Outcome following Gene Therapy in Patients with Severe Wiskott-Aldrich Syndrome

Abstract

Importance

Wiskott-Aldrich syndrome (WAS) is a rare primary immunodeficiency associated with severe microthrombocytopenia. Partially HLA-matched allogeneic hematopoietic stem cell (HSC) transplantation is associated with significant co-morbidity.

Objective

To assess the outcome and safety of autologous HSC gene therapy in WAS.

Design

Gene-corrected autologous HSC were infused in 7 consecutive WAS patients (age range: 0.8 to 15.5 years, mean 7 years) following myeloablative conditioning.

Setting

and participants: Patients with severe WAS lacking HLA-matched related or unrelated HSC donors were treated between December 2010 and January 2014. The follow up of patients in this intermediate analysis ranged from 9 to 42 months.

Intervention

A single infusion of gene-modified CD34+ cells with an advanced lentiviral vector.

Main Outcome(s) and Measure(s)

Primary outcomes were improvement at 24 months in eczema, the frequency and severity of infections, bleeding tendency, autoimmunity and reduction in disease-related days of hospitalization. Secondary outcomes were improvement in immunological and haematological parameters, and evidence for safety through vector integration analysis.

Results

Six out of the 7 patients were alive at the time of last follow-up (mean and median follow-up time: 28 and 27 months respectively) and showed sustained clinical benefit. One patient died 7 months after treatment from pre-existing drug- resistant herpes virus infections. Eczema and susceptibility to infections resolved in all 6 patients. Autoimmunity improved in 5/5 patients. No severe bleeding episodes were recorded after treatment, and at last follow up 6/6 patients were free from blood product support and thrombopoietic agonists. Hospitalization days were reduced from 25 days (median) in the 2 years pretreatment to 0 days (median) in the 2 years post treatment. All 6 surviving patients exhibited high-level, stable engraftment of functionally corrected lymphoid cells. The degree of myeloid cell engraftment and of platelet reconstitution correlated with the dose of gene-corrected cells administered. No evidence of vector-related toxicity was observed clinically or by molecular analysis.

Conclusions and Relevance

This study demonstrated the feasibility of the use of gene therapy in patients with Wiskott-Aldrich syndrome. Controlled trials with larger numbers of patients are necessary to assess long-term outcome and safety.

Introduction

Wiskott-Aldrich Syndrome (WAS, OMIM#301000) is a complex, X-linked primary immunodeficiency caused by loss-of-function mutations in the WAS gene. The condition affects the immune and haematopoietic system and has a broad spectrum of severity¹. The WAS protein (WASp) is a key regulator of the actin cytoskeleton in all hematopoietic lineages². WASp deficiency causes characteristic microthrombocytopenia and lymphoid and myeloid cell dysfunction, the severity of which is usually correlated with WASp expression levels. A clinical scoring system is used to stratify disease severity³. Patients with a score from 3 to 5 display a WAS phenotype characterized by a tendency to bleed, persistent eczema, susceptibility to severe opportunistic bacterial and viral infections, autoimmune and inflammatory complications, and an elevated risk of lymphoid malignancies^3–5. In the absence of definitive treatment, patients with classical WAS do not survive beyond their second or third decade of life. Although hematopoietic stem cell (HSC) transplantation is usually curative, the use of human leukocyte antigen (HLA) partially-matched HSCs is associated with a high incidence of complications^6–10. Gene therapy based on the transplantation of autologous, gene-corrected HSCs may be an effective and potentially safer alternative.

The first gene therapy trial for WAS used a Moloney-leukemia-virus-derived gamma-retroviral vector (MLV). Although this therapy provided significant clinical benefit as characterized by partial or complete resolution of immunodeficiency, autoimmunity, and bleeding diathesis, it was associated with an unacceptably high risk of insertional mutagenesis with activation of several proto-oncogenes leading to leukemia in 7 of the 9 evaluable patients¹¹.

We developed and tested a self-inactivating lentiviral vector for WAS gene correction (referred to below as LV-w1.6 WASp) in which a 1.6 kb fragment of the proximal promoter of the WAS gene is used to express the full-length coding sequence of the human WAS gene in cells of the hematopoietic lineage^12–14. In a recently published study, 3 young children with a moderate form of WAS were treated with this vector. They showed stable engraftment of WASp-expressing cells, and improvements in terms of immune function, platelet count and clinical score¹⁵. Here, we report the first results of a two-center study designed to assess the feasibility of HSC gene therapy in severe WAS patients.

Methods

Clinical protocol

Seven consecutive patients with confirmed WAS were enrolled at Great Ormond Street Hospital (London, UK) (EUDRACT number: 2007-004308-11) and Necker Children’s Hospital (Paris, France) (EUDRACT number: 2009-011152-22) in an open label study between December 2010 and January 2014. The final dates of follow-up were between May 28^th and November 12^th 2014.

The study protocol was approved by the UK and French drug regulatory agencies and the appropriate investigational review boards such as the Gene Therapy Advisory Committee (GTAC) in the UK and the Ethical Committee for the Protection of the Persons Submitted to a Clinical Trial (CPP idF2). Informed consent or assent was obtained after the benefits and risks of the trial were explained to the patients or their parents/legal guardians. Hematopoietic stem cells were collected from the patient bone marrow (BM) or mobilized peripheral blood (MPB) and were genetically-modified ex vivo during myeloablative conditioning of patients. All patients were placed in sterile confinement and received a low-intensity conditioning regimen with busulfan (4 mg/kg/day) and fludarabine (40 mg/m²/day) for three days. At the end of the conditioning procedure, transduced cells were infused to the patient without cryopreservation. Anti-CD20 antibody and/or alemtuzumab were added if autoimmune disease was present. Autoimmunity was defined clinically as cutaneous or large vessel vasculitis, arthritis, and cytopenia of one or more hematopoietic lineages in association with the presence of auto-antibodies. All the transduced cell products met the specifications required for product release and infusion. The synopsis of the study is provided in supplemental material.

Outcomes

Primary study objectives were to assess the outcomes following gene therapy judged by improvement in clinical manifestations including frequency and severity of infections, of bleeding episodes, of autoimmune manifestations, and eczema. Secondary objectives were based on biological tests including platelet counts, lymphocyte subset analysis, and lymphocyte function (mitogen and antigen-induced proliferation, serum Ig levels). Secondary objectives of safety were evaluated clinically (including manifestations of clonal proliferation or leukaemia) and by assessment of the frequency of vector integration sites close to relevant proto-oncogenes and their abundance within the engrafted and transduced cell population.

The severity of disease in patients was scored according to the following criteria^3,5 : A score of 1 accounts for microthrombocytopenia which is universal to all patients; a score of 2 includes mild eczema, immunodeficiency and occasional mild infections; a score of 3 refers to more severe immunodeficiency associated with recurrent and more protracted infections; a score of 4 is given if eczema or infections are persistent and do not respond easily to conventional treatments; and a score of 5 is assigned to very severe clinical forms that additionally develop autoimmune or malignant complications.

Vector production

The LV-w1.6 WASp vector has been described elsewhere^14,16. Clinical vector batches were manufactured at Genethon (Evry, France) according to Good Manufacturing Practice and were purified, concentrated and titered for infectious particles (infectious genomes (i.g.)/mL)¹⁶. The batches used in the study are described in Supplementary Table S1. Batch 1 was used in London, and batches 2 and 3 were used in Paris.

Biological analyses

Lymphocyte phenotypes, functions and T-cell receptor repertoires (beta, alpha, gamma and delta chains) were analyzed as described elsewhere^17–20. Signal joint T-cell receptor excision circles (TREC) were determined using a real-time qPCR²¹. TREC content was expressed in copies per 1×10⁵ peripheral blood mononuclear cells (PBMCs) (control range value between 150 to 2500 copies/10⁵ PBMCs). The vector copy number (VCN) per cell was measured by qPCR detection of the vector’s HIV Psi sequence with normalization against the copy number of the albumin gene, as described elsewhere²² (for more details, see supplementary methods). Lymphoid, myeloid, naive and memory T cell subpopulations were sorted by flow cytometry, using the corresponding fluorescence-labeled monoclonal antibodies. Natural killer (NK) cells cytotoxicity was evaluated against K562 target cells as described in supplementary methods.

CD34+ cell gene transfer procedure

Patients’ bone marrow cells harvested under general anesthesia, were separated with lymphoprep (Eurobio, Oslo, Norway) and centrifuged in order to collect mononuclear cells (MNC). Patient’s MPB was collected by apheresis. The positive selection of CD34+ cells from MNC or from MPB was performed using immunomagnetic beads and an immunomagnetic enrichment device (CliniMACs, Miltenyi Biotec, Bergish – Gladbach, Germany). Purified CD34+ cells were seeded on cell culture bags pre-coated with clinical grade Retronectin™ (Takara Bio Co) in serum free medium (X-Vivo 20, Biowhittacker/Lonza) and clinical grade stem cell factor (SCF) (300 ng/ml), FLT3-L (300 ng/ml), thrombopoietin (TPO) (100 ng/ml) and IL-3 (20 ng/ml) (all from Peprotech). After 24 hours of prestimulation, cells were transduced twice with LV-w1.6.WASp (1×10⁸ig/ml) each time for 18 hours. At the end of the transduction procedure, washed cells were resuspended in 4% human serum albumin and transferred in a sterile bag for infusion to the patient. Aliquots of cells were further cultured for 14 days to measure stable proviral integration by qPCR and WASp expression by flow cytometry.

Integration site analysis and clonality assays

Ligation-mediated PCR was used to sequence vector integration sites in different cell subpopulations (the cell selection procedure is provided in supplementary methods)^23–25. At least 3 independent replicates were analyzed for all samples. Deep sequencing was carried out using both the 454/Roche and Illumina techniques. Data sets analyzed are summarized in the Supplementary Report. Assays of a DNA preparation were judged to be successful if they detected at least 80 different break sites in the human genome associated with unique adaptor ligation positions. All integration site sequence data were deposited at the NCBI SRA accession: SRP050221.

Statistical analysis was carried out using R version 3.1.2. An extensive discussion of statistical methods and results is presented in the Supplementary Report. Briefly, The distributions of integration sites (relative to genomic features) were summarized using the receiver operating characteristic curve method (Supplementary Figure S1 A and B)²⁶. The abundance of cell clones was quantified using the SonicLength method²⁵. Clumping of integration site sequences was analyzed using scan statistics²⁷

Results

Clinical presentation

At enrollment, the age of the patients ranged from 0.8 to 15.5 years (median: 7). All but one of the patients (P6) had a disease score of 5 (Table 1). All patients experienced severe thrombocytopenia, which led to severe bleeding episodes in P2, P3, P4, P5 and P7 (intracerebral hemorrhage in P3, gastro-intestinal hemorrhage in P5). Furthermore, all patients had eczema and associated recurrent skin infections. Three of the 7 patients experienced recurrent, severe infections requiring hospitalization. Six patients had autoimmune disease, with P2 and P3 being most profoundly affected. In P2, a combination of severe lower limb vasculitis and arthritis became refractory to conventional treatment and prevented ambulation. Patient 3 had severe autoimmune cytopenias that led to splenectomy at the age of 3 years. He also experienced a lymphoproliferative disorder with generalized lymphoadenopathy, liver enlargement, and renal infiltration. His primary immunodeficiency was responsible for severe viral infections due to cytomegalovirus (CMV), herpes-simplex viruses (HSV1) and varicella-zoster-virus (VZV) which led to several hospitalizations from the age of 7 up to the time of gene therapy treatment including perioral herpes infections with facial cellulitis, severe respiratory tract infection to Klebsiella pneumoniae aggravated by a cerebral hematoma causing coma and requiring intensive care unit hospitalization. He developed sequelaes following his stroke requiring rehabilitation therapy. The recurrent herpes virus infections were treated with several cycles of acyclovir, ganciclovir and foscarnet requiring hospitalisation (Table 1). The HSV1 genotype was found resistant to acyclovir in the months that preceded gene therapy.

Table 1.

Characteristics of patients and the infused patient-autologous gene therapy products^(a)

	P1	P2	P3	P4	P5	P6	P7
Age at GT (years or months)	10 years	15.5 years	10 years	10 months	3 years	7 years	3.5 years
WAS Gene mutation	401C/T p.A134V	c.1453+1 G>C	c.628delT p.S210HfsX51	c.1295delG p.G432EfsX13	c.257G>A p.R86H	100C>T p.R34X	1271dup(ex10) Leu425Profsx70
Revertant cells	−	+	+	−	−	+	−
Mutant WASp expression	+	−	−	−	−	−	+
Clinical manifestations, in the 2 years preceding GT(b)
Bleeding(c)	Bruising	Severe Splenectomized	Severe Splenectomized Multiple platelet transfusions	Severe Multiple platelet transfusions	Severe Multiple platelet transfusions; N-plate	Bruising (moderate)	Severe Multiple Platelet transfusions; N-plate
Eczema	Severe	Severe	Mild	Mild	Severe	Severe	Severe
Infections	Severe	2 episodes of sepsis, severe VZV	Severe chronic VZV, HSV, CMV, EBV infections	Mild	Mild	Severe	Gastroenteritis (2 episodes)
Autoimmunity	Recurrent arthritis Renal Disease	Severe lower limbs vasculitis and arthritis, unable to walk	Pancytopenia	Severe Thrombocytopenia Mild skin vasculitis	Severe Thrombocytopenia	None	Severe Thrombocytopenia Mild cutaneous vasculitis
Days in hospital for disease-related complications	<5	47days	>6 months	26 days(b) (in 10 months)	19 days	<5	21days
Clinical score(d)	5	5	5	5	5	3	5
Date of GT	March/03/2011	May/27/2011	Nov/18/2011	April/20/2012	June/15/2012	Nov/16/2012	January/24/2014
HSC origin	BM	MPB	MPB	BM	BM	BM	MPB
Total CD34+ cell dose ×106/kg	2	11	11	7.3	6.8	3.1	15
VCN	0.7	1.3	1.2	2.8	0.6	1.7	0.6
Date of last Fup	Nov/12/2014	May/28/2014	Death July/11/2012	Oct/04/2014	June/11/2014	Nov/12/2014	Nov/03/2014
Fup (months)(d)	42	36	7	30	24	24	9
Clinical status 2 years post GT or at last Fup	A&W	A&W	Died of opportunistic viral infections	A&W	A&W	A&W	A&W
Bleeding	Bruising (resolved after splenectomy)	No	NA	Bruising	Bruising	Bruising (mild)	Bruising
Eczema	No	Very mild	NA	No	No	No	No
Infections	No	Localized zoster (1 episode)	NA	No	Mild gastroenteritis (1 episode)	No	No
Autoimmunity	No	Mild vasculitis, able to walk	NA	No	No	No	No
Days in hospital (post initial GT period), over following 2 years	0	5	Continuous hospitalization until death	0	5	0	0(d)
Clinical score at last Fup	<1	5 (mild autoimmunity)	NA	<1	<1	<1	1

^(a)P1 and P6 were included in London, and P2, P3, P4, P5 and P7 were included in Paris; P: patient; GT: gene therapy; HSC: hematopoietic stem cells; BM: bone marrow; MPB: mobilized peripheral blood; VCN: vector copy number per cell in cultured transduced CD34+ cells; Revertant cells are detected in blood and have recovered some expression of WASp as a result of selected spontaneous somatic mutations; Fup: Follow-up; A&W: alive and well; NA: not applicable; Severe eczema is defined by intense redness, thickness/swelling, and may include diffuse or superinfected lesions; Severe infection indicates infections requiring hospitalization and parenteral treatment ; Severe thrombocytopenia is defined as platelet counts below 10,000/mm³.

^(b)P4 had a shorter period of observation since he was treated at the age of 10 months

^(c)Thrombocytopenia was initially below 20,000/mm³ in P1, P2, P3 and P6 and below 10,000/mm³ in P4, P5 and P7. P2 and P3 were splenectomized prior to GT. P1 was successfully splenectomized after GT

P3, P4, P5 had more than 20 platelet transfusions, P7 more than 10, P5 and P7 received additional N-plate therapy.

^(d)P7 has a shorter follow up of 9 months.

“+” Revertant cells and mutant protein are present and detectable in blood cells

“−” Revertant cells and mutant protein are absent and not detectable in blood cells

Gene transfer

Autologous CD34⁺ HSCs (from bone marrow in 4 cases and from MPB in 3 cases; Table 1) were transduced ex vivo with the LV-w1.6 WASp lentiviral vector, and were immediately re-infused into conditioned patients. The median dose of CD34⁺ cells per kg of body weight infused was 7.3×10⁶ (range: 2×10⁶ to 15×10⁶) and the mean vector copy number (VCN) in CD34⁺ cells was 1.27 ± 0.8 (range: 0.6 to 2.8) (Table 1).

Outcome after gene therapy

Six of the 7 patients treated with gene therapy were evaluable over a period of at least 9 months (mean and median follow-up time: 28 and 27 months respectively) for primary outcomes. Patient 3 died as a consequence of opportunistic herpes viral infections that became drug-resistant post gene therapy including severe peri-oral necrotizing ulcerative lesions caused by HSV-1 and a bilateral CMV retinitis with high blood viral load (ie 4.6 log copies/ml) (Supplementary Table S2). This was associated with an inflammatory pulmonary syndrome characterized by multiple foci of bronchoalveolar parenchymal condensation responsible of oxygen dependence developed in association with diffuse aspergillus- related lesions. Clinical outcome rapidly worsened. A lung biopsy showed an extensive fibrosis. The patient eventually died of septic shock in the intensive care unit seven months post gene therapy. In December 2014, the other 6 treated patients were alive and well and displayed significant clinical improvements (Table 1). In terms of the primary outcomes, eczema and susceptibility to infections have resolved in all cases. Minor, non-recurrent only infections were observed in 2 patients (Table 1). Patient1 has not had any further episodes of arthritis. Patient 2 displayed major improvement in peripheral vasculitis and was able to return to normal physical activity without need for a wheelchair. Patient 7 recovered completely from vasculitis skin lesions. With the exception of occasional bruising, there were no post-treatment recurrences of the severe, recurrent bleeding episodes that had previously affected P2, P4, P5 and P7. From month 7 onwards, none of the 6 patients required regular blood product support or treatment with recombinant stimulators of platelet production (P4 received N-plate until month 13). Days of additional disease-related hospitalization once the initial gene therapy was completed was reduced to 0–5 days over the next 2 years for patients 1,2,4,5 and 6 and over a 9 month period for patient 7. P1 had a 7 day hospitalization for elective splenectomy in year 3 after engraftment. After gene therapy, patients remained on their pre-treatment regimen of immunoglobulin substitution and prophylactic antibiotics, although immunoglobulins have recently been discontinued in P4 and P6 pending evaluation of functional responses to a typical childhood vaccination schedule.

Gene marking and WASp expression in leukocytes

The presence of vector-positive cells in blood was readily detectable one month after treatment. Q-PCR revealed an increase over time in gene marking in peripheral blood mononuclear cells (Figure 1). Gene marking in PBMCs was primarily due to transduced lymphocytes, most prominently T cells, but multiple cell lineages were also transduced. At the last follow-up, the VCN ranged from 0.35 to 1.20 in sorted CD3⁺ T cells, from 0.1 to 1.34 in sorted CD19⁺ B cells and from 0.04 to 0.7 in sorted natural killer CD56⁺ cells. The extent of gene marking in CD14⁺ or CD15⁺ myeloid cells was more variable, with 0.4 copies per cell in P4, 0.2 copies in P6, 0.1 in P2 and P7, and between 0.01 and 0.03 in P1 and P5 (Figure 1). The intracellular expression of WASp correlated with the levels of transduction in the different cell subsets. At the last follow-up, the proportion of cells expressing WASp was highest in T cells (34% to 84%) and somewhat lower in NK cells (14% to 85%) and B cells (13% to 55%) (Figure 2A–B and Table 2).

Figure 1. Longitudinal evaluation of gene marking in blood cells after gene therapy.

(A). Gene marking in peripheral blood cells over time after gene therapy in patients P1 to P7, as expressed by vector copy numbers per cell (VCN) in PBMC and measured by q-PCR. (B). Gene marking in various blood cell subsets in each patient, expressed as VCN in: CD3+ T cells (filled squares); CD56+ NK cells (filled diamonds); CD19+ B cells (filled triangles); CD15+ neutrophils (open squares) and CD14+ monocytes (open circles).

Figure 2. Lymphoid engraftment and expression of the WASp transgene measured by flow cytometry.

(A) Time course of lymphoid cell subsets recovery in blood after gene therapy: The total (open symbols) or WASp+ (filled symbols) levels of CD3+ T cells (squares), CD19+ B cells (triangles) and CD56+ NK cells (diamonds) were measured in blood over time. Tinted areas indicate values in aged-matched individuals. (B) WASp expression in lymphoid cell subpopulations at different times after gene therapy: Expression of WASp using specific antibodies is indicated by a thick line in CD3+ T lymphocytes, CD19+ B lymphocytes, CD56+ NK cells and CD4+ CD25+ FoxP3+ regulatory T cells (Tregs). Control antibody staining is shown by a gray overlay. WASp marking in T, B and NK cells of P1 to P7 performed respectively at months 30, 30, 6, 24, 24, 12 were shown. WASp marking in Tregs of P2, P4, P5 were performed respectively at months 30, 18, 18.

Table 2.

Most recent immunological analysis of patients

Patient	P1	P2	P3†	P4	P5	P6	P7
Age at last Fup years)	14	18	11	3	5	9	4.3
Time of analysis (months following gene therapy)	42	36	6	30	24	24	9	Control range < 7 years	≥ 7 years
CD3/μl	840	715	2106	1921	1984	970	756	1400–3700	800–2200
WASp+ T cells (%)	71§	76	54	84§	34	61§	70	100	100
CD4/μl	560	440	972	1256	1271	730	434	700–2200	530–1300
CD8/μl	180	187	1053	471	620	180	196	490–1300	330–920
Naive CD4+ T cells (%)	37	23	1	27§	32§	54	22	60–72	43–55
Naïve CD8+ T cells (%)	33	17	0.3	38§	15§	70	22	52–68	52–68
TREC/105 PBMC	847§	612§	ND	1118§	1444§	130§	ND	>150	>150
PHA-induced proliferation	+	+	+	+	+	+	+	+	+
Anti-CD3 induced proliferation	+/−	+	ND	+	+	+	+	+	+
Ag-induced proliferation*	ND	2*	0	1*§	1*	ND	1*	NA	NA
CD56/μl	190	99	297	324§	496	240	210	160–950	70–480
WASp+ NK cells (%)	14 §	60	ND	60§	85	23	74	100	100
CD19/μl	70	132	270	1260§	558	170	196	610–2600	110–570
WASp+ B cells (%)	13 §	27	17	55§	15	15 §	44	100	100
Platelets ×109/L	217§§	87	71**	44	6	13	10	>100	>100
MPV (fl)	9.1§§	7.8§	ND	9.1§	ND	9.1	ND	5–12	6.5–12
WASp+ platelets	ND	+	ND	+	+	ND	ND	+	+

^†Patient 3 died at 7 months from opportunistic infections.

TREC: T-cell receptor excision circle; PHA: phytohemagglutinin; Ag: antigen; (*) positive T cell proliferation with any of the following antigens tested (tetanus toxoid, varicella zoster virus, candidin and purified protein derivative), numbers “2” and “1”, indicate the number of antigen to which the patient’s cell proliferate; (+): comparable to the control; MPV: mean platelet volume; ND: not determined; NA: not applicable.

^§value measured at the previous time point. (**)during platelet transfusion.

^§§Measured at month 42, after splenectomy. The control values for lymphocytes are from W.T. Shearer et al study (Shearer W.T. et al, J. Aller.Immunol, 2003). The standards for the other parameters have been established by our local hospital diagnostic laboratories.

+/− : proliferation below the normal value.

Immune reconstitution

Recovery of normal absolute T cell counts was achieved in 4 of the 6 evaluable patients (P1, P4, P5, and P6) while values remained just below the normal range in P2 and P7 (Table 2 and Figure 2A). Normal counts of CD4⁺ T cells were demonstrated in the same 4 patients. Low absolute CD8⁺ T cell count was observed in 4 out of 6 patients (Table 2 and Supplementary Figure S2). In all patients, ongoing thymopoiesis was evidenced by recovery of circulating naïve T-cells and detection of TRECs. One patient has normal absolute naive CD4⁺ T-cell counts, although for 5 of the 6 evaluable patients this remained below the normal range (Supplementary Figure S2, and Table 2). For P3, the follow-up period was too short to evaluate immune reconstitution, although T cell numbers recovered rapidly after treatment and were transgene-positive prior to his death 7 months after gene therapy. The presence of anti- CMV specific T cells was detected by pp65 CMV-specific tetramers as well as IFNγ+ immuno-staining (Supplementary Table S2). This is an evidence of partial, albeit functionally incomplete specific T cell response.

Expression of functional WASp is known to provide T cells with a significant growth and survival advantage; this is especially true for the memory subset and effector populations that normally express higher levels of WASp than naïve T cells^28–30. Accordingly, VCN were repeatedly found to be higher in memory T cells than in naïve T cells in P4 (12 months after gene therapy, we found: 2.1 versus 1.1 vector copies/cell respectively). Although WASp is not essential for natural regulatory T cell (nTreg) generation, it is required for nTreg function^31,32. After gene therapy, CD4⁺/CD25⁺/FoxP3⁺ Treg cells expressed the WAS transgene in all 3 patients evaluated (P2, P4 and P5) (Figure 2B).

At last follow-up absolute NK cell counts were within the normal range in all patients and the function was normal in the sample available for testing (P7) (Figure 2A, Table 2 and Supplementary Figure S3). At last follow-up, B cell counts were within the normal range in 3 out of the 6 patients (P2, P4 and P6) (Figure 2A, Table 2).

Immunoscope analysis revealed that the transduced T cells displayed a polyclonal TCR repertoire (Supplementary Figure S4). As a consequence of impaired T-cell receptor (TCR)-mediated signaling, T cells from WAS-patients typically show defective proliferation after stimulation with anti-CD3 monoclonal antibodies^33–35. After gene therapy, all patients displayed normal proliferative responses to phytohemagglutinin (PHA) and anti-CD3 antibody -mediated proliferation was also observed in all patients except P1 (Table 2). A positive response to one or more microbial antigens (tetanus toxoid, varicella-zoster virus, Candida albicans and purified protein derivative) was detected in P2, P4, P5 and P7 (Table 2).

WASp-deficient dendritic cells (DCs) and monocytes fail to form the actin-rich adhesion structures known as podosomes^36,37. After gene therapy, monocyte-derived DCs from P2, P4 and P5 were assayed for their ability to assemble podosomes upon adhesion to fibronectin. The fraction of podosome-positive DCs in P4, P2 and P5 was 50%, 20% and <5%, respectively, demonstrating that WASp expression (which was correlated with myeloid cell gene marking) restored the DCs’ ability to regulate cytoskeletal rearrangements.

As of December 2014, B-cell function could not be fully evaluated because 4 out of 6 patients were still receiving immunoglobulin (Ig) replacement therapy but 2 patients (P6 and P4) had recently stopped Ig treatment. In P4, the analysis of the B-cell phenotype 24 months after gene therapy showed B cell subsets within normal ranges compared with healthy age-matched reference values most probably due to WASp expression restoration in B cells³⁸ (Supplementary Figure S5). Moreover, serum IgE levels which are generally elevated in WAS patients, have dropped significantly in P2 and become very low in P7 (Supplementary Table S3).

Platelet reconstitution

Platelets counts and platelet volumes were variably ameliorated by gene therapy in the 6 evaluable patients (Table 2 and Figure 3A). Although all patients remained thrombocytopenic, the detected platelets were predominantly WASp-positive (Figure 3B). The patients’ mean platelet volume (range: 8.1 to 9.3 fl) was in the normal range for healthy controls in our centers (7.5 to 10.1 fl). Patients receiving the highest doses of transduced CD34⁺ cells appeared to exhibit the most pronounced increases in platelet counts but findings were not statistically tested (Table 1 and 2, Figures 3A and 3C). P1 was splenectomised for quality of life reasons 3 years after gene therapy and recovered normal platelet counts.

Figure 3. Platelet reconstitution after gene therapy.

(A) The change over time in platelet counts in each individual patient. The lower normal value is indicated by a horizontal dotted bar. Filled triangles indicate platelet transfusions and open triangles indicate administration of romiplostim. (B) WASp expression in platelets, as measured by flow cytometry and represented as in Fig. 2B. (C) The correlation between platelet reconstitution and the number of transduced cells infused. The platelet count at last follow-up (Table 2) was plotted against the number of transduced CD34+ cells infused per kg of body weight, which was calculated by taking account of VCN values below 1.0 in the infused product (Table 1).

Analysis of the vector integration site distribution

More than 5 million genomic integration site sequence reads were collected from sorted cell samples from the 6 patients, yielding over 90,000 unique integration sites. As expected for a lentiviral vector, correlation of the integration site distribution with genomic features revealed preferential integration within transcription units and association with epigenetic markers of transcription, in the infused gene therapy products (pre-transplant) and in circulating blood cells at different time points after gene therapy (post-transplant)^23,39 (Supplementary Figure S1 A–B). Analysis of genomic regions with high frequencies of integration revealed clusters of sites²⁷ in both the pre-transplant and post-transplant samples (also referred to as “clumps” in the Supplementary Report). Clusters were spread over large genomic distances (>0.5 Mb) and did not overlap with the 5′ ends of the genes implicated in the occurrence of adverse events in previous gene therapy trials (i.e. LMO2, CCND2, and MECOM/EVI1) (Supplementary Figure S1 C–D). Gene-rich regions showed more clusters, occasionally overlapping with cancer-associated genes in some patients at statistically non-significant frequency. Clones associated with genes of concern were a far smaller proportion of sites in the WAS-lentiviral trial than the WAS-gammaretroviral trial (~0.06% versus ~2% respectively; p<10e-6), and none showed persistent expansion over time (Supplementary Figure S6) (for further analysis, see the Supplementary methods and the Supplementary Report). Thus, in contrast to what has been observed in patients treated with gammaretroviral vectors^11,40,41, we did not detect an association between recurrent integration near specific cancer-associated genes and cell amplification or persistence.

Sequencing of pre-transplant integration sites from a sample of the infused cells revealed up to 2.9×10⁴ unique sites. Replicate analyses of pre-transplant samples from each patient showed relatively little overlap in the integration patterns, which is consistent with very large population sizes. Change in clonality of PBMCs, and also in sorted peripheral blood lymphoid (CD3+ T cells, CD19+, B cells and CD56+ NK cells) and myeloid cells (CD15+ neutrophils and CD14+ monocytes) were monitored over time in the 6 evaluable patients (Supplementary Figure S6). Population sizes in well-characterized samples could be modeled by comparison with the overlap in sites detected in replicate analyses (using the Chao 1 estimator with Jackknife correction, as applied to counts of linker positions)²⁵. The estimated population sizes in PBMCs corresponded to hundreds or thousands of clones. In all analyzed cell types and in all patients, no one clone accounted for more than 10% of the population detected at a given time point. All patients displayed a stable, high diversity Shannon index (Supplementary Figure S1E). These data indicate that reconstitution with gene-corrected cells was highly polyclonal, with intermittent progenitor activity and no lasting clonal expansion.

Common integration sites in myeloid and lymphoid lineages are indicative of the transduction of multipotent progenitors. We focused our analysis on highly pure (>97%) neutrophils, T and B lymphocytes sorted from P4 twelve months after transplantation (Supplementary Figure S7). These neutrophils shared 12% and 14% of their integration sites with T and B lymphocytes, respectively, suggesting that common progenitors were successfully transduced and engrafted in this patient.

Discussion

WAS is a multifaceted disease with a broad spectrum of severity¹. There is still a need for novel, effective, well-tolerated treatments – particularly in patients with advanced disease and/or who lack an HLA-matched allogeneic donor^10,42. Here, we report on the outcome of HSC gene therapy in 7 severely affected WAS patients, using a lentiviral vector to transfer a WAS expression cassette in repopulating HSC^13,14. The protocol incorporated a near-myeloablative and immunosuppressive conditioning regimen, in order to enhance the engraftment of transduced cells. Compared to a recently-reported monocentric study of three WAS patients aged 1 to 6 (and with a clinical score of 3 or 4)¹⁵, the children in the present two-center study were older and had more severe disease (with a clinical score of 5 in 6 out of 7 cases, which is a risk factor for allogeneic HSC transplantation). Among 6 of the 7 patients, there was clinical improvement after gene therapy, which was well tolerated. However, one patient died of pre-existing, treatment-refractory, infectious disease. In the 6 surviving patients the infectious complications resolved after gene therapy, and prophylactic antibiotic therapy was successfully discontinued in 3 cases. Severe eczema resolved in all affected patients as did signs and symptoms of autoimmunity. No patient developed hemorrhagic complications after withdrawal of supportive treatment where implemented. T-cell-related function was corrected in all evaluable patients, regardless of their age at the time of treatment or the dose of transduced cells received. A longer-term follow-up will be required to assess the functional reconstitution of humoral immunity, although the evidence of an accumulation of WASp-expressing B cells is encouraging.

Part of our study’s objectives was to evaluate the haematological and immunological outcomes after engraftment of gene-corrected cells in various lineages. In all patients, the degree of gene marking in lymphoid cells was greater than that achieved in myeloid cells. This is consistent with a strong proliferative and/or survival advantage conferred on the lymphoid compartment by WASp expression, as predicted from earlier observations in mice and in cells from patients^28,29. This hierarchy in gene marking has also been observed in patients included in two other gene therapy trials^11,15 and is in keeping with the results of previous studies of WASp status in patients after allogeneic HSC transplantation.

One of the major, invariable signs of WAS is a tendency to bleed as a result of intrinsic microthrombocytopenia – particularly when compounded by the development of anti-platelet antibodies. Platelet counts and the mean platelet volume increased in 3 of the 6 evaluable patients (P2, P4 and P5) but remained below normal values although no patient experienced any major bleeding episodes after gene therapy. Persistent thrombocytopenia also occurs after allogeneic HSC transplantation when associated with low myeloid chimerism¹⁰. The platelet counts measured in our patients and in other individuals treated with lentiviral gene therapy¹⁵ are at the low end of the range observed in patients with mixed chimerism after allogeneic HSC transplantation. Recovery of platelet counts may be related to the dose of transduced cells received. These findings suggest that robust engraftment of HSCs is required to fully correct the disease phenotype. Nevertheless, lower-level engraftment enables lymphoid reconstitution as a consequence of the profound selective advantage conferred on these lineages²⁹. Of note, a rapid normalization of platelet counts and platelet volume was obtained in patient P1 following splenectomy 45 months after gene therapy.

The present study is the first to our knowledge to demonstrate clinical improvement after autologous gene therapy using a lentiviral vector in severely affected children and young adult patients in whom more pronounced procedure-related complications would be expected. When considered alongside with another study in younger and less severely affected patients¹⁵, this lentiviral vector may represent a safer alternative to a MLV-derived vector used in a recently reported trial in which 7 of the 9 patients developed acute leukemia as a result of insertional oncogenesis¹¹. Interpretation of the results of this type of study is constrained by the small number of patients and the difficulty in performing randomized trials in severe orphan diseases. We therefore cannot draw conclusions on long–term outcome and safety. Further follow-up of these patients, and those reported in a similar study last year¹⁵ together with additional clinical trials of this therapy are therefore necessary.

Conclusion

This study demonstrated the feasibility of the use of gene therapy in patients with Wiskott-Aldrich syndrome. Controlled trials with larger numbers of patients are necessary to assess long-term outcome and safety.