Lentiviral Vectors for the Treatment and Prevention of Cystic Fibrosis Lung Disease

Despite the continued development of cystic fibrosis transmembrane conductance regulator (CFTR) modulator drugs for the treatment of cystic fibrosis (CF), the need for mutation agnostic treatments remains. In a sub-group of CF individuals with mutations that may not respond to modulators, such as those with nonsense mutations, CFTR gene transfer to airway epithelia offers the potential for an effective treatment. Lentiviral vectors are well-suited for this purpose because they transduce nondividing cells, and provide long-term transgene expression. Studies in primary cultures of human CF airway epithelia and CF animal models demonstrate the long-term correction of CF phenotypes and low immunogenicity using lentiviral vectors. Further development of CF gene therapy requires the investigation of optimal CFTR expression in the airways. Lentiviral vectors with improved safety features have minimized insertional mutagenesis safety concerns raised in early clinical trials for severe combined immunodeficiency using γ-retroviral vectors. Recent clinical trials using improved lentiviral vectors support the feasibility and safety of lentiviral gene therapy for monogenetic diseases. While work remains to be done before CF gene therapy reaches the bedside, recent advances in lentiviral vector development reviewed here are encouraging and suggest it could be tested in clinical studies in the near future

The Sirt1 activator SRT3025 expands hematopoietic stem and progenitor cells and improves hematopoiesis in Fanconi anemia mice

Fanconi anemia is a genetic bone marrow failure syndrome. The current treatment options are suboptimal and do not prevent the eventual onset of aplastic anemia requiring bone marrow transplantation. We previously showed that resveratrol, an antioxidant and an activator of the protein deacetylase Sirt1, enhanced hematopoiesis in Fancd2 mutant mice and improved the impaired stem cell quiescence observed in this disease. Given that Sirt1 is important for the function of hematopoietic stem cells, we hypothesized that Sirt1 activation may improve hematopoiesis. Indeed, Fancd2−/− mice and wild-type mice treated with the selective Sirt1 activator SRT3025 had increased numbers of hematopoietic stem and progenitor cells, platelets and white blood cells. SRT3025 was also protective against acetaldehyde-induced hematopoietic damage. Unlike resveratrol, however, SRT3025 did not affect stem cell quiescence, suggesting distinct mechanisms of action. Conditional deletion of Sirt1 in hematopoietic cells did not abrogate the beneficial effects of SRT3025, indicating that the drug did not act by directly stimulating Sirt1 in stem cells, but must be acting indirectly via extra-hematopoietic effects. RNA-Seq transcriptome analysis revealed the down-regulation of Egr1–p21 expression, providing a potential mechanism for improved hematopoiesis. Overall, our data indicate that SRT3025 or related compounds may be beneficial in Fanconi anemia and other bone marrow failure syndromes

Increased CFTR expression and function from an optimized lentiviral vector for cystic fibrosis gene therapy

Despite significant advances in cystic fibrosis (CF) treatments, a one-time treatment for this life-shortening disease remains elusive. Stable complementation of the disease-causing mutation with a normal copy of the CF transmembrane conductance regulator (CFTR) gene fulfills that goal. Integrating lentiviral vectors are well suited for this purpose, but widespread airway transduction in humans is limited by achievable titers and delivery barriers. Since airway epithelial cells are interconnected through gap junctions, small numbers of cells expressing supraphysiologic levels of CFTR could support sufficient channel function to rescue CF phenotypes. Here, we investigated promoter choice and CFTR codon optimization (coCFTR) as strategies to regulate CFTR expression. We evaluated two promoters—phosphoglycerate kinase (PGK) and elongation factor 1-α (EF1α)—that have been safely used in clinical trials. We also compared the wild-type human CFTR sequence to three alternative coCFTR sequences generated by different algorithms. With the use of the CFTR-mediated anion current in primary human CF airway epithelia to quantify channel expression and function, we determined that EF1α produced greater currents than PGK and identified a coCFTR sequence that conferred significantly increased functional CFTR expression. Optimized promoter and CFTR sequences advance lentiviral vectors toward CF gene therapy clinical trials

Oxymetholone Therapy of Fanconi Anemia Suppresses Osteopontin Transcription and Induces Hematopoietic Stem Cell Cycling

Summary: Androgens are widely used for treating Fanconi anemia (FA) and other human bone marrow failure syndromes, but their mode of action remains incompletely understood. Aged Fancd2−/− mice were used to assess the therapeutic efficacy of oxymetholone (OXM) and its mechanism of action. Eighteen-month-old Fancd2−/− mice recapitulated key human FA phenotypes, including reduced bone marrow cellularity, red cell macrocytosis, and peripheral pancytopenia. As in humans, chronic OXM treatment significantly improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells. RNA-Seq analysis implicated downregulation of osteopontin as an important potential mechanism for the drug’s action. Consistent with the increased stem cell proliferation, competitive repopulation assays demonstrated that chronic OXM therapy eventually resulted in stem cell exhaustion. These results expand our knowledge of the regulation of hematopoietic stem cell proliferation and have direct clinical implications for the treatment of bone marrow failure. : In this article, Zhang and colleagues show that 18-month-old Fancd2−/− mice recapitulated key human Fanconi anemia phenotypes, including peripheral pancytopenia and macrocytosis. Chronic oxymetholone treatment improved these hematological parameters and stimulated the proliferation of hematopoietic stem and progenitor cells, but eventually resulted in stem cell exhaustion. RNaseq analysis implicated downregulation of osteopontin as an important mechanism for the drug’s action

Ética de la investigación científica, humanística, tecnológica y artística universitarias

Este libro representa la vertiente del esfuerzo académico del CEI (Comité de Ética de la Investigación) para avanzar en la reflexión de la ética de la investigación universitaria que nutrirá el marco de referencia para mejorar la capacidad analítica en torno a la integridad científica. En este sentido, el lector encontrará en la presente obra aspectos conceptuales y epistémicos que permitan poner en escena la aplicación de altos valores y virtudes a la actividad de investigación a lo largo de todas sus fases y la promoción de la aplicación de sus resultados con responsabilidad social.Secretaría de Educación Pública - Subsecretaría de Educación Superior - Dirección General de Educación Superior Universitaria. Número del convenio con la SEP: 2017-15-001-017

Fancd2−/− mice have hematopoietic defects that can be partially corrected by resveratrol

Progressive bone marrow failure is a major cause of morbidity and mortality in human Fanconi Anemia patients. In an effort to develop a Fanconi Anemia murine model to study bone marrow failure, we found that Fancd2−/− mice have readily measurable hematopoietic defects. Fancd2 deficiency was associated with a significant decline in the size of the c-Kit+Sca-1+Lineage− (KSL) pool and reduced stem cell repopulation and spleen colony-forming capacity. Fancd2−/− KSL cells showed an abnormal cell cycle status and loss of quiescence. In addition, the supportive function of the marrow microenvironment was compromised in Fancd2−/− mice. Treatment with Sirt1-mimetic and the antioxidant drug, resveratrol, maintained Fancd2−/− KSL cells in quiescence, improved the marrow microenvironment, partially corrected the abnormal cell cycle status, and significantly improved the spleen colony-forming capacity of Fancd2−/− bone marrow cells. We conclude that Fancd2−/− mice have readily quantifiable hematopoietic defects, and that this model is well suited for pharmacologic screening studies