result, the normal IDS gene was transcribed to facilitate large amounts of IDS production by the extremely strong albumin promotor. This IDS was excreted to the blood stream from the liver and was able to penetrate the BBB. The third approach involves HSC-GT using a lentivirus vector. The rationale of this approach is that enzyme-competent microglial cells differentiated from genetically modified hematopoietic stem cells (HSCs) can migrate into the brain and correct the enzyme deficiency of the surrounding neural cells [30,31]. This approach is similar to HSCT, but the difference is that the microglial cells in HSC-GT produce larger amounts of the missing enzyme and that HSC-GT is carried out with autologous cells. This method is very promising and positive results have been reported in a clinical trial of metachromatic leukodystrophy [32,33]. Thus, so far, HSC-GT is the only approach for LSD that has been proven beneficial in humans. We previously reported that HSC-GT using a lentiviral vector achieved increased IDS activity and GAGs reduction in the brain and that it improved the behavioral deficits of MPS II mice [34]. A similar approach using IDS gene fusions to the receptor-binding domain of apolipoprotein E (ApoE) in a vector to facilitate BBB penetration of the enzyme was conducted and achieved normalization of brain function in MPS II mice [35,36]. LSDs cause global brain malfunction, thus HSC-GT seems to have a clear advantage compared to the local administration of AAV. However, the above-mentioned regimens required strong preconditioning methods, such as high dose chemotherapeutic agents and/or whole body irradiation to open a recipient's niche for efficient engraftment of the genetically modified cells. Strong preconditioning often results in various life-threatening complications; in humans, it is a last resort used for some patients with malignant disease, but is not appropriate for patients with MPS II. However, whether mild preconditioning for MPS II HSC-GT can be used to improve CNS symptoms is unclear.

In this study, we examined whether HSC-GT with mild conditioning also improves the CNS symptoms in MPS II mice as does HSC-GT with lethal preconditioning.

Section snippets

Animals

C57BL/6 J female mice heterogeneous for the disruption of the mouse IDS locus (exon 4 and part of exon 5) with replacement vector containing the neomycin-resistance gene were kindly gifted by Dr. Joseph Muenzer (University of North Carolina, Chapel Hill, NC). These female heterozygote mice were mated with C57BL/6 wild male mice. Hemizygous MPS II mice were identified (IDS+/°) by genotyping using polymerase chain reaction (PCR) analysis. All mice used in this study were males identified as

Ex vivo HSC-GT for MPS II using lentiviral vector increases IDS activity in various peripheral tissues

In the spleen, IDS activity levels in LDIR, ACK2/LDIR and LIR group increased compared to the WT level (Fig. 2a). However, the mean activity of IDS in the mice of the ACK2/LDIR group was significantly higher than that in the mice of the LDIR group (49.5 ± 27.7 nmol/4 h/mg vs. 19.3 ± 12.6 nmol/4 h/mg, p = .0014). As we expected, the IDS activity in the LIR group mice was also higher than LDIR group. Plasma IDS activities in three treated groups increased dramatically 4 weeks after treatment and

Discussion

ERT, which is one of the current treatments for MPS II, cannot alleviate CNS symptoms because the enzyme does not penetrate the BBB. Although HSCT is another choice of treatment, its CNS effects are unclear [5,[9], [10], [11]]. More than 60% of patients with MPS II develop CNS disease and developing treatments for these symptoms is an urgent task. To address this issue, clinical trials with brain directed ERTs are underway such as direct administration of the enzyme to the brain [15] or

Role of the funding source

This study was supported by AMED under Grant Number JP19ek109224h.

Author contribution

S.M. and T.O. designed and conducted the research and analyzed the data. S.M wrote the manuscript. Y.S., T.H., T.F. and H.K. performed the research and discussed the data. A.M.W designed the behavioral experiments. F.K. and H·I. analyzed the results and edited the manuscript.

Declaration of Competing Interest

T.O. and H.I have active research support from Sanofi-Genzyme. These activities have been fully disclosed and are managed under a Memorandum of Understanding with the Conflict of Interest Resolution Board of the Jikei University School of Medicine.

Acknowledgments

The authors thank Ms. Sayoko Iizuka, Ms. Natsumi Iishi, Ms. Hiromi Hiraki, Ms. Aimi Yuasa, and Ms. Kazuko Shibahara (the Jikei University School of Medicine) for their excellent technical assistance, and members of Laboratory Animal Facility (the Jikei University School of Medicine) for helping with animal studies. The authors are indebted to Dr. Joseph Muenzer for providing MPS II mice, to Dr. Donald B. Kohn for providing lentiviral vector, to Dr. Shinichi Nishikawa for providing ACK2.

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