Stem-cell gene therapy for the Wiskott-Aldrich syndrome

Abstract

The Wiskott-Aldrich syndrome (WAS) is an X-linked recessive primary immunodeficiency disorder associated with thrombocytopenia, eczema, and autoimmunity. We treated two patients who had this disorder with a transfusion of autologous, genetically modified hematopoietic stem cells (HSC). We found sustained expression of WAS protein expression in HSC, lymphoid and myeloid cells, and platelets after gene therapy. T and B cells, natural killer (NK) cells, and monocytes were functionally corrected. After treatment, the patients' clinical condition markedly improved, with resolution of hemorrhagic diathesis, eczema, autoimmunity, and predisposition to severe infection. Comprehensive insertion-site analysis showed vector integration that targeted multiple genes controlling growth and immunologic responses in a persistently polyclonal hematopoiesis. (Funded by Deutsche Forschungsgemeinschaft and others; German Clinical Trials Register number, DRKS00000330.).

Figure 1. Restored Expression of Wiskott–Aldrich Syndrome (WAS) Protein (WASP) after Gene Therapy

Shown is the restoration of WASP expression after gene therapy (GT) in different subgroups of leukocytes, as determined by fluorescence-activated cell sorting, in Patient 1 (Panel A) and Patient 2 (Panel B). WASP protein expression is confirmed in both patients on Western blot analyses in peripheral-blood mononuclear cells (PBMCs) (Panels C and D) and platelet protein lysates (Panels G and H), as compared with a sample from a healthy control subject (HC) and from a patient with WAS who did not undergo gene therapy. There is also a marked increase in platelet counts in Patients 1 and 2 (Panels E and F).

Figure 2. Correction of Leukocyte Function after Gene Therapy (GT)

Formation of the immunologic synapse between natural killer (NK) cells and tumor cells (K562), which is impaired in patients with the Wiskott–Aldrich syndrome (WAS), is restored after GT. The localization of perforin is restored 1 year after GT in Patient 2, with an immunologic synapse similar in appearance to that in a sample from a healthy control subject (HC) and in contrast to that from a patient with WAS who did not undergo GT (Panel A). Differential interference contrast (DIC) microscopy shows normal formation of cellular conjugates. However, an overlay view of the immunofluorescence analysis shows that the perforin staining is localized close to the adjacent cells in a healthy control and in Patient 2 after therapy, in contrast to the findings in a patient with untreated WAS. NK-cell lytic activity is at least partially restored after GT in Patients 1 and 2, as compared with an untreated patient with WAS and a control subject (Panel B). Podosome formation is restored in a fraction of monocytes from both patients after GT, as confirmed at multiple time points, in comparison with values in a control sample (Panel C). T-cell proliferative responses are normalized 2 years after GT, after stimulation with phytohemagglutinin (PHA) or CD3, as compared with unstimulated (US) samples (Panel D). The skewed Vβ family repertoire for T-cell receptors (TCRs), which was observed in both Patients 1 and 2, has normalized after GT (Panel E). (For a complete set of TCR Vβ spectratyping, see Fig. 9 in the Supplementary Appendix.) The severe and therapy-refractory eczema in Patient 2 shows complete regression 2 years after GT (Panel F). The I bars indicate standard errors.

Figure 3. Sustained Polyclonal Hematopoietic Repopulation after Gene Therapy

In Panels A and B, retroviral insertion sites (RISs) were detected by linear-amplification–mediated polymerasechain-reaction (LAM-PCR) analysis of transduced hematopoietic cells at various days after gene therapy from bone marrow cells and primary blood leukocytes obtained from Patients 1 and 2. CD3 denotes T cells, G granulocytes, HC healthy control subject, IC internal vector control, and M 100-bp marker. In Panels C and D, the relative contribution of individual clones retrieved from bone marrow cells and primary blood leukocytes is estimated in sequenced polyclonal-cell samples from Patients 1 and 2. The presence of individual gene-corrected cells is given as the percentage of all LAM-PCR amplicon sequence reads identified in a particular sample. The 10 most frequently sequenced RISs are ranked from 1 to 10 according to retrieval frequency. Genes that are highlighted in gray carry a RIS that was detected at more than one time point within the 10 most frequently sequenced sites. Also listed are the numbers of other unique, mappable RISs that were sequenced less frequently than the first 10 in the respective sample analyzed, which are labeled “others.”

Figure 4. Insertion Sites Clustered in Specific Gene Regions

The results of screening for the presence and clonal contribution of highly prominent common-insertion-site (CIS) clones are shown for Patient 1 (Panel A) and Patient 2 (Panel B). At every time point analyzed, sequence counts for all retroviral insertion sites (RISs) contributing to an individual CIS derived from bone marrow cells and primary blood leukocytes are clustered and relate to the total sequence count at the respective time point. To estimate the overall contribution, the relative sequence counts in all CIS clones are related to sequence counts of non–CIS-related genes. (An overview of CIS clones that were detected at more than one time point is provided in Table 5 in the Supplementary Appendix.) CISs of second order are defined as integration sites detected in a window of 30 kb, those of third order in a window of 50 kb, and those of fourth order in a window of 100 kb. CIS clones of fifth and higher orders were further defined in a window of 200 kb. The sequence count is given as a proportion of total sequence counts in CIS genes, as compared with the total overall sequence count, showing the number of days after gene therapy (GT). The relative clonal contribution of vector-targeted gene loci that have been shown to induce malignant clonal expansion in gene-therapy studies involving patients with chronic granulomatous disease (CGD) and severe combined immunodeficiency with γc-chain defects (γc-SCID) is shown for Patient 1 (Panel C) and Patient 2 (Panel D). In both patients, the presence of LMO2, CCND2, and BMI1 (which have been shown to trigger malignant transformation of CD3+ T cells in patients with γc-SCID) was almost exclusively restricted to CD3+ T cells. Accordingly, the presence of MDS1/EVI1, PRDM16, and SETBP1 (which have been shown to trigger myeloid clonal expansion in patients with CGD) was largely found in granulocytes (G). Numbers in parentheses indicate the number of clones containing an insertion site within or close to the respective RefSeq gene. The label “Other” indicates the number of all less frequently encountered genetic locations that carry insertion sites in the respective sample analyzed. The MDS1 clone carries an integration site at the MDS1 locus at position 169071575 bp on chromosome 3, which substantially contributes to myeloid regeneration.