Adeno-associated viral AAV vectors [67,68]. The new vector we are exploring now provided

A . Bjorklund et al. Brain Research 886 2000 82 –98 85 of the transgene or even kill the transduced cells [66]. This absence of such receptor molecules on the cell surface may is particularly well illustrated in the experiments of Bohn determine the efficiency by which the rAAV vectors are et al. [8] and Lawrence et al. [40], using injections of internalized into different types of neurons [3,54,61]. Ad-lacZ vector in the caudate nucleus of intact monkeys. In the nigrostriatal system, rAAV vectors have been In a group of 10 monkeys, all given the Ad-lacZ vector in shown to be effective in transducing neurons in both the same dose, the number of b-gal-expressing cells in the substantia nigra, globus pallidus and striatum, and high caudate varied from 0 to 600 000. Staining with inflamma- levels of transgene expression have been observed over at tory markers revealed an inverse correlation between least 6 months after vector injection [34,35,41,42] Figs. 1 transgene expression and extent of the inflammatory and 2. Over 90 of the transduced cells can, by mor- response, including demyelination [40]. Injection of vary- phological criteria, be classified as neurons. The high 7 9 ing titers of the vector from 5310 to 2310 pfu showed affinity of the rAAV vector for the pars compacta of the that the intensity of the host’s immune response increased substantia nigra makes it possible to express the transgene with increasing titers. At 1 month after vector injection, no in a high proportion of the nigral dopamine neurons with a b -gal expression was seen in the animal given the highest single 1–2 ml injection of the vector Fig. 1J–L. The titer [8]. These data clearly support the view that the host’s tranduction efficiency is, by comparison, lower in the immune response is a major limitation for high-level, striatum. In our recent study [34], using a single 3 ml stable transgene expression in the brain from the currently injection of the rAAV-GFP vector in the striatum, we used Ad vectors, and is probably a major reason why observed GFP-positive cells around the injection site GDNF expression from intracerebrally injected Ad vectors within a radius of about 0.3 mm, compared to a radius of declines and becomes more variable over time. In the about 1.5 mm in the substantia nigra after a 1 ml injection. recent study of Connor et al. [15], for example, only 10 of To reach larger areas of the striatum, therefore, we have 14 animals showed GDNF expression at 6 weeks after had to use multiple injections of the vector 333 ml, Ad-GDNF injection into striatum or nigra of aged rats, as spaced with a distance of about 1 mm between the revealed by immunohistochemistry. Ad-GDNF expression injection sites. at survival times longer than 6–7 weeks has so far not Recent improvements in rAAV vector production has been reported in the rat PD model. resulted in 100–10 000-fold higher titers, a higher propor- tion of infectious particles relative to empty ones, as well as completely Ad helper virus free vector preparations 3. Adeno-associated viral AAV vectors [67,68]. The new vector we are exploring now provided by the University of Florida vector core includes, in The recombinant AAV vectors have 96 of the viral addition, a modified promoter construct, i.e. a hybrid genome removed, leaving only the two short inverted CMV chicken b-actin CBA promoter with the wood- terminal repeats ITRs which are sufficient for packaging chuck hepatitis virus posttranscriptional element WPRE and integration. The advantage of these vectors is that they [18,69], instead of the MD promoter [53] used in our can integrate and stably express their transgene product in earlier studies [34,43,45]. The new vector has resulted in a non-diving cells, including neurons, and that the absence substantial increase in transduction efficiency in both of viral genes minimizes the expression of foreign proteins striatum and substantia nigra, both in terms of the number and hence the risk of triggering host immune responses of GFP-expressing cells and the level of GFP per cell. The [50]. The disadvantage is that the rAAV DNA packaging transduced protein is transported intra-axonally, from the capacity is small, less than 5 kb, which limits the size of nigra along the nigrostriatal pathway to the terminals in the the gene constructs that can be delivered with the rAAV striatum Fig. 1, and from the striatum along the system. Another limiting factor is that the transgene is striatonigral pathway to the terminals in the globus pal- expressed with a delay of several days, and increases lidus, entopeduncular nucleus and pars reticulata of the gradually over the first 2–3 weeks, probably due to the fact substantia nigra Fig. 2. Indeed, with the new vector that a second strand of DNA needs to be synthesized in the construct, the level of expression is such that the axonal transduced cells before the transgene can be expressed for and dendritic projections of the transduced nigral and recent reviews, see Refs. [48,54,61]. striatal neurons appear to be completely filled by the Studies using b-gal or GFP as reporter genes have transduced GFP. shown that the rAAV vector is efficient in transducing Intracerebral delivery of GDNF by means of AAV- non-dividing cells, mainly neurons, in the adult CNS GDNF vectors has so far been explored in the intrastriatal [3,31,35,43,55]. However, not all types of neurons are 6-OHDA lesion model in three studies [34,44,45], using a 12 equally good targets and the transduction efficiency varies rAAV vector of high titer 1.0310 viral particles per ml greatly between different brain regions. This is, at least in and the MD promoter a CMV immediate-early promoter part, likely to be due to the fact that AAV requires binding with an intervening b-globin intron which has been shown to heparan sulfate proteoglycans, as well as to co-receptors to support sustained, long-term expression in the rat brain such as the FGF receptor 1. Thus, the presence or [43]. In the first two studies [44,45] the AAV-GDNF vector 86 A Fig. 1. Distribution of the GFP protein in an intact animal receiving an injection of the AAV-GFP vector into the substantia nigra SN 2 ml, 5 weeks survival, CBA promoter. The transgene is highly expressed within the nigral dopamine neurons J–L and transported anterogradely in the axons to fill out virtually the entire nigrostriatal pathway and its axonal branches in the entopeduncular nucleus EP; G–I, globus pallidus GP; D–F and the striatum STR; A–C. Also, the dendrites in the substantia nigra, pars reticulata contain high levels of the transduced GFP protein L. was injected over the nigra, either 3 weeks before or and lesion was sufficient to allow GDNF to become fully immediately after the intrastriatal 6-OHDA injection. In expressed at the time of the 6-OHDA injection. In the the first case, the time interval between vector injection second case, GDNF was expressed with a delay of a few A . Bjorklund et al. Brain Research 886 2000 82 –98 87 Fig. 2. Injection of the AAV-GFP vector in the striatum 333 ml, 5 weeks survival, CBA promoter will transduce a large number of cells almost exclusively neurons in both striatum and globus pallidus B,C,E,F. The transduced protein is effectively transported anterogradely along the striatanigral pathway to the globus pallidus GP, D–F, entopeduncular nucleus EP, G–I and substantia nigra SN, J–L. days, with full expression at about 10 days after the initial starts 5–7 days after the 6-OHDA injection [63]. Both 6-OHDA-induced axon terminal damage. This means that injection paradigms were equally efficient in rescuing the the onset of GDNF production coincided with the onset of dopamine neuron cell bodies: 92–94 survival in the degeneration of the nigral dopamine cell bodies, which AAV-GDNF-injected groups, compared to 45–51 surviv- 88 A al in the AAV-lacZ-injected groups. The extent of denerva- These results are consistent with previous studies using tion of the TH-positive fibers in the striatum, however, was injections or infusions of recombinant GDNF protein, unaffected by the AAV-GDNF treatment. which have shown that survival of the nigral dopamine The level of GDNF expression, as determined in 2-mm neurons in the absence of a functional nigrostriatal projec- diameter punches from the transduced substantia nigra, tion is insufficient for functional sparing or functional remained fairly stable over the 10-week observation recovery in the intrastriatal 6-OHDA lesion model period, at a level of 0.7–1.2 ng punch approximately [33,59,65]. equivalent to 0.3–0.6 ng mg tissue; see Table 1. In the The animals receiving AAV-GDNF injections into the Choi-Lundberg et al. [12] study, using Ad-GDNF in- striatum were as impaired as the control lesion rats acutely jections into the substantia nigra, the protection of the after the lesion Fig. 4A,C, indicating that the over- nigral cell bodies was incomplete 79 despite several- expression of GDNF, at the levels obtained, was not fold higher GDNF tissue levels see Table 1. This sufficient to protect the striatal dopamine terminals against difference may be explained by the fact that the Ad vector the toxic insult. However, the TH-positive axons along the is expressed mainly outside the dopamine neurons. Thus, nigrostriatal pathway were partly preserved and sprouting over-expression of GDNF within the dopamine neurons fibers were abundant in the globus pallidus and in the themselves, as obtained with the AAV-GDNF vector, may caudal and ventral parts of the striatum Fig. 5C. The be particularly efficient for rescue of the axotomised nigral efficient striatal reinnervation seen in these animals thus cell bodies. With the new generation of AAV vector now appeared to be caused by a combination of protection of available see above the level of GDNF obtained after lesioned nigrostriatal axons followed by regeneration injection in either nigra or striatum is 7–50-fold higher that towards and into the region of high GDNF expression. that achieved with the previous vector Kirik et al., This sequence of axonal damage followed by a protracted unpublished data. remodelling of the nigrostriatal projection is consistent In the Kirik et al. [34] study, the AAV-GDNF vector was with the slow and progressive functional recovery that injected at multiple sites in the striatum, in the nigra, or in developed during the first months after the lesion. both striatum and nigra, 4 weeks before the 6-OHDA It is notable that over-expression of GDNF in the nigra injection. Expression of GDNF, as observed by immuno- failed to preserve the TH-positive axons along the nigros- histochemistry, was maintained at high levels in both sites triatal pathway. In these animals, there was massive axonal throughout the 6-month experimental period. In animals sprouting in and around the medial forebrain bundle, close receiving vector injection into the striatum, the GDNF to the rescued cell bodies. These sprouting fibers were seen protein was widely distributed throughout the striatum and to extend up to the border of the globus pallidus, but not transported along the striatonigral pathway to the globus further rostrally Fig. 5D. Indeed, the area containing pallidus Fig. 3D–F, the entopeduncular nucleus Fig. dense sprouting fibers coincided with the area of high 3G–I and the substantia nigra Fig. 3J–L. In this GDNF-immunoreactivity, as seen in sections stained with experiment, a four-site intrastriatal 6-OHDA lesion was the GDNF antibody. used 437 mg which gives a substantial, 80–90, These data are in agreement with results obtained with denervation of the striatum, sufficient to induce marked direct intracerebral injections of recombinant GDNF pro- motor impairments that are stable over time [32,34]. Vector tein [33,60]. As illustrated in Fig. 7, GDNF injected into injection into the nigra was more efficient in protecting the the striatum either 6 h before or 1 day after the intrastriatal nigral dopamine cell bodies: 91 cell survival in the nigral 6-OHDA injection is efficient in preserving the cell bodies injection group, compared to 57 in the striatal injection and the axons of the nigrostriatal pathway, as well as group and 12 in the lesion-only controls. However, only inducing axonal sprouting in the globus pallidus and the rats receiving AAV-GDNF injections into the striatum regeneration of TH-positive fibers in the striatum, while showed behavioral recovery, and this was accompanied by GDNF over the substantia nigra induces sprouting locally partial sparing of the nigrostriatal projection and reinnerva- around the injection site. In both cases, sprouting of tion of the lesioned striatum see Fig. 5C. As illustrated in TH-positive fibers occurs in the area reached by high Fig. 4, the recovery developed gradually over 4–5 months concentrations of the GDNF protein. Consistent with these after the lesion and was observed in both drug-induced morphological data, significant sparing or recovery of rotation Fig. 4A and in tests of spontaneous motor motor functions was obtained only in the animals receiving behavior, i.e. forelimb use in the so-called cylinder and GDNF injection into the striatum, i.e. in those animals staircase tests Fig. 4B–D. No functional sparing or which had significant sparing of the nigrostriatal projection recovery was seen in the animals receiving AAV-GDNF in combination with GDNF-induced sprouting of TH- injections into the substantia nigra, despite near-complete positive axons in the lesioned striatum [33]. protection of the cell bodies. In these animals, the nigros- Additionally, in the Kirik et al. [34] study, it is triatal projection and striatal TH-positive innervation was important to point out that no significant reinnervation of damaged to the same extent as in the lesion-only animals the striatum or functional recovery was seen in the animals that received injections of the control vector Fig. 5B,D. with combined AAV-GDNF injections in both nigra and A . Bjorklund et al. Brain Research 886 2000 82 –98 89 Fig. 3. Immunohistochemical visualization of the GDNF protein in an animal receiving injections of the AAV-GDNF vector 333 ml, MD promoter in the striatum, 6 months survival. The transduced GDNF is widely distributed not only throughout the striatum STR; A–C but also along the axons of the striatal projection neurons to the globus pallidus GP, D–F, entopeduncular nucleus EP; G–I and substantia nigra SN; J–L. The efficient intra-axonal transport of the expressed protein suggests that it may be able to exert effects throughout the nigrostriatal system even when the vector is injected in the striatum only. 90 A Fig. 4. Functional recovery in drug-induced rotation A, and forelimb use in the paw-reaching B and cylinder tests C and D in 6-OHDA lesioned rats that had received AAV-GDNF injections into either striatum STR, substantia nigra SN or both striatum and nigra STR SN. The vector injection was made 3 weeks before the intrastriatal 6-OHDA injection. Note that functional recovery is seen only in animals with intrastriatal vector injections modified after Kirik et al. [34]. striatum. We believe this is attributable to the intense local Expression of GDNF in the nigral dopamine neurons sprouting close to the substantia nigra seen in these themselves, therefore, may be detrimental, rather than animals, which may prevent regrowth of the lesioned positive, for the recovery of function in 6-OHDA lesioned axons towards the striatal source of GDNF see Section 5. rats. A . Bjorklund et al. Brain Research 886 2000 82 –98 91 Fig. 5. Over-expression of GDNF in the striatum by intrastriatal injections of the AAV-GDNF vector can block dopamine neuron degeneration induced by the intrastriatal 6-OHDA lesion, as illustrated in B and C. Importantly, the axons along the nigrostriatal pathway are partially preserved. These rescued axons provide the substrate for sprouting and regrowth into the area of high GDNF expression indicated by green color in C, which in turn is accompanied by a gradual functional recovery as illustrated in Fig. 4. Injection of the vector into the substantia nigra D, by contrast, protects the nigral cell bodies but is unable to preserve the axonal projection to the striatum. Instead, there is extensive local sprouting of TH-positive fibers in regions close to the rescued cell bodies, i.e. into the area of high GDNF expression green color in D. Based on data from Ref [34].

4. Lentiviral LV vectors In the current version of LV vectors, the particles are