Highly active zinc-finger nucleases by extended modular assembly.

Zinc-finger nucleases (ZFNs) are important tools for genome engineering. Despite intense interest by many academic groups, the lack of robust noncommercial methods has hindered their widespread use. The modular assembly (MA) of ZFNs from publicly available one-finger archives provides a rapid method to create proteins that can recognize a very broad spectrum of DNA sequences. However, three- and four-finger arrays often fail to produce active nucleases. Efforts to improve the specificity of the one-finger archives have not increased the success rate above 25%, suggesting that the MA method might be inherently inefficient due to its insensitivity to context-dependent effects. Here we present the first systematic study on the effect of array length on ZFN activity. ZFNs composed of six-finger MA arrays produced mutations at 15 of 21 (71%) targeted loci in human and mouse cells. A novel drop-out linker scheme was used to rapidly assess three- to six-finger combinations, demonstrating that shorter arrays could improve activity in some cases. Analysis of 268 array variants revealed that half of MA ZFNs of any array composition that exceed an ab initio B-score cutoff of 15 were active. These results suggest that, when used appropriately, MA ZFNs are able to target more DNA sequences with higher success rates than other current methods.

Enhancing the specificity of recombinase-mediated genome engineering through dimer interface redesign.

Despite recent advances in genome engineering made possible by the emergence of site-specific endonucleases, there remains a need for tools capable of specifically delivering genetic payloads into the human genome. Hybrid recombinases based on activated catalytic domains derived from the resolvase/invertase family of serine recombinases fused to Cys2-His2 zinc-finger or TAL effector DNA-binding domains are a class of reagents capable of achieving this. The utility of these enzymes, however, has been constrained by their low overall targeting specificity, largely due to the formation of side-product homodimers capable of inducing off-target modifications. Here, we combine rational design and directed evolution to re-engineer the serine recombinase dimerization interface and generate a recombinase architecture that reduces formation of these undesirable homodimers by >500-fold. We show that these enhanced recombinases demonstrate substantially improved targeting specificity in mammalian cells and achieve rates of site-specific integration similar to those previously reported for site-specific nucleases. Additionally, we show that enhanced recombinases exhibit low toxicity and promote the delivery of the human coagulation factor IX and α-galactosidase genes into endogenous genomic loci with high specificity. These results provide a general means for improving hybrid recombinase specificity by protein engineering and illustrate the potential of these enzymes for basic research and therapeutic applications.

Reducing patient restraints: a pilot approach using clinical case review.

PURPOSE: This study aims to reduce the incidents of restraints by applying a nontraditional consultation process in which a university-based team focused on patient consultations to collect data on treatment interventions and milieu approaches and conditions, as well as staff interactions. CONCLUSIONS: The efforts resulted in restraint reduction from 36 episodes per month to 0 episodes per month as well as precipitating a change in unit climate and care approaches on a specialized unit for patients with developmental disabilities and mental illness. PRACTICE IMPLICATIONS: Reducing the use of restraints involving multiple restraint incident patients is possible with a team-based approach and a specific intervention plan.

Combined buprenorphine and chlonidine for short-term opiate detoxification: patient perspectives.

The approval in 2003 for the use of buprenorphine in opiate addiction treatment has provided physicians with a new pharmacological tool to combat opiate addiction. We surveyed a sample of 100 inpatients who completed short-term opiate detoxification treatment utilizing a combination of buprenorphine and clonidine to assess patient perspectives regarding the usefulness and tolerability of this medication regimen and to compare it to their past opiate detox experiences, if any. Patients identified pain (63%), sleep problems (57%), and anxiety (56%) as the symptoms they perceived to be most helped with buprenorphine. Over 90% of patients with past detoxification treatments rated buprenorphine treatment to be as good as or better than their past treatments. Reports of a euphoric effect were minimal (7%) and no patients reported any generalized worsening of their opiate withdrawal symptoms. We conclude that based upon patient perspectives that combining buprenorphine with clonidine is a useful and well-tolerated medication regimen for the treatment of opiate withdrawal.

Redesigning Recombinase Specificity for Safe Harbor Sites in the Human Genome.

Site-specific recombinases (SSRs) are valuable tools for genetic engineering due to their ability to manipulate DNA in a highly specific manner. Engineered zinc-finger and TAL effector recombinases, in particular, are two classes of SSRs composed of custom-designed DNA-binding domains fused to a catalytic domain derived from the resolvase/invertase family of serine recombinases. While TAL effector and zinc-finger proteins can be assembled to recognize a wide range of possible DNA sequences, recombinase catalytic specificity has been constrained by inherent base requirements present within each enzyme. In order to further expand the targeted recombinase repertoire, we used a genetic screen to isolate enhanced mutants of the Bin and Tn21 recombinases that recognize target sites outside the scope of other engineered recombinases. We determined the specific base requirements for recombination by these enzymes and demonstrate their potential for genome engineering by selecting for variants capable of specifically recombining target sites present in the human CCR5 gene and the AAVS1 safe harbor locus. Taken together, these findings demonstrate that complementing functional characterization with protein engineering is a potentially powerful approach for generating recombinases with expanded targeting capabilities.

Improved cell-penetrating zinc-finger nuclease proteins for precision genome engineering.

Safe, efficient, and broadly applicable methods for delivering site-specific nucleases into cells are needed in order for targeted genome editing to reach its full potential for basic research and medicine. We previously reported that zinc-finger nuclease (ZFN) proteins have the innate capacity to cross cell membranes and induce genome modification via their direct application to human cells. Here, we show that incorporation of tandem nuclear localization signal (NLS) repeats into the ZFN protein backbone enhances cell permeability nearly 13-fold and that single administration of multi-NLS ZFN proteins leads to genome modification rates of up to 26% in CD4(+) T cells and 17% in CD34(+) hematopoietic stem/progenitor cells. In addition, we show that multi-NLS ZFN proteins attenuate off-target effects and that codelivery of ZFN protein pairs facilitates dual gene modification frequencies of 20-30% in CD4(+) T cells. These results illustrate the applicability of ZFN protein delivery for precision genome engineering.