Final Report Summary - TARGETINGGENETHERAPY (Towards Safe and Effective Hematopoietic Stem Cell Gene Therapy: Targeting Integration to Genomic Safe Harbors and Exploiting Endogenous microRNA to Regulate Transgene Expression)
Hematopoietic Stem Cell (HSC) gene therapy offers a therapeutic option for several genetic diseases. It consists of harvesting HSC from a patient, genetically modifying them ex vivo to correct the inherited defect and infusing them back into the patient, where they are able to self-maintain and give rise to a functional progeny of all lineages that can rescue the disease. In two first-in-human trials of HSC gene therapy for Wiskott-Aldrich Syndrome (WAS) and Metachromatic Leukodystrophy (MLD) using lentiviral vectors we recently reported stable multi-lineage reconstitution of hematopoiesis, with evidence of clinical benefit and a favorable safety profile (Aiuti et al., Science 2013; Biffi et al., Science 2013). These results paved the way to broadening the application of HSC gene therapy to other diseases. In order to achieve this goal, in this project we have worked on two major new strategies to improve the safety and efficacy of HSC gene therapy, i.e.: i) exploiting microRNA regulation to stringently control transgene expression and, ii) employing artificial nucleases (such as Zinc Finger Nucleases, ZFN) for in situ correction of inherited mutations and to target transgene integration into predetermined safe genomic sites.
Concerning the first strategy, starting from our initial identification of miRNA that show preferential activity in Hematopoietic Stem and Progenitor Cells (HSPC) as opposed to differentiated hematopoietic cells (Gentner et al., Sci Transl Med 2010) we designed regulated vector cassettes containing an optimized combination of target sequences complementary to such miRNA, thereby suppressing unwanted transgene expression in HSPC. We developed a candidate preclinical vector for Chronic Granulomatous Disease (CGD), combining miRNA-126-based de-targeting with a strong phagocyte-specific promoter, and proved its high specificity and efficacy in the CGD mouse model and in patients’ cells (Chiriaco et al., Mol Ther 2014). Similarly, an optimized vector was designed for the gene therapy of Globoid Cell Leukodystrophy (GLD) (Ungari et al., Mol Ther Methods Clin Dev 2015). Moreover, we implemented miRNA-126-based de-targeting in a cancer gene therapy approach for the sustained and targeted delivery of IFNa into tumors by tumor-infiltrating macrophages. We showed that this strategy results in a powerful activation of tumor-infiltrating macrophages, NK and T-cells, leading to effective anti-tumor responses (Escobar et al., Sci Transl Med 2014; Catarinella et al., EMBO Mol Med 2016). Given these encouraging results, we envision a relatively fast progression to clinical testing of HSC gene therapy for such diseases employing our miRNA-regulated lentiviral vector platform.
In addition to these application-oriented studies, we further explored the biological function of miRNA-126 in HSC and leukemia. By knocking down or overexpressing miRNA-126 in mouse and human HSPC, we demonstrated that it regulates HSC quiescence, with inhibition of miRNA-126 activity leading to a sustained expansion of long-term HSC, without exhaustion or transformation. Mechanistically, miRNA-126 downregulates PI3K/AKT signaling, thereby rendering HSC less responsive to activatory niche signals (Lechman et al., Cell Stem Cell 2012). We also established the importance of miRNA-126 in acute leukemia. In Acute Myeloid Leukemia (AML), leukemic stem cells depend on miRNA-126 expression to escape exhaustion and differentiation, and higher miRNA-126 expression in clinical samples is associated with chemotherapy resistance and worse outcome (Lechman et al., Cancer Cell 2016). We also found that ectopic miRNA-126 expression in mouse HSPC induces leukemia, preferentially B-cell Acute Lymphoblastic Leukemia (B-ALL), addicted to miRNA-126 expression. Mechanistically, miRNA-126 downregulates p53 activity and maintains blasts in a proliferative B cell progenitor stage (Nucera et al., Cancer Cell 2016). The obligate dependence of acute leukemia - in stark contrast to normal HSC - on adequate levels of miRNA-126 expression opens up a therapeutic window that can be exploited in patients with hematologic malignancies. The rationale for developing therapeutic strategies that target miRNA-126 in leukemia patients is two-fold: i) reducing miRNA-126 expression in normal HSC can expand their numbers without causing exhaustion or malignant transformation, thus boosting hematologic recovery; ii) antagonizing miRNA-126 in leukemia counteracts chemotherapy resistance, impairs leukemia propagating cells and induces apoptosis. Developing pharmacologic agents that target miRNA-126 and/or its pathways may thus have the potential to improve the outcome of patients with acute leukemia.
Concerning the second new strategy to improve the safety and efficacy of HSC gene therapy, we first exploited artificial ZFNs to target transgene integration into predetermined safe genomic sites. Specifically, we devised a “sustainable” modality of gene transfer in which transgene insertion occurs into a paradigmatic “safe genomic harbor”, the AAVS1 locus, thus obtaining robust transgene expression with no detrimental impact on endogenous gene expression (Lombardo et al., Nat Methods 2011). This approach may allow efficient and predictable levels of transgene expression while abrogating the risks associated with quasi-random vector insertional mutagenesis. We also showed that gene targeting can be used to achieve in situ correction of mutations by inserting a functional cDNA copy of the gene downstream of its own endogenous promoter. This way it is possible to correct most mutations occurring in the targeted gene, restoring both its function and physiological expression control. We are using this strategy to correct mutations in the IL2RG gene, which causes X-linked Severe Combined Immunodeficiency (SCID-X1). Specifically, we developed two new protocols which enable robust and reproducible gene editing in human HSC (Genovese et al., Nature 2014) or fibroblasts (Rio et al., EMBO Mol Med 2014). We demonstrated that gene-edited human HSC are able to repopulate transplanted mice and give rise to functional immune cells. Importantly, we also achieved the correction of an endogenous mutation in HSPC derived from a SCID-X1 patient. Based on these promising results, we are currently optimizing/scaling-up the HSPC gene editing procedure in order to develop a clinically applicable protocol. Gene-corrected induced Pluripotent Stem Cells (iPSC) derived from patients’ fibroblasts may represent an alternative source of corrected HSPC if gene targeting efficiency or ex vivo expansion of somatic HSC remain technically challenging. By using an improved reprogramming vector (Friedli et al., Genome Res 2014), we obtained gene-corrected iPSC from Fanconi Anemia A (Rio et al., EMBO Mol Med 2014) and SCID-X1 patients (Firrito et al., manuscript under revision). We expect to translate our targeted gene correction strategy in SCID-X1 patients in the near future. This could represent the first clinical trial employing a gene editing approach and may position gene editing as new gold standard for precise HSC engineering.
Concerning the first strategy, starting from our initial identification of miRNA that show preferential activity in Hematopoietic Stem and Progenitor Cells (HSPC) as opposed to differentiated hematopoietic cells (Gentner et al., Sci Transl Med 2010) we designed regulated vector cassettes containing an optimized combination of target sequences complementary to such miRNA, thereby suppressing unwanted transgene expression in HSPC. We developed a candidate preclinical vector for Chronic Granulomatous Disease (CGD), combining miRNA-126-based de-targeting with a strong phagocyte-specific promoter, and proved its high specificity and efficacy in the CGD mouse model and in patients’ cells (Chiriaco et al., Mol Ther 2014). Similarly, an optimized vector was designed for the gene therapy of Globoid Cell Leukodystrophy (GLD) (Ungari et al., Mol Ther Methods Clin Dev 2015). Moreover, we implemented miRNA-126-based de-targeting in a cancer gene therapy approach for the sustained and targeted delivery of IFNa into tumors by tumor-infiltrating macrophages. We showed that this strategy results in a powerful activation of tumor-infiltrating macrophages, NK and T-cells, leading to effective anti-tumor responses (Escobar et al., Sci Transl Med 2014; Catarinella et al., EMBO Mol Med 2016). Given these encouraging results, we envision a relatively fast progression to clinical testing of HSC gene therapy for such diseases employing our miRNA-regulated lentiviral vector platform.
In addition to these application-oriented studies, we further explored the biological function of miRNA-126 in HSC and leukemia. By knocking down or overexpressing miRNA-126 in mouse and human HSPC, we demonstrated that it regulates HSC quiescence, with inhibition of miRNA-126 activity leading to a sustained expansion of long-term HSC, without exhaustion or transformation. Mechanistically, miRNA-126 downregulates PI3K/AKT signaling, thereby rendering HSC less responsive to activatory niche signals (Lechman et al., Cell Stem Cell 2012). We also established the importance of miRNA-126 in acute leukemia. In Acute Myeloid Leukemia (AML), leukemic stem cells depend on miRNA-126 expression to escape exhaustion and differentiation, and higher miRNA-126 expression in clinical samples is associated with chemotherapy resistance and worse outcome (Lechman et al., Cancer Cell 2016). We also found that ectopic miRNA-126 expression in mouse HSPC induces leukemia, preferentially B-cell Acute Lymphoblastic Leukemia (B-ALL), addicted to miRNA-126 expression. Mechanistically, miRNA-126 downregulates p53 activity and maintains blasts in a proliferative B cell progenitor stage (Nucera et al., Cancer Cell 2016). The obligate dependence of acute leukemia - in stark contrast to normal HSC - on adequate levels of miRNA-126 expression opens up a therapeutic window that can be exploited in patients with hematologic malignancies. The rationale for developing therapeutic strategies that target miRNA-126 in leukemia patients is two-fold: i) reducing miRNA-126 expression in normal HSC can expand their numbers without causing exhaustion or malignant transformation, thus boosting hematologic recovery; ii) antagonizing miRNA-126 in leukemia counteracts chemotherapy resistance, impairs leukemia propagating cells and induces apoptosis. Developing pharmacologic agents that target miRNA-126 and/or its pathways may thus have the potential to improve the outcome of patients with acute leukemia.
Concerning the second new strategy to improve the safety and efficacy of HSC gene therapy, we first exploited artificial ZFNs to target transgene integration into predetermined safe genomic sites. Specifically, we devised a “sustainable” modality of gene transfer in which transgene insertion occurs into a paradigmatic “safe genomic harbor”, the AAVS1 locus, thus obtaining robust transgene expression with no detrimental impact on endogenous gene expression (Lombardo et al., Nat Methods 2011). This approach may allow efficient and predictable levels of transgene expression while abrogating the risks associated with quasi-random vector insertional mutagenesis. We also showed that gene targeting can be used to achieve in situ correction of mutations by inserting a functional cDNA copy of the gene downstream of its own endogenous promoter. This way it is possible to correct most mutations occurring in the targeted gene, restoring both its function and physiological expression control. We are using this strategy to correct mutations in the IL2RG gene, which causes X-linked Severe Combined Immunodeficiency (SCID-X1). Specifically, we developed two new protocols which enable robust and reproducible gene editing in human HSC (Genovese et al., Nature 2014) or fibroblasts (Rio et al., EMBO Mol Med 2014). We demonstrated that gene-edited human HSC are able to repopulate transplanted mice and give rise to functional immune cells. Importantly, we also achieved the correction of an endogenous mutation in HSPC derived from a SCID-X1 patient. Based on these promising results, we are currently optimizing/scaling-up the HSPC gene editing procedure in order to develop a clinically applicable protocol. Gene-corrected induced Pluripotent Stem Cells (iPSC) derived from patients’ fibroblasts may represent an alternative source of corrected HSPC if gene targeting efficiency or ex vivo expansion of somatic HSC remain technically challenging. By using an improved reprogramming vector (Friedli et al., Genome Res 2014), we obtained gene-corrected iPSC from Fanconi Anemia A (Rio et al., EMBO Mol Med 2014) and SCID-X1 patients (Firrito et al., manuscript under revision). We expect to translate our targeted gene correction strategy in SCID-X1 patients in the near future. This could represent the first clinical trial employing a gene editing approach and may position gene editing as new gold standard for precise HSC engineering.