The Digest

page: 897

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In this issue, Keeney and colleagues describe a novel technology to restore bioenergetic function to cells through mitochondrial gene transfer. The technique is named ProtoFection, which stands for protein-mediated transfection, and is in development by Gencia Corporation. Mitochondrial defects can lead to a wide array of diseases that affect the energy metabolism of the cell. High-energy, postmitotic tissues such as muscle, retina, and the brain are particularly sensitive to mitochondrial defects. Mutations or deletions in mitochondrial DNA (mtDNA) can lead to inborn defects; however, mtDNA mutations have also been implicated in aging and neurodegeneration. The importance of these somatic mtDNA mutations in aging or disease remains uncertain but findings in the substantia nigra of a cohort of individuals with Parkinson disease (PD) indicated an increased level of deleted mtDNA with different clonally expanded deletions in cells.

To obtain mitochondrial transduction, the authors engineered a chimeric recombinant protein that combines a mitochondrial transcription factor (TFAM), an N-terminal protein transduction domain, and a mitochondrial localization signal to constitute the ProtoFection reagent. In proof-of-concept experiments, a PD-derived cybrid (i.e., a eukaryotic cell line originating from a fusion between a cytoplast and a whole cell) line was treated with either the reagent alone or admixed with human mtDNA. Both conditions led to increased mitochondrial gene content and gene expression; yet only supplementation with mtDNA (i.e., ProtoFection and DNA) was able to restore the respiration rate of this cell line to normal. (lv)

page: 883

Several of the commonly used methods of gene transfer lead to persistent expression in vivo from episomal DNA. Three components of this phenomenon have fascinated researchers for decades. First, how and in which conformations is the genome maintained? Second, which of the various conformations serves as a template for efficient transcription that leads to maintained transgene expression? And finally, is replication of the genome necessary in order for it to persist?

In this issue, Jager and Ehrhardt pose exactly these questions for nonintegrative high-capacity adenoviral vectors (HC-AdV). These vectors are gutless, that is, devoid of all viral open reading frames, and provide up to 36 kb of space to accommodate transgene cargo on an linear genome flanked by inverted terminal repeats. Transduction with HC-AdV in hepatic tissue can lead to genome persistence and long-term transgene expression in mice and nonhuman primates. The authors demonstrate that both in vitro and in vivo HC-AdV persists as linear monomers. Moreover, a sensitive quantitative PCR protocol did not show evidence of substantial amplification of the genome, indicating that it is the input vector genomes that persist. (lv)

page: 831

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Many molecular pathways are affected in cancer, including both canonical protein-coding genes as well as noncoding genes. MicroRNAs (miRNAs) are a class of noncoding small RNAs (17 to 27 nucleotides in length) that control gene expression by regulating mRNA translation via partial Watson–Crick interactions with complementary sequences within the 3′ untranslated regions of targeted transcripts. An estimated one-third of protein-coding human mRNAs are regulated by miRNAs. An aberrant miRNA expression profile is a hallmark of several diseases, including cancer. Previous studies have shown that changes in the abundance of a single miRNA can affect the expression levels of hundreds of different proteins and push cells into a transformed state. Two new reports now demonstrate that using gene therapy-based approaches to modulate microRNA activity may be of therapeutic value for the treatment of cancers.

In the study by Torrisani and colleagues , which appears in this issue (see page 831), the authors confirm previous results showing that let-7 miRNA expression is decreased in pancreatic ductal adenocarcinoma (PDAC) cells of patients compared with normal pancreatic acini adjacent to the tumor. Given that dysregulation of let-7 expression affects mitotic signaling, cell cycling, angiogenesis, and cell adhesion in other models of cancer, the authors hypothesized that restoring let-7 miRNA levels would alter tumor progression in PDAC as well. In vitro, the authors found that transducing PDAC-derived cells with let-7-expressing lentivirus leads to the inhibition of cell proliferation, K-ras expression, and mitogen-activated protein kinase activation. In vivo, however, subcutaneous injection of let-7-expressing lentivirus restored let-7 miRNA expression levels but did not halt tumor growth in a mouse model of PDAC. As to why the treatment did not work in vivo, Dr. Joanne Weidhaas of the Yale University School of Medicine notes that “let-7 has not been previously shown to be important in pancreatic cancer, and although they [the authors] showed reduction of let-7 levels, they did not prove that let-7, specifically let-7a, is critical in pancreatic cancer.” According to Dr. Weidhaas, the lack of success in vivo “is most likely due to the miRNA that was used to conduct these studies and is not a reflection of the techniques used or the general principle of miRNA as cancer gene therapy.”

The study by Kota and colleagues, ¹ published in Cell, uses a similar strategy but achieves greater success in vivo. The authors chose to target miR-26a miRNA, which plays an important role in the normal cell cycle control of hepatocytes and is known to be downregulated in hepatocarcinomas. In vitro, Kota and colleagues demonstrate that forced expression of miR-26a in a human hepatocellular carcinoma cell line, using a murine retroviral vector, induces G₁ arrest in human liver cancer cells, thereby halting cellular proliferation. Importantly, using an inducible mouse model of hepatocarcinoma, the authors show that intravenous administration of AAV8 expressing miR-26a results in the inhibition of cancer cell proliferation, induction of tumor-specific apoptosis, and protection from tumor progression.

According to Dr. Yong Sun Lee of the University of Texas Medical Branch, the big questions remain “why was miR26a overexpression successful in vivo but not let-7? Why is let-7 overexpression successful in the suppression of lung cancer [see Ref. 2] but not PDAC?” The answer to these questions may lie in the molecular basis of each cancer and the tumor microenvironment. “Nonetheless,” states Dr. Lee, “both of these studies demonstrate that miRNA-based cancer therapy is undoubtedly a promising strategy for the future.” (sk)