extfieldsofstudy:
Computer Science
Medicine

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A Systematic Review for the Design of In Vitro Flow Studies of the Carotid Artery Bifurcation

Purpose In vitro blood flow studies in carotid artery bifurcation models may contribute to understanding the influence of hemodynamics on carotid artery disease. However, the design of in vitro blood flow studies involves many steps and selection of imaging techniques, model materials, model design, and flow visualization parameters. Therefore, an overview of the possibilities and guidance for the design process is beneficial for researchers with less experience in flow studies. Methods A systematic search to in vitro flow studies in carotid artery bifurcation models aiming at quantification and detailed flow visualization of blood flow dynamics results in inclusion of 42 articles. Results Four categories of imaging techniques are distinguished: MRI, optical particle image velocimetry (PIV), ultrasound and miscellaneous techniques. Parameters for flow visualization are categorized into velocity, flow, shear-related, turbulent/disordered flow and other parameters. Model materials and design characteristics vary between study type. Conclusions A simplified three-step design process is proposed for better fitting and adequate match with the pertinent research question at hand and as guidance for less experienced flow study researchers. The three consecutive selection steps are: flow parameters, image modality, and model materials and designs. Model materials depend on the chosen imaging technique, whereas choice of flow parameters is independent from imaging technique and is therefore only determined by the goal of the study.

INTRODUCTION
Atherosclerotic plaque formation in the carotid artery bifurcation causes narrowing of the artery (stenosis) and the plaque may rupture, which can cause stroke or transient ischemic attack (TIA). Several parameters are known to influence risk of stroke in patients with significant carotid artery stenosis, e.g. plaque vulnerability, volume and stenosis degree. 20,26,52 Also, there is an association between low and oscillating wall shear stresses (WSS) and formation and/or progression of atherosclerotic plaque. 19,41,57,76 Surgical treatment is indicated in severe symptomatic carotid artery stenosis. An alternative approach is stenting of the lesion. However, this approach is not optimized yet, since it results in higher short-term stroke risk compared to surgery. There is a need for a better understanding of the factors that influence plaque characteristics and for analysis of flow changes caused by intervention, to eventually improve treatment and stroke prevention.
Blood flow studies are excellent approaches to enhance knowledge on the relationship between blood flow dynamics and plaque formation/progression and treatment outcome. General technological development leads to improvements in imaging and postprocessing techniques, which enables quantitative and detailed blood flow studies, such as image velocimetry. These techniques are superior to flow measurement techniques that only enable qualitative investigations, such as the way ultrasound Doppler is generally used in the clinic, namely only the measurement of flow in the center of the artery.
There are three methods to perform hemodynamic flow studies: in vivo, in vitro and in silico. The benefit of in vitro and in silico over in vivo is that certain parameters can be altered in a controlled environment. Compared to in silico, in vitro studies are sometimes preferred, due to the possibility to test and validate potential use of flow imaging techniques in patient studies. Furthermore, in vitro studies can be performed in situations where it is difficult to experiment on patients, for example in cases with radiation exposure or in case of rare diseases. Therefore, this review focusses on in vitro flow studies.
Starting in vitro flow studies brings along many steps and choices. For example, which imaging technique to choose from the wide range of (clinical) imaging modalities, to measure WSS or other flow parameters, and which phantom material to use. The choices for the flow setup also have to match the clinical research question. Review articles about in vitro flow study techniques often focus on one specific technique. Therefore, the aim of this review article is to give an overview of the possibilities of the various approaches for the design of in vitro flow studies. It will serve as guidance by best practice for researchers with less experience in flow studies to get familiar with the options and opportunities in flow study design.

Search Strategy
A systematic literature search was performed in January 2017 and repeated in June 2018. The key words are combinations of 'carotid', 'flow', 'modeling', 'in vitro', and synonyms of these terms, such as 'setup'/'setup', 'blood flow velocity', 'rheology', 'wall shear stress', 'hemodynamics', 'simulation', 'phantom'. We have modified the search query to match each specific database (Scopus, Medline, Embase, and Cochrane).

Study Selection
Two authors-A.H. and E.V.-independently screened the query results on the basis of titles and abstracts. Both authors independently checked fulltext eligibility. All discrepancies regarding inclusion or exclusion were discussed until consensus was reached. The inclusion criteria were: (1) in vitro flow study; (2) carotid bifurcation models; and (3) quantitative flow imaging. The inclusion was limited to carotid bifurcation models, since the design of an in vitro flow study strongly depends on specific flow rates and vessel wall properties. Studies aiming at flow quantification in other arteries, for example abdominal aorta, might use other methods and characteristics, and are therefore not included. Thus, studies regarding intracranial carotid artery were excluded, as well as in vivo and animal studies. Also, studies using ultrasound Doppler measurement without further post processing and studies describing flow velocities only were excluded. Other exclusion criteria were: only in silico/computational fluid dynamics, full-text not in English, review articles and conference proceedings.

Data Processing
The included full-text articles were organized into four categories of imaging techniques used to visualize flow: magnetic resonance imaging (MRI), laser particle image velocimetry (PIV), ultrasound, and miscellaneous techniques. Data extraction parameters for all imaging techniques are: resolution, study type (technique development/validation, flow exploration), working fluid, fluid scatters, flow type (steady, sinusoidal, physiologic), Reynolds number, viscosity, flow rate, velocity, model materials, model design: pathology (healthy, stenosed or aneurysmatic), geometry (average or patient-specific), wall (thin-walled or wallless), origin (commercial or home-made); and flow visualization parameters. Technique-specific data extraction parameters were: -for MRI: sequence; -for optical PIV: light source; -for ultrasound: protocol/postprocessing, system type (clinical or research); -for miscellaneous techniques: methods.

RESULTS
The systematic search yields 1877 unique articles. Most articles are excluded on the basis of title and abstract screening. Full-text review includes 144 articles, of which 42 articles are selected for this review (Fig. 1).
The oldest techniques to quantify blood flow patterns in vitro in carotid artery bifurcation that are included in this review, use laser doppler anemometry 34 and digital subtraction angiography. 74 The first articles that reported the use of MRI date from 1992. Studies using ultrasound or optical PIV techniques are mostly from 2008 and later. A timeline of included articles per imaging technique is shown in Fig. 2.

MRI
MRI is used in 14/42 articles (33%) to scan the in vitro carotid bifurcation model to visualize the flow ( Table 1). One of these articles describes the use of both MRI and ultrasound. 18 Among the several imaging sequences (MRI protocols) that are used, phase contrast sequences are applied in most articles. 4,11,18,33,38,39,43,46,54,56,78 A distinction is made between two-dimensional and three-dimensional phase contrast sequences. Phase contrast MRI makes use of the phase shift of moving spinning protons. Velocity data can be computed by comparing the phase shifts between moving and stationary protons.
Three articles use other MRI-sequences to visualise flow. The first article proposes an extension of the single-bolus multi-zone adiabatic passage technique. 65 This extended version uses flow velocity profiles from several directions other than the main flow direction. The second article describes the feasibility of spiral Fourier velocity encoded MRI for measuring carotid wall shear rate. 8 Compared to standard Fourier velocity encoding, spiral Fourier velocity encoding is faster due to a higher temporal resolution, so wall shear rate can be measured not only in vitro, but also in vivo. The third article describes disordered flow, which can be visualised using temporal variations in magnetization by applying a two-dimensional Fourier Transform Gradient Echo sequence. 66 The resulting images only show disordered flow.

Optical Particle Image Velocimetry
An alternative imaging technique to visualize flow is optical PIV. It is a technique that uses one or more lasers to illuminate contrast material flowing through a transparent phantom and captures the motion using a high-frame-rate digital camera. Each image frame is divided into small, so-called interrogation areas. Subsequently, each area is compared to the corresponding area of the following frame by applying correlation techniques. Finally, a velocity field is calculated, and localized velocity vectors can be visualized. 11/42 articles (26%) report on the use of optical PIV to study flow patterns in the carotid bifurcation in an in vitro model ( Table 2). Two articles also use ultrasound. 59,77 The flow setups are equipped with a continuous wave laser, 2,30,31 a pulsed laser, 2,29,44 or a LED light source. 40 Where most of the studies uses one or two (stereo-PIV) cameras, one article describes the construction of a tomographic setup, using four digital cameras arranged at various angles. 7

Ultrasound
In 16/42 articles (38%), ultrasound is applied as imaging technique (Table 3). Three research groups contribute to this review by two or more included articles. The applied equipment is almost similar within these groups. Both clinical and research-based ultrasound systems are used.
Seven out of 16 articles within the ultrasound category perform image velocimetry. Different acquisition protocols and post processing techniques are used: echo particle image velocimetry using contrast agent (echoPIV), 77 high-frame-rate ultrasound imaging velocimetry using speckle patterns, 36,37 Vector Projectile Imaging using multi-angle Doppler analysis, 72 transverse oscillation and directional beamforming for vector velocity estimation, 28 a biomechanical method that produces a map of displacement vectors, 45 and vector flow mapping using color Doppler images from a clinical system. 59 Doppler protocols are applied in seven articles. One article uses a Doppler protocol to measure volume flow. 18 Some articles describe the use of a semiautomatic Doppler ultrasound acquisition system to obtain small sample volumes at desired spatial intervals to perform velocity measurements over time. [48][49][50]67,68 In another article, pulsed Doppler and color flow imaging are applied on a research system to investigate velocities in home-made phantoms. 35 A combination of clinical Doppler flow measurements and an advance plane wave protocol on a research-based system is also described. 9 Another technique applied in Ref. 73 called color-encoded speckle imaging, uses high-frame-rate steered plane wave imaging on a system that includes a pre-beamformed RF data acquisition tool (a channel-domain imaging research platform).

Miscellaneous Techniques
4/42 articles (10%) report miscellaneous techniques for flow visualization (Table 4). One article describes the use of digital photographic imaging in combination with photochromic dye. 12 A novel grid reconstruction technique has resulted in development of quantitative measurements. The use of Laser Doppler Anemometry (LDA) is described in two articles. 14,34 This technique is based on the Doppler shift induced by scattering of a laser beam when it hits moving fluid. Flow rate is measured using Digital Subtraction Angiography in the fourth article. 74 Time density curves are created and blood flow among the regions of interest is calculated using the obtained velocity and known radius of the vessel.

Model Characteristics
A full overview of the characteristics of the bifurcation models is presented in Table 5. Most of the MRI articles make use of a commercial model, in contrast to the majority of the optical PIV and ultrasound articles, where home-made models are used. Casting is a technique that is frequently used to obtain certain geometries in home-made models. For example, rapid prototyping or 3D printing techniques are used to retrieve molds and one article even describes a fabrication method for separate plaque inclusion in the model. 49 Between groups of imaging techniques and within the groups, different materials are used for the fabrication of the home-made models. Most optical PIV models are made of silicone. In the ultrasound articles, polyvinylalcohol (PVA) is a frequently used material, especially in the last decade.
Both healthy and diseased carotid artery bifurcations are studied. One of the models with diseased geometry is an aneurysmatic bifurcation, 40 all other diseased models are stenotic, sometimes with ulceration. 35,67,68 The geometries are based on either patientspecific geometries or averaged bifurcation dimensions. The home-made models are either 'thin-walled', meaning that the model has a certain (thin) wall thickness, or 'wall-less', that means that the fluid space is frequently surrounded by a block of material, instead of a thin wall. Thin-walled models surrounded by a tissue mimicking material (TMM) are also reported. 9, 18,35,36,49,50 The optical PIV articles mainly use wall-less models. One article describes the fabrication of a model that was compatible with x-ray, ultrasound and MRI. 18 The article illustrated a polyester resin thin-walled model with a layer of agar as tissue-mimicking-material to be compatible with the different imaging techniques.

Working Fluid, Scatter Particles and Contrast Agents
Flow studies in the human body can be performed without addition of scatter particles or contrast materials, in which case blood functions as natural contrast agent. However, for some techniques it is necessary to add contrast agents to the blood circulation, for example in some ultrasound protocols to enhance the scattering properties of blood. In case of in vitro studies, scatter particles or contrast material is regularly used, since the natural contrast enhancing properties of blood are not present.
No contrast agents or scatter particles are used in the MRI studies. The working fluid in MRI varies from commercially available blood mimicking fluid to an aqueous mixture of machine tool cutting fluid (Table 1). Optical PIV requires the addition of scatter   material. In ten of eleven studies synthetic particles are added to the working fluid (  Table 3) is based on the recipe of 51 (BMF-R in Table 3). A photochromic dye is used in one article in the 'miscellaneous' category 12 (Table 4). Another 'miscellaneous' article uses a mixture of 76% Renografin digital subtraction angiography contrast medium, sodium and meglumine to visualize flow using x-ray techniques. 74

Flow Visualization Parameters
Flow characteristics can be presented and visualized using many quantitative parameters, each accentuating different aspects of the blood flow. Some quantities need to be measured to estimate or calculate other, more relevant parameters. This review only includes articles that quantified and visualized flow characteristics and patterns.
The flow visualization parameters (Table 6) are divided into five categories: velocity, flow, shear-related, turbulent/disordered flow and other parameters. Velocity and flow are commonly used parameters and mostly presented as magnitude values, vectors, streak-/ streamlines, and profiles over time. All imaging techniques are able to measure these two parameters. Wall shear stress (WSS) is a commonly calculated parameter in quantitative flow studies. Arterial WSS is defined as: ''the drag force acting on the endothelium as a result of blood flow''. 8 WSS magnitude is calculated by multiplying wall shear rate (WSR) by fluid viscosity, 8,11 as shown in Ref. 33 with the following equation: where g is the fluid viscosity, v is the velocity, r is the radial co-ordinate, and a is the vessel radius. Oscillating wall shear stress (OSI) (

DISCUSSION
This systematic review serves as starting point for designing in vitro carotid flow studies by presenting an overview of methods that have been applied in in vitro hemodynamic studies using imaging techniques to visualize and quantify flow and flow-related parameters. The review is limited to research using carotid artery bifurcation models. The next paragraph shortly summarizes the results, followed by a discussion on considerations for in vitro flow studies, to be concluded with a section of strengths and limitations of this review.

Summary of Results
We distinguished four categories of imaging techniques used to visualize and quantify blood flow dynamics: MRI, optical PIV, ultrasound, and miscellaneous techniques. A trend towards the use of optical PIV and ultrasound is seen in the last decade (Fig. 2). Noticeable in model design is the use of commercial models in MRI, while the other studies mainly use home-made models. Furthermore, the choice of model material depends on the imaging technique (Table 5). Optical PIV and ultrasound require the use of scatter particles, whereas the use of scatter particles in MRI studies is limited. Visualization parameters are divided into four categories (Table 6). Velocity-based parameters are widely reported, flow-and shear-based parameters are frequently studied, and turbulent/disordered flow-based parameters are mostly reported for optical PIV and ultrasound studies.

Considerations for In Vitro Flow Study Design
The starting point of flow studies in general strongly depends on the goal of the study. Since there is a large variety of goals and aims in flow studies, the authors of this review article are not in the position to select one favourite flow parameter, imaging technique, model material or design for future use. This section illustrates a simplified three-step design process, combining and discussing the information from the results section.
The first step in flow study design is selection of parameters for flow visualization. This is strongly related to the goal of the study. Clinical relevance can  also be considered while selecting flow visualization parameters. However, it does not restrict the imaging modality choice. Velocity and flow-based parameters give a global impression on flow dynamics and are presented in many ways: over time, spatial, mean or peak, as contour, etc. (see Table 6). Shear-based parameters are frequently studied, however their clinical relevance has yet to be determined. It is generally accepted that regions of low WSS and oscillating WSS correlate to formation and growth of atherosclerotic plaque. 53,57 However, a review on this topic shows that there are novel studies that find an inverse relationship. 47 Moreover, there is no clear conclusion about the relation between low and high WSS in plaque vulnerability. 8,30,70 Turbulent/disordered flow-based parameters are mostly reported by ultrasound and optical PIV studies. Some of these parameters clearly show and quantify regions of disordered flow, however some other quantitative parameters are hard to interpret. The second step in flow study design is selection of an imaging technique. Generally, the choice of visualization parameters does not lead to restrictions in imaging techniques, thus the three main imaging techniques (MRI, optical PIV, ultrasound) are all available after flow parameter selection. The benefits and limitations shown in Table 7 are discussed next. It is necessary to realize that some imaging techniques require the use of (clinical) contrast agents or scatter particles when using it in vitro. In this respect, MRI is beneficial, since retrieving flow information is not dependent on scatter material. However, MRI is generally only available in a hospital. Also, the flow setup needs to be adjusted so that there are no metallic parts near the MRI-equipment, the help of a laboratory technician is preferred during measurements, and development and adjustment of a protocol or sequence has a large learning curve. The benefit of MRI is the possibility of conversion to a patient study. This translation to a clinical study is not possible in optical PIV, because laser will not pass through the human body and the region of interest inside the patient cannot be captured using digital cameras. Optical PIV is ultimately suitable for accurate and precise quantification of flow patterns in vitro. In some situations, another benefit of optical PIV is that a hospital setting is not required to perform the in vitro flow studies. Optical PIV models need to be transparent and flat laser-entry surfaces are required for preventing distortion or refraction of the laser beam. Considering the third option, ultrasound, there is a division into studies using clinical systems and studies using research-based systems. To perform detailed flow analysis using image velocimetry techniques, fast imaging is prescribed and therefore the use of a research-based ultrasound system  is necessary. However, a research-based ultrasound system has a larger learning curve. Also, researchbased systems are not marketed for direct clinical use, so translation to a clinical study is more complicated compared to a clinical system. In short, MRI and ultrasound are appropriate imaging modalities if translation to a patient study is desired. MRI requires a learning curve or specific knowledge and a metal-free setup. For in vitro flow studies, ultrasound requires contrast agents or scatter material in the working fluid. Optical PIV is preferred if there is no aim to translate it to a patient study, if no clinical equipment is available and if restrictions to transparent models are not an issue.
The third step is selection of model materials and model design. This depends on the imaging technique that will be used. For example, poly(vinyl alcohol) gelbased models are ultimately suitable for ultrasound studies. If optical PIV is considered, silicone models are preferred, such as polydimethylsiloxane (PDMS). As stated before, the models for optical PIV need to be transparent and require flat surfaces. Ultrasound models often have a tissue mimicking layer, while MRI-models frequently are thin-walled without TMM. Remarkably, the included studies barely reported on choices made in model selection. For example, only a few studies indicated that the model is elastic or rigid and moreover, elasticity was quantified in only four articles. 9, 24,48,49 These are certainly parameters that need to be considered, since rigidity or elasticity has a large influence on the similarity of the simulation with the real situation.

Strengths and Limitations of this Review
This review summarizes in vitro carotid artery flow studies according to used imaging techniques, model materials and designs. Compared to other review articles which usually focus on one specific imaging technique, this review has a wide scope as it provides an overview of multiple imaging techniques. For the design of an in vitro flow study, this wide scope is beneficial, because model materials and model designs depend on the chosen imaging techniques.
In the ultrasound category, multiple articles of the same research group are found. This is a bias to the results which we took into consideration by noting the research groups (Table 3). Furthermore, we marked the flow parameters that we found in multiple articles from the same research group (Table 6). Since our analysis does not strongly depend on the number of parameters and it only happens in the ultrasound category, this bias does not influence our conclusions.
The notations and units of several parameters, such as accuracy, resolution, viscosity, vary widely among the included articles. Therefore, interpretation of these parameters was difficult.
To the authors knowledge, imaging techniques and model parameters might have been missed in our search by restricting it to carotid artery bifurcations. Moreover, newest techniques might be tested first on 'simple' straight models, so we might have missed these novel techniques by restricting the search. Techniques and methods might have been missed as well by restricting the search to carotid arteries, since in vitro flow studies are also widely performed in intracranial, abdominal or other peripheral artery models. On the other hand, carotid artery specific flow rates, types of diseases, and vessel wall characteristics, lead to specific choices of materials and methods in the design of the flow study.
The imaging techniques reported in this review are not only used to study carotid arteries. Other parts of the cardiovascular system are studied as well. The ascending aorta, 21,25,62 aortic arch 3 and also aortic coarctation 42 are studied using three-and four-dimensional flow MRI. In addition, this technique is applied intracranially to study aneurysms, 5 55,61 Furthermore, ventricular filling is studied using four-dimensional flow MRI. 16 Ultrasound-based vector flow imaging and echoPIV are used to study flow in the ascending aorta, 22 the abdominal aorta, 17,63 and to study ventricular blood flow. 27,32 The latter is also reported in combination with optical PIV flow studies. 1,64 Moreover, optical PIV is applied in enlarged coronary artery flow phantoms. 6,60 Thus, despite the restriction of the systematic search to carotid artery bifurcation, the reported imaging techniques and considerations for the design of an in vitro flow study can generally be applied. Fabrication methods of the models for in vitro flow studies fall out of the scope of this review article. Only a small amount of the included articles reported about the fabrication method of the models. A specific search to articles concerning the process of constructing models is necessary to write a review article on that topic. Two literature reviews for ultrasound and PIV models specifically have been published already. 13,71 CONCLUSION This systematic review on in vitro flow studies aiming at quantifying and visualizing flow parameters in carotid bifurcation models shows important factors to consider when designing a flow study. In contrast to most other review articles on flow studies, this review is not restricted to one imaging modality. Therefore, it gives a complete overview of techniques for in vitro flow studies.

also in
Since the design of flow studies strongly depends on the pertinent research question at hand, there is no preferred imaging technique or design that can be selected based on the information in this review. Three important steps need to be considered while designing in vitro flow studies: (1) selection of flow visualization parameters, (2) selection of an imaging technique, (3) model materials and design.
The selection of flow visualization parameters is completely dependent on the aim and goal of the study and independent of the selected imaging modality. Flow parameters are classified into velocity, flow, shear-related and turbulent/disordered flow-based parameters. The selection of an imaging technique can roughly be categorized in MRI, optical PIV and ultrasound. Conclusions on accuracy and resolution of the imaging systems cannot be made, since these parameters are not consistently reported throughout the literature. The selection of model materials and design of the model depends on the imaging technique and it strongly depends on the goal of the study.

CONFLICT OF INTEREST
The authors affirm that there are no financial and personal relationships or involvement with any commercial organization that could inappropriately influence or bias the present manuscript.

OPEN ACCESS
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Enhancing the Therapeutic Potential of Sulfamidase for the Treatment of Mucopolysaccharidosis IIIA

Mucopolysaccharidosis type IIIA (MPS-IIIA) is a lysosomal storage disorder (LSD) caused by inherited defect of sulfamidase, a lysosomal sulfatase. MPS-IIIA is one of the most common and severe forms of LSDs with CNS involvement. Presently there is no cure. Here we have developed a new gene delivery approach for the treatment of MPS-IIIA based on the use of a modified version of sulfamidase expression cassette. This cassette encodes both a chimeric sulfamidase containing an alternative signal peptide (sp) to improve enzyme secretion and sulfatase-modifying factor 1 (SUMF1) to increase sulfamidase post-translational activation rate. We demonstrate that improved secretion and increased activation of sulfamidase act synergistically to enhance enzyme biodistribution in wild-type (WT) pigs upon intrathecal adeno-associated virus serotype 9 (AAV9)-mediated gene delivery. Translating such gene delivery strategy to a mouse model of MPS-IIIA results in a rescue of brain pathology, including memory deficit, as well as improvement in somatic tissues. These data may pave the way for developing effective gene delivery replacement protocols for the treatment of MPS-IIIA patients.

INTRODUCTION
Mucopolysaccharidosis type IIIA (MPS-IIIA) is one of the most common and severe forms of neurodegenerative lysosomal storage disorders (LSDs). 1,2 It is caused by inherited defect of the sulfamidase (SGSH), a soluble lysosomal enzyme belonging to the family of sulfatases, 3 and leads to the accumulation of heparan sulfate in cell, particularly within the CNS. Presently, there are no treatments available to treat the CNS in MPS-IIIA patients. Gene delivery aimed at correcting defective hydrolytic lysosomal defects represents the most promising replacement strategy for the CNS treatment of MPS-IIIA, as well as other MPSs with similar causes, because of its potential for a onetime treatment. 4 Among viral vectors used for gene transfer, adeno-associated viral (AAV) vectors are most commonly utilized for in vivo gene transfer because they are safe, provide significantly long transgene expression, and may be generated with variable serotypes allowing efficient delivery of therapeutic genes to different target tissues. 5,6 To date, several therapeutic approaches have been designed and developed based on AAV-mediated gene delivery of SGSH using different routes of administration to reach the CNS. [7][8][9][10][11][12] Although all of these approaches showed potential benefits in preclinical animal models, the effective therapeutic application of these protocols in the clinical management of MPS-IIIA patients is challenging because of the difficulty in achieving widespread distribution of the corrective enzyme in the CNS and in maintaining therapeutic threshold levels of them in targeted cells. 13 Phase I/II clinical trials based on either AAV9-mediated systemic (ClinicalTrials. gov: NCT02716246) or AAVrh10-mediated intra-parenchymal delivery of the SGSH gene are ongoing (ClinicalTrials.gov: NCT03612869). Although these approaches are promising, they also show drawbacks. Indeed, the first approach suffers from potential toxicity because of the high doses of systemically delivered AAV9 vectors required for efficacy, 14 whereas the second approach is invasive and preliminary efficacy data did not show evident improvement of CNS pathology, likely also due to the inefficient targeting of CNS regions at any significant distance from the injection sites. 15 Therefore, a major medical need in the clinical care of MPS-III patients is to overcome these limitations and develop safe and minimally invasive gene transfer approaches with improved CNS transduction.
Enhancing the therapeutic potential of sulfamidase, along with developing tools for efficient and safe CNS targeting, may represent a novel area of intervention to improve CNS therapy in MPS-IIIA patients. Intra-cerebrospinal fluid (CSF) administration allows circulating virus to reach a large CNS surface area, and at the same time provides access to visceral organs due to the leakage of vector into the bloodstream. 9,16,17 Among different AAV serotypes, serotype 9 has been shown to have a broad tropism, including neurons and astrocytes, when delivered either via CSF or through intravenous injection due to its capability to cross the blood-brain barrier (BBB). 18, 19 We recently demonstrated that AAV9 outperforms all other AAV serotypes tested in CNS transduction efficiency when administered via CSF even in large-animal models. 20 Lysosomal hydrolases, including SGSH, can be secreted and taken up by surrounding cells. 21 Such cross-correction capability makes gene replacement protocols potentially more effective because transduced cells (factory cells) may also correct non-transduced cells. In a previous study we demonstrated that replacing the signal peptide (sp) of sulfamidase with that belonging to another lysosomal hydrolase that is highly secreted, the iduronate 2-sulfatase (IDS), strongly improved the efficiency of SGSH secretion from multiple cell types both in vivo and in vitro, a property that enhanced the cross-correction capability of transduced cells upon gene delivery. 8 To be fully active, all sulfatases, including the SGSH, must be activated through the generation of the catalytic residue C-alpha-formylglycine (FGly). This post-translational modification is accomplished in the endoplasmic reticulum by the FGly-generating enzyme (FGE) encoded by the sulfatase-modifying factor 1 (SUMF1) gene. 22 SUMF1 is both an essential and a limiting factor for sulfatases. Co-expression of SUMF1 with sulfatases results in a synergistic increase of sulfatase activity. 22,23 Here we have generated a potentiated SGSH expression cassette by combining enhanced secretion efficiency of SGSH with improved SUMF1-mediated enzyme activation capacity and explored its therapeutic potential upon intra-CSF AAV9-mediated gene delivery. Our results demonstrate that by using this strategy it is possible to achieve a superior SGSH CNS biodistribution in a large-animal model (wild-type [WT] pigs) and to fully rescue the CNS phenotype in a mouse model of MPS-IIIA.

RESULTS
Usage of a SGSH Expression Cassette with Enhanced Enzyme Secretion and Activation Improved Enzyme Biodistribution in the CNS of Pigs upon Intra-CSF AAV9-Mediated Gene Delivery In order to improve the biodistribution potential of the SGSH enzyme, we generated a bicistronic SGSH expression cassette containing a chimeric SGSH bearing the iduronate 2-sulfatase signal peptide (IDSsp) and the gene encoding SUMF1 (IDSspSGSH-IRES-SUMF1). The SGSH biodistribution in the CNS triggered by IDSspSGSH-IRES-SUMF1 expression was then evaluated upon intra-CSF AAVmediated delivery in WT pigs (Sus Scrofa white model). Thirty days after birth (P30), WT pigs were intra-CSF injected via cisterna magna with 4.5 Â 10 12 GCs/kg of AAV9 encoding IDSspSGSH-IRES-SUMF1 under the CMV promoter. As control, pigs were injected with AAV9 encoding for only either WT SGSH (AAV9-SGSH), IDSspmodified SGSH (AAV9-IDSspSGSH), or bicistronic cassette containing SUMF1 together with WT SGSH (AAV9-SGSH-IRES-SUMF1). One month after injection, 20 CNS regions covering the main representative areas of the brain and the spinal cord were collected for both SGSH enzyme activity evaluation and immunohistochemistry (IHC) analysis. The expression of highly secreted forms of SGSH (IDSspSGSH and IDSspSGSH-IRES-SUMF1 cassettes) resulted in a remarkable and significant increase of the specific enzyme activity over the CNS as compared with control (pigs injected with unmodified SGSH constructs: SGSH and SGSH-IRES-SUMF1) ( Figure 1A; Table S1). Interestingly, the enhancing effect of SUMF1 was statistically significant only when SUMF1 was co-expressed with IDSsp-modified SGSH (IDSspSGSH-IRES-SUMF1) and not when it was co-expressed with unmodified SGSH (SGSH-IRES-SUMF1) ( Figure 1A; Table S1). Immunostaining analysis against exogenous human sulfamidase revealed a very strong SGSH expression in multiple brain areas of pigs injected with IDSspSGSH-IRES-SUMF1, which indicated that the increased enzymatic activity is associated with a strong SGSH protein signal in the same brain areas of interest ( Figure 1B). Our results showed that combining improved secretion and enhanced SUMF1-mediated enzymatic activation gave rise to a synergistic effect on SGSH activity in the CNS of a large-animal model. To evaluate the potential for therapeutic effectiveness of intra-CSF AAV9-mediated delivery of IDSspSGSH-IRES-SUMF1, we used the B6Cg-Sgsh (mps3a/PstJ) mouse (Sgsh À/À ), a spontaneous MPS-IIIA mouse model largely used in preclinical studies of this disease. 24 Initially, we performed a short-term comparative study in a small cohort of Sgsh À/À mice (hereinafter referred to as MPS-IIIA mice) to evaluate the efficacy of the IDSspSGSH-IRES-SUMF1 expression cassette on a background of disease-associated enzymatic activity (MPS-IIIA levels in this model show 3%-5% of normal SGSH activity). P60 MPS-IIIA mice were intraventricularly (i.c.v.) injected with 5.4 Â 10 12 GCs/kg of AAV9 vectors encoding WT SGSH, IDSspSGSH, or IDSspSGSH-IRES-SUMF1. One month after injection, we found a significant increase in SGSH activity in brain sections from the IDSspSGSH-injected MPS-IIIA mice and a further significant increase in enzymatic activity upon the administration of AAV9-IDSspSGSH-IRES-SUMF1 (until to 50% of WT levels) ( Figure S1). These results confirmed the data obtained in pigs showing enhanced therapeutic potential of the IDSspSGSH-IRES-SUMF1 expression cassette.
We then performed an efficacy study in a larger cohort of MPS-IIIA mice in which AAV9 encoding IDSspSGSH-IRES-SUMF1 was delivered i.c.v. Following 1 (early time point [ETP]) or 7 months (late time point [LTP]) after injection, the enzyme biodistribution and the pathological phenotype were analyzed. Consistent with previous results ( Figure S1), a significant increase of SGSH activity in the brain of injected animals with peaks of 35% of WT levels was observed at ETP ( Figure 2A). Importantly, such increased activity was sustained at LTP (25% of WT levels) (Figure 2A). Increased SGSH activity correlated with both the increase in SGSH protein expression ( Figure S2A; Figure 2B) and the number of GCs detected in the brain ( Figure S2B). Co-immunolabeling analysis of cell-specific transduction in the brain samples of MPS-IIIA mice i.c.v. injected with AAV9-encoding IDSspSGSH-IRES-SUMF1 showed that SGSH signal co-localized with both NeuN (neuronal nuclei protein) and glial fibrillary acidic protein (GFAP) (astroglial) markers in several brain regions, with NeuN colocalization being prevalent over GFAP co-localization ( Figure 2C).  flammation, both hallmarks of MPS-IIIA neuropathology. 7,24 Immunostaining for lysosomalassociated membrane protein 1 (LAMP1) on brain sections of MPS-IIIA mice injected with AAV9-IDSspSGSH-IRES-SUMF1 showed a significant reduction in the lysosomal compartment enlargement compared with control MPS-IIIA mice ( Figure 3A). Such improvement was associated to a significant reduction in the abnormal vacuolization as shown by toluidine blue staining analysis ( Figure 3B). Consistently, GAGs levels were also significantly reduced following IDSspSGSH-IRES-SUMF1 treatment ( Figure 3C). Next, to evaluate the inflammation levels in treated MPS-IIIA mice, brain sections were stained for GFAP, a marker of astrocytes. As expected, excessive astrocyte levels were seen in MPS-IIIA control mice ( Figure 3D). Remarkably, the brain of MPS-IIIA mice treated with IDSspSGSH-IRES-SUMF1 showed a strong reduction of GFAP signal as compared with control affected mice, indicating a striking reduction of inflammatory processes ( Figure 3D).
Along with the severe manifestations, MPS-IIIA also shows mild somatic pathology characterized by GAG storage in several somatic tissues, particularly in the liver, which leads to increased mass (hepatomegaly). Being that the CSF is continuously absorbed and returned to the bloodstream, AAV9 vectors may travel from the CSF to the bloodstream and eventually reach the somatic tissues. The presence of such leakage was supported by the fact that following i.c.v. AAV9-mediated delivery of IDSspSGSH-IRES-SUMF1 gene transfer, the livers of treated mice were efficiently transduced as shown by significant increased levels of vector genomes ( Figure S3A) and a subsequent significant increase in SGSH enzymatic activity ( Figure S3B). This increase was associated www.moleculartherapy.org with higher levels of SGSH protein as well ( Figure S3C). Importantly, quantitative measurement of GAGs in the liver of IDSspSGSH-IRES-SUMF1-treated MPS-IIIA mice showed an almost complete normalization of GAG content ( Figure S3D). Together these results demonstrated the effectiveness of AAV9-mediated delivery of the enhanced version of SGSH with improved secretion and activation in rescuing both CNS and somatic pathology in MPS-IIIA mice.  Figure S1) were collected at both 1 (ETP) and 7 months (LTP) after injection. Sulfamidase activity was measured in these areas and expressed as percentage of the activity found in age-matched WT injected with AAV9 encoding GFP. n = 6-7 animals for each group. Data represent mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.00 versus MPS-IIIA GFP-treated.  Figure S4C). This tendency was not evident at 9 months, likely due to the test-retest effects observed also in control animals, 25 which did not allow to properly evaluate the rescuing efficacy of IDSspSGSH-IRES-SUMF1 ( Figures  S4A-4C). Therefore, we tested the animals in a memory task that is extensively used to test contextual memory depending on the functional integrity of the medial temporal lobe, namely, the fear contextual conditioning test. 26 Using this task, we identified, for the first time in MPS-IIIA mice, an age-dependent long-term memory impairment: 6-monthsold MPS-IIIA mice show normal freezing time when they are exposed to a mild foot shock during training, as compared with control animals; similarly, exposure to the context (24 h after training) paired with the mild foot shock is sufficient to elicit freezing as well as in control mice ( Figure 3E). This suggests that at this stage MPS-IIIA mice can form emotional contextual memories as well as WT animals. However, when the same test is repeated 3 months later, although there are no differences in basal freezing during training, on the testing day MPS-IIIA mice have impaired freezing response (Figure 3E). Interestingly, IDSspSGSH-IRES-SUMF1 fully rescues the memory deficit in 9-month-old MPS-IIIA mice ( Figure 3E). These behavioral effects in MPS-IIIA mice (both treated and untreated) cannot be explained as due to reduced immobility time (a possible correlate of reduced freezing time), because in the open field test both MPS-IIIA groups show similar levels of immobility time at this testing age. Considering the specificity of this test for memory deficits occurring in animal models of neurodegenerative diseases, these results have a high relevance for the validation of the neuronal dysfunctions correction in MPS-IIIA mice treated with AAV9-IDSspSGSH-IRES-SUMF1.

DISCUSSION
Here, we developed and tested an intra-CSF AAV-mediated gene transfer approach for the MPS-IIIA based on intrathecal delivery of AAV9 bearing a modified SGSH expression cassette (IDSspSGSH-IRES-SUMF1). Such expression cassette contained an SGSH bearing the signal peptide of IDS, which confers to the engineered enzyme the capability to be secreted at higher efficiency compared with the WT enzyme. Such modification resulted in improved biodistribution of the enzyme in the CNS of both small-animal (MPS-IIIA mice) and large-animal (WT pigs) models. Very recently, Chen et al. 12 reported that intra-CSF delivery of AAV4 encoding a highly secreted variant of SGSH mutated in the mannose-6-phosphate (M6P) binding site was therapeutically superior to the intra-CSF AAV4-mediated delivery system based on the use of the WT enzyme. Both our and Chen et al.'s 12 studies support the concept that enhancing SGSH secretion improves the distribution of enzyme in the CNS because of its enhanced cross-correction capability. Of note, whereas in the approach reported by Chen et al. 12 the mutation of the M6P binding site makes SGSH uptake M6P independent, in our approach the mannose-6-phosphate receptor (M6PR)-mediated system, which underlies the uptake in disease-relevant cellular targets such as neurons and astrocytes, is preserved. 8 Moreover, an important additional feature of the SGSH expression cassette used in our strategy is the insertion of the cDNA codifying for SUMF1, the enzyme responsible for the post-translational activation of sulfatases. Such modification synergistically acts together with enhanced secretion to further improve enzyme CNS biodistribution upon intra-CSF AAV9-mediated gene delivery. The finding that the additive effect of SUMF1 is not evident in the brain of pigs treated with WT enzyme ( Figure 1A; Table S1) can be explained by the fact that the brain amount of SUMF1 becomes limiting for SGSH activation only in a context where SGSH has an enhanced cross-correction capability. Remarkably, SGSH enzyme biodistribution in the CNS was more efficient in the pig than in the MPS-IIIA mouse model, likely due to the disease state and different administration routes used.
Importantly, we demonstrated that the modified SGSH expression cassette we developed is therapeutically effective, being able to efficiently rescue CNS and somatic storage pathology, and improve memory impairment.
Despite that a direct demonstration of the therapeutic superiority of the modified SGSH construct containing both IDSsp and SUMF1 by a side-by side comparative analysis has not been provided, our data clearly show that using a modified SGSH expression cassette may have a potential therapeutic advantage. It is remarkable that the performance of MPS-IIIA mice treated with IDSspSGSH-IRES-SUMF1 in the long-term memory task was undistinguishable from that of WT animals, thus suggesting that together with enhancing the biodistribution of the therapeutic enzyme, IDSspSGSH-IRES-SUMF1 treatment also allows to obtain maximal improvement in functional tests.
Intra-CSF AAV9-mediated gene delivery using WT SGSH has previously been successfully tested in different preclinical animal models using vector doses similar to those used in our approach (i.e., 4-5 Â 10 12 GCs/kg). 9 In that study the authors also showed that reducing AAV9 vector dosage in MPS-IIIA mice below 4-5 Â 10 12 GCs/kg led to inefficient enzyme biodistribution, thus resulting in only mild neuropathology improvement. Here, we demonstrated that intra-CSF delivery of AAV9 bearing the modified SGSH expression cassette at doses of~4-5 Â 10 12 GCs/kg led to a more efficient CNS enzyme biodistribution compared with WT SGSH both in small-and in large-animal models. Therefore, although we did not evaluate SGSH enzyme distribution at a dose below 4.5 Â 10 12 GCs/kg, our data suggested that by using our approach it might be possible to achieve and maintain therapeutic threshold levels of the enzyme throughout the CNS at reduced and, therefore, safer AAV9 vector dosages, thus making our approach potentially more attractive for clinical purposes.
Overall, the results presented here pave the way for developing gene delivery replacement protocols with enhanced therapeutic potential for the treatment of MPS-IIIA patients and also provide a proof of principle for potential use of this strategy for the treatment of other forms of LSDs caused by sulfatase deficiency.

Animals Pigs
The animals enrolled were postnatal day (P) 30 castrated male WT large White Â Duroc hybrids (Sus scrofa). These animals were transferred to our facility on the day of weaning (28th day after birth) and housed in multiple stalls with infrared heating lamps and monitored for general health. They were strictly monitored in order to rule out any pathology that may have affected the entire experiment. The study was conducted in accordance with the provisions of European Economic Community Council Directive 86/609 adopted by the Italian Government (DL 27/01/1992 No. 116) under the local approval of the Ethical Committee of the University of Bologna and under the approval of the Italian Ministry of Health.
Mice P60 homozygous mutant [B6Cg-Sgsh (mps3a/PstJ) mouse (Sgsh À/À ) from Jackson Laboratory; phenotypically MPS-IIIA affected] and healthy (Sgsh +/+ ) C57BL/6 mice were used. 1,27 Experiments were conducted in accordance with the guidelines of the Animal Care and Use Committee of Cardarelli Hospital in Naples and authorized by the Italian Ministry of Health.

AAV Administration Pigs
All of the activities performed on the day of the injection have been thoroughly described by Sorrentino et al. 20 For the intra cisterna magna (ICM) injection, the dorsal area of the neck was trimmed and surgically prepared, and the puncture of the cisterna magna was performed as previously described. 3,28 In brief, animals received an intramuscular (IM) bolus of tiletamine-zolazepam (5 mg/kg) 10 min before induction; general anesthesia was achieved using sevoflurane with an induction mask. After orotracheal intubation and stabilization, venous access for fluid therapy was achieved from an auricular vein. Blood samples (6 mL) were collected through the femoral artery.
The dose of 4.5 Â 10 12 GCs/kg of viral vector in the volume range from 1.5 to 2.8 mL was injected slowly to avoid a sudden increase in intracranial pressure. Piglets were then placed in Trendelenburg position for 2 min in order to help the injected compound to spread toward the more rostral parts of the CNS. Animals were then monitored until complete recovery. During the following days, all animals were strictly monitored in order to rule out any possible side effect of the procedure and to evaluate any changes in behavior and consequentially in welfare.

Mice
Mice were anesthetized by intraperitoneal (IP) injection of ketamine (100 mg/kg) and xylazine (10 mg/kg), and placed on a stereotaxic instrument with a motorized stereotaxic injector. A midline incision was made to expose the bregma. A hole in the skull was made by a drill (anteroposterior +2.18 mm, mediolateral 0.6 mm, dorsoventral À1.7 mm). Recombinant AAV9 (rAAV9) vectors, 5.4 Â 10 12 GCs/kg, were injected in a volume of 10 mL into the lateral ventricles at a rate of 1 mL/min. After allowing the needle to remain in place for 5 min, the needle was slowly raised at a rate of 0.1 cm/min.

Tissue Collection Pigs
Animals were euthanized 1 month after injection with a single bolus (0.3 mL/kg) of Tanax, and total body perfusion with Dulbecco's PBS was started. After median sternotomy, the right atrium was opened and the left ventricle was infused with 500 mL of warm Dulbecco's PBS (+38 C) and 1,000 mL of cold Dulbecco's PBS (+4 C); blood ejected from the right atrium was drained using a surgical aspirator. As far as CNS samples, collected tissues were the whole brain and cervical region of spinal cord. Dissection was performed using the technique previously described. 20 Twelve coronal sections (0.5 cm) were cut covering the main regions of the brain (right and left hemispheres) and the cervical region of the spinal cord. Sections were then frozen in liquid nitrogen for biochemical analysis or fixed in 4% (w/v) paraformaldehyde in PBS for OCT embedding.

Mice
Mice were euthanized at 1, 6, and 7 months by injection with ketamine and xylazine before blood and CSF collection. CSF was collected by glass capillary inserted into the cisterna magna. For tissue collection, mice were intracardially perfused with PBS (pH 7.4), and brains were removed, divided into halves, and fixed for further analysis. The right half was sliced in five slices (A-E) and frozen in liquid nitrogen; www.moleculartherapy.org the left half was sliced in two coronally anterior and posterior parts fixed in 4% (w/v) paraformaldehyde in PBS and embedded in paraffin. For toluidine blue staining analysis, a coronal slice derived from the left half of the brain was fixed in 4% paraformaldehyde, 25% glutaraldehyde in phosphate buffer for plastic embedding. Additionally, the left lateral lobe of the liver from the mice was fixed in 4% (w/v) paraformaldehyde in PBS and embedded in paraffin, and the right medial lobe of the liver of the mice was frozen in liquid nitrogen.

SGSH Activity
Twenty main regions covering the entire CNS of injected pigs were dissected and homogenized with Tissue Lyser using 10 vol of H2O Milli-Q (700 mL). In mice, five different slices covering the entire brain and liver samples were homogenized separately with Tissue Lyser using 8 vol of H2O mQ. The SGSH activity was assayed by a 4-methylumbelliferone-derived fluorogenic substrate (4-MU; Moscerdam Substrates), following established protocols. 4,8 Immunoassay of SGSH Immunoquantification of SGSH was performed in brain and liver samples of mice by using an ELISA technology. A total of 100 mL of anti-SGSH antibody R3074 from Shire (1 mg/mL) diluted in 0.05 M carbonate-bicarbonate buffer (pH 9.6; Sigma-Aldrich Japan, Tokyo, Japan) was used to coat multi-well plates at 37 C for 1 h. Afterward, samples or standards were incubated at 37 C for 1 h and then with horseradish peroxidase (HRP)-conjugated rabbit anti-HNS antibody R1315 (1:4,000) at 37 C for 1 h. The plate was then incubated with the HRP substrate, 3,3',5,5'-tetramethylbenzidine (TMB) peroxidase substrate (1721066; Bio-Rad, Hercules, CA, USA), for 30 min at 37 C. This enzyme-substrate reaction was stopped using a stop buffer (2N of H 2 SO 4 ), and the absorbance of each well was measured at the absorbance wavelength of 450 nm (Lm1) with a reference wavelength of 655 nm (Lm2) using a microplate reader (GloMax-Multi+ Detection System; Promega, Madison, WI, USA). The concentrations of HNS in samples were calculated using the HNS calibration curve in the same plate.

Evaluation of AAV Vector Genome Copy Number in the CNS
Genomic DNA was extracted from mouse brain samples using a DNeasy Blood and Tissue Extraction kit (QIAGEN, Valencia, CA, USA). DNA concentration was determined by using a NanoDrop. Real-time PCR was performed on 100 ng of genomic DNA using a LightCycler SYBR green I system (Roche, Almere, the Netherlands). Amplification was run on a LightCycler 96 device (Roche) with standard cycles. The primers forward SGSH (5 0 -CATCCACTTT GCCTTTCTCTCCA-3 0 ) and reverse SGSH (5 0 -TCAAAGCCTCCG TCATCCGC-3 0 ) were used. A standard curve was generated, using the corresponding AAV vector plasmid pAAV2.1CMV-IDSspSGSH-IRES-SUMF1.

GAG Quantification
Brain samples and liver samples were lysed in water by Tissue Lyser equipment. The lysates were then digested with Proteinase K, and extracts were clarified by centrifugation and filtration. GAG levels in brain extracts were determined using the Blyscan sulfated glycosaminoglycan kit (Biocolor, Carrickfergus, UK) with chondroitin 4-sulfate as the standard.

Immunohistochemistry
IHC experiments were performed in 5-mm paraffin sections with BondRX Stainer following the standard protocol. The primary antibodies used were mouse anti-Human SGSH (Shire 2C7) 1:25 for immunofluorescence and 1:500 for IHC, rabbit anti-LAMP1 (ab24170; Abcam), rabbit anti-GFAP (ab7260; Abcam), and rabbit anti-NeuN (ab177487). Five-micrometer paraffin-embedded tissue sections were incubated overnight at 4 C with rabbit anti-GFAP (Z0334; Dako Cytomation). The detection system including secondary antibodies used for IHC was the Bond Polymer refine kit (DS9800; Leica) for the visualization of SGSH and LAMP1 signals. The secondary antibody used for the detection of GFAP signal was biotinylated universal antibody of Vectastain ABC kit (Vector Laboratories, Burlingame, CA, USA).
Bright-field sections were stained with 3,3-diaminobenzidine (Sigma-Aldrich) and counterstained with hematoxylin. The SGSH-and LAMP1-stained slides were scanned with Aperio ScanScope AT2 scanner. The whole digital slides were viewed and analyzed by Image-Scope. The positive pixel count algorithm was selected and adjusted to cover each individual positive staining of LAMP1 for analysis. The data were presented as positivity, which was obtained from the following formula: Positivity (%) = positive area (pixels)/total analyzed area (pixels) Â 100%.
The GFAP-stained slides were scanned with Hamamatsu Nanozoomer 2.0-RS scanner and viewed with NDP.view2. The total number of GFAP-positive signals was counted using the cell-counter program (ImageJ software) with a fixed threshold.
For immunofluorescence, secondary antibodies Donkey anti-mouse immunoglobulin G (IgG)-Alexa Fluor 488 (R37114; Thermo Fisher) and Goat anti-rabbit IgG-Alexa Fluor 568 (A-11011; Thermo Fisher) were used for the visualization. Stained slides were read and representative photos were taken with Nikon fluorescent microscope.

Toluidine Blue Staining
Fixed samples of specific brain regions were post-fixed in 1% osmium tetroxide, dehydrated, and embedded in resin. One-micrometer sections were stained with 1% toluidine blue and examined by light microscopy. Ultra-thin sections from the selected region of cortex were cut and stained with 0.5% uranyl acetate.

Behavioral Tests
Behavioral tests were carried out in a behavioral testing room maintained under constant light, temperature, and humidity. The mice were tested during daylight hours (between 9 a.m. and 6 p.m.). Before testing, animals were habituated to the testing room for at least 30 min. The same groups of animals were tested at 6 and 9 months of age. We performed the open field task, which in previous studies we have found to be impaired in adult MPS-IIIA mice. Additionally, we tested them in the contextual fear conditioning task, which allows to evaluate animal ability to learn and remember a Pavlovian association between a mild food shock and a specific context.

Open Field Task
The open field task was performed as previously described. 4,8 Mice were placed in the middle of a Plexiglas arena with a masonite base (43 Â 32 Â 40 cm). Animals were left free to explore the device for 10 min. The distance traveled (m), the immobility time (s), and line crossing were recorded using a video camera (WV-BP330; Panasonic) hanging over the arena that was connected to a video-tracking system (ANY-maze; Stoelting, Chicago, IL, USA).

Contextual Fear Conditioning
Each mouse was trained in a conditioning chamber (30 cm Â 24 cm Â 21 cm; Ugo Basile) that had a removable grid floor and waste pan. The grid floor contained 36 stainless steel rods (3-mm diameter) spaced 8 mm center to center. When placed in the chamber, the grid floor contacted a circuit board through which scrambled shock was delivered. The shock intensity was 0.5 mA with a duration of 2 s, and it was presented three times and was associated to a context. After 24 h after training, mice were tested without foot shock but with the same context. Freezing behavior was defined as complete lack of movement, except for respiration, and scored with a videotracking system (ANY-maze; Stoelting, USA).

Statistical Analysis
Data of behavioral tests were expressed as mean ± SEM. Two-way ANOVA for repeated measures with the factor group as independent factor and trials or test phase as repeated-measures was performed to assess significance among multiple experimental groups and at different time points, followed by Duncan post hoc test when appropriate. A p value <0.05 was considered as statistically significant.
For the enzymatic activity, ELISA, vector copy number, and GAG analysis, the data were expressed as mean ± SEM. One-way comparisons were performed to calculate the significance among the experimental groups, followed by Tukey's post hoc test. A p value <0.05 was considered as statistically significant.

AUTHOR CONTRIBUTIONS
N.C.S. co-supervised and performed research, analyzed data, and contributed to the writing of the entire manuscript. V.C., V.M., S.S., and N.T. performed research. E.N. maintained the mouse colony from TIGEM. N.R. and D.V. contributed with pig studies. Y.H. and N.L. conducted tissue sectioning, immunohistochemistry IHC, and data analysis from Shire. S.L.K. and V.W.C. supervised project direction, study designs, and data interpretation from Shire. M.D.R. performed and analyzed behavioral data. E.D.L. conceived the behavioral studies, contributed to the analysis of behavioral data, supervised behavioral studies, and contributed to the writing of the manuscript (behavioral results). A.F. designed and supervised research, and wrote the manuscript.

CONFLICTS OF INTEREST
Y.H., N.L., S.L.K., and V.W.C. are employees and stockholders of Shire. The other authors declare no competing interests.