Superior lentiviral vectors designed for BSL-0 environment abolish vector mobilization.

Lentiviral vector mobilization following HIV-1 infection of vector-transduced cells poses biosafety risks to vector-treated patients and their communities. The self-inactivating (SIN) vector design has reduced, however, not abolished mobilization of integrated vector genomes. Furthermore, an earlier study demonstrated the ability of the major product of reverse transcription, a circular SIN HIV-1 vector comprising a single- long terminal repeat (LTR) to support production of high vector titers. Here, we demonstrate that configuring the internal vector expression cassette in opposite orientation to the LTRs abolishes mobilization of SIN vectors. This additional SIN mechanism is in part premised on induction of host PKR response to double-stranded RNAs comprised of mRNAs transcribed from cryptic transcription initiation sites around 3'SIN-LTR's and the vector internal promoter. As anticipated, PKR response following transfection of opposite orientation vectors, negatively affects their titers. Importantly, shRNA-mediated knockdown of PKR rendered titers of SIN HIV-1 vectors comprising opposite orientation expression cassettes comparable to titers of conventional SIN vectors. High-titer vectors carrying an expression cassette in opposite orientation to the LTRs efficiently delivered and maintained high levels of transgene expression in mouse livers. This study establishes opposite orientation expression cassettes as an additional PKR-dependent SIN mechanism that abolishes vector mobilization from integrated and episomal SIN lentiviral vectors.

A large U3 deletion causes increased in vivo expression from a nonintegrating lentiviral vector.

The feasibility of using nonintegrating lentiviral vectors has been demonstrated by recent studies showing their ability to maintain transgene expression both in vitro and in vivo. Furthermore, human immunodeficiency virus-1 (HIV-1) vectors packaged with a mutated integrase were able to correct retinal disease in a mouse model. Interestingly, these results differ from earlier studies in which first-generation nonintegrating lentiviral vectors yielded insignificant levels of transduction. However, to date, a rigorous characterization of transgene expression from the currently used self-inactivating (SIN) nonintegrating lentiviral vectors has not been published. In this study, we characterize transgene expression from SIN nonintegrating lentiviral vectors. Overall, we found that nonintegrating vectors express transgenes at a significantly lower level than their integrating counterparts. Expression from nonintegrating vectors was improved upon introducing a longer deletion in the vector's U3 region. A unique shuttle-vector assay indicated that the relative abundance of the different episomal forms was not altered by the longer U3 deletion. Interestingly, the longer U3 deletion did not enhance expression in the corpus callosum of the rat brain, suggesting that the extent of silencing of episomal transcription is influenced by tissue-specific factors. Finally, and for the first time, episomal expression in the mouse liver was potent and sustained.

Dominant-negative androgen receptor inhibition of intracrine androgen-dependent growth of castration-recurrent prostate cancer.

BACKGROUND: Prostate cancer (CaP) is the second leading cause of cancer death in American men. Androgen deprivation therapy is initially effective in CaP treatment, but CaP recurs despite castrate levels of circulating androgen. Continued expression of the androgen receptor (AR) and its ligands has been linked to castration-recurrent CaP growth. PRINCIPAL FINDING: In this report, the ligand-dependent dominant-negative ARΔ142-337 (ARΔTR) was expressed in castration-recurrent CWR-R1 cell and tumor models to elucidate the role of AR signaling. Expression of ARΔTR decreased CWR-R1 tumor growth in the presence and absence of exogenous testosterone (T) and improved survival in the presence of exogenous T. There was evidence for negative selection of ARΔTR transgene in T-treated mice. Mass spectrometry revealed castration-recurrent CaP dihydrotestosterone (DHT) levels sufficient to activate AR and ARΔTR. In the absence of exogenous testosterone, CWR-R1-ARΔTR and control cells exhibited altered androgen profiles that implicated epithelial CaP cells as a source of intratumoral AR ligands. CONCLUSION: The study provides in vivo evidence that activation of AR signaling by intratumoral AR ligands is required for castration-recurrent CaP growth and that epithelial CaP cells produce sufficient active androgens for CaP recurrence during androgen deprivation therapy. Targeting intracrine T and DHT synthesis should provide a mechanism to inhibit AR and growth of castration-recurrent CaP.

Adeno-associated virus type 2 (AAV2) capsid-specific cytotoxic T lymphocytes eliminate only vector-transduced cells coexpressing the AAV2 capsid in vivo.

A recent clinical trial has suggested that recombinant adeno-associated virus (rAAV) vector transduction in humans induces a cytotoxic T-lymphocyte (CTL) response against the AAV2 capsid. To directly address the ability of AAV capsid-specific CTLs to eliminate rAAV-transduced cells in vitro and in vivo in mice, we first demonstrated that AAV2 capsid-specific CTLs could be induced by dendritic cells with endogenous AAV2 capsid expression or pulsed with AAV2 vectors. These CTLs were able to kill a cell line stable for capsid expression in vitro and also in a mouse tumor xenograft model in vivo. Parent colon carcinoma (CT26) cells transduced with a large amount of AAV2 vectors in vitro were also destroyed by these CTLs. To determine the effect of CTLs on the elimination of target cells transduced by AAV2 vectors in vivo, we carried out adoptive transfer experiments. CTLs eliminated liver cells with endogenous AAV2 capsid expression but not liver cells transduced by AAV2 vectors, regardless of the reporter genes. Similar results were obtained for rAAV2 transduction in muscle. Our data strongly suggest that AAV vector-transduced cells are rarely eliminated by AAV2 capsid-specific CTLs in vivo, even though the AAV capsid can induce a CTL response. In conclusion, AAV capsid-specific CTLs do not appear to play a role in elimination of rAAV-transduced cells in a mouse model. In addition, our data suggest that the mouse model may not mimic the immune response noted in humans and additional modification to AAV vectors may be required for further study in order to elicit a similar cellular immune response.