RESUMO
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.
Assuntos
Cromatina/genética , Inativação Gênica , Vetores Genéticos/genética , Lentivirus/genética , Splicing de RNA , Animais , Linhagem Celular , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Terapia Genética/instrumentação , Vetores Genéticos/metabolismo , Ribonucleoproteínas Nucleares Heterogêneas Grupo A-B/genética , Humanos , Lentivirus/metabolismo , Camundongos , Mutagênese InsercionalRESUMO
Insertional mutagenesis by viral vectors is a problem in gene therapy. We recently reported that lentiviral vectors with an intact HIV long terminal repeat (LTR) caused insertional gene activation by transcripts from the 5' LTR splicing to an adjacent gene. Here we demonstrate that the level of transcription from the 5' LTR, and also insertional gene activation, is dependent on the internal promoter in the vector. We also show that there are more transcripts originating from the 5' LTR than from, or reading through, the 3' LTR. This study will allow the design of safer lentiviral vectors for applications in which an intact HIV LTR is required.
Assuntos
Vetores Genéticos , Repetição Terminal Longa de HIV/genética , Lentivirus/genética , Mutagênese Insercional , Regiões Promotoras Genéticas , Ativação Transcricional , Linhagem Celular , Perfilação da Expressão Gênica , Terapia Genética/efeitos adversos , Humanos , Transcrição GênicaRESUMO
Gammaretroviral and lentiviral vectors are promising tools for gene therapy, but they can be oncogenic. The development of safer vectors depends on a quantitative assay for insertional mutagenesis. Here we report a rapid, inexpensive, and reproducible assay which uses a murine cell line to measure the frequency of interleukin-3 (IL-3)-independent mutants. Lentiviral and gammaretroviral vectors cause insertional mutagenesis at similar frequencies; however, they use different mechanisms. Human immunodeficiency virus (HIV)-based vectors generate mutants by insertion only into the growth hormone receptor (Ghr) locus. The HIV enhancer/promoter is active in the absence of the HIV Tat protein in this locus, and an HIV/Ghr spliced transcript expresses GHR and cells respond to GH. Deletion of the enhancer/promoter in a self-inactivating HIV-based vector prevents this mechanism of insertional mutagenesis. In contrast, gammaretroviral vectors insert into other loci, including IL-3 and genes identified as common insertion sites in the Retroviral Tagged Cancer Gene Database (RTCGD).