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769. Correction of Canine XSCID by Gene Therapy Using Preclinical SIV Lentiviral Vectors Expressing Human or Canine Gamma Chain
769. Correction of Canine XSCID by Gene Therapy Using Preclinical SIV Lentiviral Vectors Expressing Human or Canine Gamma Chain
Morvarid Moayeri,' Douglas R. Kennedy- Nora Naumann; Yasuhiro Ikeda,' Felsburg J. Peter/ Harry L. Malech,' Suk See De Ravin. '
'Laboratory ofHost Defenses, NIAID, NIH, Bethesda, MD; "School of J!i?terinGlJ' Medicine, University ofPennsylvania, Philadelphia, PA; 'Molecular Medicine Program, Mayo Clinic College ofMedicine, Rochester, MN.
X-linkedsevere combined immunodeficiency (XSCID) is caused by mutations in the common gamma chain (ye) of receptors for interleukins -2, -4, -7, -9, -15 and -21 and characterized by profound immunodeficiency and early mortality. Infants with XSCID arc conventionally transplanted by T cell depleted bone marrow with very modest or no conditioning. While 5-yr survival is >95% with HLA-matched sibling donors, most patients receive a haploidentical graft from a parent, resulting in lower survival and less robust immune reconstitution. An alternative treatment in infants without a sibling donor is ex vivo autologous stem cell gene therapy, capable of achieving substantial to complete immune reconstitution. However, emergence of leukemia in a few children undergoing gene therapy with gammaretroviral vectors warrants development of safer gene delivery systems. The gammaretrovirusescurrently in use have potent enhancers in the LTRand a predilectionto insert at the 5' end of genes, potentially increasingthe chance of insertional mutagenesis. On the other hand, self-inactivating (SIN) lentiviral vectors seem to be safer as they do not show this property and can be constructed with internal promotors having less potent enhancer activity and also insulators. We have constructed 3 SIN simian immunodeficiency viral vectors(SIV ) encodingeither the human or copy of the chicken insulator dog yc, two of which have a doubl~ core element in the 3' LTR. We pseudotyped these vectors with a chimeric RD114 envelope to enhance targeting of hematopoietic stem cells (HSC) and avoid the cytotoxicityof the VSV-G envelope traditionally used with lentivectors.Wehave previouslyshown that it is possibleto correctan XSCIDdog model by in vivo gene therapy using an RDl l-l-pseudctyped gammaretroviral vector encoding dye. Unlike the XSCID mouse model which lacks 8 cells, the dog model more closely resemblesthe phenotype in humansand is ideal for preclinical testing of viral vectors. In our study, ten 2-5 day-old pups were injected intravenously with 24-30 mls virus (2.2-7x107 infectious viral partielcs per dog). Improvement of absolute lymphocytc counts has been achieved in dogs treated with each of the 3 vectors up to 45 wks post-gene therapy, associated with variable yc expression in their lymphocytes «5% up to 80%) and an average viral copy number of<3 in these cells. Furthermore, in some dogs up to 6% of the myeloid lineage showed gene marking 20-24 wks after viral delivery, indicating that early committed progenitors or HSCs had been targeted, Genomic insertionanalysis shows a polyclonal pattern and individual insertion sites arc currently being evaluated. Our finding that both dyc and hyc can efficiently reconstitute the 'f-lymphoid compartment in XSCID dogs makes it an excellent large animal model for preclinical evaluation of those SIV vectors encoding the human gene, the actual candidate vectors being considered for the clinic.
S296
770. Self-Inactivating Retroviral Vectors Expressing hlL2RG from an Internal SFFV Enhancer-Promoter Induce Leukemia by Insertional Mutagenesis
Ute Modlich,' Axel Schambach,' Daniel Wicke,' Sabine Knoess,' Christopher Baurn.':'
I Department ofExperimental Hematology, Hannover Medical School, Hannover; Germany; lDivision ofExperimental Hematology, Cincinnati Children Hospital Medical Center; Cincinnati.
s
To address the potential of leukemia induction by retroviral vectors expressing the human IL2-receptor common gamma chain (hIL2RG), we transplanted bone marrow cells transduced by retroviral SIN vectors containing the SFFV promoter/enhancer in C57BI6 mice. In the first experiment recipients had a low average transgene copy number in peripheral leukocytes(0.12 +/- 0.2, n=5), in the second the marking was significantly higher (IL2RG: 0.57 +/- 004, n=6, and dsRED; 0.2 +/- 0.25, n=6). No severe adverse events caused bygene-modifiedcells (SAE-GMC)were detected in primary recipients during an observation time of II months. Mice (n=9) transplanted with MFG-yC (encoding hIL2RG) transduced cellsor with cellstransducedby a lentiviral vectorencodinghIL2RG (SFFV promoter) also remained free of SAE-GMC(mean marking 0.2 +/- 0.25, n=6, and 0.8 +/- 0.2, n=6, respectively).Thus, hlL2RG expression from a strong enhancer-promoterwas not sufficient for transformation in mice with significant gene marking and a long follow-up (> 12 months). This is in contrast to previous findings in mice transplanted with lentivirally transduced hematopoietic cells that expressed the murine I12rg and developed hematopoietic tumors with high frequency after 6 to 12 months (Woods et al., Nature 2006). However,when we performedserial transplantations with bone marrow of selected primary hlL2RG recipients, two cases of lymphoid and myeloid leukemia arose with a latency of more than 4 months (>15 months since first transplantation). Both leukemias contained SIN retroviral insertions in the murine Evil proto-oncogene and were positive for hIL2RG surface expression by FACS, but noneof the leukemiccells neededreceptorstimulation by recombinant IL-2, 4, 7, 9 or 15 to grow in culture. In line with these findings, insertion site analysis in transplanted mice, including an additional cohort transplanted with dsRed2-expressingcells, revealedfrequentinsertionsin proto-oncogenesand signalinggenes by SIN vectors containing the internal SFFV enhancer-promoter. Selection for dominant clones occurred. These findings reveal that SIN vectors with strong internal enhancer-promoters may induce clonal dominanceand even malignanttransformationby insertional activationof cellularproto-oncogenesand additional hematopoietic stress induced by the secondary transplantation. In conclusion, SIN retroviral vectors containing the internal SFFV promoter are still able to transactivate cellular genes and trigger clonal outgrowth and malignant transformation in vivo. Insertional mutagenesis is essential for leukemia development induced by IL2RG vectors, as we have previously demonstrated in mice transplanted with LTR vectors encoding other transgenes (Li et al., Science2002; Modlich et al., Blood 2005). Our recent insights from cell culture models suggestthat these side effects can be prevented by further improved vector design and the use of internal cellular promoters(sec abstract by Zychlinski et al.).
Molecular Therapy Volume15. Supplement I, .\by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr
Morvarid Moayeri,' Douglas R. Kennedy- Nora Naumann; Yasuhiro Ikeda,' Felsburg J. Peter/ Harry L. Malech,' Suk See De Ravin. '
'Laboratory ofHost Defenses, NIAID, NIH, Bethesda, MD; "School of J!i?terinGlJ' Medicine, University ofPennsylvania, Philadelphia, PA; 'Molecular Medicine Program, Mayo Clinic College ofMedicine, Rochester, MN.
X-linkedsevere combined immunodeficiency (XSCID) is caused by mutations in the common gamma chain (ye) of receptors for interleukins -2, -4, -7, -9, -15 and -21 and characterized by profound immunodeficiency and early mortality. Infants with XSCID arc conventionally transplanted by T cell depleted bone marrow with very modest or no conditioning. While 5-yr survival is >95% with HLA-matched sibling donors, most patients receive a haploidentical graft from a parent, resulting in lower survival and less robust immune reconstitution. An alternative treatment in infants without a sibling donor is ex vivo autologous stem cell gene therapy, capable of achieving substantial to complete immune reconstitution. However, emergence of leukemia in a few children undergoing gene therapy with gammaretroviral vectors warrants development of safer gene delivery systems. The gammaretrovirusescurrently in use have potent enhancers in the LTRand a predilectionto insert at the 5' end of genes, potentially increasingthe chance of insertional mutagenesis. On the other hand, self-inactivating (SIN) lentiviral vectors seem to be safer as they do not show this property and can be constructed with internal promotors having less potent enhancer activity and also insulators. We have constructed 3 SIN simian immunodeficiency viral vectors(SIV ) encodingeither the human or copy of the chicken insulator dog yc, two of which have a doubl~ core element in the 3' LTR. We pseudotyped these vectors with a chimeric RD114 envelope to enhance targeting of hematopoietic stem cells (HSC) and avoid the cytotoxicityof the VSV-G envelope traditionally used with lentivectors.Wehave previouslyshown that it is possibleto correctan XSCIDdog model by in vivo gene therapy using an RDl l-l-pseudctyped gammaretroviral vector encoding dye. Unlike the XSCID mouse model which lacks 8 cells, the dog model more closely resemblesthe phenotype in humansand is ideal for preclinical testing of viral vectors. In our study, ten 2-5 day-old pups were injected intravenously with 24-30 mls virus (2.2-7x107 infectious viral partielcs per dog). Improvement of absolute lymphocytc counts has been achieved in dogs treated with each of the 3 vectors up to 45 wks post-gene therapy, associated with variable yc expression in their lymphocytes «5% up to 80%) and an average viral copy number of<3 in these cells. Furthermore, in some dogs up to 6% of the myeloid lineage showed gene marking 20-24 wks after viral delivery, indicating that early committed progenitors or HSCs had been targeted, Genomic insertionanalysis shows a polyclonal pattern and individual insertion sites arc currently being evaluated. Our finding that both dyc and hyc can efficiently reconstitute the 'f-lymphoid compartment in XSCID dogs makes it an excellent large animal model for preclinical evaluation of those SIV vectors encoding the human gene, the actual candidate vectors being considered for the clinic.
S296
770. Self-Inactivating Retroviral Vectors Expressing hlL2RG from an Internal SFFV Enhancer-Promoter Induce Leukemia by Insertional Mutagenesis
Ute Modlich,' Axel Schambach,' Daniel Wicke,' Sabine Knoess,' Christopher Baurn.':'
I Department ofExperimental Hematology, Hannover Medical School, Hannover; Germany; lDivision ofExperimental Hematology, Cincinnati Children Hospital Medical Center; Cincinnati.
s
To address the potential of leukemia induction by retroviral vectors expressing the human IL2-receptor common gamma chain (hIL2RG), we transplanted bone marrow cells transduced by retroviral SIN vectors containing the SFFV promoter/enhancer in C57BI6 mice. In the first experiment recipients had a low average transgene copy number in peripheral leukocytes(0.12 +/- 0.2, n=5), in the second the marking was significantly higher (IL2RG: 0.57 +/- 004, n=6, and dsRED; 0.2 +/- 0.25, n=6). No severe adverse events caused bygene-modifiedcells (SAE-GMC)were detected in primary recipients during an observation time of II months. Mice (n=9) transplanted with MFG-yC (encoding hIL2RG) transduced cellsor with cellstransducedby a lentiviral vectorencodinghIL2RG (SFFV promoter) also remained free of SAE-GMC(mean marking 0.2 +/- 0.25, n=6, and 0.8 +/- 0.2, n=6, respectively).Thus, hlL2RG expression from a strong enhancer-promoterwas not sufficient for transformation in mice with significant gene marking and a long follow-up (> 12 months). This is in contrast to previous findings in mice transplanted with lentivirally transduced hematopoietic cells that expressed the murine I12rg and developed hematopoietic tumors with high frequency after 6 to 12 months (Woods et al., Nature 2006). However,when we performedserial transplantations with bone marrow of selected primary hlL2RG recipients, two cases of lymphoid and myeloid leukemia arose with a latency of more than 4 months (>15 months since first transplantation). Both leukemias contained SIN retroviral insertions in the murine Evil proto-oncogene and were positive for hIL2RG surface expression by FACS, but noneof the leukemiccells neededreceptorstimulation by recombinant IL-2, 4, 7, 9 or 15 to grow in culture. In line with these findings, insertion site analysis in transplanted mice, including an additional cohort transplanted with dsRed2-expressingcells, revealedfrequentinsertionsin proto-oncogenesand signalinggenes by SIN vectors containing the internal SFFV enhancer-promoter. Selection for dominant clones occurred. These findings reveal that SIN vectors with strong internal enhancer-promoters may induce clonal dominanceand even malignanttransformationby insertional activationof cellularproto-oncogenesand additional hematopoietic stress induced by the secondary transplantation. In conclusion, SIN retroviral vectors containing the internal SFFV promoter are still able to transactivate cellular genes and trigger clonal outgrowth and malignant transformation in vivo. Insertional mutagenesis is essential for leukemia development induced by IL2RG vectors, as we have previously demonstrated in mice transplanted with LTR vectors encoding other transgenes (Li et al., Science2002; Modlich et al., Blood 2005). Our recent insights from cell culture models suggestthat these side effects can be prevented by further improved vector design and the use of internal cellular promoters(sec abstract by Zychlinski et al.).
Molecular Therapy Volume15. Supplement I, .\by 2007 Copyright © '111C American Societyo f Gene TIICr.lpr