Adenovirus-retrovirus hybrid vectors achieve highly enhanced tumor transduction and antitumor efficacy in vivo

Abstract

Murine leukemia virus (MLV)-based replication-competent retrovirus (RCR) vectors have been shown to mediate efficient, selective, and persistent tumor transduction, thereby achieving significant therapeutic benefit in a wide variety of cancer models. To further augment the efficiency of this strategy, we have developed a delivery method employing a gutted adenovirus encoding an RCR vector (AdRCR); thus, tumor cells transduced with the adenoviral vector transiently become RCR vector producer cells in situ. As expected, high-titer AdRCR achieved significantly higher initial transduction levels in human cancer cells both in vitro and in vivo, as compared to the original RCR vector itself. Notably, even at equivalent initial transduction levels, more secondary RCR progeny were produced from AdRCR-transduced cells as compared to RCR-transduced cells, resulting in further acceleration of subsequent RCR replication kinetics. In pre-established tumor models in vivo, prodrug activator gene therapy with high-titer AdRCR could achieve enhanced efficacy compared to RCR alone, in a dose-dependent manner. Thus, AdRCR hybrid vectors offer the advantages of high production titers characteristic of adenovirus and secondary production of RCR in situ, which not only accelerates subsequent vector spread and progressive tumor transduction, but can also significantly enhance the therapeutic efficacy of RCR-mediated prodrug activator gene therapy.

Figure 1

Adenovirus–RCR hybrid vector (AdRCR) design and validation. (a) Structure of AdRCR vectors. Upper: hybrid vector AdRCR-GFP, a helper-dependent adenoviral vector encoding RCR-GFP. Lower: hybrid vector AdRCR-CD, encoding RCR-CD. The adenoviral backbone of both AdRCR vectors contains a βgal reporter cassette situated outside the RCR sequence, which therefore serves as an independent marker of adenoviral transduction. (b) Principle of two-stage transduction with AdRCR vectors. AdRCR-GFP is shown as an example: the first-stage HDAd vector infects primary target cells efficiently as an adenovirus, and transiently produces RCR vectors in situ (indicated by βgal⁺ GFP⁺ cells). The released second-stage RCR vector then infects surrounding secondary target cells and undergoes permanent genomic integration, resulting in stable gene expression of its associated transgene (indicated by βgal⁻ GFP⁺ cells), and further replicative spread. (c) Experimental design to validate RCR production following AdRCR infection. MDA-MB-231 cells inoculated with AdRCR-GFP vector (multiplicity of infection of 0.1) were maintained in replicate culture and passaged, and examined for expression of βgal and GFP at serial time points. (d) Analysis of transduced cells following AdRCR infection. At serial time points as indicated, aliquots of transduced cells were analyzed for βgal expression by X-gal staining, and for GFP by fluorescence microscopy and flow cytometry. Inset numbers indicate quantitation of βgal⁺ cells by imaging software or GFP⁺ cells by flow cytometry. Ad-ITR, adenovirus inverted terminal repeat sequence; βgal, β-galactosidase gene; CD, cytosine deaminase; CMV, cytomegalovirus promoter; gag-pol/env, amphotropic MLV coding sequences; GFP, green fluorescent protein; IRES, internal ribosome entry site; PGK, phosphoglycerate kinase promoter; ψ, packaging signal; RCR, replication-competent retrovirus.

Figure 2

In vitro production and spread of RCR following AdRCR infection. (a) Time course of RCR production from AdRCR-GFP-infected MDA-MB-231 cells. Conditioned medium from cells infected with AdRCR-GFP (MOI = 0.125, 0.25, and 0.5) or RCR (MOI =0.3) was harvested and replenished daily for 6 days, and GFP titers on naive MDA-MB-231 cells were determined in the presence of AZT. Data shown are mean ± SD from experiments performed in triplicate. (b) Replication kinetics of secondary RCR vectors following RCR versus AdRCR infection. MDA-MB-231 cells were inoculated with RCR-GFP versus AdRCR-GFP vector at MOI 0.01 or 0.1. On the days indicated, cells were passaged and analyzed for GFP expression by flow cytometry. Data shown are mean ± SD, experiments performed in triplicate. AdRCR, adenovirus–RCR hybrid vector; GFP, green fluorescent protein; MOI, multiplicity of infection; RCR, replication-competent retrovirus; TU, transducing units.

Figure 3

AdRCR-mediated gene transfer in vivo in a subcutaneous breast cancer model. (a) Green fluorescent protein (GFP) versus β-galactosidase (βgal) expression in vivo after intratumoral injection of AdRCR-GFP into subcutaneous MDA-MB-231 human breast cancer xenografts on day 0. On day 4 and 10, tumors digested into cell suspensions were immediately analyzed for expression of βgal (bar graph) and GFP (line graph). Data shown are mean ± SD. (b) GFP expression in vivo on day 4 and day 10 after intratumoral injection of AdRCR-GFP (open circles) versus RCR-GFP (solid circles) determined by flow cytometry of disaggregated tumor cells. Data shown are mean ± SD. AdRCR, adenovirus–RCR hybrid vector; RCR, replication-competent retrovirus.

Figure 4

Prodrug activator gene-mediated cell killing effect of RCR following AdRCR infection in vitro. (a) Cell viability of MDA-MB-231 human mammary carcinoma cells after AdRCR-CD transduction (MOI 0.01, 0.1), and secondary RCR vector spread in vitro for 4, 7, and 10 days, as indicated. x axis: days postinitiation of 5-FC prodrug treatment at 0.5 mmol/l or 2 mmol/l, as indicated; y axis: percentage of viable cells by MTS assay. (b) Cell viability versus βgal expression in vitro after AdRCR-CD infection. Left vertical axis: Cell killing (reciprocal of cell viability by MTS assay) of MDA-MB-231 cells by day 4 after initiation of 0.5 mmol/l 5-FC, after prior AdRCR-CD transduction (MOI 0.01) and secondary RCR spread for 4, 7, or 10 days, as indicated by bar graph. Right vertical axis: Percentage of adenovirus-infected cells by X-gal staining at each time point, indicated by line graph. AdRCR, adenovirus–RCR hybrid vector; 5-FC, 5-fluorocytosine; MOI, multiplicity of infection; RCR, replication-competent retrovirus.

Figure 5

In vivo antitumor effect of AdRCR-CD in human breast cancer xenograft model. (a) Bioluminescence imaging of AdRCR-mediated prodrug activator gene therapy in a human breast cancer xenograft model. Subcutaneous MDA-MB-231-luc tumors were injected intratumorally with PBS vehicle or AdRCR-CD on day 0, followed by intraperitoneal administration of 5-FC from day 8 to 18, and monitored by optical bioluminescence imaging as indicated. Images of representative mice from both groups are shown. (b) Subcutaneous MDA-MB-231 tumors established in nude mice were injected intratumorally with PBS vehicle control, RCR-CD, or AdRCR-CD on day 0, followed by intraperitoneal administration of 5-FC or PBS from day 8 until the end of the experiment (n = 5/group). Tumor volumes were measured every other day, and data shown are mean ± SD. AdRCR, adenovirus–RCR hybrid vector; CD, cytosine deaminase; 5-FC, 5-fluorocytosine; MOI, multiplicity of infection; PBS, phosphate-buffered saline; RCR, replication-competent retrovirus.