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659. The Plasticity of Hematopoietic Stem Cells (HSC): Rat HSC Transduced In Situ by rSV40 Vectors Differentiate into Multiple Lineages of CNS Cells
NEUROLOGIC: ADVANCES IN VECTORS, DELIVERY AND IMAGING at 5.5 weeks post-infusion. Clinical observations of these monkeys after vector infusions revealed no behavioral abnormalities during the study period. Histopathology revealed only minimal localized inflammation along the needle track in response to cannula placement and vector infusion. AADC immunohistochemistry demonstrated that vector was distributed evenly throughout the putamen. The device is manufactured according to device quality system regulations and is being used successfully in a human clinical trial. This device could be used in a number of CNS applications where small volume delivery and precise targeting of therapeutics is needed.
659. The Plasticity of Hematopoietic Stem Cells (HSC): Rat HSC Transduced In Situ by rSV40 Vectors Differentiate into Multiple Lineages of CNS Cells Jean-Pierre Louboutin,1 Bianling Liu,1 Beverley S. Reyes,2 Elizabeth J. Van Bockstaele,2 David S. Strayer.1 1 Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, PA; 2Neurosurgery, Farber Institute for Neurosciences, Philadelphia, PA. An increasing number of reports suggest that hematopoietic stem cells can migrate into the brain and differentiate into cell types only present in the CNS. In situ gene transfer to hematopoietic stem/ progenitor cells by intramarrow injection of viral vectors has rarely been rarely reported and the consequent distribution of transgenepositive derivative cells in different organs has never been assessed. We injected recombinant Tag-deleted SV40 vectors (rSV40s) into the femoral bone marrow of rats. Test rats were given a rSV40 bearing a marker transgene, the FLAG epitope appended to the carboxyl terminus of HIV-1 Nef, which was used as a carrier protein. Control animals received an unrelated rSV40. FLAG expression was detected by FACS or fluorescence microscopy following intracellular immunostaining. Up to 12% (average, 5%) of circulating peripheral blood cells expressed the transgene through the end of the experiment at 18 months post injection. The distribution of FLAG-positive cells in the brain was extensively studied using serial cryostat sections of the brain taken at different times (< 1 month, 4 months, and 18 months) post-intramarrow injection. FLAG-positive cells were mainly found in the dentate gyrus (DG) of the hippocampus, either 4 or 18 months after the injection of SV40 (Nef-FLAG). Transgene-positive cells were found in the hilus, as well as in the inner/outer blades of the DG, with similar percentages for the 2 areas (5.2, and 5.5% of the total number of cells respectively). No expression of FLAG was seen in the brains of the control animals. Analysis of DG sections taken within 1 month of intra-marrow injection showed no evidence of such positive cells, indicating that direct transduction of the brain did not occur and suggesting that the FLAG-positive cells seen were the result of migration into the DG of transduced cells or their derivatives. The nature of the transgene-positive cells was assessed by double immunofluorescence using markers for neurons (neuN), microglia (ED1), and astrocytes (GFAP). The percentages of FLAG-positive cells expressing neuN, ED1, and GFAP, were respectively 48.6, 49.7, and 1.6%. FLAG expression was rarely seen in the few proliferating cells stained for PCNA. These data suggest that hematopoietic stem cells, or their derivatives, can migrate into the brain and differentiate into cells specific for the CNS.
Molecular Therapy Volume11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
660. Quantitative Comparison of AAV Serotypes AAV2, AAV5, and AAVrh.10 Efficiency for CNS Gene Therapy Following Intracranial Gene Delivery Dolan Sondhi*,1 Daniel A. Peterson*,2 Elizabeth Vassallo,1 Christine T. Sanders,2 Jamie A. Stratton,1 Neil R. Hackett,1 Guangping Gao,3 James M. Wilson,3 Ronald G. Crystal.1 1 Genetic Med, Weill Medical College of Cornell University, NY, NY; 2Chicago Medical School, North Chicago, IL; 3School of Medicine, University of Pennsylvania, Philadelphia, PA *Both authors contributed equally. Late infantile neuronal ceroid lipofuscinosis (Batten disease) is a fatal pediatric neurodegenerative disease resulting from mutation of the CLN2 gene encoding for a tripeptidyl peptidase (TPP-I). Deficiency of TPP-I results in aberrant degradation of membrane proteins in the lysosome with development of inclusion bodies which lead to neuronal cell death throughout the brain. We have previously shown that AAV2 based vectors expressing human CLN2 cDNA driven by the CMV-enhanced chicken-b-actin hybrid promoter can mediate long term expression of TPP-I in rat brain. In the present study, we have compared the ability of an AAV2-based vector to mediate distribution of TPP-I production in the rat CNS to that of recombinant AAV2-based vectors pseudotyped with capsids derived from human AAV5 and rh.10, a rhesus macaque derived AAV. Vectors (2.5x109 genome copies) were injected into the striatum of 8 wk old male F344 rats. Fluorometric enzyme assay for TPP-I in the injected region after 4 wk revealed that while AAV2- and AAV5-mediated gene delivery produced TPP-I enzyme levels that were 1.2 and 1.8 fold of endogenous (p<0.01 compared to naive) respectively, AAVrh.10-mediated delivery provided TPPI levels that approached 17-fold above endogenous levels (p<0.01 compared to either naive or AAV5-treated animals). Immunohistochemical staining showed vector-derived TPP-I in the striatum of rats injected with each of the three vectors, with AAV5 and AAVrh.10 producing the greatest striatal filling. Volumetric analysis showed that while infection by AAV2-derived vector lead to TPP-I detection in 5.1±0.4% of the striatum, AAV5 and AAVrh.10 resulted in TPP-I expression in 46.9±2.9% and 51.2±6.1% of the striatum respectively. Co-immunofluorescence studies demonstrated that virtually all neurons in the region of AAVrh.10 injection were TPP-I positive but no glia stained TPP-I positive. When the whole brain was examined, AAVrh.10 mediated more extensive TPP-I expression, with TPP-I being detected in the globus pallidus and basal forebrain. There was also prominent substantia nigra staining at 4 wk with AAVrh.10 delivery, suggesting retrograde distribution. In addition TPP-I positive cells were readily detected in the frontal cortex and the thalamus, both of which project to the location of the injection in the striatum. When non-striatal staining was quantitated, AAV2 administration resulted in TPP-I positive cells in 0.4±0.1% of the hemisphere compared to 4.8±0.6% for AAV5 and 8.6±1.4% for AAVrh.10. The data show that capsids from other AAV serotypes give more widespread and higher levels of gene transfer than AAV2 vectors and suggests that high transgene expression levels may correlate with a wider distribution of transgene product. In particular, AAV vectors pseudotyped with the capsid from AAVrh.10 may be applicable to gene therapy for diffuse neurological diseases.
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659. The Plasticity of Hematopoietic Stem Cells (HSC): Rat HSC Transduced In Situ by rSV40 Vectors Differentiate into Multiple Lineages of CNS Cells Jean-Pierre Louboutin,1 Bianling Liu,1 Beverley S. Reyes,2 Elizabeth J. Van Bockstaele,2 David S. Strayer.1 1 Pathology, Anatomy, and Cell Biology, Thomas Jefferson University, Philadelphia, PA; 2Neurosurgery, Farber Institute for Neurosciences, Philadelphia, PA. An increasing number of reports suggest that hematopoietic stem cells can migrate into the brain and differentiate into cell types only present in the CNS. In situ gene transfer to hematopoietic stem/ progenitor cells by intramarrow injection of viral vectors has rarely been rarely reported and the consequent distribution of transgenepositive derivative cells in different organs has never been assessed. We injected recombinant Tag-deleted SV40 vectors (rSV40s) into the femoral bone marrow of rats. Test rats were given a rSV40 bearing a marker transgene, the FLAG epitope appended to the carboxyl terminus of HIV-1 Nef, which was used as a carrier protein. Control animals received an unrelated rSV40. FLAG expression was detected by FACS or fluorescence microscopy following intracellular immunostaining. Up to 12% (average, 5%) of circulating peripheral blood cells expressed the transgene through the end of the experiment at 18 months post injection. The distribution of FLAG-positive cells in the brain was extensively studied using serial cryostat sections of the brain taken at different times (< 1 month, 4 months, and 18 months) post-intramarrow injection. FLAG-positive cells were mainly found in the dentate gyrus (DG) of the hippocampus, either 4 or 18 months after the injection of SV40 (Nef-FLAG). Transgene-positive cells were found in the hilus, as well as in the inner/outer blades of the DG, with similar percentages for the 2 areas (5.2, and 5.5% of the total number of cells respectively). No expression of FLAG was seen in the brains of the control animals. Analysis of DG sections taken within 1 month of intra-marrow injection showed no evidence of such positive cells, indicating that direct transduction of the brain did not occur and suggesting that the FLAG-positive cells seen were the result of migration into the DG of transduced cells or their derivatives. The nature of the transgene-positive cells was assessed by double immunofluorescence using markers for neurons (neuN), microglia (ED1), and astrocytes (GFAP). The percentages of FLAG-positive cells expressing neuN, ED1, and GFAP, were respectively 48.6, 49.7, and 1.6%. FLAG expression was rarely seen in the few proliferating cells stained for PCNA. These data suggest that hematopoietic stem cells, or their derivatives, can migrate into the brain and differentiate into cells specific for the CNS.
Molecular Therapy Volume11, Supplement 1, May 2005 Copyright The American Society of Gene Therapy
660. Quantitative Comparison of AAV Serotypes AAV2, AAV5, and AAVrh.10 Efficiency for CNS Gene Therapy Following Intracranial Gene Delivery Dolan Sondhi*,1 Daniel A. Peterson*,2 Elizabeth Vassallo,1 Christine T. Sanders,2 Jamie A. Stratton,1 Neil R. Hackett,1 Guangping Gao,3 James M. Wilson,3 Ronald G. Crystal.1 1 Genetic Med, Weill Medical College of Cornell University, NY, NY; 2Chicago Medical School, North Chicago, IL; 3School of Medicine, University of Pennsylvania, Philadelphia, PA *Both authors contributed equally. Late infantile neuronal ceroid lipofuscinosis (Batten disease) is a fatal pediatric neurodegenerative disease resulting from mutation of the CLN2 gene encoding for a tripeptidyl peptidase (TPP-I). Deficiency of TPP-I results in aberrant degradation of membrane proteins in the lysosome with development of inclusion bodies which lead to neuronal cell death throughout the brain. We have previously shown that AAV2 based vectors expressing human CLN2 cDNA driven by the CMV-enhanced chicken-b-actin hybrid promoter can mediate long term expression of TPP-I in rat brain. In the present study, we have compared the ability of an AAV2-based vector to mediate distribution of TPP-I production in the rat CNS to that of recombinant AAV2-based vectors pseudotyped with capsids derived from human AAV5 and rh.10, a rhesus macaque derived AAV. Vectors (2.5x109 genome copies) were injected into the striatum of 8 wk old male F344 rats. Fluorometric enzyme assay for TPP-I in the injected region after 4 wk revealed that while AAV2- and AAV5-mediated gene delivery produced TPP-I enzyme levels that were 1.2 and 1.8 fold of endogenous (p<0.01 compared to naive) respectively, AAVrh.10-mediated delivery provided TPPI levels that approached 17-fold above endogenous levels (p<0.01 compared to either naive or AAV5-treated animals). Immunohistochemical staining showed vector-derived TPP-I in the striatum of rats injected with each of the three vectors, with AAV5 and AAVrh.10 producing the greatest striatal filling. Volumetric analysis showed that while infection by AAV2-derived vector lead to TPP-I detection in 5.1±0.4% of the striatum, AAV5 and AAVrh.10 resulted in TPP-I expression in 46.9±2.9% and 51.2±6.1% of the striatum respectively. Co-immunofluorescence studies demonstrated that virtually all neurons in the region of AAVrh.10 injection were TPP-I positive but no glia stained TPP-I positive. When the whole brain was examined, AAVrh.10 mediated more extensive TPP-I expression, with TPP-I being detected in the globus pallidus and basal forebrain. There was also prominent substantia nigra staining at 4 wk with AAVrh.10 delivery, suggesting retrograde distribution. In addition TPP-I positive cells were readily detected in the frontal cortex and the thalamus, both of which project to the location of the injection in the striatum. When non-striatal staining was quantitated, AAV2 administration resulted in TPP-I positive cells in 0.4±0.1% of the hemisphere compared to 4.8±0.6% for AAV5 and 8.6±1.4% for AAVrh.10. The data show that capsids from other AAV serotypes give more widespread and higher levels of gene transfer than AAV2 vectors and suggests that high transgene expression levels may correlate with a wider distribution of transgene product. In particular, AAV vectors pseudotyped with the capsid from AAVrh.10 may be applicable to gene therapy for diffuse neurological diseases.
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