In Vivo Transduction of Cerebellar Purkinje Cells Using Adeno-Associated Virus Vectors

ARTICLE

doi:10.1006/mthe.2000.0134, available online at http://www.idealibrary.com on IDEAL

In Vivo Transduction of Cerebellar Purkinje Cells Using Adeno-Associated Virus Vectors William F. Kaemmerer,* Rukmini G. Reddy,† Christopher A. Warlick,*,† Seth D. Hartung,*,† R. Scott McIvor,*,†,‡ and Walter C. Low§,¶,1 *Department of Laboratory Medicine and Pathology, †Gene Therapy Program, Institute of Human Genetics, ‡Department of Genetics, Cell Biology and Development, §Graduate Program in Neuroscience, and ¶Department of Neurosurgery, University of Minnesota, Minneapolis, Minnesota 55455 Received for publication May 24, 2000, and accepted in revised form August 23, 2000; published online October 7, 2000

We investigated whether adenovirus or adeno-associated virus vectors can transduce cerebellar Purkinje cells (PCs) in vivo. Mice were injected in the deep cerebellar nuclei (DCN) with lacZ-transducing adenovirus (Ad.RSV-βgal) or a recombinant AAV serotype 2 (rAAV2) vector (vTR-CMVβ) mixed with wild-type adenovirus type 5 (Ad5). One week later, Ad.RSV-βgal transduced cells were found throughout the cerebellar white matter in a dose-dependent manner, but few transduced PCs were evident. In contrast, vTR-CMVβ with Ad5 transduced several hundred PCs throughout the injected hemisphere. Using an rAAV2 vector transducing a CMV-regulated green fluorescent protein gene, we again found PC transduction, but only with Ad5 coinjection. To assess the effect of injection site and to determine whether the apparent requirement for Ad5 coinfection is observed with other promoters, a β-actin-regulated vector was injected with or without Ad5 to DCN or cerebellar cortical sites. Thousands of transduced PCs were observed under each condition. Cortical injection yielded greater numbers of transduced cells. Injection of rAAV2 without Ad5 led to greater specificity for PC transduction. We conclude that injection of rAAV2 vectors into the cerebellum is an effective means for transferring genes into substantial numbers of Purkinje cells in vivo. Key Words: Purkinje cells; transduction; gene transfer; in vivo; adeno-associated virus; adenovirus; deep cerebellar nuclei.

INTRODUCTION In both rodents and primates, the cerebellum plays an essential role in motor control, balance, coordination, and the timing and accuracy of movements. Disorders of the cerebellum manifest as disturbances of gait, postural instability, and ataxia. While there are five types of neurons in the cerebellum, Purkinje cells provide the sole neuronal output from the cerebellum to the rest of the central nervous system. These cells are found throughout the cerebellar hemispheres, organized in a single cell layer that parallels the convolutions of the cerebellar cortex. They are distinguished by their extensive, planar dendritic arbors that project toward the cortical surface, and their large, pear-shaped cell bodies. All Purkinje cell axons project to and synapse in the deep cerebellar nuclei, located in the interior of each hemisphere.

1To whom correspondence and reprint requests should be addressed at Department of Neurosurgery, University of Minnesota, 2001 Sixth Street S.E., Minneapolis, MN 55455. Fax: 1-(612) 626-9201. E-mail: [email protected].

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In several hereditary disorders, Purkinje cells are particularly affected by the disease processes. For example, in spinocerebellar ataxias, expansion of a CAG repeat in the coding region of a gene results in expression of an expanded protein that produces Purkinje cell dysfunction (1). While various regions of the spinal cord and brainstem are also affected in these diseases, the ability to transfer therapeutic constructs into Purkinje cells will be one essential aspect of any somatic gene therapy for these fatal disorders. The goal of this study was to establish conditions that will lead to reliable Purkinje cell transduction in vivo. Adenovirus (Ad) vectors can transfer genetic material to a variety of cell types in the central nervous system (CNS), including microglia, astrocytes, and neurons [see (2) for a review]. However, previous studies of Purkinje cell transduction using Ad vectors (3, 4) have shown limited Purkinje cell transduction, primarily in the Purkinje cell layers directly intersected by the injection needle. In addition to adenovirus, we chose to focus on adenoassociated virus vectors because of their demonstrated ability to deliver foreign genes into neurons in other MOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright  The American Society of Gene Therapy 1525-0016/00 $35.00

ARTICLE regions of the CNS (5, 6). Recombinant AAV vectors (rAAV) are attractive because of their nonpathogenicity and ability to integrate with the host genome, potentially resulting in long-term expression of the foreign gene. Injection of rAAV into the CNS has been found to produce a minimal host reaction as indicated by changes in astrocytic or microglial markers or by leukocytic invasion (7). Several studies have shown that rAAV vectors transcriptionally regulated by the cytomegalovirus (CMV) promoter can provide neuronal expression of transgenes in the hippocampus, olfactory tubercle, piriform cortex, inferior colliculus (5), and striatum (5, 8, 9). Also, rAAV has been shown to have a specific tropism for binding and uptake by subtypes of neurons (10). Here we report the efficient transduction of Purkinje cells by injection of rAAV serotype 2 (rAAV2) vector into the cerebellar cortex or the deep cerebellar nuclei. We chose the latter site because we reasoned that, similar to neurons of the precerebellar nuclei whose axonal projections to the cerebellar cortex take up adenoviral vector (4), Purkinje cells might take up viral vector from their axon terminals in the DCN. These vectors might then travel retrogradely through the axons to the cell bodies, resulting in foreign gene expression in Purkinje cells throughout the cerebellar hemisphere. The results of this study show that transduction occurred effectively with an rAAV2 vector regulated by a chicken β-actin promoter/CMV enhancer combination (CBA). Vectors regulated by the CMV promoter alone required coinfection with wild-type adenovirus for transduction to be observed. Furthermore, rAAV2 vectors regulated by CBA effectively transduced thousands of Purkinje cells when injected into either the DCN or the cerebellar cortex.

MATERIALS

AND

METHODS

Viral Vector and Virus Preparation Diagrams of each of the vector constructs used are shown in Fig. 1.TRUF5 and TR-UF11 vector preparations were kindly provided by the Vector Production Facility of the Gene Therapy Center, University of Florida (Gainesville, FL). vTR-CMVβ. Adeno-associated vector was generated from plasmid pTR-CMVβ containing AAV terminal repeat elements, and β-galactosidase (β-gal) under control of the CMV promoter. Recombinant AAV vectors

FIG. 2. Diagram of a sagittal view of a mouse cerebellum illustrating some injection sites used (dark circles). Depending on syringe depth, injections were to the deep cerebellar nuclei (DCN) or the cerebellar cortex. PCL, Purkinje cell layer.

were packaged as described previously (11). Briefly, 293 cells at passage number 45 or lower were plated at 40–60% confluence in 15-cm tissue culture plates approximately 16 h before transfection. For each plate, 25 µg rAAV2 plasmid and 25 µg pAAVAd (kindly provided by Somatix Corp.) were combined in a solution of 250 mM CaCl2 in a final volume of 2.25 ml. DNA-CaCl2 was added dropwise to an equal volume of 2× Hepesbuffered saline and immediately added to the culture plate. After 6–12 h, the transfection mixture was replaced with DMEM and adenovirus 5 at a multiplicity of infection of 0.1. After 48–60 h, cells were harvested and subjected to three cycles of freeze-thaw to release virions from the cells. Virions were precipitated by adding ice-cold, saturated (NH4)2SO4 to 50%, resuspended, and then banded on a CsCl density gradient. Fractions were assessed for AAV vector by dot blot hybridization as described (12) and quantitated using a cDNA probe. Recombinant AAV titer was determined by comparing the hybridization signal with a dilution series of plasmid standard. Positive fractions were pooled and incubated at 55ºC for 45 min to inactivate adenovirus. To assay for contaminating adenovirus, 293 cells were transduced and monitored for cytopathic effects; they showed no cytopathic effects after 6 weeks. The presence of wildtype AAV or wild-type AAV-like particles (13) was assessed in separate dot blot hybridizations of the final vector preparations using a Rep probe derived from a BamHI–HindIII fragment of pAAV/Ad. Vector preparations commonly contained approximately 0.08% contamination with particles that hybridize to the Rep probe. The titer of vTR-CMVβ in functional units (fu) was determined by inoculating serial dilutions onto 293 cells followed by enumeration of positively stained cells 2 days later. Stained cells were enumerated in multiple fields (>10) using a conventional light microscope with eyepiece grid. No background β-gal activity was observed in negative controls during any titering assay. vTR-UF5. Adeno-associated vector containing the enhanced green fluorescent protein (eGFP) under control of the CMV promoter was generated as previously described (14). The functional titer was assayed just prior to use in the same manner as vTR-CMVβ. Titer was estimated by enumerating eGFP-positive cells in multiple fields within each cell culture well. No background fluorescence was observed in negative controls in any titering assay.

FIG. 1. Diagrams of the AAV vector constructs used. See Materials and Methods for a description of the reporter genes and their promoters. PA, polyadenylation signal; neo, neomycin resistance coding sequence under control of the thymidine kinase (TK) promoter; arrows, transcription start sites. MOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright  The American Society of Gene Therapy

vTR-UF11. Plasmid vector pTR-UF11 was derived from the pTR-UF5 (15) vector backbone and the chicken β-actin (CBA) promoter fragment from pBacMam (Novagen) using standard cloning techniques and will be described in detail elsewhere (N. Muzyczka, personal communication). rAAV-UF5 and UF11 were packaged and purified as described (14). Briefly, DNA–CaPO4 coprecipitate was prepared by adding 12.5 ml of 2× HBS pH 7.05 (prewarmed to 37C) to a mixture of 0.7 mg of pDG (16) and 180 µg rAAV vector plasmid in a total volume of 12.5 ml 0.25 M CaCl2, added to 200 ml of prewarmed DMEM–10% FBS, and then distributed into ten 15-

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ARTICLE TABLE 1 Injection Types, Amounts, and Locations Exp. No. 1

2

Number of Mice

Amount per site 1.7 

104

Volume per site

AP

4 µl

-3.00

DV

2.25

-1 .00

vTR-CMV plus Ad5

2

Ad.RSV-gal

3.0  108 fu

3 µl

same as above

1

Ad.RSV-gal

3.0  107 fu

3 µl

same as above

1

Ad.RSV-gal

3.0  106 fu

3 µl

same as above

1

-gal protein

100 units

3 µl

3

vTRUF5 plus Ad5

7.5  106 fu of each

4 µl

fu of each

2

vTRUF5

5.0  106 fu

2 µl

3

vTRUF5

5.0  106 fu

2 µl

4 4

vTRUF5 vTRUF11 vTRUF11 plus Ad5

5.0  106 fu 2.7  108 fu of vTRUF11 2.7  108 fu of vTRUF11

2 µl 4 µl 4 µl

plus 1.0  105 fu Ad5 2 2

vTRUF11 vTRUF11 plus Ad5

2.7  108 fu of vTRUF11 2.7  108 fu of vTRUF11

4 µl 4 µl

plus 1.0  105 fu Ad5 a All

ML

2

1 3

Stereotactic coordinatesa Injection type

same as above -2.75

2.25

-1.00

-2.75

1.50

-1.50

same as above -2.50

2.00

-1.50

-2.50

1.25

-2.00

-3.00

2.25

-1.50

-3.00

1.25

-2.00

-3.00

2.25

-1.00

-3.00

1.25

-1.50

-3.00

2.25

-1.00

-3.00

1.25

-1.50

-3.00

2.25

-0.50

-3.00

1.25

-0.50

-3.00

2.25

-0.50

-3.00

1.25

-0.50

coordinates are in millimeters from lambda, with the incisor bar at -5.0 mm.

cm plates of 293 cells at 75–80% confluence. Forty-eight hours posttransfection, the cells were harvested by centrifugation at 1140g for 10 min and lysed in 15 ml of 0.15 M NaCl, 50 mM Tris–HCl, pH 8.5, by three cycles of freeze/thaw. The lysate was supplemented with Benzonase (Nycomed Pharma A/S, pure grade, 50 U/ml final concentration), incubated for 30 min at 37C and clarified by centrifugation at 3700g for 20 min. The recovered virus was further purified by iodixanol discontinuous density gradient centrifugation and heparin-agarose column chromatography, and concentrated by centrifugation through a BIOMAX 100 K filter (Millipore) according to the manufacturer’s instructions. Virus titer was determined by dot blot and by quantitative competitive polymerase chain reaction (PCR). Ad.RSV-βgal. Ad.RSV-βgal was kindly provided by Dr. Mark Kay (Stanford University School of Medicine) and has been previously described (17). Expansion and titering of recombinant adenovirus vector was performed by standard techniques. Briefly, adenovirus vector was harvested from infected cultures of 293 cells by freeze-thaw and purified by CsCl density gradient centrifugation. A titer of 1 × 1011 plaque-forming units (pfu) per milliliter was assessed by plaque assay. The stock was stored at −80ºC until use, then thawed and optionally diluted 1:10 or

1:100 in sterile phosphate buffered saline (PBS) prior to injection. Ad5. Propagation and titering of wild-type adenovirus 5 (Ad5) was according to Xiao et al. (12).

Cerebellar Injections FVB/N mice between 6 and 16 weeks of age were used for three studies. Vector amounts, injection volumes, and stereotactic coordinates are presented in Table 1. Figure 2 illustrates the location of various injection sites with respect to the gross anatomy of the cerebellum. Each mouse was injected intraperitoneally with 6 µl of ketamine/xylazine mixture (36 mg/ml ketamine, 5.5 mg/ml xylazine) to produce deep anesthesia. The mouse was mounted in a stereotactic frame (Kopf Model 900) and its head shaved. A midline sagittal incision was made and the cranium over the right cerebellar hemisphere was exposed. For each injection site, a burr hole was drilled and a Hamilton syringe (Model 701) was inserted to the depth described below dura, plus an additional 0.5 mm. After 2 min, the syringe was retracted 0.5 mm, to form a slight pocket in the parenchyma. After a pause of at least 2 min for pressure equalization, the injection was performed manually at an approxi-

FIG. 3. β-Galactosidase gene transfer and expression in mouse cerebellum. (a) Sagittal section of the cerebellum of a mouse injected with β-gal protein. Brightfield image showing β-gal activity by X-gal staining merged with image under fluorescent illumination for Cy2 showing calbindin immunoreactivity of Purkinje cells; arrows indicate the Purkinje cell layer; no Purkinje cell uptake of β-gal protein was evident; scale bar = 300 µm. (b) Sagittal section of the cerebellum of a mouse injected with 3 × 108 pfu Ad.RSV-βgal per site. In addition to β-gal activity in cell bodies throughout the white matter (white arrow), intensive β-gal activity in ependymal cells lining the fourth ventricle was visible (black arrow, bottom); scale bar = 300 µm. (c) Coronal section of the cerebellum of a mouse injected with 3 × 107 pfu Ad.RSV-βgal per site; β-gal activity was visible in cells in the white matter and in a sulcus. Much of the β-gal activity appearing in the Purkinje cell layer (white arrow) was not colocalized with the interior of Purkinje cells as visualized by calbindin (inset, white arrow); scale bar = 300 µm. (d) Mouse injected with 3 × 108 pfu Ad.RSV-βgal per site, sagittal section; 4× objective, scale bar = 300 µm. Inset: β-gal activity colocalized with the interior of Purkinje cell soma could occasionally be detected (white arrow); 20× objective. (e) Brightfield image of the cerebellum of a mouse injected with 1.7 × 104 fu vTR-CMVβ and Ad5 per site, showing β-gal activity in cell soma in the Purkinje cell layer (black arrows) and dendritic arbors projecting into the molecular layer. Some cells in the DCN are also transduced (asterisk); very little β-gal activity was detectable in the white matter; sagittal section, scale bar = 500 µm. (f) High magnification view of calbindin and X-gal stained section from same mouse as (e), showing detailed morphology of Purkinje cell soma and dendrites (arrows) containing β-gal activity transduced by AAV; inset: brightfield view of same Purkinje cell dendrites; scale bar = 50 µm.

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ARTICLE mate rate of 0.5 µl per minute. Afterwards, the syringe was left in place an additional 3 min, then withdrawn over a period of 2 min or more. Once injections were complete, the scalp was sutured and the mouse kept under a warming lamp until recovered from the anesthesia, then returned to standard housing.

ME] were included in all X-gal assays. Hippocampal neurons in this mouse robustly express the lacZ transgene following in vivo induction with cadmium sulfate, injected ip. Following X-gal incubation, sections were immunostained for calbindin to aid in the identification of Purkinje cells.

Tissue Processing

Immunohistochemistry

One week after the cerebellar injections, mice were deeply anesthetized by intraperitoneal injection of 12 µl sodium pentobarbital and transcardially perfused with PBS for several minutes, followed by perfusion with 4% formaldehyde for 10 to 15 min. The brain was removed and postfixed for 1 to 2 h in 4% formaldehyde, then transferred to a 30% solution of sucrose and stored at 4C until it sank. Then, the brain was frozen in dry ice, and cut into 30-µm coronal or sagittal sections using a sliding microtome. Sections were stored in an antifreeze solution [30% ethylene glycol and 30% glycerol (v/v) in PBS] at 20ºC until further processing. For visualization of eGFP expression, tissue sections were rinsed 3 × 20 min in PBS, mounted on glass slides, coverslipped with an aqueous mounting solution (Shandon Immumount, No. 9990402), then viewed by fluorescence microscopy. Sections from mice coinfected with vTR-UF5 plus Ad5 were immunostained for calbindin prior to mounting.

Sections were rinsed 3 × 20 min in PBS, then transferred to a solution containing 2% normal goat serum (NGS) and 0.3% Triton X-100 for a minimum of 1 h. Sections were then transferred to a solution of 2% NGS, 0.3% Triton X-100 and 1:500 antibody to calbindin-D-28k (Sigma, No. C8666), and incubated at 4C with gentle agitation for at least 48 h. Sections were rinsed 3 × 20 min at room temperature in PBS, then incubated for at least 24 h at 4C, with gentle agitation, in a solution of 2% NGS, 0.3% Triton X-100 and 1:400 goat anti-mouse IgG antibody conjugated to Cy3 fluorophore (Jackson ImmunoLabs No. 115-165-146) or 1:400 goat anti-mouse IgG antibody conjugated to Cy2 fluorophore (Jackson ImmunoLabs No. 115-225-146). After incubation with the secondary antibody, sections were washed 3 × 20 min, mounted on slides and coverslipped as described.

β-Galactosidase Visualization

To quantify the extent of Purkinje cell transduction in vivo, every fourth tissue section (Study 1) or every second tissue section (Study 3) throughout the injected cerebellar hemisphere was examined by light and fluorescence microscopy. Since Purkinje cell soma are a maximum of 50 µm in diameter, examining every other section among 30-µm-thick sections minimizes the chance of double-counting a given Purkinje cell, while not undercounting smaller cell types. Because a total count of cells in each section was feasible, stereologic sampling methods were not used.

For visualization of β-gal activity, tissue sections were rinsed 3 × 20 min in PBS, then incubated overnight at 37C in an X-gal solution consisting of 4 mM K4Fe(CN)6, 4 mM K3Fe(CN)6, 2 mM MgCl2, and 0.4 mg/ml X-gal (5-bromo-4-chloro-3-indolyl-β-D-galactoside, GibcoBRL No. 15520-034) in PBS. As a positive control, sections of the cerebrum of a lacZ transgenic mouse [TgN(MtnlacZ)204Bri, Jackson Labs, Bar Harbor,

Cell Counting

FIG. 4. Distribution of cells with β-gal activity in the cerebellar hemisphere of a mouse injected with vTR-CMVβ plus Ad5. Although the majority of Purkinje cells with β-gal activity were near the sagittal planes containing the injection sites (1250 and 2250 µm from medial), Purkinje cells with β-gal activity were detected throughout the medial–lateral extent of the hemisphere. In the hemisphere as a whole, the majority of cells with β-gal activity were Purkinje cells.

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ARTICLE To be identified as a transduced Purkinje cell, the cell soma had to be in the Purkinje cell layer, the appropriate size, intensely stained by β-gal (or provide an intense eGFP signal), and had to have a portion of its dendritic arbor visible in the molecular layer. Other transduced cells (nonPurkinje cells) were identified by morphology and intensity of staining or signal.

PCR Assay DNA from mouse cerebellum was analyzed by PCR to detect β-gal sequence. Sections of the cerebrum of a lacZ transgenic mouse [TgN(MtnlacZ)204Bri] were included as a positive control, and sections from the cerebellum of a noninjected wild-type FVB/N mouse served as a negative control. To extract the DNA, 10 tissue sections (30 µm thick) were incubated at 55C for 4 h in a solution containing 400 µl Tens (50 mM Tris–HCl pH 8.0, 1 mM EDTA, 20 mM NaCl, and 1% SDS) and 20 µl proteinase K (GibcoBRL No. 25530-049), with gentle vortexing once per hour. Following phenol–chloroform extraction and ethanol precipitation, the DNA was spooled onto a glass pipette, and resuspended in Tris–EDTA buffer. PCR was conducted using primers that amplify a 316bp region of β-gal sequence (5′-ATC CTC TGC ATG GTC AGG TC-3′ and 5′-CGT GGC CTG ATT CAT TCC-3′) in a reaction mix assembled according to the manufacturer’s protocol (Promega PCR Core Kit No. M7665). The reaction was carried out using an initial melting at 94C for 7 min, 30 cycles consisting of melting at 94C for 1 min, annealing at 55C for 1 min, and extension at 72C for 1 min, and a final extension of 72C for 8 min. Reaction products were separated by electrophoresis in a 1% agarose gel, and visualized by ethidium bromide staining.

RESULTS Examination of the mouse cerebellar sections, as well as preliminary studies in which 6-week-old FVB/N mice were immediately sacrificed following injection of thymidine dye into their cerebella, indicated that the stereotactic coordinates used appropriately target the DCN and cerebellar cortex in this age and strain of mouse.

Study 1: AAV Vectors Effectively Transduce Purkinje Cells To determine the ability of rAd and rAAV to transduce Purkinje cells, two mice were injected in each of two sites in the DCN with vTR-CMVβ mixed 1:1 with Ad5, and four mice were injected with Ad.RSV-βgal only. As a control for potential residual β-gal contamination in viral preparations, an additional mouse received an injection of 100 U of β-gal protein (Sigma, No. G5635) in each site.

FIG. 5. PCR assay for lacZ sequences. PCR products were generated using primers that amplify a 316-base-pair segment. Lanes: 1, Ad.RSV-βgal injected mouse; 2, vTR-CMVβ plus Ad5-injected mouse; 3 positive control [TgN(MTnlacZ)204Bri mouse]; 4, negative control (wild-type mouse); 5, 100bp ladder; 6, HindIII-digested lambda phage marker. A product of the expected size was detected in mice injected with Ad.RSV-βgal, vTR-CMVβ plus Ad5, and the positive control.

X-gal processing of the latter mouse showed that a large amount of active β-gal protein remained in the cerebellum 1 week after injection. However, calbindin immunostaining revealed that none of this β-gal activity colocalized with Purkinje cell soma or dendritic arbors (Fig. 3a). Furthermore, while colocalization of the β-gal activity with other cell bodies cannot be completely ruled out, the spatial pattern and regional distribution of the β-gal activity is distinctly different from that seen in the animals injected with vTR-CMVβ plus Ad5, or with Ad.RSV-βgal. This result indicates that β-gal activity seen in Purkinje cells and other cells of mice injected with Ad.RSV-βgal or with vTR-CMVβ plus Ad5, and in cells within the white matter of mice injected with Ad.RSV-βgal is not a pseudotransduction effect due to any residual β-gal protein potentially remaining in the vector stocks. All four mice injected with Ad.RSV-βgal showed extensive, punctate β-gal activity throughout the cerebellar white matter in the injected hemisphere (Figs. 3b–3d). However, β-gal activity was seldom seen in Purkinje cells. Close examination of regions in which β-

FIG. 6. eGFP expression in vTR-UF5 injected mice. (a) Sagittal section from a mouse injected with vTR-UF5 alone (5 × 106 fu in 2 µl per site) showing calbindin–Cy3 immunofluorescence (red) and eGFP expression. No eGFP expression was evident in Purkinje cells or in the white matter. Scale bar = 100 µm. Image is representative of results obtained from six mice. (b) Higher magnification image of the boxed portion in (a), showing the eGFP signal from the region of the deep cerebellar nuclei. Cellular morphology indicative of neurons within the nuclei is apparent. Scale bar = 20 µm. (c–f) Fluorescence microscopy of sagittal cerebellar sections from two mice injected with 7.5 × 106 fu each of vTR-UF5 and Ad5 in 4 µl per site. (c) Sagittal section showing eGFP-positive Purkinje cells surrounding the primary fissure; scale bar = 50 µm. (d) Section from a second mouse, with numerous eGFP-positive Purkinje cells rostral to the primary fissure in a section 1.5 mm from lateral, scale = 50 µm. (e) Same section as (d), showing eGFP-positive cells in the region of the DCN (asterisk), and eGFP signal visible in axons coursing through the white matter to the deep nuclei (arrow); scale bar = 100 µm. (f) Same mouse as (d), 3.2 mm from lateral, showing eGFPpositive Purkinje cells near dorsal surface; scale = 50 µm. This section is 0.6 mm from the plane of the nearest needle track; the inset diagram shows the location of these cells (box) in the sagittal plane relative to the needle track and DCN injection site. FIG. 7. Sagittal sections of mice injected with vTR-UF11 with or without Ad5, to cortical or DCN sites; all scale bars = 100 µm. (a) Mouse injected with vTRUF11 and Ad5 to DCN sites. Arrow shows angle of needle track approaching the DCN (asterisk); note transduction of Purkinje cells distal from needle path, and numerous eGFP-positive cells in white matter and granular layer as well as Purkinje cell layer. (b) Mouse injected with vTR-UF11 alone to DCN sites; (c) Mouse injected with vTR-UF11 and Ad5 to cortical sites; (d) Mouse injected with vTR-UF11 alone to cortical sites. Coinjection with Ad5 (a, c) yields less specificity of transduction to Purkinje cells than does vTR-UF11 alone (b, d). MOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright  The American Society of Gene Therapy

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ARTICLE gal activity appeared to be colocalized with Purkinje cells often revealed that the activity colocalized with other cell types but not with Purkinje cell soma (Fig. 3c). However, some colocalization of β-gal activity and Purkinje cell soma was identified (Fig. 3d). This β-gal activity was not distributed throughout the cell soma, consistent with the nuclear-localized β-gal transduced by the Ad.RSV-β-gal vector (17). The mice that received 108 pfu of Ad.RSV-βgal per injection site exhibited more extensive β-gal activity than those mice receiving 106 or 107 pfu of vector, but did not show a correspondingly greater frequency of Purkinje cell transduction. These results indicate that Purkinje cells are not highly susceptible to transduction using the Ad.RSV-βgal vector delivered to the DCN. In one of the two mice injected with the vTRCMVβ/Ad5 mixture, no β-gal activity was detected anywhere in the cerebellum. However, in the other mouse, intense β-gal activity was detected in numerous Purkinje cells as well as cells local to the DCN injection sites (Fig. 3e). While β-gal-positive cells in the DCN themselves were confined to the apparent injection sites ± 4 sampled sections (±120 µm), other β-gal-positive cells and, notably, β-gal-positive Purkinje cells were readily detected in virtually all sections. In many Purkinje cells, β-gal activity could be detected in the dendritic arbor of the cell as well as the cell soma (Fig. 3f). This is consistent with the nonnuclear form of β-gal transduced by the vTR-CMVβ vector, and aided in the definitive identification of Purkinje cells as being among the transduced cells. The count of β-gal-positive Purkinje cells in each sampled section of this mouse indicated that the majority of these Purkinje cells were in the middle to lateral sagittal planes of the hemisphere; however, substantial numbers of Purkinje cells remote from the sagittal planes containing the needle tracks were also β-gal positive (Fig. 4). Thus, Purkinje cell transduction by vTRCMVβ vector was not limited to planes containing the needle tracks. Almost all of the other transduced cells were located within the deep nuclei, which extend medially and laterally through the cerebellar hemisphere. PCR does not distinguish between single-stranded rAAV genomes and those that have undergone the second-strand synthesis necessary for transgene expression. However, PCR can indicate persistence of the reporter gene sequence in vector-injected mouse tissue. A PCR assay conducted on DNA extracted from the mouse cerebellar tissue revealed a PCR product of the expected size in a mouse injected with vTR-CMVβ plus Ad5, one injected with Ad.RSV-βgal, and in a lacZ transgenic mouse used as a positive control, but not in an FVB/N wild-type mouse (Fig. 5).

Study 2: Purkinje Cell Expression from vTR-UF5 Requires Adenovirus Coinfection To replicate the result obtained with vTR-CMVβ plus Ad5 injection and to assess the importance of coinfection

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with Ad5 for Purkinje cell transduction, six mice were injected with vTR-UF5 vector without coinjection of Ad5. In addition, to determine whether the precise anatomical site within the DCN affects Purkinje cell transduction efficiency, the stereotactic coordinates were varied slightly among these six littermates. Three additional mice were injected with vTR-UF5 vector in 1:1 ratio with Ad5. Surprisingly, despite the fact that the amount of vTR-UF5 vector injected was two orders of magnitude higher in functional titer than the vTR-CMVβ of Study 1, none of the six mice injected with vTR-UF5 alone provided more than a few isolated eGFP-positive Purkinje cells. Rather, in all six mice, the vast majority of eGFP-positive cells observed 7 days after injection was confined to the immediate region surrounding the injection sites in the DCN (Fig. 6a). The eGFP signals observed were intense, completely confined to the gray matter of the DCN, and had morphology consistent with intranuclear neurons (Fig. 6b). In contrast, in two of the three mice injected with vTR-UF5 plus Ad5, eGFP-positive Purkinje cells were readily identifiable, with the signal visible in many cases in both Purkinje cell dendritic arbors (Figs. 6c and 6d) and axons coursing through the cerebellar white matter toward the DCN (Fig. 6e). Cells in the DCN themselves were also eGFP positive. Purkinje cells expressing eGFP were visible in regions proximal to the dura of the cerebellar hemisphere as well as deeper in the cerebellar lobules, and in sagittal sections both near and remote from the apparent needle track. Isolated Purkinje cells in dorsal regions remote from the injection sites and needle tracks were visible in a Golgi-like manner while the Purkinje cells arrayed between the transduced Purkinje cells in the same lobule, and Purkinje cells in lobules lying between the transduced cells and the DCN, remained untransduced (Fig. 6f). This suggests that transduction of some cells was not a result of vector diffusion to the cell soma in the Purkinje cell layer, but rather from retrograde axonal transport of vector from the DCN. In the third mouse injected with the vTR-UF5/Ad5 mixture, the distal two-thirds of the needle tracks coincided with the primary fissure, suggesting that the vector was inadvertently injected into the subdural space, from which it diffused without any detectable cell transduction.

Study 3: DCN Delivery and Ad Coinjection Are Not Necessary for Purkinje Cell Transduction To determine whether the necessity for adenovirus coinfection to achieve foreign gene expression from rAAV2 in Purkinje cells is a general phenomenon, a third study was undertaken using vTR-UF11, in which eGFP expression is regulated by the CBA rather than the CMV promoter. This vector was used at a higher titer than vTRUF5, and a pilot study showed that coinjection of Ad5 in a 1:1 ratio yielded extensive tissue damage in the cerebellum. Therefore, in the mice coinjected with Ad5, the quantity of Ad5 was limited to the amount used in Studies 1 and 2. In addition, to determine the importance MOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright  The American Society of Gene Therapy

ARTICLE of vector delivery to the DCN, we varied the sites of injection between cortical and DCN targets, in a 2 × 2 study design. Eight mice were injected at DCN sites (4 with coinjection of Ad5) and 4 mice were injected at cerebellar cortical sites (2 with coinjection of Ad5). Large numbers of eGFP-positive Purkinje cells were observed in all animals in this study, indicating that neither coinjection with Ad5 nor delivery of vector to the DCN is necessary for Purkinje cell transduction by rAAV2. Figure 7 shows results for one mouse from each of the four experimental groups. Extensive eGFP expression in Purkinje cell soma and dendritic arbors is apparent in all four cases. In all mice, a total count of eGFP-positive cells was obtained from every other sagittal section throughout the injected cerebellar hemisphere, with cells identified as Purkinje cells or some other cell type. An analysis of variance of cell counts showed that, compared to DCN injection, cortical injection yielded a greater number of eGFP-positive Purkinje cells (Fig. 8a, F (1,8) = 23.2, P ≤ 0.001), a

greater number of eGFP-positive other cell types (F(1,8) = 12.4, P ≤ 0.008), and a greater number of eGFP-positive cells overall (F(1,8) = 22.2, P ≤ 0.002). Compared to coinjection with Ad5, injection of vTRUF11 alone yielded a greater percentage of Purkinje cells among total eGFP-positive cells (Fig. 8b, F(1,8) = 7.5, P ≤ 0.025). No interaction effects of coinjection status with injection site were statistically significant. This indicates that the effect of the presence or absence of Ad5 was not different for cortical versus DCN sites. Figures 8c–8f show the medial–lateral distribution of the average number eGFP-positive Purkinje cells and other cells observed in animals injected with vTRUF11 under each of the four experimental conditions. Cortical injection yielded a broader distribution of eGFP-positive cells throughout the hemisphere, while injection of vTR-UF11 in the absence of Ad5 yielded relatively greater specificity of transduction for Purkinje cells in both DCN and cortical sites.

FIG. 8. Counts of eGFP-positive Purkinje cells and other cell types in mice 7 days after injection of vTR-UF11 with or without Ad5, to cortical or DCN sites. (a) Coinjection of Ad5 has no effect on Purkinje cell transduction by vTR-UF11, but cortical injection yields a greater number of transduced Purkinje cells than DCN injection (**P ≤ 0.001). (b) In the absence of Ad5 coinjection, a greater percentage of the cells transduced by vTR-UF11 are Purkinje cells (*P ≤ 0.025). (c–f) medial–lateral distribution of eGFP-positive Purkinje cells and other cells, averaged across mice; (c) vTR-UF11 plus Ad5 to DCN, (d) vTR-UF11 plus Ad5 to cortex, (e) vTR-UF11 alone to DCN, (f) vTR-UF11 alone to cortex.

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ARTICLE DISCUSSION Using three different rAAV2 constructs, two different promoters, and two different reporter genes, we found rAAV2 to be capable of in vivo transduction of Purkinje cells. In contrast, injection of Ad.RSV-βgal vector suggested that even at high titer, this particular adenovirus vector is poor at transducing Purkinje cells in vivo. The widespread distribution of β-gal activity in the cerebellar white matter of Ad.RSV-βgal injected mice indicates that the limited transduction of Purkinje cells by this vector was not due to limited spread of the vector from the DCN delivery sites. One possible reason for the low transduction may be minimal uptake of Ad vector by Purkinje cells. Alternatively, there may be limited activity of the RSV promoter in Purkinje cells. The results from our study demonstrate that rAAV2 vectors are capable of mediating effective transduction of substantial numbers of cerebellar Purkinje cells when introduced into either the cerebellar cortex or the DCN. In our best case, the number of Purkinje cells transduced (3050) represents approximately 3.4% of the total Purkinje cell population, which is about 88,500 per hemisphere (18). Furthermore, rAAV2 appears to have a specific affinity for Purkinje cell transduction. In Study 3, we found that a disproportionately large fraction of cells transduced by rAAV2, particularly when delivered without Ad5, are Purkinje cells. Other types of cerebellar cells exist in far greater numbers. When mixed with Ad5, rAAV2 transduces proportionately more of these other cell types, including cells in the cerebellar white matter, consistent with the pattern of cell transduction seen with Ad.RSV-βgal. This suggests that in the absence of coinfection with Ad5, other cells are less able than Purkinje cells to express the foreign gene, or Purkinje cells have a specific susceptibility to rAAV2 infection. The receptor for AAV serotype 2 (AAV2) has not been definitively identified, but evidence suggests that heparan sulfate on the cell surface may contribute to the binding of AAV2 to permissive cells (19). In HeLa cells, AAV uptake is dependent upon dynamin, suggesting internalization by receptor-mediated endocytosis from clathrin-coated pits (20). The presence of αVβ5 integrin in cell membranes has been found to facilitate AAV2 internalization (21). Qing et al. (22) found that cells must express both heparan sulfate proteoglycan and basic fibroblast growth factor receptor 1 to bind AAV. There is evidence suggesting that each of these molecular factors is present in Purkinje cells. The product of the gene for Kallman’s syndrome, expressed by Purkinje cells, is bound to cell surfaces by heparan sulfate (23). Immunostaining has shown that dynamin is abundant in Purkinje cells (24) while almost entirely absent from the cerebellar medulla (25). The messenger RNA for the αV integrin subunit and the β5 subunit have each been detected in Purkinje cells (26). Finally, a high density of bFGF immunoreactivity is found in the Purkinje cell layer of the rodent brain (27) and fresh frozen normal

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human brain tissue (28). Matsuda et al. (29) found evidence suggesting that bFGF in cerebellar Purkinje cells undergoes two modes of transport, one to axon terminals and the other to nuclear euchromatin. Thus, if rAAV2 enters Purkinje cells via a bFGF receptor-mediated route, internal mechanisms exist that might lead to efficient transport of the vector to the Purkinje cell nucleus. In mice coinjected with vTR-UF5 and Ad5 in the DCN, we frequently observed transduced Purkinje cells that were strikingly isolated and remote from the needle tracks and injection sites, with little or no transduced cells in the lobules intervening between the injection site and the observed cells. Our studies did not include any experimental manipulations pertaining to retrograde axonal transport mechanisms. However, the most parsimonious explanation for these findings may be vector uptake at the injection site in the DCN, to which all Purkinje cells throughout the cerebellum project their axons, followed by retrograde axonal transport of the vector within the Purkinje cell to its soma. The cerebellar tissue architecture is highly organized, with a regular distribution of Purkinje cell bodies in a single convoluted layer and little variation in Purkinje cell size and density or in surrounding tissue density. Thus, it seems unlikely that these factors, coupled with vector diffusion, could account for the isolated transduction events observed. An alternative explanation might be cell-to-cell variation in receptor expression levels, but these differences would have to be locally extreme to account for the highly discrete pattern of isolated transduced cells seen. Pre-injection of the DCN with a microtubule inhibiting agent such as colchicine, vinblastine, or nocodazole prior to rAAV injection may be one means of experimentally determining whether retrograde axonal transport of AAV vectors occurs in Purkinje cells. The results showing that Purkinje cell transduction with vTR-UF5 requires coinjection with Ad5 were unexpected. In contrast, our results using vTR-UF11 alone show that adenoviral coinjection is by no means a general requirement for Purkinje cell uptake of rAAV2, movement of the rAAV2 to the cell soma, and foreign gene expression. Together, these results suggest that in the absence of adenovirus coinfection, the CMV promoter is transcriptionally inactive in these cells, or gene expression using the CMV promoter does not occur in the time frame examined. The expression observed when Ad5 was coinjected may have been the result of transactivation of the CMV promoter within Purkinje cells by the adenovirus E1a protein (30). The E1a 13S gene product has been found to increase CMV regulated reporter gene expression in some cell lines [18- to 19-fold increase in Jurkat and macrophage lines (31)] though not others [BHK-21 cells (32)]. Further study is needed before the mechanism by which adenovirus facilitates CMV-regulated expression in Purkinje cells can be defined. However, to the extent that other studies of rAAV2 transduction in the central nervous system have focused on constructs using the CMV promoter, our findings may MOLECULAR THERAPY Vol. 2, No. 5, November 2000 Copyright  The American Society of Gene Therapy

ARTICLE account for the paucity of reports on Purkinje cell transduction to date. It is important to note that the greater extent of Purkinje cell and other cell transduction observed in Study 3 versus Study 2 most likely resulted from the higher titer of vTR-UF11 compared to vTR-UF5, rather than promoter differences. Study 3 was not designed to compare the transduction or expression levels obtained using different promoters, but to rule out the necessity for adenovirus coinjection to achieve substantial in vivo Purkinje cell transduction by rAAV. All results presented are limited to assessment of reporter gene expression 1 week postinjection. Research is underway to assess the persistence of reporter gene expression in Purkinje cells over longer periods of time. Transduction of other neuronal cell types in vivo by rAAV has led to persistent gene expression, lasting as long as 6 months in rat medial septum neurons (33), as long as 1 year in rat striatal neurons (9), and up to 15 weeks in neurons of adult rat spinal cord (34). Thus, persistent gene expression in Purkinje cells may be anticipated. We have shown that transfer and subsequent expression of foreign genes in substantial numbers of Purkinje cells can be achieved in vivo through the use of rAAV2 vectors delivered to the cerebellar cortex or deep cerebellar nuclei. This approach may be useful as a research tool for investigating the effects of newly expressed gene products in Purkinje cells in vivo. With further improvements in vector production and delivery methods, and studies of the safety of the vectors with regard to immune response and neurotoxicity, these results may also have implications for future molecular therapies to treat diseases involving Purkinje cell dysfunction. ACKNOWLEDGMENTS The authors thank Sergei Zolotukhin and Nicholas Muzyczka of the Gene Therapy Center, Department of Molecular Genetics and Microbiology, University of Florida (Gainesville, FL) for graciously providing the vTR-UF5 and vTR-UF11 used. This investigation was supported in part by National Research Service Award 5 F31 MH11640 from the National Institute of Mental Health to W.F.K., P01 HD32652, and the Lyle French Fund.

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