Feline leukemia virus integrase and capsid packaging functions do not change the insertion profile of standard Moloney retroviral vectors

Abstract

Adverse events linked to perturbations of cellular genes by vector insertion reported in gene therapy trials and animal models have prompted attempts to better understand the mechanisms directing viral vector integration. The integration profiles of vectors based on MLV, ASLV, SIV and HIV have all been shown to be non-random, and novel vectors with a safer integration pattern have been sought. Recently, we developed a producer cell line called CatPac that packages standard MoMLV vectors with feline leukemia virus (FeLV) gag, pol and env gene products. We now report the integration profile of this vector, asking if the FeLV integrase and capsid proteins could modify the MoMLV integration profile, potentially resulting in a less genotoxic pattern. We transduced rhesus macaque CD34+ hematopoietic progenitor cells with CatPac or standard MoMLV vectors, and determined their integration profile by LAM-PCR. We obtained 184 and 175 unique integration sites (ISs) respectively for CatPac and standard MoMLV vectors, and these were compared with 10 000 in silico-generated random IS. The integration profile for CatPac vector was similar to MoMLV and equally non-random, with a propensity for integration near transcription start sites and in highly dense gene regions. We found an IS for CatPac vector localized 715 nucleotides upstream of LMO-2, the gene involved in the acute lymphoblastic leukemia developed by X-SCID patients treated by gene therapy using MoMLV vectors. In conclusion, we found that replacement of MoMLV env, gag and pol gene products with FeLV did not alter the basic integration profile. Thus, there appears to be no safety advantage for this packaging system. However, considering the stability and efficacy of CatPac vectors, further development is warranted, using potentially safer vector backbones, for instance those with a SIN configuration.

Figure 1

Distribution of integration sites determined experimentally or in silico-generated in a 60 Kb window centered on Transcription Start Sites (TSS). The window of 60 Kb is subdivided into regions of 5 Kb and percentage of integration sites are reported for CatPac (black) and MoMLV (grey) vectors.

Figure 2

Occurrence of Common Integration Sites (CIS) in CD34+ cells transduced with CatPac or MoMLV vectors. According to the definition of Suzuki and coworkers, a second order CIS is defined as 2 or more IS located within a 30 kb window; a third order CIS as 3 or more IS within a 50 kb window; and a fourth order CIS as 4 or more IS within a 100 kb window. The black solid arrow indicates the number of second order CIS found in the experimental dataset obtained for CatPac vector (7). There was no third order CIS found in the CatPac experimental dataset. The solid grey and empty grey arrows respectively indicate the number of second (3) and third (1) order CIS found in the experimental dataset for MoMLV. These numbers can be compared to the CIS obtained for the 10 000 independent random datasets generated in silico to match the CatPac and MoMLV datasets. The X-axis gives the number of CIS and the Y-axis gives the number of random datasets, out of 10 000, that included second order (solid boxes) or third order (empty boxes) CIS. Black and grey boxes respectively represent data obtained for random sets matching CatPac and MoMLV vectors. There was no fourth order CIS found in any set (random or experimental).