- Email: [email protected]
651. Gene Therapy for Beta-Thalassemia: Studies on Human Cells
tion efficiency in the imatinib-resistant cell line LAMA84-R than their sensitive counterpart LAMA84-S was particularly striking, as the difference in susceptibility for the capsid mutants vs rAAV2 of LAMA84-S. On solid tumor cell lines rAAV2 was more efficient, suggesting the increased specificity of our capsid mutants for CML and PBPC. On primary human PBPC (up to 16%; 9-fold) as well as on primary human CML cells (up to 5%; 5-fold) significantly higher gene transfer could be obtained with the newly generated vectors compared to standard rAAV2. Conclusion: These novel vectors may finally enable efficient gene transfer into primary human blood progenitor cells using rAAV-based vectors with a future clinical application. Table 1:.Gene transferefficlency (GI'P) --,-,----:-:-:-:--_ _ Cell line lJest cal1sid mutant S t a n dar d -.:. r ' -,\-, ~ \.:.: ' . :' 2 _ ---i BVI73 (CML) : 4 8 ,4 ~ f 4 ' ,3,0.63' 1..,8,- - - - - - i EM3 (CML) i3.9 ± 0.7' 0.2 ± 0.2 --i LAMA84-S(CML) 178.2 ± 7.9' 7.5± 3.2 L ~M J\84-R ( CM L) --f. 8 , 0 ~ 0 .3· 92.4 ± 0.9 ----' HL-60 (AMI.) 54.0 ± 5.4 53 ~6 ±8 .7 KGla (AML) '25,4 ± 2.9' 12.8 ± 5.0 Data given as mean ± SO. ·p<0.05
650. In Vivo Selection of Hematopoietic Cells Following Retroviral Gene Transfer in a Dog with Mucopolysaccharidosis VII
Thomas M. O'Malley,' Xiucui Ma,2 Uimook Chol,' Patricia O'Donnell,' Ping Wang,' Douglas R. Kennedy,' James Rhodes ,' Caroline Bryan,' Peter J. Felsburg,' Harry L. Malech, ' Katherine P. Ponder,' Mark E. Haskins.' 'School of Veterinary Medicine, University ofPennsylvania, Philadelphia, PA; 2School ofMedicine, Washington University, St. Louis, MO; WIAID, National Institutes ofHealth, Bethesda, MD. MPS VII is a lysosomal storage disorder caused by deficient activity of beta-glucuronidase (GUSB). Neonatal retroviral (RV) gene therapy has been successful in dramatically reducing the clinical disease through cross correction from GUSB secreted by transduced hepatocytes, In addition , a low percentage ofperipheral whitc blood cells «1% ofcclls at >6 years post-treatment) were also stably transduced and expressed GUSB. Here we present an approach to explore the significance of localized white blood cell-based GUSB expression against a background of circulating serum activity by selecting for an increased number of transduced hematopoietic cells. A three day-old dog received an intravenous injection of7.5xl 09 transducing units/kg ofan amphotropic gamma RV with the canine GUSB and a mutant murine methylguanine rnethyltransfcrasc (MGMT)-GFP fusion protein downstream of IRES, followed by the WPRE. Transcription was under control of the viral LTR and human alpha-1 antitrypsin promoter. The P144K MGMT component of the fusion protein is a DNA repair enzyme insensitive to inactivation by 06-benzylguanine (BG) allowing for in vivo selection of transduced hematopoietic cells with the methylating agent, Temozolomide (TMZ) , after infusion ofBG. Serum GUSB, the majority of which was likely secreted by the liver, rose rapidly and has averaged 37.6 +/- 7.0 Ulml (14% of normal) over eight months. The number of GFP+ peripheral blood monocytcs, lymphocytes, and neutrophils initially stabilized at <0.1% for each lineage. Administration of BGrrMZ commenced at eleven weeks of age as a single day treatment in 21-day cycles, consisting of an intravenous infusion ofBG over 40 minutes immediately followed by oral TMZ. The dog has received eight treatments in a gradual dose escalation of BGITMZ from 80/37 to 120/600 mg/m-, The 120/600 mg/rn? dose was biologically active as seen by transient increases in each lineage. The absolute number ofGFP+ monocytes and lymphocytes peaked 17 days after treatment at 5.6/ul (1.20%) and 5.8/ul (0.29%), respectively. Maximum marking ofneutrophils occurred 21 days after treatment at 130/ul (1.86%). For the subsequent cycle, the 120/600 mg/m? dose was repeated and the response Molecular 'Therapy Volume 15. Supplement l, ~ br 2007 Copyright © T he American Soci ety o r Gen e "1l1f:r.lpy
was morc pronounced with absolute numbers ofGFP+ monocytes, lymphocytes, and neutrophils at 14 days after treatment reaching 27.7/ul (5.44%), 22.8/ul (1.55%), and 193.5/ul (5.33%), respectively. Serum GUSB activity has increased 2-fold during the course of the BGITMZ selection. Clinically, the dog is similar to those previously seen with relatively low serum GUSB activity: he is able to stand, but has corneal clouding and mitral valve insufficiency.
651. Gene Therapy for Beta-Thalassemia: Studies on Human Cells Emanuela A. Roselli,' Rossano Ccsari,' Annarita Miccio,' Francesca Tiboni, ' Paola Corbella,' Claudia Rossi,' Erika Biral,' Sarah Marktcl,' Michael Antoniou/ Marco Andrcani ,~ Guido Lucarelli,~ Giuliana Ferrari.' 1H. San Raffaele Telethon Institute for Gene Therapy (HSR- TIGET), H.S. Raffaele, Milan, Italy; 2Pediatric Clinical Research Unit (PCRU-TIGET), H'S. Raffaele, Milan, Italy ; 3King's College London, Guy's Campus. London, United Kingdom; "Mediterranean Institute ofHematology. Policlinico di Tor Ti!rgata, Rome, Italy. Beta-thalassemia is a common monogenic disorder and by affecting thousands of annually born children represents an increasing world health problem. So far, allogeneic bone marrow (BM) transplantation from HLA-matched donors represents the only curative treatment, although limited to patients with compatible donors. A gene therapy approach based on autologous transplantation ofgenetically corrected hematopoietic stem cells (HSCs) remains therefore a desirable goal. However, the validation ofa gene therapy approach requires to obtain results of gene correction in a broad number of patients ' cells, since different parameters, such as the heterogeneity in the molecular defects and in the proportion of alpha and nonalpha chains, will represent a key element to set a threshold in the amount of vector-derived beta-chain required to correct the chains imbalance. In order to address these issues, we collected samples from BM aspirates and isolated CD34 +cells from 58 beta" and beta? thalassem ic patients. The phenotype analysis on total mononucleated cells, tested by FACS analysis for the expression oflineage surface markers (CD3 , CD 13, CDI9, GpA and CD71), showed expansion of the erythroid compartment and a reduction in the myeloid and lymphoid sub-populations. Indeed, FACS analysis of erythroid markers expression in thalassemic CD34+ progenitors revealed a normal proportion of GlycophorinA+ cells and a decreased level ofCD71 +progenitors, and the c1onogenic potential of thalassemic CD34 +cells , tested in comparison to normal samples, resulted to be comparable. Following pre-activation and transduction by Icntiviral vectors, analysis of hematopoietic surface markers showed that transduced BM-progenitors maintain the CD34+phenotype. An erythroid-specific lentiviral vector (LV) expressing human beta-globin (GLOBE) was used to transduce BM derived CD34+ cells at high efficiency (37-95%, mean value: 58%). The efficacy ofthe GLOBE LV in correcting the human thalassemic phenotype was tested in an in vitro model of erythropoiesis. Upon transduction, normal level of HbA expression was achieved in erythroblastic cultures and in single BFU-E, associated with a progression towards erythroid maturation, which was impaired in mock-transduced thalassemic cells. Taqrnan and FACS analysis, performed on BFU-Es, showed that all transduced colonies express the transgene, thus indicating absence of position effect variegation and silencing. Molecular analysis on bulk population showed proviral integrity, with no detectable rearrangements and an average proviral copy number ranging from 1.3 to 2.4. Microarray analysis ofthe thalassemic CD34 +cells expression profile and molecular analysis of LV integrations are under evaluation.
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650. In Vivo Selection of Hematopoietic Cells Following Retroviral Gene Transfer in a Dog with Mucopolysaccharidosis VII
Thomas M. O'Malley,' Xiucui Ma,2 Uimook Chol,' Patricia O'Donnell,' Ping Wang,' Douglas R. Kennedy,' James Rhodes ,' Caroline Bryan,' Peter J. Felsburg,' Harry L. Malech, ' Katherine P. Ponder,' Mark E. Haskins.' 'School of Veterinary Medicine, University ofPennsylvania, Philadelphia, PA; 2School ofMedicine, Washington University, St. Louis, MO; WIAID, National Institutes ofHealth, Bethesda, MD. MPS VII is a lysosomal storage disorder caused by deficient activity of beta-glucuronidase (GUSB). Neonatal retroviral (RV) gene therapy has been successful in dramatically reducing the clinical disease through cross correction from GUSB secreted by transduced hepatocytes, In addition , a low percentage ofperipheral whitc blood cells «1% ofcclls at >6 years post-treatment) were also stably transduced and expressed GUSB. Here we present an approach to explore the significance of localized white blood cell-based GUSB expression against a background of circulating serum activity by selecting for an increased number of transduced hematopoietic cells. A three day-old dog received an intravenous injection of7.5xl 09 transducing units/kg ofan amphotropic gamma RV with the canine GUSB and a mutant murine methylguanine rnethyltransfcrasc (MGMT)-GFP fusion protein downstream of IRES, followed by the WPRE. Transcription was under control of the viral LTR and human alpha-1 antitrypsin promoter. The P144K MGMT component of the fusion protein is a DNA repair enzyme insensitive to inactivation by 06-benzylguanine (BG) allowing for in vivo selection of transduced hematopoietic cells with the methylating agent, Temozolomide (TMZ) , after infusion ofBG. Serum GUSB, the majority of which was likely secreted by the liver, rose rapidly and has averaged 37.6 +/- 7.0 Ulml (14% of normal) over eight months. The number of GFP+ peripheral blood monocytcs, lymphocytes, and neutrophils initially stabilized at <0.1% for each lineage. Administration of BGrrMZ commenced at eleven weeks of age as a single day treatment in 21-day cycles, consisting of an intravenous infusion ofBG over 40 minutes immediately followed by oral TMZ. The dog has received eight treatments in a gradual dose escalation of BGITMZ from 80/37 to 120/600 mg/m-, The 120/600 mg/rn? dose was biologically active as seen by transient increases in each lineage. The absolute number ofGFP+ monocytes and lymphocytes peaked 17 days after treatment at 5.6/ul (1.20%) and 5.8/ul (0.29%), respectively. Maximum marking ofneutrophils occurred 21 days after treatment at 130/ul (1.86%). For the subsequent cycle, the 120/600 mg/m? dose was repeated and the response Molecular 'Therapy Volume 15. Supplement l, ~ br 2007 Copyright © T he American Soci ety o r Gen e "1l1f:r.lpy
was morc pronounced with absolute numbers ofGFP+ monocytes, lymphocytes, and neutrophils at 14 days after treatment reaching 27.7/ul (5.44%), 22.8/ul (1.55%), and 193.5/ul (5.33%), respectively. Serum GUSB activity has increased 2-fold during the course of the BGITMZ selection. Clinically, the dog is similar to those previously seen with relatively low serum GUSB activity: he is able to stand, but has corneal clouding and mitral valve insufficiency.
651. Gene Therapy for Beta-Thalassemia: Studies on Human Cells Emanuela A. Roselli,' Rossano Ccsari,' Annarita Miccio,' Francesca Tiboni, ' Paola Corbella,' Claudia Rossi,' Erika Biral,' Sarah Marktcl,' Michael Antoniou/ Marco Andrcani ,~ Guido Lucarelli,~ Giuliana Ferrari.' 1H. San Raffaele Telethon Institute for Gene Therapy (HSR- TIGET), H.S. Raffaele, Milan, Italy; 2Pediatric Clinical Research Unit (PCRU-TIGET), H'S. Raffaele, Milan, Italy ; 3King's College London, Guy's Campus. London, United Kingdom; "Mediterranean Institute ofHematology. Policlinico di Tor Ti!rgata, Rome, Italy. Beta-thalassemia is a common monogenic disorder and by affecting thousands of annually born children represents an increasing world health problem. So far, allogeneic bone marrow (BM) transplantation from HLA-matched donors represents the only curative treatment, although limited to patients with compatible donors. A gene therapy approach based on autologous transplantation ofgenetically corrected hematopoietic stem cells (HSCs) remains therefore a desirable goal. However, the validation ofa gene therapy approach requires to obtain results of gene correction in a broad number of patients ' cells, since different parameters, such as the heterogeneity in the molecular defects and in the proportion of alpha and nonalpha chains, will represent a key element to set a threshold in the amount of vector-derived beta-chain required to correct the chains imbalance. In order to address these issues, we collected samples from BM aspirates and isolated CD34 +cells from 58 beta" and beta? thalassem ic patients. The phenotype analysis on total mononucleated cells, tested by FACS analysis for the expression oflineage surface markers (CD3 , CD 13, CDI9, GpA and CD71), showed expansion of the erythroid compartment and a reduction in the myeloid and lymphoid sub-populations. Indeed, FACS analysis of erythroid markers expression in thalassemic CD34+ progenitors revealed a normal proportion of GlycophorinA+ cells and a decreased level ofCD71 +progenitors, and the c1onogenic potential of thalassemic CD34 +cells , tested in comparison to normal samples, resulted to be comparable. Following pre-activation and transduction by Icntiviral vectors, analysis of hematopoietic surface markers showed that transduced BM-progenitors maintain the CD34+phenotype. An erythroid-specific lentiviral vector (LV) expressing human beta-globin (GLOBE) was used to transduce BM derived CD34+ cells at high efficiency (37-95%, mean value: 58%). The efficacy ofthe GLOBE LV in correcting the human thalassemic phenotype was tested in an in vitro model of erythropoiesis. Upon transduction, normal level of HbA expression was achieved in erythroblastic cultures and in single BFU-E, associated with a progression towards erythroid maturation, which was impaired in mock-transduced thalassemic cells. Taqrnan and FACS analysis, performed on BFU-Es, showed that all transduced colonies express the transgene, thus indicating absence of position effect variegation and silencing. Molecular analysis on bulk population showed proviral integrity, with no detectable rearrangements and an average proviral copy number ranging from 1.3 to 2.4. Microarray analysis ofthe thalassemic CD34 +cells expression profile and molecular analysis of LV integrations are under evaluation.
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