Epigenetic control of multiple genes with a lentiviral vector encoding transcriptional repressors fused to compact zinc finger arrays.

Gene silencing without gene editing holds great potential for the development of safe therapeutic applications. Here, we describe a novel strategy to concomitantly repress multiple genes using zinc finger proteins fused to Krüppel-Associated Box repression domains (ZF-Rs). This was achieved via the optimization of a lentiviral system tailored for the delivery of ZF-Rs in hematopoietic cells. We showed that an optimal design of the lentiviral backbone is crucial to multiplex up to three ZF-Rs or two ZF-Rs and a chimeric antigen receptor. ZF-R expression had no impact on the integrity and functionality of transduced cells. Furthermore, gene repression in ZF-R-expressing T cells was highly efficient in vitro and in vivo during the entire monitoring period (up to 10 weeks), and it was accompanied by epigenetic remodeling events. Finally, we described an approach to improve ZF-R specificity to illustrate the path toward the generation of ZF-Rs with a safe clinical profile. In conclusion, we successfully developed an epigenetic-based cell engineering approach for concomitant modulation of multiple gene expressions that bypass the risks associated with DNA editing.

Drive-thru anticoagulation clinic: Can we supersize your care today?

Warfarin therapy requires maintenance of a therapeutic international normalized ratio (INR) and thus requires routine monitoring to ensure benefits of anticoagulation, while avoiding complications. As the pharmacist's role evolves from traditional medication dispensing towards direct patient care, many anticoagulation management services are pharmacist-managed. Due to the coronavirus disease 2019 (COVID-19) pandemic, healthcare providers were faced with re-evaluating anticoagulation management practices to minimize person-to-person exposure risk. Although being anticoagulated is not considered high risk for illness from the coronavirus, these patients are often of advanced age and frequently have multiple comorbidities, putting them at increased risk. Consequently, two hospital-based, pharmacist-managed outpatient anticoagulation management services developed drive-thru curbside clinics to continue providing care to warfarin patients. The services utilized universal COVID-19 precautions to conduct curbside appointments where pharmacists determined patient's warfarin therapy plan, scheduled timely follow-up, and provided dosing instructions. With the unexpected coronavirus outbreak, this immediate change to traditional anticoagulation management was essential for safe and effective anticoagulation therapy. Implementing a curbside clinic allowed for safe distancing while managing warfarin appropriately.

Diversifying the structure of zinc finger nucleases for high-precision genome editing.

Genome editing for therapeutic applications often requires cleavage within a narrow sequence window. Here, to enable such high-precision targeting with zinc-finger nucleases (ZFNs), we have developed an expanded set of architectures that collectively increase the configurational options available for design by a factor of 64. These new architectures feature the functional attachment of the FokI cleavage domain to the amino terminus of one or both zinc-finger proteins (ZFPs) in the ZFN dimer, as well as the option to skip bases between the target triplets of otherwise adjacent fingers in each zinc-finger array. Using our new architectures, we demonstrate targeting of an arbitrarily chosen 28 bp genomic locus at a density that approaches 1.0 (i.e., efficient ZFNs available for targeting almost every base step). We show that these new architectures may be used for targeting three loci of therapeutic significance with a high degree of precision, efficiency, and specificity.

Expression, purification, and renaturation of bone morphogenetic protein-2 from Escherichia coli.

Homodimeric bone morphogenetic protein-2 (BMP-2) is a member of the transforming growth factor beta superfamily that has been used for bone grafting. We were interested in exploring the functions of BMP-2 in other disease areas and focused on expressing and purifying active BMP-2 proteins. We have developed a new approach which involves using FoldIt refolding buffer to refold BMP-2 followed by a heparin affinity column to separate correctly folded dimer from monomer. A high yield of 29.4 mg BMP-2 dimer per gram cell wet weight was achieved. The purified BMP-2 dimer was shown to possess the same level of activity as BMP-2 from CHO cells as tested by the induction of alkaline phosphatase activity in C2C12 cells. This approach has potential application in refolding and purifying other homodimeric proteins.

The development and applications of nonradioactive plate-formatted DNA-binding assay for Ku70/80, a multifunctional DNA-binding protein complex.

Ku is a heterodimer composed of p70 and p80, and is the regulatory subunit of DNA-dependent protein kinase. As a multifunctional DNA-binding protein complex, Ku plays important roles in DNA damage repair through non-homologous end joining and in V(D)J recombination. In addition, Ku has also been implicated in various biological functions including growth control, cell proliferation, cell cycle, chromosome maintenance, transcriptional regulation, apoptosis, and viral infection. In particular, using our Inverse Genomics (Immusol, Inc., San Diego, CA) platform technology, we recently identified Ku80 as an essential co-factor for human immunodeficiency virus replication. Although Ku has been studied extensively in the past years, its in-depth study as well as development as a drug target has been limited by conventional DNA-binding activity assay. Here we describe the development and applications of a nonradioactive DNA binding assay in the 96-well format. We show that this plate-formatted assay is more sensitive and allows for direct quantification when compared with an electrophoretic mobility shift assay. The establishment of this assay will not only facilitate structure and function studies on Ku, but also help the development of Ku protein or its DNA repair enzyme complex as a drug target.

Molecular immunolabeling with recombinant single-chain variable fragment (scFv) antibodies designed with metal-binding domains.

To study the molecular structure and function of gene products in situ, we developed a molecular immunolabeling technology. Starting with cDNA from hybridomas producing monoclonal antibodies against biotin, catalase, and superoxide dismutase, we bioengineered recombinant single-chain variable fragment antibodies (scFv) and their derivatives containing metal-binding domains (scFv:MBD). As tested with surface plasmon resonance and enzyme-linked immunosorbent assay, affinity binding constants of the scFv (5.21 x 10(6) M(-1)) and scFv:MBD (4.17 x 10(6) M(-1)) were close to those of Fab proteolytic fragments (9.78 x 10(6) M(-1)) derived from the parental IgG antibodies. After saturation of MBD with nickel or cobalt, scFv:MBD was imaged with electron spectroscopic imaging at each element's specific energy loss, thus generating the element's map. Immunolabeling with scFv:MBD resulted in a significant improvement of the labeling fidelity over that obtained with Fab or IgG derivatives, as it produced a much heavier specific labeling and label-free background. As determined with radioimmunoassay, labeling effectiveness with scFv:MBD was nearly the same as with scFv, but much higher than with scFv conjugated to colloidal gold, Nanogold, or horseradish peroxidase. This technology opens possibilities for simultaneous imaging of multiple molecules labeled with scFv:MBD at the molecular resolution within the same sample with electron spectroscopic imaging. Moreover, the same scFv:MBD can also be imaged with fluorescence resonance energy transfer and lifetime imaging as well as positron emission tomography and magnetic resonance imaging. Therefore, this technology may serve as an integrative factor in life science endeavors.