Development of an adenoviral vector for intracerebral delivery of the dopamine D2 receptor

Mechanisms of Ageing and Development 116 (2000) 77 – 93 www.elsevier.com/locate/mechagedev

Development of an adenoviral vector for intracerebral delivery of the dopamine D2 receptor Donald K. Ingram a,*, George S. Roth a, Hiroyuki Umegaki b, Hiroyuki Ikari c a Molecular Physiology and Genetics Section, Laboratory of Cellular and Molecular Biology, Gerontology Research Center, National Institute on Aging, 5600 Nathan Shock Lane, Baltimore, MD 21224, USA b Department of Geriatrics, Uni6ersity of Nagoya School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagayo 466 -8550, Japan c Fukushimura Hospital, 19 -14 Aza-nakayama, Noyori-cho, Toyohashi, Aichi 441 -8124, Japan

Received 3 December 1999; accepted 26 March 2000

Abstract The age-related loss of striatal dopamine D2 receptors (D2R) has been observed in numerous species, including rodents, monkeys, and man, and is partly responsible for impaired motor function in aged mammals. We have developed an adenoviral vector designed for intracerebral transfer of cDNA for D2R. Results of in vitro studies demonstrated that the vector produced abundant message for D2R and that the vector was membrane bound and capable of binding appropriate ligand. Results of in vivo studies provided clear evidence of D2R production when injected into the striatum of rats. The D2R produced were capable of binding appropriate ligand. In addition, evidence of functional receptors was produced by demonstrating apomorphine-induced rotational behavior in rats receiving a unilateral injection of the vector. Despite these successes, we have been unable to demonstrate improvement in the motor behavior of aged rats receiving bilateral injections of the vector. A major problem with this vector as with similar adenoviral vectors is the loss of expression beginning 3–5 days after injection to undetectable levels at 21 days. Because of the lack of motor functional effects in aged rats and the loss of expression of the vector, other strategies for development of the vector are being pursued. Regarding functional effects, we have examined the feasibility of manipulating hippocampal acetylcholine (ACh)

* Corresponding author. Tel.: +1-410-5588180; fax: +1-410-5588323. 0047-6374/00/$ - see front matter © 2000 Published by Elsevier Science Ireland Ltd. PII: S 0 0 4 7 - 6 3 7 4 ( 0 0 ) 0 0 1 1 3 - 5

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release through D2R manipulation to improve memory performance. Using microdialysis, we have demonstrated in vivo in rats that treatment with a D2R agonist increases hippocampal ACh release while treatment with a D2R antagonist attentuates this effect as well as impairs performance in a complex maze task. In addition, a D2R null mutant mouse is being used to examine possible therapeutic effects of the vector. These mice show specific motor deficits. Recent studies using positron emission tomography have also demonstrated the feasibility of in vivo imaging of the vector. Thus, use of adenoviral vectors specific for neurotransmitter receptors can provide a highly useful research tool for examining age-related alterations in behavioral function and a possible strategy for therapeutic intervention. © 2000 Published by Elsevier Science Ireland Ltd. Keywords: Gene therapy; Parkinson’s disease; Huntington’s disease; Motor performance; Neurotransmitter receptor; Striatum; Memory; Acetylcholine; Mutant mice

1. Introduction During the last decade strategies to produce viral vectors for brain-targeted gene therapy have emerged (Suhr and Gage, 1993; Lesch, 1999). Vectors have included those made from herpes simplex virus (HSV), vaccinia virus, adenovirus (Ad), and adenoassociated virus (AAd). The use of Ad vectors offered several advantages over other types of viral vectors developed for gene therapy, including the following: (1) the ability to be rendered replication-deficient; (2) the ability to accept large pieces of DNA; (3) production in high titers; (4) extrachromosomal nuclear location; and (5) lack of association with human neoplasms (Crystal, 1992). The feasibility of using Ad vectors for intracerebral gene transfer was also supported in many studies. For example, several early studies using Ad vectors constructed with lacZ as a reporter gene documented high levels of expression in brain that were maintained several weeks after injection (Akli et al., 1993; Bajocchi, et al., 1993; Davidson et al., 1993; Le Gal La Salle et al., 1993). Functional effects could also be demonstrated following intracerebral gene transfer using Ad vectors. For example, an Ad vector producing human glial cell line-derivied neurotrophic factor injected into rat substantia nigra was shown to protect against a neurotoxic insult directed at dopamine neurons (Choi-Lundberg et al., 1997). Our efforts have been directed toward developing an adenoviral vector capable of intracerbral delivery of a functional neurotransmitter receptor (Ikari et al., 1995, 1999; Umegaki et al., 1997; Ingram et al., 1998). We have focused on the dopamine D2 receptor (D2R). The age-related loss of dopamine D2 receptors (D2R) in the striatum is one of the most well documented features of brain aging (Morgan and Finch, 1988; Joseph et al., 1990). Decreased motor function is associated with a decline in this receptor (Morgan and Finch, 1988; Joseph et al., 1990). D2R loss is also observed in age-related neurodegenerative diseases, such as Huntington’s disease (Reisine et al., 1977) and in the late stages of Parkinson’s disease (Rinne et al., 1979, 1991). Increasing the concentration of striatal D2R by pharmacological means can produce beneficial effects (Joseph and Roth, 1993). For example, treatment with

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estrogen or prolactin produces D2R upregulation and partial restoration of motor function in aged rodents (Joseph and Roth, 1988, 1993). While the feasibility of such manipulations has been established, systemic treatments of this type do not provide a long-term theraputic strategy because of possible side effects associated with their lack of specificity for D2R in targeted brain regions. It was the hoped that intracerebral gene therapy could provide a more specifically targeted treatment and a potentially longer duration of action after a single treatment compared to continual application of pharmacological treatments. With this objective, we have investigated the use of an Ad vector encoding the cDNA for the rat D2R (Ikari et al., 1995, 1999; Umegaki et al., 1997; Ingram et al., 1998)

2. Vector construction Details of the vector construction are provided in an earlier report (Ikari et al., 1995). In general, the recombinant Ad vectors were produced using standard procedures described previously (Rosenfeld et al., 1991, 1992; Maeda et al., 1994). The vector was derived from an Ad type 5 in which the majority of the E1 and a portion of the E3 regions had been deleted, and an expression cassette containing the rat D2R cDNA (Bunzow et al., 1988) along with the cytomegalovirus (CMV) immediate early promotor and enhancer was inserted at the site of the E1 deletion. Two vectors were constructed: (1) AdCMV.DopD2R, containing the rat D2R cDNA, and (2) AdCMV.Null, a similar construct, missing the D2R cDNA, as a control.

3. In vitro expression By first using HeLa and HS24 cell lines, which normally do not express D2R, the viability of the vectors could be confirmed in vitro (Ikari et al., 1995). RNA was extracted from the infected cultures at 3 or 5 days post-infection. Northern analysis was used to demonstrate abundant mRNA for D2R 3–5 days after infection with AdCMV.DopD2R in both cell lines, while no message was detectable in control cultures infected with AdCMV.Null. The ability of the AdCMV.DopD2R to produce a neurotransmitter receptor could also be established in vitro. Expression of protein for D2R was examined in membrane preparations extracted from HeLa cell cultures 4 days after vector infection (Ikari et al., 1995). A membrane binding assay was completed using the D2R ligand, [3H]spiperone. In membranes taken from cultures infected with AdCMV.DopD2R, specific binding for [3H]spiperone was very evident but could not be detected in uninfected cultures nor in control cultures infected with AdCMV.Null. Thus, high levels of protein targeted to the cytoplasmic membrane and capable of binding a D2R specific ligand could be demonstrated.

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4. In vivo expression Several experiments were next accomplished to demonstrate the in vivo utility of the vector. As in the first experiment, the vector (5 ml) was injected into the striatum of young (3 months) male Sprague–Dawley (SD) rats (Ikari et al., 1995). Five days after injection, the rats were sacrificed, and their brains were prepared for [3H]spiperone autoradiography. As observed in Fig. 1, a marked increase in [3H]spiperone binding was detected at the site of striatal injection and along the injection tract in rats injected with AdCMV.DopD2R. In the contralateral striatum of the same rats, no alteration in [3H]spiperone binding could be observed following injections of AdCMV.Null or vehicle. Following histological examination of the striatal tissue, some degree of degeneration associated with the injections could be detected in both sides, but no gross pathological abnormalities were evident. However, the pattern of striatal expression of D2R revealed a lack of cellular specificity as binding of [3H]spiperone was observed in both neurons and glia. Previous studies of

Fig. 1. Autoradiographic images of [3H] spiperone binding at 5 days after injection of AdCMV.DopD2R, AdCMV.Null, or cannula implantation in young Sprague – Dawley rats. Intense concentration of D2R (A, B, C) is noted at site of injection (dark arrows) and in neocortex along cannula track (white arrow in A) of AdCMV.DopD2R injected sites. Contralateral sites receiving AdCMV.Null (A) or no treatment (B, C) exhibit no increased concentration of [3H] spiperone binding at site of injection or cannula track. Displacement of [3H] spiperone binding by (+)-butaclamol confirms specificity of [3H] spiperone for D2R binding sites.

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intracerebral gene transfer had noted this lack of cellular specificity using an Ad vector driven by the CMV promoter (Davidson et al., 1993).

5. Functional assessment in young Sprague–Dawley rats Despite the lack of neuronal specificity of the AdCMV.DopD2R vector, relevant D2R function could be assessed in a well-established behavioral paradigm, the apomorphine-induced rotational model (Pycock, 1980). This model has been used to demonstrate assymetrical distribution of striatal D2R in rats, typically following a unilateral injection of 6-hydroxydopamine (6-OHDA) into the substantia nigra. This neurotoxin markedly reduces dopaminergic innervation to the striatum. Ipsilateral upregulation of striatal D2R occurs after a few days. Following a peripheral injection of the dopamine agonist, apomorphine, a 6-OHDA-lesioned rat will display a stereotypic rotational behavior in the direction contralateral to the lesioned side where an upregulation of D2R is observed. The rotational model had been used to demonstrate the functional viability of an Ad vector delivering the cDNA for human tyrosine hydroxylase, which is a synthetic enzyme for the production of dopamine (Horellou et al., 1994). Unilateral injections of the AdCMV.DopD2R vector were made into the striatum of young (3 months), male SD rats with a control injection of either vehicle or AdCMV.Null made into the contralateral site. The side of injection of the AdCMV.DopD2R (2 ml) into each rat was selected randomly such that about half received injections into right striatum and half into left striatum. The predicted effect was to observe the rat rotating in a contralateral rotation when injected with apomorphine. In the first studies injection of AdCMV.DopD2R vector was targeted at the dorsomedial striatum. This site was selected to achieve maximum diffusion of the vector throughout the striatum without infecting other brain structures. When apomorphine injections (1 mg/kg) were begun about a week later and repeated on a weekly basis, rotational behavior became evident beginning  4 weeks after injection in the groups injected with AdCMV.DopD2R, and the rotations were in the predicted directions (Ingram et al., 1998). A control group that had received bilateral striatal injections of either AdCMV.Null or vehicle displayed no lateralized rotational behavior. Although some individual rats in the group showed lateralized rotations, the control group did not exhibit the rotational bias clearly observed in the experimental groups. Although this first set of results appeared to be a successful demonstration of adenoviral-mediated receptor function, other results did not support this view. After these rats were sacrificed 9 weeks following vector injection and their brains were processed for [3H]spiperone autoradiography, no evidence of a D2R upregulation could be observed in the striatum injected with AdCMV.DopD2R. Instead there was evidence of limited neurodegeneration in the lesion sites in many striatal slices. Thus, it was possible that the rotational behavior observed could have resulted from vector-induced lesions and not from the direct action of vector-generated D2R.

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Fig. 2. Autoradiographic images of [125I]iodosulpride binding in striatal slices from Fischer-344 (F344) and Sprague–Dawley (SD) rats. Greater binding (darker images) is observed near striatal sites of AdCMV.DopD2R injection (left side for F344 rats; right side for SD rats on this figure) compared with contralateral AdCMV.Null striatal injection. Reduced D2R expression is observed as a function of day after injection.

6. Time-course of vector expression in young Sprague-Dawley and Fischer-334 rats The time-course of D2R expression following vector injection was investigated in young rats from two strains, SD and Fischer-344 (F344). Fig. 2 presents the changes in expression as visualized using autoradiography with [125I]iodosulpride, which is a more specific D2R ligand than [3H]spiperone. D2R expression was quantitated by densitometry over several striatal slices, and these results are presented in Fig. 3 (Umegaki et al., 1997). D2R expression was highest  3–5 days

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after injection of AdCMV.DopD2R, after which expression fell to baseline levels by 21 days after injection. Results from several earlier studies of intracerebral Ad vector administration documented prolonged expression of protein over several months (Akli et al., 1993; Le Gal La Salle et al., 1993); however, other studies had noted a more limited period of vector expression. It appeared that expression of a targeted protein endured a few weeks at best (Davidson et al., 1993; Byrnes et al., 1995). Among the reasons suggested for the rapid loss of expression are cellular immunity (Yang et al., 1994), downregulation of viral promoters (Choi-Lundberg et al. 1997), and neurotoxicity induced by viral particles (Peltekian et al., 1997).

7. Further functional assessment in young Fischer-344 rats To validate the function of the vector-produced D2R, it thus became necessary to demonstrate a functional effect within 3–14 days after vector injection. As a change to the previous protocol, a more ventral striatal site was selected for vector injection. Based on a systematic analysis of the 6-OHDA model, Koshikawa et al. (1990) had noted the ventral striatal region was more sensitive to apomorphine-in-

Fig. 3. D2R expression in Sprague–Dawley (SD) and Fischer-344 (F344) rats as function of days after vector injection. For each time-point, the mean density of grains in the autoradiographic images using [125I]iodosulpride as a ligand was estimated. The ratio of the means (AdCMV.DopD2R-injected striatum/AdCMV.Null-injected striatum) 9 S.E.M. is presented. Two representative striatal slices that exhibited the highest levels of expression were taken from four rats of each strain for each time-point.

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Fig. 4. Apomorphine-induced rotational behavior reflects the functionality of increased D2R expression in F344 rats. Represented are group means (9S.E.M.) for the net rotations (number of turns ipsilateral to the AdCMV.DopD2R-injected striatum subtracted from the number of turns contralateral to the AdCMV.DopD2R-injected striatum) induced by apomorphine (1 mg/kg s.c.). Experimental groups (n = 10) received unilateral striatal injection of AdCMV.DopD2R and contralateral injection of AdCMV.Null. R: D2R refers to AdCMV.DopD2R injected into right striatum and AdCMV.Null injected into left striatum. L: D2R refers to AdCMV.DopD2R injected into left striatum and AdCMV.Null injected into right striatum. Control refers to AdCMV.Null injected bilaterally (n =11). *PB 0.05 compared with control according to two-tailed t-test.

duced rotational behavior compared to other striatal sites. To reduce the risk of procedure-induced lesions at this site, a slower infusion of the vectors (2 ml over 10 min compared to 2 ml over 5 min in previous studies) was implemented in young (2–3 months) F344 rats (Umegaki et al., 1997). The side of the striata injected with AdCMV.DopD2R was balanced within experimental groups with the contralateral side receiving an injection of AdCMV.Null. Control groups were injected bilaterally with the AdCMV.Null vector. Apomorphine injections were administered 3 and 7 days after vector injection. As presented in Fig. 4, a lateralized rotational pattern in the predicted contralateral direction was evident in the experimental group receiving unilateral injections of AdCMV.DopD2R; whereas, in the control groups, no directional rotational bias was recorded.

8. Functional assessment and expression in aged rats The next challenge was to determine if motor function in aged rats could be improved following intracerebral delivery of the AdCMV.DopD2R vector. To meet this objective, a preliminary experiment was conducted in which aged F344 rats (21– 25 months) received bilateral striatal injections of either AdCMV.DopD2R or AdCMV.Null vectors or served as unoperated controls (Ingram et al., 1998). The

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rats were assessed in a motor battery 4–6 days following vector injection. Tests in this battery have proven highly useful for demonstrating age-related declines in performance of rats (Spangler et al. 1994). As observed in Fig. 5, no evidence emerged of significant performance improvments following striatal injection of the AdCMV.DopD2R vector. Compared with the unoperated controls in the rotarod (Fig. 5A) and inclined screen (Fig. 5B) tests,

Fig. 5. Comparison of motor performance (rotarod, inclined screen, and wire hang) of aged (21 – 25 months) Fischer-344 rats receiving bilateral striatal injections of AdCMV.DopD2R (n =7) and controls receiving AdCMV.Null (n= 4) or unoperated controls (n =4). Dependent variable in the rotarod test was the number of falls made during a 3-min period of placement on a cylinder rotating at 3 rpm. In the inclined screen test, dependent variable was the time spent clinging with all four paws to a wire mesh screen titled 60° from horizontal. For the wire hang test, the dependent variable was the mean time spent suspended by the front paws from a small metal rod during three trials.

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Fig. 5. (Continued)

no significant improvement was observed in groups receiving bilateral striatal injections of either AdCMV.DopD2R or AdCMV.Null. In the wire hang test (Fig. 5C), however, the two groups receiving Ad vector injections experienced significantly improved performance compared to unoperated controls, but did not differ significantly from each other. Several factors could account for the failure to observe functional improvement in aged rats injected with AdCMV.DopD2R, including the following: (1) lack of specificity for cell type infected; (2) lack of sufficient expression in the appropriate striatal loci; (3) or the reduced expression over time observed previously may be been even greater in the aged brain. To determine the extent that reduced expression was a factor, another time-course experiment was conducted to compare D2R expression in young (3 months) and aged (21–22 months) F344 rats. These results are presented as a linear regression analysis in Fig. 6. The regression across time was significant only for young rats; thus, it was apparent that the aged rats exhibited a longer duration of D2R expression compared to the young group over this period of observation. During the time of behavioral assessment shown in Fig. 5, expression of vector-produced D2R would have been maintained in the aged brain. Explanations for this age difference in duration of expression are not forthcoming. One possibility is that the inflammatory response to the vector in aged rats was less than that mounted in the brains of young rats.

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9. Hippocampal D2R involvement in acetylcholine release and learning Given the failure to improve motor performance in aged rats through striatal injections of the AdCMV.DopD2R vector, we have been examining new sites for assessment in rats. One possible target is the ventral hippocampus where D2R involvement in acetylcholine (ACh) release had been demonstrated previously (Wilkerson and Levin, 1999). Recently in a study using microdialysis, we have confirmed that infusion of the D2R, agonist, quinpirole, into the ventral hippocampus of young F344 rats will increase ACh release, and that this increase can be attenuated by treatment with the D2R antagonist, eticlopride (Umegaki et al., 1999). As a functional assay for this new line of research, we have used the Stone maze, which is a shock-motivated 14-unit T-maze that has proven to be highly sensitive to aging and manipulation of the cholinergic system in rats (Ingram et al. 1994; Ingram et al., 1996). Infusion of the D2R antagonist, raclopride, into the ventral hippocampus of young F344 rats will impair learning in this maze task, and the impairment can be attenuated by infusion of quinpirole (Umegaki et al., 1999). Thus, we have shown that the ventral hippocampus could be a potential target for intracerebral gene transfer of D2R.

Fig. 6. Time-course of increased D2R expression in young (3 months) and aged (22 months) Fischer-344 rats receiving unilateral striatal injection of AdCMV.DopD2R. At each time point, the mean density of grains in the autoradiographic images using [125I]iodosulpride as a ligand was estimated. The ratio of the means (AdCMV.DopD2R-injected striatum/AdCMV.Null-injected striatum) is presented. Three representative striatal slices that exhibited the highest levels of expression were taken from two to three rats of each age for each time-point. Linear regression analysis suggested that only the young group showed a significant linear decline across time, P B0.05, but the regression was not significant for the aged group, P\ 0.05.

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What we nor anyone else, to our knowledge, have established is whether there is in fact an age-related loss of D2R in this brain region, but these studies are also being planned.

10. D2R null mutant mice New experiments planned in a D2R null mutant (knock-out) mouse will provide additional opportunities for assessment of the AdCMV.DopD2R vector. Use of this mouse will permit us to evaluate whether increased D2R expression can be linked to specific behavioral performance that could then be evaluated in normal, aged rats. Two such D2R null mutant mice have been produced. One group has reported that D2R null mice exhibit akinesia and bradykinesia in motor tests and reduced spontaneous locomotion (Baik et al., 1995). Another has reported chronic hyperprolactinemia related to loss of dopamine inhibition over pituitary hormone secretion (Kelly et al., 1997) as well as impaired motor function that was somewhat dependent upon genetic background (Kelly et al., 1999). In preliminary studies with a D2R knock-out mouse on a 129/Sv × C57BL/6 background, we have observed distinct motor performance deficits that resemble age-related impairments previously noted in rodents. Specifically, as presented in Fig. 7, the D2R null mutant mouse exhibit decreased levels of locomotor activity and an increased number of falls in a rotarod task compared to wild-type controls. These observations are consistent with previous observations in these mice (Kelly et al., 1999). If we can demonstrate that striatal injections of the D2R can restore motor function in these D2R null mice, then such evidence would provide additional rationale for examining more specific responses in aged rats. However, it should be noted that the deficit in rotarod performance in these mice is dependent upon other genes from the 129/Sv background (Kelly et al., 1999). Before proceeding with this plan to use D2R knock-out mice, however, it is important to confirm that a vector engineered with the rat cDNA for would be expressed appropriately in mouse brain. To this end, we can show in Fig. 8 that a high level of expression of D2R can be obtained in mouse brain (Ingram et al., 1998). Additional time-course studies to determine how long this expression can be maintained will need to be conducted. The D2R null mutant mouse would be highly beneficial for such studies because expression detected by [125I]iodosulpride binding can be measured in the absence of native receptors, which is a methodological problem encountered in our current studies with normal rats.

11. Other developments Other recent successes in experimental procedure will permit further development of the AdCMV.DopD2R vector for intracerebral delivery. First, in vivo imaging of the vector has now been made possible (Ogawa et al., 2000). When the vector was injected into rat striatum, D2R binding coud be clearly detected using positron

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Fig. 7. Comparison of a D2R null mutant mice (knock-out) and the wild-type in locomotor activity in the home cage (A) and the number of falls from a rotarod rotating at 3 rpm (B). In both tests, a significant difference between the groups was observed according to a two-tailed t-test (P B0.05).

emission tomography (PET) and several D2R ligands, including [11C]methylspiperone, [11C]raclopride, and [11C]nemonapride. Second, the development of so-called ‘gutless’ Ad vectors will permit intracerebral delivery that produces much less inflammation and thus longer expression (Hammerschmidt, 1999; Morsy and Caskey, 1999). This third generation of Ad vectors has been made with greater deletions of the viral genome but still have proven to be highly

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effective for gene transfer. The ability to examine PET images of the D2R produced by new vectors will further enhance the development effort. Rather than rely upon in vitro binding analysis in separate groups of animals sacrificed at different times, PET images can track protein expression longitudinally in the same animal.

12. Summary An Ad vector has been developed for intracerebral transfer of the rat cDNA for the D2R. The vector-produced protein is apparently transported to the cellular membrane where it binds appropriate ligand in vitro and in vivo. Functional effects of the vector-produced D2R can also be demonstrated in vivo. Specifically, rats receiving unilateral striatal injections of the vector exhibit rotational behavior in a predicted direction when injected s.c. with the dopamine agonist, apomorphine. Despite this progress in vector development, several caveats have emerged. First, the duration of D2R expression is no longer than  3–5 weeks following injection, but duration of expression appears longer in aged rats than in young rats. Second, no significant evidence of improved motor performance has been demonstrated in preliminary experiments in which aged rats received bilateral striatal injections of the vector. Several new research directions have been charted. First, we are examining the feasibility of enhancing hippocampal acetylcholine release and

Fig. 8. Autoradiographic image indicates increased expression of [125I]iodosulpride binding in mouse (C57BL/6J) striatum receiving striatal injection of AdCMV.DopD2R.

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learning performance through application of the vector. Second, we are investigating the application of the vector to D2R null mutant mice. Third, we are examining the feasibility of in vivo imaging of the vector using positron emission tomography. Fourth, we are planning the construction of new vectors that might be more specifically targeted, exhibit longer duration of D2R expression, and contain the human cDNA for D2R. Fifth, we have begun to examine the feasibility of evaluating the vector in a nonhuman primate model. Previous studies of intracerebral gene transfer using Ad vectors have demonstrated limited success in nonhuman primates with the main problem being length of protein expression limited by immunogenicity (Bohn et al., 1999; Lawrence et al., 1999). Our long-term goals are to achieve additional success in several different models, and thus gain confidence in the therapeutic potential of Ad vectors for treating age- and disease-related behavioral impairments mediated by loss of D2R. Acknowledgements The authors acknowledge the valuable assistance and advice of the following colleagues in completing this research: Y. Asai, J. Bunzow, J. Chernak, R. Crystal, D. Grandy, A. Iguchi, K. Ishiwata, S. Kato, H. Kuo, G. Martin, R. Meyer, A. Mastrangeli, K. Oda, O. Ogawa, A. M. Senda, A. Shimada, E. Spangler, H. Toyama, J. Yoshimura, L. Zhang.

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