Long-term follow-up of foamy viral vector-mediated gene therapy for canine leukocyte adhesion deficiency

Abstract

The development of leukemia following gammaretroviral vector-mediated gene therapy for X-linked severe combined immunodeficiency disease and chronic granulomatous disease (CGD) has emphasized the need for long-term follow-up in animals treated with hematopoietic stem cell gene therapy. In this study, we report the long-term follow-up (4-7 years) of four dogs with canine leukocyte adhesion deficiency (CLAD) treated with foamy viral (FV) vector-mediated gene therapy. All four CLAD dogs previously received nonmyeloablative conditioning with 200 cGy total body irradiation followed by infusion of autologous, CD34(+) hematopoietic stem cells transduced by a FV vector expressing canine CD18 from an internal Murine Stem Cell Virus (MSCV) promoter. CD18(+) leukocyte levels were >2% following infusion of vector-transduced cells leading to ongoing reversal of the CLAD phenotype for >4 years. There was no clinical development of lymphoid or myeloid leukemia in any of the four dogs and integration site analysis did not reveal insertional oncogenesis. These results showing disease correction/amelioration of disease in CLAD without significant adverse events provide support for the use of a FV vector to treat children with leukocyte adhesion deficiency type 1 (LAD-1) in a human gene therapy clinical trial.

Figure 1

Myeloid and lymphoid chimerism after gene therapy or transplantation. The percentage of CD18⁺ neutrophils (square), CD14⁺ monocytes (triangle), and CD3⁺ T-lymphocytes (circle) was determined by flow cytometry at the designated time points after treatment. Y axis represents the percentage of CD18⁺ corrected cells, whereas the X axis represents the time (years) after infusion. Double-crosses represent end-of-experiment time points for the designated animals.

Figure 2

Clinical course and white blood counts of treated animals. (a) The times at which each transplanted animal had infectious episodes, as indicated by fever, intensive care, and/or parenteral antibiotics, are shown from 90 days before receiving gene therapy until the latest time point or the end of study (indicated by a double-cross) and indicated by the shaded boxes. (b) The peripheral blood white blood cell (WBC) counts are shown for each transplanted animal over the same time period. The normal WBC range for dogs is shaded in the figure. Each horizontal line represents the follow-up of an animal (ar row represents ongoing follow-up), with the day of infusion indicated by the filled triangle.

Figure 3

Bone marrow aspirate and biopsy taken from FV vector-treated CLAD dogs FD1 (a) and FD2 (b) at 4–5 years after therapy. Bone marrow aspirates show normal myeloid and erythroid development with no increase in immature myeloid cells (Wright Giemsa, 500X). Bone marrow biopsies have normal cellularity with megakaryocytes present in the center of each section (hematoxylin and eosin, 500X).

Figure 4

Analysis for the presence of FV vector DNA in carcinoma and control tissues. PCR of DNA from tissue samples from pancreatic carcinoma (carcinoma), prostate (prostate), mandibular lymph node (lymph node) and peripheral blood leukocytes (PBL) from FD1 at time of necropsy, and from peripheral blood leukocytes from an untreated dog (untreated). PCR was performed with canine CD4 specific primers (CD4) or FV vector-specific primers (FV). Water (H₂O) was used as a cross-contamination control. M indicates 100 bp ladder. The gel picture was inverted and levels were globally adjusted to darken the image.

Figure 5

T-cell subpopulations in the peripheral blood after infusion. Peripheral blood obtained from (a) FD1 and (b) FD2 was obtained at 6.5 years and 6 years after infusion, respectively, and immunostained for CD18 expression and one of four T-cell markers: CD4, CD8, CD45RA, and TCRαβ. Lines indicate positive or negative populations with percentages noted for each quadrant.

Figure 6

Integration sites in FV vector-treated dogs. The percentage of all integration sites within 15 kb of transcriptional (Tx) start sites, within genes, and within 30 kb of human oncogenes are shown for (a) peripheral blood leukocytes (PBL) 1 year after transplant (1 year), 4–6 years after transplant (4–6 years), and computer-generated random sites (RND); (b) sorted neutrophils (PMN); and (c) sorted lymphocytes. IS, insertion sites.