Prolonged Transgene Expression Mediated by a Helper-Dependent Adenoviral Vector (hdAd) in the Central Nervous System

doi:10.1006/mthe.2000.0104, available online at http://www.idealibrary.com on IDEAL

ARTICLE

Prolonged Transgene Expression Mediated by a HelperDependent Adenoviral Vector (hdAd) in the Central Nervous System Linglong Zou,*,†,1 Heshan Zhou,†,‡,§,1 Lucio Pastore,‡ and Keyi Yang*,†,2 *Department of Neurosurgery, †Center for Cell and Gene Therapy, and ‡Department of Molecular and Human Genetics and §Department of Pediatrics, Baylor College of Medicine, Houston, Texas 77030 Received for publication April 10, 2000, and accepted in revised form June 27, 2000

Conventional adenoviral vectors such as E1-deleted first-generation adenovirus (fgAd) elicit striking host immune response, resulting in limited expression of the transgene. A recently described helper-dependent, or gutless, adenoviral vector (hdAd) can promote stable transgene expression in peripheral organs, including the liver. We therefore investigated the safety and durability of hdAd-mediated gene transfer to the central nervous system (CNS) of rats compared with gene delivery by fgAd. Equal amounts of either fgAd or hdAd carrying the geo transgene were stereotactically injected into the right hippocampus of adult rats. Transgene expression was assessed by histochemical staining, transgene stability by PCR analysis, and immune infiltration of T lymphocytes and macrophages by immunocytochemical methods. Strong transgene expression from either vector was detected in brain tissue examined on day 6 postinoculation. Thereafter, fgAdmediated gene expression rapidly decreased, becoming undetectable by day 66, while expression from the hdAd vector persisted throughout the test period. PCR confirmed the presence of hdAdassociated DNA at 66 days postinoculation. The hdAd injection elicited apparently lower numbers of brain-infiltrating macrophages and T cells than did administration of fgAd. These results indicate improved transgene expression and reduced immunogenicity with use of hdAd to deliver genes to the CNS. Key Words: gene transfer; adenovirus; gutless vector; brain; immunogenicity.

INTRODUCTION Replication-deficient adenoviruses (Ad), derived from serotype 5 Ad, have been widely and conventionally used in gene transfer studies (1–5) because of their favorable characteristics. These include a relatively large cloning capacity, a well-characterized genetic background, ease of growth to high titers, and ability to transduce a wide range of cell types, both dividing and nondividing, with high efficiency (2). However, the initial enthusiasm over these conventional Ad vectors has been tempered by their failure to mediate persistent transgene expression and by their toxicity in preclinical and clinical studies (6–9), which included first-genera-

1These authors contributed equally. 2To whom correspodence should be addressed at Department of

Neurosurgery and Center for Cell and Gene Therapy, Baylor College of Medicine, One Baylor Plaza, Houston, TX 77030. Fax: 713-798-1230. E-mail: [email protected]..

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tion Ad (fgAd) containing an E1 deletion in the viral genome. For example, fgAd-mediated transgene expression in liver cells lasts only 2–3 weeks (10). It has also been reported that multiple transduction of single cells with Ad induces apoptosis (6). Although traditionally believed to be an immuneprivileged site, the brain can generate significant immune responses against fgAd vectors (11–16), limiting the duration of transgene expression. In one recent study performed in primates, the immune response induced by fgAd-mediated gene transfer led to severe consequences, including localized demyelination, parenchymal necrosis, and apoptosis of transduced cells in the central nervous system (CNS) (17). Many factors contribute to the adverse effects of conventional Ad-based gene therapy, including host inflammatory responses to the vector, acute and chronic toxicity induced by Ad transduction, and generation of anti-Ad cytotoxic T lymphocytes. Thus, conventional Ad vectors are inadequate for CNS applications, which generally require safe gene transfer with stable long-term gene expression.

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ARTICLE It has been suggested that the toxicity and immunogenicity of conventional Ad could be caused, at least in part, by leaky expression of viral genes. Thus, further genetic modifications of these vectors are required and potentially could benefit clinical applications in the CNS (18, 19). A new generation of adenoviral vectors, socalled helper-dependent, or gutless, adenoviral vectors (hdAd), were developed to meet this need (20–25). These novel constructs lack almost the entire viral genome, excluding the noncoding sequences responsible for packaging and terminal repeat sequences, thereby avoiding any possibility of viral gene expression and extending the cloning capacity of the vector (up to 36 kb). The hdAd can be propagated in the presence of helper virus and grown to high titers, and their use in peripheral gene transfer studies has resulted in persistent transgene expression, leading to therapeutic effects without appreciable toxicity (23, 24, 26). For example, systemically administered hdAd indicated decreased toxicity toward the liver, the organ showing the greatest susceptibility to fgAd (21). Intravenous injection of mice with hdAd expressing human α1-antitrypsin (hAAT) led to stable serum levels of the protein for more than 10 months (23, 27), while hdAd-mediated expression of the leptin gene resulted in high serum levels of leptin for a longer period than could be achieved with fgAd (24). The effects of hdAd vectors in the CNS have not been characterized. We therefore studied hdAd-mediated gene transfer and expression in the brain, comparing the duration of transgene expression, transgene stability, and immune responses with results for a fgAd vector. The hippocampus was chosen as the virus inoculation site because of its relevance for therapeutic gene delivery to correct or alleviate aging- or disease-related memory loss. Our results indicate, apparently for the first time, that much stable and less toxic gene expression can be achieved in the CNS with hdAd-mediated gene transfer.

MATERIALS

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METHODS

Vector construction. To develop a fgAd vector harboring the βgeo gene, a product of β-galactosidase (βgal) and neomycin resistance gene fusion, we cleaved the βgeo expression cassette containing a strong mammalian pro-

moter, SRα (28), from the plasmid pSRα-βgeo (22), using the SalI restriction enzyme, and then inserted it into p∆E1sp1A (29) at the XhoI and SalI sites. The resulting shuttle plasmid, p∆E1sp1A/SRα-βgeo, was cotransfected with the DNA of Ad genomic plasmid pBHG10 into 293 cells as previously described (30). Homologous recombination between the shuttle DNA and pBHG10 DNA produced the fgAd vector Ad∆E1E3/SRα-βgeo (Fig. 1). The hdAd vector, Ad∆SRα-βgeo, was rescued in the recently developed E2T-Cre6 cell line (complementing Ad E1 and E2a, expressing Cre; Heshan Zhou, Lucio Pastore, and Arthur L. Beaudet, unpublished data). Briefly, the SRα-βgeo cassette was inserted into the pDelta28 plasmid, resulting in p∆SRα-βgeo, which was then converted into a hdAd vector in E2T-Cre6 cells by previously described methods (25) (Fig. 1). The fgAd was propagated in 293 cells, and hdAd was amplified in E2T-Cre6 cells. Both vectors were purified by two rounds of CsCl centrifugation (27) and passage through desalting columns (Econo-Pac, 10 DG; Bio-Rad, Hercules, CA). All vector preparations were evaluated by particle count, as determined by optical density measurements of DNA, and by their ability to express galactosidase activity (blue-forming unit, BFU) in 293 cells. The quantity of contaminated helper viruses in the hdAd final purification was determined by plaque-forming units according to the published method (31). Brain injection. Male Fischer-344 rats (200–250 g) were anesthetized with intramuscular injections of a combination of anesthetics (0.5 ml/kg body wt) that included 42.8 mg ketamine, 8.6 mg xylazine, and 1.4 mg acepromazine per milliliter. To prepare rats for injection of viral vectors, we drilled a burr hole into the head (secured in a stereotaxic frame) according to the coordinates of the hippocampus: 4.3 mm posterior to the bregma and 3.0 mm lateral to the sagittal suture. The needle was then inserted 3.0 mm below the dura, and viral vectors (5 µl, 1  108 particles per microliter diluted in PBS) were injected into the right hippocampus. In control animals, 5 µl PBS (vehicle) was injected. Histological preparation. Rats were deeply anesthetized with sodium pentobarbital and perfused transcardially with 200 ml PBS (0.1 M, pH 7.4), followed by 150 ml of fixative (4% paraformaldehyde in PBS). The brains were removed and postfixed overnight in the same fixative at 4C, followed by a 2- or 3-day immersion in 25% sucrose in PBS until the tissues completely sank. Serial coronal sections (30 µm) were cut at −20C in a cryostat. X-gal histochemistry. Free-floating brain sections (30 µm) were washed with three changes of PBS and then incubated at 37C for 2 h with 0.05% X-gal in 0.5 M Hepes buffer (pH 7.4) containing 1 M MgCl2, 5 M NaCl, 50 mM K3Fe(CN)6, and 50 mM K4Fe(CN)6. The incubation was stopped with PBS. The sections were counterstained with 0.05% cresyl violet for morphological observation. After slide mounting, the sections were examined under a microscope, and transgene expression was quantified. Briefly, every fourth section through the hippocampus along rostral to caudal orientation in each animal was analyzed. Images were captured into a computer that was equipped with image-analyzing software Optimas 6.5 (Optimas Development Group, Bothell, WA). Staining (dots or areas) in the images was automatically spotted according to a prede-

FIG. 1. Structure of the fgAd (Ad∆E1E3/SRαβgeo) and the hdAd (Ad∆SRα-βgeo). The Ad∆E1E3/SRα-βgeo construct is based on the Ad genomic plasmid pBHG10 with deletions of the E1 and E3 genes. The Ad∆SRα-βgeo contains only the inverted terminal redundancies (ITRs) and package region (Ψ) of adenovirus with all coding sequences deleted.

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ARTICLE fined threshold and the values (area size and density) were given by the software. Immunohistochemistry. Mouse anti-rat monoclonal antibodies ED1 (Serotec Inc., Raleigh, NC), CD3 (Pharmingen, San Diego, CA), and NeuN (Chemicon, Temecula, CA) were used as primary antibody to identify macrophages, T lymphocytes, and neurons, respectively. Sections of hippocampus were incubated overnight at 4C with primary antibody diluted in PBS containing 1.5% normal horse serum and 0.1% Triton X-100. Following three washes in PBS, the tissues were incubated at room temperature for 1 h with biotin-labeled horse anti-mouse antibody (Vector Labs Inc., Burlingame, CA) as the secondary antibody. After three PBS washes, the sections were incubated with ABC reagent for 1 h at room temperature. Positive cells were observed after 3 to 5 min of incubation in freshly prepared 3,3′-diaminobenzidine solution containing 0.03% hydrogen peroxide. Cell profiles for infiltrated macrophages and T lymphocytes within the hippocampus were counted. Viral DNA analysis. To monitor viral DNA after Ad transduction, hippocampus was dissected, removed, and homogenized for DNA isolation. DNA was purified by a standard technique (26) and subjected to polymerase chain reaction (PCR) amplification: two rounds of 40 cycles at 94C for 30 s, 60C for 30 s, and 72C for 60 s with AmpliTaq Gold enzyme in a PTC-200 Peltier thermal cycler (M. J. Research, Inc., Watertown, MA). One primer in the βgal coding sequence (βgal 2861, 5′ACT TCC AGT TCA ACA TCA GC) and another starting at the fusion site of βgal and neomycin (geofus, 5′-TCC CAT ATT GGC TGC AGC CC) were used to amplify the βgeo gene in viral vectors. The expected PCR product size is 236 bp. Statistics. The results of X-gal staining were quantified by determining the densities and areas of staining in each section. Staining intensity for each section was obtained by multiplying of these two values, and total staining intensity for each animal was produced by summarizing the staining intensity value of each section. Data were presented as percentages of the maximal staining intensity achieved with the hdAd vector. Mean staining intensities (with standard deviation) were calculated from pooled data from four or five rats. For quantification of macrophages and T lymphocytes, cell profiles were counted in eight sections, collected around the needle tracks, from each animal (total 32–40 sections from each group comprising four or five rats). Differences between groups were evaluated with an unpaired t test. A two-tailed P value of less than 0.05 was considered statistically significant.

RESULTS Titer and Purity of Adenoviral Vectors Produced in the E2T-Cre6 Cell Line After purification of fgAd and hdAd, titration studies showed that both vectors were concentrated at about 1.0  1012 particles per milliliter (fgAd 9.7  1011 and hdAd 1.1  1012). BFU numbers were 1.1  1011 and 1.0  1011 for fgAd and hdAd, respectively; that is, about 10 particles of both vectors represented one BFU in 293 cells after overnight infection. The helper virus (E1-deleted adenovirus) contamination in the hdAd preparation was only 4  104 plaque-forming units (PFU) per milliliter. In other words, for every 1  106 BFU vectors, there was less than one PFU of helper virus. These results indicate that the hdAd vector prepared from E2T-Cre6 cells substantially decreased helper virus contamination, compared with a previously published study in which use of the 293Cre4 cell line yielded 1  102 helper viruses for every 1  106 vectors (25). MOLECULAR THERAPY Vol. 2, No. 2, August 2000 Copyright  The American Society of Gene Therapy

Pattern of hdAd-Mediated Transgene Expression in the Hippocampus Since transgene expression peaked at 6 days after hdAd-mediated βgeo gene transfer in our experiments, we chose this time point to examine the distribution and pattern of transgene expression. After hdAd injection into the hippocampus, the βgal activity was found in the hippocampus, with most staining concentrated in the hilus and granular cell layer of the dentate gyrus. A typical distribution pattern is shown in Figs. 2A and 2B. Some cells in the pyramidal cell layer of the CA1 region also become stained (Figs. 2C and 2D). Because of the lack of a nuclear localization sequence in the βgeo expression cassette, the transgene product heavily diffused outside the cells, making it difficult to identify transduced cell types in the intensely stained areas (Fig. 2A). However, according to their anatomical locations, the transduced cells should include neurons and glial cells. Indeed, neuronal involvement was demonstrated by immunohistochemical staining with neuron-specific NeuN (Fig. 2E). Inoculation of fgAd led to a similar distribution of βgal activity (data not shown). Although staining was observed most often in the hippocampus, some positive cells were also seen in distal areas, such as the basal magnocellular nucleus (Fig. 2F), which may send axonal projections to the hippocampus.

Duration of Transgene and the Presence of Transgene DNA We next studied the duration and temporal changes of transgene expression mediated by fgAd and hdAd. Following Ad inoculation, transgene expression was assessed by X-gal histochemical staining at 3, 6, 16, 33, and 66 days. On day 3, the earliest time point studied, three of four rats injected with fgAd showed X-gal staining, compared with two of four receiving hdAd. Overall, the staining intensity was much weaker at this time point relative to that on day 6, in agreement with the recognized delay in Ad-mediated transgene expression (Ref. 13; also our unpublished data). Six days after virus injection, transgene expression increased dramatically, with all animals showing X-gal staining, whether they received fgAd or hdAd (Fig. 3). Quantitative analysis of transgene expression on day 6, based on staining intensity, indicated an approximate 10-fold increase compared to day 3 in transgene expression mediated by either fgAd or hdAd (Fig. 4A). Over the subsequent time points, fgAd-induced transgene expression diminished rapidly and disappeared altogether by day 66. By contrast, hdAdmediated transgene expression remained fairly strong through day 33, with a decline to approximately 50% of the maximal value apparent at 66 days postinoculation (Figs. 3 and 4A). Differences in X-gal staining between the hdAd- and fgAd-injected groups on days 16, 33, and 66 were statistically significant. To further characterize transgene duration, we determined the presence of transgene DNA by PCR analysis. As

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FIG. 2. Microphotographs showing histochemical staining for β-galactosidase in the hippocampus of Fischer-344 rats 6 days after hdAd-mediated βgeo gene transfer. (A) X-gal staining in dentate gyrus of the hippocampal region, double stained with NeuN immunohistochemistry. Note stained cells in the upper granular cell layer (comprising neuron) and innermost region (comprising primarily glial cells) of the lower granular cell layer. (B) Staining in the granular cell layer and transduced cells in the hilus. The section was counterstained with cresyl violet. (C) NeuN immunohistochemistry demonstrated transduced neurons (indicated by arrows) in the pyramidal cell layer of the CA1 region. (D) Staining cells in the pyramidal cell layer of the hippocampal CA1 region, counterstained with cresyl violet. (E) Stained cells in hilus; some of them (indicated by arrows) exhibit neuronal phenotype as shown by NeuN immunohistochemistry. (F) Scattered cells in the distal region, basal magnocellular nucleus, counterstained with cresyl violet. Scale bars, 100 µm in C and 50 µm in F. A–D are at the same magnification, and E and F are at the same magnification. G, granular cell layer. H, hilus.

shown in Fig. 4B, at 3 days postinoculation, strong DNA signals were generated in both the fgAd- and hdAd-injected groups. However, at 66 days following virus inoculation, the fgAd DNA band was no longer detectable, while the hdAdderived DNA signal remained relatively strong, although it

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was somewhat attenuated compared to the result obtained with day 3 samples. These findings indicate that both types of Ad vectors were subject to DNA clearance after transduction. Nonetheless, transgene DNA associated with the hdAd vector was much more stable than its fgAd counterpart. MOLECULAR THERAPY Vol. 2, No. 2, August 2000 Copyright  The American Society of Gene Therapy

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FIG. 3. Microphotographs showing transgene expression in the hippocampus of Fischer-344 rats after inoculation of the fgAd (A, C, E) or hdAd (B, D, F). Transgene expression was visualized with X-gal staining at various time points: 6 days (A, B), 33 days (C, D), and 66 days (E, F). The sections were counterstained with cresyl violet. Note that fgAd-mediated transgene expression diminished rapidly over time and was undetectable at day 66 postinoculation. By contrast, hdAd-mediated transgene expression remained relatively stable over the same periods. All panels were photographed at the same magnification. Scale bar, 200 µm.

Immune Responses and Tissue Damage Induced by Ad Inoculation Ad-induced host immune responses, especially cellular immune responses, are major factors limiting the application of Ad as gene transfer vectors (13, 14, 17). To characterize fgAd- and hdAd-mediated immune responsMOLECULAR THERAPY Vol. 2, No. 2, August 2000 Copyright  The American Society of Gene Therapy

es, we monitored the infiltration of macrophages and T lymphocytes in rat brain, using ED1- and CD3-based immunohistochemical methods, respectively. After Ad inoculation into brain, significant numbers of macrophages were apparent as early as 3 days in both fgAd- and hdAd-injected animals compared to PBS control (Figs. 5A, 5C, and 5E). On day 6, when transgene

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doi:10.1006/mthe.2000.0057, available online at http://www.idealibrary.com on IDEAL

early as 16 days after fgAd but not hdAd inoculation. These cells had increased in number by day 33 in fgAdinjected animals (Fig. 5D), declining to lower but still detectable levels by day 66 (Fig. 6B). In contrast, only one animal inoculated with hdAd showed lower-level infiltration of T lymphocytes on day 33 (Fig. 5F). No T lymphocytes were observed in the PBS-injected samples (Fig. 5B). Counterstaining of brain sections with cresyl violet revealed tissue damage and gliosis around the transduced area at 33 and 66 days postinoculation of fgAd (Fig. 5H), contrasting with intact tissues exposed to hdAd (Fig. 5G), excluding an injection-related trauma lesion.

FIG. 4. Duration of transgene expression and transgene DNA. (A) Timedependent transgene expression in rat hippocampus. The transgene was delivered by either fgAd or hdAd vector. X-gal staining was quantified by determining and converting the data into percentages of the maximum staining value obtained with hdAd-injected samples. Four or five rats were used in each group. Data were presented as means ± SD. *P < 0.05 and **P < 0.01 for hdAd vs fgAd. (B) Detection of transgene DNA in the hippocampus after fgAd- or hdAd-mediated βgeo gene transfer. At either 3 or 66 days after virus inoculation, the hippocampus was harvested by dissection. Total DNA was subjected to two rounds of PCR amplification with primers corresponding to one fragment (236 bp) of the βgeo gene. Lane descriptions: P (positive control), DNA prepared from hdAd particles; N (negative control), DNA isolated from the contralateral, uninjected side of the hippocampus 3 days after hdAd injection; Day 3, DNA isolated from the hippocampus 3 days after injection of the indicated virus types (three rats); Day 66, DNA isolated from the hippocampus 66 days after injection of the indicated virus types (two rats). Note that transgene DNA was not apparent on day 66 after fgAd-mediated gene transfer, whereas a DNA band remained at this time point in hdAd-injected samples.

expression peaks, macrophage numbers were strikingly increased in hippocampus injected with fgAd, but not hdAd (Fig. 6A). At later time points, hdAd-induced macrophage infiltration dramatically declined, whereas fgAd induced slightly decreased numbers of macrophage after 16 or 33 days postinoculation. On day 66, macrophages were still observed in fgAd-inoculated rat brains, but had disappeared in tissue inoculated with hdAd (Fig. 6A). Starting day 6, the numbers of infiltrated macrophages between animals inoculated with fgAd or hdAd were statistically different. CD3 immunostaining demonstrated the presence of infiltrating T lymphocytes around the injection site as

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FIG. 6. Quantification of immune infiltration in hippocampus after injection of 5 µl PBS, fgAd, or hdAd. Macrophages (A) and T lymphocytes (B) are counted as profiles present in a 30-µm-thick section at indicated time points. Eight sections were counted in each animal, and values were means ± SD of the profile numbers from four to five rats. *P < 0.05, **P < 0.01 vs fgAd.

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ARTICLE DISCUSSION Our results demonstrate that prolonged transgene expression can be achieved in rat brain with use of the hdAd vector. Based on the results of X-gal histochemical staining, hdAd-mediated βgeo gene expression lasted for at least 66 days; even longer periods of expression might be expected since the βgal activity remained half at the peak level. However, fgAd-mediated βgeo gene expression was not detectable on day 66, although it was readily found on day 33, suggesting a shorter expression period associated with this vector type. These results agree with other studies (32) demonstrating limited duration of fgAd-mediated transgene expression. Stable long-term transgene expression is critical for the development of gene therapy to treat CNS structural and functional deficits. For example, the recovery of aging-related memory loss requires at least 2 months of stable expression of the neurotrophin gene (33), while the correction of behavioral abnormalities in animal models of Parkinson’s disease, Alzheimer’s disease, or amyotrophic lateral sclerosis requires even longer-term expression of therapeutic transgenes (34). Thus, prolonged transgene expression mediated by hdAd should benefit the development of gene therapy for CNS disorders. Safety has become an extremely important issue in current gene therapy protocols for clinic applications (35). Consistent with other reports of negligible hdAdinduced toxicity in peripheral organs (23), our results indicate improved safety with hdAd vectors. That is, the numbers of macrophages and T lymphocytes infiltrating the brain were greatly reduced in hdAd-treated versus fgAd-treated animals, and there was no demonstrable tissue damage after injection of the hdAd vector. Although virus-related macrophage infiltration was observed (compared to the PBS control, additional macrophages were present) after hdAd inoculation, such infiltration was attenuated dramatically and only a small number of macrophages were seen after day 6. According to Smith and Eck (36), immunity to Ad-mediated gene transfer develops in two stages: an initial inflammatory response characterized with macrophage recruitment and a late antigen-specific cellular and humoral response involving T and B cell activation. It has been further suggested that the initial viral entry or virion contact with target cells activates early recruitment of macrophages (37), while the novel epitopes, either from a certain transgene product or from leaky expression of viral genes, are recognized as antigens and initiate a cellular immune response (38). Therefore, early macrophage infiltration observed in this study may reflect a response of host cells to viral contact and entry. Furthermore, since transgene expression is increased, rather than decreased, within the immediate postinoculation period (up to day 6), it can be reasonably hypothesized that the early recruitment of macrophages does MOLECULAR THERAPY Vol. 2, No. 2, August 2000 Copyright  The American Society of Gene Therapy

not strongly impact transgene expression. In contrast to hdAd, fgAd inoculation caused persistent macrophage infiltration, up to 66 days, and the numbers of macrophages were significantly greater than that in hdAd-inoculated animals. This indicates a severe immune response after fgAd inoculation. Indeed, T lymphocytes were observed in the injected sites beginning on day 16. These reactions are presumably due to expression of viral genes (36) and responsible for the rapid elimination of transgene DNA and the decline of transgene expression associated with the vector type. Despite removal of all viral coding sequences, small numbers of immune cells were still observed in hdAdinjected brain tissue after 6 days. This suggests that immune responses may have occurred against the transgene product (βgeo) in addition to viral proteins. βgal, one component of βgeo, has been reported to be immunogenic in rodent and contributes to immune responses after Ad-mediated gene transfer (15, 20). Replacement of the βgeo reporter gene with an endogenous therapeutic gene would likely decrease or eliminate immune responses against the transgene product. Alternatively, the level of helper-virus contamination associated with the hdAd vector used in the present study, which was purified from a new Cre-expressing cell line, E2T-Cre6, is estimated to be about 1  10−6. The presence of trace amounts of helper virus may have contributed to part of the observed immunity. This obstacle could be circumvented by further refinement of the vector-producing system to remove all helper viruses from the hdAd preparation. Some authors (39, 40) have reported that transgene expression from fgAd inoculated in brain lasts for more than 2 months, in apparent contrast to our observation and another study (32) of a shorter duration of fgAdmediated transgene expression. The discrepancy can be attributed to the dosage difference of inoculated viral vectors (39) or the difference in promoter used to drive the transgene (40). It has been suggested that a higher dose of viral vector induces a more striking immune response, leading to shorter duration of transgene expression (41). Because of the frequent need to induce high levels of transgene expression in clinical gene therapy, it is necessary to study the effect of gene transfer in animal models using high dosages of vectors. We used 5  108 viral particles of both fgAd and hdAd in this study, a dose which is higher than that used in another report (39). One of the important findings is a prolonged transgene expression from hdAd compared to results with fgAd at the same (high) dosage of vectors. Furthermore, there was no demonstrable tissue damage after hdAd inoculation, even at such a high dose, indicating a higher tolerance for this vector. The higher hdAd tolerance provides a significant “Ad”vantage for future clinical applications. Taken together, our results indicate that hdAd vectors, as described in this report, would lessen the risk of serious toxicity associated with clinical applications of

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FIG. 5. Microphotographs showing infiltration of immune cells and tissue damage. Three days postinoculation of PBS (A), fgAd (C), and hdAd (E), macrophage infiltration was seen at the injection site within the injected hippocampus. T lymphocytes infiltrate into the injected side of the hippocampus 33 days after inoculation of fgAd (D) or hdAd (F), but not in the hippocampus receiving PBS injection (B). Note that the numbers of infiltrating macrophages and T lymphocytes were substantially greater after fgAd inoculation than the hdAd-inoculated samples. There are no detected T lymphocytes in the PBS-injected animals. Tissue damage (marked by arrowheads) and gliosis (indicated by arrows) were also evident 33 days after inoculation of fgAd (H), but not hdAd (G). Scale bar, 50 µm.

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ARTICLE conventional Ad vectors. The immune responses induced by Ad are a critical factor limiting transgene expression in the brain. The prolonged transgene expression and significantly attenuated immune response observed in hdAd-mediated gene transfer to the CNS lead us to conclude that hdAd is an improved type of adenoviral vector for CNS gene transfer. ACKNOWLEDGMENTS We thank Xiaoqing Yuan for assistance in animal surgery and Yayun Zheng and Tiejun Zhao for preparation of the vectors. We are grateful to Dr. Malcolm K. Brenner and Dr. David H. Shine for critical reading of the manuscript and John Gilbert for valuable editing of the manuscript. This work was supported by grants from the National Institutes of Health (RO1-NS35502-02 and RO3AG16033-01 to K.Y.) and Texas Higher Education Coordinating Board Grants ATP 004949-049 and 0159 (to K.Y.) and ATP 004949-0119 (to H.Z.).

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