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Open Access Publications from the University of California

Scholarly Works (2 results)

  • Thesis
  • Peer Reviewed
UCLA Electronic Theses and Dissertations (2014)

An improved Retroviral Replicating Vector (RRV), Toca 511, encoding an optimized yeast cytosine deaminase prodrug activator gene, is currently under investigation for recurrent high-grade glioma in Phase I/II clinical trials. RRVs exhibit inherent tumor selectivity, due to their intrinsic inability to enter post-mitotic cells, and unlike replication defective vectors, are capable of efficient gene transfer to tumors, as the virus continues to spread through the tumor mass after initial infection1. However, tumor transduction may still be limited by cellular anti-viral immune factors. Moreover, concerns remain about the use of RRVs for gene therapy given the possibility that viral integration might lead to off-target effects, or genotoxicity, in non-cancerous cells. Therefore, we investigated mechanisms to improve both the safety and efficiency of RRV-mediated gene therapy.

Enhancing the tumor specificity of RRVs

We examined whether the incorporation of miRNA target sequences (miRTs) into the RRV genome could restrict viral replication in a tissue-specific manner. Accordingly, we developed novel RRVs with one to four copies of the target sequence for human miR142-3p, a lymphohematopoietic-specific miRNA, inserted into the ψ packaging signal, the 3' LTR and/or the transgene cassette of the RRV. We showed that incorporation of the miR142-3p target sequence (miRT142) significantly reduced viral spread to hematopoietic tissues, without inhibiting intratumoral replication or cytotoxic effect. However, the genomic location of the miRT affected viral replication kinetics, transgene stability, and miRT sequence integrity, in some cases. Therefore careful placement of miRTs is necessary to preserve efficient viral replication and genomic stability in non-restricted tissues, while maximizing suppression of RRV replication in lymphohematopoietic cells. MicroRNA-based restriction can effectively suppress RRV replication in a tissue-specific manner, and this approach may add an additional safeguard to gene therapy applications.

Improving the efficiency of RRV-mediated gene transfer

To explore the effect of cellular factors on RRV replication, we monitored RRV spread in a panel of primary human glioblastoma (glioma) cell lines, and quantified the expression, in these same cells, of various cellular factors that may play a role in RRV replication. We confirmed the finding, by another group, that tetherin (BST2, CD317) is strongly overexpressed in glioma cells, and identified an additional antiviral factor, protein kinase R, whose expression may also be dysregulated. We demonstrated that tetherin expression is correlated with slower RRV spread and reduced viral titers in the supernatants of infected cells, and suggest a possible antitumor effect of tetherin knockdown in glioma. Our results can be extended to future studies of the effect of tetherin on RRV replication or glioma tumor progression, or may be used to inform either the development of novel RRVs with enhanced infectivity, or the stratification of patients in clinical trials.

    Cover page: Enhancing the Safety and Efficiency of Retroviral Replicating Vectors for Cancer Gene Therapy
    • Article
    • Peer Reviewed
    UC San Francisco Previously Published Works (2014)
    We are developing a retroviral replicating vector (RRV) encoding cytosine deaminase as an anticancer agent for gliomas. Despite its demonstrated natural selectivity for tumors, and other safety features, such a virus could potentially cause off-target effects by productively infecting healthy tissues. Here, we investigated whether incorporation of a hematopoietic lineage-specific microRNA target sequence in RRV further restricts replication in hematopoietic lineage-derived human cells in vitro and in murine lymphoid tissues in vivo. One or four copies of a sequence perfectly complementary to the guide strand of microRNA 142-3p were inserted into the 3' untranslated region of the RRV genome expressing the transgene encoding green fluorescent protein (GFP). Viral spread and GFP expression of these vectors in hematopoietic lineage cells in vitro and in vivo were measured by qPCR, qRT-PCR, and flow cytometry. In hematopoietic lineage-derived human cell lines and primary human stimulated peripheral blood mononuclear cells, vectors carrying the 142-3pT sequence showed a remarkable decrease in GFP expression relative to the parental vector, and viral spread was not observed over time. In a syngeneic subcutaneous mouse tumor model, RRVs with and without the 142-3pT sequences spread equally well in tumor cells; were strongly repressed in blood, bone marrow, and spleen; and generated antiviral immune responses. In an immune-deficient mouse model, RRVs with 142-3pT sequences were strongly repressed in blood, bone marrow, and spleen compared with unmodified RRV. Tissue-specific microRNA-based selective attenuation of RRV replication can maintain antiviral immunity, and if needed, provide an additional safeguard to this delivery platform for gene therapy applications.
      Cover page: MicroRNA 142-3p Attenuates Spread of Replicating Retroviral Vector in Hematopoietic Lineage-Derived Cells While Maintaining an Antiviral Immune Response
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