Compositions useful in treatment of metachromatic leukodystrophy

Abstract

A recombinant adeno-associated virus (rAAV) having an AAVhu68 capsid and a vector genome containing a nucleic acid sequence encoding a functional human arylsulfatase A (ARSA) is also provided. A production system for producing rAAV is also provided; a pharmaceutical composition containing rAAV; and a method for treating a subject with metachromatic leukodystrophy, improving symptoms of metachromatic leukodystrophy, or slowing the progression of metachromatic leukodystrophy by administering an effective amount of rAAV to a desired subject.

Description

[Technical Field] This invention relates to a recombinant adeno-associated virus (rAAV) for treating metachromatic leukodystrophy, a pharmaceutical composition comprising the rAAV, and an rAAV production system for producing the rAAV. [Previous Technology] Metachromatic leukodystrophy (MLD) is a monosomic recessive neurosphingolipid storage disorder caused by mutations in the gene encoding the lysosomal enzyme ARSA (Von Figura et al., 2001; Gieselmann and Krageloh-Mann, 2010). ARSA deficiency leads to the accumulation of its native receptors, which are sulfated galactosylsphingolipids (galactosylceramide-3-O-sulfate and galactosylsphingosine-3-O-sulfate), commonly known as sulfatides. In addition to Schwann cells and macrophages in the peripheral nervous system (PNS), sulfatides also accumulate in oligodendritic cells, microglia, and lysosomes of certain types of neurons in the central nervous system (CNS) (Peng and Suzuki, 1987). Although it primarily affects the PNS and CNS, sulfadiazine accumulation also occurs in visceral organs; most notably the kidneys, liver (Toda et al., 1990), and gallbladder (Rodriguez-Waitkus et al., 2011; McFadden and Ranganathan, 2015). MLD patients (i.e., those with mutations in both of their paired genes) typically have ARSA enzyme activity of 0-10% of the control value in assays based on synthetic substrates. Individuals with an ARSA mutation, having a single mutated ARSA paired gene and a normal paired gene, are clinically unaffected and typically have approximately 10% of the control value of ARSA enzyme activity, while asymptomatic individuals with a pseudodeficiency (PD, another genetically different form of ARSA deficiency) paired gene have approximately 10-20% of the ARSA enzyme activity of healthy controls (Gomez-Ospina, 2017). Clinically, MLD can be differentiated into three forms based on the age of onset of symptoms across a broad, continuous range spanning disease severity: rapidly progressive severe late infantile MLD, juvenile MLD, and late-onset, slowly progressive adult MLD, accounting for 50-60%, 20-30%, and 15-20% of MLD diagnoses, respectively (Gomez-Ospina, 2017; Wang et al., 2011). Infant MLD is considered an orphan disease. Late infantile MLD, which occurs before 30 months of age, is the most severe form. Late infantile MLD presents with uniform clinical manifestations and a rapidly progressing, predictable disease course. Juvenile MLD is characterized by an age of onset between 30 months and 16 years, with a median age of onset of 6 years and 2 months (Kehrer et al., 2011a) to 10 years (Mahmood et al., 2010), depending on research. To better characterize the clinical phenotype, a subgroup of juvenile MLD patients has been described as early juvenile MLD, characterized by a clinical age of onset ≤6 years and similar initial disease progression, albeit less rapid, compared to children with late infancy MLD (Biffi et al., 2008; Chen et al., 2016; Sessa et al., 2016). The early juvenile and late infancy phenotypes are collectively referred to as early-onset MLD (Sessa et al., 2016). In late juvenile MLD patients (i.e., those with symptoms onset between 7 and 16 years of age), behavioral problems, attention deficit, or cognitive decline typically appear first, sometimes accompanied by gait disturbances. There are no approved therapies for curing or improving the progression of MLD (disease-modifying therapy). Since MLD is caused by defective ARSA, various research approaches target correcting biochemical defects by replacing functional ARSA in the affected neural tissue of the CNS. Enzyme replacement therapy (ERT) and hematopoietic stem cell transplantation (HSCT) rely on providing normal enzymes to ARSA-deficient cells, while gene therapy approaches are based on the overexpression of wild-type ARSA in different cell types (Patil and Maegawa, 2013). The efficacy of hematopoietic stem cell transplantation (HSCT) using umbilical cord blood (UCB), allogeneic peripheral blood stem cells, or allogeneic bone marrow depends on the MLD phenotype and the timing of intervention relative to the patient's disease state (Patil and Maegawa, 2013; van Rappard et al., 2015). Bone marrow transplantation (BMT) requires a usable, HBeAg-matched sibling donor for optimal outcomes (Boucher et al., 2015) and carries the risk of transplant- and conditioning-related complications, such as graft-versus-host disease (GvHD), infection, and death. Umbilical cord blood (UCB) transplantation offers an alternative to BMT with advantages including faster availability, lower risk of GvHD, lower mortality, higher overall donor chimerism, and better correction of enzyme deficiencies (Batzios and Zafeiriou, 2012; Martin et al., 2013). However, BMT is not widely available in Europe. Brain implantation is slow, typically requiring months for cells to implant, migrate to the central nervous system, differentiate, and restore enzyme levels. Furthermore, the physiological enzyme levels achieved with HSCT may not be sufficient to correct deficiencies across the entire CNS. This could explain why transplantation is ineffective for rapidly progressing early-onset MLD, and even if performed before symptoms appear, it may not be able to correct or stabilize all aspects of the disease (de Hosson et al., 2011; Martin et al., 2013; Boucher et al., 2015). Thus, there is a substantial unmet need for fast-onset therapies that can stop or prevent disease progression in such patients. Besides HSCT, various other cell-based pathways exist that (over-)express ARSA and deliver the enzyme to affected cells to treat the neurological manifestations of MLD. These pathways include microencapsulated recombinant cells, oligodendrocytes and neural precursor cells, as well as embryonic stem cells. These cell therapies have shown considerable clearance of thiolipin deposits in animal models (Patil and Maegawa, 2013), but have not yet been tested in humans. Ex vivo lentiviral gene therapy has been explored, which involves transducing autologous CD34+ cells into a lentiviral vector encoding human ARSA and re-infusing the patient with genetically corrected cells, combined with hematopoietic stem cell transplantation and gene therapy (HSC-GT) (Biffi et al., 2013). While this therapy shows promise for patients identified before symptom onset (after diagnosis of older siblings with the disease), it has not yet been shown to be effective for symptomatic patients. Unfortunately, because newborn screening is not yet feasible, and most new MLD diagnoses are made after symptom onset, it is an unlikely treatment option for many MLD patients. Furthermore, there are inherent risks associated with myeloablative conditioning regimens and the risk of insertional mutagenesis associated with these integrative vectors. Pharmacological and toxicological studies conducted at the NHP have demonstrated significant dose-limiting toxicity due to inflammation of the brain around the injection site (encephalitis) (Zerah et al., 2015). A phase 1 / 2 clinical trial is underway to evaluate the safety and efficacy of AAVrh10-mediated ARSA gene transfer into the brains of children with early-onset MLD (NCT01801709) (Aubourg, 2016), also involving intrabrain delivery of the vector at 12 sites in the white matter of the brain (Zerah et al., 2015). Results of this trial have not been published except in abstract form, with preliminary reports suggesting a lack of efficacy in preventing onset or halting disease progression (Sevin et al., 2018). The trial organizers have not discussed the reasons for this lack of efficacy. In addition to AAVrh10-mediated gene therapy, intrabrain-delivered lentiviral gene therapy has also recruited patients with any form of MLD (NCT03725670). Enzyme replacement therapy (ERT) is now the standard of care (SOC) for several lysosomal storage diseases (LSDs) (Sands, 2014) and depends on the ability of cells to absorb injected enzymes via mannose-6-phosphate receptors (Ghosh et al., 2003). In MLD, ERT reduces thiolipin storage in the kidneys, peripheral nerves, and CNS of Arsa- / - mice (Matzner et al., 2005). In a mouse model of advanced MLD with immune tolerance to human ARSA and unusually high thiolipin synthesis, improvement in MLD symptoms and reduction in thiolipin storage were observed only in mice treated at an early time point, suggesting that intravenous ERT may not be suitable for patients with late-stage symptoms (Matthes et al., 2012). In the same modality, continuous IT infusion of ARSA to bypass the BBB (Stroobants et al., 2011) led to complete reversal of thiolipin accumulation and correction of CNS dysfunction, while other non-clinical studies in mice resulted in reduced thiolipin accumulation and improved function (Matzner et al., 2009; Piguet et al., 2012). However, in humans, the metabolic correction provided by ERT is unlikely to adequately and immediately prevent rapid cerebral demyelination in early-onset MLD (Rosenberg et al., 2016). Because the BBB restricts the entry of most large proteins into the CNS, it is believed that ERT may only be effective when delivered directly to the CNS (Abbott, 2013), and its short half-life will necessitate frequent administration. This hypothesis was confirmed in ERT clinical trials that attempted to overcome these limitations through frequent high-dose intravenous administration (NCT00681811) or IT injections (Giugliani et al., 2018). However, in early-onset and late-adolescent MLD (NCT01510028), the results of ERT administered intravenously in infants with late-adolescent MLD were disappointing (NCT00418561). Small molecule-based therapies have the potential to overcome the limitations of current MLD treatments (e.g., by crossing the BBB) and may also reveal different pathogenic mechanisms of the disease. Warfarin (Coumadin), an anticoagulant, has been tested as a receptor-reducing agent in a small cohort of infants with late-stage MLD. It had no beneficial effect on urinary thiolipin levels or brain biomarkers N-acetylaspartate and myo-inositol levels (Patil and Maegawa, 2013). The limited benefits, restricted patient populations, short therapeutic window, and associated risks of HSCT and HSC-GT, combined with disappointing overall nonclinical results obtained through other research pathways, indicate a significant unmet clinical need for other viable treatment options, particularly for patients with early-onset MLD. The hope is for an alternative therapy to treat conditions associated with abnormal ARSA genes and / or metachromatic leukodystrophy. [Summary of the Invention] This article provides a therapeutic, recombinant, and replication-defective adeno-associated virus (rAAV) for treating diseases associated with mutations in the arylsulfatase A (ARSA) gene (e.g., metachromatic leukodystrophy, i.e., MLD or ARSA pseudodeficiency) in desired subjects. Ideally, the rAAV is replication-defective and carries a vector genome containing an inverted terminal repeat (ITR) sequence and a nucleic acid sequence encoding a functional human arylsulfatase A (hARSA) under the control of regulatory sequences that guide the expression of hARSA in target cells. In some specific embodiments, the rAAV further comprises an AAVhu68 capsid in which the vector genome is packaged. In some specific embodiments, the vector genome is entirely exogenous to the AAVhu68 capsid because it does not contain the AAVhu68 genome sequence. In some specific embodiments, the functional hARSA has a message peptide and the sequence of amino acids (aa) 19 to aa 507 of SEQ ID NO: 2. In some specific embodiments, a natural hARSA message peptide is used, for example, aa 1 to aa 18 of SEQ ID NO: 2. In some specific embodiments, the message peptide is aa 1 to aa 20 of SEQ ID NO: 4. In some specific embodiments, the functional hARSA has the amino acid sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In some specific embodiments, the hARSA coding sequence is approximately 95% to 100% identical to nucleotides (nt) 55 to nt 1521 of SEQ ID NO: 1. In some specific embodiments, the hARSA coding sequence is SEQ ID NO: 1 or SEQ ID NO: 3. In another specific embodiment, the hARSA coding sequence encodes the sequence of amino acids (aa) 19 to aa 507 of SEQ ID NO: 2. In yet another specific embodiment, the hARSA coding sequence encodes the sequence of SEQ ID NO: 2 or SEQ ID NO: 4. In certain embodiments, the regulatory sequence comprises one or more of the following: regulatory elements derived from the chicken β-actin (BA) promoter and the human cytomegalovirus immediate early enhancer (CMV IE) (e.g., CB7 promoter, nt 198 to nt 862 of SEQ ID NO: 5), chimeric introns composed of chicken BA splice donor and rabbit β-globulin (rBG) splice acceptor elements (e.g., CI, nt 956 to nt 1928 of SEQ ID NO: 5), and polyadenylation (PolyA) messages derived from the rBG gene (e.g., rBG, nt 3539 to nt 3665 of SEQ ID NO: 5). In certain embodiments, the vector genome has the sequence of nucleotides (nt) 1 to nt 3883 of SEQ ID NO: 5. In certain specific embodiments, rAAV or a composition containing rAAV is administered to a subject in need to improve symptoms of an ARSA mutation-related disease (e.g., MLD) and / or delay the progression of an ARSA mutation-related disease (e.g., MLD). In another embodiment, a production system for producing rAAV is provided. In this system, cells containing a nucleic acid sequence encoding the AAVhu68 capsid protein, a vector genome as described herein, and sufficient AAV rep and auxiliary functions to allow the vector genome to be packaged into an AAV capsid. Similarly, this document provides a vector for treating ARSA mutation-related diseases (e.g., MLD) in desired subjects. The vector carries a nucleic acid sequence encoding a functional human arylsulfatase A (hARSA) under the control of a regulatory sequence that guides the expression of hARSA in target cells. In some embodiments, the hARSA encoding sequence is approximately 95% to 100% identical to SEQ ID NO: 1. Alternatively or additionally, the functional hARSA protein has the amino acid sequence of SEQ ID NO: 2. In some embodiments, the hARSA encoding sequence is SEQ ID NO: 1. In some embodiments, the vector or a composition containing the vector is administered to a desired subject to improve symptoms of ARSA mutation-related diseases (e.g., MLD) and / or delay the progression of ARSA mutation-related diseases (e.g., MLD). In another embodiment, this document provides a composition comprising the rAAV or carrier as described herein and an aqueous suspension medium. In certain embodiments, the aqueous composition comprises a formulation buffer and the rAAV or carrier as described herein. In certain embodiments, the formulation buffer comprises: artificial cerebrospinal fluid comprising buffered saline and one or more sodium, calcium, magnesium, potassium, or mixtures thereof; and a surfactant. In certain embodiments, the formulation buffer comprises about 0.0005% to about 0.001% of a surfactant. In certain embodiments, the composition has a pH of 7.2 to 7.8. In another embodiment, a method is provided for treating a subject with an ARSA mutation-related disease (e.g., MLD), or for improving symptoms of an ARSA mutation-related disease (e.g., MLD), or for delaying the progression of an ARSA mutation-related disease (e.g., MLD). This method comprises administering an effective amount of rAAV or a vector as described herein to the subject in need. In some embodiments, the vector or rAAV is administered to the patient via intra-cisterna magna injection (ICM), for example, CT-guided suboccipital injection into the cisterna magna. In some embodiments, a vector or composition is provided for administration to a patient aged 7 years or younger, or 6 years or younger, with metachromatic leukodystrophy. In some embodiments, the method involves delivering rAAV or a vector to a human patient in a single dose. These and other aspects of the invention will become apparent from the following detailed description of the invention.

Implementation Method

Claims

1. A recombinant adeno-associated virus (rAAV) comprising: (a) an AAVhu68 capsid; and (b) a vector genome in the AAV capsid of (a), wherein the vector genome comprises an inverted terminal repeat (ITR) and a nucleic acid sequence encoding a functional hARSA operatively linked to a regulatory sequence of human arylsulfatase A (hARSA), wherein the nucleic acid sequence encoding the functional hARSA comprises: the sequence of nucleotides (nt)1 to nt 1527 of SEQ ID NO: 3; or a sequence at least 99% identical thereto, the sequence encoding amino acids 1 to 509 of SEQ ID NO: 4, wherein the regulatory sequence comprises a CB7 promoter comprising a cytomegalovirus immediate early enhancer, a chicken β-actin promoter, and an intron.

2. The rAAV of claim 1, wherein the cytomegalovirus immediate early enhancer has the sequence of nucleotides 198 to 579 of SEQ ID NO:

5.

3. The rAAV of claim 1, wherein the chicken β-actin promoter has the sequence of nucleotides 582 to 862 of SEQ ID NO:

5.

4. rAAV as claimed in claim 1, wherein the intron has the sequence of nucleotides 956 to 1928 of SEQ ID NO:

5.

5. rAAV as claimed in claim 1, wherein the vector genome contains polyadenylation information of rabbit globulin.

6. The rAAV of claim 5, wherein the polyadenylation message has the sequence of nucleotides 3539 to 3665 of SEQ ID NO:

5.

7. rAAV as requested in claim 1, wherein the nucleic acid sequence encoding functional hARSA is SEQ ID NO:

3.

8. rAAV as claimed in claim 1, wherein the vector genome has the sequence nt 1 to nt 3883 of SEQ ID NO:

5.

9. An aqueous pharmaceutical composition comprising rAAV as claimed in claim 1 and a formulation buffer.

10. The aqueous pharmaceutical composition of claim 9, wherein the formulation buffer comprises: artificial cerebrospinal fluid comprising buffered saline and one or more of sodium, calcium, magnesium, potassium, or mixtures thereof; and a surfactant.

11. The aqueous pharmaceutical composition of claim 10, wherein the formulation buffer is suitable for intracerebral injection (ICM), intravenous delivery, intrathecal administration, or intraventricular administration.

12. An rAAV production system for producing rAAV, wherein the production system comprises a cell culture comprising: (a) a nucleic acid sequence encoding an AAVhu68 capsid protein; (b) a recombinant nucleic acid molecule comprising a vector genome comprising an AAV 5'ITR, an expression cassette, and an AAV 3'ITR, wherein the expression cassette comprises a nucleic acid sequence encoding a functional hARSA operatively linked to a regulatory sequence of human arylsulfatase A (hARSA), wherein the nucleic acid sequence encoding the functional hARSA comprises the sequence of nucleotides (nt) 1 to nt 1527 of SEQ ID NO: 3; or a sequence at least 99% identical thereto, the sequence encoding amino acids 1 to 509 of SEQ ID NO: 4; and (c) sufficient AAV rep function and auxiliary function to allow the vector genome to be packaged into the AAVhu68 capsid.

13. The rAAV production system of claim 12, wherein the vector genome comprises a human cytomegalovirus immediate early enhancer (CMV IE), a chicken β-actin promoter, a chicken β-actin splice donor, and a rabbit β-globulin splice acceptor element, the nucleic acid sequence encoding a functional hARSA, and a polyadenylation message of rabbit β-globulin.

14. The rAAV production system of request item 12, wherein the recombinant nucleic acid molecule is a plastid.

15. The rAAV production system of claim 12, wherein the vector genome has the sequence of nt 1 to nt 3883 of SEQ ID NO:

5.

16. The rAAV production system of claim 12, wherein the cell culture is a human embryonic kidney 293 cell culture.

17. The rAAV production system as described in request item 12, wherein the AAV rep originates from AAV2.

18. The rAAV production system of claim 12, wherein the AAV rep coding sequence and the cap gene are located on the same nucleic acid molecule.

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