Stuart E. Leff

Viral gene delivery selectively restores feeding and prevents lethality of dopamine-deficient mice.

Dopamine-deficient mice (DA-/- ), lacking tyrosine hydroxylase (TH) in dopaminergic neurons, become hypoactive and aphagic and die by 4 weeks of age. They are rescued by daily treatment with L-3,4-dihydroxyphenylalanine (L-DOPA); each dose restores dopamine (DA) and feeding for less than 24 hr. Recombinant adeno-associated viruses expressing human TH or GTP cyclohydrolase 1 (GTPCH1) were injected into the striatum of DA-/- mice. Bilateral coinjection of both viruses restored feeding behavior for several months. However, locomotor activity and coordination were partially improved. A virus expressing only TH was less effective, and one expressing GTPCH1 alone was ineffective. TH immunoreactivity and DA were detected in the ventral striatum and adjacent posterior regions of rescued mice, suggesting that these regions mediate a critical DA-dependent aspect of feeding behavior.

Recombinant adeno-associated viral vector-mediated glial cell line-derived neurotrophic factor gene transfer protects nigral dopamine neurons after onset of progressive degeneration in a rat model of Parkinson's disease.

Previous work has demonstrated that viral vector mediated gene transfer of glial cell line-derived neurotrophic factor (GDNF), when administered prior to a striatal injection of the specific neurotoxin, 6-hydroxydopamine (6-OHDA), can protect nigral dopamine (DA) neurons from cell death. When considering gene therapy for Parkinsons disease (PD), vector delivery prior to the onset of neuropathology is not possible and chronic delivery will likely be necessary in a GDNF-based PD therapy. The present study was undertaken to determine if GDNF delivered via a recombinant adeno-associated viral vector (rAAV) could affect nigral DA cell survival when initiated just after the administration of striatal 6-OHDA. The onset of rAAV-mediated GDNF transgene expression near the substantia nigra was determined to begin somewhere between 1 and 7 days after the 6-OHDA injection and subsequent vector administration. The cell survival data indicate that rAAV-GDNF delivery results in a highly significant sparing of nigral DA neurons. These data indicate that a single delivery of rAAV encoding GDNF is efficacious when delivered after the onset of progressive degeneration in a rat model of PD.

Long-term restoration of striatal l-aromatic amino acid decarboxylase activity using recombinant adeno-associated viral vector gene transfer in a rodent model of Parkinson's disease

As a potential treatment for Parkinsons disease, viral vector-mediated over-expression of striatal L-aromatic amino acid decarboxylase was tested in an attempt to facilitate the production of therapeutic levels of dopamine after peripheral L-dihydroxyphenylalanine administration. The results of microdialysis and enzyme activity assays indicate that striatal decarboxylation of peripherally administered L-dihydroxyphenylalanine was enhanced by recombinant adeno-associated virus-mediated gene transfer of L-aromatic amino acid decarboxylase in unilateral 6-hydroxydopamine-lesioned rats. This gene transfer-induced increase in striatal decarboxylase activity was shown to remain undiminished over a six-month period and transgene expression was demonstrated to persist for at least one year. Unlike previous approaches involving delivery of either tyrosine hydroxylase, or tyrosine hydroxylase and L-aromatic amino acid decarboxylase transgenes together to accomplish unregulated dopamine delivery, the current study proposes a pro-drug strategy (peripheral L-dihydroxyphenylalanine administration after L-aromatic amino acid decarboxylase transduction). This strategy for dosage control could potentially allow lowered L-dihydroxyphenylalanine doses and potentially obviate complicated transcriptional regulation paradigms. These data suggest that the use of the non-pathogenic adeno-associated virus to transfer the L-aromatic amino acid decarboxylase gene into the striatum of Parkinsons disease patients may be an attractive gene therapy strategy.

Nerve Growth Factor Expressed in the Medial Septum Followingin VivoGene Delivery Using a Recombinant Adeno-Associated Viral Vector Protects Cholinergic Neurons from Fimbria-Fornix Lesion-Induced Degeneration

Nerve growth factor (NGF) has been shown to support the survival of axotomized medial septal cholinergic neurons after aspirative lesions of the fimbria-fornix (FF). This survival effect has been achieved utilizing intraventricular and intraparenchymal delivery of the NGF protein. While the use of NGF for the treatment of the cholinergic deficits present in Alzheimers disease shows promise based on its efficacy in animal models, concerns about side-effects of intraventricular NGF delivery in humans have been raised. In the present study, NGF was delivered directly to the medial septum via a recombinant adeno-associated viral vector (rAAV) encoding the cDNA for human NGF prior to a FF lesion in rats. This rAAV-mediated NGF delivery was shown to significantly attenuate the medial septal cholinergic cell loss observed in animals receiving an equivalent injection of a control rAAV vector.

In VivoL-DOPA Production by Genetically Modified Primary Rat Fibroblast or 9L Gliosarcoma Cell Grafts via Coexpression of GTPcyclohydrolase I with Tyrosine Hydroxylase

To investigate the biochemical requirements for in vivo L-DOPA production by cells genetically modified ex vivo in a rat model of Parkinsons disease (PD), rat syngeneic 9L gliosarcoma and primary Fischer dermal fibroblasts (FDFs) were transduced with retroviral vectors encoding the human tyrosine hydroxylase 2 (hTH2) and human GTP cyclohydrolase I (hGTPCHI) cDNAs. As GTPCHI is a rate-limiting enzyme in the pathway for synthesis of the essential TH cofactor, tetrahydrobiopterin (BH4), only hTH2 and GTPCHI cotransduced cultured cells produced L-DOPA in the absence of added BH4. As striatal BH4 levels in 6-hydroxydopamine (6-OHDA)-lesioned rats are minimal, the effects of cotransduction with hTH2 and hGTPCHI on L-DOPA synthesis by striatal grafts of either 9L cells or FDFs in unilateral 6-OHDA-lesioned rats were tested. Microdialysis experiments showed that those subjects that received cells cotransduced with hTH2 and hGTPCHI produced significantly higher levels of L-DOPA than animals that received either hTH2 or untransduced cells. However, animals that received transduced FDF grafts showed a progressive loss of transgene expression until expression was undetectable 5 weeks after engraftment. In FDF-engrafted animals, no differential effect of hTH2 vs hTH2 + hGTPCHI transgene expression on apomorphine-induced rotation was observed. The differences in L-DOPA production found with cells transduced with hTH2 alone and those cotransduced with hTH2 and hGTPCHI show that BH4 is critical to the restoration of the capacity for L-DOPA production and that GTPCHI expression is an effective means of supplying BH4 in this rat model of PD.

Practical Aspects of the Development ofex Vivoandin VivoGene Therapy for Parkinson's Disease ☆

Current approaches to gene therapy of CNS disorders include grafting genetically modified autologous cells or introducing genetic material into cells in situ using a variety of viral or synthetic vectors to produce and deliver therapeutic substances to specific sites within the brain. Here we discuss issues related to the application of ex-vivo and in-vivo gene therapies as possible treatments for Parkinsons disease. Autologous monkey fibroblasts engineered ex-vivo to express tyrosine hydroxylase were grafted into MPTP-treated monkeys and found to express for up to 4 months. Adeno-associated (AAV) viral vectors expressing beta-galactosidase or tyrosine hydroxylase were introduced into monkey brains to determine the extent of infection and the types of cells infected by the vector at 21 days and 3 months. Gene expression was detected at both time points and was restricted to neurons in the striatum. These experiments demonstrate that two different approaches can be used to deliver proteins into the CNS. However, further technological advances are required to optimize gene delivery, regulation of gene expression, and testing in appropriate functional models before gene therapy can be considered for treating human disease.

Progress in Direct Striatal Delivery of l-Dopa via Gene Therapy for Treatment of Parkinson's Disease Using Recombinant Adeno-Associated Viral Vectors

Viral vectors have recently been used successfully to transfer genes and express different proteins in the brain. This review discusses the requirements to consider human clinical trials in which recombinant adeno-associated virus vectors are used to transfer the genes necessary to produce l-dihydroxyphenylalanine (l-dopa) directly into the striatum of Parkinsons patients. Preclinical data that apply to the criteria defined as prerequisite for clinical trials are discussed. Thus, in animal models using recombinant adeno-associated virus vectors it has been demonstrated that l-dopa can be synthesized in the striatum after in vivo transduction. In addition, these l-dopa levels are sufficient to affect behavior in a dopamine-deficient animal model, the expression is extremely long-lasting, and the ability to transcriptionally regulate tyrosine hydroxylase has been demonstrated but not fully characterized. However, while immune responses to recombinant adeno-associated virus infection in the periphery have been studied, direct assessment of the potential immune response in the brain has not been sufficiently defined. Therefore, the rationale for delivering l-dopa directly to the striatum to treat Parkinsons disease is sound and the preclinical data are promising but all the issues surrounding this strategy are not resolved.

Age-related decreases in GTP-cyclohydrolase-I immunoreactive neurons in the monkey and human substantia nigra

Guanosine triphosphate cyclohydrolase I (GTPCHI) is a critical enzyme in catecholamine function and is rate limiting for the synthesis of the catecholamine co‐factor tetrahydrobiopterin. The present study assessed the distribution of GTPCHI immunoreactivity (‐ir) within the monkey and human ventral midbrain and determined whether its expression is altered as a function of age. Light and confocal microscopic analyses revealed that young monkeys and humans displayed GTPCHI‐ir within melanin‐containing and tyrosine‐hydroxylase‐ir neurons in primate substantia nigra. Stereological counts revealed that there was a 67.4% reduction in GTPCHI‐ir neuronal number, a 63.5% reduction in GTPCHI‐ir neuronal density, and a 37.6% reduction in neuronal volume in aged monkeys relative to young cohorts. Similar age‐related changes were seen in humans, in whom there were significant reductions in the number of GTPCHI‐ir nigral neurons in middle age (58.4%) and aged (81.5%) cases relative to young cohorts. The density of GTPCHI‐ir neurons within the nigra was similarly reduced in middle‐aged (63.0%) and aged (81.8%) cases. In contrast to monkeys, aged humans did not display shrinkage in the volume of GTPCHI‐ir nigral neurons. The presence of numerous melanin‐positive, but GTPCHI‐ir immunonegative, neurons in the aged monkey and human nigra indicates that these decreases represent an age‐related phenotypic downregulation of this enzyme and not a loss of neurons per se. These data indicate that there is a dramatic decrease in GTPCHI‐ir in nonhuman primates and humans as a function of age and that loss of this enzyme may be partly responsible for the age‐related decrease in dopaminergic tone within nigrostriatal systems. J. Comp. Neurol. 426:534–548, 2000.

Intracerebral Gene Transfer Using Viral Vectors

The recent development of recombinant viral vectors that are capable of transducing postmitotic cells may provide a powerful new tool for studying brain function, as well as ameliorative strategies in models of neurological disease. Some of these vectors have recently demonstrated direct expression of biologically relevant levels of protein expression (Mandel et al., 1997, 1998; Left et al., 1999; B16mer et al., 1998) for varying periods of time of up to 1 yr after direct intracerebral injection (Mandel et al., 1997, 1998; B16mer et al., 1997; Naldini et al., 1996a). Moreover, vectors injected into the nervous system have been shown, not only to be capable of expressing de novo transgenes, but also to be able to regionally suppress gene expression by delivery of antisense oligonucleotides (Xiao et al., 1997), by expression of a dominant-negative protein, by local knock-out of genes in transgenic animals carrying a transgene surrounded by lox-P sites (Donello et al., 1998), or by the production of molecules capable of inhibiting specific mRNAs, called ribozymes (Lewin et al., 1998). Thus, the use of viral vectors has great potential, not only for the study of gene therapy (GT), but also for the study of the molecular basis of certain brain functions. This chapter provides information about a range of practical issues involved in the use of recombinant vectors, and may be used as a guide for laboratories that intend to use viral vectors in their research. This chapter focuses on the three recombinant vectors that are among the most commonly currently used in the literature, i.e., recombinant adeno-associated virus (rAAV) vectors, recombinant adenovirus (rAd), and recombinant lentivirus (rLV) vectors. Other promising vector systems are under development for direct gene transfer, e.g., vectors based on herpes simplex virus (HSV) (During et al., 1994; Bowers et al., 1997). HSV vectors will not be discussed here, but this does not reflect any judgement on the quality or the potential of HSV vectors