Comparison of Immortalization Potential of Gamma-Retroviral, Lentiviral and Foamy Virus Gene Transfer Vectors
Authors: Ikawa Yasuhiro , Uchiyama Toru , Jagadeesh Guridevi Jayashree , Candotti Fabio 
Published: October 2019

Abstract 3116

Therapeutic gene transfer has been used successfully to treat a variety of human genetic diseases. Although protocols have shown positive clinical outcomes, the success of clinical trials were tempered by adverse events, in which integration of the viral vectors increased transcription of cancer-related genes and thereby contributed to development of leukemia. In all documented cases of insertional mutagenesis, the viral vectors used contained full-length, gamma-retrovirus long terminal repeats (LTRs) that are able to exert strong promoter and enhancer activity driving not only the expression of the transgene carried by the vector, but also that of genes neighboring the insertion site. Assessing safety of integrating viral vectors for future clinical use is therefore of paramount importance.

In preparation for gene therapy approaches for the Wiskott-Aldrich syndrome (WAS), we used an in vitro assay of murine bone marrow (BM) cell immortalization to compare the consequences of transduction by four different kinds of viral vectors, including a full-length LTR Moloney leukemia virus (MLV), a self-inactivating MLV carrying a 3' LTR deleted of the viral enhancer region (SIN), a lentivirus (LV), and a foamy virus (FV) construct. All vectors carried EGFP under the control of a ubiquitously acting chromatin opening element (UCOE) or the WAS endogenous promoter (WASp). In this assay, BM cells are harvested from C57BL6 mice, exposed to retroviral supernatants and cultured long-term. Derived lines are considered immortalized based on their ability to continue to grow in vitro for more than 6 weeks in the presence of interleukin-3 and stem cell factor.

To date, MLV and SIN transduction of 123 and 132 cultures, respectively, gave rise to 48 and 43 immortalized lines (39.0% and 32.6% immortalization rate). The difference in immortalization rate between MLV and SIN vectors was not statistically significant (Chi square: p=0.30). As expected, immortalized cells were negative/low for IgER, cKit and Sca1 expression, and positive to different degrees for expression of the myeloid markers CD11b/Mac1 and Ly6g/Gr1. Transduction of 114 and 62 cultures with LV and FV vectors, respectively, resulted in no immortalized lines.

Real-time PCR was performed to evaluate transduction efficiency of bone marrow cells and immortalized lines. Integrated vector sequences in bone marrow cells transduced by LV and FV were detected in significantly higher quantities than in cells transduced with MLV and SIN vectors. However, expression of the EGFP transgene was markedly reduced in LV- and FV-transduced cells compared to cells exposed to MLV vectors (MFI: 14.0, 1.88, 93.2, respectively).

These preliminary results confirm that gamma-retroviral gene transfer vectors are prone to causing immortalization of hematopoietic cells and suggest the vectors based on LV and FV backbones may be safer alternatives for WAS and other genetic disorders, provided that effective gene expression levels can be achieved in the biological model system under study.Table.Summary of immortalization results using MLV, SIN, LV and FV vectorsVirusMOITransduction efficiencyTransduction experiments% ImmortalizationMLV/UCOE/EGFP2067%5643MLV/WASp/EGFP2065%6736SIN/UCOE/EGFP532%7236SIN/WASp/EGFP537%6028LV/UCOE/EGFP1040%570LV/WASp/EGFP1049%570FV/UCOE/EGFP1027%280FV/WASp/EGFP1022%340negativeN.A.N.A.720

Disclosures:

No relevant conflicts of interest to declare.