Cell Stem Cell
In Translation Clinical Translation of TALENS: Treating SCID-X1 by Gene Editing in iPSCs Alessandra Biffi1,* 1San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, 20132 Milan, Italy *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2015.03.009
Mutations causing X-linked severe combined immunodeficiency (SCID-X1) reduce immune cell populations and function and may be amenable to targeted gene correction strategies. Now in Cell Stem Cell, Menon et al. (2015) correct SCID-X1-related blood differentiation defects by TALEN-mediated genome editing in patientderived iPSCs, suggesting a possible strategy for autologous cell therapy of SCID-X1. X-linked severe combined immunodeficiency (SCID-X1) is a primary immunodeficiency with severe prognosis of death occurring within a few years after birth. Affected patients carry inactivating mutations in the interleukin-2 receptor gamma chain (IL2RG) gene, resulting in immune defects including lack of T cells, functional B cells, and NK cells. Transplantation of HLA-matched hematopoietic stem cells (HSCs) from healthy donors provides substantial clinical benefit, with an overall survival of 85%–90% in subjects receiving transplants from matched-sibling donors (Antoine et al., 2003; Gennery et al., 2010; Brown et al., 2011). Survival rates are significantly lower, and transplant-related morbidity is increased, when cells from mismatched related donors, matched unrelated donors, or umbilical-cord-blood donors are employed or patients experience severe infections at time of transplantation (Brown et al., 2011). Now in Cell Stem Cell, Menon et al. (2015) suggest an innovative strategy for the development of an autologous cell therapy for SCID-X1. SCID-X1 represents a suitable and attractive setting for the clinical translation of targeted gene correction strategies and adoptive transfer of genecorrected cells, as cells bearing a functional gamma chain show a positive selective advantage in vivo in the affected patients (Hacein-Bey-Abina et al., 2002). Previously, gene therapy based on the transplantation of autologous gene-corrected HSCs has been attempted in SCID-X1 patients who did not have matched family donors. These patients received autologous HSCs transduced with a first-generation Moloney murine leukemia virus vector (Hacein-Bey-Abina
et al., 2002, 2003) and, more recently, with a self-inactivating (SIN) retroviral vector (Hacein-Bey-Abina et al., 2014) expressing IL2RG cDNA. In both cases, gene therapy resulted in a favorable clinical outcome, although patients receiving the first-generation retroviral vector developed acute lymphoblastic leukemia with an incidence rate of 25%, arising from insertional mutagenesis of the vector (Hacein-Bey-Abina et al., 2003). The limited follow-up of the patients treated with the SIN vector does not yet allow definitive conclusions on the long-term risk of vector-related leukemogenesis in this latter case. One goal of gene therapy is to restore not only the function but also the physiological levels of expression of the target gene. In situ replacement of diseasecausing gene mutations by homologous recombination is expected to achieve this goal and provide therapeutic benefit without potential accompanying complications such as insertional oncogenesis, transgene silencing, or inappropriate gene expression. In a recent proof of concept study, exon 5 of the IL2RG gene, a hotspot for SCID-X1 mutations, was targeted in HSCs and progenitor cells from healthy donors and an SCIDX1 patient using zinc finger nucleases (ZFNs) (Genovese et al., 2014). This resulted in successful reconstitution of a functional IL2RG gene with remarkably high efficiency (Genovese et al., 2014) and encouraged potentially moving this approach toward the clinic. However, issues such as the availability of a potentially limiting number of gene-corrected stem cells may constitute a challenge for the clinical translation of this strategy for SCID-X1 correction. Moreover, ZFNs
348 Cell Stem Cell 16, April 2, 2015 ª2015 Elsevier Inc.
may have potential off-target activity that could be of clinical relevance. Now in Cell Stem Cell, Menon, Firth, and colleagues (Menon et al., 2015) demonstrate that genetically corrected mature lymphoid cells can be generated in vitro with transcription activatorlike effector nuclease (TALEN)-mediated genome editing in induced pluripotent stem cells (iPSCs) derived from a SCIDX1 subject. iPSC lines were generated from mesenchymal bone marrow cells of the subject using Cre-excisable lentiviral vectors encoding six reprogramming factors, and then the subject’s specific mutation was corrected using TALEN pairs targeting genomic sequences proximal to the mutation. Of note, such an approach allows the reprogramming factors to be eventually excised before gene correction, strengthening its clinical transferability. The use of TALENs for gene correction may further improve the path to clinical translation of this approach, as the ease and speed of genome editing achieved with this technology may alleviate concerns such as off-target effects. Importantly, correction of IL2RG mutations in subject iPSCs allowed generation of T cell precursors and mature NK cells after long-term differentiation in vitro. Significantly, these cell populations were abnormal or absent in the subject and could not be obtained by differentiating non-corrected control iPSCs from the same individual. In addition to the technical improvements resulting from optimized in vitro differentiation protocols, these results are of great clinical relevance. The data from Menon, Firth, and colleagues demonstrate that engineered iPSCs could be a valuable source of autologous,
Cell Stem Cell
In Translation gene-corrected, and mature immune cells for adoptive transfer, as a supportive and/or possibly curative treatment for SCID-X1. The T and NK populations in SCID-X1 subjects are numerically and functionally reduced, and supplementation of these populations with their gene-corrected, iPSC-derived counterparts could provide substantial clinical benefit to subjects, including prevention and treatment of infections. Importantly, the survival advantage conferred onto the adoptively transferred in vitro progeny of the iPSCs by gene correction would constitute a critical advantage of this approach when tested in subjects. Beyond SCID-X1, adoptive transfer of NK and T-precursor cells is being evaluated in individuals with cancer and could potentially provide great benefit to immunodeficient patients, as shown for adenosine deaminase deficiency (Aiuti et al., 2002). Derivation of mature immune cells from stable, gene corrected, autologous iPSCs could successfully address many of the issues raised by clinical experience of adoptive cell transfer. Such an approach could resolve issues related to the availability of allogeneic cells, the lack of adequate cell numbers, and the need for efficient gene correction into mature cells
while removing the risks related to the use of integrating vectors in traditional gene therapy approaches. However, several challenges must be addressed to translate these findings to the clinic. Critical issues in iPSC generation and differentiation (i.e., reproducibility and consistency across subjects and the need for functional characterization of the mature gene-corrected cells to be employed for adoptive transfer) and in gene correction (i.e., the efficiency by which the desired genetic modification can be achieved in the target cell population and the limited tolerance of the cells to DNA doublestrand breaks) must be resolved, and optimizing protocols to scale up processes to obtain the amounts of cells required to treat patients is of the utmost importance. With these challenges in mind, the findings of Menon et al. provide exciting perspectives for the development of new therapies based on adoptive transfer of autologous cells fixed in their mutations for people affected by genetic disorders.
REFERENCES Aiuti, A., Vai, S., Mortellaro, A., Casorati, G., Ficara, F., Andolfi, G., Ferrari, G., Tabucchi, A., Carlucci, F., Ochs, H.D., et al. (2002). Nat. Med. 8, 423–425.
Antoine, C., Mu¨ller, S., Cant, A., Cavazzana-Calvo, M., Veys, P., Vossen, J., Fasth, A., Heilmann, C., Wulffraat, N., Seger, R., et al.; European Group for Blood and Marrow Transplantation; European Society for Immunodeficiency (2003). Lancet 361, 553–560. Brown, L., Xu-Bayford, J., Allwood, Z., Slatter, M., Cant, A., Davies, E.G., Veys, P., Gennery, A.R., and Gaspar, H.B. (2011). Blood 117, 3243–3246. Gennery, A.R., Slatter, M.A., Grandin, L., Taupin, P., Cant, A.J., Veys, P., Amrolia, P.J., Gaspar, H.B., Davies, E.G., Friedrich, W., et al.; Inborn Errors Working Party of the European Group for Blood and Marrow Transplantation; European Society for Immunodeficiency (2010). J. Allergy Clin. Immunol. 126, 602–610, e1–e11. Genovese, P., Schiroli, G., Escobar, G., Di Tomaso, T., Firrito, C., Calabria, A., Moi, D., Mazzieri, R., Bonini, C., Holmes, M.C., et al. (2014). Nature 510, 235–240. Hacein-Bey-Abina, S., Le Deist, F., Carlier, F., Bouneaud, C., Hue, C., De Villartay, J.P., Thrasher, A.J., Wulffraat, N., Sorensen, R., Dupuis-Girod, S., et al. (2002). N. Engl. J. Med. 346, 1185–1193. Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., McCormack, M.P., Wulffraat, N., Leboulch, P., Lim, A., Osborne, C.S., Pawliuk, R., Morillon, E., et al. (2003). Science 302, 415–419. Hacein-Bey-Abina, S., Pai, S.Y., Gaspar, H.B., Armant, M., Berry, C.C., Blanche, S., Bleesing, J., Blondeau, J., de Boer, H., Buckland, K.F., et al. (2014). N. Engl. J. Med. 371, 1407–1417. Menon, T., Firth, A.L., Scripture-Adams, D.D., Galic, Z., Qualls, S.J., Gilmore, W.B., Ke, E., Singer, O., Anderson, L.S., Bornzin, A.R., et al. (2015). Cell Stem Cell 16, this issue, 367–372.
Cell Stem Cell 16, April 2, 2015 ª2015 Elsevier Inc. 349
In Translation Clinical Translation of TALENS: Treating SCID-X1 by Gene Editing in iPSCs Alessandra Biffi1,* 1San Raffaele Telethon Institute for Gene Therapy, San Raffaele Scientific Institute, 20132 Milan, Italy *Correspondence: [email protected] http://dx.doi.org/10.1016/j.stem.2015.03.009
Mutations causing X-linked severe combined immunodeficiency (SCID-X1) reduce immune cell populations and function and may be amenable to targeted gene correction strategies. Now in Cell Stem Cell, Menon et al. (2015) correct SCID-X1-related blood differentiation defects by TALEN-mediated genome editing in patientderived iPSCs, suggesting a possible strategy for autologous cell therapy of SCID-X1. X-linked severe combined immunodeficiency (SCID-X1) is a primary immunodeficiency with severe prognosis of death occurring within a few years after birth. Affected patients carry inactivating mutations in the interleukin-2 receptor gamma chain (IL2RG) gene, resulting in immune defects including lack of T cells, functional B cells, and NK cells. Transplantation of HLA-matched hematopoietic stem cells (HSCs) from healthy donors provides substantial clinical benefit, with an overall survival of 85%–90% in subjects receiving transplants from matched-sibling donors (Antoine et al., 2003; Gennery et al., 2010; Brown et al., 2011). Survival rates are significantly lower, and transplant-related morbidity is increased, when cells from mismatched related donors, matched unrelated donors, or umbilical-cord-blood donors are employed or patients experience severe infections at time of transplantation (Brown et al., 2011). Now in Cell Stem Cell, Menon et al. (2015) suggest an innovative strategy for the development of an autologous cell therapy for SCID-X1. SCID-X1 represents a suitable and attractive setting for the clinical translation of targeted gene correction strategies and adoptive transfer of genecorrected cells, as cells bearing a functional gamma chain show a positive selective advantage in vivo in the affected patients (Hacein-Bey-Abina et al., 2002). Previously, gene therapy based on the transplantation of autologous gene-corrected HSCs has been attempted in SCID-X1 patients who did not have matched family donors. These patients received autologous HSCs transduced with a first-generation Moloney murine leukemia virus vector (Hacein-Bey-Abina
et al., 2002, 2003) and, more recently, with a self-inactivating (SIN) retroviral vector (Hacein-Bey-Abina et al., 2014) expressing IL2RG cDNA. In both cases, gene therapy resulted in a favorable clinical outcome, although patients receiving the first-generation retroviral vector developed acute lymphoblastic leukemia with an incidence rate of 25%, arising from insertional mutagenesis of the vector (Hacein-Bey-Abina et al., 2003). The limited follow-up of the patients treated with the SIN vector does not yet allow definitive conclusions on the long-term risk of vector-related leukemogenesis in this latter case. One goal of gene therapy is to restore not only the function but also the physiological levels of expression of the target gene. In situ replacement of diseasecausing gene mutations by homologous recombination is expected to achieve this goal and provide therapeutic benefit without potential accompanying complications such as insertional oncogenesis, transgene silencing, or inappropriate gene expression. In a recent proof of concept study, exon 5 of the IL2RG gene, a hotspot for SCID-X1 mutations, was targeted in HSCs and progenitor cells from healthy donors and an SCIDX1 patient using zinc finger nucleases (ZFNs) (Genovese et al., 2014). This resulted in successful reconstitution of a functional IL2RG gene with remarkably high efficiency (Genovese et al., 2014) and encouraged potentially moving this approach toward the clinic. However, issues such as the availability of a potentially limiting number of gene-corrected stem cells may constitute a challenge for the clinical translation of this strategy for SCID-X1 correction. Moreover, ZFNs
348 Cell Stem Cell 16, April 2, 2015 ª2015 Elsevier Inc.
may have potential off-target activity that could be of clinical relevance. Now in Cell Stem Cell, Menon, Firth, and colleagues (Menon et al., 2015) demonstrate that genetically corrected mature lymphoid cells can be generated in vitro with transcription activatorlike effector nuclease (TALEN)-mediated genome editing in induced pluripotent stem cells (iPSCs) derived from a SCIDX1 subject. iPSC lines were generated from mesenchymal bone marrow cells of the subject using Cre-excisable lentiviral vectors encoding six reprogramming factors, and then the subject’s specific mutation was corrected using TALEN pairs targeting genomic sequences proximal to the mutation. Of note, such an approach allows the reprogramming factors to be eventually excised before gene correction, strengthening its clinical transferability. The use of TALENs for gene correction may further improve the path to clinical translation of this approach, as the ease and speed of genome editing achieved with this technology may alleviate concerns such as off-target effects. Importantly, correction of IL2RG mutations in subject iPSCs allowed generation of T cell precursors and mature NK cells after long-term differentiation in vitro. Significantly, these cell populations were abnormal or absent in the subject and could not be obtained by differentiating non-corrected control iPSCs from the same individual. In addition to the technical improvements resulting from optimized in vitro differentiation protocols, these results are of great clinical relevance. The data from Menon, Firth, and colleagues demonstrate that engineered iPSCs could be a valuable source of autologous,
Cell Stem Cell
In Translation gene-corrected, and mature immune cells for adoptive transfer, as a supportive and/or possibly curative treatment for SCID-X1. The T and NK populations in SCID-X1 subjects are numerically and functionally reduced, and supplementation of these populations with their gene-corrected, iPSC-derived counterparts could provide substantial clinical benefit to subjects, including prevention and treatment of infections. Importantly, the survival advantage conferred onto the adoptively transferred in vitro progeny of the iPSCs by gene correction would constitute a critical advantage of this approach when tested in subjects. Beyond SCID-X1, adoptive transfer of NK and T-precursor cells is being evaluated in individuals with cancer and could potentially provide great benefit to immunodeficient patients, as shown for adenosine deaminase deficiency (Aiuti et al., 2002). Derivation of mature immune cells from stable, gene corrected, autologous iPSCs could successfully address many of the issues raised by clinical experience of adoptive cell transfer. Such an approach could resolve issues related to the availability of allogeneic cells, the lack of adequate cell numbers, and the need for efficient gene correction into mature cells
while removing the risks related to the use of integrating vectors in traditional gene therapy approaches. However, several challenges must be addressed to translate these findings to the clinic. Critical issues in iPSC generation and differentiation (i.e., reproducibility and consistency across subjects and the need for functional characterization of the mature gene-corrected cells to be employed for adoptive transfer) and in gene correction (i.e., the efficiency by which the desired genetic modification can be achieved in the target cell population and the limited tolerance of the cells to DNA doublestrand breaks) must be resolved, and optimizing protocols to scale up processes to obtain the amounts of cells required to treat patients is of the utmost importance. With these challenges in mind, the findings of Menon et al. provide exciting perspectives for the development of new therapies based on adoptive transfer of autologous cells fixed in their mutations for people affected by genetic disorders.
REFERENCES Aiuti, A., Vai, S., Mortellaro, A., Casorati, G., Ficara, F., Andolfi, G., Ferrari, G., Tabucchi, A., Carlucci, F., Ochs, H.D., et al. (2002). Nat. Med. 8, 423–425.
Antoine, C., Mu¨ller, S., Cant, A., Cavazzana-Calvo, M., Veys, P., Vossen, J., Fasth, A., Heilmann, C., Wulffraat, N., Seger, R., et al.; European Group for Blood and Marrow Transplantation; European Society for Immunodeficiency (2003). Lancet 361, 553–560. Brown, L., Xu-Bayford, J., Allwood, Z., Slatter, M., Cant, A., Davies, E.G., Veys, P., Gennery, A.R., and Gaspar, H.B. (2011). Blood 117, 3243–3246. Gennery, A.R., Slatter, M.A., Grandin, L., Taupin, P., Cant, A.J., Veys, P., Amrolia, P.J., Gaspar, H.B., Davies, E.G., Friedrich, W., et al.; Inborn Errors Working Party of the European Group for Blood and Marrow Transplantation; European Society for Immunodeficiency (2010). J. Allergy Clin. Immunol. 126, 602–610, e1–e11. Genovese, P., Schiroli, G., Escobar, G., Di Tomaso, T., Firrito, C., Calabria, A., Moi, D., Mazzieri, R., Bonini, C., Holmes, M.C., et al. (2014). Nature 510, 235–240. Hacein-Bey-Abina, S., Le Deist, F., Carlier, F., Bouneaud, C., Hue, C., De Villartay, J.P., Thrasher, A.J., Wulffraat, N., Sorensen, R., Dupuis-Girod, S., et al. (2002). N. Engl. J. Med. 346, 1185–1193. Hacein-Bey-Abina, S., Von Kalle, C., Schmidt, M., McCormack, M.P., Wulffraat, N., Leboulch, P., Lim, A., Osborne, C.S., Pawliuk, R., Morillon, E., et al. (2003). Science 302, 415–419. Hacein-Bey-Abina, S., Pai, S.Y., Gaspar, H.B., Armant, M., Berry, C.C., Blanche, S., Bleesing, J., Blondeau, J., de Boer, H., Buckland, K.F., et al. (2014). N. Engl. J. Med. 371, 1407–1417. Menon, T., Firth, A.L., Scripture-Adams, D.D., Galic, Z., Qualls, S.J., Gilmore, W.B., Ke, E., Singer, O., Anderson, L.S., Bornzin, A.R., et al. (2015). Cell Stem Cell 16, this issue, 367–372.
Cell Stem Cell 16, April 2, 2015 ª2015 Elsevier Inc. 349
