Alpharetroviral self-inactivating vectors: long-term transgene expression in murine hematopoietic cells and low genotoxicity

Abstract

Comparative integrome analyses have highlighted alpharetroviral vectors with a relatively neutral, and thus favorable, integration spectrum. However, previous studies used alpharetroviral vectors harboring viral coding sequences and intact long-terminal repeats (LTRs). We recently developed self-inactivating (SIN) alpharetroviral vectors with an advanced split-packaging design. In a murine bone marrow (BM) transplantation model we now compared alpharetroviral, gammaretroviral, and lentiviral SIN vectors and showed that all vectors transduced hematopoietic stem cells (HSCs), leading to comparable, sustained multilineage transgene expression in primary and secondary transplanted mice. Alpharetroviral integrations were decreased near transcription start sites, CpG islands, and potential cancer genes compared with gammaretroviral, and decreased in genes compared with lentiviral integrations. Analyzing the transcriptome and intragenic integrations in engrafting cells, we observed stronger correlations between in-gene integration targeting and transcriptional activity for gammaretroviral and lentiviral vectors than for alpharetroviral vectors. Importantly, the relatively "extragenic" alpharetroviral integration pattern still supported long-term transgene expression upon serial transplantation. Furthermore, sensitive genotoxicity studies revealed a decreased immortalization incidence compared with gammaretroviral and lentiviral SIN vectors. We conclude that alpharetroviral SIN vectors have a favorable integration pattern which lowers the risk of insertional mutagenesis while supporting long-term transgene expression in the progeny of transplanted HSCs.

Figure 1

Vectors used in this study (edited from ref. 13). (a) Nucleotide sequences of the unique 3′ (U3) region of alpharetroviral self-inactivating (SIN) vectors used in a previous study and in this study (noTATA). The site of sequence deletion is indicated by a dashed line. The nucleotide sequences of the introduced SnaBI restriction site, as well as those of the TATA box and the polyadenylation signal (pA signal), are shown. (b) Schema of lentiviral, gammaretroviral, and alpharetroviral SIN vectors used in this experiment. Indicated are the long-terminal repeat (LTR) (ΔU3, R, and U5), direct repeat element (DRE), packaging signal (ψ), splice donor, and acceptor site (SD and SA), spleen focus-forming virus (SFFV) promoter, enhanced green fluorescent protein (EGFP), and woodchuck hepatitis virus post-transcriptional regulatory element (wPRE), Rev-responsive element (RRE), polypurine tract (PPT), and remaining viral gag-coding sequences (Δgag).

Figure 2

Enhanced green fluorescent protein (EGFP) expression in transduced cells. (a) EGFP expression in transduced murine lineage-negative cells. Indicated are percentages of EGFP⁺ cells and mean fluorescence intensities (MFIs). Exemplary flow-cytometry plots of untransduced cells are shown in comparison to cells transduced with the alpharetroviral self-inactivating (SIN) vector 1 day and 9 days after transduction. Also provided are time-series of flow-cytometric analyses of transduced cells by all three vectors from 1 day to 9 days after transduction. (b) EGFP⁺ cells in different lineages of peripheral blood. From left to right: EGFP⁺ cells in CD11b⁺ cells, in CD19⁺ cells, or in CD3ε⁺ cells. Depicted are mean values + SD; n = 8 for primary recipients and n = 5 for secondary recipients. (c) EGFP MFI in different lineages of peripheral blood. From left to right: EGFP MFI in CD11b⁺ cells, in CD19⁺ cells, or in CD3ε⁺ cells. Depicted are mean values + SD; n = 8 for primary recipients and n = 5 for secondary recipients. (d) EGFP⁺ cells and EGFP MFI in transduced human X-CGD PLB985 cells during long-term culture in vitro.

Figure 3

Integration site analyses. (a) Integrations near transcription start sites. Shown are the percentages of integration sites near transcriptions start sites of adjacent protein-coding genes within a distance of 10 kb. (b) Integrations near CpG islands. Shown are the percentages of integration sites near CpG islands within a distance of 10 kb. (c) Integrations in genes. (d) Integrations in proximity of genes with implications in cancer. Shown are the percentages of integrations in 250 kb proximity to retrovirus tagged cancer gene database (RTCGD) and cancer-gene census database (CGC) annotated cancer genes. Vertical lines indicate percentages of integrations for a random reference sample provided by QuickMap. n = numbers of nonredundant reads: alpha pre n = 107; alpha in vitro n = 68; alpha 1°tx n = 165; alpha 1°tx*** n = 125; alpha 2°tx n = 46; gamma pre n = 74; gamma in vitro n = 79; gamma 1°tx n = 67; gamma 1°tx*** n = 39; gamma 2°tx n = 33; lenti pre n = 39; lenti in vitro n = 70; lenti 1°tx n = 442; lenti 1°tx*** n = 206; lenti 2°tx n = 93. Statistical significance was tested with Fisher's exact test, corrected for multiple testing by the Benjamini–Hochberg method. Indicated are gammaretroviral or lentiviral data bars which are significantly different from alpharetroviral data bars. *P = 0.01–0.05, **P = 0.01–0.001, ***P < 0.001.

Figure 4

Integrations in highly expressed genes. Shown are integrations in highly expressed genes (Log2 expression value >6.5). Statistical significance was tested with Fisher's exact test. The vertical line indicates the percentage of integrations in highly expressed genes for random integration targeting. n = numbers of intragenic integrations/numbers of intragenic integrations on array: alpha 1°tx n = 78/73; alpha 2°tx n = 22/19; gamma 1°tx n = 26/25; gamma 2°tx n = 11/10; lenti 1°tx n = 295/266; lenti 2°tx n = 74/69. Indicated are gammaretroviral or lentiviral data bars which are significantly different from alpharetroviral data bars. **P = 0.01–0.001, ***P < 0.001.

Figure 5

IVIM assay. (a) Replating frequencies normalized to mean vector copy numbers. We analyzed the replating frequencies (RF) of cells transduced with alpharetroviral, gammaretroviral, and lentiviral self-inactivating (SIN) vectors. Plotted are the RFs corrected for mean vector copy numbers (mVCNs) measured 4 days after transduction. Data points were either obtained in this study (black) or have been published previously (gray). Numbers next to alpharetroviral data-points are identifiers of wells with cells recovered after replating (Table 2). Horizontal bars indicate the median of all positive assays for a given vector. Statistical significance was tested with Fisher's exact test on the number of positive and negative assays. *P = 0.01–0.05. The horizontal dashed line at RF₁₀₀/mVCN = 0.0001 separates robustly replating from weakly replating clones. (b) Expression levels of Evi1 or Prdm16 in recovered cells after replating. RNA was purified from expanded cells after replating, reverse transcribed, and subjected to quantitative PCR (qPCR) for detection of Evi1 or Prdm16 transcripts. Target gene expression levels were determined, normalized to actin expression levels, and are depicted in reference to untransduced cells harvested before replating. Data not in measurable range; dashed lines separate clones recovered from independent plates/assays. Depicted are technical triplicate mean values + SD.