Lentivirus vectors in ß-thalassemia.

Patients with ß-thalassemia major require lifelong transfusions and iron chelation, regardless of the type of causative mutations (e.g., ß°, ß(E)/ß°). The only available curative therapy is allogeneic hematopoietic transplantation, although most patients do not have an HLA-matched, geno-identical donor, and those who do still risk graft-versus-host disease. Hence, gene therapy by ex vivo transfer of a functional ß-globin gene is an attractive novel therapeutic modality. In ß-thalassemia, transfer of a therapeutic globin gene does not confer a selective advantage to transduced stem cells, and complex DNA regulatory sequences have to be present within the transfer vector for proper expression. This is why lentiviral vectors have proven especially suited for this application, and the first Phase I/II human clinical trial was initiated. Here, we report on the first gene therapy patient with severe ß(E)/ß°-thalassemia, who has become transfusion-independent, and provide methods and protocols used in the context of this clinical trial.

Distribution of lentiviral vector integration sites in mice following therapeutic gene transfer to treat ß-thalassemia.

A lentiviral vector encoding ß-globin flanked by insulator elements has been used to treat ß-thalassemia (ß-Thal) successfully in one human subject. However, a clonal expansion was observed after integration in the HMGA2 locus, raising the question of how commonly lentiviral integration would be associated with possible insertional activation. Here, we report correcting ß-Thal in a murine model using the same vector and a busulfan-conditioning regimen, allowing us to investigate efficacy and clonal evolution at 9.2 months after transplantation of bone marrow cells. The five gene-corrected recipient mice showed near normal levels of hemoglobin, reduced accumulation of reticulocytes, and normalization of spleen weights. Mapping of integration sites pretransplantation showed the expected favored integration in transcription units. The numbers of gene-corrected long-term repopulating cells deduced from the numbers of unique integrants indicated oligoclonal reconstitution. Clonal abundance was quantified using a Mu transposon-mediated method, indicating that clones with integration sites near growth-control genes were not enriched during growth. No integration sites involving HMGA2 were detected. Cells containing integration sites in genes became less common after prolonged growth, suggesting negative selection. Thus, ß-Thal gene correction in mice can be achieved without expansion of cells harboring vectors integrated near genes involved in growth control.

Transfusion independence and HMGA2 activation after gene therapy of human ß-thalassaemia.

The ß-haemoglobinopathies are the most prevalent inherited disorders worldwide. Gene therapy of ß-thalassaemia is particularly challenging given the requirement for massive haemoglobin production in a lineage-specific manner and the lack of selective advantage for corrected haematopoietic stem cells. Compound ß(E)/ß(0)-thalassaemia is the most common form of severe thalassaemia in southeast Asian countries and their diasporas. The ß(E)-globin allele bears a point mutation that causes alternative splicing. The abnormally spliced form is non-coding, whereas the correctly spliced messenger RNA expresses a mutated ß(E)-globin with partial instability. When this is compounded with a non-functional ß(0) allele, a profound decrease in ß-globin synthesis results, and approximately half of ß(E)/ß(0)-thalassaemia patients are transfusion-dependent. The only available curative therapy is allogeneic haematopoietic stem cell transplantation, although most patients do not have a human-leukocyte-antigen-matched, geno-identical donor, and those who do still risk rejection or graft-versus-host disease. Here we show that, 33 months after lentiviral ß-globin gene transfer, an adult patient with severe ß(E)/ß(0)-thalassaemia dependent on monthly transfusions since early childhood has become transfusion independent for the past 21 months. Blood haemoglobin is maintained between 9 and 10 g dl(-1), of which one-third contains vector-encoded ß-globin. Most of the therapeutic benefit results from a dominant, myeloid-biased cell clone, in which the integrated vector causes transcriptional activation of HMGA2 in erythroid cells with further increased expression of a truncated HMGA2 mRNA insensitive to degradation by let-7 microRNAs. The clonal dominance that accompanies therapeutic efficacy may be coincidental and stochastic or result from a hitherto benign cell expansion caused by dysregulation of the HMGA2 gene in stem/progenitor cells.

A potential antitumor peptide therapeutic derived from antineoplastic urinary protein.

New therapies in cancer treatment are focusing on multifaceted approaches to starve and kill tumors utilizing both antiangiogenic and chemotherapeutic compounds. Antineoplastic Urinary Protein (ANUP), a 32k Da protein normally secreted in human urine, has been previously described as a molecule possessing both antiproliferative and antiangiogenic activities. Two synthetic peptides complimentary to the N-terminus of ANUP were designed to test their ability to reproduce these beneficial effects but ultimately to provide a more useful small molecule therapeutic. The results show that the peptides reduced tumor burden by up to 70% in a nude mouse model and demonstrated the ability to inhibit blood vessel formation in a chick chorioallantoic membrane assay (CAM).