- Email: [email protected]
640. CNS-Directed Gene Delivery To Treat Experimental Parkinson's Disease
GENE THERAPY FOR THE NERVOUS SYSTEM II 640. CNS-Directed Gene Delivery To Treat Experimental Parkinson’s Disease David S. Strayer,1 David W. Anderson,1 Pierre Cordelier,1 Jay S. Schneider.1 1 Pathology, Anatomy and Cell Biology, Jefferson Medical College, Philadelphia, PA. Background: Parkinson’s disease is characterized by death of dopaminergic neurons in the substantia nigra and loss of dopamine in the striatum. Loss of dopamine in the striatum results in excessive activity of GABA-ergic neurons in the internal globus pallidus (GPi) and substantia nigra pars reticulata (SNr), which appear to drive the expression of Parkinsonian symptoms. One might thus consider approaching treatment of Parkinson’s disease by inhibiting the activity of these overactive GABAergic neurons. We had previously shown that administering antisense oligoribonucleotides directed against mRNAs for enzymes involved in GABA synthesis, GAD67 and GAD65, transiently (up to 7 days) improved Parkinsonian symptomatology in rodent and primate models of Parkinsonism. In the present studies, we tested whether recombinant SV40-derived gene delivery vectors could deliver these antisense molecules constitutively and adequately to provide longer-lasting improvement. Methods: SV40 vectors were designed to express either the antisense GAD67 oligonucleotide or both antisense GAD65 + GAD67. In both SV(GAD67) and GAD(65+67), expression of each antisense transgenes was driven separately by adenoviral VA pol III promoters. This promoter directs small RNA transcripts to the cytosol. Rats were unilaterally lesioned with 6-OHDA. Their rotational responses to d-amphetamine were measured 4 and 6 weeks post-lesion to assess the extent of the 6-OHDA lesion. Asymmetrical rotational responses to d-amphetamine are characteristic of this experimental model of Parkinson’s disease. The rats then received a single stereotaxic injection into the globus pallidus (GP) of 10 9 infectious units of SV(GAD67), SV(GAD65+67), or SV(nef-FLAG) as a control vector. Animals were tested 1, 2 and 4 weeks post-injection to see if delivery of the antisense transgenes reduced the rotational responses to damphetamine. Brains were processed for routine histology and in situ PCR to verify the presence of the virus. Results: Animals receiving SV(GAD67) showed 30%, 59% and 60% average decreases in rotation at 1, 2 and 4 weeks respectively. Rats receiving (SVGAD65+67) also showed decreases in amphetamine-induced rotations, while those receiving the control rSV40 or no vector had unchanged responses to d-amphetamine. Histologically, slight tissue damage was seen around the needle tracts, but there was no evidence of inflammation at the injection sites. In situ PCR to detect vector DNA showed many rSV40-positive cells at the vector delivery site on the injected side, but none on the uninjected side. Conclusions: Recombinant SV40-mediated gene delivery can effectively transduce neurons in vivo when injected stereotaxically. rSV40 gene delivery of antisense against GAD 67 or GAD65 + GAD67 to inhibit GABA synthesis can lead to improvement in Parkinsonian symptomatology in this experimental model of Parkinson’s disease. Thus, recombinant Tag-deleted SV40-derived vectors may be effective tools for gene delivery to the central nervous system.
641. Gene Therapy to Brain Microglia Using rSV40 Vectors Pierre Cordelier,1 David S. Strayer.1 1 Pathology, Jefferson Medical College, Philadelphia, PA. Objectives: Therapeutic gene delivery to CNS cells continues to present challenges. In these studies, we asked whether a particular CNS population, microglia, could be transduced efficiently by S250
recombinant Tag-deleted SV40 vectors (rSV40s), using as a test system the inhibition of HIV in these cells. Antiretroviral agents, however effective in the periphery, penetrate the CNS poorly. CNS HIV, sheltered from chemotherapy, may thus cause CNS disease. Microglia play a major role in viral persistence in the CNS. Recombinant SV40 vectors (rSV40) transduce both dividing and quiescent cells efficiently, and so were tested for their ability to deliver anti-HIV transgenes to primary human microglia. Methods: Enriched microglia were prepared from second trimester human fetal brains. We used rSV40 vectors constitutively expressing either RevM10, an inhibitor of HIV-1 Rev (SV(RevM10), or human α1antitrypsin (α1AT), which inhibits processing of both HIV-1 Env and Gag (SV(AT)). In addition, we used α1AT and human IFN-α2, a powerful inhibitor of HIV integration, which expression would be driven by the HIV-1 LTR and so be trans-activated by HIV infection (SV[LTR](AT) and SV[LTR](IFN), respectively). HIV replication was measured by ELISA as supernatant p24 antigen. Results: SV(RevM10), SV[LTR](IFN), SV[LTR](AT) and SV(AT) transduced microglia efficiently and without detectable toxicity. By immunostaining, 95% of unselected cells expressed the transgenes. Fetal microglia treated with SV(RevM10), SV[LTR](IFN), SV[LTR](AT) or SV(AT) strongly resisted challenge with HIV-1 BaL, an R5-tropic strain of HIV that infects microglia. In addition, we tested whether transduction with SV(AT) or SV(RevM10) could affect HIV replication if the gene delivery was performed after HIV infection was established in microglia. We found that rSV40 transduction strongly inhibited HIV replication in microglia that were transduced with SV(AT) or SV(RevM10) well after HIV infection was established in these cells. Conclusions: rSV40 vectors transduced primary human microglia, a potential CNS cellular target for HIV, effectively and without selection. Several different potent anti-HIV transgenes delivered by rSV40s strongly inhibited HIV replication in these microglia. rSV40s even blocked HIV replication in culture transduced after HIV infection was established. Thus gene delivery to microglia may have a role to play in inhibiting HIV replication in CNS AIDS.
642. Fetal Brain Injections with AAV Vectors Results in Widespread Enzyme Activity in the Postnatal Mouse Brain Brian A. Karolewski,1 Marco A. Passini,2 John H. Wolfe.1,3 1 Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA, United States; 2Neurology, Genzyme, Cambridge, MA, United States; 3Department of Pathobiology and Center for Comparative Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States. Mucopolysaccharidosis (MPS) VII is a heritable lysosomal storage disease caused by the deficiency of beta-glucuronidase (GUSB) enzyme. MPS VII is a chronic and progressive multiorgan disorder with signs of pathology already present during fetal life. Most therapeutic approaches for MPS VII have focused on postnatal treatments. These approaches have been shown to reverse pathology in most organs; however, a more complete reversal of disease is seen when therapy is performed in neonates compared to later in life. Genetically modified fetal liver cells have been used to deliver GUSB to the mouse fetus. Even though fetal liver cell engraftment was very low in these experiments, the disease progression was delayed in transplanted mice. This suggests that early treatment with even low levels of enzyme can result in clinical improvement. Neonatal brains treated with adeno-associated vectors (AAV) can result in widespread pattern of viral transduction and enzyme expression when delivered into the lateral ventricle. To investigate patterns of transduction in the fetal mouse brain, we tested AAV1 and AAV2 vectors expressing the human GUSB promoter and cDNA. We are characterizing the patterns of transduction and enzyme activity Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy
641. Gene Therapy to Brain Microglia Using rSV40 Vectors Pierre Cordelier,1 David S. Strayer.1 1 Pathology, Jefferson Medical College, Philadelphia, PA. Objectives: Therapeutic gene delivery to CNS cells continues to present challenges. In these studies, we asked whether a particular CNS population, microglia, could be transduced efficiently by S250
recombinant Tag-deleted SV40 vectors (rSV40s), using as a test system the inhibition of HIV in these cells. Antiretroviral agents, however effective in the periphery, penetrate the CNS poorly. CNS HIV, sheltered from chemotherapy, may thus cause CNS disease. Microglia play a major role in viral persistence in the CNS. Recombinant SV40 vectors (rSV40) transduce both dividing and quiescent cells efficiently, and so were tested for their ability to deliver anti-HIV transgenes to primary human microglia. Methods: Enriched microglia were prepared from second trimester human fetal brains. We used rSV40 vectors constitutively expressing either RevM10, an inhibitor of HIV-1 Rev (SV(RevM10), or human α1antitrypsin (α1AT), which inhibits processing of both HIV-1 Env and Gag (SV(AT)). In addition, we used α1AT and human IFN-α2, a powerful inhibitor of HIV integration, which expression would be driven by the HIV-1 LTR and so be trans-activated by HIV infection (SV[LTR](AT) and SV[LTR](IFN), respectively). HIV replication was measured by ELISA as supernatant p24 antigen. Results: SV(RevM10), SV[LTR](IFN), SV[LTR](AT) and SV(AT) transduced microglia efficiently and without detectable toxicity. By immunostaining, 95% of unselected cells expressed the transgenes. Fetal microglia treated with SV(RevM10), SV[LTR](IFN), SV[LTR](AT) or SV(AT) strongly resisted challenge with HIV-1 BaL, an R5-tropic strain of HIV that infects microglia. In addition, we tested whether transduction with SV(AT) or SV(RevM10) could affect HIV replication if the gene delivery was performed after HIV infection was established in microglia. We found that rSV40 transduction strongly inhibited HIV replication in microglia that were transduced with SV(AT) or SV(RevM10) well after HIV infection was established in these cells. Conclusions: rSV40 vectors transduced primary human microglia, a potential CNS cellular target for HIV, effectively and without selection. Several different potent anti-HIV transgenes delivered by rSV40s strongly inhibited HIV replication in these microglia. rSV40s even blocked HIV replication in culture transduced after HIV infection was established. Thus gene delivery to microglia may have a role to play in inhibiting HIV replication in CNS AIDS.
642. Fetal Brain Injections with AAV Vectors Results in Widespread Enzyme Activity in the Postnatal Mouse Brain Brian A. Karolewski,1 Marco A. Passini,2 John H. Wolfe.1,3 1 Neurology, Children’s Hospital of Philadelphia, Philadelphia, PA, United States; 2Neurology, Genzyme, Cambridge, MA, United States; 3Department of Pathobiology and Center for Comparative Medical Genetics, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, United States. Mucopolysaccharidosis (MPS) VII is a heritable lysosomal storage disease caused by the deficiency of beta-glucuronidase (GUSB) enzyme. MPS VII is a chronic and progressive multiorgan disorder with signs of pathology already present during fetal life. Most therapeutic approaches for MPS VII have focused on postnatal treatments. These approaches have been shown to reverse pathology in most organs; however, a more complete reversal of disease is seen when therapy is performed in neonates compared to later in life. Genetically modified fetal liver cells have been used to deliver GUSB to the mouse fetus. Even though fetal liver cell engraftment was very low in these experiments, the disease progression was delayed in transplanted mice. This suggests that early treatment with even low levels of enzyme can result in clinical improvement. Neonatal brains treated with adeno-associated vectors (AAV) can result in widespread pattern of viral transduction and enzyme expression when delivered into the lateral ventricle. To investigate patterns of transduction in the fetal mouse brain, we tested AAV1 and AAV2 vectors expressing the human GUSB promoter and cDNA. We are characterizing the patterns of transduction and enzyme activity Molecular Therapy Vol. 7, No. 5, May 2003, Part 2 of 2 Parts
Copyright © The American Society of Gene Therapy