Correction of the disease phenotype in canine leukocyte adhesion deficiency using ex vivo hematopoietic stem cell gene therapy

Abstract

Canine leukocyte adhesion deficiency (CLAD) represents the canine counter-part of the human disease leukocyte adhesion deficiency (LAD). Defects in the leukocyte integrin CD18 adhesion molecule in both CLAD and LAD lead to recurrent, life-threatening bacterial infections. We evaluated ex vivo retroviral-mediated gene therapy in CLAD using 2 nonmyeloablative conditioning regimens--200 cGy total body irradiation (TBI) or 10 mg/kg busulfan--with or without posttransplantation immunosuppression. In 6 of 11 treated CLAD dogs, therapeutic levels of CD18(+) leukocytes were achieved. Conditioning with either TBI or busulfan allowed long-term engraftment, and immunosuppression was not required for efficacy. The percentage of CD18(+) leukocytes in the peripheral blood progressively increased over 6 to 8 months after infusion to levels ranging from 1.26% to 8.37% at 1-year follow-up in the 6 dogs. These levels resulted in reversal or moderation of the severe CLAD phenotype. Linear amplification-mediated polymerase chain reaction assays indicated polyclonality of insertion sites. These results describe ex vivo hematopoietic stem cell gene transfer in a disease-specific, large animal model using 2 clinically applicable conditioning regimens, and they provide support for the use of nonmyeloablative conditioning regimens in preclinical protocols of retroviral-mediated gene transfer for nonmalignant hematopoietic diseases such as LAD.

Figure 1.

Diagram of experimental design. Bone marrow (BM) was harvested from CLAD dogs and enriched for CD34⁺ cells. CD34⁺ cells were used immediately, or cryopreserved and thawed 2 to 3 weeks later. After transduction, cells were harvested, washed, and reinfused into CLAD pups that had received conditioning with either 200 cGy TBI on day 0 or –1 (n = 9), or 10 mg/kg busulfan intravenously on day –2(n = 2). Four dogs (A1-A4) received posttransplantation immunosuppression with CSP starting on day –1 and continuing until day +65 (35 days at 30 mg/kg, 30 days at 15 mg/kg), along with MMF from day 0 to day +28 (at 20 mg/kg). Seven dogs received no posttransplantation immunosuppression. LTR, retroviral long terminal repeat; Ψ+, extended packaging signal; gln tRNA PBS, glutamine tRNA primer binding site; PCMV, PCC4-cell passaged myeloproliferative sarcoma virus.

Figure 2.

Flow cytometric analysis of CD18⁺ leukocytes in the peripheral blood following infusion of gene-corrected cells. Peripheral-blood leukocytes from the 6 CLAD dogs surviving long-term were isolated, stained with an anti-CD18 antibody, and examined by flow cytometry. The percentage of CD18⁺ leukocytes was determined in comparison to isotype stained controls. Prior to the infusion of the gene-corrected CD34⁺ cells, there were no CD18⁺ leukocytes (not different from isotype control staining) in the peripheral blood. The CD18⁺ leukocyte percentages over time in the peripheral blood are shown.

Figure 3.

Assessment of CD18⁺ peripheral-blood leukocyte subsets in the 6 treated CLAD dogs with 1-year follow-up. The contribution of each subset was assessed in each of the 6 long-term treated dogs by flow cytometric analysis of CD18 expression on (A) granulocytes, (B) CD14⁺ monocytes, and (C) lymphocytes. Each subset was analyzed using a specific subset antibody, or was determined by the absence of CD14 staining and side scatter (SSC) profile (high SSC, granulocytes; low SSC, lymphocytes), along with an anti-CD18 antibody.

Figure 4.

In vitro detection of adhesion by activated CD18⁺ leukocytes. Peripheral-blood leukocytes from dogs A1, A2, B1, B2, and C1 were added to fibrinogen-coated wells with or without stimulation by PMA or blocking by the anti-CD18 antibody 60.3 antibody. (A) Photo-multiplier tube (PMT) reconstructed images of field images (500 μm × 192 μm) of representative areas of the wells from dog B1 using scatter, CD18 green fluorescence, and blue fluorescence for Hoechst-stained nucleated cells. Without PMA stimulation, few of the adherent leukocytes are CD18⁺ (without PMA; top panel). Activated CD18⁺ leukocytes are present following PMA stimulation (with PMA; middle panel). Blocking of PMA-stimulated CD18⁺ leukocytes was accomplished using the anti-CD18 monoclonal antibody 60.3 (with PMA + 60.3; bottom panel). (B) Cell adherence was assessed using total peripheral-blood leukocytes from 5 treated CLAD dogs. The number of adherent leukocytes that are CD18⁺, divided by the total number of adherent CD18⁺ and CD18^– leukocytes present, indicates the percentage of adherent leukocytes that are CD18⁺. The percentage of nonspecific adherence is influenced by washing stringency, hence the ratio of the stimulated-to-nonstimulated percentages is a more accurate indicator of preferential adhesion than the total number of cells binding. The average of 2 experiments is shown with the range indicated.

Figure 5.

Selective migration of CD18⁺ leukocytes in vivo. Analysis of CD18⁺ leukocytes in the peripheral-blood and pus collected at the same time in 2 CLAD dogs after infusion of CD18⁺ gene-corrected cells demonstrated in vivo migration of CD18⁺ leukocytes. Leukocytes were isolated from the peripheral blood and pus from infected tissue (dog A3, week 3; dog A4, week 9) and stained for CD18. Dotplots represent CD18 expression (x-axis) versus side-scatter (y-axis). Percentages of CD18⁺ leukocytes are shown at the lower right corner.

Figure 6.

Clinical course of 6 long-term surviving CLAD dogs following gene therapy. The clinical course of the 6 long-term CLAD survivors treated with gene therapy is shown prior to and following infusion of gene-corrected cells. The line and arrow indicate the period of follow-up for each dog. Episodes of fever (≥ 102.4°F) necessitating a more aggressive treatment (increased antibiotics/fluids) are shown by the vertical lines. The shaded boxes indicate periods during which the animal was in intensive care. The appearance of an episode of HOD (lameness/joint swelling/leg pain) or CMO (bony jaw/cranial swelling/soreness) is indicated by the closed diamonds. The day of infusion (day 0) is indicated. Attainment of a CD18⁺ ANC level of 100 ± 5 cells/μL is indicated by the inverted pyramid. Time in days in relationship to the infusion is shown on the x-axis in the panel.

Figure 7.

Clonality analysis by linear amplification–mediated PCR (LAM-PCR). Genomic DNA was isolated from peripheral-blood leukocytes of 6 dogs at 2 time points (6 and 12 months after infusion) and subjected to LAM-PCR to examine clonality of retroviral insertions. The presence of a band likely indicates a unique insertion site. Samples were electrophoresed on a Spreadex gel and photographed by a digital camera. M indicates marker; bp, base pairs.