Summary Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multi-finger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1 to 50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally-integrated EGFP reporter gene. In summary, OPEN provides an “open-source” method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.

Background: HIV-1 recombination between different subtypes has a major impact on the global epidemic. The generation of these intersubtype recombinants follows a defined set of events starting with dual infection of a host cell, heterodiploid virus production, strand transfers during reverse transcription, and then selection. In this study, recombination frequencies were measured in the C1-C4 regions of the envelope gene in the presence (using a multiple cycle infection system) and absence (in vitro reverse transcription and single cycle infection systems) of selection for replication-competent virus. Ugandan subtypes A and D HIV-1 env sequences (115-A, 120-A, 89-D, 122-D, 126-D) were employed in all three assay systems. These subtypes co-circulate in East Africa and frequently recombine in this human population.

In vitro reaction conditions using HIV reverse transcriptase (RT) and nucleocapsid protein (NC) that allowed efficient synthesis of single-stranded DNA products over a thousand nucleotides in length from genomic HIV RNA were characterized. Consistent with previous reports, the reactions required high concentrations of NC and RT. Long products were produced as a result of frequent strand transfer between RNA templates, averaging at least one transfer per 300 nucleotides synthesized. No change in RT processivity was observed in the reactions in the presence versus absence of NC. Synthesis of long products required formation of a high molecular mass aggregate between NC and nucleic acids. The aggregate formed rapidly and pelleted with low speed centrifugation. The aggregate was accessible to RT as pre-formed aggregates synthesized long products when RT was added. NC finger mutants lacking either finger one or two or with the finger positions switched were all effective in promoting long products. This suggests that the aggregation/condensation but not helix-destabilizing activity of NC was required. We propose that these high molecular mass aggregates promote synthesis of long reverse transcription products in vitro by concentrating nucleic acids, RT enzyme and NC to close proximity, thereby mimicking the role of the capsid environment within the host cell.

The aim of gene therapy for cystic fibrosis (CF) lung disease is to efficiently and safely express the CF transmembrane conductance regulator (CFTR) in the appropriate pulmonary cell types. Although CF patients experience multi-organ disease, the chronic bacterial lung infections and associated inflammation are the primary cause of shortened life expectancy. Gene transfer-based therapeutic approaches are feasible, in part, because the airway epithelium is directly accessible by aerosol delivery or instillation. Improvements in standard delivery vectors and the development of novel vectors, as well as emerging technologies and new animal models, are propelling exciting new research forward. Here, we review recent developments that are advancing this field of investigation.

Diminished appetite and poor eating behavior accompanied by weight loss or cachexia are often reported in dogs living with cancer. This study was conducted to determine the acceptance and eating enthusiasm in dogs with cancer for a new therapeutic, nutritionally balanced, and calorically dense food designed for dogs with cancer. Adult dogs with diagnosis of cancer were recruited from general and oncology practices and were fed the study food for 28 days. Evaluations included physical examination, body weight, food intake, caloric intake, hematology and serum biochemistry, and owner assessments, namely food evaluation, quality of life, and stool scores. The dogs transitioned smoothly and tolerated the food very well. The results showed high food acceptance within the first day, with continued eating enthusiasm over the 28 days. Significant increases in food and caloric intake were observed, with the study food having a positive impact on body weight in dogs that were losing weight and helping to maintain a high quality of life. Blood laboratory parameters remained within reference ranges. Thus, the therapeutic study food was well accepted and efficacious in supporting continued eating and required caloric intake, promoting a healthy weight gain and maintaining a high quality of life in dogs with cancer.