ABSTRACT
Comparative integrome analysis has revealed that the most neutral integration pattern among retroviruses is attributed to alpharetroviruses. We chose X-linked chronic granulomatous disease (X-CGD) as model to evaluate the potential of self-inactivating (SIN) alpharetroviral vectors for gene therapy of monogenic diseases. Therefore, we combined the alpharetroviral vector backbone with the elongation factor-1α short promoter, both considered to possess a low genotoxic profile, to drive transgene (gp91(phox)) expression. Following efficient transduction transgene expression was sustained and provided functional correction of the CGD phenotype in a cell line model at low vector copy number. Further analysis in a murine X-CGD transplantation model revealed gene-marking of bone marrow cells and oxidase positive granulocytes in peripheral blood. Transduction of human X-CGD CD34+ cells provided functional correction up to wild-type levels and long-term expression upon transplantation into a humanized mouse model. In contrast to lentiviral vectors, no aberrantly spliced transcripts containing cellular exons fused to alpharetroviral sequences were found in transduced cells, implying that the safety profile of alpharetroviral vectors may extend beyond their neutral integration profile. Taken together, this highlights the potential of this SIN alpharetroviral system as a platform for new candidate vectors for future gene therapy of hematopoietic disorders.
Subject(s)
Alpharetrovirus/genetics , Genetic Therapy/methods , Genetic Vectors , Granulomatous Disease, Chronic/therapy , RNA Splicing , Animals , Bone Marrow Cells , Cell Line, Tumor , DNA Copy Number Variations , Disease Models, Animal , Granulocytes , Granulomatous Disease, Chronic/genetics , Humans , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , NADPH Oxidase 2 , NADPH Oxidases/genetics , NADPH Oxidases/metabolism , Phenotype , Reverse Transcriptase Polymerase Chain Reaction , Transduction, Genetic , TransgenesABSTRACT
Gammaretroviral and lentiviral vectors have been used successfully in several clinical gene therapy trials, although powerful enhancer elements have caused insertional mutagenesis and clonal dysregulation. Self-inactivating vectors with internal heterologous regulatory elements have been developed as potentially safer and more effective alternatives. Lentiviral vectors containing a ubiquitous chromatin opening element from the human HNRPA2B1-CBX3 locus (A2UCOE), which allows position-independent, long-term transgene expression, are particularly promising. In a recently described assay, aberrantly spliced mRNA transcripts initiated in the vector A2UCOE sequence were found to lead to upregulation of growth hormone receptor gene (Ghr) expression in transduced murine Bcl-15 cells. Aberrant hybrid mRNA species formed between A2UCOE and a number of other cellular genes were also detected in transduced human PLB-985 myelomonocytic cells. Modification of the A2UCOE by mutation or deletion of recognized and potential cryptic splice donor sites was able to abrogate these splicing events and hybrid mRNA formation in Bcl-15 cells. This modification did not compromise A2UCOE regulatory activity in terms of resistance to CpG methylation and gene silencing in murine P19 embryonic carcinoma cells. These refined A2UCOE regulatory elements are likely to improve intrinsic biosafety and may be particularly useful for a number of clinical applications where robust gene expression is desirable.