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Published in final edited form as: Science. 1994 Nov 25;266(5189):1399–1403. doi: 10.1126/science.266.5189.1399

Long-Term Behavioral Recovery in Parkinsonian Rats by an HSV Vector Expressing Tyrosine Hydroxylase

Matthew J During 1,✉, Janice R Naegele 1, Karen L O’Malley 1, Alfred I Geller 1
PMCID: PMC2638002  NIHMSID: NIHMS10231  PMID: 7669103

Abstract

One therapeutic approach to treating Parkinson’s disease is to convert endogenous striatal cells into levo-3,4-dihydroxyphenylalanine (l-dopa)–producing cells. A defective herpes simplex virus type 1 vector expressing human tyrosine hydroxylase was delivered into the partially denervated striatum of 6-hydroxydopamine–lesioned rats, used as a model of Parkinson’s disease. Efficient behavioral and biochemical recovery was maintained for 1 year after gene transfer. Biochemical recovery included increases in both striatal tyrosine hydroxylase enzyme activity and in extracellular dopamine concentrations. Persistence of human tyrosine hydroxylase was revealed by expression of RNA and immunoreactivity.

Parkinson’s disease (PD), a neurodegenerative disorder, is characterized by the progressive loss of the dopaminergic neurons in the substantia nigra pars compacta that project to the corpus striatum (1). The principal therapy for PD is the oral administration of l-dopa (2), which is converted to dopamine (DA) by endogenous striatal aromatic amino acid decarboxylase (AADC) (3). Although it is initially effective, l-dopa therapy loses efficacy over a period of several years (1). Transplantation of cells that produce l-dopa or DA into the striatum can correct animal models of PD (4) but has not been a viable therapy in most human trials (5). Peripheral cell types that are genetically modified to express tyrosine hydroxylase (TH) and produce l-dopa have supported only short-term improvement (less than 2 months) in animal models of PD (6, 7). Genetically modified muscle cells support longer improvements (6 months) (8), but the viability of a muscle cell graft in the human striatum is not yet clear. An alternative therapeutic strategy is to convert a fraction of the striatal cells into l-dopa–producing cells by expression of TH in striatal cells (9) from a defective herpes simplex virus type 1 (HSV-1) vector (10). Potential advantages of this approach include production of l-dopa at the required site of action, so that diffusion over substantial distances is not necessary, and alleviation of potential problems caused by graft rejection or tumor formation. To test this strategy, a human TH complementary DNA (cDNA) (form II) (11, 12) was inserted into an HSV-1 vector (pHSVth). Infection of cultured striatal cells with pHSVth resulted in expression of human TH RNA, TH immunoreactivity, and the release of l-dopa into the culture medium (13). The amounts of l-dopa released per infected cell suggested that pHSVth might be evaluated in the 6-hydroxydopamine (6-OHDA)–lesioned rat, a model of PD.

pHSVth virus or pHSVlac virus or vehicle alone [phosphate-buffered saline (PBS)], was delivered by stereotactic injection into the partially denervated striatum of unilaterally 6-OHDA–lesioned rats (14). The apomorphine-induced rotation rate was measured as an index of behavioral recovery. The average decrease in the rotation rate caused by pHSVth was 64 ± 6% at 2 weeks after gene transfer. This value remained relatively constant over a 1-year period, and the decrease remained statistically significant at both 6 months (P < 0.01) and 1 year (P < 0.05) after gene transfer as compared with the control groups (Fig. 1A and Table 1). The rotation rate of each rat in the pHSVth group remained relatively constant and was similar to the rotation rate in the final test (Table 1).

Fig. 1.

Fig. 1

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Rotation rates and striatal l-dopa or DA concentrations after stereotactic injection of pHSVth, pSHVlac, or PBS into the partially denervated striatum (14). (A) The rats were tested at various times (14) for the apomorphine-induced rotation rate, and the values shown are the average percent of the baseline rotation rate for each group. (B) Striatal l-dopa concentrations were measured by microdialysis (15) after perfusion with NSD 1015 as an indication of striatal TH activity (16). (C) Striatal DA levels were measured by microdialysis in low K+ (3 mM) and after perfusion with high K+ (56 mM) (15).

Table 1.

Summary of rotation rates and histological analysis of rats receiving pHSVth, pHSVlac, or PBS.

Histological analysis
Decrease in baseline rotation rate* (%)
TH-IR§
X-Gal
TH PCR||
Rat Injection Average Final Time of autopsy† Microdialysis‡ S C GP S RS LS CB
2 pHSVth 57 56 16 N + + + + + − −
3 pHSVth 93 97 12 N + − + ND
4 pHSVth 46 29 11 N + + − + + ND
9 pHSVth 62 62 15.5 N + − − + − −
26 pHSVth 39 56 5.5 Y PP + − ND
27 pHSVth 34 26 5.5 Y + + + + + + + − ND
30 pHSVth 26 16 6 N − + + − ND
31 pHSVth 41 46 8 Y + + + + − − ND
33 pHSVth 66 70 6 Y + − − + − ND
1 pHSVlac −7 −31 12 Y +
6 pHSVlac −11 0 12 N − − − + +
8 pHSVlac −2 −14 10 Y ND
20 pHSVlac 11 21 13 Y − − − + − − ND
32 pHSVlac −10 13 11 Y ND
12 PBS 14 20 13 Y − − − −
14 PBS 8 0 6 N
17 PBS 29 32 12 Y − − − −
19 PBS −13 0 12 Y − − − −
25 PBS −7 −9 8 Y PP/ND
38 PBS −5 3 12 Y − − − −
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*

The two tests from each month of the first 3 months were averaged; those three values and the other monthly values (14) were used to calculate the average.

†

Months after gene transfer.

‡

Microdialysis was done (Y, yes; N, no).

§

−, 0; +, 1 to 3; or + +, 4 to 20 cells contained TH immunoreactivity (TH-IR) in one or more sections (18). S, striatum; C, cortex; GP, globus pallidus; PP, poor perfusion; ND or no entry, not done.

||

pHSVth DNA was detected with PCR (23); RS, injected right striatum; LS, uninjected left striatum; Cb, cerebellum.

Presumably because of poor tissue preservation, attempts to detect cells with TH immunoreactivity were unsuccessful, although pHSVth DNA was detected with PCR.

TH enzyme activity and extracellular DA concentrations in the injected striatum were evaluated in selected rats 4 to 6 months after gene transfer by means of in vivo microdialysis (15) (Table 1). An inhibitor of AADC [100 μM NSD 1015 (3-hydroxybenzylhydrazine)] was added to the microdialysis perfusate, and accumulation of l-dopa was measured (15) as an indication of TH enzyme activity (16) (Fig. 1B). Ninety minutes after addition of NSD 1015, pHSVth directed a 60% average increase in striatal l-dopa concentrations as compared with those of the control groups (pHSVlac or PBS; P < 0.05), whereas normal (unlesioned and uninjected) rats showed 180% higher striatal l-dopa concentrations as compared with those of the control groups (P < 0.01).

To determine whether or not pHSVth directed an increase in DA production, extracellular DA levels were measured (15). In the basal state (3 mM K+), pHSVth mediated a 120% (P < 0.05) increase in striatal DA concentrations as compared with those of the control groups, whereas normal rats showed DA concentrations that were 250% (P < 0.01) above those of the control groups (Fig. 1C). After depolarization of neurons with high K+ (56 mM K+ in microdialysis perfusate), pHSVth mediated a 310% (P < 0.05) increase in DA concentrations as compared with those of the control groups, whereas normal rats showed an 1150% (P < 0.005) increase in DA concentrations. Concentrations of γ-aminobutyric acid and acetylcholine, the predominant neurotransmitters of striatal neurons, were unaltered in the pHSVth group as compared with concentrations in the control groups.

Histological analysis was done 6 to 16 months after gene transfer. Because these defective HSV-1 vectors should not replicate in vivo (17), gene transfer is most likely to occur in striatal neurons and glia that are proximal to the injection site. However, HSV-1 particles can also diffuse through the extracellular space or be retrogradely transported through processes to the cell bodies of striatal projection neurons. TH immunoreactivity was detected with an antibody to TH (18). The normal rat striatum lacks cells with TH immunoreactivity (19). Striata injected with pHSVth, but not pHSVlac or PBS, contained immunoreactive cells, frequently in clusters spread over 200 to 300 μm (Fig. 2, A through C, and Table 1), and many of these cells displayed neuronal morphology (Fig. 2C). Again, only in the pHSVth group, two striatal projection areas that normally lack cells with TH immunoreactivity, namely the pallidum (20) and the medial agranular cortex (bilateral, layers 3 and 5) (21), contained cells with TH immunoreactivity (Fig. 3, D and E, respectively, and Table 1). One rat (pHSVth no. 30) lacked immunoreactive striatal cells, although cortical cells with immunoreactivity were detected, and this was the only rat in the group that did not show a decrease in the rotation rate.

Fig. 2.

Fig. 2

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TH immunoreactivity was detected with an antibody to TH (78), and β-Gal was detected with X-Gal (22). (A) through (C) show rat pHSVth no. 27. (A) Composite drawing of charted sections, showing the positions of 48 cells containing TH immunoreactivity in the striatum and neocortex. Every third section was analyzed. L, lateral; R, rostral; scale bar, 2 mm. (B) Low-magnification photomicrograph of clusters of striatal cells containing TH immunoreactivity. Arrowheads point to two clusters, and the arrow indicates a third cluster [boxed in (A)]; scale bar, 500 μm. (C) High-magnification view of a cluster of striatal cells containing TH immunoreactivity with neuronal morphology [boxed (A)]; scale bar, 50 μm. (D) and (E) show rat pHSVth no. 31. (D) A cluster of pallidal neurons containing TH immunoreactivity; scale bar, 50 μm (E) A cluster of cortical neurons (agranular frontal cortex, layers 3 and 5) containing TH immunoreactivity; scale bar, 100 μm (F) High-magnification view of X-Gal–positive striatal cells from rat pHSVlac no. 1; scale bar, 50 μm.

Fig. 3.

Fig. 3

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Persistence of pHSVth DMA and expression of human TH RNA. (A) DMA was extracted from sections and subjected to PCR with the use of primers specific to the human TH gene, and the products were electrophoresed (23). Age is the time after gene transfer a rat was analyzed (6 months, rat pHSVth no. 27; 16 months, rats pHSVth no. 2, and no. 9). Brain areas: R, right injected striatum; L, left uninjected striatum; Cb, cerebellum. Minus sign indicates no DMA; plus sign indicates pHSVth DMA isolated from Escherichia coli, which should direct production of a 186-bp fragment (number of base pairs is shown at left). (B) RT-PCR analysis of RNA isolated from specific brain areas 1 month after injection of pHSVth (14) into the right striatum. Brain areas: St, striatum; Ct, cortex; SN, midbrain (substantia nigra); Cb, cerebellum. Minus sign indicates no RNA; plus sign indicates pHSVth DNA isolated from E. coli; the methods used (24) should generate a 160-bp fragment (number of base pairs is shown at left).

In the pHSVth group, the total number of cells containing TH immunoreactivity (18) ranged from approximately 5 to 10 to 200 to 300, the majority of which were striatal cells. The number of immunoreactive cells did not correlate with the extent of behavioral recovery, possibly because multiple cell types contained TH immunoreactivity and the amount of l-dopa produced by recombinant TH is cell-type–dependent (6–8). Cells expressing β-galactosidase (β-Gal) were detected with X-Gal (22) in striata injected with pHSVlac but not with pHSVth or PBS (Fig. 2F and Table 1).

pHSVth DNA was detected by means of polymerase chain reaction (PCR) (23) in pHSVth-injected striata for up to 16 months after gene transfer (Fig. 3A and Table 1). pHSVth DNA was detected in the uninjected contralateral striatum in two rats (pHSVth no. 4 and the rat analyzed 3 months after gene transfer in Fig. 3A). This could be due to pHSVth virus rising up a needle track to infect axons projecting from the contralateral striatum or neocortex [some samples contained small amounts of neocortex (23)] or to a projection from the contralateral neocortex to the injected striatum (21). pHSVth DNA was not detected in the cerebellum, which does not project to the striatum, or in a striatum that received pHSVlac.

Because the brains of the rats used for rotation rate analysis were fixed with 4% paraformaldehyde, this tissue was not suitable for reverse transcriptase–PCR (RT-PCR) analysis of human TH RNA. To investigate long-term expression of human TH RNA, pHSVth was injected into the striatum (14) of normal rats, and 1 month later human TH RNA was detected (24) in specific brain areas in 3 of 10 rats (Fig. 3B). The negative rats may be due to the limitations of the assay or to other possibilities, including inefficient gene transfer or loss of expression. Typically, 1 week is required before HSV-1 particles are absent from the brain and a persistent infection is established (25). Thus, expression of human TH RNA 1 month after gene transfer indicates that the IE 4/5 promoter can direct persistent expression in this HSV-1 vector (26).

The present configuration of the vector system has limitations. Virus prepared with this packaging system contains wild-type HSV-1 (frequency ~10−5) (17), and HSV-1 particles contain specific HSV-1–encoded proteins that mediate acute cytopathic effects. These factors may have contributed to a number of rat deaths (<10%) that occurred within 2 weeks after gene transfer (27); however, the majority of the rats (>90%) were healthy and gained weight until deliberately killed. The brains from the pHSVth and pHSVlac groups were of normal size and showed normal morphology except for a small zone of necrosis around the injection sites (pHSVth group, approximately ≤1 × 104 to 5 × 105 μm2; PBS group, approximately 1 × 105 μm2). No brain tumors were observed, and no cells contained HSV-1 particle immunoreactivity (28). Expression of TH in striatal projection neurons could potentially interfere with other brain functions, although ingestive and gross motor behaviors appeared unaltered (29). In an initial comparison of short- and long-term expression, significantly more positive cells were observed at 4 days than at 1 to 3 months after gene transfer (14) (pHSVth or pHSVlac), although expression occurred in the same cell types. The decrease in expression could be due to the acute cytopathic effects associated with gene transfer, to downregulation of the IE 4/5 promoter, or to other properties of the vector system. Thus, whereas improvements in the vector system are needed, these studies demonstrate the feasibility of this approach.

An alternative explanation for the results is that trauma to the partially denervated rat striatum induced trophic factor–mediated growth of remaining TH axons, resulting in behavioral recovery (30). This is unlikely, because the pHSVlac group did not show behavioral recovery, and because the number of axons with TH immunoreactivity in the injected striatum was similar in the pHSVth, pHSVlac, and PBS groups and was low. It is also unlikely that the vector system induced expression of endogenous TH, because the pHSVlac group lacked striatal, pallidal, or cortical cells with TH immunoreactivity, and because human TH RNA was expressed.

The pHSVth group showed persistence of vector DNA, long-lasting expression of TH, increased striatal TH activity and extracellular DA concentrations, and long-term behavioral recovery. The capability of a limited number of cells expressing TH to support sustained responses might be explained by the relatively wide dispersion of the cells, potentially elevating l-dopa concentrations over an extended area, and by observations suggesting that increased diffusion of DA occurs in the partially denervated striatum because of reduced concentrations of DA transporters (31).

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  • 32.We thank K. Burns, K. Davis, S. Harmon, M. Kake, P. Leone, E. Lukacsi, G. Mirchandani, and D. Ullrey for technical assistance. We thank J. Haycock and R. Roth for critical readings of the manuscript. Supported by NIH grants NS28227 and NS06208 and the Parkinson’s Disease Foundation (M.J.D.); by NIH grants EY09749 and MH49351 and the Tourette Association (J.R.N.); by the American Parkinson’s Disease Association and NIH grant AG10827 (A.I.G. and K.L.O’M.); by NIH grant 50081 (K.L.O’M.) and by Alkermes Inc., the American Health Assistance Foundation, the Burroughs Wellcome Fund, and the National Parkinson Foundation (A.I.G.).

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