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1094. Biological Identification of Ankyrin-1 Promoters Transcribed at Higher Rates for Globin Gene Therapy
1094. Biological Identification of Ankyrin-1 Promoters Transcribed at Higher Rates for Globin Gene Therapy
1095. Neuronal Apoptosis Related to HIV-1 gp120 Can Be Inhibited by Different Strategies Using rSV40-Delivery of Antioxidant Enzymes
Karina Laflamme; Laura Elnitski.' Patrick G. Gallagher,' David M. Bodine.' 'Genetic and Molecular Branch. National Human Genome Research Institute, Bethesda, MD; 2Genollle Technology, National Human Genome Research Institute, Rockville, MD; JPediatrics, Yale University, New Haven, CT.
Jean-Pierre Louboutin, I Lokesh Agrawal, I Beverly A. S. Reyes.? Elisabeth 1. van Bockstaele.' David S. Strayer.' I Pathology; Thomas Jefferson University, Philadelphia, PA; 'Neurosurgery and Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, PA.
Gene Therapy for the hemoglobin chain disorders, Sickle Cell Disease (SCD) or beta thalassemia (B-thal), requires both efficient globin gene transfer into hematopoietie stem cells (HSC) , as well as expression of globin mRNA and protein at levels that are >20% of the level of a-globin. Expression of the B-likc globin genes in either lenti- or gamma-RNA virus vectors requires enhancer elements from the Locus Control Region (LCR). However, the powerful LCR enhancer elements pose a significant risk of insertional activation ofleukemiagenes. We have taken an alternative approach, using enhancer-independent promoters from genes expressed in erythrocytes, to express sufficient levels of B-like globin. We have focused on the erythroid ankyrin (ANK-I E) promoter, a compact GC-rich promoter with no conserved sequences. We have previously demonstrated that a double copy gamma-MLY vector containing the ANK-I E promoter linked to a y-globin gene was produced at high titer. In mice repopulated with HSC transduced with theANKI Ely-globin double copy vector, y-globin mRNA and protein were expressed at a uniform level of 7.5% of a-globin per vector copy. To obtain the 3-to-4-fold increase in y-globin expression needed to reach therapeutic levels, we have taken advantage of our recent demonstration that patients with a deletion of a TG dinucleotide at position -73/-72 in the transcribed, but not translated, region of the ANK-I E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID. We tested whether altering the sequence in the TFIID-binding region of the ANK-IE promoter would increase TFIID binding and result in increased ANK-IE/y-globin transcription. We generated a library of ANKIE promoters with degenerate sequence in the TrllD-binding region , preserving the critical TG dinucleotide (NNNNNTGNN). This library of promoters was transcribed in nuclear extract from erythroid K562 cells. The RNA transcripts were cloned by 5'RACE and analyzed by sequencing. Five different sequences were obtained: GGCGGTGAG, GCGGGTGAG, GGGGGTGAG, CGGGGTGAG, and the wild type sequence TGCGGTGAG. The consensus sequence derived from these clones , (GIT)(G/C)(G/C)GGTGAG, was found at two other locations in the ANK-I E promoter, as well as in 25% of 5'UTRs across the human genome (p=2.2e-16). ANK-IE promoters containing the novel sequences were tested in a cell-free in vitro transcription assay. Transcription products generated by the GCGGGTGAG promoter were increased -1.2-fold in K562 nuclear extracts when compared to wild type. ANK-I E promoters containing the novel sequences were also linked to a luciferase reporter gene and tested individually in transient and stable transfection assays in K562 cells. The GCGGGTGAG and GGCGGTGAG promoters expressed 7- and 2.5-fold higher levels ofluciferase mRNAand protein than the wild type promoter (p=0.00 I; 0.006, respectively). \\'e are evaluating the ability of these promoters to direct higher levels ofy-globin expression in primary mouse erythroid cells.
S418
HIV-I infection is the most common cause of dementia in adults under 40 years of age. mV-I coat glycoprotein gp 120 has been implicated as a mediator of neurotoxicity, since it elicits neuron apoptosis in culture. However, studies testing direct injection of gp 120 into the brain have yielded conflicting results. We tested the neurotoxic potential of gp 120 in vivo, and evaluated the cfIectiveness of gene delivery of antioxidant molecules in protecting neurons from gpl20-induced apoptosis. We injected different doses (100 ng/microl, 250 ng/microl, 500 ng/microl) of gp 120 unilaterally into the caudate-putamen (CP) of the rat brain using stereotaxic conditions. Saline , and thc contralateral CP, were used as controls. Brains were harvested at different time points after injection (6 hours to 14 days) and processed for TUNEL assay. Recombinant, Tag-deleted SV40-derived vectors were designed to carry CulZn superoxide dismutase (SY(SOD I» or glutathione peroxidase (SV(GPx 1». These enzymes convert the free radical oxygen species superoxide (02-) to peroxide (02-2) and peroxide to water respectively. SY(I3UGT) was used as an unrelated rSY40 control. Rats were given vectors stereotaxically, either intraparenchyrnally into the caudate-putamen (CP) or into the lateral ventricle (LY). To study ifrSY40 gene delivery could be enhanced by blood brain barrier (BBB) disruption, we administered 30% mannitol intraperitoneally (i.p.) prior injecting the vector into the LV. For all doses ofgp 120, peak cellular apoptosis was seen 1 dayafterthe injection. The number ofapoptotic cells was greater after injection of 500 and 250 ng/microl , than after injection of 100 ng/microl. In all cases, TUNEL-positive cells were mostly of neuronal origin (stained by Neurotracc); a few apoptotic cells were microglial cells immunostained by CD68 antibody. Injection of either SV(SODI) or SV(GPx I) into the CP, from 4 to 24 weeks before injection of 500 ngimicrol gpl20 into the same area significantly reduced the number ofapoptotic cells. Numbers ofTUNEL-positive cells were also substantially reduced when 500 ng/microl gpl20 was injected into the CP I or 2 months after SY(SODI) was administered into the LY. A similar reduction in gpl20-induced apoptosis was seen 2 months (but not I month) after injection of SY(GPx I) in thc LV. Injecting mannitol i.p. prior to injection ofSY(SOD I) or SV(GPx I) into the LV further improved SV(SOD I) and SV(GPx I) protection from neuron apoptosis elicited when 500 ng/microl gp 120 was injected into the CP, compared to intaventricular injection without mannitol. No protection was observed using SY(BUGT). These data document the in vivo neurotoxicity ofHIY-1 gp 120and suggest that diverse gene delivery strategies ofantioxidant molecules using rSY40 vectors may be effective in protecting neurons from oxidant damage due to HIY-I gp 120 and other causes .
Molecular Therapy Yofume 15. Supplement I, .\by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr
1095. Neuronal Apoptosis Related to HIV-1 gp120 Can Be Inhibited by Different Strategies Using rSV40-Delivery of Antioxidant Enzymes
Karina Laflamme; Laura Elnitski.' Patrick G. Gallagher,' David M. Bodine.' 'Genetic and Molecular Branch. National Human Genome Research Institute, Bethesda, MD; 2Genollle Technology, National Human Genome Research Institute, Rockville, MD; JPediatrics, Yale University, New Haven, CT.
Jean-Pierre Louboutin, I Lokesh Agrawal, I Beverly A. S. Reyes.? Elisabeth 1. van Bockstaele.' David S. Strayer.' I Pathology; Thomas Jefferson University, Philadelphia, PA; 'Neurosurgery and Farber Institute for Neurosciences, Thomas Jefferson University, Philadelphia, PA.
Gene Therapy for the hemoglobin chain disorders, Sickle Cell Disease (SCD) or beta thalassemia (B-thal), requires both efficient globin gene transfer into hematopoietie stem cells (HSC) , as well as expression of globin mRNA and protein at levels that are >20% of the level of a-globin. Expression of the B-likc globin genes in either lenti- or gamma-RNA virus vectors requires enhancer elements from the Locus Control Region (LCR). However, the powerful LCR enhancer elements pose a significant risk of insertional activation ofleukemiagenes. We have taken an alternative approach, using enhancer-independent promoters from genes expressed in erythrocytes, to express sufficient levels of B-like globin. We have focused on the erythroid ankyrin (ANK-I E) promoter, a compact GC-rich promoter with no conserved sequences. We have previously demonstrated that a double copy gamma-MLY vector containing the ANK-I E promoter linked to a y-globin gene was produced at high titer. In mice repopulated with HSC transduced with theANKI Ely-globin double copy vector, y-globin mRNA and protein were expressed at a uniform level of 7.5% of a-globin per vector copy. To obtain the 3-to-4-fold increase in y-globin expression needed to reach therapeutic levels, we have taken advantage of our recent demonstration that patients with a deletion of a TG dinucleotide at position -73/-72 in the transcribed, but not translated, region of the ANK-I E promoter are ankyrin deficient due to reduced binding of the transcription initiation complex, TFIID. We tested whether altering the sequence in the TFIID-binding region of the ANK-IE promoter would increase TFIID binding and result in increased ANK-IE/y-globin transcription. We generated a library of ANKIE promoters with degenerate sequence in the TrllD-binding region , preserving the critical TG dinucleotide (NNNNNTGNN). This library of promoters was transcribed in nuclear extract from erythroid K562 cells. The RNA transcripts were cloned by 5'RACE and analyzed by sequencing. Five different sequences were obtained: GGCGGTGAG, GCGGGTGAG, GGGGGTGAG, CGGGGTGAG, and the wild type sequence TGCGGTGAG. The consensus sequence derived from these clones , (GIT)(G/C)(G/C)GGTGAG, was found at two other locations in the ANK-I E promoter, as well as in 25% of 5'UTRs across the human genome (p=2.2e-16). ANK-IE promoters containing the novel sequences were tested in a cell-free in vitro transcription assay. Transcription products generated by the GCGGGTGAG promoter were increased -1.2-fold in K562 nuclear extracts when compared to wild type. ANK-I E promoters containing the novel sequences were also linked to a luciferase reporter gene and tested individually in transient and stable transfection assays in K562 cells. The GCGGGTGAG and GGCGGTGAG promoters expressed 7- and 2.5-fold higher levels ofluciferase mRNAand protein than the wild type promoter (p=0.00 I; 0.006, respectively). \\'e are evaluating the ability of these promoters to direct higher levels ofy-globin expression in primary mouse erythroid cells.
S418
HIV-I infection is the most common cause of dementia in adults under 40 years of age. mV-I coat glycoprotein gp 120 has been implicated as a mediator of neurotoxicity, since it elicits neuron apoptosis in culture. However, studies testing direct injection of gp 120 into the brain have yielded conflicting results. We tested the neurotoxic potential of gp 120 in vivo, and evaluated the cfIectiveness of gene delivery of antioxidant molecules in protecting neurons from gpl20-induced apoptosis. We injected different doses (100 ng/microl, 250 ng/microl, 500 ng/microl) of gp 120 unilaterally into the caudate-putamen (CP) of the rat brain using stereotaxic conditions. Saline , and thc contralateral CP, were used as controls. Brains were harvested at different time points after injection (6 hours to 14 days) and processed for TUNEL assay. Recombinant, Tag-deleted SV40-derived vectors were designed to carry CulZn superoxide dismutase (SY(SOD I» or glutathione peroxidase (SV(GPx 1». These enzymes convert the free radical oxygen species superoxide (02-) to peroxide (02-2) and peroxide to water respectively. SY(I3UGT) was used as an unrelated rSY40 control. Rats were given vectors stereotaxically, either intraparenchyrnally into the caudate-putamen (CP) or into the lateral ventricle (LY). To study ifrSY40 gene delivery could be enhanced by blood brain barrier (BBB) disruption, we administered 30% mannitol intraperitoneally (i.p.) prior injecting the vector into the LV. For all doses ofgp 120, peak cellular apoptosis was seen 1 dayafterthe injection. The number ofapoptotic cells was greater after injection of 500 and 250 ng/microl , than after injection of 100 ng/microl. In all cases, TUNEL-positive cells were mostly of neuronal origin (stained by Neurotracc); a few apoptotic cells were microglial cells immunostained by CD68 antibody. Injection of either SV(SODI) or SV(GPx I) into the CP, from 4 to 24 weeks before injection of 500 ngimicrol gpl20 into the same area significantly reduced the number ofapoptotic cells. Numbers ofTUNEL-positive cells were also substantially reduced when 500 ng/microl gpl20 was injected into the CP I or 2 months after SY(SODI) was administered into the LY. A similar reduction in gpl20-induced apoptosis was seen 2 months (but not I month) after injection of SY(GPx I) in thc LV. Injecting mannitol i.p. prior to injection ofSY(SOD I) or SV(GPx I) into the LV further improved SV(SOD I) and SV(GPx I) protection from neuron apoptosis elicited when 500 ng/microl gp 120 was injected into the CP, compared to intaventricular injection without mannitol. No protection was observed using SY(BUGT). These data document the in vivo neurotoxicity ofHIY-1 gp 120and suggest that diverse gene delivery strategies ofantioxidant molecules using rSY40 vectors may be effective in protecting neurons from oxidant damage due to HIY-I gp 120 and other causes .
Molecular Therapy Yofume 15. Supplement I, .\by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr