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Human Molecular Genetics Advance Access originally published online on June 16, 2005
Human Molecular Genetics 2005 14(15):2113-2123; doi:10.1093/hmg/ddi216
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© The Author 2005. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oupjournals.org

A direct gene transfer strategy via brain internal capsule reverses the biochemical defect in Tay–Sachs disease

S. Martino1,{dagger}, P. Marconi2,{dagger}, B. Tancini1,{ddagger}, D. Dolcetta3,{ddagger}, M.G. Cusella De Angelis4,{ddagger}, P. Montanucci1, G. Bregola5, K. Sandhoff6, C. Bordignon3, C. Emiliani1, R. Manservigi2 and A. Orlacchio1,*

1Dipartimento di Medicina Sperimentale e Scienze Biochimiche, Sezione di Biochimica e Biologia Molecolare University of Perugia, Via del Giochetto, 06126 Perugia, Italy, 2Dipartimento di Medicina Sperimentale e Diagnostica, Sezione di Microbiologia, University of Ferrara, Ferrara, Italy, 3San Raffaele Telethon Institute for Gene Therapy (HSR-TIGET), Milano, Italy, 4Dipartimento di Medicina Sperimentale, University of Pavia, Pavia, Italy, 5Dipartimento di Medicina Clinica e Sperimentale, Sezione di Farmacologia, University of Ferrara, Ferrara, Italy and 6Kekulè-Institut f. Organische Chemie und Biochemie, University of Bonn, Bonn, Germany

* To whom correspondence should be addressed. Tel: +39 755852187; Fax: +39 755852185/7443; Email: orly{at}unipg.it

Received March 2, 2005; Revised May 4, 2005; Accepted June 10, 2005


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Therapy for neurodegenerative lysosomal Tay–Sachs (TS) disease requires active hexosaminidase (Hex) A production in the central nervous system and an efficient therapeutic approach that can act faster than human disease progression. We combined the efficacy of a non-replicating Herpes simplex vector encoding for the Hex A alpha-subunit (HSV-T0alphaHex) and the anatomic structure of the brain internal capsule to distribute the missing enzyme optimally. With this gene transfer strategy, for the first time, we re-established the Hex A activity and totally removed the GM2 ganglioside storage in both injected and controlateral hemispheres, in the cerebellum and spinal cord of TS animal model in the span of one month's treatment. In our studies, no adverse effects were observed due to the viral vector, injection site or gene expression and on the basis of these results, we feel confident that the same approach could be applied to similar diseases involving an enzyme defect.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
RESULTS
 DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Tay–Sachs (TS) disease is a GM2 gangliosidosis due to the deficiency of the {alpha}-subunit of ß-hexosaminidase A (1Go,2Go) (HEXA gene). In the most severe and typical clinical form, rapid mental and motor deterioration, starting within the first year of life, leads to death within 2–3 years. Pathological features include wide neurodegeneration and neuronal lipid storages. Currently, TS treatment is restricted to supportive care and appropriate management of intervening problems (3Go).

In mammalian tissues, ß-hexosaminidase (Hex, EC 3.2.1.52 [EC] ) exists in two major forms: Hex A ({alpha}ß structure) and Hex B (ßß structure) (1Go,2Go). The homodimer {alpha}{alpha}, Hex S, represents the residual Hex activity in Sandhoff disease patients, a type 0 GM2 gangliosidosis due to inherited defects in the HEXB gene (4Go–7Go). Only Hex A, in the presence of the GM2-activator protein, hydrolyses the ß-GalNAc-(1-4)-ß-Gal glycosidic linkage of the GM2 ganglioside effectively (1Go,7Go–10Go).

Therapy for lysosomal storage disorders with neurological involvement such as TS disease requires production and distribution of the missing enzyme in the central nervous system (CNS). Several therapeutic approaches have succeeded in restoring the enzymatic activity to many key tissues (kidney, liver, spleen, etc.) but reduction of GM2 ganglioside deposits in the CNS is difficult to achieve (11Go–16Go). In fact, the blood–brain barrier represents a general obstacle to therapy (13Go,17Go,18Go). A further obstacle stems from our observation that the fibroblasts from TS patients are not cross-corrected in vitro by simple administration of the missing enzyme (19Go). Moreover, because pre-clinical diagnosis of TS is both a fortunate and a sporadic event, a therapeutic approach could be effective if it is able to delay the acute phase of the disease. Therefore, restoration of Hex A activity and reduction of GM2 ganglioside storage are primary goals.

Here, we propose an in vivo gene transfer strategy for the production and distribution of the HEXA gene in the CNS of a TS animal model (20Go). We used a non-replicating Herpes simplex viral vector because HSV-1 has the ability to infect a wide variety of cell types in the non-replicating phase, e.g. neurons, as well as the intrinsic capacity to be transported in a retrograde manner to motor and/or sensory neuronal cell bodies following peripheral inoculation (21Go–23Go). We identified the internal capsule as a suitable site of injection. This is a connecting structure mainly involved in motor pathways, which are highly impaired in human TS, which is also affected in amyotrophic lateral sclerosis (24Go–26Go), and in other genetic neurodegenerative diseases (27Go).

Our data clearly show that the combination of the internal capsule anatomic features, together with the described viral properties, allows a wide distribution of the Hex A activity and the disappearance of the GM2 ganglioside storage in both injected and controlateral brain hemispheres. Interestingly, these results were also obtained for the first time in the cerebellum and in the spinal cord suggesting that this approach is effective in the retardation of the progression of the disease. In our studies, we did not observe adverse effects due to the viral vector, injection site or gene expression and on the basis of the results that we have obtained, we feel confident that the same approach could be applied to similar diseases involving an enzyme defect.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
DISCUSSION
 MATERIALS AND METHODS
 REFERENCES
 
Construction of herpes simplex viral vector encoding for Hex {alpha}-subunit cDNA
We produced the non-replicating herpes simplex viral vector containing the human Hex {alpha}-subunit cDNA under the control of the ICP0 promoter (28Go,29Go) (HSV-T0{alpha}Hex) (Fig. 1A). ICP0 promoter, which is an immediate early viral promoter, was up-regulated to extremely high levels in a background of a triple mutant replication-deficient vector. The expression cassette, containing the cDNA surrounded by UL41 flanking sequences of HSV, was recombined to UL41 locus of the T0Z viral vector (ICP4–, 27–, 22–, UL41–/LacZ) (Fig. 1A). We evaluated the insertion of the Hex {alpha}-subunit transgene into the viral DNA by Southern blot (data not shown). We used HSV-T0{alpha}Hex (therapeutic viral vector) and HSV-T0Z (control viral vector) to infect the cells of the CNS of TS mice.



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  		Figure 1. Production and validation of recombinant HSV-1 non-replicating viral vector containing Hex-{alpha}-subunit cDNA. (A) Recombinant HSV-1 non-replicating viral vector was produced as described in Materials and Methods; {alpha}-subunit Hex cDNA or ß-Galactosidase cDNA (LacZ) were expressed under the ICP0 promoter. (B) Hex activity was assayed towards the synthetic substrate MUGS in transduced organotypic brain slices. One unit (U) is the amount of enzyme that hydrolyses 1 µmol/min of substrate at 37°C. {ddi21606} TS, Tay–Sachs mice; {ddi21607} TS-T0{alpha}Hex, Tay–Sachs mice transduced with the HSV-T0{alpha}Hex; {blacksquare} WT, C57/Bl6 mice; {ddi21608} TS-T0Z, Tay–Sachs mice transduced with the HSV-T0Z.

 
Validation of HSV-T0{alpha}Hex viral vector by transduction of TS organotypic brain slices
To test the efficacy of HSV-T0{alpha}Hex viral vector, we transduced organotypic brain slices from TS mice either with the therapeutic or with the control vector, as described in Materials and Methods. We evaluated the Hex specific activity in slice extracts using the fluorogenic substrate MUGS, which is only hydrolysed by Hex isoenzymes containing the {alpha}-subunit. Three days TS organotypic brain slices transduced with the therapeutic viral vector displayed MUGS activity comparable with that found in wild-type slices (Fig. 1B). No MUGS activity was detected either in the untransduced slices or in the slices transduced with control vector from TS mice (Fig. 1B).

In vivo gene transfer strategy for TS disease
We injected the vector into the internal capsule of the left-brain hemisphere (coordinates: –0.34 mm to bregma, 1.4 mm mediolateral and 3.8 mm depth). We designed an experimental plan comprised of five groups of 5-month-old TS mice. Group 1: TS+HSV-T0{alpha}Hex, 2.5x106 total p.f.u.; group 2: TS+HSV-T0{alpha}Hex, 5x106 total p.f.u.; group 3: TS+HSV-T0Z, 5x106 total p.f.u.; group 4: untreated TS mice; group 5: WT mice.

Therapeutic effect of the HSV-T0{alpha}Hex viral vector in treated TS mice
Four weeks after injection, we sacrificed the mice, separated the brain hemispheres, the cerebellum and the spinal cord. We dissected each brain hemisphere into four rostro-caudal 2.5 mm thick sections and the spinal cord into three antero-posterior segments. We analyzed each brain section, as well as the cerebellum and spinal cord, for Hex A activity and for GM2 ganglioside content.

Brain hemisphere analysis
We evaluated the Hex specific activity in the brain section using the fluorogenic substrate MUGS. Figure 2A presents the Hex specific activity toward the MUGS substrate in each brain section of both treated and untreated animals. All TS mice injected with the therapeutic viral vector displayed MUGS activity in the range of normal WT. The injection of TS mice with the highest p.f.u. resulted in the highest increase of the MUGS specific activity. We detected no MUGS activity either in TS mice brain slices infected with the control viral vector or in untreated TS mice. Interestingly, MUGS activity was restored both in the inoculated hemisphere and in the controlateral one.



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  		Figure 2. Hex A activity in TS mice brain transduced with HSV-{alpha}Hex viral vector. Hex activity was assayed towards the synthetic substrate MUGS in brain section extracts (A) and cerebellum (B); L1, L2, L3, L4 and R1, R2, R3, R4 are the left and right brain hemisphere sections, respectively. {ddi21606} Tay–Sachs mice; {ddi21609} Tay–Sachs mice injected with the lower HSV-T0{alpha}Hex p.f.u.; {ddi21607} Tay–Sachs mice injected with the highest HSV-T0{alpha}Hex p.f.u.; {blacksquare} WT, C57/Bl6 mice; {ddi21608} Tay–Sachs mice infected with the HSV-T0Z. (C) DEAE-cellulose chromatography analysis of Hex A in a representative injected mouse brain hemisphere sections TS, Tay–Sachs mice; TS-T0{alpha}Hex*, Tay–Sachs mice injected with the lower HSV-T0{alpha}Hex p.f.u.; TS-T0{alpha}Hex**, Tay–Sachs mice injected with the highest HSV-T0{alpha}Hex p.f.u.; WT, C57/Bl6 mice. Fractions (1 ml) were collected and assayed for Hex activity towards the two substrates MUG (•) and MUGS ({circ}).

 
We verified the correct formation of Hex A isoenzyme in the brain of treated mice by combining the chromatographic analysis on DEAE-cellulose with a specific enzymatic assay employing two different substrates, MUG, hydrolyzed by both {alpha}- and ß-subunits, and MUGS, hydrolyzed only by {alpha}-subunit of hexosaminidases yielding information on the subunit composition of the formed Hex isoenzymes. Under our experimental conditions, Hex B, unretained by the column, was simply eluted with void volume, whereas Hex A and Hex S were eluted by a linear gradient of NaCl (6Go,19Go,30Go). Figure 2C provides a representative Hex isoenzyme pattern of the brain sections of all the mice. Murine TS brain sections, transduced with HSV-T0{alpha}Hex viral vector doses (2.5x106 p.f.u. and 5x106 p.f.u.) have Hex A and a Hex isoenzyme pattern comparable with that found in WT mice (Fig. 2C). All transduced brain sections displayed similar Hex isoenzyme patterns demonstrating the restoration of the enzymatic defect throughout the brain hemisphere. Chromatography also showed the restoration of Hex A isoenzyme in the controlateral TS hemisphere (data not shown).

We analyzed each hemisphere section for the GM2 ganglioside content with thin layer chromatography (TLC). A representative ganglioside chromatographic pattern of treated and untreated brain sections is reported (Fig. 3A).



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  		Figure 3. Decrease of GM2 ganglioside in TS brain sections transduced with HSV-T0{alpha}Hex viral vector. Gangliosides TLC pattern of a representative injected mouse brain hemisphere sections (A) and cerebellum (B) of treated and untreated mice. Lines: std, Ganglioside Standards from bovine brain supplemented with GM2 and GM3; GM2, pure GM2; TS-T0{alpha}Hex*, Tay–Sachs mice injected with the lowest HSV-T0{alpha}Hex p.f.u.; TS-T0{alpha}Hex**, Tay–Sachs mice injected with the highest HSV-T0{alpha}Hex p.f.u.; WT, C57/Bl6 mice; TS, Tay–Sachs mice. (C) Densitometric 1D Image Analysis Software of the GM2 ganglioside content in hemisphere brain sections and cerebellum. {ddi21609} Tay–Sachs mice injected with the lower dose of HSV-T0{alpha}Hex p.f.u., {ddi21606} Tay–Sachs mice injected with the higher HSV-T0{alpha}Hex p.f.u. dose. L1, L2, L3, L4 and R1, R2, R3, R4 are the left and right brain hemisphere sections, respectively; C is the cerebellum. Results are expressed as percentage of removed GM2 ganglioside when compared with the GM2 content of the corresponding TS brain section. Data are representative of three independent experiments.

 
No variation in the ganglioside pattern was detected in the brain hemispheres of TS mice treated with the higher dose of HSV-T0Z control vector (data not shown). Densitometric analysis of the TLC by 1D Image analysis Software of all the mice investigated demonstrated the disappearance of the accumulated GM2 ganglioside in TS mice treated with the higher dose of HSV-T0{alpha}Hex viral vector and a decrease of the stored ganglioside in animals treated with the lower dose of the therapeutic viral vector (Fig. 3C and D). These data were observed in all brain sections (both the injected and the controlateral hemispheres) and suggest a dose-dependent gene transfer reversion of the altered phenotype. Moreover, these data are consistent with the distribution of the Hex A activity.

Cerebellum analysis
We also found the presence of MUGS activity (Fig. 2B) and Hex A isoenzymes (data not shown) in the cerebellum of TS mice treated with the therapeutic viral vector. As for the brain hemispheres, the GM2 ganglioside storage disappeared in the cerebellum of TS mice treated with the higher dose of the HSV-T0{alpha}Hex viral vector and it was partially removed in TS mice treated with the lower dose of the same vector (Fig. 3B and C).

Spinal cord analysis
No clear restoration of the Hex A activity was detected in the spinal cord probably because the infected cells (Fig. 5C) are distributed along the whole spine and the total number of these cells is rather low.



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  		Figure 5. HSV-T0Z distribution in the mouse CNS: relevant areas. High magnification of significant brain area (A and B) and representative spinal cord sections (C). (A) A and A1: tela choroidea of third ventricule, thalamus (pulvinar); B and B1: anterior horn of the lateral ventricule; C and C1: corpus callosum, tela chorioidea of third ventricule, thalamus; D and D1: Fourth ventricle and cerebellar vermis; E and E1: genu of the internal capsule; FF1: cerebellar peduncle, ependimal channel, pyramis. (B) A and A1: thalamus, internal capsule, posterior thalamic radiation; B and B1: white anterior commissure, C and C1: corpus callosum; D and D1: putamen, nucleo caudato, anterior arm internal capsule; E and E1: third ventricle, globus pallidum, internal capsule; F and F1: mid brain; G and G1: hippocampus, controlateral hemisphere; H and H1: fimbria, controlateral hemisphere. (C) Typical spine transversal section. A: Spinal cord is surrounded by muscoloskeletal apparatus (40x, dark field). Representative sections of spinal cord at the cervical level (C3 in B and B1, C7 in C and C1) or thoracic (T3 in D and D1, T8 E and E1); structures reacting with the X-Gal substrate are shown at 40x (B, C, D, E) and 200x (B1, C1, D1, E1) magnification.

 
Distribution of the HSV-T0Z viral vector in treated TS mice
We analyzed the diffusion of the vector in the brain after treating TS mice with a number of p.f.u. of the HSV-T0Z viral vector corresponding to the higher dose of the therapeutic viral vector used. Animals were sacrificed after 72 h and 1 month.

We followed the viral vector distribution by monitoring the X-Gal staining in coronal (Fig. 4A), transversal (Fig. 4B) and sagittal (Fig. 4C) brain serial sections. The results demonstrate a wide viral vector spreading in both the injected and uninjected hemispheres. We observed blue cells in all the brain areas. The signal started from second section on glass A2 and extended through all sections collected on glass A14, this means that signals lasted for 6 mm in coronal sectioning (Materials and Methods). Interestingly, brain sections showed blue cells in the brain internal capsule and encephalic trunk, which morphologically were undoubtedly of neuronal identity. Positive cells were also located in the cerebellum and controlateral hemisphere (Figs 4C and 5A). The presence of X-Gal staining in the ependimal layer of lateral ventricles and third and fourth ventricles, Silvius aqueductus, indicated that a small part of the vector also spread from the site of injection to other brain areas (Fig. 5B). We also observed blue cells in the spinal cord. In Fig. 5C, we report representative X-Gal staining of spinal cord transversal sections of treated TS mice. We observed blue cells in the encephalic trunk; moreover, a few cells (from 12 to 20) were also identified in the distal part of the spinal cord (T8 level the farthest) (Fig. 5C).




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  		Figure 4. HSV-T0Z distribution in the mouse CNS. Serial brain sections were produced dissecting animals in coronal, transversal and sagittal orientation. Sections were stained with the X-Gal substrate. (A) Coronal brain sections: a1–a8, represent coronal serial sections; a4: site of injection (IC, internal capsule). (B) Transversal brain sections: b1–b7, represent transversal serial sections (C) Sagittal brain sections: c1–c8, represent sagittal serial sections. DF, dark field; BF, bright field.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
MATERIALS AND METHODS
 REFERENCES
 
In this study, we delineate a new therapeutic approach for the genetic lysosomal storage disorder TS disease. Bearing in mind the rapid neurodegeneration and the high lethality of this disease, we propose a plan able to delay the acute phase of the disease and to enhance the emergency service gap. We focused our attention on the rapid restoration of hexosaminidase A activity and the reduction of GM2 ganglioside storage in the CNS because these represent the principle cause of this metabolic alteration. We hypothesize that reduction/prevention of the neural deposits obtained by direct delivery of the missing enzyme into the CNS could slow down the clinical features of TS.

We designed a strategy which combines the potentiality of HSV-1 non-replicating viral vector and the anatomic structure of a chosen injection site to allow wide distribution of Hex A into the CNS.

We delivered a non-replicating herpes-derived viral vector encoding for the Hex A {alpha}-subunit cDNA into the brain internal capsule of TS mice to transduce the CNS cells in vivo. This injection site seemed particularly suitable because it contains numerous fiber bundles which make it possible to reach several CNS areas with a single vector administration. Pyramidal tract connects the frontal motor cortex to the midbrain, spinal cord and cerebellum, whereas the ‘higher thalamic peduncle’ connects the cortex to the thalamus. Other ‘minor’ components connect capsular fibers to the parietal, occipital and temporal cortex (31Go). We combined anatomical features of the internal capsule with properties of the herpetic vectors: (i) intrinsic capacity to be transported in a retrograde manner to neuronal cell bodies; (ii) ability to infect a wide variety of cell types in the non-replicating phase, especially neurons; (iii) ability to allow a sustained transgene expression, because the transgene is under the control of ICP0 promoter (21Go,22Go,32Go–34Go), which is a target for activation by neuronal transcription factors in neurons that have undergone damage (34Go). These features may provide this HSV promoter with the ability to enhance transgene expression under pathological conditions.

All of these HSV features, along with the chosen site of injection, have allowed a long and strong transgene expression and a wide spreading of the missing enzyme into the brain after a single injection (Figs 2–5).

We evaluated the efficacy of our strategy by injecting the animal model of TS disease (20Go) with HSV-T0{alpha}Hex. We used 5-months-old TS mice because they clearly present GM2 ganglioside storage in the brain. In contrast to human patients, due to the hydrolysis of GM2 ganglioside to GA2 by a murine sialidase, TS mice express a mild form of the disease (35Go,36Go). They do not show neurologic symptoms; however, they are lacking in Hex A activity (Fig. 2) and present a mild storage of GM2 ganglioside in the brain (Fig. 3). Therefore, these mice represent a suitable experimental model for testing the effectiveness of our approach on slowing down the progression of the disease.

We found a decrease in GM2 ganglioside storage in the brain of all TS mice treated with the therapeutic vector, which was consistent with the viral vector administration dose. One month after injection, we observed the absence of the GM2 ganglioside storage only in TS mice injected with the higher dose of HSV-T0{alpha}Hex (Fig. 3). We assume that results obtained with the two different viral doses are due to the different amounts of enzyme production. In fact, TS mice inoculated with the lower dose of HSV-T0{alpha}Hex only had 40% of MUGS activity levels when compared with normal WT mice, whereas the MUGS activity produced in the animals treated with the higher dose of HSV-T0{alpha}Hex was 60–70% when compared with the WT value. The lower level of Hex A activity was insufficient to hydrolyze all the stored GM2 ganglioside. We suspect that the lower dose did not transfect enough cells, suggesting more time is required to remove the storage material completely.

All treated TS mice showed the depletion of GM2 ganglioside in both injected and non-injected brain hemispheres and in the cerebellum, thus indicating a wide diffusion of the viral construct and/or the therapeutic enzyme. Interestingly, our gene transfer strategy restores the enzymatic activity in TS mice to a range of values comparable with that observed in the WT mice.

We found the presence of the HSV-T0Z control vector in both injected and non-injected hemispheres (Figs 4 and 5). More importantly, the distribution of X-Gal positive brain cells indicated that the viral vector distribution mimics the internal capsule architecture (e.g. positive blue cells within the thalumus, corpus callosum and optic chiasma) (31Go). Moreover, the presence of X-Gal stained cells in the ventricular regions (lateral ventricle, third ventricle and Silvius aqueduct) (Fig. 5B) could indicate that the diffusion of the therapeutic viral vector into the controlateral hemisphere is, in part, via cerebro-spinal fluid flow (37Go,38Go). Cellular phenotype suggested that viral vector transduced to most neurones and astroglial cells in the injection site. However, some positive cells far from the injection site showed a similar neuronal phenotype (Fig. 5A), whereas some are ependymal cells (Fig. 5B).

Interestingly, few X-Gal positive cells were present in the cervical and thoracic tract of the spinal cord (Fig. 5C). This phenomenon strongly reveals the potentiality of this site of injection for the distribution of the transgene throughout the CNS.

According to the vector distribution, Hex A activity was restored in all treated TS mouse brain areas analyzed (Figs 2, 4 and 5). However, we cannot exclude that some secreted enzyme from transduced cells could be taken up by neighboring cells in vivo contributing to the diffusion of the recombinant enzyme into the different brain areas. This suggestion is particularly attractive in that we have previously demonstrated, in vitro, the absence of cross-correction in TS fibroblasts (19Go). Secreted recombinant Hex A was able to hydrolyze the GM2 ganglioside to GM3 in an in vitro assay, but it did not correct the metabolic defect of TS fibroblasts. It would appear that a specific neural endocytic pathway of the enzyme may occur in vivo. A previous study by Consiglio et al. (39Go) demonstrated the presence and the distribution of the recombinant arylsulfatase A in all tested brain areas and suggested that the enzyme is transported from the site of injection to the other hemisphere through cerebral commissures. According to these data, Passini et al. (40Go) demonstrated the presence of the ß-glucuronidase in the hippocampus area of controlateral hemisphere in MPSVII animals treated with an adeno-associated viral vector encoding for the missing enzyme.

Our data are novel because they present the first evidence of the distribution of a therapeutic viral vector throughout the entire CNS and suggest that the anatomic structure of the brain may be a useful tool in therapy for genetic neurodegenerative disorders. In our studies, we did not observe adverse effects due to the viral vector, injection site or gene expression and on the basis of the results that we have obtained, we feel confident in expressing the opinion that the same approach could be applied to similar diseases involving an enzyme defect.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 RESULTS
 DISCUSSION
 MATERIALS AND METHODS
REFERENCES
 
Materials
The animal model of TS disease was generated by Yamanaka et al. (20Go) and bred in our laboratory. C57/bl6 mice were from Charles-River, Italy. The fluorogenic substrates 4-methylumbelliferyl derivative of ß-N-acetylglucosaminide (MUG), of ß-N-acetylglucosamine-6-sulfate (MUGS) and of 4-methyl-umbelliferone, 5-bromo-4-chloro-2-indolyl-ß–D-galactopiranoside (X-Gal), NP-40, agarose gel for electrophoresis, the 2,2,2-tribromoethanol, the 2-methyl-2-butanol, GM2, GM3, and gangliosides mix type III from bovine brain, resorcinol, were from Sigma Chemical Co. Bovine seric albumin and Bio-Rad protein assay reagents were from Bio-Rad Laboratories, DE-52 DEAE-cellulose was from Whatman Biochemicals. Thin-layer 20x20 CM plates TLC, were from E. Merck A.G. (Darmstadt, Germany). The medium for tissue culture was from Euro-Clone, Celbio Laboratory, fetal calf serum was from Mascia Brunelli, penicillin/streptomycin was from Gibco BRL. All other reagents were of analytical grade.

Construction of a suitable herpes vector for Hex {alpha}-subunit cDNA transfer and transduction
The non-replicating HSV-1 viral vector, which expresses the {alpha}-subunit cDNA of Hex A, was created using the PacI recombination system according to the methods described by Krisky et al. (28Go). The {alpha}-subunit cDNA of Hex A was cloned under the transcriptional control of ICP0 promoter. The expression cassette, containing the cDNA surrounded by UL41 flanking sequences of HSV, was recombined into UL41 locus of T0Z viral vector (ICP4–, 27–, 22–, UL41–/LacZ). T0Z vector derived from the replication-deficient T.1, defective for ICP4, ICP27, ICP22 immediate early genes and for UL41 gene where the ß-Galactosidase cDNA under the control of the ICP0 IE promoter and flanked by PacI restriction sites (which are not present in the viral genome), was inserted into the vhs (UL41) locus. Potential recombinant virus (T0{alpha}Hex) was identified by a ‘clear plaque’ phenotype following X-Gal staining because the insertion of the transgene construct into the viral genome eliminated the LacZ gene. The T0{alpha}Hex recombinant virus was purified by three rounds of limiting dilution and verified by Southern blot analysis for the presence of the {alpha}-subunit cDNA of Hex A and for the correct insertion of the named gene in the viral genome.

TS organotypic brain slice preparation and transduction with the HSV-T0{alpha}Hex
The organotypic brain slices were produced and cultured under standard conditions as defined in Malgaroli's laboratories. For each experiment, at least three animals and 18 slices were used. After decapitation, 5-month-old TS and wild-type mice (C57/bl6) brains were dissected out into cold Gey's balanced salt solution containing 5 mg/ml glucose. Brain coronal slices (500 µm thick) were cut on a McIlwain tissue chopper and transferred into membranes of 30 mm Millipore culture inserts with 0.45 µm pore size (Millicell; Millipore, Bedford, MA, USA). Slices were maintained in culture in six-well plates containing 1 ml of medium at 37°C in an atmosphere of humidified 5% CO2. The medium was comprised of 50% basal medium with Earle's salts (Invitrogen, Gaithersburg, MD, USA), 25% HBSS (Invitrogen), 25% horse serum (Invitrogen), L-glutamine (1 mM) and 5 mg/ml glucose.

After 24 h, the medium was changed with new fresh medium and 1 µl either of HSV-T0{alpha}Hex (p.f.u.=1x106) or of HSV-T0Z (p.f.u.=1x106) was delivered as a drop on the top of each organotypic slice. Slices were then maintained in culture and collected at different time points (third, seventh, 14th days). Slices were homogenized in 10 mM Na/phosphate buffer pH 6.0 for the determination of the Hex activity.

T0{alpha}Hex direct injection into the brain internal capsule of TS mice
Five groups of 5-month-old animals were injected with two different doses of T0{alpha}Hex into the internal capsule of the left-brain hemisphere of the TS mice. Mice were anesthetized with 0.02 ml/g body weight of 2,2,2-tribromoethanol and 2-methyl-2-butanol and placed on the Styrofoam platform of a stereotaxic injection apparatus (David Kopf Instruments, Tujunga, CA, USA). The skull was exposed following a 10 mm incision in the midline. The injection coordinates for the internal capsule were –0.34 mm to bregma, 1.4 mm mediolateral and 3.8 mm depth. These coordinates were chosen in order to minimize vector leakage into the ventricular space. Each injection was 5 µl total, and the injection speed was 0.1 µl/min. The injections were carried out using a needle capillary (1.2 mmx0.6 mm) attached to a Hamilton syringe. The injections were delivered at a rate of 0.1 µl/min, and the needle was slowly withdrawn after an additional 5 min. The scalp was closed by suture.

HSV-T0Z viral vector distribution
One month after injection, some mice were sacrificed by cardiac perfusion. The left ventricle was cannulated, an incision was made in the right atrium and the animals were perfused with 2% paraformaldehyde in PBS until the outflow ran clear then the brain was included in ornithyne carbamoyl transferase (OCT TM Compound TISSUE-TEK, Sakamura, The Netherlands) after exposure at 5–30% glucose gradient and finally sectioned on a cryostat into 15 µm thick serial sections. ß-Gal positive cells were assayed through X-Gal staining (41Go) to develop ß-Gal positive cells. Animal experimentation protocols were approved by the HSR Institutional Animal Care. We collected brain serial sections in four series of glasses (A–B–C–D) so that: section no. 1 on glass B3 was collected immediately after section no. 1 on glass A3 and immediately before section no. 1 on glass C3. Thus, staining only one-fourth of the sections (A), we checked the beta-gal staining distribution (one section every 60 µm) along the whole brain extension.

After perfusion, whole spines were removed, and after decalcification in 3% Trifluoracetic acid (Merck), spinal cords, surrounded by vertebrae and remains of skeletal muscles, were cut into blocks containing a known number of vertebrae (four or five). Each block was sectioned on a cryostat into 10 µm serial sections. Beta-gal distribution was checked along the whole spinal cord column.

Brain extract
Some mice were decapitated and the brain and cerebellum collected. Brain hemispheres were dissected in four rostro-caudal 2.5 mm sections and cerebellum. Organotypic brain slices and brain sections were homogenized in a potter Elvehjem type homogenizer in 10 mM sodium phosphate buffer, pH 6.0, containing 0.1% (v/v) NP-40 detergent and sonicated. The lysates were centrifuged at 12 000 r.p.m. Eppendorf microfuge for 20 min and supernatants used as tissue extracts for enzyme analysis. All procedures were carried out at 4°C.

ß-Hexosaminidase activity assay
Enzyme activity was determined using two fluorogenic substrates: 3 mM MUG or MUGS in 0.1 M citrate/0.2 M disodium phosphate buffer at pH 4.5 (6Go,19Go,31Go). Fluorescence of the liberated 4-methylumbelliferone was measured on a Perkin Elmer LS3 fluorimeter (excitation 360 nm, emission 446 nm).

ß-Hexosaminidase isoenzymes analysis
Tissue extracts were analyzed with ionic-exchange chromatography on DEAE-cellulose (6Go,17Go,30Go). The chromatography was performed using a 1 ml column equilibrated with 10 mM Na-phosphate buffer, pH 6.0 (buffer A). The flow rate was 0.5 ml/min. Enzyme activity retained by the column was eluted by a linear gradient of NaCl (0.0–0.5 M in 40 ml of buffer A). Finally, the column was eluted with 1.0 M NaCl in the same buffer. Fractions (1 ml) were collected and assayed for Hex activity.

Gangliosides extraction and quantitative determination
Gangliosides were extracted from the mouse brain and cerebellum (10–70 mg of tissue) using the method of Folch et al. (42Go) as modified by Hess and Rolde (43Go). Briefly, the weighted frozen tissue was thawed, manually homogenized in a 1 ml potter-Elvehjem homogenizer with a Teflon pestle and 2:1 chloroform–methanol mixture (v/v) in a volume 20-fold the tissue weight was added to the mash. The obtained homogenate was centrifuged for 10 min at 5000 r.p.m. in an Eppendorf Centrifuge 5415D and the supernatant was recovered. This crude extract was partitioned by adding 20% of its volume of re-distilled water. The two phases were separated by centrifugation for 15 min at 3000 r.p.m. in a microfuge: the upper phase was carefully recovered and the interface rinsed with a few-tenths of microliters of theoretical upper phase (3:48:47 chloroform–methanol–water); the lower phase was re-extracted with a volume of theoretical upper phase containing 0.015 M of KCl in water. After centrifugation, the recovered upper phase was combined with the first one and dried.

The total ganglioside concentration was determined using the resorcinol–HCl method according to Svennerholm with 85:15 butyl-acetate–butyl alcohol as extractant (44Go) and added to an eppendorf tube containing an equal volume of appropriately diluted upper phase re-suspended in water, mixed well and heated to 100°C in a thermo-block for 15 min. After addition of 1 ml of extractant and mixing, the samples were cooled on ice water and centrifuged for 3 min at 5000 r.p.m. The solvent layer was recovered and measured at 580 nm wavelengths using a Shimadzu UV-Visible Recording Spectrophotometer (UV-160A). The sialic acid concentration was determined by comparison with a standard curve.

TLC analysis of gangliosides
Aliquots of each sample corresponding to 3 µg of sialic acid were re-suspended in chloroform–methanol (1:1 v/v) and spotted on a TLC Silica gel plate previously washed in acetone. The chromatography was carried out using three successive runs with different migration solvents according to Dreyfus et al. (45Go), with plates being thoroughly dried between each run. The ganglioside pattern was quantified by densitometry scanning of the TLC plates stained with the resorcinol–HCl reagent (Svennerholm), using 1D Image Analysis Software (Amersham Pharmacia Biotech).

Other analytical methods
Proteins were measured by the method of Bradford (46Go) using serum bovine albumin as standard. X-Gal staining was carried out according to the method described in the manufacturer's procedure (Boheringer Lab).


   ACKNOWLEDGEMENTS
 
We thank Professor Glorioso J. and Dr Sampaolesi M. for helpful comments and critical reading of the manuscript; Dr Magaroli for organotypic brain slice preparation; Dr Benedetti L. Risso A. and Ghelfi C. for histology and technical animal care; Mattoli F. for computer assistance. The paper has been read by a native English speaker (H. Giles, MA). This work was supported in part by Italian grants: FIRB-2001 ‘Programma Nazionale Strategico di Neuroscienze’ no. RBNE012LW8; FIRB 2001 ‘Progetto Nazionale Nuova Ingegnaeria Medica’ no. RBAU01M5FR; Consorzio Internazionale di Biotecnologie 2004-2005; Pietro Vigezzi's family Foundation.

Conflict of Interest statement. None declared.


   FOOTNOTES
 
{dagger} The authors wish it to be known that, in their opinion, the first two authors should be regarded as joint First Authors. Back

{ddagger} The authors contributed equally to this paper. Back


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 MATERIALS AND METHODS
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