Successful treatment of canine leukocyte adhesion deficiency by foamy virus vectors

Abstract

Recent successes in treating genetic immunodeficiencies have demonstrated the therapeutic potential of stem cell gene therapy. However, the use of gammaretroviral vectors in these trials led to insertional activation of nearby oncogenes and leukemias in some study subjects, prompting studies of modified or alternative vector systems. Here we describe the use of foamy virus vectors to treat canine leukocyte adhesion deficiency (CLAD). Four of five dogs with CLAD that received nonmyeloablative conditioning and infusion of autologous, CD34+ hematopoietic stem cells transduced by a foamy virus vector expressing canine CD18 had complete reversal of the CLAD phenotype, which was sustained more than 2 years after infusion. In vitro assays showed correction of the lymphocyte proliferation and neutrophil adhesion defects that characterize CLAD. There were no genotoxic complications, and integration site analysis showed polyclonality of transduced cells and a decreased risk of integration near oncogenes as compared to gammaretroviral vectors. These results represent the first successful use of a foamy virus vector to treat a genetic disease, to our knowledge, and suggest that foamy virus vectors will be effective in treating human hematopoietic diseases.

Figure 1

In vitro analysis of foamy virus vector ΔΦMscvCD18. (a) Schematic diagrams of foamy virus vector ΔΦMscvCD18 and wild-type foamy virus (FV). The locations of gag, pol, env and bel gene sequences are labeled in the wild-type FV and are indicated by shading in the vector. All viral genes are deleted or truncated in the vector, and the LTR contains a deletion in the U3 region (ΔU3). The packaging signal (Ψ), translation termination codons (asterisks), central polypurine tracts (cPPTs; filled circles), MSCV promoter and canine CD18 cDNA are also shown. (b) CD18 expression levels in FV vector–transduced CLAD CD34⁺ cells (middle) compared to normal CD34⁺ cells (right) and untransduced CLAD CD34⁺ cells (left). The percentage of CD18⁺ cells within the stained population is indicated in the upper right corner of each panel. CD18⁺ cells appearing in the CLAD populations represent background staining due to cytokine expansion.

Figure 2

Expression and function of transduced CLAD peripheral blood cells after infusion. (a) The percentages of CD18⁺ PBLs, neutrophils, monocytes and lymphocytes determined by flow cytometry for the first 24 months after treatment are shown for each animal. The y axis has a different scale for the lymphocytes. (b) CD18 expression in FV vector–transduced PBLs compared to PBLs from a normal dog, as determined by flow cytometry. The percentage of CD18⁺ cells is indicated in the upper right corner of each panel. (c) Comparison of FV vector provirus copy number in CD18⁻ and CD18⁺ PBLs from FV vector–treated CLAD dogs 12 months after infusion, as determined by quantitative PCR. (d) PMA-stimulated adhesion of CD18⁺ leukocytes. PBLs from an untreated CLAD dog, an FV vector–treated CLAD dog (FD3) and a normal dog were added to fibrinogen-coated wells with or without stimulation by PMA or blocking by an antibody to CD18. Representative images are shown with blue fluorescence for Hoechst-stained nuclei and green fluorescence for CD18 expression. (e) CD18 expression and CFSE fluorescence in lymphocyte proliferation assays of cells from a CLAD dog, an FV vector–treated CLAD dog (FD3) and a normal (carrier) dog, at the indicated dose of SEA. Proliferation results in decreased fluorescence of the CFSE label. The percentages of CD18⁻ and CD18⁺ proliferating cells are indicated in the lower left and right quadrants, respectively.

Figure 3

Correction of the CLAD phenotype in vivo. (a) The clinical course of the four FV vector–treated CLAD dogs is compared to that of four CLAD dogs that did not receive FV vector–treated cells (UD1–4). Each horizontal line represents the life of a dog (arrow represents ongoing follow-up), with the day of infusion indicated by the filled triangle. Evidence of fever and infection are indicated by the shaded boxes. A dagger indicates the death of the dog. (b) WBC counts for the four FV vector–treated CLAD dogs. (c) WBC counts for four untreated CLAD dogs until the time of death.

Figure 4

Integration sites in FV vector–treated dogs. (a) The chromosomal positions (distance from the centromere) of FV vector integration sites are shown for every chromosome except the Y chromosome. Each dot is a different integration site from four different cell populations (the number of sites for each group is shown in parentheses). (b) Percentage of all integration sites (IS) within 15 kilobases (kb) of transcriptional (Tx) start sites, within genes, and within 30 kb of human oncogenes is shown for a PG13/MSCV-cCD18 gammaretroviral vector used to treat CLAD dogs (RV), the FV vector integration sites from PBLs 1 year after transplant (FV) and computer-generated random sites (RND). Significant P values are shown (**) as ascertained by a two-tailed Fisher’s exact test. (c) Ontologies of genes within 30 kb of FV vector integration sites are shown for preinfusion and postinfusion (PBLs, lymphocytes and neutrophils combined) cell sources and an averaged set of random sites. Percentages represent the number of genes for each particular gene class divided by the total number of genes found in or near insertion sites. The asterisk (*) indicates those classes overrepresented after analysis with a modified one-tailed Fisher’s exact test.