- Email: [email protected]
In Vivo Selection for Gene-Corrected HSPCs Advances Gene Therapy for a Rare Stem Cell Disease
Cell Stem Cell
In Translation In Vivo Selection for Gene-Corrected HSPCs Advances Gene Therapy for a Rare Stem Cell Disease Bernhard Gentner1,2,* and Luigi Naldini1,3,* 1San
Raffaele Telethon Institute for Gene Therapy, Milan, Italy and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy 3Vita Salute San Raffaele University, Milan, Italy *Correspondence: [email protected] (B.G.), [email protected] (L.N.) https://doi.org/10.1016/j.stem.2019.10.004 2Hematology
Two recent papers (one by Roma´n-Rodrı´guez et al., 2019 in this issue of Cell Stem Cell) highlight how the power of biological selection on hematopoietic stem cell fitness can facilitate gene therapies for Fanconi Anemia. A clinical trial using lentiviral gene replacement and a proof-of-concept targeted genome editing study show robust engraftment and expansion of gene-corrected cells at levels reaching therapeutic relevance. Fanconi anemia (FA), the most common inherited bone marrow failure syndrome, has long intrigued researchers and challenged clinicians (Garaycoechea and Patel, 2014). Loss-of-function mutations in any one of at least 17 genes in the FA pathway result in the inability to repair interstrand DNA crosslinks (ICLs), highly toxic adducts that form from exposure to exogenous and endogenous mutagens. ICL accumulation prevents DNA transcription and replication, leading to cell death. Hematopoiesis, a process characterized by extensive, lifelong proliferation of stem and progenitor cells, is particularly sensitive to unrepaired ICLs. FA patients progressively exhaust their hematopoietic reserve and carry an increased risk of developing myelodysplasia, acute leukemia, and solid cancers. Case reports from patients with rarely occurring somatic reversion of the mutation suggest that one or a few functionally corrected hematopoietic stem cell (HSC) clones may be sufficient to rescue the hematologic manifestations of FA (Mankad et al., 2006). Two recent papers from partly overlapping teams report the early results of a clinical trial of HSC gene therapy for FA using gene replacement by lentiviral vectors (Rı´o et al., 2019) and the design of a gene correction strategy for FA using targeted genome editing (Roma´n-Rodrı´guez et al., 2019). Both studies show a powerful effect of biological selection at the HSC level, where the growth advantage provided by genetic correction of FA deficiency allows expansion of rare corrected cells and he-
matopoietic repopulation to levels reaching therapeutic relevance in patients, even without conditioning. The team led by Juan Bueren (Rı´o et al., 2019) undertook the challenging attempt to alleviate bone marrow failure in FA complementation group A (FANCA) patients by infusing autologous HSCs following lentiviral transfer of functional FANCA cDNA. Collection of HSCs early in life when their reservoir is not yet exhausted, the use of a very short ex vivo manipulation protocol to minimize perturbation, and the capacity of lentiviral vectors to efficiently transduce non-dividing cells were key for obtaining sufficient numbers of functionally corrected HSCs upon FANCA gene replacement. Remarkably, the authors demonstrate engraftment of vector-marked cells in the four treated FANCA patients. Engraftment typically appeared late, around 6 to 12 months after HSC administration, and was variable but progressively increased in all patients to reach proportions ranging from 4% to 55% at the latest follow-up (18–30 months). Donor cells contributed to multi-lineage and oligoclonal hematopoiesis, with a tendency toward increasing numbers of clones over time, suggesting delayed and progressive expansion of a sizable number of engrafted gene-corrected HSCs. A proportion of hematopoietic progenitors collected from the treated patients, corresponding to that estimated by vector marking, were resistant to ex vivo challenge with DNA crosslinking agents, indicating functional rescue of
592 Cell Stem Cell 25, November 7, 2019 ª 2019 Published by Elsevier Inc.
the repair defect. Patients with the highest levels of reconstitution displayed stabilization of blood cell counts, indicating therapeutic benefit on the otherwise progressive hematopoietic exhaustion. Clinical studies of HSC gene therapy for other inherited diseases typically show stable and highly polyclonal engraftment with up to 10,000 active HSC clones (Sessa et al., 2016; Marktel et al., 2019). This FA trial infused corrected HSPCs without conditioning and up to 2 logs lower than the average dose usually administered in the aforementioned studies, suggesting that the oligoclonal composition detected in FA patients reflects the number of infused HSCs. Moreover, the delayed activation kinetics of gene-corrected FA HSCs is reminiscent of what has been described in other studies of clonal dynamics after HSC gene therapy (Scala et al., 2018). The concordant results among the different studies imply that not only acute (conditioning-based) but also chronic depletion of endogenous HSCs allows engraftment of exogenous HSCs, at least when they have a growth advantage. These findings raise several questions: do the corrected FA HSCs require the presence of empty bone marrow niches at the time of infusion, or can infused HSCs persist for some time in the body until niche space becomes available as the disease progresses? Can advantaged HSCs actively displace endogenous HSCs, and what is the cell-dose-relationship of this hypothetic process? Recent evidence in mice
Cell Stem Cell
In Translation suggests that mega-doses of ex-vivoexpanded HSCs can out-compete endogenous HSCs, leading to donor-cellderived hematopoiesis in the absence of conditioning (Wilkinson et al., 2019), and FA represents an intriguing model where the size and fitness of the endogenous HSC pool are reduced to such levels that low numbers of corrected cells have the opportunity to persist and expand in what appears to be a physiologic process of in vivo HSC dynamics. In another paper, published in this issue of Cell Stem Cell, a team led by Paula Rio leveraged the clinically validated positive selection concept and tested a gene-editing-based technology to restore the function of hematopoietic cells carrying FA mutations (Roma´n-Rodrı´guez et al., 2019). While this approach may seem counter-intuitive in FA, where cells are hypersensitive to DNA damage and have impaired homology-based repair, authors smartly exploited the non-homologous end joining (NHEJ) repair pathway, which is reported to be increased in FA cells and does not require cell division, to randomly induce compensatory mutations that restore FA protein function. Following a DNA double-strand break induced by CRISPR/Cas9 nucleases targeted to the vicinity of the FA mutation, the authors show a low but reproducible proportion of cells harboring therapeutic indels (insertion/deletion of few bases at the repair site) introduced upon NHEJ-mediated repair, alongside cells carrying other indels which did not restore the reading frame. However, due to the strong selective growth advantage conferred to the FA-corrected cells, their proportion significantly increased upon culture and in murine xenografts and showed evidence of functional rescue of some characteristic FA phenotypic features. This strategy worked for several FANCA mutations and other FA complementation groups. It is thus plausible that such a strategy may eventually also be pursued clinically, although the yield of corrected cells might be rate-limiting for achieving efficacy. How does this gene editing approach compare to lentiviral-vector-based gene replacement? The latter offers higher
yields and correction efficiencies. Both lentiviral vectors and site-specific nucleases carry a risk of mutagenesis, the former by semi-randomly integrating into the genome, the latter by off-target DNA cuts or large-scale genomic alterations. While the vector integration analyses from this and other clinical studies of HSC gene therapy suggest low mutagenic risk for lentiviral vectors containing moderate internal promoters (as the one used in this study), it is difficult to estimate such risk for gene editing, given the early stage of development of the platform, its currently limited clinical testing, and the dependence on the specific guide sequences and reagents used to induce the DNA break. A low mutagenic footprint is particularly important in FA, where a limited number of corrected HSC clones are subjected to substantial replication stress while sustaining hematopoiesis (Walter et al., 2015). It is therefore prudent to await longer patient follow-up in the FA clinical study to assess safety and efficacy of HSC gene replacement therapy, while further optimizing the editing protocol for follow-up studies. Key points of actions are earlier collection of higher numbers of HSCs, higher rates of transduction/correction, and earlier treatment of patients, possibly paired with strategies to deplete non-corrected HSCs in the patients. Recent studies have highlighted the occurrence of oligoclonal hematopoiesis with a dominant HSC clone in aging people and the association of this condition with increased risk of developing MDS/ leukemia (Steensma, 2018). One thus wonders whether oligo/monoclonal drive might impair the long-term resilience of hematopoiesis because of stress-driven HSC exhaustion and accumulation/selection of growth-promoting mutations, eventually leading to transformation. Thus, long-term monitoring of the clonal composition and evolution of reconstituted hematopoiesis in FA patients will inform on the long-term safety of gene therapy strategies whose yield of corrected HSCs is limiting. Overall, FA will continue to shed new lights on fundamental aspects of HSC biology and represent a favorable
context for introducing innovations into cell and gene therapy platforms based on this highly clinically relevant cell type. REFERENCES Garaycoechea, J.I., and Patel, K.J. (2014). Why does the bone marrow fail in Fanconi anemia? Blood 123, 26–34. Mankad, A., Taniguchi, T., Cox, B., Akkari, Y., Rathbun, R.K., Lucas, L., Bagby, G., Olson, S., D’Andrea, A., and Grompe, M. (2006). Natural gene therapy in monozygotic twins with Fanconi anemia. Blood 107, 3084–3090. Marktel, S., Scaramuzza, S., Cicalese, M.P., Giglio, F., Galimberti, S., Lidonnici, M.R., Calbi, V., Assanelli, A., Bernardo, M.E., Rossi, C., et al. (2019). Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent ß-thalassemia. Nat. Med. 25, 234–241. Rı´o, P., Navarro, S., Wang, W., Sa´nchezDomı´nguez, R., Pujol, R.M., Segovia, J.C., Bogliolo, M., Merino, E., Wu, N., Salgado, R., et al. (2019). Successful engraftment of gene-corrected hematopoietic stem cells in non-conditioned patients with Fanconi anemia. Nat. Med. 25, 1396–1401. Roma´n-Rodrı´guez, F.J., Ugalde, L., A´lvarez, L., Dı´ez, B., Ramı´rez, M.J., Risuen˜o, C., Co´rton, M., Bogliolo, M., Bernal, S., et al. (2019). NHEJMediated Repair of CRISPR-Cas9-Induced DNA Breaks Efficiently Corrects Mutations in HSPCs from Patients with Fanconi Anemia. Cell Stem Cell 25, this issue, 607–621. Scala, S., Basso-Ricci, L., Dionisio, F., Pellin, D., Giannelli, S., Salerio, F.A., Leonardelli, L., Cicalese, M.P., Ferrua, F., Aiuti, A., and Biasco, L. (2018). Dynamics of genetically engineered hematopoietic stem and progenitor cells after autologous transplantation in humans. Nat. Med. 24, 1683–1690. Sessa, M., Lorioli, L., Fumagalli, F., Acquati, S., Redaelli, D., Baldoli, C., Canale, S., Lopez, I.D., Morena, F., Calabria, A., et al. (2016). Lentiviral haemopoietic stem-cell gene therapy in earlyonset metachromatic leukodystrophy: an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial. Lancet 388, 476–487. Steensma, D.P. (2018). Clinical consequences of clonal hematopoiesis of indeterminate potential. Blood Adv. 2, 3404–3410. Walter, D., Lier, A., Geiselhart, A., Thalheimer, F.B., Huntscha, S., Sobotta, M.C., Moehrle, B., Brocks, D., Bayindir, I., Kaschutnig, P., et al. (2015). Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells. Nature 520, 549–552. Wilkinson, A.C., Ishida, R., Kikuchi, M., Sudo, K., Morita, M., Crisostomo, R.V., Yamamoto, R., Loh, K.M., Nakamura, Y., Watanabe, M., et al. (2019). Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature 571, 117–121.
Cell Stem Cell 25, November 7, 2019 593
In Translation In Vivo Selection for Gene-Corrected HSPCs Advances Gene Therapy for a Rare Stem Cell Disease Bernhard Gentner1,2,* and Luigi Naldini1,3,* 1San
Raffaele Telethon Institute for Gene Therapy, Milan, Italy and Bone Marrow Transplantation Unit, IRCCS San Raffaele Scientific Institute, Milan, Italy 3Vita Salute San Raffaele University, Milan, Italy *Correspondence: [email protected] (B.G.), [email protected] (L.N.) https://doi.org/10.1016/j.stem.2019.10.004 2Hematology
Two recent papers (one by Roma´n-Rodrı´guez et al., 2019 in this issue of Cell Stem Cell) highlight how the power of biological selection on hematopoietic stem cell fitness can facilitate gene therapies for Fanconi Anemia. A clinical trial using lentiviral gene replacement and a proof-of-concept targeted genome editing study show robust engraftment and expansion of gene-corrected cells at levels reaching therapeutic relevance. Fanconi anemia (FA), the most common inherited bone marrow failure syndrome, has long intrigued researchers and challenged clinicians (Garaycoechea and Patel, 2014). Loss-of-function mutations in any one of at least 17 genes in the FA pathway result in the inability to repair interstrand DNA crosslinks (ICLs), highly toxic adducts that form from exposure to exogenous and endogenous mutagens. ICL accumulation prevents DNA transcription and replication, leading to cell death. Hematopoiesis, a process characterized by extensive, lifelong proliferation of stem and progenitor cells, is particularly sensitive to unrepaired ICLs. FA patients progressively exhaust their hematopoietic reserve and carry an increased risk of developing myelodysplasia, acute leukemia, and solid cancers. Case reports from patients with rarely occurring somatic reversion of the mutation suggest that one or a few functionally corrected hematopoietic stem cell (HSC) clones may be sufficient to rescue the hematologic manifestations of FA (Mankad et al., 2006). Two recent papers from partly overlapping teams report the early results of a clinical trial of HSC gene therapy for FA using gene replacement by lentiviral vectors (Rı´o et al., 2019) and the design of a gene correction strategy for FA using targeted genome editing (Roma´n-Rodrı´guez et al., 2019). Both studies show a powerful effect of biological selection at the HSC level, where the growth advantage provided by genetic correction of FA deficiency allows expansion of rare corrected cells and he-
matopoietic repopulation to levels reaching therapeutic relevance in patients, even without conditioning. The team led by Juan Bueren (Rı´o et al., 2019) undertook the challenging attempt to alleviate bone marrow failure in FA complementation group A (FANCA) patients by infusing autologous HSCs following lentiviral transfer of functional FANCA cDNA. Collection of HSCs early in life when their reservoir is not yet exhausted, the use of a very short ex vivo manipulation protocol to minimize perturbation, and the capacity of lentiviral vectors to efficiently transduce non-dividing cells were key for obtaining sufficient numbers of functionally corrected HSCs upon FANCA gene replacement. Remarkably, the authors demonstrate engraftment of vector-marked cells in the four treated FANCA patients. Engraftment typically appeared late, around 6 to 12 months after HSC administration, and was variable but progressively increased in all patients to reach proportions ranging from 4% to 55% at the latest follow-up (18–30 months). Donor cells contributed to multi-lineage and oligoclonal hematopoiesis, with a tendency toward increasing numbers of clones over time, suggesting delayed and progressive expansion of a sizable number of engrafted gene-corrected HSCs. A proportion of hematopoietic progenitors collected from the treated patients, corresponding to that estimated by vector marking, were resistant to ex vivo challenge with DNA crosslinking agents, indicating functional rescue of
592 Cell Stem Cell 25, November 7, 2019 ª 2019 Published by Elsevier Inc.
the repair defect. Patients with the highest levels of reconstitution displayed stabilization of blood cell counts, indicating therapeutic benefit on the otherwise progressive hematopoietic exhaustion. Clinical studies of HSC gene therapy for other inherited diseases typically show stable and highly polyclonal engraftment with up to 10,000 active HSC clones (Sessa et al., 2016; Marktel et al., 2019). This FA trial infused corrected HSPCs without conditioning and up to 2 logs lower than the average dose usually administered in the aforementioned studies, suggesting that the oligoclonal composition detected in FA patients reflects the number of infused HSCs. Moreover, the delayed activation kinetics of gene-corrected FA HSCs is reminiscent of what has been described in other studies of clonal dynamics after HSC gene therapy (Scala et al., 2018). The concordant results among the different studies imply that not only acute (conditioning-based) but also chronic depletion of endogenous HSCs allows engraftment of exogenous HSCs, at least when they have a growth advantage. These findings raise several questions: do the corrected FA HSCs require the presence of empty bone marrow niches at the time of infusion, or can infused HSCs persist for some time in the body until niche space becomes available as the disease progresses? Can advantaged HSCs actively displace endogenous HSCs, and what is the cell-dose-relationship of this hypothetic process? Recent evidence in mice
Cell Stem Cell
In Translation suggests that mega-doses of ex-vivoexpanded HSCs can out-compete endogenous HSCs, leading to donor-cellderived hematopoiesis in the absence of conditioning (Wilkinson et al., 2019), and FA represents an intriguing model where the size and fitness of the endogenous HSC pool are reduced to such levels that low numbers of corrected cells have the opportunity to persist and expand in what appears to be a physiologic process of in vivo HSC dynamics. In another paper, published in this issue of Cell Stem Cell, a team led by Paula Rio leveraged the clinically validated positive selection concept and tested a gene-editing-based technology to restore the function of hematopoietic cells carrying FA mutations (Roma´n-Rodrı´guez et al., 2019). While this approach may seem counter-intuitive in FA, where cells are hypersensitive to DNA damage and have impaired homology-based repair, authors smartly exploited the non-homologous end joining (NHEJ) repair pathway, which is reported to be increased in FA cells and does not require cell division, to randomly induce compensatory mutations that restore FA protein function. Following a DNA double-strand break induced by CRISPR/Cas9 nucleases targeted to the vicinity of the FA mutation, the authors show a low but reproducible proportion of cells harboring therapeutic indels (insertion/deletion of few bases at the repair site) introduced upon NHEJ-mediated repair, alongside cells carrying other indels which did not restore the reading frame. However, due to the strong selective growth advantage conferred to the FA-corrected cells, their proportion significantly increased upon culture and in murine xenografts and showed evidence of functional rescue of some characteristic FA phenotypic features. This strategy worked for several FANCA mutations and other FA complementation groups. It is thus plausible that such a strategy may eventually also be pursued clinically, although the yield of corrected cells might be rate-limiting for achieving efficacy. How does this gene editing approach compare to lentiviral-vector-based gene replacement? The latter offers higher
yields and correction efficiencies. Both lentiviral vectors and site-specific nucleases carry a risk of mutagenesis, the former by semi-randomly integrating into the genome, the latter by off-target DNA cuts or large-scale genomic alterations. While the vector integration analyses from this and other clinical studies of HSC gene therapy suggest low mutagenic risk for lentiviral vectors containing moderate internal promoters (as the one used in this study), it is difficult to estimate such risk for gene editing, given the early stage of development of the platform, its currently limited clinical testing, and the dependence on the specific guide sequences and reagents used to induce the DNA break. A low mutagenic footprint is particularly important in FA, where a limited number of corrected HSC clones are subjected to substantial replication stress while sustaining hematopoiesis (Walter et al., 2015). It is therefore prudent to await longer patient follow-up in the FA clinical study to assess safety and efficacy of HSC gene replacement therapy, while further optimizing the editing protocol for follow-up studies. Key points of actions are earlier collection of higher numbers of HSCs, higher rates of transduction/correction, and earlier treatment of patients, possibly paired with strategies to deplete non-corrected HSCs in the patients. Recent studies have highlighted the occurrence of oligoclonal hematopoiesis with a dominant HSC clone in aging people and the association of this condition with increased risk of developing MDS/ leukemia (Steensma, 2018). One thus wonders whether oligo/monoclonal drive might impair the long-term resilience of hematopoiesis because of stress-driven HSC exhaustion and accumulation/selection of growth-promoting mutations, eventually leading to transformation. Thus, long-term monitoring of the clonal composition and evolution of reconstituted hematopoiesis in FA patients will inform on the long-term safety of gene therapy strategies whose yield of corrected HSCs is limiting. Overall, FA will continue to shed new lights on fundamental aspects of HSC biology and represent a favorable
context for introducing innovations into cell and gene therapy platforms based on this highly clinically relevant cell type. REFERENCES Garaycoechea, J.I., and Patel, K.J. (2014). Why does the bone marrow fail in Fanconi anemia? Blood 123, 26–34. Mankad, A., Taniguchi, T., Cox, B., Akkari, Y., Rathbun, R.K., Lucas, L., Bagby, G., Olson, S., D’Andrea, A., and Grompe, M. (2006). Natural gene therapy in monozygotic twins with Fanconi anemia. Blood 107, 3084–3090. Marktel, S., Scaramuzza, S., Cicalese, M.P., Giglio, F., Galimberti, S., Lidonnici, M.R., Calbi, V., Assanelli, A., Bernardo, M.E., Rossi, C., et al. (2019). Intrabone hematopoietic stem cell gene therapy for adult and pediatric patients affected by transfusion-dependent ß-thalassemia. Nat. Med. 25, 234–241. Rı´o, P., Navarro, S., Wang, W., Sa´nchezDomı´nguez, R., Pujol, R.M., Segovia, J.C., Bogliolo, M., Merino, E., Wu, N., Salgado, R., et al. (2019). Successful engraftment of gene-corrected hematopoietic stem cells in non-conditioned patients with Fanconi anemia. Nat. Med. 25, 1396–1401. Roma´n-Rodrı´guez, F.J., Ugalde, L., A´lvarez, L., Dı´ez, B., Ramı´rez, M.J., Risuen˜o, C., Co´rton, M., Bogliolo, M., Bernal, S., et al. (2019). NHEJMediated Repair of CRISPR-Cas9-Induced DNA Breaks Efficiently Corrects Mutations in HSPCs from Patients with Fanconi Anemia. Cell Stem Cell 25, this issue, 607–621. Scala, S., Basso-Ricci, L., Dionisio, F., Pellin, D., Giannelli, S., Salerio, F.A., Leonardelli, L., Cicalese, M.P., Ferrua, F., Aiuti, A., and Biasco, L. (2018). Dynamics of genetically engineered hematopoietic stem and progenitor cells after autologous transplantation in humans. Nat. Med. 24, 1683–1690. Sessa, M., Lorioli, L., Fumagalli, F., Acquati, S., Redaelli, D., Baldoli, C., Canale, S., Lopez, I.D., Morena, F., Calabria, A., et al. (2016). Lentiviral haemopoietic stem-cell gene therapy in earlyonset metachromatic leukodystrophy: an ad-hoc analysis of a non-randomised, open-label, phase 1/2 trial. Lancet 388, 476–487. Steensma, D.P. (2018). Clinical consequences of clonal hematopoiesis of indeterminate potential. Blood Adv. 2, 3404–3410. Walter, D., Lier, A., Geiselhart, A., Thalheimer, F.B., Huntscha, S., Sobotta, M.C., Moehrle, B., Brocks, D., Bayindir, I., Kaschutnig, P., et al. (2015). Exit from dormancy provokes DNA-damage-induced attrition in haematopoietic stem cells. Nature 520, 549–552. Wilkinson, A.C., Ishida, R., Kikuchi, M., Sudo, K., Morita, M., Crisostomo, R.V., Yamamoto, R., Loh, K.M., Nakamura, Y., Watanabe, M., et al. (2019). Long-term ex vivo haematopoietic-stem-cell expansion allows nonconditioned transplantation. Nature 571, 117–121.
Cell Stem Cell 25, November 7, 2019 593