RESUMO
Ex vivo gene therapy procedures targeting hematopoietic stem and progenitor cells (HSPCs) predominantly utilize lentivirus-based vectors for gene transfer. We provide the first pre-clinical evidence of the therapeutic utility of a foamy virus vector (FVV) for the genetic correction of human leukocyte adhesion deficiency type 1 (LAD-1), an inherited primary immunodeficiency resulting from mutation of the ß2 integrin common chain, CD18. CD34+ HSPCs isolated from a severely affected LAD-1 patient were transduced under a current good manufacturing practice-compatible protocol with FVV harboring a therapeutic CD18 transgene. LAD-1-associated cellular chemotactic defects were ameliorated in transgene-positive, myeloid-differentiated LAD-1 cells assayed in response to a strong neutrophil chemoattractant in vitro. Xenotransplantation of vector-transduced LAD-1 HSPCs in immunodeficient (NSG) mice resulted in long-term (â¼5 months) human cell engraftment within murine bone marrow. Moreover, engrafted LAD-1 myeloid cells displayed in vivo levels of transgene marking previously reported to ameliorate the LAD-1 phenotype in a large animal model of the disease. Vector insertion site analysis revealed a favorable vector integration profile with no overt evidence of genotoxicity. These results coupled with the unique biological features of wild-type foamy virus support the development of FVVs for ex vivo gene therapy of LAD-1.
Assuntos
Síndrome da Aderência Leucocítica Deficitária , Spumavirus , Humanos , Camundongos , Animais , Spumavirus/genética , Vetores Genéticos/genética , Síndrome da Aderência Leucocítica Deficitária/genética , Síndrome da Aderência Leucocítica Deficitária/terapia , Células-Tronco Hematopoéticas , Antígenos CD18/genética , Antígenos CD34/genéticaRESUMO
RATIONALE: Pulmonary hypertension (PH) is characterized by progressive elevation in pulmonary pressure and loss of small pulmonary arteries. As bone morphogenetic proteins promote pulmonary angiogenesis by recruiting the Wnt/ß-catenin pathway, we proposed that ß-catenin activation could reduce loss and induce regeneration of small pulmonary arteries (PAs) and attenuate PH. OBJECTIVE: This study aims to establish the role of ß-catenin in protecting the pulmonary endothelium and stimulating compensatory angiogenesis after injury. METHODS AND RESULTS: To assess the impact of ß-catenin activation on chronic hypoxia-induced PH, we used the adenomatous polyposis coli (Apc(Min/+)) mouse, where reduced APC causes constitutive ß-catenin elevation. Surprisingly, hypoxic Apc(Min/+) mice displayed greater PH and small PA loss compared with control C57Bl6J littermates. PA endothelial cells isolated from Apc(Min/+) demonstrated reduced survival and angiogenic responses along with a profound reduction in adhesion to laminin. The mechanism involved failure of APC to interact with the cytoplasmic domain of the α3 integrin, to stabilize focal adhesions and activate integrin-linked kinase-1 and phospho Akt. We found that PA endothelial cells from lungs of patients with idiopathic PH have reduced APC expression, decreased adhesion to laminin, and impaired vascular tube formation. These defects were corrected in the cultured cells by transfection of APC. CONCLUSIONS: We show that APC is integral to PA endothelial cells adhesion and survival and is reduced in PA endothelial cells from PH patient lungs. The data suggest that decreased APC may be a cause of increased risk or severity of PH in genetically susceptible individuals.