In Vivo Gene Therapy for Mucopolysaccharidosis Type III (Sanfilippo Syndrome): A New Treatment Horizon

Abstract

For most lysosomal storage diseases (LSDs), there is no cure. Gene therapy is an attractive tool for treatment of LSDs caused by deficiencies in secretable lysosomal enzymes, in which neither full restoration of normal enzymatic activity nor transduction of all cells of the affected organ is necessary. However, some LSDs, such as mucopolysaccharidosis type III (MPSIII) diseases or Sanfilippo syndrome, represent a difficult challenge because patients suffer severe neurodegeneration with mild somatic alterations. The disease's main target is the central nervous system (CNS) and enzymes do not efficiently cross the blood–brain barrier (BBB) even if present at very high concentration in circulation. No specific treatment has been approved for MPSIII. In this study, we discuss the adeno-associated virus (AAV) vector-mediated gene transfer strategies currently being developed for MPSIII disease. These strategies rely on local delivery of AAV vectors to the CNS either through direct intraparenchymal injection at several sites or through delivery to the cerebrospinal fluid (CSF), which bathes the whole CNS, or exploit the properties of certain AAV serotypes capable of crossing the BBB upon systemic administration. Although studies in small and large animal models of MPSIII diseases have provided evidence supporting the efficacy and safety of all these strategies, there are considerable differences between the different routes of administration in terms of procedure-associated risks, vector dose requirements, sensitivity to the effect of circulating neutralizing antibodies that block AAV transduction, and potential toxicity. Ongoing clinical studies should shed light on which gene transfer strategy leads to highest clinical benefits while minimizing risks. The development of all these strategies opens a new horizon for treatment of not only MPSIII and other LSDs but also of a wide range of neurological diseases.

Introduction

Mucopolysaccharidosis type III disease

Mucopolysaccharidoses (MPSs) are a group of rare, inherited lysosomal storage diseases (LSDs) caused by specific lysosomal enzyme deficiencies that lead to intracellular accumulation of partially degraded glycosaminoglycans (GAGs, formerly called mucopolysaccharides) within cells.^1,2 Depending on the nature of stored material and the deficient enzyme, MPSs have been classified into different types.³

MPS type III (MPSIII), also known as Sanfilippo syndrome, is an autosomal recessive disease characterized by intralysosomal accumulation of the GAG, heparan sulfate (HS).^1,3,4 It is caused by deficiency in one of the four enzymes involved in lysosomal degradation of HS: deficiency in N-sulfoglucosamine sulfohydrolase or sulfamidase (SGSH, EC 3.10.1.1) causes type IIIA (OMIM No. 252900)⁵; in α-N-acetylglucosaminidase (NAGLU, EC 3.2.1.50), type IIIB (OMIM No. 252920)⁶; in acetyl CoA α-glucosaminide acetyltransferase (HGSNAT, EC 2.3.1.78), type IIIC (OMIM No. 252930)⁷; and in N-acetylglucosamine-6-sulfatase (GNS, EC 3.1.6.14), type IIID (OMIM No. 252940).⁸

MPSIII is one of the most common types of MPSs.⁴ MPSIIIA is the most frequent subtype of Sanfilippo syndrome found in Northwest Europe, North America, and Australia,^9

–14 and type B has the highest prevalence in Southeast Europe and Brazil.^{13,15

–19} MPSIIIC and D are much rarer diseases with few cases reported in the literature.^{4,9,16,20
–22}

GAGs, including HS, play an important role in the mechanical support of tissues and are also involved in regulating cell growth and development, cell–cell interactions, immunity and defense against viral infections, coagulation, and lipid metabolism.²³ The excess of HS fragments and HS-derived oligosaccharides that accumulate in lysosomes and are released into the extracellular medium in MPSIII disease could interfere with all of these processes.^3,4 However, the exact cascade of pathological events that leads to cell dysfunction and death is not completely understood. In addition to HS, neurons from Sanfilippo patients accumulate GM2 and GM3 gangliosides and, in some cases, also unesterified cholesterol.^24

–30 This secondary accumulation also appears to play an important role in the neurological pathology characteristic of the disease.³¹

Clinical manifestations appear once HS accumulation causes cellular damage, after which symptoms progressively worsen. MPSIII is generally characterized by severe, progressive central nervous system (CNS) degeneration. Affected children appear normal at birth, and the first symptoms are detected around 1–4 years, generally under the form of delayed intellectual development.^{4,20,22,32,33} The disease progresses to severe neurological manifestations, including hyperactivity, sleeping problems, loss of speech, and epilepsy.^{4,20,22,32,33} At later stages of the disease, affected individuals develop profound dementia and progressive loss of motor functions, most patients being wheelchair-bound and fully dependent by the second decade of their lives.^{4,20,22,32
–34}

In contrast to the majority of other MPS disorders, children with MPSIII have relatively mild somatic symptoms.^4,35 Facial dysmorphisms, although usually slight, are detected in most individuals.^{4,20,22,32,36} Patients frequently present visceromegaly, mainly hepatosplenomegaly, and mild skeletal alterations.^20,32,33,36 Recurrent ear, nose, throat, and chest infections are commonly found in young patients, as well as frequent diarrhea.^{4,20,32,33,37} Death usually occurs in the mid-late teenage years.^{4,22,32,33,36
–38}

Treatment of MPSIII disease

Presently, there is no specific treatment approved for MPSIII. Control of the disease is merely symptomatic, aimed at improving the quality of life of patients and their families.^3,39 Several new therapies are being developed to treat Sanfilippo syndrome and some of them have already reached the clinical stage.

Most of these therapies are forms of enzyme replacement therapy (ERT), in which the enzyme is provided periodically as a recombinant protein, or a classic example of gene augmentation gene therapy, in which the enzyme is endogenously synthetized by cells that have received a correct copy of the mutated gene. Underlying all these strategies is the principle of cross-correction, whereby a soluble lysosomal enzyme present in the extracellular compartment can be taken up by endocytosis through binding to mannose-6-phosphate receptors present on the plasmatic membrane of cells.^40

–43 Therefore, the enzyme present in the extracellular milieu after recombinant protein administration or produced by engineered/corrected cells can cross-correct other cells.^41
–43

All these novel therapeutic approaches need, however, to overcome a major challenge in drug development for LSDs with CNS involvement: the existence of the blood–brain barrier (BBB), which limits efficient access of compounds to the CNS after systemic administration. To bypass the BBB and allow for repeated drug administrations, the use of a drug delivery device permanently implanted in the intrathecal space for delivery of recombinant proteins directly to the cerebrospinal fluid (CSF) has been tested for MPSIIIA (ClinicalTrials.gov; NCT01155778, NCT01299727, NCT02060526, and NCT02350816)^44,45 and MPSIIIB (ClinicalTrials.gov; NCT02754076 and NCT03784287).

Although technically feasible, permanent implantation of intrathecal devices is associated with complications, such as implant site infections or device malfunction.^44
–46 Despite these shortcomings, clinical studies of intrathecal ERT with recombinant human sulfamidase for MPSIIIA (ClinicalTrials.gov; NCT01155778 and NCT02060526) have consistently demonstrated declines in HS levels in the CSF, although clear evidence of clinically meaningful improvements in neurocognitive function is still missing.^44,45

An investigational, intrathecal ERT drug for the treatment of MPSIIIB (NCT02754076) consisting in a recombinant chimeric protein in which human NAGLU is fused to a truncated form of human insulin-like growth factor 2 has shown HS reduction in the CSF and stabilization of development quotient.⁴⁷

In Vivo Gene Therapy for MPSIII Diseases

To overcome the need for periodic administration of the therapeutic protein of classic ERT approaches, gene therapies for MPSIII are being developed to achieve constant production of active enzymes available to cross-correct cells. A key advantage of gene therapy strategies is that correction of the genetic defect in all cells of an organ is not required since few corrected cells could, in principle, produce sufficient amount of the active enzyme to become available to neighboring cells or to other cells within the target organ or nontargeted organs in the body if the enzyme reaches main fluids, for example, CSF and serum.^{48

–58}

Since the CNS is the most affected organ in MPSIII, and the CNS is anatomically isolated from the rest of the body, a sufficient degree of gene correction has to be achieved in this organ. CNS-targeted gene therapy is a very active field of research. Recent years have witnessed the publication of numerous preclinical and clinical studies that demonstrate the potential of gene therapy for treating neurological conditions, particularly monogenic hereditary diseases.^59,60

Most of the gene therapy strategies that are being developed for MPSIII are based on in vivo gene transfer mediated by adeno-associated virus (AAV)-derived vectors. These vectors have shown high transduction efficiencies in vivo as well as excellent safety profiles in clinical studies.^61,62 In addition, preclinical studies in large animal models as well as clinical data have provided strong evidence supporting long-term expression in the CNS mediated by AAV vectors in the absence of clinically significant adverse events.^{48,49,51,63

–67}

This overview will focus on in vivo gene therapies for MPSIII. Nevertheless, ex vivo gene therapies have also been developed for MPSIIIA and MPSIIIB.^68

–71 They are based on the autologous transplantation of hematopoietic stem cells genetically modified using lentiviral vectors to express SGSH or NAGLU for the treatment of MPSIIIA or MPSIIIB, respectively.^68
–70 The progeny of transplanted gene-corrected cells traffics to the brain, bypassing the BBB, to become resident cells that produce the therapeutic protein in the CNS. Proof-of-concept studies demonstrated normalization of brain HS, secondary GM2 storage and neuroinflammation, and improvement of behavioral deficits in MPSIIIA and MPSIIIB mice.^68
–70 A clinical trial is to be initiated soon in MPSIIIA patients.^*

Similar ex vivo gene therapy approaches have demonstrated clinical efficacy for the treatment of other neurometabolic storage disorders, such as adreno and metachromatic leukodystrophies, as well as other indications such as MPS type I.^72

–78 These studies are discussed in detail in the review by Poletti and Biffi included in this special issue of Human Gene Therapy.⁷²

Routes of administration to deliver AAV vectors to the CNS

MPSs are diseases in which the neurodegenerative process affects the whole CNS, although little information is available from human specimens on whether there are structures that are more affected than others.^27,79,80 Several AAV-based strategies for delivery of genes to extensive areas of the CNS have been described.

Initial efforts administered the AAV through multiple direct injections to the brain parenchyma. Other systems followed that exploited the ability of certain AAV serotypes, such as serotype 9 (AAV9), to cross the BBB after intravenous administration, resulting in widespread transduction of the brain as well as of the liver and other peripheral organs through a noninvasive procedure. More recently, in an effort to maximize gene transfer to the most affected organ while minimizing vector dose, direct delivery of AAV vectors to the CSF has been proposed as an alternative for global CNS gene transfer. Additionally, at least in animal models, this approach results in transduction of the peripheral nervous system and the liver, providing for somatic disease correction.

Figure 1 depicts a schematic representation of potential vector and enzyme biodistribution after AAV vector delivery through these different routes of administration.^{49,51,54,81

–85} Several clinical trials are currently testing the safety and efficacy of these approaches in MPSIII disease and are listed in Table 1.

Figure 1.

Possible routes of vector administration and subsequent enzyme biodistribution, in vivo, AAV-mediated gene therapy strategies for Sanfilippo syndrome. Based on results obtained in proof-of-concept studies in small and large animal models of MPSIII,^{49,51,53,54,56,81

–84,91,96,97,99,102,117

–120,125,126} a schematic representation of the possible biodistribution of the gene therapy product in patients is presented. (A) The intraparenchymal route of administration consists in multiple direct injections into the brain parenchyma through six or eight burr holes (3/4 per hemisphere) at two different depths per site of injection. This route of administration has limited AAV diffusion in the CNS with very high local concentrations of vector genomes at the site of injection, which progressively decrease with distance. Cells around the sites of injections can be corrected by locally expressed enzymes, but release of the expressed protein into the CSF is low. Vector transduction of peripheral organs, for example, the liver, is very low. (B) Systemic administration through the intravenous route is a noninvasive technique to efficiently transduce somatic organs, especially the liver, depending on the serotype of AAV used. Among them, AAV9 has demonstrated the ability to cross the blood–brain barrier and transduce the CNS. Administration of very high vector doses is required since injection of lower doses results in fractional delivery of the vector to the CNS, insufficient to support therapeutically meaningful levels of enzyme production in the brain. CNS efficacy is expected to be abolished by the presence of pre-existing immunity against the AAV as serum NAb would block the entrance of the vector to the CNS. (C) Delivery of AAV9 vectors to the CSF through unilateral administration to the lateral ventricle or through the cisterna magna leads to widespread and even distribution of AAV vectors throughout the whole brain and spinal cord as the CNS is bathed by the CSF. Moreover, some of the AAV9 vector reaches the circulation, leading to transduction of the liver. As a result of this profile of vector distribution, enzyme activity increases throughout the CNS, in the CSF and in serum, the liver being the most important source of circulating enzyme. Moreover, studies in seropositive animal models suggest that CNS efficacy would not be compromised by the presence of anti-AAV9 NAbs in serum. AAV, adeno-associated virus; CSF, cerebrospinal fluid; CNS, central nervous system; NAbs, neutralizing antibodies.

Table 1.

In vivo gene therapy clinical trials for Sanfilippo patients

Disease	Study Phase	Route of Administration	Type of Vector	Outcome	Sponsor	Clinical Trial Identifier	Publications
MPSIIIA	Phase I/II (completed)	Intraparenchymal	AAVrh10-hSGSH-SUMF1	Safe and well tolerated Moderate behavioral improvement	Lysogene	NCT01474343^a NCT02053064^a (LT)	Tardieu et al. ⁹²
	Phase II/III (recruiting)	Intraparenchymal	AAVrh10-hSGSH	NA	Lysogene	NCT03612869^a 2018-000195-15^b	—
	Phase I/II (recruiting)	Intravenous	scAAV9-hSGSH	NA	Abeona Therapeutics	NCT02716246^a 2015-003904-21^b	—
	Phase I/II (active)	Intra-CSF	AAV9-hSGSH	NA	Esteve	2015-000359-26^b	—
MPSIIIB	Phase I/II (active)	Intraparenchymal	AAV5-hNAGLU	Safe and well tolerated Sustained enzyme production in the CSF Improved neurocognitive progression	UniQure Biopharma B.V./Institut Pasteur	NCT03300453^a 2012-000856-33^b	Tardieu et al. ⁹³
MPSIIIB	Phase I/II (recruiting)	Intravenous	AAV9-hNAGLU	NA	Abeona Therapeutics	NCT03315182^a 2014-001411-39^b	—
MPSIIIC	Phase I/II (in preparation)^c	Intraparenchymal	AAV-TT-hHGSNAT ^d	NA	Phoenix Nest	Not yet registered	—

Clinical trial registered in www.ClinicalTrials.gov.

Clinical trial registered in www.ClinicalTrialsregister.eu.

www.phoenixnestbiotech.com.

Information based on Tordo, 2018.

AAV, adeno-associated virus; CSF, cerebrospinal fluid; LT, long-term follow-up; NA, nonavailable; NAGLU, α-N-acetylglucosaminidase; SGSH, N-sulfoglucosamine sulfohydrolase or sulfamidase; SUMF1, sulfatase-modifying factor 1; HGSNAT, acetyl CoA alpha-glucosaminide acetyltransferase.

Direct administration of AAV vectors to the brain parenchyma

Vectors can be delivered directly to the CNS by intraparenchymal injection, and this approach has been used safely in patients to target the putamen in the context of investigational gene therapies for Parkinson's disease.^{65,86

–90} MPSIII, however, affects the whole of the CNS.

Given the limited diffusion of AAV vectors from the site of injection,^67,91 the applicability of this method to global neurodegenerative diseases has required the development of a rather complex surgical procedure by which small volumes of vectors are simultaneously deposited in the brain white matter at different sites.^64,92
–94 There is a limit to the number of injections and to the locations at which these injections can be safely performed.^67,92,94 Moreover, intraparenchymal injection fails to transduce deep CNS structures, a fact that has been considered the culprit of the limited efficacy in previous studies in humans using this approach for other neurodegenerative diseases.⁶⁴ In the original procedure, a total of six burr holes were drilled laterally from the midline and the vector was administered at two depths per site.⁹⁴

Intraparenchymal administration of AAVs at multiple sites has been tested in the clinic for MPSIIIA (NCT01474343) and MPSIIIB (NCT03300453), using AAVs of serotypes rh10 and 5, respectively.^92,93 In the case of the phase I/II trial for MPSIIIA, the vector encoded for SGSH as well as for the sulfatase-modifying factor 1 (SUMF1) protein. SUMF1 post-translationally generates C-alpha-formylglycine (FGly), the catalytic residue in the active site of eukaryotic sulfatases, from a cysteine.⁹⁵ The rationale behind SUMF1 supplementation was to avoid potential toxicity derived from competition for SUMF1 sequestration at sites with very high levels of expression of sulfamidase (e.g., injection sites).

Preliminary results of the 1-year follow-up of this trial reported moderate improvements in behavior, attention, and sleep disturbances.⁹² Nevertheless, these patients have remained under long-term immunosuppression since receiving AAV vectors, and the potentially beneficial effect of immunosuppression on the observed clinical benefit cannot be ruled out because neuroinflammation is a main player in disease pathology.⁹² The ongoing long-term follow-up of these MPSIIIA patients (NCT02053064) should provide further information regarding efficacy. Recently, a new, open-label, single-arm, multicenter, phase II/III clinical trial has started in MPSIIIA patients (NCT03612869) administered a simplified version of the therapeutic vector, which does not include the SUMF1 gene present in the original vector.⁸⁵

As a technical improvement to the approach used for the original MPSIIIA study, a more recent trial for MPSIIIB was conducted with 2 additional deposits in the cerebellum, resulting in vector deposition simultaneously in 16 sites (8 burr holes).⁹³ Four young MPSIIIB patients have been treated thus far using this method, with concomitant immunosuppression.⁹³ The procedure was well tolerated and initial evidence of improved cognitive outcomes is suggestive of efficacy, with the same caveat than for the MPSIIIA trial regarding the use of chronic immunosuppression.⁹³

Interestingly, a similar intraparenchymal approach using a new AAV vector variant with a putative better pharmacokinetic profile, AAV-TT, is being developed for MPSIIIC, although in this case, the deficient enzyme is not secretable and cross-correction cannot occur.⁹⁶ Treatment with AAV-TT-HGSNAT was able to correct the neurological phenotype of a mouse model of MPSIIIC.⁹⁶ A clinical trial has been planned to test the approach in MPSIIIC patients.^†

Intravascular delivery of AAV vectors to treat MPSIII

Of the several ways of delivering genes to the CNS, the simplest approach exploits the ability of certain AAV serotypes, such as AAV9, to cross BBB and reach the CNS when delivered intravenously (IV).^97,98

Systemic delivery of AAV9 has proven successful in the treatment of spinal muscular atrophy type 1 (SMA1). SMA1 is caused by a mutation in the survival motor neuron 1 (SMN1) gene that leads to severe degeneration and loss of lower motor neurons, resulting in muscle atrophy.⁹⁹ Death occurs during infancy (before 2 years of age).¹⁰⁰ Intravascular AAV9-SMN1 gene therapy delivered the SMN1 gene to motor neurons of SMA1 patients, was safe, and resulted in longer survival, superior achievement of motor milestones, and better motor function than in historical cohorts.⁹⁹ The highly positive results obtained in these clinical studies (NCT02122952 and NCT03955679)⁹⁹ have led to the recent marketing approval by the Food and Drug Administration (FDA) of the new gene therapy product Zolgensma.

Treatment of MPSIII disease requires targeting of the whole CNS, and this may be more difficult to achieve after systemic delivery of AAV9 vectors than targeting of motor neurons, as in the case of SMA. Proof-of-concept studies in mouse models of MPSIIIA and MPSIIIB following IV delivery of AAV9 vectors encoding for deficient enzymes have shown reduction in brain GAG accumulation and neuroinflammation, as well as correction of behavioral deficits.^{82,84,101,102} Based on the demonstrated efficacy in rodent models, clinical studies are presently underway for MPSIIIA and MPSIIIB.

In an open-label, dose-escalation, phase I/II gene transfer trial, MPSIIIA patients were IV administered self-complementary AAV9 vectors carrying the hSGSH gene under the control of the ubiquitous U1a promoter (scAAV9.U1A.hSGSH) (NCT02716246). All subjects received immunosuppression, consisting in a tapering course of prophylactic enteral prednisone or prednisolone.

It has been shown in several clinical trials that systemic exposure to high doses of AAV vectors may trigger activation of CD8⁺ T cell responses directed against the viral capsid in a dose-dependent manner.^103

–106 Short-term (few weeks) steroid treatment is used to prevent the immune-mediated elimination of vector-transduced cells, resulting in long-term expression of therapeutic genes.^52,103

No product-related, serious adverse events have been reported to date in this MPSIIIA trial. Efficacy data have not been published yet, but some interim data have been made available by the sponsor Abeona Therapeutics. A dose-dependent and sustained reduction in levels of heparan sulfate in the CSF has been reported for all three cohorts of patients treated with low (5 × 10¹² vg/kg), medium (1 × 10¹³ vg/kg), or high (3 × 10¹³ vg/kg) vector doses. Moreover, the three youngest patients enrolled in the high-dose cohort (cohort 3)—14–26 months of age at dosing—continued to track within normal age-equivalent development 12–18 months post-treatment^‡.

Based on these encouraging results, the same sponsor has initiated a new trial using the IV AAV9 approach for treatment of MPSIIIB (NCT03315182). This is an open-label, dose-escalation phase I/II trial in MPSIIIB patients aged 6 months to 2 years, or older than 2 years with a minimum cognitive development quotient of 60 or above. In this case, the AAV9 vector carried the hNAGLU gene under control of the strong viral cytomegalovirus promoter (AAV9.CMV.hNAGLU). All dosed subjects have received immunosuppression, consisting in oral prednisolone from the day before gene transfer until at least 60 days after.¹⁰⁷ Two doses of AAV vectors are being tested, 2 × 10¹³ vg/kg (cohort 1) and 5 × 10¹³ vg/kg (cohort 2), which are higher than those administered in cohorts 1 and 2 of the MPSIIIA trial. Dosing of cohort 1 has been completed and the first patient in cohort 2 has received the vector.^§

Although clinical evidence supports that systemic administration of AAV vectors results in therapeutic benefit for MPSIII patients, this route of administration may not be of choice to reach the CNS in the subset of patients who are seropositive for anti-AAV antibodies, which can greatly limit the efficacy of in vivo gene transfer upon systemic administration.^104,108,109

Serum neutralizing antibodies (NAbs) to AAVs are highly prevalent in humans.¹¹⁰ The prevalence and magnitude of seropositivity, however, vary with the AAV serotype. While about 60% of the adult population has anti-AAV2 NAbs at high titers, only about 30% of healthy individuals have detectable anti-AAV9 antibodies.¹¹⁰ Moreover, there is a pattern of seroconversion with age since children under 1 year are seronegative, or seropositive at very low titers, but then titers progressively increase up to around 5–6 years of age, reflecting an increase in socialization, which favors natural infection by wild-type AAVs.^111,112 The same tendency was observed in healthy and MPSIIIA- and MPSIIIB-affected children. Serum NAb titers against AAV2 were generally higher than those against AAV9 and they tended to increase with age.^51,101

Aside from the limitations imposed by anti-AAV pre-existing immunity and the potential risk of cytotoxic CD8⁺ T cell responses, the consistently high doses required for brain transduction and efficacy following IV administration of AAV vectors^{38,82,84,113,114} may suppose a manufacturing challenge from the technical and economic points of view, although great efforts are being devoted to scaling up and improving the yield of AAV manufacturing by different platforms.

Intra-CSF administration of AAV vectors to transduce the CNS

As an alternative to local delivery of vectors to the CNS, several groups have begun to administer AAV vectors—particularly AAV9—directly to the CSF and have achieved efficient CNS transduction in different animal species (mice, rats, cats, dogs, pigs, and nonhuman primates [NHPs]) at relatively lower vector doses when compared with those used when the vector is injected systemically.^{49,51,53,54,56,115

–119} Upon administration to the CSF, the vector is diluted in a fluid that circulates through CNS compartments, bathing the whole encephalon and spinal cord, allowing for widespread vector distribution, including deep structures.^51,119 As a result, the pattern of vector transduction differs considerably from that obtained following intraparenchymal injection at multiple sites, which results in uneven distribution of vector genomes, with high copy numbers at the point of injection quickly decreasing with distance.^83,91,120

When vectors carry genes encoding for secretable proteins, such as in MPSIIIA and IIIB, transduced cells secrete these proteins to the CSF at relatively high levels,^49,51 which could further increase efficacy by distribution of the therapeutic protein throughout the whole CNS. Furthermore, at least in animal models, upon intra-CSF AAV9 delivery, a portion of the vector escapes to the circulation and transduces the liver, which can secrete the therapeutic protein to the bloodstream.^{49,51,53,54,56,116}

Whole-body correction of LSD was observed following delivery of AAV9 vectors to the CSF of several MPS mouse models.^{49,51,53,54,56} In all cases, administration of the therapeutic vector mediated clearance of accumulated GAGs, resolution of lysosomal pathology and neuroinflammation in the brain, correction of behavioral deficits, and significant extension of life span.

Proof-of-concept studies in dogs and pigs have shown that the delivery of vectors to the CSF is scalable to larger brains.^49,51,119 In most of these studies, vectors were administered to the cisterna magna, a large subarachnoid space between the caudal part of the cerebellum and the medulla oblongata, filled with CSF produced in the fourth ventricle that will distribute the vectors to the whole CNS.¹²¹ Intracisternal injection, however, is not a common route of administration to the CSF in clinical pediatric practice because of the relatively smaller size of the cisterna magna in humans compared with animals and its proximity to vital centers, which adds risk to the procedure in case of accidental puncture.¹²¹

An alternative route for CSF delivery is lumbar puncture. Although this is a common clinical procedure, limited distribution of products in widespread CNS areas is achieved through this route.^122,123 Preclinical data in pigs show that AAV9-derived gene expression following local lumbar intrathecal administration remains restricted to areas of injection and that widespread spinal cord transduction requires several administrations of the vector at the cervical, thoracic, and lumbar regions, suggesting that the vector penetrated mostly at the vicinity of the catheter tip used for delivery.¹²² Global CNS gene delivery following intrathecal AAV administration was reported in cynomolgus macaques, an NHP of small size (∼3–7 kg), using AAV vectors diluted in a hyperosmotic buffer.¹¹⁶ Although this study provides evidence that the approach is potentially feasible, detailed confirmatory studies in larger animal models are needed.

Ventriculostomy is a standard surgical procedure to deliver drugs (oncology drugs, antibiotics, and antifungal agents, etc.) to the CSF for treatment of different diseases or for management of hydrocephalus.^123,124 Although intracerebroventricular (ICV) access does require unilateral trepanation of the skull, the trajectory to reach the ventricle is well defined and goes through mute areas of the brain.¹²³ Using a surgical procedure that has been in place for many years has the advantage of providing a solid safety record as well as a well-characterized list of potential complications and protocols for their management. It also simplifies clinical translation as the technique is known to pediatric neurosurgeons worldwide and would not require specific training.

Compared with intraparenchymal administration, another advantage of ICV administration is that by delivering the vector to the CSF, a relatively large volume of vector can be supplied within a brief period of time, thus shortening the duration of surgery and providing flexibility in terms of vector concentration and formulation. In dogs, ICV administration demonstrated to be highly efficacious in delivering AAV vectors to the whole encephalon and spinal cord.⁵¹

An open-label, dose-escalation, phase I/II clinical trial was initiated recently in MPSIIIA patients older than 2 years, which uses the ICV procedure to deliver AAV9 vectors carrying the human SGSH gene (AAV9-CAG-cohSGSH) into the CSF (EU Clinical Trials Register 2015-000359-26). This is a safety, tolerability, and initial efficacy clinical trial.

Although it has been suggested that the BBB is disrupted in patients affected by LSDs,⁴¹ asymmetrical distribution of anti-AAV NAbs across the BBB of patients suffering from Sanfilippo syndrome is preserved even in severely affected children.⁵¹

The evidence showing that very low titers of NAbs are sufficient to completely block transduction upon systemic administration^104,108,109 prompted several laboratories, including ours, to evaluate CNS transduction after intra-CSF delivery in large animals with anti-AAV pre-existing immunity. In NHPs with serum AAV9 NAb titers of 1:128, successful CNS gene transfer was observed after intra-CSF administration of vectors.¹¹⁶ Similarly, in healthy dogs preimmunized by IV administration of noncoding AAV9 vectors leading 1 month later to high anti-AAV9 NAb serum titers (1:100–1:1000), intra-CSF delivery of AAV9 vectors encoding for green fluorescent protein resulted in efficient transduction of the brain and spinal cord at levels comparable with those achieved in animals naïve to AAV9.⁵¹

These findings were further confirmed following intra-CSF administration of a therapeutic transgene (canineNaglu) to seropositive dogs with high AAV9 NAb titers in plasma (1:100–1:1000).⁴⁹ Again, similarly high levels of NAGLU activity were detected in the CSF of both naïve and seropositive animals upon intra-CSF AAV-mediated gene transfer.⁴⁹ These studies suggest that CNS efficacy would not be compromised in seropositive patients when vectors are delivered to the CSF. As expected, transduction of the liver is completely abolished following vector delivery to the CSF of animals with circulating NAbs.⁵¹

Given that the main target organ for MPSIII is the CNS, the lack of peripheral efficacy would still allow for a significant degree of disease correction. Further clinical studies should establish the safety of gene transfer to the CSF of seropositive individuals.

Concluding Remarks

Several AAV vector-mediated, in vivo, gene therapy approaches for treatment of MPSIII have been developed in recent years, some of which are already under clinical investigation. Different therapeutic strategies use different routes of vector administration to reach the CNS, the most affected organ.

Studies in small and large animal models of MPSIII diseases have provided evidence supporting the efficacy and safety of these strategies and are expected to be predictive of possible efficacy in humans. Results from the ongoing clinical trials should confirm, or not, these expectations. Although direct comparisons of clinical outcomes of these studies cannot be made, due in part to differences in inclusion criteria, protocol design, or vector manufacturing, the ongoing studies should shed light on which gene transfer strategy maximizes efficacy in the CNS while minimizing delivery-associated risks, leading to higher clinical benefits.

Of importance, clinical trials in which the vector is delivered directly to the CNS (intraparenchymal or intra-CSF) should inform on the potential to achieve amelioration of somatic disease in humans, a fact that would become more relevant as patients live longer due to improved neurological outcomes.

Finally, these studies should provide information on the tolerability, safety, and efficacy of AAV-mediated CNS gene transfer not only for treatment of MPSIII but also of other neurodegenerative diseases.

Footnotes

Author Disclosure

F.B., V.H., and S.M. are coinventors on patent applications for the use of AAV vectors for treatment of MPSIII.

Funding Information

Work in the authors' laboratory relevant to this review has been supported by grants from Plan Nacional I+D+I from the Ministerio de Ciencia, Innovación y Universidades, and the European Union through Regional Development Funds (ERDF) (INNPACTO IPT-2012-0772-300000, SAF 2014-54866-R, and SAF2017-86166-R), Generalitat de Catalunya (ICREA Academia Award to F.B.), MPS España Foundation, and it is part of a public–private partnership on gene therapy between UAB and ESTEVE Pharmaceuticals, Spain.

References

Neufeld

, Muenzer

. The mucopolysaccharidoses. In: Schriver

, Beaudet

, Sly

, et al., eds. Metabolic and Molecular Bases of Inherited Disease. New York, McGraw-Hill, 2001:3421–3452.

Parenti

, Andria

, Ballabio

. Lysosomal storage diseases: from Pathophysiology to Therapy. Annu Rev Med, 2015; 66:471–486.

Fedele

. Sanfilippo syndrome: causes, consequences, and treatments. Appl Clin Genet, 2015; 8:269.

Valstar

, Ruijter

, van Diggelen

, et al. Sanfilippo syndrome: a mini-review. J Inherit Metab Dis, 2008; 31:240–252.

Kresse

, Neufeld

. The Sanfilippo A corrective factor. Purification and mode of action. J Biol Chem, 1972; 247:2164–2170.

von Figura

, Kresse

. The sanfilippo B corrective factor: a N-acetyl-alpha-D-glucosamindiase. Biochem Biophys Res Commun, 1972; 48:262–269.

Klein

, Kresse

, von Figura

. Sanfilippo syndrome type C: deficiency of acetyl-CoA:alpha-glucosaminide N-acetyltransferase in skin fibroblasts. Proc Natl Acad Sci U S A, 1978; 75:5185–5189.

Kresse

, Paschke

, von Figura

, et al. Sanfilippo disease type D: deficiency of N-acetylglucosamine-6-sulfate sulfatase required for heparan sulfate degradation. Proc Natl Acad Sci U S A, 1980; 77:6822–6826.

Meikle

, Hopwood

, Clague

, et al. Prevalence of lysosomal storage disorders. JAMA, 1999; 281:249–254.

10.

Poorthuis

, Wevers

, Kleijer

, et al. The frequency of lysosomal storage diseases in The Netherlands. Hum Genet, 1999; 105:151–156.

11.

Baehner

, Schmiedeskamp

, Krummenauer

, et al. Cumulative incidence rates of the mucopolysaccharidoses in Germany. J Inherit Metab Dis, 2005; 28:1011–1017.

12.

Malm

, Lund

, Månsson

J-E

, et al. Mucopolysaccharidoses in the Scandinavian countries: incidence and prevalence. Acta Paediatr, 2008; 97:1577–1581.

13.

Héron

, Mikaeloff

, Froissart

, et al. Incidence and natural history of mucopolysaccharidosis type III in France and comparison with United Kingdom and Greece. Am J Med Genet, 2011; 155A:58–68.

14.

Noh

, Lee

. Current and potential therapeutic strategies for mucopolysaccharidoses. J Clin Pharm Ther, 2014; 39:215–224.

15.

Michelakakis

, Dimitriou

, Tsagaraki

, et al. Lysosomal storage diseases in Greece. Genet Couns, 1995; 6:43–47.

16.

Coelho

, Wajner

, Burin

, et al. Selective screening of 10,000 high-risk Brazilian patients for the detection of inborn errors of metabolism. Eur J Pediatr, 1997; 156:650–654.

17.

Weber

, Guo

, Kleijer

, et al. Sanfilippo type B syndrome (mucopolysaccharidosis III B): allelic heterogeneity corresponds to the wide spectrum of clinical phenotypes. Eur J Hum Genet, 1999; 7:34–44.

18.

Emre

, Terzioglu

, Tokatli

, et al. Sanfilippo syndrome in Turkey: identification of novel mutations in subtypes A and B. Hum Mutat, 2002; 19:184–185.

19.

Pinto

, Caseiro

, Lemos

, et al. Prevalence of lysosomal storage diseases in Portugal. Eur J Hum Genet, 2004; 12:87–92.

20.

Ruijter

, Valstar

, van De Kamp

, et al. Clinical and genetic spectrum of Sanfilippo type C (MPS IIIC) disease in The Netherlands. Mol Genet Metab, 2008; 93:104–111.

21.

Canals

, Elalaoui

, Pineda

, et al. Molecular analysis of Sanfilippo syndrome type C in Spain: seven novel HGSNAT mutations and characterization of the mutant alleles. Clin Genet, 2011; 80:367–374.

22.

Valstar

, Bertoli-Avella

, Wessels

, et al. Mucopolysaccharidosis type IIID: 12 new patients and 15 novel mutations. Hum Mutat, 2010; 31:E1348–E1360.

23.

Dreyfuss

, Regatieri

, Jarrouge

, et al. Heparan sulfate proteoglycans: structure, protein interactions and cell signaling. An Acad Bras Cienc, 2009; 81:409–429.

24.

Jones

, Alroy

, Rutledge

, et al. Human mucopolysaccharidosis IIID: clinical, biochemical, morphological and immunohistochemical characteristics. J Neuropathol Exp Neurol, 1997; 56:1158–1167.

25.

McGlynn

, Dobrenis

, Walkley

. Differential subcellular localization of cholesterol, gangliosides, and glycosaminoglycans in murine models of mucopolysaccharide storage disorders. J Comp Neurol, 2004; 480:415–426.

26.

Walkley

. Secondary accumulation of gangliosides in lysosomal storage disorders. Semin Cell Dev Biol, 2004; 15:433–444.

27.

Constantopoulos

, Eiben

, Schafer

. Neurochemistry of the mucopolysaccharidoses: brain glycosaminoglycans, lipids and lysosomal enzymes in mucopolysaccharidosis type III B (alpha-N-acetylglucosaminidase deficiency). J Neurochem, 1978; 31:1215–1222.

28.

Constantopoulos

, Iqbal

, Dekaban

. Mucopolysaccharidosis types IH, IS, II, and IIIA: glycosaminoglycans and lipids of isolated brain cells and other fractions from autopsied tissues. J Neurochem, 1980; 34:1399–1411.

29.

Hadfield

, Ghatak

, Nakoneczna

, et al. Pathologic findings in mucopolysaccharidosis type IIIB (Sanfilippo's sydnrome B). Arch Neurol, 1980; 37:645–650.

30.

Dawson

, Fuller

, Helmsley

, et al. Abnormal gangliosides are localized in lipid rafts in Sanfilippo (MPS3a) mouse brain. Neurochem Res, 2012; 37:1372–1380.

31.

Bigger

, Begley

, Virgintino

, et al. Anatomical changes and pathophysiology of the brain in mucopolysaccharidosis disorders. Mol Genet Metab, 2018; 125:322–331.

32.

Valstar

, Neijs

, Bruggenwirth

, et al. Mucopolysaccharidosis type IIIA: clinical spectrum and genotype-phenotype correlations. Ann Neurol, 2010; 68:876–887.

33.

Valstar

, Bruggenwirth

, Olmer

, et al. Mucopolysaccharidosis type IIIB may predominantly present with an attenuated clinical phenotype. J Inherit Metab Dis, 2010; 33:759–767.

34.

Valstar

, Marchal

, Grootenhuis

, et al. Cognitive development in patients with Mucopolysaccharidosis type III (Sanfilippo syndrome). Orphanet J Rare Dis, 2011; 6:43.

35.

de Ruijter

, Valstar

, Wijburg

. Mucopolysaccharidosis type III (Sanfilippo Syndrome): emerging treatment strategies. Curr Pharm Biotechnol, 2011; 12:923–930.

36.

Buhrman

, Thakkar

, Poe

, et al. Natural history of Sanfilippo syndrome type A. J Inherit Metab Dis, 2014; 37:431–437.

37.

Cleary

, Wraith

. Management of mucopolysaccharidosis type III. Arch Dis Child, 1993; 69:403–406.

38.

Meyer

, Kossow

, Gal

, et al. Scoring evaluation of the natural course of mucopolysaccharidosis type IIIA (Sanfilippo syndrome type A). Pediatrics, 2007; 120:e1255–e1261.

39.

Rohrbach

, Clarke

JTR

. Treatment of lysosomal storage disorders: progress with enzyme replacement therapy. Drugs, 2007; 67:2697–2716.

40.

Enns

, Huhn

. Central nervous system therapy for lysosomal storage disorders. Neurosurg Focus, 2008; 24:E12.

41.

Begley

, Pontikis

, Scarpa

. Lysosomal storage diseases and the blood-brain barrier. Curr Pharm Des, 2008; 14:1566–1580.

42.

Calias

, Banks

, Begley

, et al. Intrathecal delivery of protein therapeutics to the brain: a critical reassessment. Pharmacol Ther, 2014; 144:114–122.

43.

Sands

, Davidson

. Gene therapy for lysosomal storage diseases. Mol Ther J Am Soc Gene Ther, 2006; 13:839–849.

44.

Jones

, Breen

, Heap

, et al. A phase 1/2 study of intrathecal heparan-N-sulfatase in patients with mucopolysaccharidosis IIIA. Mol Genet Metab, 2016; 118:198–205.

45.

Wijburg

, Whitley

, Muenzer

, et al. Intrathecal heparan-N-sulfatase in patients with Sanfilippo syndrome type A: a phase IIb randomized trial. Mol Genet Metab, 2019; 126:121–130.

46.

Muenzer

, Hendriksz

, Fan

, et al. A phase I/II study of intrathecal idursulfase-IT in children with severe mucopolysaccharidosis II. Genet Med, 2016; 18:73–81.

47.

Cleary

, Muschol

, Couce

, et al. ICV-administered tralesinidase alfa (BMN 250 NAGLU-IGF2) is well-tolerated and reduces heparan sulfate accumulation in the CNS of subjects with Sanfilippo syndrome type B (MPS IIIB). Mol Genet Metab, 2019; 126:S40.

48.

Hordeaux

, Hinderer

, Buza

, et al. Safe and sustained expression of human iduronidase after adeno-associated virus 9 intrathecal administration in infant rhesus monkeys. Hum Gene Ther, 2019; 30:957–966.

49.

Ribera

, Haurigot

, Garcia

, et al. Biochemical, histological and functional correction of mucopolysaccharidosis Type IIIB by intra-cerebrospinal fluid gene therapy. Hum Mol Genet, 2015; 24:2078–2095.

50.

Brown

, Wright

, Zhou

, et al. Bioengineered coagulation factor VIII enables long-term correction of murine hemophilia A following liver-directed adeno-associated viral vector delivery. Mol Ther Methods Clin Dev, 2014; 1:14036.

51.

Haurigot

, Marcó

, Ribera

, et al. Whole body correction of mucopolysaccharidosis IIIA by intracerebrospinal fluid gene therapy. J Clin Invest, 2013; 123:3254–3271.

52.

Nathwani

, Reiss

, Tuddenham

EGD

, et al. Long-term safety and efficacy of factor IX gene therapy in hemophilia B. N Engl J Med, 2014; 371:1994–2004.

53.

Motas

, Haurigot

, Garcia

, et al. CNS-directed gene therapy for the treatment of neurologic and somatic mucopolysaccharidosis type II (Hunter syndrome). JCI Insight, 2016; 1:e86696.

54.

Roca

, Motas

, Marcó

, et al. Disease correction by AAV-mediated gene therapy in a new mouse model of mucopolysaccharidosis type IIID. Hum Mol Genet, 2017; 26:1535–1551.

55.

Colella

, Sellier

, Costa Verdera

, et al. AAV gene transfer with tandem promoter design prevents anti-transgene immunity and provides persistent efficacy in neonate Pompe mice. Mol Ther Methods Clin Dev, 2019; 12:85–101.

56.

Hinderer

, Bell

, Gurda

, et al. Intrathecal gene therapy corrects CNS pathology in a feline model of mucopolysaccharidosis I. Mol Ther, 2014; 22:2018–2027.

57.

Hinderer

, Bell

, Gurda

, et al. Liver-directed gene therapy corrects cardiovascular lesions in feline mucopolysaccharidosis type I. Proc Natl Acad Sci U S A, 2014; 111:14894–14899.

58.

George

, Sullivan

, Giermasz

, et al. Hemophilia B gene therapy with a high-specific-activity factor IX variant. N Engl J Med, 2017; 377:2215–2227.

59.

Hocquemiller

, Giersch

, Audrain

, et al. Adeno-associated virus-based gene therapy for CNS diseases. Hum Gene Ther, 2016; 27:478–496.

60.

Gessler

, Gao

. Gene therapy for the treatment of neurological disorders: metabolic disorders. Methods Mol Biol, 2016; 1382:429–465.

61.

Mingozzi

, High

. Therapeutic in vivo gene transfer for genetic disease using AAV: progress and challenges. Nat Rev Genet, 2011; 12:341–355.

62.

Naldini

. Gene therapy returns to centre stage. Nature, 2015; 526:351–360.

63.

Jaén

, Vilà

, Elias

, et al. Long-term efficacy and safety of insulin and glucokinase gene therapy for diabetes: 8-year follow-up in dogs. Mol Ther Methods Clin Dev, 2017; 6:1–7.

64.

Leone

, Shera

, McPhee

, et al. Long-term follow-up after gene therapy for canavan disease. Sci Transl Med, 2012; 4:165ra163.

65.

Mittermeyer

, Christine

, Rosenbluth

, et al. Long-term evaluation of a phase 1 study of AADC gene therapy for parkinson's disease. Hum Gene Ther, 2012; 23:377–381.

66.

Buchlis

, Podsakoff

, Radu

, et al. Factor IX expression in skeletal muscle of a severe hemophilia B patient 10 years after AAV-mediated gene transfer. Blood, 2012; 119:3038–3041.

67.

Sondhi

, Johnson

, Purpura

, et al. Long-term expression and safety of administration of AAVrh.10hCLN2 to the brain of rats and nonhuman primates for the treatment of late infantile neuronal ceroid lipofuscinosis. Hum Gene Ther Methods, 2012; 23:324–335.

68.

Sergijenko

, Langford-Smith

, Liao

, et al. Myeloid/microglial driven autologous hematopoietic stem cell gene therapy corrects a neuronopathic lysosomal disease. Mol Ther, 2013; 21:1938–1949.

69.

Holley

, Ellison

, Fil

, et al. Macrophage enzyme and reduced inflammation drive brain correction of mucopolysaccharidosis IIIB by stem cell gene therapy. Brain, 2018; 141:99–116.

70.

Ellison

, Liao

, Wood

, et al. Pre-clinical safety and efficacy of lentiviral vector-mediated ex vivo stem cell gene therapy for the treatment of mucopolysaccharidosis IIIA. Mol Ther Methods Clin Dev, 2019; 13:399–413.

71.

Langford-Smith

, Wilkinson

, Langford-Smith

, et al. Hematopoietic stem cell and gene therapy corrects primary neuropathology and behavior in mucopolysaccharidosis IIIA mice. Mol Ther J Am Soc Gene Ther, 2012; 20:1610–1621.

72.

Poletti

, Biffi

. Gene-based approaches to inherited neurometabolic diseases. Hum Gene Ther, 2019; 30:1222–1235.

73.

Sessa

, Lorioli

, Fumagalli

, et al. Lentiviral haemopoietic stem-cell gene therapy in early-onset metachromatic leukodystrophy: an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial. Lancet (London, England), 2016; 388:476–487.

74.

Aiuti

, Biasco

, Scaramuzza

, et al. Lentiviral hematopoietic stem cell gene therapy in patients with Wiskott-Aldrich syndrome. Science (80-), 2013; 341:1233151.

75.

Eichler

, Duncan

, Musolino

, et al. Hematopoietic stem-cell gene therapy for cerebral adrenoleukodystrophy. N Engl J Med, 2017; 377:1630–1638.

76.

Visigalli

, Delai

, Politi

, et al. Gene therapy augments the efficacy of hematopoietic cell transplantation and fully corrects mucopolysaccharidosis type I phenotype in the mouse model. Blood, 2010; 116:5130–5139.

77.

Biffi

, Montini

, Lorioli

, et al. Lentiviral hematopoietic stem cell gene therapy benefits metachromatic leukodystrophy. Science (80-), 2013; 341:1233158.

78.

Squeri

, Passerini

, Ferro

, et al. Targeting a pre-existing anti-transgene T cell response for effective gene therapy of MPS-I in the mouse model of the disease. Mol Ther, 2019. [Epub ahead of print]; DOI: 10.1016/j.ymthe.2019.04.014.

79.

Constantopoulos

, McComb

, Dekaban

. Neurochemistry of the mucopolysaccharidoses: brain glycosaminoglycans in normals and four types of mucopolysaccharidoses. J Neurochem, 1976; 26:901–908.

80.

Tamagawa

, Morimatsu

, Fujisawa

, et al. Neuropathological study and chemico-pathological correlation in sibling cases of Sanfilippo syndrome type B. Brain Dev, 1985; 7:599–609.

81.

Winner

, Beard

, Hassiotis

, et al. A preclinical study evaluating AAVrh10-based gene therapy for Sanfilippo syndrome. Hum Gene Ther, 2016; 27:363–375.

82.

Ruzo

, Marcó

, García

, et al. Correction of pathological accumulation of glycosaminoglycans in central nervous system and peripheral tissues of MPSIIIA mice through systemic AAV9 gene transfer. Hum Gene Ther, 2012; 23:1237–1246.

83.

Ellinwood

, Ausseil

, Desmaris

, et al. Safe, efficient, and reproducible gene therapy of the brain in the dog nodels of Sanfilippo and Hurler Syndromes. Mol Ther, 2011; 19:251–259.

84.

, DiRosario

, Killedar

, et al. Correction of neurological disease of mucopolysaccharidosis IIIB in adult mice by rAAV9 trans-blood-brain barrier gene delivery. Mol Ther, 2011; 19:1025–1033.

85.

Gray

, O'Leary

, Liao

, et al. An improved adeno-associated virus vector for neurological correction of the mouse model of mucopolysaccharidosis IIIA. Hum Gene Ther, 2019; 30:1052–1066.

86.

Niethammer

, Tang

, LeWitt

, et al. Long-term follow-up of a randomized AAV2-GAD gene therapy trial for Parkinson's disease. JCI Insight, 2017; 2:e90133.

87.

Marks

, Bartus

, Siffert

, et al. Gene delivery of AAV2-neurturin for Parkinson's disease: a double-blind, randomised, controlled trial. Lancet Neurol, 2010; 9:1164–1172.

88.

Bartus

, Baumann

, Siffert

, et al. Safety/feasibility of targeting the substantia nigra with AAV2-neurturin in Parkinson patients. Neurology, 2013; 80:1698–1701.

89.

Christine

, Starr

, Larson

, et al. Safety and tolerability of putaminal AADC gene therapy for Parkinson disease. Neurology, 2009; 73:1662–1669.

90.

Eberling

, Jagust

, Christine

, et al. Results from a phase I safety trial of hAADC gene therapy for Parkinson disease. Neurology, 2008; 70:1980–1983.

91.

Ciron

, Cressant

, Roux

, et al. AAV1-, AAV2- and AAV5-mediated human alpha-iduronidase gene transfer in the brain of nonhuman primate: vector diffusion and bio distribution. Hum Gene Ther, 2009; 20:350–360.

92.

Tardieu

, Zérah

, Husson

, et al. Intracerebral administration of adeno-associated viral vector serotype rh.10 carrying human SGSH and SUMF1 cDNAs in children with Mucopolysaccharidosis type IIIA disease: results of a phase I/II trial. Hum Gene Ther, 2014; 25:506–516.

93.

Tardieu

, Zérah

, Gougeon

M-L

, et al. Intracerebral gene therapy in children with mucopolysaccharidosis type IIIB syndrome: an uncontrolled phase 1/2 clinical trial. Lancet Neurol, 2017; 16:712–720.

94.

Souweidane

, Fraser

, Arkin

, et al. Gene therapy for late infantile neuronal ceroid lipofuscinosis: neurosurgical considerations. J Neurosurg Pediatr, 2010; 6:115–122.

95.

Dierks

, Miech

, Hummerjohann

, et al. Posttranslational formation of formylglycine in prokaryotic sulfatases by modification of either cysteine or serine. J Biol Chem, 1998; 273:25560–25564.

96.

Tordo

, O'Leary

, Antunes

ASLM

, et al. A novel adeno-associated virus capsid with enhanced neurotropism corrects a lysosomal transmembrane enzyme deficiency. Brain, 2018. [Epub ahead of print]; DOI: 10.1093/brain/awy126.

97.

Foust

, Nurre

, Montgomery

, et al. Intravascular AAV9 preferentially targets neonatal neurons and adult astrocytes. Nat Biotechnol, 2009; 27:59–65.

98.

Duque

, Joussemet

, Riviere

, et al. Intravenous administration of self-complementary AAV9 enables transgene delivery to adult motor neurons. Mol Ther, 2009; 17:1187–1196.

99.

Mendell

, Al-Zaidy

, Shell

, et al. Single-dose gene-replacement therapy for spinal muscular atrophy. N Engl J Med, 2017; 377:1713–1722.

100.

Finkel

, McDermott

, Kaufmann

, et al. Observational study of spinal muscular atrophy type I and implications for clinical trials. Neurology, 2014; 83:810–817.

101.

, Meadows

, Pineda

, et al. Differential prevalence of antibodies against adeno-associated virus in healthy children and patients with mucopolysaccharidosis III: perspective for AAV-mediated gene therapy. Hum Gene Ther Clin Dev, 2017; 28:187–196.

102.

Meadows

, Duncan

, Camboni

, et al. A GLP-compliant toxicology and biodistribution study: systemic delivery of an rAAV9 vector for the treatment of mucopolysaccharidosis IIIB. Hum Gene Ther Clin Dev, 2015; 26:228–242.

103.

Nathwani

, Tuddenham

, Rangarjan

, et al. Adenovirus-associated virus vector-mediated gene transfer in Hemophilia B. N Engl J Med, 2011; 365:2357–2365.

104.

Manno

, Pierce

, Arruda

, et al. Successful transduction of liver in hemophilia by AAV-Factor IX and limitations imposed by the host immune response. Nat Med, 2006; 12:342–347.

105.

Mingozzi

, Maus

, Hui

, et al. CD8(+) T-cell responses to adeno-associated virus capsid in humans. Nat Med, 2007; 13:419–422.

106.

Mingozzi

, High

. Immune responses to AAV in clinical trials. Curr Gene Ther, 2011; 11:321–330.

107.

Flanigan

, Truxal

, McBride

, et al. A phase 1/2 clinical trial of systemic gene transfer of rAAV9.CMV.hNAGLU for MPS IIIB: safety, tolerability, and preliminary evidence of biopotency. Mol Ther, 2018; 26:311–312.

108.

Scallan

, Jiang

, Liu

, et al. Human immunoglobulin inhibits liver transduction by AAV vectors at low AAV2 neutralizing titers in SCID mice. Blood, 2006; 107:1810–1817.

109.

Jiang

, Couto

, Patarroyo-White

, et al. Effects of transient immunosuppression on adenoassociated, virus-mediated, liver-directed gene transfer in rhesus macaques and implications for human gene therapy. Blood, 2006; 108:3321–3328.

110.

Boutin

, Monteilhet

, Veron

, et al. Prevalence of serum IgG and neutralizing factors against adeno-associated virus (AAV) types 1, 2, 5, 6, 8, and 9 in the healthy population: implications for gene therapy using AAV vectors. Hum Gene Ther, 2010; 21:704–712.

111.

Calcedo

, Morizono

, Wang

, et al. Adeno-associated virus antibody profiles in newborns, children, and adolescents. Clin vaccine Immunol, 2011; 18:1586–1588.

112.

, Narkbunnam

, Samulski

, et al. Neutralizing antibodies against adeno-associated virus examined prospectively in pediatric patients with hemophilia. Gene Ther, 2012; 19:288–294.

113.

Meyer

, Ferraiuolo

, Schmelzer

, et al. Improving single injection CSF delivery of AAV9-mediated gene therapy for SMA: a dose-response study in mice and nonhuman primates. Mol Ther, 2015; 23:477–487.

114.

Ruzo

, Garcia

, Ribera

, et al. Liver production of sulfamidase reverses peripheral and ameliorates CNS pathology in mucopolysaccharidosis IIIA mice. Mol Ther J Am Soc Gene Ther, 2012; 20:254–266.

115.

Samaranch

, Salegio

, San Sebastian

, et al. Adeno-associated virus serotype 9 transduction in the central nervous system of nonhuman primates. Hum Gene Ther, 2012; 23:382–389.

116.

Gray

, Nagabhushan Kalburgi

, McCown

, et al. Global CNS gene delivery and evasion of anti-AAV-neutralizing antibodies by intrathecal AAV administration in non-human primates. Gene Ther, 2013; 20:450–459.

117.

Donsante

, McEachin

, Riley

, et al. Intracerebroventricular delivery of self-complementary adeno-associated virus serotype 9 to the adult rat brain. Gene Ther, 2016; 23:401–407.

118.

Gurda

, De Guilhem De Lataillade

, Bell

, et al. Evaluation of AAV-mediated gene therapy for central nervous system disease in canine mucopolysaccharidosis VII. Mol Ther, 2016; 24:206–216.

119.

Sorrentino

, Maffia

, Strollo

, et al. A comprehensive map of CNS transduction by eight recombinant adeno-associated virus serotypes upon cerebrospinal fluid administration in pigs. Mol Ther, 2016; 24:276–286.

120.

Ciron

, Desmaris

, Colle

, et al. Gene therapy of the brain in the dog model of Hurler's syndrome. Ann Neurol, 2006; 60:204–213.

121.

Standring

Gray's Anatomy: The Anatomical Basis of Clinical Practice, 40th ed. Churchill Livingstone: Elsevier, 2008.

122.

Federici

, Taub

, Baum

, et al. Robust spinal motor neuron transduction following intrathecal delivery of AAV9 in pigs. Gene Ther, 2012; 19:852–859.

123.

Cook

, Mieure

, Owen

, et al. Intracerebroventricular administration of drugs. Pharmacotherapy, 2009; 29:832–845.

124.

Piatt

. Technique of ventriculostomy. In: Rengachary

, Wilkins

, eds. Neurosurgical Operative Atlas. Chicago: The American Association of Neurological Surgeons, 1991:171–175.

125.

Duque

, Arnold

, Odermatt

, et al. A large animal model of spinal muscular atrophy and correction of phenotype. Ann Neurol, 2015; 77:399–414.

126.

, Meadows

, Pineda

, et al. Serum global metabolomics profiling reveals profound metabolic impairments in patients with MPS IIIA and MPS IIIB. Metab Brain Dis, 2017. [Epub ahead of print]; DOI: 10.1007/s11011-017-0009-1.