- Email: [email protected]
Conditional Negative Selection of Gene-Modified Hematopoietic Stem Cells
© The American Society of Gene & Cell Therapy
13726–13731. 21. Buzzard, JJ, Gough, NM, Crook, JM and Colman, A (2004). Karyotype of human ES cells during extended culture. Nat Biotechnol 22: 381–382; author reply 382.
commentary
22. Taylor, CJ, Bolton, EM, Pocock, S, Sharples, LD, Pedersen, RA and Bradley, JA (2005). Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching. Lancet 366: 2019–2025.
See page 1932
Conditional Negative Selection of GeneModified Hematopoietic Stem Cells Christopher Baum1 doi:10.1038/mt.2012.195
I
n this issue of Molecular Therapy, Barese and colleagues from the National Institutes of Health present a landmark study conducted in a clinically relevant largeanimal model to evaluate the utility of the herpes simplex virus thymidine kinase (HSVtk) gene for conditional elimination of transplanted long-term repopulating hematopoietic cells, potentially representing true hematopoietic stem cells (HSCs).1 This paper provides proof of concept for this approach, is rich in highly relevant experimental findings, and presents a stimulating discussion that will certainly motivate the field to pay even more attention to the important principle of conditional negative selection of gene-modified cells. Indeed, despite the remarkable success achieved with the negative selection of gene-modified T lymphocytes in clinical trials,2–4 the application of this principle to gene-modified stem cells has been far less explored. Although some studies have addressed suicide gene transfer as a safety mechanism to eliminate potential tumors occurring following the transplantation of pluripotent stem cell progeny (see references in Barese et al.1), studies addressing the utility of this principle for the more realistic and increasingly successful genetic
1 Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany Correspondence: Christopher Baum, Institute of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Straße 1, D-30625 Hannover, Germany. E-mail: [email protected]
Molecular Therapy vol. 20 no. 10 october 2012
modification of HSCs have been lacking. This is remarkable given that suicide gene transfer has been discussed over many years as a potential solution for the control of serious adverse events originating from the transformation of gene-modified hematopoietic cells since the first discovery of leukemias and premalignant clonal expansion induced by insertional mutagenesis.5–7 One explanation for the lack of experimental studies testing the utility of the suicide principle for HSC modification may be related to negative expectations. The HSVtk-mediated mechanism of suicide was known to be largely restricted to proliferating cells; human HSCs, however, typically spend weeks in quiescence before undergoing cell division, at least in homeostatic conditions. In addition, ganciclovir (GCV), the prodrug used for suicide induction of HSVtk-expressing cells, is myelotoxic when administered for prolonged periods of time, thus potentially also eliminating untransduced hematopoietic cells. Moreover, studies with HSVtk-modified tumor cells had suggested that epigenetic and genetic escape mechanisms, such as vector silencing, rearrangement of the transgene or the flanking chromosomal sequences, or point mutations, may constitute major limitations for the complete elimination of the target population.8,9 The new article from the Dunbar lab addressed all these limitations, with truly encouraging results.1 First, this work revealed that the restriction of GCV-mediated cell killing to proliferating cells does not limit the potential to eliminate long-term repopulating hematopoietic cells, not even
in the clinically relevant rhesus model that is far closer than any small-animal model to recapitulating the complex dynamics of human hematopoiesis, with coexistence of cycling and quiescent cells. The remarkable complete elimination of vector-transduced cell populations was assessed by very sensitive assays and persisted for at least 18 months after GCV application. These results imply the elimination of the pool of quiescent, long-lived lymphocytes, which must have originated from the transplanted HSCs and progenitor cells in the long time before administration of GCV. Whether GCV/HSVtk-mediated killing of quiescent hematopoietic cells is caused by mitochondrial damage, as speculated by Barese et al., remains to be addressed. Second, this study identified at least two drug regimens that allow killing of longterm repopulating, HSVtk-transduced hematopoietic cells while avoiding major unspecific myelotoxicity. These drug regimens are another important component of this study, with direct translational implications. The good news is that a single course of GCV over a period of five days was sufficient for cell elimination. Another, more continued course lasting for three weeks was also well tolerated and highly efficient. Thus, even if elimination would be more difficult, as in the case of a larger population of transduced cells and/or malignant transformation, the therapeutic index of GCV might be sufficient to be clinically useful in patients receiving gene-modified HSCs. Third, the authors of this study found no evidence for escape mechanisms by genetic instability, but did report evidence suggesting that the specific activity of the HSVtk has an impact on the completeness of cell killing in their model. Although not directly addressed, this implies that a given threshold of HSVtk expression and activity has to be overcome to exploit this suicide principle in HSCs and their progeny cells. Engineering the HSVtk expression cassette and coding sequences provides room for further improvements (e.g., Preuss et al.10). Furthermore, Barese and colleagues make the interesting and somewhat challenging suggestion that promoters be designed that work only in transformed cells, which would be of special interest when a malignant gene-modified clone is to be eliminated while sparing the untransformed yet gene-modified cell population. 1841
© The American Society of Gene & Cell Therapy
commentary Finally, Barese and colleagues also addressed whether cell elimination involved an immune response to the transgene products encoded by the gene vectors, for which they found no evidence. This was not unexpected, given that the transplantation of gene-modified primitive hematopoietic cells is known to potentially induce central tolerance to transgene products. In any case, this represents another important observation, as many suicide genes contain nonhuman sequences. In summary, the article by Barese et al. marks major progress on the path toward conditional stem cell therapy, relevant for numerous applications beyond hematopoiesis. This work will certainly revive the concept of conditional, optional suicide induction to control the fate of gene-modified stem cells and their progeny in vivo. ACKNOWLEDGMENTS The author is grateful for support by the German Ministry for Research and Education (programs PIDNET, REGENE, and IGENE), the Deutsche Forschungsgemeinschaft (research Priority Program 1230, SFB738 and Cluster of Excellence REBIRTH), and the European Union (Integrated Projects PERSIST and CELL-PID).
REFERENCES
1. Barese, CN, Krouse, AE, Metzger, ME, King, CA, Traversari, C, Marini, FC et al. (2012). Thymidine kinase suicide gene–mediated ganciclovir ablation of autologous gene-modified rhesus hematopoiesis. Mol Ther 20: 1932–1943. 2. Bonini, C, Brenner, MK, Heslop, HE and Morgan, RA (2011). Genetic modification of T cells. Biol Blood Marrow Transplant 17: S15–S20. 3. Di Stasi, A, Tey, SK, Dotti, G, Fujita, Y, Kennedy-Nasser, A, Martinez, C et al. (2011). Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med 365: 1673–1683. 4. Bonini, C, Ferrari, G, Verzeletti, S, Servida, P, Zappone, E, Ruggieri, L et al. (1997). HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 276: 1719–1724. 5. Li, Z, Düllman, J, Schiedlmeier, B, Schmidt, M, von Kalle, C, Meyer, J et al. (2002). Murine leukemia induced by retroviral gene marking. Science 296: 497. 6. Hacein-Bey-Abina, S, von Kalle, C, Schmidt, M, McCormack, MP, Wulffraat, N, Leboulch, P et al. (2003). LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302: 415–419. 7. Ott, MG, Schmidt, M, Schwarzwaelder, K, Stein, S, Siler, U, Koehl, U et al. (2006). Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 12: 401–409. 8. Frank, O, Rudolph, C, Heberlein, C, von Neuhoff, N, Schröck, E, Schambach, A et al. (2004). Tumor cells escape suicide gene therapy by genetic and epigenetic instability. Blood 104: 3543–3549. 9. Blumenthal, M, Skelton, D, Pepper, KA, Jahn, T, Methangkool, E and Kohn, DB (2007). Effective suicide gene therapy for leukemia in a model of insertional oncogenesis in mice. Mol Ther 15: 183–192. 10. Preuss, E, Treschow, A, Newrzela, S, Brücher, D, Weber, K, Felldin, U et al. (2010). TK.007: a novel, codon-optimized HSVtk(A168H) mutant for suicide gene therapy. Hum Gene Ther 21: 929–941.
1842
See pages 1902 and 1912
AAV Vectors for the Nucleolus David W Russell1 doi:10.1038/mt.2012.193
D
uring the replication of wild-type adeno-associated virus (AAV), viral capsids are assembled in the nucleolus,1 and the capsid proteins interact with the nucleolar proteins nucleolin (NCL) and nucleophosmin (NPM1), possibly promoting their transport from the cytoplasm into the nucleolus.2,3 In the case of replication-incompetent AAV vectors, the nucleolus is also important because vector virions can be found inside the nucleolus soon after entering the cell, and both NCL and NPM1 are involved in this trafficking.4 The nucleolar localization of vector virions is probably responsible for the unusual preference of AAV vectors for integration within the ribosomal DNA (rDNA) repeats, which accounts for 3–8% of all nonhomologous integration events,5,6 or 10- to 30-fold more than expected based on the amount of rDNA per cell. In this issue of Molecular Therapy, two reports from the labs of Markus Grompe and Mark Kay exploit this tendency of AAV virions to localize to the nucleolus by incorporating rDNA sequences into the vector genome and transducing mouse hepatocytes with these “rDNA vectors.”7,8 This simple trick produced dramatic effects, resulting in a 30-fold increase in chromosomal integration, about one third of which occurred within the rDNA repeats.8 The nucleolus is a multifunctional nuclear organelle best known for its role in the synthesis of ribosomes. It assembles around the tandem rDNA repeats present on multiple chromosomes that encode the rRNAs, is organized into morphologically distinct regions, contains its own cohort of proteins, small RNAs, and ribonucleoproteins, and plays an important role in the 1 Departments of Medicine and Biochemistry, University of Washington, Seattle, Washington, USA Correspondence: David W Russell, Department of Medicine and Biochemistry, Mailstop 357720, University of Washington, Seattle, Washington 98195, USA. E-mail: [email protected]
life cycles of many viruses,9 including AAV, as noted above. Figure 1 diagrams the numerous potential transduction pathways of both rDNA and non-rDNA AAV vectors and highlights possible interactions with the nucleolus. Existing data suggest that a large proportion of input genomes are initially delivered to the nucleolus regardless of their sequence, perhaps by capsid interactions with NCL and NPM1 (ref. 4). Although the exact timing of capsid disassembly and uncoating of the vector genome is not clear, some of the non-rDNA vector genomes integrate in rDNA, but most exit the nucleolus and are converted to double-stranded episomal forms through second-strand synthesis, annealing, and/or circularization.10–13 Some of these nuclear vector genomes may integrate into host chromosomes at random locations, or into homologous sites at about a 10-fold lower frequency if designed for gene targeting.14 By contrast, most of the rDNA vector genomes remain in the nucleolus, presumably by binding the nucleolar factors that recognize rDNA. This results in a much higher frequency of both nonhomologous and homologous integration into the rDNA repeats. Although distinguishing these two types of integrants was technically difficult, the two studies estimate that homologous integration at the rDNA repeats was 5- to 50-fold lower than nonhomologous integration.7,8 The rDNA vectors were able to produce therapeutic levels of factor IX and fumarylacetoacetate hydrolase (Fah) in murine models of hemophilia and hereditary tyrosinemia, respectively. Although this had previously been achieved with conventional AAV vectors, the rDNA vectors have potential advantages. Because the integration frequencies are so much higher, one would expect stable, long-term transgene expression. This could allow a single rDNA vector treatment to last for a patient’s entire lifetime, produce more consistent levels of the transgene product, and ultimately translate into lower vector doses, making gene
www.moleculartherapy.org vol. 20 no. 10 october 2012
13726–13731. 21. Buzzard, JJ, Gough, NM, Crook, JM and Colman, A (2004). Karyotype of human ES cells during extended culture. Nat Biotechnol 22: 381–382; author reply 382.
commentary
22. Taylor, CJ, Bolton, EM, Pocock, S, Sharples, LD, Pedersen, RA and Bradley, JA (2005). Banking on human embryonic stem cells: estimating the number of donor cell lines needed for HLA matching. Lancet 366: 2019–2025.
See page 1932
Conditional Negative Selection of GeneModified Hematopoietic Stem Cells Christopher Baum1 doi:10.1038/mt.2012.195
I
n this issue of Molecular Therapy, Barese and colleagues from the National Institutes of Health present a landmark study conducted in a clinically relevant largeanimal model to evaluate the utility of the herpes simplex virus thymidine kinase (HSVtk) gene for conditional elimination of transplanted long-term repopulating hematopoietic cells, potentially representing true hematopoietic stem cells (HSCs).1 This paper provides proof of concept for this approach, is rich in highly relevant experimental findings, and presents a stimulating discussion that will certainly motivate the field to pay even more attention to the important principle of conditional negative selection of gene-modified cells. Indeed, despite the remarkable success achieved with the negative selection of gene-modified T lymphocytes in clinical trials,2–4 the application of this principle to gene-modified stem cells has been far less explored. Although some studies have addressed suicide gene transfer as a safety mechanism to eliminate potential tumors occurring following the transplantation of pluripotent stem cell progeny (see references in Barese et al.1), studies addressing the utility of this principle for the more realistic and increasingly successful genetic
1 Institute of Experimental Hematology, Hannover Medical School, Hannover, Germany Correspondence: Christopher Baum, Institute of Experimental Hematology, Hannover Medical School, Carl-Neuberg-Straße 1, D-30625 Hannover, Germany. E-mail: [email protected]
Molecular Therapy vol. 20 no. 10 october 2012
modification of HSCs have been lacking. This is remarkable given that suicide gene transfer has been discussed over many years as a potential solution for the control of serious adverse events originating from the transformation of gene-modified hematopoietic cells since the first discovery of leukemias and premalignant clonal expansion induced by insertional mutagenesis.5–7 One explanation for the lack of experimental studies testing the utility of the suicide principle for HSC modification may be related to negative expectations. The HSVtk-mediated mechanism of suicide was known to be largely restricted to proliferating cells; human HSCs, however, typically spend weeks in quiescence before undergoing cell division, at least in homeostatic conditions. In addition, ganciclovir (GCV), the prodrug used for suicide induction of HSVtk-expressing cells, is myelotoxic when administered for prolonged periods of time, thus potentially also eliminating untransduced hematopoietic cells. Moreover, studies with HSVtk-modified tumor cells had suggested that epigenetic and genetic escape mechanisms, such as vector silencing, rearrangement of the transgene or the flanking chromosomal sequences, or point mutations, may constitute major limitations for the complete elimination of the target population.8,9 The new article from the Dunbar lab addressed all these limitations, with truly encouraging results.1 First, this work revealed that the restriction of GCV-mediated cell killing to proliferating cells does not limit the potential to eliminate long-term repopulating hematopoietic cells, not even
in the clinically relevant rhesus model that is far closer than any small-animal model to recapitulating the complex dynamics of human hematopoiesis, with coexistence of cycling and quiescent cells. The remarkable complete elimination of vector-transduced cell populations was assessed by very sensitive assays and persisted for at least 18 months after GCV application. These results imply the elimination of the pool of quiescent, long-lived lymphocytes, which must have originated from the transplanted HSCs and progenitor cells in the long time before administration of GCV. Whether GCV/HSVtk-mediated killing of quiescent hematopoietic cells is caused by mitochondrial damage, as speculated by Barese et al., remains to be addressed. Second, this study identified at least two drug regimens that allow killing of longterm repopulating, HSVtk-transduced hematopoietic cells while avoiding major unspecific myelotoxicity. These drug regimens are another important component of this study, with direct translational implications. The good news is that a single course of GCV over a period of five days was sufficient for cell elimination. Another, more continued course lasting for three weeks was also well tolerated and highly efficient. Thus, even if elimination would be more difficult, as in the case of a larger population of transduced cells and/or malignant transformation, the therapeutic index of GCV might be sufficient to be clinically useful in patients receiving gene-modified HSCs. Third, the authors of this study found no evidence for escape mechanisms by genetic instability, but did report evidence suggesting that the specific activity of the HSVtk has an impact on the completeness of cell killing in their model. Although not directly addressed, this implies that a given threshold of HSVtk expression and activity has to be overcome to exploit this suicide principle in HSCs and their progeny cells. Engineering the HSVtk expression cassette and coding sequences provides room for further improvements (e.g., Preuss et al.10). Furthermore, Barese and colleagues make the interesting and somewhat challenging suggestion that promoters be designed that work only in transformed cells, which would be of special interest when a malignant gene-modified clone is to be eliminated while sparing the untransformed yet gene-modified cell population. 1841
© The American Society of Gene & Cell Therapy
commentary Finally, Barese and colleagues also addressed whether cell elimination involved an immune response to the transgene products encoded by the gene vectors, for which they found no evidence. This was not unexpected, given that the transplantation of gene-modified primitive hematopoietic cells is known to potentially induce central tolerance to transgene products. In any case, this represents another important observation, as many suicide genes contain nonhuman sequences. In summary, the article by Barese et al. marks major progress on the path toward conditional stem cell therapy, relevant for numerous applications beyond hematopoiesis. This work will certainly revive the concept of conditional, optional suicide induction to control the fate of gene-modified stem cells and their progeny in vivo. ACKNOWLEDGMENTS The author is grateful for support by the German Ministry for Research and Education (programs PIDNET, REGENE, and IGENE), the Deutsche Forschungsgemeinschaft (research Priority Program 1230, SFB738 and Cluster of Excellence REBIRTH), and the European Union (Integrated Projects PERSIST and CELL-PID).
REFERENCES
1. Barese, CN, Krouse, AE, Metzger, ME, King, CA, Traversari, C, Marini, FC et al. (2012). Thymidine kinase suicide gene–mediated ganciclovir ablation of autologous gene-modified rhesus hematopoiesis. Mol Ther 20: 1932–1943. 2. Bonini, C, Brenner, MK, Heslop, HE and Morgan, RA (2011). Genetic modification of T cells. Biol Blood Marrow Transplant 17: S15–S20. 3. Di Stasi, A, Tey, SK, Dotti, G, Fujita, Y, Kennedy-Nasser, A, Martinez, C et al. (2011). Inducible apoptosis as a safety switch for adoptive cell therapy. N Engl J Med 365: 1673–1683. 4. Bonini, C, Ferrari, G, Verzeletti, S, Servida, P, Zappone, E, Ruggieri, L et al. (1997). HSV-TK gene transfer into donor lymphocytes for control of allogeneic graft-versus-leukemia. Science 276: 1719–1724. 5. Li, Z, Düllman, J, Schiedlmeier, B, Schmidt, M, von Kalle, C, Meyer, J et al. (2002). Murine leukemia induced by retroviral gene marking. Science 296: 497. 6. Hacein-Bey-Abina, S, von Kalle, C, Schmidt, M, McCormack, MP, Wulffraat, N, Leboulch, P et al. (2003). LMO2-associated clonal T cell proliferation in two patients after gene therapy for SCID-X1. Science 302: 415–419. 7. Ott, MG, Schmidt, M, Schwarzwaelder, K, Stein, S, Siler, U, Koehl, U et al. (2006). Correction of X-linked chronic granulomatous disease by gene therapy, augmented by insertional activation of MDS1-EVI1, PRDM16 or SETBP1. Nat Med 12: 401–409. 8. Frank, O, Rudolph, C, Heberlein, C, von Neuhoff, N, Schröck, E, Schambach, A et al. (2004). Tumor cells escape suicide gene therapy by genetic and epigenetic instability. Blood 104: 3543–3549. 9. Blumenthal, M, Skelton, D, Pepper, KA, Jahn, T, Methangkool, E and Kohn, DB (2007). Effective suicide gene therapy for leukemia in a model of insertional oncogenesis in mice. Mol Ther 15: 183–192. 10. Preuss, E, Treschow, A, Newrzela, S, Brücher, D, Weber, K, Felldin, U et al. (2010). TK.007: a novel, codon-optimized HSVtk(A168H) mutant for suicide gene therapy. Hum Gene Ther 21: 929–941.
1842
See pages 1902 and 1912
AAV Vectors for the Nucleolus David W Russell1 doi:10.1038/mt.2012.193
D
uring the replication of wild-type adeno-associated virus (AAV), viral capsids are assembled in the nucleolus,1 and the capsid proteins interact with the nucleolar proteins nucleolin (NCL) and nucleophosmin (NPM1), possibly promoting their transport from the cytoplasm into the nucleolus.2,3 In the case of replication-incompetent AAV vectors, the nucleolus is also important because vector virions can be found inside the nucleolus soon after entering the cell, and both NCL and NPM1 are involved in this trafficking.4 The nucleolar localization of vector virions is probably responsible for the unusual preference of AAV vectors for integration within the ribosomal DNA (rDNA) repeats, which accounts for 3–8% of all nonhomologous integration events,5,6 or 10- to 30-fold more than expected based on the amount of rDNA per cell. In this issue of Molecular Therapy, two reports from the labs of Markus Grompe and Mark Kay exploit this tendency of AAV virions to localize to the nucleolus by incorporating rDNA sequences into the vector genome and transducing mouse hepatocytes with these “rDNA vectors.”7,8 This simple trick produced dramatic effects, resulting in a 30-fold increase in chromosomal integration, about one third of which occurred within the rDNA repeats.8 The nucleolus is a multifunctional nuclear organelle best known for its role in the synthesis of ribosomes. It assembles around the tandem rDNA repeats present on multiple chromosomes that encode the rRNAs, is organized into morphologically distinct regions, contains its own cohort of proteins, small RNAs, and ribonucleoproteins, and plays an important role in the 1 Departments of Medicine and Biochemistry, University of Washington, Seattle, Washington, USA Correspondence: David W Russell, Department of Medicine and Biochemistry, Mailstop 357720, University of Washington, Seattle, Washington 98195, USA. E-mail: [email protected]
life cycles of many viruses,9 including AAV, as noted above. Figure 1 diagrams the numerous potential transduction pathways of both rDNA and non-rDNA AAV vectors and highlights possible interactions with the nucleolus. Existing data suggest that a large proportion of input genomes are initially delivered to the nucleolus regardless of their sequence, perhaps by capsid interactions with NCL and NPM1 (ref. 4). Although the exact timing of capsid disassembly and uncoating of the vector genome is not clear, some of the non-rDNA vector genomes integrate in rDNA, but most exit the nucleolus and are converted to double-stranded episomal forms through second-strand synthesis, annealing, and/or circularization.10–13 Some of these nuclear vector genomes may integrate into host chromosomes at random locations, or into homologous sites at about a 10-fold lower frequency if designed for gene targeting.14 By contrast, most of the rDNA vector genomes remain in the nucleolus, presumably by binding the nucleolar factors that recognize rDNA. This results in a much higher frequency of both nonhomologous and homologous integration into the rDNA repeats. Although distinguishing these two types of integrants was technically difficult, the two studies estimate that homologous integration at the rDNA repeats was 5- to 50-fold lower than nonhomologous integration.7,8 The rDNA vectors were able to produce therapeutic levels of factor IX and fumarylacetoacetate hydrolase (Fah) in murine models of hemophilia and hereditary tyrosinemia, respectively. Although this had previously been achieved with conventional AAV vectors, the rDNA vectors have potential advantages. Because the integration frequencies are so much higher, one would expect stable, long-term transgene expression. This could allow a single rDNA vector treatment to last for a patient’s entire lifetime, produce more consistent levels of the transgene product, and ultimately translate into lower vector doses, making gene
www.moleculartherapy.org vol. 20 no. 10 october 2012