- Email: [email protected]
47. Cytokine gene-modified mesenchymal stem cells promote functional recovery and reduce infarct size in the rat middle cerebral artery occlusion model
NEURO GENE THERAPY I: NEW APPROACHES FOR DOMINANT, RECESSIVE, DEGENERATIVE AND AUTOIMMUNE DISEASES (p<0.05). In mice were MEP were recordable, central conduction time was significant decreased in IL-4 treated vs control mice (p<0.05). IL-4 gene therapy by intracisternal administration of HDAd vectors therefore induces clinical and functional recovery in a murine model of MS. Biodistribution, immunological and toxicity studies in non-human primates are currently in progress.
47. Cytokine Gene-Modified Mesenchymal Stem Cells Promote Functional Recovery and Reduce Infarct Size in the Rat Middle Cerebral Artery Occlusion Model 1,2
2
Kazuhiko Kurozumi, Kiminori Nakamura, Tomotsugu Ichikawa,1 Yasuhiro Ono,1 Takashi Tamiya,3 Yoshinori Ito,2 Osamu Honmou,4 Kiyohiro Houkin,4 Hirofumi Hamada,2 Isao Date.1 1 Department of Neurological Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Okayama, Japan; 2Department of Molecular Medicine, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan; 3 Department of Neurological Surgery, Kagawa University, Takamatsu, Kagawa, Japan; 4Department of Neurosurgery, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan. Background: Examination of the clinical therapeutic efficacy of using mesenchymal stem cells (MSC) has recently been the focus in many studies. MSC were reported to ameliorate functional deficits after stroke in rats, with some of this improvement possibly resulting from the role of cytokines secreted by these cells. In order to enhance such cytokine effects, we transfected telomerized human MSC with a variety of cytokine gene using a fiber-mutant F/RGD adenovirus vector and investigated whether these cells contributed to functional recovery in a rat transient middle cerebral artery occlusion (MCAO) model. Methods and Results: First, we have constructed and tested the adenoviral vector which expresses brain-derived neurotrophic factor (BDNF) (Kurozumi et al. Mol Ther. 2004). BDNF production by MSC-BDNF cells was 23-fold greater than that observed in noninfected MSC. Following MCAO, rats that received MSC-BDNF showed more significant functional recovery than did control rats in the limb placement test and the treadmill test. We summed the areas of hyperintensity on T2-weighted images over the central six MR images and expressed lesion volume as a percentage of contralateral hemispheric volume (%HLV). Seven days and fourteen days after MCAO, there was a significant reduction in %HLV in rats in the MSC-BDNF group compared with rats in the control groups. The number of TUNEL positive cells in the ischemic boundary zone was significantly less in animals treated with MSC-BDNF compared to animals in the control group. MSC in the ischemic area were examined morphologically 7 days after MCAO to determine whether they exhibited a neuronal phenotype. Some transplanted MSC were immunopositive for the neuronal maker NeuN and the astrocytic maker GFAP. Secondly, we investigated which cytokine most enhances the effect of MSC among BDNF, glial derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), or neurotrophin 3 (NT3), and whether MSC infected with each gene contributed to improved functional recovery in the rat transient MCAO model. Rats that received MSC-GDNF (P<0.05) as well as MSC-BDNF (P<0.05) showed significantly more functional recovery than did control rats following MCAO in the limb placement test after 7 and 14 days. MRI analysis also revealed that the rats in the MSC-GDNF group (P<0.05) as well as MSC-BDNF group (P<0.05) exhibited more significant recovery from ischemia after 7 and 14 days. On the other
S20
hand, the rats that received MSC-CNTF or MSC-NT3 showed neither functional recovery nor ischemic damage reduction compared with the control rats following MCAO. Conclusions: The intracerebral injection of MSC transfected with the BDNF or GDNF gene using a fiber-mutant adenovirus vector resulted in improved function and reduced ischemic damage in a rat model of MCAO. These data indicate a possible usefulness of this gene-modified cell therapy as an approach for the treatment of stroke.
48. In Vivo and In Situ Delivery of HuntingtinSpecific Single-Chain Fv Intrabody Genes as Therapeutic Agents for Huntington’s Disease Todd W. Miller,1,2 William J. Wolfgang,1,2 Kevin Manley,1,2 Yangsheng Zhou,1,2 Chun Zhou,1,2 Jack M. Webster,1,2 Nicholas D. Mazarakis,3 Anne Messer.1,2 1 Wadsworth Center, NY State Dept. of Health, Albany, NY; 2Dept. of Biomedical Sciences, Univ. at Albany, Albany, NY; 3 Neurobiology Group, Oxford Biomedica, Ltd., Oxford, United Kingdom. Huntington’s disease (HD) is one of several neurodegenerative disorders caused by expansion of a CAG repeat. The resulting expanded polyglutamine tract causes the huntingtin protein to form insoluble aggregates and participate in aberrant protein-protein interactions. Therapeutic intervention to prevent aggregation, reduce abnormal interactions, and allow proper proteolytic processing may reduce HD pathogenesis. Intrabodies (single-chain Fv antibody fragments) can bind with high specificity and affinity to intracellular targets, potentially altering their folding and interactions, and serving as curative agents and/or tools for drug discovery in an array of neurological disorders associated with altered protein structures. We have previously demonstrated that an intrabody targeting the N-terminus of huntingtin (upstream of the polyglutamine tract) dramatically reduces aggregate formation in HEK-293, BHK-21, and COS-7 cell lines, and provides functional protection against expanded polyglutamine toxicity in organotypic brain slice cultures. Transient dual expression of both intrabody and huntingtin exon 1 (httex1)-GFP proteins from a single vector in a rat striatal cell model (ST14A) shows that the intrabody enhances protein turnover, measured as a decrease in the amount of soluble httex1-GFP; effects were even more dramatic in the HEK293T cell line. We are now using recombinant equine infectious anemia virus (EIAV; pONY8.7), pseudotyped with either VSV-G or Rabies-G, encoding intrabodies alone or in bicistronic vectors to stably transduce neuronal cell lines and brain cells. Viral titers are 108-9IU/ml for all constructs. We have generated stably transfected striatal cell lines previously derived from wild-type, heterozygous, and homozygous htt Q111 knock-in mice (Trettel, 2000). Coexpression of the anti-htt intrabody (or control intrabody) and a GFP marker from a bicistronic cassette allows flow-sorting of positive cells expressing different levels of intrabody. There is no sign of toxicity, even at high levels of the antihtt intrabody, which was selected against an epitope common to both mutant and wild-type htt. These lines will allow further analysis of intrabody effects in the context of full-length, endogenous htt, which may better simulate aspects of the human disease. Intrastriatal injection of HD R6/1 transgenic mice with Rabies-Gpseudotyped EIAV encoding the anti-htt intrabody shows widespread, long-term (>3 months) neuronal expression in the striatum, with cortical cell labeling at higher viral concentrations. Untreated HD transgenic mice have a severe reduction in DARPP32, a D1 dopamine receptor-associated signaling protein and common marker of HD pathology. Intrabody-positive cells in the striatum of injected R6/1 mice show increased DARPP-32 expression, suggesting
Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy
47. Cytokine Gene-Modified Mesenchymal Stem Cells Promote Functional Recovery and Reduce Infarct Size in the Rat Middle Cerebral Artery Occlusion Model 1,2
2
Kazuhiko Kurozumi, Kiminori Nakamura, Tomotsugu Ichikawa,1 Yasuhiro Ono,1 Takashi Tamiya,3 Yoshinori Ito,2 Osamu Honmou,4 Kiyohiro Houkin,4 Hirofumi Hamada,2 Isao Date.1 1 Department of Neurological Surgery, Okayama University Graduate School of Medicine and Dentistry, Okayama, Okayama, Japan; 2Department of Molecular Medicine, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan; 3 Department of Neurological Surgery, Kagawa University, Takamatsu, Kagawa, Japan; 4Department of Neurosurgery, Sapporo Medical University School of Medicine, Sapporo, Hokkaido, Japan. Background: Examination of the clinical therapeutic efficacy of using mesenchymal stem cells (MSC) has recently been the focus in many studies. MSC were reported to ameliorate functional deficits after stroke in rats, with some of this improvement possibly resulting from the role of cytokines secreted by these cells. In order to enhance such cytokine effects, we transfected telomerized human MSC with a variety of cytokine gene using a fiber-mutant F/RGD adenovirus vector and investigated whether these cells contributed to functional recovery in a rat transient middle cerebral artery occlusion (MCAO) model. Methods and Results: First, we have constructed and tested the adenoviral vector which expresses brain-derived neurotrophic factor (BDNF) (Kurozumi et al. Mol Ther. 2004). BDNF production by MSC-BDNF cells was 23-fold greater than that observed in noninfected MSC. Following MCAO, rats that received MSC-BDNF showed more significant functional recovery than did control rats in the limb placement test and the treadmill test. We summed the areas of hyperintensity on T2-weighted images over the central six MR images and expressed lesion volume as a percentage of contralateral hemispheric volume (%HLV). Seven days and fourteen days after MCAO, there was a significant reduction in %HLV in rats in the MSC-BDNF group compared with rats in the control groups. The number of TUNEL positive cells in the ischemic boundary zone was significantly less in animals treated with MSC-BDNF compared to animals in the control group. MSC in the ischemic area were examined morphologically 7 days after MCAO to determine whether they exhibited a neuronal phenotype. Some transplanted MSC were immunopositive for the neuronal maker NeuN and the astrocytic maker GFAP. Secondly, we investigated which cytokine most enhances the effect of MSC among BDNF, glial derived neurotrophic factor (GDNF), ciliary neurotrophic factor (CNTF), or neurotrophin 3 (NT3), and whether MSC infected with each gene contributed to improved functional recovery in the rat transient MCAO model. Rats that received MSC-GDNF (P<0.05) as well as MSC-BDNF (P<0.05) showed significantly more functional recovery than did control rats following MCAO in the limb placement test after 7 and 14 days. MRI analysis also revealed that the rats in the MSC-GDNF group (P<0.05) as well as MSC-BDNF group (P<0.05) exhibited more significant recovery from ischemia after 7 and 14 days. On the other
S20
hand, the rats that received MSC-CNTF or MSC-NT3 showed neither functional recovery nor ischemic damage reduction compared with the control rats following MCAO. Conclusions: The intracerebral injection of MSC transfected with the BDNF or GDNF gene using a fiber-mutant adenovirus vector resulted in improved function and reduced ischemic damage in a rat model of MCAO. These data indicate a possible usefulness of this gene-modified cell therapy as an approach for the treatment of stroke.
48. In Vivo and In Situ Delivery of HuntingtinSpecific Single-Chain Fv Intrabody Genes as Therapeutic Agents for Huntington’s Disease Todd W. Miller,1,2 William J. Wolfgang,1,2 Kevin Manley,1,2 Yangsheng Zhou,1,2 Chun Zhou,1,2 Jack M. Webster,1,2 Nicholas D. Mazarakis,3 Anne Messer.1,2 1 Wadsworth Center, NY State Dept. of Health, Albany, NY; 2Dept. of Biomedical Sciences, Univ. at Albany, Albany, NY; 3 Neurobiology Group, Oxford Biomedica, Ltd., Oxford, United Kingdom. Huntington’s disease (HD) is one of several neurodegenerative disorders caused by expansion of a CAG repeat. The resulting expanded polyglutamine tract causes the huntingtin protein to form insoluble aggregates and participate in aberrant protein-protein interactions. Therapeutic intervention to prevent aggregation, reduce abnormal interactions, and allow proper proteolytic processing may reduce HD pathogenesis. Intrabodies (single-chain Fv antibody fragments) can bind with high specificity and affinity to intracellular targets, potentially altering their folding and interactions, and serving as curative agents and/or tools for drug discovery in an array of neurological disorders associated with altered protein structures. We have previously demonstrated that an intrabody targeting the N-terminus of huntingtin (upstream of the polyglutamine tract) dramatically reduces aggregate formation in HEK-293, BHK-21, and COS-7 cell lines, and provides functional protection against expanded polyglutamine toxicity in organotypic brain slice cultures. Transient dual expression of both intrabody and huntingtin exon 1 (httex1)-GFP proteins from a single vector in a rat striatal cell model (ST14A) shows that the intrabody enhances protein turnover, measured as a decrease in the amount of soluble httex1-GFP; effects were even more dramatic in the HEK293T cell line. We are now using recombinant equine infectious anemia virus (EIAV; pONY8.7), pseudotyped with either VSV-G or Rabies-G, encoding intrabodies alone or in bicistronic vectors to stably transduce neuronal cell lines and brain cells. Viral titers are 108-9IU/ml for all constructs. We have generated stably transfected striatal cell lines previously derived from wild-type, heterozygous, and homozygous htt Q111 knock-in mice (Trettel, 2000). Coexpression of the anti-htt intrabody (or control intrabody) and a GFP marker from a bicistronic cassette allows flow-sorting of positive cells expressing different levels of intrabody. There is no sign of toxicity, even at high levels of the antihtt intrabody, which was selected against an epitope common to both mutant and wild-type htt. These lines will allow further analysis of intrabody effects in the context of full-length, endogenous htt, which may better simulate aspects of the human disease. Intrastriatal injection of HD R6/1 transgenic mice with Rabies-Gpseudotyped EIAV encoding the anti-htt intrabody shows widespread, long-term (>3 months) neuronal expression in the striatum, with cortical cell labeling at higher viral concentrations. Untreated HD transgenic mice have a severe reduction in DARPP32, a D1 dopamine receptor-associated signaling protein and common marker of HD pathology. Intrabody-positive cells in the striatum of injected R6/1 mice show increased DARPP-32 expression, suggesting
Molecular Therapy Volume 9, Supplement 1, May 2004
Copyright The American Society of Gene Therapy