Mobilization and mechanism of transcription of integrated self-inactivating lentiviral vectors

Abstract

Permanent genetic modification of replicating primitive hematopoietic cells by an integrated vector has many potential therapeutic applications. Both oncoretroviral and lentiviral vectors have a predilection for integration into transcriptionally active genes, creating the potential for promoter activation or gene disruption. The use of self-inactivating (SIN) vectors in which a deletion of the enhancer and promoter sequences from the 3' long terminal repeat (LTR) is copied over into the 5' LTR during vector integration is designed to improve safety by reducing the risk of mobilization of the vector genome and the influence of the LTR on nearby cellular promoters. Our results indicate that SIN vectors are mobilized in cells expressing lentiviral proteins, with the frequency of mobilization influenced by features of the vector design. The mechanism of transcription of integrated vector genomes was evaluated using a promoter trap design with a vector encoding tat but lacking an upstream promoter in a cell line in which drug resistance depended on tat expression. In six clones studied, all transcripts originated from cryptic promoters either upstream or within the vector genome. We estimate that approximately 1 in 3,000 integrated vector genomes is transcribed, leading to the inference that activation of cryptic promoters must depend on local features of chromatin structure and the constellation of nearby regulatory elements as well as the nature of the regulatory elements within the vector.

FIG. 1.

Mobilization of a SIN lentiviral vector genome. (A.) Medium conditioned by 293T cells which had been multiply transduced with MpGFP vector particles at a high multiplicity of infection after transfection with the helper packaging plasmids. (B.) Medium conditioned by control cells which had not been transduced with the vector particles. (C.) Medium conditioned by transduced cells which were not transfected with the plasmids was titrated on HeLa cells. Vector mobilization was accessed by flow cytometry 5 days or 14 days after exposure of the HeLa cells to the conditioned medium.

FIG. 2.

Impact of modifications of the HIV-1 LTR-U3 region of the SIN vector on the apparent mobilized titer. Shown above is the diagram of the wild-type U3 region showing the protein binding sites that have been defined (5). Additional deletions of the residual sequences of the U3 region in the SIN vector were performed as described in Materials and Methods. A 75-bp fragment from the SV40 late polyadenylation region containing the upstream efficiency enhancer for polyadenylation was added to the lower vector as described in the text. n is the number of replicates for each determination.

FIG. 3.

Influence of vector design on mobilization of an integrated SIN lentiviral vector. Aliquots of 293T cells were transduced multiple times with each of the vectors individually at high multiplicities of infection to develop subpopulations containing 80 to 112 copies of the vector genome. After passage, these cell populations were transfected with the viral packaging plasmids and conditioned medium was titrated on HeLa cells. In each case, the mobilized titer was corrected for copy number in the 293T cells to yield the data displayed in the right panel. Duplicate populations of 293T cells were derived for each of the vectors, and the transfection experiment and evaluation of the mobilized titer were performed on each three separate times. Thus, the data displayed represent the mean and standard deviation of six independent determinations of the mobilized titer for each vector. The data are expressed as the titer of conditioned medium in transducing units/ml/100 copies of the vector genome.

FIG. 4.

Mobilized vector particles integrate an intact proviral genome in transduced cells. Southern blot analysis was performed on DNA samples from individual clones. The DNA samples were digested with BbsI, which cuts once in each proviral LTR, yielding a 3.3-kb fragment.

FIG. 5.

The 5′ LTRs of integrated proviral genomes derived from mobilized vector particles are intact. PCR analysis was performed on DNA extracted from individual clones using primers positioned as shown on the diagram on the bottom of the figure. In each case, a 417-bp band predicted by the primer set was obtained. The control DNA was derived from a pool of 293T cells which had been transduced with vector particles at an MOI of 1.

FIG. 6.

Transcription of lentiviral self-inactivating genomes. Segments of the HIV genome encoding tat were introduced into several vectors as shown upstream from the internal promoter. Conventional titers of preparations of the individual vectors were determined by transfer of the GFP marker into naïve HeLa cells, whereas the tat titers of these preparations were determined by scoring puromycin-resistant colonies after transduction of a HeLa cell line in which the puromycin expression cassette was under the control of the wild-type HIV LTR. n is the number of independent replicators of each assay.

FIG. 7.

Influence of vector design on genome transcription as reflected by the production of tat. The panel on the left shows the mean and standard deviation of the titers of preparations of each of the vectors (n = 6). The parental vector is MpGFP and the enhancer-deleted vector is mMpGFP, whereas the insulator vectors containing the chicken hypersensitive site 4 (cHS4) from the β-globin locus are designated INS1R MpGFP or INS1L MpGFP for the forward and reverse orientations, respectively. The vector containing the EF1α promoter plus intron is designated EF1αGFP. Shown on the right is the capacity of vector preparations of each to generate puromycin-resistant colonies when used to transduce the tat expression indicator cell line, HeLa-PUR. In each case the normalized tat titer reflects the number of puromycin-resistant colonies observed with serial dilutions of each vector preparation normalized, based on the GFP titer, to yield the data shown in the right panel. Six replicates were performed to derive the tat titers.

FIG. 8.

Mapping of the 5′ ends of tat-encoding transcripts. (A.) Analysis on the 2% agarose gel detected longer transcripts in clones 4, 5, and 7 as well as shorter species in clones 2 through 6. (B.) The SpreadexEX800 gel further defined multiple shorter species in all clones.

FIG. 9.

Localization of the 5′ start sites for tat-encoding transcripts. Multiple start sites were identified for each clone by shotgun cloning of the 5′-RACE products followed by sequencing of the junctions. The sizes shown for clones 4, 5, and 7 are the distances from the transcriptional start site to the 5′ end of the LTR.