Tissue- and tumor-specific targeting of murine leukemia virus-based replication-competent retroviral vectors

Abstract

Replication-competent retrovirus vectors based on murine leukemia virus (MLV) have been shown to effectively transfer therapeutic genes over multiple serial infections in cell culture and through solid tumors in vivo with a high degree of genomic stability. While simple retroviruses possess a natural tumor selectivity in that they can transduce only actively dividing cells, additional tumor-targeting strategies would nevertheless be advantageous, since tumor cells are not the only actively dividing cells. In this study, we used the promiscuous murine cytomegalovirus promoter, a chimeric regulatory sequence consisting of the hepatitis B virus enhancer II and the human alpha1-antitrypsin (EII-Pa1AT) promoter, and a synthetic regulatory sequence consisting of a series of T-cell factor binding sites named the CTP4 promoter to generate replicating MLV vectors, whereby the last two are transcriptionally restricted to liver- and beta-catenin/T-cell factor-deregulated cells, respectively. When the heterologous promoters were used to replace almost the entire MLV U3 region, including the MLV TATA box, vector replication was inefficient since nascent virus particle production from infected cells was greatly decreased. Fusion of the heterologous promoters lacking the TATA box to the MLV TATA box, however, generated vectors which replicated with almost-wild-type kinetics throughout permissive cells while exhibiting low or negligible spread in nonpermissive cells. The genomic stability of the vectors was shown to be comparable to that of a similar vector containing wild-type MLV long terminal repeats, and tropism analysis over repeated infection cycles showed that the targeted vectors retained their original specificity.

FIG. 1.

Transcriptional activities of mCMV, EII-Pa1AT, and CTP4 promoters in human cell lines. HepG2, HeLa, and 293 cells were stably transfected with plasmids driving eGFP expression under the control of the mCMV, EII-Pa1AT (E2), and CTP4 promoters, and expression levels were quantified by FACS analysis. The MFI values depicted are the means from three independent FACS measurements. Plasmid pEGFP1 is the promoterless plasmid control.

FIG. 2.

Vector construction. All constructs are based on the parental vector ACE-GFP, which is comprised of Moloney MLV containing the amphotropic 4070A env gene and from which eGFP expression is mediated by the encephalomyocarditis virus internal ribosome entry site (IRES) fused to the 3′ end of the 4070A env gene. The position of the inserted heterologous promoters is depicted by a black box, and its insertion is designed such that either the heterologous promoter lacking its TATA box is fused to the MLV TATA box (TF) or almost the entire MLV U3 region is deleted and replaced by the heterologous promoter, including its TATA box and transcriptional start site (TR). As a result of these modifications, the original 448-bp MLV U3 region in the parental vector ACE-GFP is either shortened to 299 bp and 402 bp in the case of vectors CTP4-TF and CTP4-TR, respectively, or lengthened to 492 bp and 489 bp, in the case of vectors E2-TF and E2-TR, respectively, and to 538 bp and 615 bp, in the case of vectors mCMV-TF and mCMV-TR, respectively.

FIG. 3.

Replication kinetics, virus production, infectivity, and transgene expression of targeted vectors in HepG2 cells. (A) HepG2 cells were infected with RCR vectors, and spread of virus was monitored by serial passaging and FACS analysis at each passage. The mean values from three independent experiments of the percentage of eGFP-positive cells at each passage are depicted. (B) Virus-containing supernatant was harvested from infected HepG2 cells and used to infect new HepG2 cells, and 50 μM azidothymidine was applied at 24 h postinfection to prevent secondary infection events. The quantity of integrated proviral copies per infected producer cell and the quantity of nascent virus released into the supernatant from each infected producer cell were determined by real-time PCR and real-time RT-PCR, respectively, using primers and probes binding in the rRNA genes and eGFP genes. Values shown indicate the number of nascent virions released per proviral copy in infected producer cells relative to the value obtained for ACE-GFP (black bars). The infectivity of nascent virus particles was quantified by FACS analysis at 2 days postinfection of HepG2 cells. The quantity of eGFP-positive cells generated per virus particle was calculated for each vector relative to the value obtained for ACE-GFP (gray bars). White bars represent the MFI of cells at 48 h postinfection relative to the values obtained for ACE-GFP.

FIG. 4.

Replication kinetics and transgene expression of targeted vectors. HepG2, SW480, DLD-1, AKH12, AKH13, HuH-7, HeLa, and 293 cells were infected with vectors (A) ACE-GFP, (B) CTP4-TF, and (C) E2-TF. Virus-containing supernatant (500 μl) was used to infect each cell line. Infected cells were passaged at regular intervals, and FACS analysis was performed at each passage. The values shown are the percentage of eGFP-positive cells at each passage. (D) The MFIs of eGFP-positive cells infected with ACE-GFP (black bars), CTP4-TF (gray bars), and E2-TF (white bars) are shown as the means from three independent experiments. Error bars represent the standard deviations between experiments.

FIG. 5.

Genomic stability of targeted vectors. HepG2 cells were infected with vectors ACE-GFP, CTP4-TF, and E2-TF at a multiplicity of infection of 0.01 and passaged until a maximum percentage of eGFP-positive cells was reached. The supernatant was harvested from infected cells at the second passage, diluted 100-fold, and used to initiate a new infection cycle. This was repeated for 13 serial infection cycles. (A) The values shown indicate the maximum percentage of eGFP-expressing cells reached in each infection cycle. (B to D) Genomic DNA was extracted from HepG2 cells infected with ACE-GFP (B), CTP4-TF (C), and E2-TF (D) at cycles 1 to 7, and PCR was performed using primers binding in the 3′ end of the env gene and 3′ U3 regions flanking the transgene cassette. Lane M, marker.

FIG. 6.

Promoter sequence and specificity over multiple infection cycles. (A) PCR was performed on genomic DNA extracted from HepG2 cells infected with ACE-GFP, CTP4-TF, and E2-TF at cycles 1 and 7, using primers flanking the heterologous promoter in the MLV LTR. The expected fragment sizes are indicated. Lane M, marker. (B) Vector tropism remains unchanged following multiple serial infection cycles. HepG2, 293, and HeLa cells were infected with the supernatant from the seventh infection cycle of HepG2 cells infected with ACE-GFP, CTP4-TF, and E2-TF. The cells were passaged every 3 days, and spread of virus was monitored by FACS analysis. The percentage of eGFP-positive cells at each passage is shown.