Research ArticleGene Therapy

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

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Science Translational Medicine  20 Apr 2016:
Vol. 8, Issue 335, pp. 335ra57
DOI: 10.1126/scitranslmed.aad8856
  • 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, phytohemagglutinin. (B) Emergence of transitional T1 B cells (CD10++CD21lo) in P3 at 12 weeks, progressing to T2/3 B cells (CD10+CD21hi) 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 CD3CD19+ 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.

  • Table 1. Patient characteristics and treatment.

    All patients received allogeneic stem cell transplant (HSCT) from haploidentical (haplo) parent donor once or repeated (booster). IL2RG, interleukin 2 receptor γ; CFU, colony-forming units; PLE, protein-losing enteropathy; IVIG, intravenous immunoglobulin; AUC, area under the curve.

    P1P2P3P4P5
    IL2RG mutation823T>G447 deIA923C>Ac341G>A31T>A
    Age (years)232271510
    Prior HSCTHaplo, boosterHaploHaplo, boosterHaploHaplo, booster
    Immunophenotype↓ T, B, NK↓ B, NK↓ T, B, NK↓ T, B, NK↓ T, B, NK
    Medical problemsNorovirus, infections,
    PLE, IVIG
    Norovirus, infections,
    IVIG, warts, molluscum,
    bronchiectasis, bronchiolitis
    obliterans
    Norovirus, infections,
    PLE, IVIG, bronchiectasis,
    growth failure
    Norovirus, infections,
    PLE, IVIG, bronchiectasis
    Norovirus, PLE,
    IVIG, molluscum,
    bronchiectasis
    Busulfan AUC (min*∗μM)3603.14528.92519.64523.63096.6
    CD34+ cells infused (×106/kg)181620.421.725
    Bulk CD34 in vitro CFU (%)27172257.736.1
    Follow-up (months)3624633
    Current statusCleared norovirus,
    off IVIG
    Cleared norovirus,
    off IgG supplement,
    fatal bronchial bleed
    StableStableStable

Supplementary Materials

  • www.sciencetranslationalmedicine.org/cgi/content/full/8/335/335ra57/DC1

    Materials and Methods

    Fig. S1. Summary of patient and product treatment.

    Fig. S2. Surveillance of lymphocytes, platelets, and absolute neutrophil count to monitor effects of busulfan conditioning before cell infusion in patients (P1 to P5).

    Fig. S3. Flow cytometric evaluation for CD3 T, CD19 B, and CD56 NK cells in purified PMN.

    Fig. S4. Analysis of T cell receptor repertoire and TRECs.

    Fig. S5. Absolute numbers of immune cells in subjects P3 to P5 after gene therapy.

    Fig. S6. Class-switched memory B cells.

    Fig. S7. NK cell phenotype.

    Fig. S8. Chest computed tomography.

    Fig. S9. Similarity index of vector integration sites.

    Fig. S10. Vector insulator PCR.

    Table S1. Protocol schedule for clinical procedures and follow-up.

    Table S2. IS summary in P1 and P2.

    Table S3. Combined IS from P1 up to 30m.

    Table S4. Combined IS from P2 up to 24m.

    Table S5. Lentivector integration sites in human CD34+ cells cells in vitro, n = 259648.

    Table S6. Comparison of in vitro and in vivo integration sites.

    Table S7. Gene ontology KEGG PATHWAY.

    References (32, 33)

  • Supplementary Material for:

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

    Suk See De Ravin,* Xiaolin Wu, Susan Moir, Lela Kardava, Sandra Anaya-O'Brien, Nana Kwatemaa, Patricia Littel, Narda Theobald, Uimook Choi, Ling Su, Martha Marquesen, Dianne Hilligoss, Janet Lee, Clarissa M. Buckner, Kol A. Zarember, Geraldine O'Connor, Daniel McVicar, Douglas Kuhns, Robert E. Throm, Sheng Zhou, Luigi D. Notarangelo, I. Celine Hanson, Mort J. Cowan, Elizabeth Kang, Coleen Hadigan, Michael Meagher, John T. Gray, Brian P. Sorrentino, Harry L. Malech*

    *Corresponding author. E-mail: sderavin{at}niaid.nih.gov (S.S.D.R.); hmalech{at}niaid.nih.gov (H.L.M.)

    Published 20 April 2016, Sci. Transl. Med. 8, 335ra57 (2016)
    DOI: 10.1126/scitranslmed.aad8856

    This PDF file includes:

    • Materials and Methods
    • Fig. S1. Summary of patient and product treatment.
    • Fig. S2. Surveillance of lymphocytes, platelets, and absolute neutrophil count to monitor effects of busulfan conditioning before cell infusion in patients (P1 to P5).
    • Fig. S3. Flow cytometric evaluation for CD3 T, CD19 B, and CD56 NK cells in purified PMN.
    • Fig. S4. Analysis of T cell receptor repertoire and TRECs.
    • Fig. S5. Absolute numbers of immune cells in subjects P3 to P5 after gene therapy.
    • Fig. S6. Class-switched memory B cells.
    • Fig. S7. NK cell phenotype.
    • Fig. S8. Chest computed tomography.
    • Fig. S9. Similarity index of vector integration sites.
    • Fig. S10. Vector insulator PCR.
    • Table S1. Protocol schedule for clinical procedures and follow-up.
    • References (32, 33)

    [Download PDF]

    Other Supplementary Material for this manuscript includes the following:

    • Table S2 (Microsoft Excel format). IS summary in P1 and P2.
    • Table S3 (Microsoft Excel format). Combined IS from P1 up to 30m.
    • Table S4 (Microsoft Excel format). Combined IS from P2 up to 24m.
    • Table S5 (Microsoft Excel format). Lentivector integration sites in human CD34+ cells in vitro, n = 259648.
    • Table S6 (Microsoft Excel format). Comparison of in vitro and in vivo integration sites.
    • Table S7 (.pdf format). Gene ontology KEGG PATHWAY.

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