Lentiviral hematopoietic stem cell gene therapy for X-linked severe combined immunodeficiency

Abstract

X-linked severe combined immunodeficiency (SCID-X1) is a profound deficiency of T, B, and natural killer (NK) cell immunity caused by mutations inIL2RGencoding the common chain (γc) of several interleukin receptors. Gamma-retroviral (γRV) gene therapy of SCID-X1 infants without conditioning restores T cell immunity without B or NK cell correction, but similar treatment fails in older SCID-X1 children. We used a lentiviral gene therapy approach to treat five SCID-X1 patients with persistent immune dysfunction despite haploidentical hematopoietic stem cell (HSC) transplant in infancy. Follow-up data from two older patients demonstrate that lentiviral vector γc transduced autologous HSC gene therapy after nonmyeloablative busulfan conditioning achieves selective expansion of gene-marked T, NK, and B cells, which is associated with sustained restoration of humoral responses to immunization and clinical improvement at 2 to 3 years after treatment. Similar gene marking levels have been achieved in three younger patients, albeit with only 6 to 9 months of follow-up. Lentiviral gene therapy with reduced-intensity conditioning appears safe and can restore humoral immune function to posthaploidentical transplant older patients with SCID-X1.

Fig. 1. Immune cell gene marking and numbers after gene therapy

(A) Gene marking in sorted cell lineages as vector copy number (VCN) per genome after treatment in P1 (to 36 months) and P2 (to 24 months). (B) Early gene marking in first 6 months in myeloid and B cells in P1 to P5. (C) Percent autologous (corrected) host cells as determined by restriction fragment length polymorphism assay of T cell chimerism. (D) Immune cell numbers in P1 and P2 after treatment. K/μl, thousands per cubic milliliter.

Fig. 2. Functional correction of T and B cells with SIN-LV gene therapy

(A) CD3 T cell proliferative responses to indicated stimuli. CPM, counts per minute; PHA, phyto-hemagglutinin. (B) Emergence of transitional T1 B cells (CD10⁺⁺CD21^lo) in P3 at 12 weeks, progressing to T2/3 B cells (CD10⁺CD21^hi) by 16 weeks after gene therapy. (C) Changes in B cell subset profiles over time in P3. (D) Serum immunoglobulin M (IgM) and IgG over time in P1 and P2 after treatment. Withdrawal of supplemental IgG is indicated by the arrow. Dotted lines indicate respective normal reference ranges. (E) Early increases in IgM, comparing P1 to P5. (F) A summary of VCN in flow-sorted T1, T2/3, and naïve B cells compared to CD3 T cells from P3, P4, and P5.

Fig. 3. Restoration of B cell signaling and specific antibody production

(A) B cell responses to IL21 and CD40 ligand (CD40L) stimulation. Peripheral blood mononuclear cells (PBMCs) from P1 (bottom) and a healthy control (HC, top) were stained with carboxyfluorescein diacetate succinimidyl ester (CFSE) and stimulated with IL21 and CD40L. Gated CD3⁻CD19⁺ B cells that have undergone division are shown in left upper area showing CD27⁺-expressing (left panels), IgG⁺-expressing (middle panels), and IgM⁺-expressing (right panels) B cells. (B) ELISpot of P1 B cells before and after vaccination. Numbers of Ig antibody-secreting cells (ASC) (left), influenza-specific ASC per 10⁶ B cells, and influenza-specific as a fraction of total Ig ASC (right) detected by ELISpot of peripheral blood B cells from P1 before and after influenza vaccination to determine memory B cell responses.

Fig. 4. Clinical progress after gene therapy

(A) Photographs demonstrating human papilloma virus warts (top) and molluscum contagiosum on P2 (bottom) before and 15 months after gene therapy as indicated. (B) Serial serum albumin for P1 to P5 after gene therapy. Upper and lower reference ranges are indicated by dotted lines. (C) Body mass measurements for P1 to P5 after treatment.

Fig. 5. Vector integration site analysis

(A) Total unique integration sites, shown in proportion to their representation of the total diversity in P1 and P2 to 30 and 24 months (30 m, 24 m), respectively (left). Clonal composition for sorted cell lineages for P1 and P2 (right). Each horizontal bar represents clonal frequency, from most abundant on the top. The number of unique clones in the top 50% of the cells, UC₅₀, is listed above each sample. (B) Serial quantitative ddPCR tracking of the four most frequent clones [TNFSF12 (tumor necrosis factor ligand superfamily, member 12), TNFSF12-TNFSF13, chr6 (chromosome 6), and PIM1 (proto-oncogene serine/threonine-protein kinase)] in P2 is shown as a percentage of vector-marked cells (left) or of total cells in each lineage (right). GRx, gene therapy. (C) Schematic of unique integrations of HMGA2 in P1 and P2. Most clones (enumerated next to the arrow) are in the same orientation as the gene (blue), with a few in the reverse orientation (red). Clones are seen in all lineages: CD34, CD14, CD19, NK and PMN, and CD3 (summarized below).

Fig. 6. Circular projection of the human genome with integration sites from P1 and P2 (in vivo, orange and red), in vitro LV-transduced CD34 cells (green), and in vitro mγRV-transduced CD34 cells (red)

The top 100 target genes (in vitro) are listed on the outside, with the top 10 target genes (in vitro) in bold. MLV, murine leukemia virus.