- Email: [email protected]
TAGTC meets across the pond
meeting report
© The American Society of Gene Therapy
doi:10.1038/mt.2008.276
doi:10.1038/mt.2008.276
TAGTC meets across the pond 3rd Transatlantic Gene Therapy Consortium London, UK, 2–3 October 2007 Robert M Frederickson1
T
he third Transatlantic Gene Therapy Consortium (TAGTC) workshop took place on 2–3 October 2007 at the Royal Society of Medicine in London. This workshop brings together leading investigators from the United States, United Kingdom, and Germany to discuss recent developments in gene therapy relevant to vector development and clinical trials. The workshop represents the culmination of multiple collaborative efforts organized as the TAGTC. The first retreat was held in 2005 at the Eberbach Monastery (Germany) in conjunction with the annual Georg-Speyer-Haus Symposium in Frankfurt, Germany. The second retreat took place in Baltimore in 2006 in conjunction with the annual meeting of the American Society of Gene Therapy. Approximately 30–40 individuals from five to seven laboratories attended each of these retreats so as to facilitate shared vector development, preclinical trials, and clinical protocols. The most recent workshop involved around 43 participants from nine groups representing some of the leading laboratories in the world in gene therapy, insertional mutagenesis, and leukemogenesis. The retreat was organized around short, data-driven talks and included equal time for discussions and planning of collaborative efforts. One outcome of these efforts was the development of a safety-improved, self-inactivating, long-terminal-repeat (LTR) enhancer-deleted, γ-retrovirus– expressing interleukin-2 receptor common γ-chain by the cellular elongation
factor 1–short promoter (pSRS11.EFS. IL2RG.pre*) for use in a severe combined immunodeficiency X1 (SCID-X1) gene therapy trial proposed for multiple sites in Europe and the United States. The vector was produced according to Good Manufacturing Practices (GMP) by transient transfection at Cincinnati Children’s Hospital Medical Center, and the collaborating sites are currently proposed to be (in the United States) Children’s Hospital Boston, Children’s Hospital–Los Angeles, Cincinnati Children’s Hospital Medical Center; (internationally) Necker Hospital (Paris) and Great Ormond Street Children’s Hospital (London). Much of the vector development was done at Hannover Medical School, Hannover, Germany. The first session of the meeting was devoted to new vector development. Boris Fehse kicked off the session with a discussion of the LeGo vector system. The LeGo vectors are a set of modular vectors characterized by efficient gene transfer and stable expression of transgenes as well as short hairpin RNAs that are aimed to facilitate functional gene analyses. Incorporation of a wide panel of fluorescent markers (with or without drug selectability) in these vectors allows identification of multiply transduced cells. Melanie Galla discussed recent results using so-called retroviral particle–mediated mRNA transfer, demonstrating the use of such reverse-transcription mutants for low, transient expression of genes of interest. Alex Schambach described work on a
1 Division of Medical Genetics, Department of Medicine, University of Washington School of Medicine, Seattle, Washington, USA Correspondence: Robert M Frederickson, Division of Medical Genetics, Suite K236D, University of Washington School of Medicine, Box 357720, Seattle, Washington 98195, USA. E-mail: [email protected]
10
modular self-inactivating γ-retroviral vector platform being developed for clinical application, reporting improved efficacy, safety, and production parameters for these vectors. Conrad Vink described efforts to engineer lentiviral vectors with a substitution of the human immunodeficiency virus type 1 (HIV-1) integrase by the sleeping beauty (SB) transposase, so as to direct integration away from active genes. SB integration is known to occur less frequently within genes than HIV-1. They show that integrase-deficient, nonintegrating vectors can deliver transgene templates for transposition by SB transposase and have used integration site analysis to confirm integration-altered patterns. This approach will be tested in models such as SCID-X1, wherein small numbers of corrected stem cells have an important survival advantage and can support effective reconstitution. Tomayasu Higashimoto described efforts to develop a lentiviral vector–based RNA interference approach to treat sickle-cell anemia, by inhibiting expression of the mutant sickle-cell hemoglobin. Though tested successfully in cell lines and in primary cells in vitro, the approach has yet to be tested in an animal model of sickle-cell disease. Stefan Stein wrapped up the session with a discussion of new SIN γ-retroviral vectors for the gene therapy of chronic granulomatous disease (CGD) that seem to show advantages over the vector used in the recent CGD trial, which has shown some side effects in affected individuals. Manuel Grez provided an update on the results of this phase I–II CGD trial in the first talk of the second session, devoted to a discussion of recent clinical results. The two adults with CGD treated in Frankfurt with gene-modified cells showed increased numbers of gene-marked cells, leading to an unbalanced dominance of clones containing integrations into the MDS1/EVI1 and PRDM16 genes. He reported the occurrence of chromosomal abnormalities in both patients, most probably caused by the transcriptional activation of MDS1 and EVI1. He also described an epigenetic inactivation of the viral promoter driving gp91phox expression leading to a lack of superoxide activity in gene-transduced www.moleculartherapy.org vol. 17 no. 1 jan. 2009
© The American Society of Gene Therapy
cells in both patients. Patient 1 died 27 months after gene therapy. Patient 2 underwent stem cell transplantation from a matched unrelated donor and is recovering from the treatment. In a trial of retroviral-mediated gene therapy for SCID-X1, 10 patients have been treated in London, and all have shown significant improvement in T-cell numbers and function. Waseem Qassim reported that half of these affected individuals have also discontinued immunoglobulin replacement therapy. At the time of writing there were no adverse events seen in this cohort (although one individual developed a T-cell leukemia as a result of vector-mediated insertional mutagenesis). For adenosine deaminase SCID, three evaluable affected individuals were treated using a spleen focus-forming virus−based retroviral vector. Melphalan conditioning was given to these individuals before infusion of gene-modified cells. In the first two individuals immune and metabolic recovery has been seen, with reinitiation of thymopoiesis in both individuals. Patient 3 had a poor stem cell harvest and did not receive sufficient cells to allow effective reconstitution; this individual has again begun receiving enzyme replacement therapy. David Williams reported on the clinical gene transfer trial in individuals with Fanconi anemia (FA) complementation group A. This trial was run concurrently with a trial seeking to collect CD34+ cells (as targets) from individuals with FA. Three individuals with FA were enrolled in the gene transfer protocol. Transduction efficiency of CD34+ cells using the γ-retrovirus–expressing FA complementation group A was 40–60% as assayed by both colony assay and quantitative PCR. Two individuals with FA received genemodified cell infusions but with low doses of cells. Both showed transient increases in hemoglobin and platelet counts. One of these two patients showed transient gene marking, albeit a small amount. A third affected individual was not infused because of the small number of cells. No adverse events were noted. Overall, the conclusion of this study was that methods to increase the number of target CD34+ cells were needed so that cell doses approaching successful immunodeficiency trials could be used. Molecular Therapy vol. 17 no. 1 jan. 2009
meeting report Lilith Reeves described efforts by the Cincinnati Children’s Research Foundation Vector Production Facility to produce a retroviral vector using a transient transfection-based protocol for a planned multi-institutional SCID-X1 trial. Large-scale GMP production of this vector has now begun. Diana Nordling then discussed the quality-control, logistical, and regulatory challenges associated with transatlantic vector production— which must adhere to standards imposed by distinct regulatory agencies. Leukemia caused by retroviral insertional mutagenesis after stem cell gene transfer has been reported in several experimental animals and in individuals treated for X-linked SCID. Dorothee von Laer presented data analyzing whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental “worst-case scenario,” her group transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of γ-retroviral vectors encoding the potent T-cell oncogenes LMO2, TCL1 (now TCL1A), or ΔTRKA, a constitutively active mutant of TRKA (now NTRK1). After transplantation into RAG-1–deficient recipients (Ly5.2), stem cell–transplanted animals developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated PCR analysis revealed mono- or oligoclonality of the malignancies. In contrast, none of the mice receiving transplanted T cells transduced with the same vectors developed leukemia/lymphoma, despite persistence of gene-modified cells. The data provide direct evidence that mature T cells are less prone to transformation than hema topoietic progenitor cells. Hans Stauss tested human T-cell receptor (TCR) variants with cysteine modifications in the constant region, or with murine constant regions. The variants generate a higher frequency of antigenspecific T cells when transduced into primary human T cells. T cells transduced with the variants bind tetramer more efficiently than T cells transduced with the wild-type TCR. T-cell avidity is the same, despite the differences in tetramer binding. TCR variants can be dominant and
completely suppress the surface expression of endogenous TCR. TCR variants are expected to improve safety and efficacy of TCR gene therapy in the future. The third session covered issues of genotoxicity and integration analysis. Chris Baum opened the session with a discussion of studies investigating the target population of insertional transformation in the hematopoietic system. He showed that insertional upregulation of proto-oncogenes is not sufficient to overcome the engraftment barrier that limits the establishment of clonal dominance in more mature hema topoietic progenitor cells. Using his recently established in vitro immortalization assay, he obtained evidence that the enhancer-promoter sequences of integrating vectors are more important risk factors of transformation than the integration profile (γ-retroviral or lentiviral). Anjali Mishra provided an update of continuing studies that address the impact of vector design on clonal selection in vivo using the C57IB bone marrow transplantation model. Sebastian Newrzela reported data showing that mature T-cell populations are less susceptible than stem cell populations to transformation by known T-cell protooncogenes, such as LMO2. Christof Kalle reported modeling and verification data that uncontrolled use of restriction enzymes can significantly bias against or completely blind the detection of insertions in up to 80% of the genome. The choice of restriction motifs is key to obtaining a repertoire of insertions covering all regions of the genome. A thorough comparison of insertions from all international immunodeficiency retrovirus vector trials demonstrates that more than 40% of all vector insertions are in frequently affected, nonrandomly distributed common insertion sites, indicating that the insertion of first-generation clinical retrovirus trials using retrovirus promoter-enhancers has subtle but measurable influence on about every second engrafted cell clone. Interestingly, the most frequently retrieved (i.e., the most intensely inserted and/or selected) genes were identical to the ones that have created clonal proliferation serious adverse effects in the X-SCID and CGD trials. Kalle demonstrated that new high-throughput methodologies allow assessment of entire repertoires of vector insertions in clinical trials on a routine 11
© The American Society of Gene Therapy
meeting report basis. Insertion distribution analysis should be considered the genomic equivalent of pharmacokinetics for vector applications in humans and is indispensable for responsible drug development in phase I gene therapy trials. Ali Nowrouzi described the use of multiplex-PCR and linear amplification-mediated-PCR to identify concatameric and integrated forms of adeno-associated virus, in an effort to clarify the genetic safety of this common vector for clinical use. Anna Schnieder next described efforts to evaluate the influence of vector integration on global gene expression using microarrays, demonstrating little or no effect of integ ration of lentivectors in HeLa cell clones. The fourth session was devoted to a discussion of strategies to enhance engraftment of hematopoietic stem cells (HSCs). Lars Müller discussed an approach to gene transfer in the context of FA, a pediatric bone marrow failure and cancer predisposition syndrome. Müller highlighted two earlier clinical trials using γ-retroviral vectors for the transduction of autologous FA HSCs. In these previous trials extensive in vitro cell manipulation had been required during transduction, probably contributing to the lack of detectable long-term engraftment of genemodified cells in the recipient individuals. As a strategy for minimizing ex vivo manipulation, a “rapid” lentiviral transduction protocol was proposed. In a mu-
12
rine Fanca–/– model, rapid transduction of Fanca–/– HSCs preserved engraftment to the level achieved in wild-type cells, resulting in long-term multilineage engraftment of gene-modified cells. Mick Milsom discussed the use of the O6-methylguanine-DNA methyltransferase (MGMT) drug resistance gene in an in vivo model of HSC selection using the alkylating agent temozolomide. His work focused upon whether, in the context of a self-inactivating γ-retroviral vector, weaker human cellular promoters could drive the expression of the MGMTP140K mutant at sufficient levels to protect and select gene-modified HSCs. The driving force of this work was that the combination of weaker promoters in a SIN configuration probably composes a safer vector configuration than an LTR vector in the context of insertional mutagenesis. Milsom demonstrated that not only were the weaker human cellular promoters able to express MGMTP140K at levels that allowed robust HSC selection, but that a vector that expressed very high levels of MGMTP140K courtesy of a viral LTR promoter conveyed a pronounced growth defect onto transduced HSCs. The final session discussed the development of new gene therapy strategies. Dao Pan reported that co-expression of MGMTP140K and a-L-iduronidase in primary hepatocytes from MPS1 mice permits efficient selection with metabolic
correction as a proof of principle for the prospect of gene therapy for this disease. Ajay Perumbeti described continuing efforts to correct sickle-cell anemia with a γ-globulin transgene delivered with a lentiviral vector into the Berkeley transgenic sickle mouse, reporting functional correction and an inprovement of hematological indices. X-linked lymphoproliferative disease is another target disease for ex vivo HSC gene therapy. Defective expression of SLAM-associated protein (SAP) makes individuals susceptible to Epstein-Barr virus–mediated malignancies. Previous studies have shown that expression of SAP can correct cytotoxic responses, and there are now efforts to develop lentiviral vectors encoding SAP endogenous promoter elements to regulate expression through ontogeny, as described in Christin Rivat’s talk. Mary Collins reported that lentiviral vectors encoding antigens are potent immunogens. Antigen expression can be targeted to dendritic cells in vivo using the dectin-2 promoter. By co-expressing activators of mitogen-activated protein kinase or interferon signaling pathways, the response to the antigen could either be enhanced or suppressed. Funding for the retreat was provided by the Leukemia Lymphoma Society (United States). The next meeting is being planned for May 2009 in Boston, Massachusetts.
www.moleculartherapy.org vol. 17 no. 1 jan. 2009
© The American Society of Gene Therapy
doi:10.1038/mt.2008.276
doi:10.1038/mt.2008.276
TAGTC meets across the pond 3rd Transatlantic Gene Therapy Consortium London, UK, 2–3 October 2007 Robert M Frederickson1
T
he third Transatlantic Gene Therapy Consortium (TAGTC) workshop took place on 2–3 October 2007 at the Royal Society of Medicine in London. This workshop brings together leading investigators from the United States, United Kingdom, and Germany to discuss recent developments in gene therapy relevant to vector development and clinical trials. The workshop represents the culmination of multiple collaborative efforts organized as the TAGTC. The first retreat was held in 2005 at the Eberbach Monastery (Germany) in conjunction with the annual Georg-Speyer-Haus Symposium in Frankfurt, Germany. The second retreat took place in Baltimore in 2006 in conjunction with the annual meeting of the American Society of Gene Therapy. Approximately 30–40 individuals from five to seven laboratories attended each of these retreats so as to facilitate shared vector development, preclinical trials, and clinical protocols. The most recent workshop involved around 43 participants from nine groups representing some of the leading laboratories in the world in gene therapy, insertional mutagenesis, and leukemogenesis. The retreat was organized around short, data-driven talks and included equal time for discussions and planning of collaborative efforts. One outcome of these efforts was the development of a safety-improved, self-inactivating, long-terminal-repeat (LTR) enhancer-deleted, γ-retrovirus– expressing interleukin-2 receptor common γ-chain by the cellular elongation
factor 1–short promoter (pSRS11.EFS. IL2RG.pre*) for use in a severe combined immunodeficiency X1 (SCID-X1) gene therapy trial proposed for multiple sites in Europe and the United States. The vector was produced according to Good Manufacturing Practices (GMP) by transient transfection at Cincinnati Children’s Hospital Medical Center, and the collaborating sites are currently proposed to be (in the United States) Children’s Hospital Boston, Children’s Hospital–Los Angeles, Cincinnati Children’s Hospital Medical Center; (internationally) Necker Hospital (Paris) and Great Ormond Street Children’s Hospital (London). Much of the vector development was done at Hannover Medical School, Hannover, Germany. The first session of the meeting was devoted to new vector development. Boris Fehse kicked off the session with a discussion of the LeGo vector system. The LeGo vectors are a set of modular vectors characterized by efficient gene transfer and stable expression of transgenes as well as short hairpin RNAs that are aimed to facilitate functional gene analyses. Incorporation of a wide panel of fluorescent markers (with or without drug selectability) in these vectors allows identification of multiply transduced cells. Melanie Galla discussed recent results using so-called retroviral particle–mediated mRNA transfer, demonstrating the use of such reverse-transcription mutants for low, transient expression of genes of interest. Alex Schambach described work on a
1 Division of Medical Genetics, Department of Medicine, University of Washington School of Medicine, Seattle, Washington, USA Correspondence: Robert M Frederickson, Division of Medical Genetics, Suite K236D, University of Washington School of Medicine, Box 357720, Seattle, Washington 98195, USA. E-mail: [email protected]
10
modular self-inactivating γ-retroviral vector platform being developed for clinical application, reporting improved efficacy, safety, and production parameters for these vectors. Conrad Vink described efforts to engineer lentiviral vectors with a substitution of the human immunodeficiency virus type 1 (HIV-1) integrase by the sleeping beauty (SB) transposase, so as to direct integration away from active genes. SB integration is known to occur less frequently within genes than HIV-1. They show that integrase-deficient, nonintegrating vectors can deliver transgene templates for transposition by SB transposase and have used integration site analysis to confirm integration-altered patterns. This approach will be tested in models such as SCID-X1, wherein small numbers of corrected stem cells have an important survival advantage and can support effective reconstitution. Tomayasu Higashimoto described efforts to develop a lentiviral vector–based RNA interference approach to treat sickle-cell anemia, by inhibiting expression of the mutant sickle-cell hemoglobin. Though tested successfully in cell lines and in primary cells in vitro, the approach has yet to be tested in an animal model of sickle-cell disease. Stefan Stein wrapped up the session with a discussion of new SIN γ-retroviral vectors for the gene therapy of chronic granulomatous disease (CGD) that seem to show advantages over the vector used in the recent CGD trial, which has shown some side effects in affected individuals. Manuel Grez provided an update on the results of this phase I–II CGD trial in the first talk of the second session, devoted to a discussion of recent clinical results. The two adults with CGD treated in Frankfurt with gene-modified cells showed increased numbers of gene-marked cells, leading to an unbalanced dominance of clones containing integrations into the MDS1/EVI1 and PRDM16 genes. He reported the occurrence of chromosomal abnormalities in both patients, most probably caused by the transcriptional activation of MDS1 and EVI1. He also described an epigenetic inactivation of the viral promoter driving gp91phox expression leading to a lack of superoxide activity in gene-transduced www.moleculartherapy.org vol. 17 no. 1 jan. 2009
© The American Society of Gene Therapy
cells in both patients. Patient 1 died 27 months after gene therapy. Patient 2 underwent stem cell transplantation from a matched unrelated donor and is recovering from the treatment. In a trial of retroviral-mediated gene therapy for SCID-X1, 10 patients have been treated in London, and all have shown significant improvement in T-cell numbers and function. Waseem Qassim reported that half of these affected individuals have also discontinued immunoglobulin replacement therapy. At the time of writing there were no adverse events seen in this cohort (although one individual developed a T-cell leukemia as a result of vector-mediated insertional mutagenesis). For adenosine deaminase SCID, three evaluable affected individuals were treated using a spleen focus-forming virus−based retroviral vector. Melphalan conditioning was given to these individuals before infusion of gene-modified cells. In the first two individuals immune and metabolic recovery has been seen, with reinitiation of thymopoiesis in both individuals. Patient 3 had a poor stem cell harvest and did not receive sufficient cells to allow effective reconstitution; this individual has again begun receiving enzyme replacement therapy. David Williams reported on the clinical gene transfer trial in individuals with Fanconi anemia (FA) complementation group A. This trial was run concurrently with a trial seeking to collect CD34+ cells (as targets) from individuals with FA. Three individuals with FA were enrolled in the gene transfer protocol. Transduction efficiency of CD34+ cells using the γ-retrovirus–expressing FA complementation group A was 40–60% as assayed by both colony assay and quantitative PCR. Two individuals with FA received genemodified cell infusions but with low doses of cells. Both showed transient increases in hemoglobin and platelet counts. One of these two patients showed transient gene marking, albeit a small amount. A third affected individual was not infused because of the small number of cells. No adverse events were noted. Overall, the conclusion of this study was that methods to increase the number of target CD34+ cells were needed so that cell doses approaching successful immunodeficiency trials could be used. Molecular Therapy vol. 17 no. 1 jan. 2009
meeting report Lilith Reeves described efforts by the Cincinnati Children’s Research Foundation Vector Production Facility to produce a retroviral vector using a transient transfection-based protocol for a planned multi-institutional SCID-X1 trial. Large-scale GMP production of this vector has now begun. Diana Nordling then discussed the quality-control, logistical, and regulatory challenges associated with transatlantic vector production— which must adhere to standards imposed by distinct regulatory agencies. Leukemia caused by retroviral insertional mutagenesis after stem cell gene transfer has been reported in several experimental animals and in individuals treated for X-linked SCID. Dorothee von Laer presented data analyzing whether gene transfer into mature T cells bears the same genotoxic risk. To address this issue in an experimental “worst-case scenario,” her group transduced mature T cells and hematopoietic progenitor cells from C57BL/6 (Ly5.1) donor mice with high copy numbers of γ-retroviral vectors encoding the potent T-cell oncogenes LMO2, TCL1 (now TCL1A), or ΔTRKA, a constitutively active mutant of TRKA (now NTRK1). After transplantation into RAG-1–deficient recipients (Ly5.2), stem cell–transplanted animals developed T-cell lymphoma/leukemia for all investigated oncogenes with a characteristic phenotype and after characteristic latency periods. Ligation-mediated PCR analysis revealed mono- or oligoclonality of the malignancies. In contrast, none of the mice receiving transplanted T cells transduced with the same vectors developed leukemia/lymphoma, despite persistence of gene-modified cells. The data provide direct evidence that mature T cells are less prone to transformation than hema topoietic progenitor cells. Hans Stauss tested human T-cell receptor (TCR) variants with cysteine modifications in the constant region, or with murine constant regions. The variants generate a higher frequency of antigenspecific T cells when transduced into primary human T cells. T cells transduced with the variants bind tetramer more efficiently than T cells transduced with the wild-type TCR. T-cell avidity is the same, despite the differences in tetramer binding. TCR variants can be dominant and
completely suppress the surface expression of endogenous TCR. TCR variants are expected to improve safety and efficacy of TCR gene therapy in the future. The third session covered issues of genotoxicity and integration analysis. Chris Baum opened the session with a discussion of studies investigating the target population of insertional transformation in the hematopoietic system. He showed that insertional upregulation of proto-oncogenes is not sufficient to overcome the engraftment barrier that limits the establishment of clonal dominance in more mature hema topoietic progenitor cells. Using his recently established in vitro immortalization assay, he obtained evidence that the enhancer-promoter sequences of integrating vectors are more important risk factors of transformation than the integration profile (γ-retroviral or lentiviral). Anjali Mishra provided an update of continuing studies that address the impact of vector design on clonal selection in vivo using the C57IB bone marrow transplantation model. Sebastian Newrzela reported data showing that mature T-cell populations are less susceptible than stem cell populations to transformation by known T-cell protooncogenes, such as LMO2. Christof Kalle reported modeling and verification data that uncontrolled use of restriction enzymes can significantly bias against or completely blind the detection of insertions in up to 80% of the genome. The choice of restriction motifs is key to obtaining a repertoire of insertions covering all regions of the genome. A thorough comparison of insertions from all international immunodeficiency retrovirus vector trials demonstrates that more than 40% of all vector insertions are in frequently affected, nonrandomly distributed common insertion sites, indicating that the insertion of first-generation clinical retrovirus trials using retrovirus promoter-enhancers has subtle but measurable influence on about every second engrafted cell clone. Interestingly, the most frequently retrieved (i.e., the most intensely inserted and/or selected) genes were identical to the ones that have created clonal proliferation serious adverse effects in the X-SCID and CGD trials. Kalle demonstrated that new high-throughput methodologies allow assessment of entire repertoires of vector insertions in clinical trials on a routine 11
© The American Society of Gene Therapy
meeting report basis. Insertion distribution analysis should be considered the genomic equivalent of pharmacokinetics for vector applications in humans and is indispensable for responsible drug development in phase I gene therapy trials. Ali Nowrouzi described the use of multiplex-PCR and linear amplification-mediated-PCR to identify concatameric and integrated forms of adeno-associated virus, in an effort to clarify the genetic safety of this common vector for clinical use. Anna Schnieder next described efforts to evaluate the influence of vector integration on global gene expression using microarrays, demonstrating little or no effect of integ ration of lentivectors in HeLa cell clones. The fourth session was devoted to a discussion of strategies to enhance engraftment of hematopoietic stem cells (HSCs). Lars Müller discussed an approach to gene transfer in the context of FA, a pediatric bone marrow failure and cancer predisposition syndrome. Müller highlighted two earlier clinical trials using γ-retroviral vectors for the transduction of autologous FA HSCs. In these previous trials extensive in vitro cell manipulation had been required during transduction, probably contributing to the lack of detectable long-term engraftment of genemodified cells in the recipient individuals. As a strategy for minimizing ex vivo manipulation, a “rapid” lentiviral transduction protocol was proposed. In a mu-
12
rine Fanca–/– model, rapid transduction of Fanca–/– HSCs preserved engraftment to the level achieved in wild-type cells, resulting in long-term multilineage engraftment of gene-modified cells. Mick Milsom discussed the use of the O6-methylguanine-DNA methyltransferase (MGMT) drug resistance gene in an in vivo model of HSC selection using the alkylating agent temozolomide. His work focused upon whether, in the context of a self-inactivating γ-retroviral vector, weaker human cellular promoters could drive the expression of the MGMTP140K mutant at sufficient levels to protect and select gene-modified HSCs. The driving force of this work was that the combination of weaker promoters in a SIN configuration probably composes a safer vector configuration than an LTR vector in the context of insertional mutagenesis. Milsom demonstrated that not only were the weaker human cellular promoters able to express MGMTP140K at levels that allowed robust HSC selection, but that a vector that expressed very high levels of MGMTP140K courtesy of a viral LTR promoter conveyed a pronounced growth defect onto transduced HSCs. The final session discussed the development of new gene therapy strategies. Dao Pan reported that co-expression of MGMTP140K and a-L-iduronidase in primary hepatocytes from MPS1 mice permits efficient selection with metabolic
correction as a proof of principle for the prospect of gene therapy for this disease. Ajay Perumbeti described continuing efforts to correct sickle-cell anemia with a γ-globulin transgene delivered with a lentiviral vector into the Berkeley transgenic sickle mouse, reporting functional correction and an inprovement of hematological indices. X-linked lymphoproliferative disease is another target disease for ex vivo HSC gene therapy. Defective expression of SLAM-associated protein (SAP) makes individuals susceptible to Epstein-Barr virus–mediated malignancies. Previous studies have shown that expression of SAP can correct cytotoxic responses, and there are now efforts to develop lentiviral vectors encoding SAP endogenous promoter elements to regulate expression through ontogeny, as described in Christin Rivat’s talk. Mary Collins reported that lentiviral vectors encoding antigens are potent immunogens. Antigen expression can be targeted to dendritic cells in vivo using the dectin-2 promoter. By co-expressing activators of mitogen-activated protein kinase or interferon signaling pathways, the response to the antigen could either be enhanced or suppressed. Funding for the retreat was provided by the Leukemia Lymphoma Society (United States). The next meeting is being planned for May 2009 in Boston, Massachusetts.
www.moleculartherapy.org vol. 17 no. 1 jan. 2009