Double Transduction with GTP Cyclohydrolase I and Tyrosine Hydroxylase Is Necessary for Spontaneous Synthesis ofl-DOPA by Primary Fibroblasts

Abstract

Gene transfer of tyrosine hydroxylase (TH) in animal models of Parkinson's disease (PD), using either genetically modified cells or recombinant virus vectors, has produced partial restoration of behavioral and biochemical deficits. The limited success of this approach may be related to the availability of the cofactor, tetrahydrobiopterin (BH4), because neither the dopamine-depleted striatum nor the cells used for gene transfer possess a sufficient amount of BH4 to support TH activity. To determine the role of BH4 in gene therapy, fibroblast cells transduced with the gene for TH were additionally modified with the gene for GTP cyclohydrolase l; an enzyme critical for BH4 synthesis. In contrast to cells transduced with only TH, doubly transduced fibroblasts spontaneously produced both BH4 and 3, 4-dihydroxy-L-phenylalanine. To examine further the importance of GTP cyclohydrolase I in gene therapy for PD, in vivo micro-dialysis was used to assess the biochemical changes in the dopamine-denervated striatum containing grafts of genetically modified fibroblasts. Only denervated striata grafted with fibro-blasts possessing both TH and GTP cyclohydrolase I genes displayed biochemical restoration. However, no significant differences from controls were observed in apomorphine-induced rotation. This is partly attributable to a limited duration of gene expression in vivo. These differences between fibroblasts transduced with TH alone and those additionally modified with the GTP cyclohydrolase I gene indicate that BH4 is critical for biochemical restoration in a rat model of PD and that GTP cyclohydrolase I is sufficient for production of BH4.

Part I: Minicircle vector technology limits DNA size restrictions on ex vivo gene delivery using nanoparticle vectors: Overcoming a translational barrier in neural stem cell therapy

“…Therefore, for subsequent experiments, this copy number was used for double transfection (multifection) in order to yield maximal gene transfer. Double transduction/transfection is routinely used for many viral as well as non-viral genetic modification approaches such as lipid-mediated gene delivery and electroporation in order to enhance transfection efficiency and to prolong gene expression [37][38][39]. We previously showed that multifection of NSCs (cultured as 3-D neurospheres) resulted in higher transfection levels (~27%) compared to single transfection (~13%) with no effect on cell physiology and health [22].…”

Advances in Gene Therapy for Movement Disorders

“…In addition, instead of integrating with host brain circuitry, they tend to displace the brain parenchyma by forming a globular clump of cells, which itself can disrupt rotational behavior in rodents. 13 Astrocytes also potentially represent an autologous source of cell vehicles. 14 They have been tested owing to their natural supportive role in the brain, their efficient secretory mechanisms, transducibility in vitro, and tendency to migrate from the graft site, thus minimizing mass effect.…”

“…30 -33 Early approaches to delivering TH cDNA in the striatum of animal models of PD have included the in vivo injection of viral vectors HSV-1, AAV, and adenovirus. 34 -37 Ex vivo engineering of cell lines, primary cells, 5,13,17 and immortalized cell lines generated from embryonic mesencephalic cultures have also been used in ex vivo gene transfer studies, 9 with various degrees of functional effects, none sufficient to advance to clinical testing.…”

“…The expression of both TH and GTP-CH1 in fibroblasts grafted in denervated striata is reportedly required for the development of detectable basal L-dopa levels. 13 In vivo microdialysis studies after gene transfer with AAV have shown that TH alone is not enough for L-dopa production and that GTH-CH1 is required in the absence of exogenous BH4. 39 Thus, the role of GTH-CH1 appears to apply to both in vivo and ex vivo gene transfer.…”

Gene therapy in the CNS

“…Procedures include direct transfer in vivo of the TH cDNA to striatal cells via viral vectors, 54,135,[142][143][144] transplanting cells which have been transduced ex vivo to express TH, 87,[145][146][147][148][149][150] and administration of a DNA-liposome complex encoding TH. [151][152][153] More recently the full pathway of DA synthesis been included in this approach, with the additional transfer of the synthetic enzyme GTPcyclohydrolase (GTPCH) to generate the TH cofactor, tetrahydrobiopterin, 55,[154][155][156] as well as transfer of aromatic amino acid decorboxylase (AADC) to facilitate conversion of L-Dopa to DA. [157][158][159] Together these genes may maximize production of DA in the striatum.…”