Tissue-specific transcriptional targeting of a replication-competent retroviral vector

Abstract

The inability of replication-defective viral vectors to efficiently transduce tumor cells in vivo has prevented the successful application of such vectors in gene therapy of cancer. To address the need for more efficient gene delivery systems, we have developed replication-competent retroviral (RCR) vectors based on murine leukemia virus (MLV). We have previously shown that such vectors are capable of transducing solid tumors in vivo with very high efficiency. While the natural requirement of MLV infection for cell division imparts a certain degree of specificity for tumor cells, additional means for confining RCR vector replication to tumor cells are desirable. Here, we investigated the parameters critical for successful tissue-specific transcriptional control of RCR vector replication by replacing various lengths of the MLV enhancer/promoter with sequences derived either from the highly prostate-specific probasin (PB) promoter or from a more potent synthetic variant of the PB promoter. We assessed the transcriptional specificity of the resulting hybrid long terminal repeats (LTRs) and the cell type specificity and efficiency of replication of vectors containing these LTRs. Incorporation of PB promoter sequences effectively restricted transcription from the LTR to prostate-derived cells and imparted prostate-specific RCR vector replication but required the stronger synthetic promoter and retention of native MLV sequences in the vicinity of the TATA box for optimal replicative efficiency and specificity. Our results have thus identified promoter strength and positioning within the LTR as important determinants for achieving both high transduction efficiency and strict cell type specificity in transcriptionally targeted RCR vectors.

FIG. 1.

Constructs used in this study. (A) Sequences used in generating hybrid LTRs. The proximal rat PB promoter (left) contains CAAT and TATA box homologies and an ARR (shown as a hatched box) important for androgen induction of transcription. ARR₂PB (center) is a synthetic variant of the PB promoter and contains two copies of the ARR. The MLV LTR (right) comprises the U3, R, and U5 regions. The transcriptional control sequences of MLV are located primarily in the U3 region, which also contains CAAT and TATA box sequences. (B) Hybrid LTRs containing the wt PB promoter. LTRs Pr, Pt, and Pc contain PB promoter sequences from position −383 to the transcription start site (TSS), TATA box, and CAAT box, respectively. (C) Hybrid LTRs containing ARR₂PB. LTRs Ar, At, and Ac contain ARR₂PB sequences from the 5′ end of the upstream ARR to the TSS, TATA box, and CAAT box, respectively. In each of the six hybrid LTRs, MLV U3 sequences from the NheI site to the TSS, TATA box, or CAAT box were replaced with the corresponding PB or ARR₂PB sequences. (D) Sequence details of the hybrid LTRs. Shown are the nucleotide sequences at the 3′ borders between the PB and MLV sequences. TATA and CAAT boxes are underlined. TSS, transcription start site. (E) Luciferase reporter constructs containing hybrid LTRs. (F) Structure of replication-competent MLV vectors containing hybrid LTRs. Each vector contains an IRES-GFP cassette positioned immediately downstream of the env gene and a 5′ LTR in which the U3 region was replaced by the CMV immediate-early promoter. The 3′ LTR is used to form the 5′ LTR during MLV replication. We therefore replaced the 3′ LTR of the RCR vector with the hybrid LTRs.

FIG. 2.

Cell type specificity and androgen inducibility of transcription from hybrid LTRs containing wt PB promoter or ARR₂PB. Each of the hybrid LTRs was cloned upstream of the luciferase gene in plasmid pGL2 and transfected into two prostate (A and B) and two nonprostate (C and D) cell lines as indicated. Transcriptional activity was determined in the absence (black bars) and presence (open bars) of androgen. All cells were cultured in media containing serum that was charcoal stripped to remove endogenous steroids, and androgen induction was carried out by the addition of 1 nM DHT. Arbitrary relative light unit (RLU) levels were normalized for transfection efficiency, and the level determined for the promoterless control reporter plasmid in each cell line was assigned a value of 1. Results are the means obtained from at least three independent experiments. Error bars indicate standard deviations.

FIG. 3.

Replication of wt PB promoter-targeted vectors in prostate and nonprostate cells. GFP expression in infected cells was assessed by flow cytometry on the indicated days postinfection. Results from infections of LNCaP (A), MDA PCa 2b (B), HeLa (C), and NMU (D) cells are shown. All cells were grown in nonstripped serum, and no exogenous androgen was added. Values are the means of three experiments.

FIG. 4.

Replication of ARR₂PB-targeted vectors in prostate and nonprostate cells. GFP expression in infected cells was assessed by flow cytometric analysis on the indicated days postinfection. Results from infections of LNCaP (A), MDA PCa 2b (B), HeLa (C), and NMU (D) cells are shown. All cells were grown in medium containing nonstripped serum, and no exogenous androgen was added. Values are the means of three experiments.

FIG. 5.

Southern blot analysis of genomic DNA from LNCaP cells infected with ARR₂PB-targeted vectors. Each DNA sample was digested with NheI and SphI, separated on an agarose gel, and blotted onto a nylon membrane. The membrane was hybridized to a radiolabeled pol-env fragment of MLV. mock, DNA from mock-infected cells; control, NheI/SphI-digested pAZE-GFP plasmid with DNA from mock-infected cells. A schematic diagram of integrated vector provirus indicating the size of restriction fragments for each vector and the location of the probe is shown.

FIG. 6.

Hybrid LTR of ACE-GFP-At remains unchanged through multiple serial passages. A PCR-based assay designed to assess the stability of the hybrid LTRs by amplification and sequencing of the 5′ LTR from proviral DNA was utilized. Amplification of a cloned proviral copy of ACE-GFP-At as a positive control resulted in products of approximately 720 and 510 bp (left gel). As depicted schematically, sequencing demonstrated that these products corresponded to the full-length, intact At LTR and an artifactual form of the At LTR missing one of the two ARRs, respectively. ACE-GFP-At was subjected to seven cell-free serial passages through LNCaP cultures, and genomic DNA from each culture was used as the template in PCR amplification and sequencing of the LTR (right gel). Sequencing of the two resulting products from selected passages demonstrated that their sequences were identical to those of the products generated by amplification of the positive control template. Abbreviations: L, 100-bp ladder; P, amplification using a plasmid containing a cloned proviral copy of ACE-GFP-At as the template; N, amplification using DNA from uninfected cells.