Inefficient gene transfer and low virion concentrations are common limitations of retroviral transduction. We and others have previously shown that peptides derived from human semen form amyloid fibrils that boost retroviral gene delivery by promoting virion attachment to the target cells. However, application of these natural fibril-forming peptides is limited by moderate efficiencies, the high costs of peptide synthesis, and variability in fibril size and formation kinetics. Here, we report the development of nanofibrils that self-assemble in aqueous solution from a 12-residue peptide, termed enhancing factor C (EF-C). These artificial nanofibrils enhance retroviral gene transfer substantially more efficiently than semen-derived fibrils or other transduction enhancers. Moreover, EF-C nanofibrils allow the concentration of retroviral vectors by conventional low-speed centrifugation, and are safe and effective, as assessed in an ex vivo gene transfer study. Our results show that EF-C fibrils comprise a highly versatile, convenient and broadly applicable nanomaterial that holds the potential to significantly facilitate retroviral gene transfer in basic research and clinical applications.
Severe adverse events related to insertional mutagenesis have reinforced interest in self-inactivating (SIN) retroviral vectors lacking enhancer-promoter sequences in the long terminal repeats (LTRs). Here, we have compared the potency of gammaretroviral and lentiviral vectors expressing the P140K mutant of O(6)-methylguanine-DNA methyltransferase (MGMT). MGMT-P140K is a clinically relevant selection marker that mediates a strong survival advantage in hematopoietic cells exposed to alkylating agents. We designed gammaretroviral and lentiviral vectors that contained identical enhancer-promoter sequences located either in the LTR or downstream of the packaging region, for internal initiation of transcription from SIN backbones. Gammaretroviral vectors with intact LTRs containing enhancer-promoter sequences showed both higher titers and higher expression levels than the lentiviral counterparts, likely a result of suboptimal RNA processing of the lentiviral leader region. In the SIN context, gammaretroviral and lentiviral vectors with comparable internal cassettes had similar expression properties. Interestingly, gammaretroviral SIN vectors pseudotyped with RD114/TR had a higher transduction efficiency on proliferating human CD34(+) cells than lentiviral counterparts. These results encourage further investigations into the formation of retroviral hybrid vectors that combine the desired properties of high efficiency and increased biosafety.
T-cell receptor (TCR) signal strength determines selection and lineage fate at the CD4 +
CD8+ double-positive stage of intrathymic T-cell development. Members of the miR-181 family constitute the most abundantly expressed microRNA at this stage of T-cell development. Here we show that deletion of miR-181a/b-1 reduced the responsiveness of double-positive thymocytes to TCR signals and virtually abrogated early invariant natural killer T (iNKT) cell development, resulting in a dramatic reduction in iNKT cell numbers in thymus as well as in the periphery. Increased concentrations of agonist ligand rescued iNKT cell development in miR-181a/b-1 −/− mice. Our results define a critical role of miR-181a/b-1 in early iNKT cell development and show that miR-181a/b-1 sets a TCR signaling threshold for agonist selection.
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