The Role of Conditioning in Hematopoietic Stem-Cell Gene Therapy

Abstract

Gene therapy (GT) approaches based on autologous hematopoietic stem cells (HSC) corrected ex vivo have shown therapeutic benefit in a number of inherited disorders. GT bares the advantage of allowing each patient to be her/his own donor while reducing the risks of immune-mediated complications as compared with allogeneic hematopoietic stem-cell transplantation (HSCT). In order to achieve stable engraftment of HSC, patients undergoing transplantation of allogeneic or autologous HSC receive a chemotherapy- and/or radiotherapy-based preparation. With regard to HSC-GT for inherited genetic disorders, the ideal conditioning regimen should aim to contain toxicity by reducing the dosage and/or the number of chemotherapeutic agents administered, in comparison to fully myeloablative preparations employed in conventional allogeneic HSCT. To meet this aim, a profound knowledge of the disease-specific biological background and of the therapeutic transgene levels, as well as of the key principles of transplantation, are required. While low-dose conditioning is sufficient to create a mixed chimerism when gene-corrected cells are endowed with a natural selective advantage, such as in the case of immune deficiencies, myeloablative doses are necessary when high levels of engraftment are required in disease such as lysosomal storage disorders and beta thalassemia. Therefore, the intensity and type of conditioning regimen administered to patients undergoing HSC-GT should be tailored to reach a minimal efficacious therapeutic target level while sparing toxicity. Novel strategies based on monoclonal antibodies selectively depleting blood cells and associated with limited extramedullary toxicity might be successfully employed in the context of HSC-GT in the near future. This review focuses on the role of the conditioning regimen in HSC-GT, and in particular, it highlights the importance of modulating the preparative chemotherapy based on disease biology and transgene expression in order to optimize outcome.

Introduction

Hematopoietic stem cell (HSC) gene therapy (GT) has become in recent years an attractive therapeutic strategy for inherited genetic disorders thanks to the remarkable therapeutic benefits and favorable safety record, offering, in specific diseases, potential advantages over allogeneic HSC transplantation (HSCT).^1

–5 Early-generation gamma-retroviral vectors (RV) have shown important limitations and risks, with the exception of adenosine deaminase deficient severe combined immunodeficiency (ADA-SCID), for which the cumulative experience of several groups have established its long-term efficacy and safety.^6

–9 The vast majority of HSC-GT trials are currently conducted with self-inactivating (SIN) lentiviral vectors (LV), which have shown improved efficacy and safety in preclinical models and clinical trials⁵ for peroxysomal and lysosomal storage disorders (LSD) such as adrenoleukodystrophy (ALD)³ and metachromatic leukodystrophy (MLD),¹⁰ beta thalassemia (βThal),⁴ Wiskott–Aldrich syndrome (WAS),^2,11 and, more recently, ADA-SCID.¹² Results of these clinical trials demonstrate that genetically modified HSC engraft long-term and express the therapeutic gene, achieving evidence of clinical benefit and a favorable safety profile.

In order to achieve stable engraftment of HSC, patients undergoing transplantation of allogeneic or autologous HSC receive a chemotherapy- and/or radiotherapy-based preparation. The aim of this treatment is to deplete highly proliferating hematopoietic stem progenitor cells (HSPC) residing in the bone marrow (BM), thus making space and favoring engraftment of donor HSC.^13,14 In case of allogeneic HSC transplantation, immunosuppressive drugs are added in order to prevent immunological complications such as graft rejection and graft-versus-host disease. In case of hematological malignancies, the conditioning regimen also includes drugs with specific antitumor effect with the aim of eliminating residual tumor cells.¹³ While being very effective at depleting host HSC, full-intensity myeloablative conditioning is associated with substantial short- and long-term toxicity in hematopoietic and non-hematopoietic tissues, which might represent a hurdle to the wider application of HSCT to non-malignant genetic diseases. As far as autologous HSC-GT for inherited genetic disorders is concerned, the ideal conditioning regimen should aim to mitigate toxicity by lowering the dosage and/or reducing the number of chemotherapeutic and immunosuppressant drugs that are administered, in comparison to the regimens used in conventional allogeneic HSCT. This requires profound knowledge of the disease-specific biological background and of the therapeutic levels of transgene expression, as well as of the key principles and phases of transplantation techniques. For example, an important favorable factor in HSC-GT is represented by the presence of a natural selective advantage for gene-corrected cells over their uncorrected counterpart, such as the one occurring in ADA-SCID and WAS for lymphoid cells, in WAS for platelets, and in βThal for erythroid cells.^2,7 Based on these premises and on results obtained in patients undergoing both allogeneic HSCT and HSC-GT for non-malignant disorders, the intensity and type of conditioning regimen administered in HSC-GT should be designed and tailored to reach a minimal efficacious therapeutic target level.

This review focuses on the role of the conditioning regimen in HSC-GT approaches as they move forward clinical application, and in particular will highlight the importance of modulating the preparative chemotherapy based on disease biology and transgene expression in order to optimize outcome.

Choice of Appropriate Conditioning According to Disease ADA-SCID

In the first ADA-SCID HSC-GT trials, a conditioning regimen was not administered to patients, resulting in low or absent engraftment.^15,16 Moreover, in these first attempts, a low transduction rate was also responsible for the unsatisfactory outcome of the approach.^8,15 The introduction of a mild conditioning regimen based on the alkylating agent busulfan, together with amelioration in transduction efficiency, allowed an improvement in the engraftment of gene-modified cells.⁶ These adjustments enabled both the creation of space in the BM for corrected progenitors through the administration of the conditioning regimen and an improvement in the selective pressure for corrected cells, ultimately translating into clinical benefit for treated patients.^6,8,9

At the authors' institute, a minimal dose intensity conditioning with intravenous (i.v.) busulfan (0.5 mg/kg/doses × eight doses in 2 days), representing about 25% of the one employed in standard allogeneic HSCT, was introduced, allowing good levels of multilineage engraftment of corrected cells to be obtained in the long term (up to 13 years after treatment)^7,17
–19 while reducing potential toxicity, which is expected to be higher due to the metabolic nature of the disease and the known involvement of the microenvironment and other hematopoietic lineages.^18,20,21 This conditioning scheme allowed treated patients to obtain ADA levels sufficient to gain a decrease of toxic metabolites, functional immune recovery, discontinuation of immunoglobulin therapy, and production of antibodies after immunization.¹⁸ Moreover, no leukemic or adverse events related to the therapy were observed in these patients treated with HSPC transduced with a γ-RV vector encoding the human ADA gene.⁷ Indeed, the presence of shared vector integrations among multiple lineages demonstrates stable engraftment of multipotent HSPC.^17,19

Other patients affected by ADA-SCID have been successfully treated with autologous CD34⁺ cells transduced with a γ-RV vector in the United States and in the United Kingdom after a busulfan-based reduced-intensity conditioning regimen, leading to long-term multilineage engraftment, discontinuation of enzyme replacement therapy (ERT), persistent immune reconstitution, and sustained systemic detoxification.^8,9 In the U.S. study, Candotti et al. compared patients treated without or with chemotherapy (low-dose i.v. busulfan 65–90 mg/m², equivalent to 1.9–4.9 mg/kg), confirming the importance of conditioning with busulfan in favoring long-term HSC engraftment. Moreover, withdrawal of ERT in the group of children who received cytoreductive treatment contributed to the beneficial effect of the treatment, whereas ERT was continued in patients without chemotherapy.⁸

The studies from Aiuti et al. ⁷ and Candotti et al. ⁸ also underline the impact of measuring busulfan levels in order to calculate the area under the curve (AUC) for busulfan exposure. The U.S. study found a correlation between the administered dose (mg/m²) and the resulting busulfan AUC. In the San Raffaele-Telethon Institute for Gene Therapy (SR-TIGET) study, the duration of neutropenia correlated with busulfan AUC, and the dose of busulfan was reduced in case of elevated AUC in order to reduce nonhematopoeitic toxicity.

To improve efficacy and safety further, a SIN LV with a codon-optimized human ADA gene under the control of the short-form elongation factor-1α promoter was developed by groups in the United Kingdom and the United States. In ADA(−/−) mice, this vector displayed high-efficiency gene transfer and sufficient ADA expression to rescue ADA(−/−) mice from their lethal phenotype with good thymic and peripheral T- and B-cell reconstitution.¹² Clinical trials are ongoing to assess the safety and efficacy of this approach, with results so far looking promising (NCT02022696, NCT01852071).

SCID-X1

X-linked SCID is the most common form of SCID, accounting for 40–50% of SCID cases worldwide. Twenty patients with SCID-X1 lacking a HLA-identical sibling donor were treated between 1999 and 2006 in Paris and London with autologous CD34⁺ BM-derived HSC transduced with a first-generation Moloney murine leukemia virus vector expressing the γc complementary DNA, without any conditioning regimen. While the treatment resulted in the correction of the immunodeficiency with polyclonal and functional T-lymphocytes in the vast majority of patients, engraftment and correction of NK and B cells was lower due to the decision not to administer any preparatory regimen to the patients. Five patients treated in this trial developed T-cell leukemia between 2 and 5 years after GT due to insertional oncogenesis caused by aberrant expression of LMO2 or cyclin D2 oncogenes induced by the integration of the γc RV in the proximity of the gene regulatory regions.^22
–24

With the aim of improving safety, a SIN γ-RV with deleted Moloney murine leukemia virus LTR U3 enhancer was developed and employed to treat nine patients lacking a HLA-identical sibling in parallel phase I/II trials in Europe and the United States without preparative conditioning.²⁵ Immune reconstitution occurred in seven patients (one died) and was comparable to that of the previous trial; integration analysis showed a polyclonal profile with a reduced number of clones near known lymphoid proto-oncogenes and genes. However, also in this case, absent or very low engraftment of B cells and HSC was obtained.^25,26

Another approach based on a SIN LV expressing a γc gene has been recently developed and employed after the administration of a non-myeloablative conditioning regimen based on i.v. busulfan (6 mg/kg) with the aim of improving engraftment of gene-corrected cells.^27,28 Results of five SCID-X1 patients with persistent immune dysfunction despite haploidentical HSCT and follow-up data from two older patients have been very recently published and confirm the selective expansion of gene-marked T, NK, and B cells, which is associated with sustained restoration of humoral responses to immunization and clinical improvement after autologous HSC-GT with nonmyeloablative busulfan.²⁸

Altogether, these data indicate that reduced-intensity conditioning based on busulfan is safe and allows for stable multilineage engraftment of gene-corrected HSPC in SCID patients.^7,8,27,28

WAS

While at present allogeneic HSCT from a HLA-identical sibling donor remains the treatment of choice for WAS patients, HSC-GT with WAS gene-corrected cells represents a valid alternative for patients lacking a suitable donor or in those aged >5 years. A Phase I/II study was conducted in Germany and enrolled WAS patients treated with WASp expressing LTR-driven γ-RV using peripheral blood stem cells (PBSC) following a reduced-intensity conditioning regimen based on i.v. busulfan (8 mg/kg).^29,30 The procedure lead to stable engraftment of gene-corrected cells in multiple lineages and restoration of WASp expression with a selective proliferative advantage of corrected lymphoid cells over myeloid lineages. Unfortunately, the analysis of vector common insertion sites revealed a marked clustering between patients, which translated into the development of hematological malignancies in the majority of treated patients.³⁰

At the authors' institute, an alternative approach based on a SIN-LV in which the own WAS promoter was chosen to drive the WASp expression and infusion of autologous gene-corrected CD34⁺ cells was combined with the reduced-intensity regimen. The preparation was based on i.v. busulfan (range 0.8–1.2 mg/kg/dose × eight doses over 2 days, targeted for AUC), fludarabine (30 mg/m²/day for 2 days), and single-dose rituximab (375 mg/m²).² This regimen was designed to: (i) create space in the BM (busulfan) for the gene-corrected stem cells, exploiting the selective advantage for corrected lymphoid cells and platelets; (ii) deplete the lymphoid compartment of potentially autoreactive lymphocytes, which may be present in WAS patients displaying autoimmune/autoinflammatory manifestations at time of HSC-GT (fludarabine and rituximab); and (iii) prevent lymphoproliferative disorders due to EBV reactivation (rituximab) in such immunocompromised patients while favoring the establishment of a pool of corrected naïve T cells in the periphery. After the procedure, all patients showed multilineage engraftment of vector-transduced cells both in the BM and PB, stable levels of WASp expression, immunological function restoration, and increased platelet counts, as compared with the pre-GT situation. Gene-corrected HSC represented 20–50% of the stem-cell compartment in the BM, and this proportion increased in lymphoid cells and platelets in line with a selective advantage for WASP-expressing cells. Patients showed a reduction in severity and frequency of infections and bleeding episodes, and an absence of autoimmune manifestations. In terms of safety, analysis of the LV insertion profile in vivo showed polyclonality and no clonal expansion.^2,31 LV genome marking allowed the number of tracked clones to be followed, which homogeneously contributed to hematopoiesis in a highly polyclonal fashion.³¹

A more intense conditioning regimen was adopted for a study conducted at Necker Hospital and Great Ormond Street Hospital using a similar SIN LV encoding WASp. Gene-corrected HSC were administered to seven children with severe WAS lacking a suitable related or unrelated HLA-matched donor following preparation with i.v. busulfan (4 mg/kg/day for 3 days) and fludarabine (40 mg/m²/day for 3 days; rituximab and/or alemtuzumab was used if autoimmune disease was present). Six of the seven patients are alive and show sustained clinical benefit; one patient died due to pre-existing infection. In all six surviving children, cutaneous eczema and predisposition to develop infections resolved, whereas autoimmune manifestations ameliorated in five out of five patients. All surviving patients became transfusion independent and showed persistent high-level engraftment of gene-corrected cells. A correlation between the level of myeloid engraftment and platelet values was found with the dose of gene-corrected cells received by the patients, and no vector-related adverse events were registered.¹¹

Collectively, these results demonstrate that a reduced-intensity conditioning based on an alkylating agent (busulfan) and an immunosuppressive drug (fludarabine), in combination with B-cell depletion (especially in the presence of autoimmune manifestations), allows for sustained clinical benefit and stable multilineage engraftment of gene-corrected HSPC in WAS patients.

Neurodegenerative metabolic disorders: Metachromatic leukodystrophy and adrenoleukodystrophy

LSD are a group of inherited metabolic disorders characterized by disruption of normal lysosomal function and consequent accumulation of incompletely degraded molecules. MLD is an autosomal recessive LSD determined by mutations in the gene that encodes for the arylsulfatase A (ARSA) enzyme and translates into insufficiency of the enzyme and accumulation of sulfatides in the central nervous system (CNS) and peripheral nervous system (PNS). HLA-matched allogeneic HSCT may defer disease onset or slow down the development of new CNS manifestations if performed at a pre-symptomatic stage or before the occurrence of major symptoms. However, patients receiving HSCT manifest severe peripheral neuropathy.^32,33

A Phase I/II trial of HSC-GT for MLD patients has been conducted at the authors' institute by employing a myeloablative regimen based on i.v. busulfan. The aim of this strategy was to achieve optimal HSC engraftment and transgene overexpression in multiple lineages and to favor replacement of endogenous microglia progenitors with gene-corrected cells, based on preclinical data demonstrating that busulfan allows the blood–brain barrier (BBB) to be crossed by HSPC and provides a favored setting for microglia reconstitution.³⁴ The modified cells are expected to release functional lysosomal enzyme for the cross-correction of other resident cells in the CNS, and eventually lead to restored clearance of sulfatide storage. The myeloablative dose of busulfan was achieved by targeting the actual drug exposure to a predefined AUC, identified on the basis of previous experimental data derived from allo-HSCT. Results from the first nine patients treated at the pre or early symptomatic stage have been recently published.³⁵

The median dose of busulfan target in this first group of patients was 14 mg/kg, administered over 4 days in 14 doses. High-level and persistent engraftment of gene-corrected HSC was found, with median levels on clonogenic progenitors of about 60%. After treatment, ARSA activity could be measured both in hematopoietic cells and in the cerebrospinal fluid in all treated children, whereas a reduction in the accumulation of sulfatides was found in the peripheral nerves. Prevention of disease onset was observed in pre-symptomatic late infantile patients, as compared with untreated patients. In some patients, treatment resulted in the prevention of CNS demyelination and in improvements of PNS neuropathy with signs of remyelination. Multivariate analysis showed that parameters determining engraftment included the number of days of absolute neutropenia and the vector copy number (VCN) of the infused product. Comparison with the LV WAS studies indicates that the use of a more intense conditioning regimen resulted in higher and stable LV engraftment, as also assessed by the presence of BM progenitor cells carrying integrations, which were detected persistently (self-renewal) and in multiple mature lineages (multilineage capacity).^10,35

X-linked ALD is a severe genetic demyelinating disease caused by a deficiency in ALD protein, an adenosine triphosphate-binding cassette transporter encoded by the ABCD1 gene. Allogeneic HSCT can arrest the progression of cerebral demyelinating lesions if performed at an early stage of the disease. HSC-GT represents an alternative and potentially more effective strategy. A LV-based HSC-GT approach was employed for the treatment of two X-ALD children who manifested with progressive cerebral demyelination and did not identify an HLA-matched donor. After G-CSF stimulation, autologous CD34⁺ PBSC were collected, genetically corrected ex vivo with the LV vector encoding wild-type ABCD1 cDNA, and reinfused into the patients after a fully myeloablative conditioning with busulfan and cyclophosphamide. In the 3 years following treatment, the two patients showed polyclonal engraftment, with 7–14% of granulocytes, monocytes, and T and B lymphocytes exhibiting the missing protein. There were no signs of vector-related clonal dominance in the hematopoietic cells. Cerebral demyelination was arrested in both treated patients. To determine the efficacy of this approach, a longer observation of these two children is necessary. Moreover, other patients treated with HSC-GT are being evaluated.^3,36

These data indicate that myeloablative doses of alkylating agents are necessary when high levels of engraftment are required in diseases, such as LSD, in which a natural selective advantage for the gene corrected is not present and crossing of the BBB by hematopoietic cells is required.

β-Hemoglobinopathies

Sickle cell disease (SCD) and βThal are the most common monogenic disorders worldwide, and are caused by mutations in the β-globin gene that result in either abnormal hemoglobin structure (SCD) or reduced/absent production of β-globin chains (βThal). HSC-GT by ex-vivo LV transfer of a therapeutic β-globin gene has been evaluated in human clinical trials in recent years both in Europe and in the United States. Proof of principle of efficacy and safety was first obtained in the LG001 Phase I/II clinical trial conducted in France with the LV vector HPV569.⁴ Three subjects with βThal were treated, and one of them became transfusion independent. After the treatment of these first three subjects, the HPV569 vector was replaced with the improved BB305 vector, and a new clinical trial (HGB-205), which is currently ongoing in France, was started to treat seven patients affected by either SCD or βThal. In addition to the characteristics of the vector, key parameters for the success of the therapeutic approach are the dose and the VCN in CD34⁺ cells and an efficient myeloablative conditioning regimen. Patients enrolled in these two trials received i.v. busulfan at a starting dose of 3.2 mg/kg/day for 4 days with pharmacokinetics (PK) analysis by which dose and schedule of busulfan were monitored daily and adjusted based on plasma levels in order to maintain myeloablation (AUC exposure of 4,500–5,000 μM/min/day for a daily dosing regimen over 4 days). In the HGB-205 clinical trial, four subjects with βThal and one with SCD have been treated, and preliminary results have been reported at scientific meetings. The four βThal patients have so far become transfusion independent, and the SCD patient shows early clinical benefit.^37,38

Other patients affected by βThal and SCD are being treated in two separate international and U.S. clinical trials (HGB-204 and HGB-206) after a myeloablative conditioning based on busulfan. Results will hopefully become available soon.

At the authors' institute, a Phase I/II HSC-GT trial has recently started for adult and pediatric βThal patients in which a novel regimen has been adopted with a reduced intensity and toxicity conditioning including treosulfan (42 g/m²) and thiotepa (6–8 mg/kg; NCT02453477). The use of treosulfan, an alkylating agent with reduced toxicity when compared with busulfan, has already been reported in cohorts of thalassemic pediatric and adult patients undergoing allogeneic HSCT with a favorable safety profile and engraftment kinetic.³⁹ Treosulfan also has a potent effect in ablating extramedullary hematopoiesis, resulting in reduced hepato-splenomegaly and, therefore, expected better marrow engraftment. Data on PK of treosulfan are being collected and will be correlated with engraftment and efficacy, as there are also currently limited data in allogeneic HSCT.

Novel Approaches to Optimize the Outcome of HSC-GT

Novel conditioning regimens

While HSCT is a well-established therapeutic option for patients affected by both malignant and non-malignant disorders, its application is limited by the associated substantial morbidity, which is partly due to the toxicity of the preparative regimen, especially in patients with non-malignant disorders and pre-existing organ toxicity.^13,14 Novel strategies under investigation are aimed at reducing regimen-related toxicity, while remaining sufficiently myelosuppressive and immunoablative to allow for sustained donor engraftment and chimerism. Monoclonal antibodies directed against cell surface antigens expressed by all blood cells, such as CD45, or by subpopulations enriched for HSC, such as c-KIT, constitute potential targets to deplete resident HSC and have demonstrated preliminary efficacy in murine models.^40,41 Anti-CD45 antibodies have already been employed successfully as a preparative regimen, in combination with immune suppressive drugs, in a clinical trial of allogeneic HSCT for high-risk patients with primary immunodeficiency.⁴² In a very recent study, Palchaudhuri et al. ⁴³ developed a novel approach in which saporin, a ricin family toxin able to halt protein synthesis, was coupled to an anti-CD45 antibody and was administered to wild-type mice as a conditioning regimen before HSCT. The treatment was nearly as effective as total body irradiation (TBI) at establishing long-term stable donor chimerism, but was associated with less toxicity, as shown by the faster recovery of myeloid and B and T cells in the blood, and better preservation of BM and thymus architecture. HSC-GT might represent a unique clinical context in which this new targeted conditioning strategy can be tested and successfully developed, given that autologous cells do not need to overcome immune barriers in the recipient and that a mixed chimerism might be sufficient for therapeutic benefit in many diseases that are candidates for GT.

Choice of stem-cell source and administration route

While BM remains the standard HSPC source for GT approaches in the majority of current protocols, mobilized PBSC are emerging as a preferential source, especially in children >1–2 years of age and in adults. The BM harvest for clinical GT (usually 15–30 mL/kg of a patient's body weight) allows large numbers of HSPC to be collected that are purified for CD34 and LV-transduced. In other trials (e.g., the HSC-GT study currently enrolling at the authors' institute), peripheral blood CD34⁺ cells after mobilization with G-CSF or G-CSF + Plerixafor are being employed with the double aim of collecting high numbers of CD34⁺ cells and securing patient hematological recovery. Moreover, cryopreservation of the mobilized medicinal product will facilitate future applicability in the context of multicenter trials.

Umbilical cord blood (UCB) may become an alternative source for HSC-GT in newborns/toddlers thanks to the implementation of neonatal screening, which allows for early diagnosis and potentially timely treatment of congenital disorders. Neonatal screening programs have already been developed in the United States for SCID, and some LSD and pilot studies are also ongoing in selected regions of Italy (Tuscany) for ADA-SCID and mucopolysaccharidosis type I Hurler (MPS-IH).^44,45 Indeed, UCB has become the preferential HSC source for allogeneic HSCT in specific metabolic disorders, such as MPS-IH, thanks to easy availability, high donor chimerism, and good enzyme recovery.⁴⁶ The biological characteristics of UCB-derived HSC could also be favorably employed in HSC-GT in a neonatal setting thanks to the dedicated storage of UCB units and the future availability of neonatal screening.

While the i.v. route has been historically used in allogeneic HSCT and in previous HSC-GT studies, the intrabone injection of the graft has become of great interest following the studies conducted in the context of allogeneic HSCT employing UCB-derived cells to favor engraftment and reduce cell loss.^47,48 The intraosseous infusion is being adopted in our βThal HSC-GT protocol as an innovative route of gene-modified cell administration with the aim of both speeding engraftment and minimizing first-pass i.v. filter (NCT02453477). This route also has the advantage of balancing the potential cell loss due to the freeze–thaw of transduced CD34⁺ cells.

Conclusions and Future Directions

GT with ex vivo genetically modified HSC represents a valid alternative to allogeneic HSCT for a number of inherited disorders, allowing each patient to be his/her own donor and reducing the risks of immune-mediated complications. Administration of a conditioning regimen based on chemotherapy is currently required in order to achieve stable engraftment of gene-corrected HSC.

In the context of SCID or certain primary immunodeficiencies, low-dose chemotherapy is sufficient to create a mixed chimerism, since gene-corrected cells are endowed with a selective advantage. In terms of metabolic disorders and β-hemoglobinopathies, myeloablative doses are necessary to achieve high levels of engraftment when the immune system is competent.

While the actual chemotherapy exposure (AUC) was measured in most cases in the studies presented, showing variability among patients, the AUC was only targeted to a predefined range in selected cases.^2,35 For future studies, it will be important to predefine a target AUC to reduce variability and to allow a thorough comparison between different GT studies and/or with allogeneic transplantation.

The ideal conditioning regimen for patients with non-malignant disorders who are candidates for HSC-GT should be tailored with the aim of mitigating toxicity, especially when pre-existing organ damage is present, while reaching an efficacious therapeutic level of transgene expression. The development of novel strategies, for example based on monoclonal antibodies selectively depleting blood cells in the BM, might open new frontiers to enlarge the application of HSC-GT while sparing toxicity.

Footnotes

Acknowledgments

A.A. and M.E.B. are recipients of Telethon Foundations grants. A.A. is a recipient of grants from the FP7 Project CELL-PID HEALTH-2010-261387 and E-Rare-3 JTC 2015, (EURO-CID), Ministry of Health (NET-2011-02350069). We thank all the scientists and clinicians from SR-TIGET and San Raffaele Hospital who have contributed to the design of the gene therapy studies and conditioning protocol in the past 15 years and in particular Dr. Roncarolo, Bordignon, Naldini, Biffi, Chiesa, Ferrua, Cicalese, Ferrari, Ciceri, and Marktel.

Author Disclosure

A.A. is the PI of the SR-TIGET clinical trials for ADA-SCID, WAS, and MLD initially sponsored by Telethon and currently sponsored by GSK following inlicensing of the gene therapy. M.E.B. declares no competing financial interests.

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