Intravenous nonviral gene therapy causes normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental parkinsonism.

Brain gene-targeting technology is used to reversibly normalize tyrosine hydroxylase (TH) activity in the striatum of adult rats, using the experimental 6-hydroxydopamine model of Parkinson's disease. The TH expression plasmid is encapsulated inside an 85-nm PEGylated immunoliposome (PIL) that is targeted with either the OX26 murine monoclonal antibody (MAb) to the rat transferrin receptor (TfR) or with the mouse IgG2a isotype control antibody. TfRMAb-PIL, or mIgG2a-PIL, is injected intravenously at a dose of 10 microg of plasmid DNA per rat. TfRMAb-PIL, but not mIgG2a-PIL, enters the brain via the transvascular route. The targeting TfRMAb enables the nanocontainer carrying the gene to undergo both receptor-mediated transcytosis across the blood-brain barrier (BBB) and receptor-mediated endocytosis into neurons behind the BBB by accessing the TfR. With this approach, the striatal TH activity ipsilateral to the intracerebral injection of the neurotoxin was normalized and increased from 738 +/- 179 to 5486 +/- 899 pmol/hr per milligram of protein. The TH enzyme activity measurements were corroborated by TH immunocytochemistry, which showed that the entire striatum was immunoreactive for TH after intravenous gene therapy. The normalization of striatal biochemistry was associated with a reversal of apomorphine-induced rotation behavior. Lesioned animals treated with the apomorphine exhibited 20 +/- 5 and 6 +/- 2 rotations/min, respectively, after intravenous administration of the TH plasmid encapsulated in mIgG2a-PIL and TfRMAb-PIL. These studies demonstrate that it is possible to normalize brain enzyme activity by intravenous administration and nonviral gene transfer.

Normalization of striatal tyrosine hydroxylase and reversal of motor impairment in experimental parkinsonism with intravenous nonviral gene therapy and a brain-specific promoter.

The goal of this work was to normalize striatal tyrosine hydroxylase (TH) activity with intravenous nonviral TH gene therapy and at the same time eliminate ectopic TH gene expression in peripheral organs such as liver in the rat. TH-expression plasmids, containing either the SV40 promoter or the glial fibrillary acidic protein (GFAP) gene promoter, were globally delivered to the brain across the blood-brain barrier (BBB) after intravenous administration of pegylated immunoliposomes (PILs). The GFAP-TH- or SV40-TH-expression plasmids were encapsulated in the interior of 85-nm PILs, which were targeted across both the BBB and the neuronal cell membrane with a monoclonal antibody (mAb) to the transferrin receptor (TfR). Striatal TH activity was 98% depleted with the unilateral intracerebral injection of 6-hydroxydopamine. TH in the striatum ipsilateral to the lesion was normalized 3 days after the intravenous injection of 10 microg per rat of either the SV40-TH or the GFAP-TH plasmid DNA. Whereas the SV40-TH gene caused a 10-fold increase in hepatic TH activity, there was no increase in liver TH with the GFAP-TH gene. The GFAP-TH gene therapy caused an 82% reduction in apomorphine-induced rotation in the lesioned rats. Confocal microscopy using antibodies to TH, GFAP, and neuronal nuclei (NeuN) showed the GFAP-TH gene was selectively expressed in nigra-striatal neurons, with no expression in either cortical neurons, or astrocytes. These studies demonstrate that global delivery of exogenous genes to the brain is possible with intravenous nonviral gene transfer, and that ectopic gene expression is eliminated with the use of brain-specific gene promoters.

U1 adaptors for the therapeutic knockdown of the oncogene pim-1 kinase in glioblastoma.

U1 small nuclear interference (U1i) has recently been described as a novel gene silencing mechanism. U1i employs short oligonucleotides, so-called U1 adaptors, for specific gene knockdown, expanding the field of current silencing strategies that are primarily based on RNA interference (RNAi) or antisense. Despite the potential of U1 adaptors as therapeutic agents, their in vivo application has not yet been studied. Here we explore U1i by analyzing U1 adaptor-mediated silencing of the oncogene Pim-1 in glioblastoma cells. We have generated Pim-1-specific U1 adaptors comprising DNA, locked nucleic acids (LNA), and 2'-O-Methyl RNA and demonstrate their ability to induce a Pim-1 knockdown, leading to antiproliferative and pro-apoptotic effects. For the therapeutic in vivo application of U1 adaptors, we establish their complexation with branched low molecular weight polyethylenimine (PEI). Upon injection of nanoscale PEI/adaptor complexes into subcutaneous glioblastoma xenografts in mice, we observed the knockdown of Pim-1 that resulted in the suppression of tumor growth. The absence of hepatotoxicity and immune stimulation also demonstrates the biocompatibility of PEI/adaptor complexes. We conclude that U1i represents an alternative to RNAi for the therapeutic silencing of pathologically upregulated genes and demonstrate the functional relevance of Pim-1 oncogene knockdown in glioblastoma. We furthermore introduce nanoscale PEI/adaptor complexes as efficient and safe for in vivo application, thus offering novel therapeutic approaches based on U1i-mediated gene knockdown.