Gene therapy for adenosine deaminase-deficient severe combined immune deficiency: clinical comparison of retroviral vectors and treatment plans

Abstract

We conducted a gene therapy trial in 10 patients with adenosine deaminase (ADA)-deficient severe combined immunodeficiency using 2 slightly different retroviral vectors for the transduction of patients' bone marrow CD34(+) cells. Four subjects were treated without pretransplantation cytoreduction and remained on ADA enzyme-replacement therapy (ERT) throughout the procedure. Only transient (months), low-level (< 0.01%) gene marking was observed in PBMCs of 2 older subjects (15 and 20 years of age), whereas some gene marking of PBMC has persisted for the past 9 years in 2 younger subjects (4 and 6 years). Six additional subjects were treated using the same gene transfer protocol, but after withdrawal of ERT and administration of low-dose busulfan (65-90 mg/m(2)). Three of these remain well, off ERT (5, 4, and 3 years postprocedure), with gene marking in PBMC of 1%-10%, and ADA enzyme expression in PBMC near or in the normal range. Two subjects were restarted on ERT because of poor gene marking and immune recovery, and one had a subsequent allogeneic hematopoietic stem cell transplantation. These studies directly demonstrate the importance of providing nonmyeloablative pretransplantation conditioning to achieve therapeutic benefits with gene therapy for ADA-deficient severe combined immunodeficiency.

Figure 1

Experimental schema and maps of retroviral vectors carrying the normal human ADA cDNA. (A) The experimental schema for the clinical trial is shown. Flt-3L indicates Flt-3 ligand; MGDF, megakaryocyte growth and differentiation factor; rFBN, recombinant fibronectin; and SCF, stem cell factor. (B) The elements of the proviral forms of the 2 retroviral vectors used to transfer normal human ADA cDNA are depicted. A indicates common qPCR primer; B, vector-specific qPCR primer; dl587, endogenous murine retrovirus dl587rev; LTR, long terminal repeat; MMLV, Moloney murine leukemia virus; MND, MPSV LTR, ncr-deleted, coupled to dl587rev pbs; MPSV, myeloproliferative sarcoma virus; P, common qPCR probe; pbs, primer-binding site; Ψ, packaging signal; SA, splice acceptor site; and SD, splice donor site.

Figure 2

Hematologic values and serum transaminase levels after busulfan administration. (A) ANC, (B) platelet counts, (C) serum alanine aminotransferase, and (D) serum aspartate aminotransferase levels over 2 months.

Figure 3

qPCR measurements of the average vector copy/cell in blood cell samples obtained after transplantation. Separate qPCR analyses were performed on granulocytes (Grans) and PBMC fractions with primer/probe sets specific for the MND-ADA vector or the GCsapM-ADA vector provirus. (A) Results from subjects not receiving busulfan conditioning and remaining on ERT (201-204) and (B) results from subjects having ERT withdrawn and receiving busulfan before transplantation (301-306).

Figure 4

ADA enzymatic activity in PBMC and % deoxyadenine nucleotides in erythrocytes. ADA enzyme activity in PBMC was measured biochemically. The graphs show (A) the values from subjects not receiving busulfan conditioning and remaining on ERT (201-204) and (B) the values from subjects having ERT withdrawn and receiving busulfan before transplantation (301-306). The normal reference range for the ADA enzyme assay in human PBMC is indicated (gray-shaded horizontal bar). (C) Adenine and deoxyadenine metabolites were measured in erythrocytes by high-pressure liquid chromatography and the percentage that were deoxyadenosine nucleotides (dAMP + dADP + dATP) were plotted as %dAXP for the subjects in the group receiving busulfan (301-306). The times when ERT was resumed for subjects 304 and 306 are indicated.

Figure 5

Absolute lymphocyte counts and proliferative responses after gene transfer. (A) Results from subjects not receiving busulfan conditioning and remaining on ERT (201-204) and (B) results from subjects having PEG-ADA ERT withdrawn and receiving busulfan before transplantation (301-306). n.v. indicates normal values for age range. The time when ERT was resumed for subjects 304 and 306 is indicated. (C) Proliferative responses to PHA of PBMC from subjects over time. Open symbols represent subjects remaining on ERT and not receiving busulfan; filled symbols represent subjects receiving busulfan with ERT stopped.

Figure 6

Serum IL-7 levels. Stored serum samples were used to measure levels of IL-7 by ELISA. The average IL-7 levels for the subjects remaining on continuous ERT (201-204) and for the subjects withdrawn for ERT and receiving busulfan (300 series) are shown.