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.

Primary and immortalized cultured Schwann cells are commonly utilized in analyses of myelin gene promoters, but few lines are well-characterized in terms of their endogenous expression of myelin genes. This is particularly significant in that cultured Schwann cells typically do not express myelin genes at levels comparable to those observed in vivo. In this study, the steady-state levels of mRNA and protein for five Schwann cell markers (PMP22, P0, MBP, MAG, and LNGF-R) were assessed in primary Schwann cells and six representative Schwann cell lines (RT4-D6P2T, JS-1, RSC96, R3, S16, and S16Y). RT4-D6P2T and S16 cells were the most similar to myelinating Schwann cells based on their comparatively high expression of PMP22 and P0 mRNA. Both RT4-D6P2T and S16 also expressed P0 protein. In addition, the previously reported P1-A positive regulatory region from the myelination-specific PMP22 promoter demonstrated significant activity in both these cell lines. However, nuclear proteins that interacted with P1-A were different in extracts prepared from RT4-D6P2T and S16 cells. Primary Schwann cells expressed myelin proteins at levels that were equal or less than those observed with the RT4-D6P2T and S16 lines, indicating that primary Schwann cells are not necessarily better than immortalized Schwann cells as model systems for the study of myelin gene regulation. The data presented here demonstrate that cultured Schwann cells used to study myelin gene promoters have to be carefully selected on the basis of the endogenous level of expression of the myelin gene under study.

We describe the molecular analysis of three families with hypodontia involving primarily molar teeth and report two novel mutational mechanisms. Linkage analysis of two large families revealed that the hypodontia was linked to the PAX9 locus. These two families revealed missense mutations consisting of a glutamic acid substitution for lysine and a proline substitution for leucine within the paired domain of PAX9. A pair of identical twins affected with hypodontia in a third family demonstrated a 288-bp insertion within exon 2 that resulted in a putative frameshift mutation and a premature stop codon. The insertion was associated with the loss of 7-bp from exon 2. A block of 256-bp of sequence within the insertion was completely identical to downstream sequence from the second intron of the PAX9 gene. These studies extend the spectrum of mutations in PAX9 associated with hypodontia to include heretofore undescribed categories, including missense mutations.

Canine leukocyte adhesion deficiency (CLAD) represents the canine counterpart 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. (Blood. 2006;108: 3313-3320)

Leukocyte adhesion deficiency (LAD)-1, a primary immunodeficiency disease caused by molecular defects in the leukocyte integrin CD18 molecule, is characterized by recurrent, life-threatening bacterial infections. Myeloablative hematopoietic stem cell transplantation is the only curative treatment for LAD-1. Recently, canine LAD (CLAD) has been shown to be a valuable animal model for the preclinical testing of nonmyeloablative transplantation regimens for the treatment of children with LAD-1. To develop new allogeneic transplantation approaches for LAD-1, we assessed a nonmyeloablative conditioning regimen consisting of busulfan as a single agent before matched littermate allogeneic bone marrow transplantation in CLAD. Three CLAD dogs received busulfan 10 mg/kg intravenously before infusion of matched littermate bone marrow, and all dogs received posttransplantation immunosuppression with cyclosporin A and mycophenolate mofetil. Initially, all 3 dogs became mixed chimeras, and levels of donor chimerism sufficient to reverse the CLAD phenotype persisted in 2 animals. The third dog maintained donor microchimerism with an attenuated CLAD phenotype. These 3 dogs have all been followed up for at least 1 year after transplantation. These results indicate that a nonmyeloablative conditioning regimen with chemotherapy alone is capable of generating stable mixed chimerism and reversal of the disease phenotype in CLAD.

Over- and under expression of the 22 kDa peripheral myelin protein (PMP22) results in dysmyelinating peripheral neuropathies, such as Charcot-Marie-Tooth disease type 1A (CMT1A) and hereditary neuropathy, with the liability to pressure palsies (HNPP). Expression of the PMP22 gene is driven by two alternative promoters, P1 and P2, with transcripts originating from P1 associated with peripheral nerve myelination by Schwann cells. Transient transfections of constructs containing P1 (3.5 kb) or P2 (2.5 kb) resulted in high levels of reporter gene expression in the RT4-D6P2T schwannoma cell line. Serial deletions of P1 revealed that region P1-A (-105 to -43), situated upstream of the minimal promoter, contained a positive regulatory element. The 62 bp P1-A region conferred in cis a sevenfold increase in expression of luciferase driven by a heterologous promoter in an orientation-dependent manner. Interspecies comparison of the P1-A region revealed a 98% degree of identity between the human, mouse, and rat sequences. A prominent sequence-dependent DNA-protein complex (C-I) was detected in electrophoretic mobility shift assays with P1-A using RT4-D6P2T nuclear extract and was localized to a minimal 21 bp region within P1-A. Site-directed mutagenesis of this region revealed nucleotides at positions -46 to -43 as being necessary for formation of C-I. Functional analysis of the mutated P1-A element indicated that positions -46 and -45 were essential for transactivation mediated by this element. Characterization of the transacting factor(s) interacting with this key regulatory element will shed light on its role in regulating peripheral nerve myelination.

Overexpression of the 22-kDa peripheral myelin protein (PMP22) causes the inherited peripheral neuropathy, Charcot-Marie-Tooth disease type 1A (CMT1A). In an attempt to alter PMP22 gene expression as a possible therapeutic strategy for CMT1A, antiparallel triplex-forming oligonucleotides (TFO) were designed to bind to purine-rich target sequences in the two PMP22 gene promoters, P1 and P2. Target region I in P1 and region V in P2 were also shown to specifically bind proteins in mammalian nuclear extracts. Competition for binding of these targets by TFO vs. protein(s) was compared by exposing proteins to their target sequences after triplex formation (passive competition) or by allowing TFO and proteins to simultaneously compete for the same targets (active competition). In both formats, TFO were shown to competitively interfere with the binding of protein to region I. Oligonucleotides directed to region V competed for protein binding by a nontriplex-mediated mechanism, most likely via the formation of higher-order, manganese-destabilizable structures. Given that the activity of the P1 promoter is closely linked to peripheral nerve myelination, TFO identified here could serve as useful reagents in the investigation of promoter function, the role of PMP22 in myelination, and possibly as rationally designed drugs for the therapy of CMT1A. The nontriplex-mediated action of TFO directed at the P2 promoter may have wider implications for the use of such oligonucleotides in vivo.

Children with the severe deficiency phenotype of leukocyte adhesion deficiency (LAD-1) suffer recurrent, life-threatening bacterial infections due to defective adherence and migration of their leukocytes. LAD-1 is caused by heterogeneous molecular defects in the leukocyte integrin CD18 molecule. Dogs with the canine form of leukocyte adhesion deficiency (CLAD), like children with severe deficiency LAD-1, experience severe bacterial infections, and typically die within the first few months of life from infection. CLAD represents a disease-specific, large animal model for evaluating new therapeutic approaches for the human disease LAD. In these studies, we tested a retroviral-vector mediated gene therapy approach in CLAD. Autologous CLAD CD34+ bone marrow hematopoietic stem cells were pre-stimulated overnight with growth factors cIL-6, cSCF, hFlt3-L, and hTPO, then incubated with retroviral vector PG13/MSCV-cCD18 over 48 hours on recombinant fibronectin. Transduction of the CLAD CD34+ cells was measured by flow cytometry for CD18+ cells and ranged from 11% to 21%. The transduced cells were re-infused (0.26 − 1.49 x 106 CD18+ cells / kg) into the dogs following the administration of two different non-myeloablative conditioning regimens: 5 CLAD dogs received autologous, gene-corrected CD34+ cells following 200 cGy total body irradiation (TBI) and 2 CLAD dogs received autologous, gene-corrected CD34+ cells following 10 mg/kg busulfan. Peripheral blood samples were analyzed by flow cytometry for CD18 expression following the re-infusion of the transduced CD34+ cells. The frequency of CD18+ gene-corrected leukocytes in the peripheral blood ranged from 0.04% to a high of 4.44% at 6 – 11 months post-gene transfer. Two of the five dogs in the first group and one of the two dogs in the second group that received CD18+ gene-corrected cells are alive and well on no prophylactic treatment at 9 – 14 months of age. Of note, the CLAD dog receiving busulfan conditioning has the highest level of CD18+ gene-corrected cells (4.44% at 6 months post-infusion), with the levels increasing at monthly intervals since the second month following re-infusion. These results contrast markedly with those seen in untreated CLAD dogs that die or are euthanized within the first few months of life due to intractable infection. These studies indicate that a clinically applicable non-myeloablative regimen of either 200 cGy TBI or 10 mg/kg busulfan facilitates the engraftment of sufficient autologous, CD18-gene corrected cells to correct the lethal disease phenotype in CLAD. No evidence of monoclonality has been detected by LAM-PCR in any of the dogs with therapeutic levels of gene-corrected cells. In future studies we will optimize the transduction protocol in order to increase the number of CD34+ gene-corrected cells for infusion, as well as closely monitor the gene-corrected animals for any evidence of insertional mutagenesis or other complications related to the therapy. Together, these findings support the use of either of two clinically applicable, non-myeloablative conditioning regimens prior to the infusion of autologous, CD18 gene-corrected cells in gene therapy clinical trials for LAD.