Twenty-Five Years of Gene Therapy for ADA-SCID: from Bubble Babies to an Approved Drug

Twenty-Five Years of Gene Therapy for ADA-SCID: From Bubble Babies to an Approved Drug

Francesca Ferrua1,2 and Alessandro Aiuti1,2,* 1San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), Pediatric Immunohematology and Bone Marrow Transplantation Unit, San Raffaele Scientiﬁc Institute, Milan, Italy;2Vita-Salute San Raffaele University, Milan, Italy.

Twenty-five years have passed since first attempts of gene therapy (GT) in children affected by severe combined immunodeficiency (SCID) due to adenosine deaminase (ADA) defect, also known by the general public as bubble babies. ADA-SCID is fatal early in life if untreated. Unconditioned hematopoietic stem cell (HSC) transplant from matched sibling donor represents a curative treatment but is available for few patients. Enzyme replacement therapy can be life-saving, but its chronic use has many drawbacks. This review summarizes the history of ADA-SCID GT over the last 25 years, starting from first pioneering studies in the early 1990s using gamma-retroviral vectors, based on multiple infusions of genetically corrected autologous peripheral blood lymphocytes. HSC represented the ideal target for gene correction to guarantee production of engineered multi-lineage progeny, but it required a decade to achieve therapeutic benefit with this approach. Introduction of low-intensity conditioning represented a crucial step in achieving stable gene-corrected HSC engraftment and therapeutic levels of ADA-expressing cells. Recent clinical trials demonstrated that gamma-retroviral GT for ADA-SCID has a favorable safety profile and is effective in restoring normal purine metabolism and immune functions in patients >13 years after treatment. No abnormal clonal proliferation or leukemia development have been observed in >40 patients treated experimentally in five different centers worldwide. In 2016, the medicinal product StrimvelisÔ received marketing approval in Europe for patients affected by ADA-SCID without a suitable human leukocyte antigen–matched related donor. Positive safety and efficacy results have been obtained in GT clinical trials using lentiviral vectors encoding ADA. The results obtained in last 25 years in ADA-SCID GT development fundamentally contributed to improve patients’ prognosis, together with earlier diagnosis thanks to newborn screening. These advances open the way to further clinical development of GT as treatment for broader applications, from inherited diseases to cancer.

Keywords: gene therapy, ADA-SCID, clinical trial, primary immunodeﬁciency, adenosine deaminase

INTRODUCTION was discovered that the enzyme was absent in

Downloaded by UNIV STUDI LA SAPIENZA from www.liebertpub.com at 12/12/18. For personal use only. ADENOSINE DEAMINASE (ADA) deficiency is a rare, several immunodeficient patients being considered autosomal recessive severe combined immunode- for bone-marrow transplantation (BMT). This ficiency (SCID; OMIM#102700).1 ADA-SCID is finding showed the crucial role of the enzyme in- caused by mutations in the ADA gene and repre- volved in purine metabolism in the development of sents the cause of 10–15% of all SCIDs, with an immune system, which was later confirmed by overall prevalence in Europe ranging between studies in murine models of immunodeficiency.2 1:375,000 and 1:660,000 live births. In 1972, it be- The main features of the disease are impaired dif- came the first immunodeficiency for which a spe- ferentiation and functions of T, B, and natural cific molecular defect was identified, at both the killer (NK) cells, predisposing patients to develop genetic and biochemical level. The importance of severe, opportunistic, and recurrent infections and ADA for immune function was revealed when it failure to thrive.1 Similarly to other SCIDs, ADA-

*Correspondence: Prof. Alessandro Aiuti, San Raffaele Telethon Institute for Gene Therapy (SR-Tiget), Pediatric Immunohematology and Bone Marrow Transplantation Unit, San Raffaele Scientiﬁc Institute, Milan, Italy, Via Olgettina, 60, 20132, Milan, Italy. E-mail: [email protected]

972 HUMAN GENE THERAPY, VOLUME 28 NUMBER 11 DOI: 10.1089/hum.2017.175 j ª 2017 by Mary Ann Liebert, Inc. TWENTY-FIVE YEARS OF GENE THERAPY FOR ADA-SCID 973

SCID is a fatal disease, usually leading to death in vent chronic complications and can amount to ex- the first year of life, if not treated. Among SCIDs, tremely high costs if maintained long term. A cohort it is one of the most difficult to handle clinically due of patients (from infant age to young adults) was to the non-immunological abnormalities (mainly reported to continue PEG-ADA as chronic treat- neurodevelopmental, behavioral,3 skeletal, and ment in the absence of a matched donor.1,11 hepatic), which occur as a consequence of the sys- Because of the limitations of these therapies, temic metabolic defect.4 However, in the last years, gene correction of autologous hematopoietic stem/ the prognosis for ADA-SCID infants has improved progenitor cells (HSPC) has been explored as a greatly. potentially curative treatment for ADA-deficient SCID in the past 25 years and has now become an approved treatment in Europe.12 AVAILABLE TREATMENTS FOR ADA-SCID At present, treatment options for ADA-SCID RATIONALE FOR GT IN ADA-SCID include allogeneic hematopoietic stem-cell trans- ADA-SCID was the first inherited disease in plantation (HSCT), enzyme replacement therapy which ex vivo gene transfer in peripheral blood (ERT), or autologous ex vivo gene therapy (GT).5,6 lymphocytes (PBL) or HSPC was performed. Some HSCT from a human leukocyte antigen (HLA)- evidence supported the use of autologous gene- matched related sibling or family donor without corrected cells for ADA-SCID.13 First, ADA gene is conditioning is recommended as first-line curative a ubiquitously expressed housekeeping gene, with treatment, being associated with good survival and no need of fine regulation and therefore suitable to effective immune reconstitution. Current evidence6,7 be expressed under viral promoters as those pres- supports HSCT as early as possible after diagnosis in ent in gamma-retroviral vectors (RV). Moreover, order to prevent the development of infections and to ADA cDNA could be easily cloned and expressed by reduce systemic adenine metabolite-related toxicity, such vectors, being relatively small (1.5 kb). Sec- but this therapeutic option is only available for <25% ond, relatively low enzymatic levels allow normal of patients. immune functions in healthy individuals carry- Allogeneic HSCT from alternative donors (mat- ing a normal ADA gene. Third, normal or gene- ched unrelated, haploidentical) is still associated corrected cells have a strong selective survival with an increased risk of morbidity and mortality, advantage over ADA-deficient cells because they with survival falling significantly as HLA match- can more easily eliminate toxic metabolites from ing decreases, ranging from 67% to 43%, albeit inside the cell. This was demonstrated both in improving in most recent years.8,9 New approaches HSCT and in preclinical GT models.14 Thus, even have been recently developed to ameliorate sur- a relatively low amount of correction and/or of en- vival and immune reconstitution in these trans- grafted HSPC might have resulted in successful plant settings, such as improved methods for graft therapy. In addition, a decline in revertant T lym- manipulation (e.g., selective a/b T-cell depletion10) phocytes during ERT was observed in a delayed- and improved supportive measures, but no specific onset patient with mosaicism for a ‘‘second-site outcome data for ADA-SCID patients have been suppressor’’ of a splicing mutation,15 stressing the Downloaded by UNIV STUDI LA SAPIENZA from www.liebertpub.com at 12/12/18. For personal use only. reported to date. importance of the discontinuation of ERT in GT Newly diagnosed patients are usually treated preparatory phase in order not to compromise the with weekly intramuscular injections of ERT with selective advantage of genetically corrected cells. exogenous polyethylene-glycol-conjugated bovine Finally, the target tissue, hematopoietic cells, is ADA (PEG-ADA). PEG-ADA has proved a very relatively easy to obtain and to culture in vitro useful way to stabilize patients as a bridging ther- before reinfusion in the patients. apy to promote immune recovery before a definitive procedure can be implemented. This treatment is licensed in the United States but not in Europe ADVANTAGES AND DISADVANTAGES where it is provided under special provisions. While OF GT OVER OTHER TREATMENTS PEG-ADA is highly effective in the short term, GT has several potential advantages over other rescuing the immunological defects through extra- existing treatment options, and is suitable for pa- cellular detoxification, these effects are often partial tients who do not have an available HLA-identical and poorly sustained over time. ERT efficacy tends sibling donor.13 With respect to HSCT, autologous to decrease over time, with an often inadequate transplantation of gene-corrected HSPC is poten- long-term immune reconstitution11 and survival tially applicable to all patients and immediately rate of 78% at 20 years.5 Moreover, it does not pre- available, with no delay due to donor search. 974 FERRUA AND AIUTI

Moreover, using autologous gene-corrected HSPC transduction procedures, and clinical protocols. abolishes the risk of rejection and graft-versus- Patients who were eligible for GT were chosen host disease due to HLA mismatches or minor among those lacking a matched familiar donor or antigen incompatibility. Finally, use of immuno- displaying inadequate immune reconstitution af- suppressive prophylaxes or high-dose conditioning ter ERT. All patients enrolled in these studies regimens are not required in the GT setting, continued to receive PEG-ADA in order to avoid the thereby decreasing treatment-related organ toxic- risk of a further deterioration of immune function, ity, prolonged myelosuppression, and increased and did not receive any preparatory conditioning infection risk. regimen. GT has also a great advantage over ERT. A single injection of gene-corrected HSPC may be suffi- PBL GT cient to treat a patient lifelong, avoiding the need Based on the observation that patients receiving for chronic replacement therapy with its extremely BMT achieved immune reconstitution by sole en- high costs and burden on patients’ quality of life. graftment of donor T cells, PBL GT was used ini- Moreover, there is evidence from studies in mice tially for the treatment of ADA deficiency. Nine and HSCT that endogenous ADA is more effective ADA-SCID patients have been reported to be than exogenously administered ADA.14 However, treated with multiple infusions of gene-corrected GT can also present some peculiar limitations and PBL in three different clinical trials.23–27 Auto- risks. Being an autologous procedure, low au- logous T lymphocytes were transduced with tologous bone marrow (BM) HSPC content or gamma-RV derived from Moloney Murine Leuke- pre-existing chromosomal alterations in BM pro- mia Virus (MMLV), veiculating ADA gene cDNA. genitors may lead to the exclusion of patients from The marker gene coding for Neomycin resistance, the procedure.16 Moreover, the efficiency of gene NeoR, was inserted inside the vector for identifi- transfer and correction represents an important cation and selection of transduced cells. The limiting factor for the procedure outcome. Finally, available follow-up reported in the literature ex- a potential serious adverse event related to gene tend to >15 years in the first-treated children, transfer can be represented by insertional muta- with no adverse events or toxicity observed. The genesis caused by RV ‘‘turning on’’ cellular proto- majority of patients showed improvements in im- oncogenes adjacent to where they integrate into mune function, and transduced T cells persisted the target cell chromosomes, leading to the ap- >12 years after infusions of gene-corrected lym- pearance of clonal expansion or even leukemic phocytes were discontinued, including functional proliferation. This event is known to have occurred decade-long surviving genetically engineered T in some patients treated with RV GT for X-linked memory stem cells,28,29 which were demonstrated SCID,17 Wiskott–Aldrich syndrome (WAS),18 and to be able to persist in circulation long term. This chronic granulomatous disease (CGD)19 who de- demonstrated the safety and the therapeutic po- veloped acute leukemia or myelodysplasia after tential of PBL GT with the sustained engraftment the procedure. So far, the cumulative experience of of ADA-expressing T cells.27 However, the real five different centers performing various GT for therapeutic impact was difficult to evaluate be-

Downloaded by UNIV STUDI LA SAPIENZA from www.liebertpub.com at 12/12/18. For personal use only. ADA-SCID did not reveal the occurrence of such cause all subjects continued to receive ERT, which complications, indicating that it has a favorable might have impaired the selective growth advan- risk–benefit profile. tage for gene-corrected cells. Indeed, in one patient in whom PEG-ADA was discontinued at San Raf- faele Telethon Institute for Gene Therapy (SR- GT PILOT STUDIES Tiget) in Milan (Italy), gene-corrected T cells pro- More than 25 years ago, fundamental preclinical gressively replaced the defective cells, resulting in proof-of-concept about GT feasibility and adequate restoration of normal T cell function and antibody safety profile was provided by the pioneering work responses to neoantigen. However, infusion of T of researchers in Italy and in the United States. In cells was not sufficient to allow full correction of the the early 1990s, several GT pilot studies were ini- metabolic defect,26 likely because of the limited tiated using gamma-RV that were previously mass of detoxifying cells. evaluated in murine14,20 and primate21 models as well as in ADA patients’ BM cells,22 in which ADA HSPC GT cDNA transgene expression was regulated by ret- A parallel strategy was developed aimed at in- roviral long terminal repeats (LTR). These studies fusing BM or umbilical cord blood (UCB) progenitors differed considerably in terms of target cells, to patients without any preparative conditioning. TWENTY-FIVE YEARS OF GENE THERAPY FOR ADA-SCID 975

Autologous HSPC have been always considered the patients continued to receive, supporting the need optimal target cells for long-term, full correction of for the implementation of new and improved clin- ADA-SCID defect. The rationale for GT in HSPC ical trials. was based on the assumption that the engineered HSPC would stably engraft, self-renew, and differentiate in multiple lineages, including mature lym- GT WITH AUTOLOGOUS BM CD34+ CELLS phocytes. These newly generated gene-corrected FOLLOWING NON-MYELOABLATIVE lymphocytes should produce adequate levels of en- CONDITIONING: THE SR-TIGET dogenous ADA enzyme to sustain their growth and CLINICAL PROTOCOL function, independently from exogenous sources. Subsequent studies using autologous CD34+ On the other hand, production of ADA in all hema- cells transduced with gamma-RV introduced some topoietic lineages should contribute to an overall key changes, including PEG-ADA withdrawal improvement in systemic detoxification. prior to BM harvest to enhance the selective ad- Eight patients have been treated with en- vantage of corrected cells and, most importantly, gineered HSPC in the first clinical trials24,30,31 the use of reduced-intensity conditioning with the after collection of BM- or UCB-derived cells fol- alkylating agent busulfan to promote engraftment lowed by transduction and infusion without any of HSPC. myeloablative conditioning. In particular, the In particular, a breakthrough was achieved in patients enrolled at SR-Tiget received multiple 2000, when these changes, combined to improved infusions of BM progenitors and peripheral T transduction protocol for BM HSPC, were intro- lymphocytes, transduced with similar vectors but duced in two successful pilot studies that were distinguished by a different marker gene.24,32 This started at Hadassah Hospital (Jerusalem, Israel) first study reported the progressive expansion of and SR-Tiget (Milan, Italy). Notably, the trans- circulating corrected T cells, derived from the in- duction protocol was optimized by the use of a ret- fusion of engineered PBL or of BM precursors dif- roviral supernatant produced in conditions adapted ferentiating in lymphoid lineage. Moreover, it for human CD34+ cells, and cells were transduced in showed that ex vivo transduced BM progenitors the presence of retronectin and of a culture medium were able to differentiate in vivo into multiple lin- containing a cytokine cocktail (including FLT3- eages, even if not achieving therapeutic levels of ligand, stem-cell factor, thrombopoietin, and IL-3) ADA expression.24 Two years after the procedure, that preserved the ability to engraft efficiently into BM-derived corrected cells were present at a low immunodeficient mice and give rise to B and T cell level and thereafter were no longer detected. progeny.35–37 In a study conducted at the Children’s Hospital The use of non-myeloablative conditioning regi- of Los Angeles in three ADA-SCID patients, CD34+ men with busulfan in advance of GT was intro- progenitor cells were collected at birth from the duced to favor corrected cells’ engraftment, making umbilical cord of newborn babies, transduced, and space for the transduced progenitors in patients’ re-infused after 4 days. In these patients, a 4-year BM.37 The rationale for its adoption originated follow-up indicated the presence and the expres- from gene-marking studies in animal models and 31,33 Downloaded by UNIV STUDI LA SAPIENZA from www.liebertpub.com at 12/12/18. For personal use only. sion of ADA in BM and peripheral blood (PB). from transplantation experiences in the field of Furthermore, lymphocytes derived from gene- hematological pediatric disorders. In particular, corrected cord blood HSPC accumulated over time, busulfan, used alone, was chosen as chemother- still being detectable at least 8 years after the apeutical agent based on its wide use in pediatric procedure,34 suggesting T cell lineage selective settings, including SCID patients, and its effects on advantage. However, due to the fact that no con- primitive HSPC. The administered dose represented ditioning was administered, the level of engraft- approximately 25% of the total dose usually used ment of transduced cells was not sufficient to in totally myeloablative protocols. Compared to the produce therapeutic ADA expression.33 Moreover, combination of cytoreduction or myeloablative che- the procedure was performed with concomitant motherapy and pre-/post-HSCT immunosuppres- PEG-ADA administration. sion, this regimen showed low toxicity in the post-GT Together, the results of these early studies clinical course. Initially, two ADA-SCID patients, showed the feasibility and safety rationale of BM who lacked an HLA-identical sibling donor and could HSPC infusion, but they resulted in low vector not access to PEG-ADA therapy, were enrolled in the marking of progenitor-derived cells with insuffi- pilot clinical trials. Since none of them received con- cient ADA production, and failed to provide clear current ERT, the efficacy of GT as single treatment clinical benefit over and above that of ERT, which could be fully assessed, exploiting at the same time 976 FERRUA AND AIUTI

the growth advantage for ADA-transduced cells. tion in severe infection rate after GT, continued Thanks to the new protocol, GT resulted in engraft- physical growth, and improved quality of life. On ment sustained by gene corrected CD34+ BM pro- the other hand, there was no indication that GT genitor cells, with multi-lineage differentiation, had an impact on incidence of neurological issues39 increase of lymphocyte counts, improvement of typically present in ADA-SCID patients.3 On the cellular and humoral responses, including antigen- whole, the safety profile was in line with what can specific responses, and a substantial reduction of be expected in an ADA-SCID population receiving toxic metabolites.37 These results represented the busulfan conditioning and undergoing immune re- first demonstration of the clinical efficacy of HSPC covery. Importantly, no serious adverse events were GT for ADA-SCID. attributable to GT, and no events indicative of myelodysplasia or leukemic transformation of transduced cell clones were reported. All but one patient had viral DEVELOPMENT OF ADA-SCID GT integrations’ insertion site within and/or near poten- AT SR-TIGET: FROM CLINICAL TRIALS tially oncogenic loci (e.g., CCND2, LMO2), but clones TO EUROPEAN UNION MARKETING with these inserts have been present for several years APPROVAL OF STRIMVELISÔ and are stable.41 An abdominal adipose tumor was Following these initial studies, results were found in one patient42 but was not related to GT. confirmed and extended in a Phase I/II pivotal Based on the positive and promising safety and clinical trial with a long-term follow-up component efficacy data collected from these 18 ADA-SCID and a compassionate use program, conducted at children treated from 2000 to 2011,39 ex vivo HSPC SR-Tiget (Milan, Italy) for a total of 18 patients GT for the treatment of ADA-SCID, based on the treated according the same strategy, including the single infusion of an ‘‘autologous CD34+ enriched pilot studies.38,39 Children with ADA-SCID lacking cell fraction containing CD34+ cells transduced a healthy HLA-identical sibling donor were en- with a gamma-RV encoding for the human ADA rolled. In addition, patients for whom ERT was not cDNA sequence,’’ was approved for licensure by the a lifelong therapeutic option or for whom PEG- European Medicines Agency (EMA) in May 2016, ADA treatment administered for at least 6 months under the commercial name of StrimvelisÔ (Glaxo- was ineffective or harmful were eligible. SmithKline [GSK]). This advanced therapy me- Preliminary results from the first 10 treated dicinal product (ATMP) is the first ex vivo HSPC patients were reported in 2009,38 showing im- GT to receive regulatory approval anywhere in the provement in immune functions, ranging from world. Its market approval in the European Union partial to full reconstitution, with adequate sys- (EU) arrived 25 years after the first pioneering GT temic detoxification in most patients. Recently, trials in ADA-SCID patients.12 This fundamental results of all 18 patients treated with this approach milestone was achieved thanks to a joint effort were reported39 for a median follow-up period of 6.9 among GSK, the Italian Telethon Foundation, and years (range 2.3–13.4 years at data cutoff of May San Raffaele Scientific Institute, at SR-Tiget, 2014) with 100% survival. The first patient treated where the medicinal product was originally devel- is alive and well and has now reached 16.5 years oped. The alliance signed by these three stake-

Downloaded by UNIV STUDI LA SAPIENZA from www.liebertpub.com at 12/12/18. For personal use only. of follow-up (P. Stepensky, pers. commun.). GT holders in 2010 was crucial to prepare for the resulted in sustained lymphoid reconstitution40 launch of this new medical product, providing the with evidence of long-term gene correction in expertise, economic resources, and infrastructure T lymphocytes, improvement of immune functions, required to complete nonclinical studies43 and restoration of functional thymopoiesis, and effec- clinical development and to establish pharmaceu- tive metabolic detoxification due to restoration of tical production. The biotech company MolMed ADA activity in the majority of patients, who re- S.p.A. produced vector and cells under Good Man- mained off ERT (15/18). Most could stop immuno- ufacturing Practices and applied its know-how in globulin replacement therapy, showing ability to product development to reach robustness and suit- mount specific antibody response after vaccination ability for commercial supply. Despite the complex- and/or infections (e.g., chickenpox). Multi-lineage ity and the many challenges faced by such a highly engraftment of gene-corrected cells stably per- innovative product, EU marketing was approved by sisted throughout long-term follow-up in the ma- the EU Commission within 12 months. The Italian jority of patients, indicating that, although at low Medicines Agency (AIFA) approved StrimvelisÔ levels, correction of multipotent stem cells was pricing and reimbursement in Italy in <2months achieved. The treatment provided clinical benefit through an accelerated procedure. StrimvelisÔ is to patients, with significant and sustained reduc- now available for ADA-SCID patients lacking a TWENTY-FIVE YEARS OF GENE THERAPY FOR ADA-SCID 977

suitable HLA-matched related donor and is cur- were reported in 201145 (all but one received con- rently administered only at San Raffaele Hospital ditioning with melphalan), showing engraftment of in Milan due to the short product shelf life. The first gene-corrected cells resulting in immune reconsti- patient was treated with commercial StrimvelisÔ in tution in four subjects. All showed effective meta- March 2017. Long-term monitoring after GT (>10–15 bolic detoxification and remained infection-free. years) will be performed to follow up patients treated None developed any leukemic side effect. with the experimental and approved product. GSK is Another clinical trial was conducted in the Uni- implementing a prospective long-term observational ted States at the National Institutes of Health and study according to EMA recommendations in order Children’s Hospital of Los Angeles.46 Ten ADA- to monitor the long-term risks of insertional muta- SCID patients were enrolled, and two slightly dif- genesis, oncogenesis, immunogenicity, and hepatic ferent RV were used to transduce CD34+ cells. toxicity, as well as the persistence of efficacy of ADA- Four subjects were treated with no myelosuppres- SCID GT over time. sion and continued to receive ERT throughout the Despite being based on the first generation procedure. Only transient, low levels of gene of vectors for HSPC GT, the safety and efficacy marking were detected in two older patients who track record of StrimvelisÔ represents a model for did not show sustained improvement in immuno- ATMP development, from early clinical experi- logical parameters during the follow-up, while mentation to drug registration and marketing some gene marking persisted in two younger pa- approval. The relevant results of this process go tients. On the contrary, six additional patients well beyond the benefit of ADA-SCID patients withdrew ERT and received low-dose busulfan be- and include development of strategies to support fore GT. This approach led to improved immuno- ATMP quality assessment, specifications, and logical and metabolic outcome in three patients manufacturing, as well as its administration to who remained off ERT. Two subjects restarted ERT patients in the hospital, and, finally but not less due to poor gene marking and immune reconsti- notably, the policies adopted for establishing its tution. Another patient experienced a prolonged cost and reimbursement. pancytopenia16 following busulfan conditioning, which was related to a pre-existing cytogenetic abnormality, indicating an important limitation to GT WITH AUTOLOGOUS BM CD34+ CELLS be considered for autologous HSPC gene transfer. FOLLOWING NON-MYELOABLATIVE This subject underwent allogeneic HSCT 8 months CONDITIONING: POSITIVE RESULTS after GT. Subsequently, the clinical safety and IN OTHER CLINICAL TRIALS therapeutic efficacy of this last GT approach were The positive outcome and the key role of non- evaluated in a Phase II study performed in the myeloablative conditioning in favoring multi- same centers,47 enrolling 10 ADA-SCID patients lineage engraftment of gene-corrected HSPC and between 2009 and 2012. All subjects, except for an achieving therapeutic benefit were also reported by adolescent, remained off ERT with normalized PB subsequent GT studies for ADA-SCID performed at mononuclear cell ADA activity, improved lympho- other centers using other gamma-RV.44–47 cyte counts, and normal proliferative responses to

Downloaded by UNIV STUDI LA SAPIENZA from www.liebertpub.com at 12/12/18. For personal use only. In 2006, researchers from Great Ormond Street mitogens. Three patients discontinued immuno- Hospital (GOSH) in London, United Kingdom, re- globulin replacement therapy. None developed ported the success of GT for ADA-SCID based on lymphoproliferative disorders or other GT-related the adoption of a different conditioning (single dose complications. These results further confirmed the of melphalan, instead of busulfan) and a different therapeutic efficacy and favorable safety profile of RV in an ADA-SCID patient44 with an inadequate HSPC GT in this disease. response to ERT, which was ceased before GT. Two Finally, the results of a Japanese clinical trial years after the procedure, metabolic and immuno- based on infusion of autologous gene-corrected BM logical corrections were maintained, with cellular CD34+ cells in two ADA-SCID children with no immunity reconstitution, recovery of thymopoiesis, cytoreductive conditioning48 strengthened the and systemic detoxification, with no adverse events importance of preparatory regimen to ensure ade- related to gene transfer. Subsequently, seven quate HSPC in vivo engraftment. In spite of PEG- additional patients were treated with the same ADA discontinuation to enhance the selective approach and are all reported to be alive. Four advantage of gene-corrected cells, delayed, partial patients remained off ERT and discontinued im- immune reconstitution and moderate detoxifica- munoglobulin replacement.6 Immune recovery and tion of purine metabolites were observed, allowing metabolic correction in the first six patients treated only temporary ERT withdrawal. 978 FERRUA AND AIUTI

Autologous ex vivo lentiviral GT for ADA-SCID ment. This is in contrast to previous experience in From first proof-of-concept in humans, vector autologous GT with gamma-RV,37,38 which fore- technology has advanced considerably in the past sees stopping ERT 1–2 weeks before HSC harvest 25 years. In particular, human immunodeficiency in order to produce a lymphopenic state/environ- virus–based lentiviruses have been adopted as the ment to drive production of de novo lymphocytes vectors of choice for new programs of HSPC gene from gene-corrected progenitors, also exploiting transfer because of their advantageous safety and their selective advantage. efficacy profile. Self-inactivating lentiviral vectors (LV) appear to have a more favorable genome in- Safety of GT for ADA-SCID sertion profile and a higher gene transfer efficiency Since 1990, >80 ADA-SCID patients have been in comparison to LTR gamma-RVs.49 Following treated with GT worldwide in different centers6 several years of preclinical studies,50,51 clinical with experimental GT based on RV or LV, and to studies using LV are currently ongoing for ADA- date no genotoxic event has been observed. The SCID.6,52 They are based on the use of a self- cumulative experience gained in the past 15 years inactivating LV with a codon-optimized human from HSPC GT with RV indicates that GT for ADA- cADA gene under the control of the short form SCID has a favorable risk–benefit profile, since no elongation factor-1a promoter (LV EFS ADA). The events of insertional oncogenesis or leukemic pro- vector showed decreased transformation poten- liferation have been observed over a long period of tial in in vitro immortalization assays compared follow-up. This is consistent with the results of to the gamma-RV used in previous British and U.S. detailed analyses of retroviral integration sites studies,51 and in the murine model allowed good in cells from ADA-SCID patients treated with immune reconstitution of treated ADA–/– mice. LTR-intact gamma-RV performed in past years, Currently, Orchard Therapeutics in partnership documenting a polyclonal pattern of vector inte- with GOSH and Children’s Hospital Los Angeles grations and T-cell repertoire,56 lack of in vivo are developing an ex vivo LV GT to restore normal skewing for potentially dangerous insertions,38 gene function in patients with ADA-SCID by con- and the absence of transcriptional perturbation of ducting a Phase I/II clinical trial to assess the cellular proto-oncogenes.57 Interestingly, Cooper safety and efficacy of a LV EFS ADA, combined et al.58 recently reported a significant correlation with low-dose busulfan conditioning. Results in the between the intensity of busulfan conditioning and first five patients treated were reported at the 16th CD34+ cell dose with clonal diversity and T-cell Biennial Meeting of the European Society for Im- repertoire in a cohort of ADA-SCID patients trea- munodeficiencies (ESID) in 2014,52 showing sig- ted with RV-GT, underscoring the relevance of cy- nificant immunological and metabolic recovery, toreductive conditioning in this type of GT setting. with improved T cell counts and normalization of The observed integration pattern included vari- PHA responses. Integration site analysis showed ous common IS near proto-oncogenes such as some expansions but no persistence of expanded LMO2, MECOM, BCL2, and CCND2, together clones. All patients were clinically well, and the with indications of mild mutagenic clonal distur- procedure was well tolerated with no significant bances that could be interpreted as molecular evi-

Downloaded by UNIV STUDI LA SAPIENZA from www.liebertpub.com at 12/12/18. For personal use only. adverse events. As of March 2016, >30 patients, dence of genotoxicity from gamma-RV GT. some of whom were identified by newborn screen- However, these findings did not lead to any gene ing, have been treated with this approach in Lon- dysregulation or frank malignant phenomena so don and Los Angeles, with promising efficacy far.41,53,56–58 Indeed, these clones remained stable results and no associated toxicity.53,54 Survival was over several years and never dominantly expanded reported to be 100% with a follow-up of 2–49 or degenerated in dysplasia. Only a benign clonal months, with evidence of immune reconstitution in dominance was reported in a patient, likely due to most patients6 in the absence of persistent clonal a gene-corrected NK cell-mediated response to dominance or insertional mutagenesis.54 Of note, chronic Epstein–Barr virus viremia.58 in these current LV GT trials, ERT is continued These findings are in sharp contrast with those through the first month after GT based on findings from GT trials for other primary immune defi- in the murine model, demonstrating equal or even ciencies, such as X-linked SCID, WAS, and CGD, improved engraftment of gene-corrected cells with presumably thanks to peculiar disease-specific 1 month of ERT after GT compared to no ERT,6,55 factors that may have contributed to this different and on the assumption that ERT in the very early outcome and toxicity profile (e.g., lower T-cell rep- follow-up after GT may help in maintaining a de- licative stress during immune reconstitution in the toxified environment that may improve engraft- thymus), suggesting that disease background may TWENTY-FIVE YEARS OF GENE THERAPY FOR ADA-SCID 979

be a critical factor in influencing leukemogenic when combined with reduced intensity condition- potential of RV GT. ing and ERT withdrawal. More than 50 ADA-SCID patients have been treated to date in various cen- Current limitations and future challenges ters with different gamma-RV GT and are all re- Results obtained so far with GT for ADA-SCID ported to be alive.6 In most patients, engraftment are very promising, but additional improvements of gene-corrected cells led to effective immunolog- could be achieved in the near future. ical and metabolic correction, without the need of Conditioning has been demonstrated to be fun- PEG-ADA or allogeneic HSCT. Importantly, to damental for appropriate engraftment of gene- date, no clinical events of abnormal clonal prolif- corrected cells.59 The low-intensity conditioning eration and/or leukemic transformation have been used in GT trials for ADA-SCID so far has been well observed, but safety monitoring will be continued tolerated by patients. However, to reduce the risk long term in all patients. of long-term sequelae of chemotherapy, novel con- The clinical management of ADA-SCID patients ditioning approaches are currently under investi- has changed, thanks to the progress in GT for ADA- gation. They are based on less harmful monoclonal SCID and the increased rate of early diagnosis due antibodies, targeted to HSPC in order to deplete to more widespread newborn screening for SCIDs. them selectively in the BM (e.g., immunotoxin Following these results, ESID and EBMT have CD45-SAP or anti-c-Kit antibodies).60 Potentially, recently updated the guidelines recommending GT these strategies could allow robust, multi-lineage as the first option to be considered for all ADA- engraftment of autologous gene-corrected HSPC SCID patients lacking an HLA-identical sibling with minimal toxicity to non-hematopoietic tis- donor,6,7 while allogeneic HSCT from unrelated or sues. This will be of particular relevance for more haploidentical donors or long-term ERT are sub- compromised patients, with pre-existing organ sequent alternative options. damage (lung, liver) or for those with a milder The advances in GT for ADA-SCID and other phenotype, for which risks of conventional condi- primary immune deficiencies have now opened the tioning regimen would not be acceptable. way to exploit HSPC GT as a treatment for a A current limitation of GT is represented by its broader list of applications, from inborn error dis- present availability in only few centers worldwide. eases to cancer. This is mainly due to the highly specialized expertise required to conduct clinical trials and the ACKNOWLEDGMENTS use of cells manufactured with a short shelf life. We thank all the researchers, clinicians, nurses, Studies are ongoing to evaluate the efficacy and the regulatory personnel, and management who have safety of a cryopreserved formulation of LV-based worked with great dedication throughout the past product (ClinicalTrials.gov #NCT02999984), with 25 years. We acknowledge in particular C. Bor- the aim of allowing patients to be treated in a dignon, G. Ferrari, M.G. Roncarolo, L. Naldini, F. higher number of clinical centers with expertise in Ciceri, M.P. Cicalese, M. Casiraghi, M. Gabaldo, HSCT on a wider geographical area. Autologous and many others who have contributed to advanc- HSPC to be corrected could be shipped to central- ing the GT field. We are grateful to the patients and

Downloaded by UNIV STUDI LA SAPIENZA from www.liebertpub.com at 12/12/18. For personal use only. ized pharmaceutical facilities for standardized their families who have participated in the clinical manufacturing, processing, and quality controls, trials. and then frozen and shipped back to the patient for re-infusion. AUTHOR DISCLOSURE A.A. is the principal investigator of the ADA- CONCLUSIONS SCID gene therapy trial sponsored by GSK. GSK A crucial milestone toward the goal of delivering acquired the license from Telethon and Ospedale new medicines for orphan diseases has recently San Raffaele, which are entitled to receive mile- been reached by GT research. Autologous trans- stone payments and royalties upon commerciali- plantation of gene-modified HSPC has shown to zation of this and other gene therapies for genetic provide clinical benefit for ADA-SCID patients diseases. F.F. has no conflict of interest to declare. 980 FERRUA AND AIUTI

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