Efficient nonviral Sleeping Beauty transposon-based TCR gene transfer to peripheral blood lymphocytes confers antigen-specific antitumor reactivity.

Genetically engineered lymphocytes hold promise for the treatment of genetic disease, viral infections and cancer. However, current methods for genetic transduction of peripheral blood lymphocytes rely on viral vectors, which are hindered by production and safety-related problems. In this study, we demonstrated an efficient novel nonviral platform for gene transfer to lymphocytes. The Sleeping Beauty transposon-mediated approach allowed for long-term stable expression of transgenes at approximately 50% efficiency. Utilizing transposon constructs expressing tumor antigen-specific T-cell receptor genes targeting p53 and MART-1, we demonstrated sustained expression and functional reactivity of transposon-engineered lymphocytes on encountering target antigen presented on tumor cells. We found that transposon- and retroviral-modified lymphocytes had comparable transgene expression and phenotypic function. These results demonstrate the promise of nonviral ex vivo genetic modification of autologous lymphocytes for the treatment of cancer and immunologic disease.

Development of optimal bicistronic lentiviral vectors facilitates high-level TCR gene expression and robust tumor cell recognition.

In human gene therapy applications, lentiviral vectors may have advantages over gamma-retroviral vectors in several areas, including the ability to transduce nondividing cells, resistance to gene silencing and a potentially safer integration site profile. However, unlike gamma-retroviral vectors it has been problematic to drive the expression of multiple genes efficiently and coordinately with approaches such as internal ribosome entry sites or dual promoters. Using different 2A peptides, lentiviral vectors expressing two-gene T-cell receptors directed against the melanoma differentiation antigens gp100 and MART-1 were constructed. We demonstrated that addition of amino-acid spacer sequences (GSG or SGSG) before the 2A sequence is a prerequisite for efficient synthesis of biologically active T-cell receptors and that addition of a furin cleavage site followed by a V5 peptide tag yielded optimal T-cell receptor gene expression. Furthermore, we determined that the furin cleavage site was recognized in lymphocytes and accounted for removal of residual 2A peptides at the post-translational level with an efficiency of 20-30%, which could not be increased by addition of multiple furin cleavage sites. The novel bicistronic lentiviral vector developed herein afforded robust anti-melanoma activities to engineered peripheral blood lymphocytes, including cytokine secretion, cell proliferation and lytic activity. Such optimal vectors may have immediate applications in cancer gene therapy.

Specific triggering of the Fas signal transduction pathway in normal human keratinocytes.

The epidermis is continually exposed to genotoxic injury and requires an efficient mechanism to eliminate genetically altered cells. The membrane receptor, Fas, initiates apoptosis in many cell types, including keratinocytes. Receptor cross-linking is the vital post-ligand binding step in Fas signal transduction, and we have utilized FK1012, capable of oligomerizing proteins engineered to contain the FK506 binding protein (FKBP), to trigger Fas via FKBP-linked receptor cytoplasmic domains in human keratinocytes. An FKBP chimera containing the Fas cytoplasmic domain targeted to the plasma membrane induced an up to 89% decrease in viability of keratinocytes, as reflected by the activity of constitutive promoters, in response to FK1012. Oligomerization of Fas, either with engineered Fas.FKBP by FK1012 or via antibody cross-linking of full-length Fas-induced cellular changes consistent with apoptosis. The lpr Fas point mutation abolished this effect. A Fas.FKBP construct unlinked to the membrane was fully active in this assay. Early developmental age or pre-treatment of cells with GM-CSF, TGF-beta, EGF, KGF, IFN-gamma, or phorbol ester failed to protect against Fas effects. These findings reveal that the Fas signal transduction pathway is active in keratinocytes, requires no induction, and dominantly overrides growth stimuli.