Adoptive immunotherapy with functional T cells is potentially an effective therapeutic strategy for combating many types of cancer and viral infection. However, exhaustion of antigen-specific T cells represents a major challenge to this type of approach. In an effort to overcome this problem, we reprogrammed clonally expanded antigen-specific CD8(+) T cells from an HIV-1-infected patient to pluripotency. The T cell-derived induced pluripotent stem cells were then redifferentiated into CD8(+) T cells that had a high proliferative capacity and elongated telomeres. These "rejuvenated" cells possessed antigen-specific killing activity and exhibited T cell receptor gene-rearrangement patterns identical to those of the original T cell clone from the patient. We also found that this method can be effective for generating specific T cells for other pathology-associated antigens. Thus, this type of approach may have broad applications in the field of adoptive immunotherapy.

The plasma membrane of mammalian cells is one of the tight barriers against gene transfer by synthetic delivery systems. Various agents have been used to facilitate gene transfer by destabilizing the endosomal membrane under acidic conditions, but their utility is limited, especially for gene transfer in vivo. In this article, we report that the protein transduction domain of human immunodeficiency virus type 1 Tat protein (Tat peptide) greatly facilitates gene transfer via membrane destabilization. We constructed recombinant phage particles displaying Tat peptide on their surfaces and carrying mammalian marker genes as part of their genomes (Tat-phage). We demonstrate that, when animal cells are briefly exposed to Tat-phage, significant expression of phage marker genes is induced with no harmful effects to the cells. In contrast, recombinant phage displaying other functional peptides, such as the integrin-binding domain or a nuclear localization signal, could not induce detectable marker gene expression. The expression of marker genes induced by Tatphage is not affected by endosomotropic agents but is partially impaired by inhibitors of caveolae formation. These data suggest that Tat peptide will become a useful component of synthetic delivery vehicles that promote gene transfer independently of the classical endocytic pathway.

Addition of antisense oligonucleotides to cell cultures has been used to specifically inhibit gene expression. We have investigated the mechanism by which oligonucleotides enter living cells. These compounds are taken up by cells in a saturable, size-dependent manner compatible with receptormediated endocytosis. Polynucleotides of any length are competitive inhibitors of oligomer transport, providing they possess a 5'-phosphate moiety. Using oligo(dT)-cellulose for affinity purification, we have identified an 80-kDa surface protein that may mediate transport. Knowledge of the oligonucleotide transport mechanism should facilitate the design of more effective synthetic antisense oligomers as potential clinical agents.When oligodeoxynucleotides [oligo(dN)s] complementary to the 5' region of c-myc mRNA are added to cells in culture, c-myc protein synthesis is specifically inhibited (1)(2)(3)(4)(5). Furthermore, addition ofantisense oligo(dN)s to cultures inhibits intracellular viral replication (6)(7)(8) Fig. 1 Top depicts a typical fluorescence histogram comparing cells incubated with no oligo(dN) to those incubated for 24 hr with either acridine-labeled oligo(dN) alone or in the presence of excess unlabeled oligo(dN). Intracellular localization of fluorescence was confirmed by fluorescence microscopy of similarly treated cells (Fig. 1 Middle and Bottom). When we examined the rates of accumulation of variously sized acridine-labeled oligo(dN)s we found that the accumulated intracellular fluorescence after incubation of HL60 cells with 12.5 AtM acridine-labeled oligomers [ranging in size from oligo(dT)3 to oligo(dT)20] increased gradually, plateauing within -50 hr after addition of acridine-labeled oligo(dN) to the culture medium ( Fig. 2A). This is in contrast to the 90 min required

Background: The mononegavirus superfamily (Mononegavirales) comprises three families, Rhabdoviridae, Paramyxoviridae and Filoviridae. These viruses possess a single stranded negative sense RNA as the genome. Recentsuccessintherecoveryofinfectiousvirusfroma transfected cDNA of mononegaviruses including Sendai virus, a prototypic paramyxovirus, is opening the possibility of their genetic engineering. However, infectious viruses have been recovered only by initiating the infectious cycle with cDNA directing the synthesis of antigenomic positive sense ( ) RNA. Starting with genomic negative sense (ÿ) RNA has been unsuccessful. Furthermore, the recovery efficiency has often been extremely low.

A specific kinetic pattern of c-MYC expression is essential for optimal generation of functional platelets from human induced pluripotent stem cells.