An effective method for direct chemical control over the production of specific proteins would be widely useful. We describe Small Molecule-Assisted Shutoff (SMASh), a technique in which proteins are fused to a degron that removes itself in the absence of drug, leaving untagged protein. Clinically tested HCV protease inhibitors can then block degron removal, inducing rapid degradation of subsequently synthesized protein copies. SMASh allows reversible and dose-dependent shutoff of various proteins in multiple mammalian cell types and in yeast. We also used SMASh to confer drug responsiveness onto a RNA virus for which no licensed inhibitors exist. As SMASh does not require permanent fusion of a large domain, it should be useful when control over protein production with minimal structural modification is desired. Furthermore, as SMASh only involves a single genetic modification and does not rely on modulating protein-protein interactions, it should be easy to generalize to multiple biological contexts.

Precise genetic manipulation of human pluripotent stem cells will be required to realize their scientific and therapeutic potential. Here, we show that adeno-associated virus (AAV) gene targeting vectors can be used to genetically engineer human embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs). Different types of sequence-specific changes, including the creation and correction of mutations, were introduced into the human HPRT1 and HMGA1 genes (HPRT1 mutations being responsible for Lesch-Nyhan syndrome). Gene targeting occurred at high frequencies in both ESCs and iPSCs, with over 1% of all colony-forming units (CFUs) undergoing targeting in some experiments. AAV vectors could also be used to target genes in human fibroblasts that were subsequently used to derive iPSCs. Accurate and efficient targeting took place with minimal or no cytotoxicity, and most of the gene-targeted stem cells produced were euploid and pluripotent.

A simple, quantitative assay for measuring the oncogenic potential of integrating vectors is needed in order to improve vector design and safety. In this study, we have developed a transient plasmid-based assay to measure the activation of a reporter gene by an adjacent vector provirus. Plasmid pACT contains a luciferase cassette driven by a minimal, enhancerless promoter, into which vector proviruses are inserted upstream for evaluation by luciferase assays and northern blots. In a comparison of analogous vectors based on murine leukemia virus (MLV), human immunodeficiency virus (HIV), and foamy virus (FV), we observed significant enhancer activity and read-through transcription from MLV proviruses, and significant read-through transcription from HIV proviruses. HIV and FV proviruses containing an internal MLV long-terminal repeat (LTR) promoter also had significant enhancer activity, which was not observed with an internal promoter from the murine phosphoglycerate kinase-1 gene, PGK. These results demonstrate that neighboring gene activation can be limited by using internal promoter(s) lacking enhancer activity, especially when present in an FV vector backbone that prevents read-through transcription. Although the pACT assay does not measure oncogenesis directly, it should be useful for screening vectors before more time-consuming and costly animal studies are undertaken.

Serum specimens from 1,109 individuals at different ages in Beijing, China, were collected between April 1996 and March 1997 and tested for IgG antibodies against human caliciviruses using enzyme immune assays (EIAs). Baculovirus-expressed recombinant Norwalk virus (rNV) and Mexican virus (rMxV) capsid proteins were used as antigens. The seroprevalence was 89% for rNV and 91% for rMxV. Similar seroprevalence between the two antigens was observed in individual age groups and both genders. Infants had a high seroprevalence (99% for NV and 94% for MxV) at birth. The lowest seroprevalence (41% for rNV and 36% for rMxV) was at 7-11 months of age. A sharp increase in seroprevalence occurred in early childhood, with 65% and 70% at one, 85% and 90% at three, and 100% and 98% at 8-9 years of age for rNV and rMxV, respectively. Forty-three individuals had antibody against rNV but not rMxV and 63 individuals had antibody against rMxV but not rNV, indicating different levels of exposure to the two strains in these individuals. This is the first report of surveillance of antibodies against NV-like viruses in China. The observed high prevalence and early age antibody acquisition suggest that infection by these two human calicivirus strains is common in this population.

An important goal in synthetic biology is to engineer biochemical pathways to address unsolved biomedical problems. One long-standing problem in molecular medicine is the specific identification and ablation of cancer cells. Here, we describe a method, named Rewiring of Aberrant Signaling to Effector Release (RASER), in which oncogenic ErbB receptor activity, instead of being targeted for inhibition as in existing treatments, is co-opted to trigger therapeutic programs. RASER integrates ErbB activity to specifically link oncogenic states to the execution of desired outputs. A complete mathematical model of RASER and modularity in design enable rational optimization and output programming. Using RASER, we induced apoptosis and CRISPR-Cas9–mediated transcription of endogenous genes specifically in ErbB-hyperactive cancer cells. Delivery of apoptotic RASER by adeno-associated virus selectively ablated ErbB-hyperactive cancer cells while sparing ErbB-normal cells. RASER thus provides a new strategy for oncogene-specific cancer detection and treatment.