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High purity chromosome sorting can be performed on instruments such as MoFlo MLS and BD influx,which are stream-in-air sorters equipped with water-cooled high power lasers. The FACSAria is a true fixed alignment,low laser powered instrument with a quartz flow cell gel-coupled to the collection optics. However,whether high purity mouse and human chromosomes can be obtained by sorting on the BD FACSAria(TM) Special Order Research Product (FACSAria SORP) remains to be determined. Here,we report that the high resolution flow karyotype of mouse lymphocytes and normal male human peripheral blood mononuclear cells (hPBMCs) can be obtained on the FACSAria SORP using laser power settings of 50 mW for 355 nm and 20 mW for 444 nm excitation. Furthermore,the use of Fluorescence in situ hybridization (FISH) confirmed that chromosome paints prepared from the sorted chromosomes demonstrated high purity and signal specificity. Notably,human chromosome 12 was separated from the chromosome 9-12 cluster in the flow karyotype,and its identity was confirmed using FISH in trisomy 12 human ES cell lines B2-C7 and B2-B8. In addition,multicolor FISH (mFISH) with human chromosome painting probes to 13,18,21,and sex chromosomes X and Y showed high signal specificity in hPBMCs. Taken together,our findings demonstrated that high resolution flow karyotype can be obtained using FACSAria SORP. Moreover,a FISH analysis confirmed high purity of the sorted chromosomes. Additionally,in contrast to centromeric satellite probes,chromosome painting probes with high specificity are more suitable for detection of chromosome aberrations,such as deletions and translocations,in prenatal diagnosis. textcopyright 2016 International Society for Advancement of Cytometry. View Publication

Selective protein kinase inhibitors were developed on the basis of the unexpected binding mode of 2,6,9-trisubstituted purines to the adenosine triphosphate-binding site of the human cyclin-dependent kinase 2 (CDK2). By iterating chemical library synthesis and biological screening,potent inhibitors of the human CDK2-cyclin A kinase complex and of Saccharomyces cerevisiae Cdc28p were identified. The structural basis for the binding affinity and selectivity was determined by analysis of a three-dimensional crystal structure of a CDK2-inhibitor complex. The cellular effects of these compounds were characterized in mammalian cells and yeast. In the latter case the effects were characterized on a genome-wide scale by monitoring changes in messenger RNA levels in treated cells with high-density oligonucleotide probe arrays. Purine libraries could provide useful tools for analyzing a variety of signaling and regulatory pathways and may lead to the development of new therapeutics. View Publication

Gaucher disease is caused by an inherited deficiency of glucocerebrosidase that manifests with storage of glycolipids in lysosomes,particularly in macrophages. Available cell lines modeling Gaucher disease do not demonstrate lysosomal storage of glycolipids; therefore,we set out to develop two macrophage models of Gaucher disease that exhibit appropriate substrate accumulation. We used these cellular models both to investigate altered macrophage biology in Gaucher disease and to evaluate candidate drugs for its treatment. We generated and characterized monocyte-derived macrophages from 20 patients carrying different Gaucher disease mutations. In addition,we created induced pluripotent stem cell (iPSC)-derived macrophages from five fibroblast lines taken from patients with type 1 or type 2 Gaucher disease. Macrophages derived from patient monocytes or iPSCs showed reduced glucocerebrosidase activity and increased storage of glucocerebroside and glucosylsphingosine in lysosomes. These macrophages showed efficient phagocytosis of bacteria but reduced production of intracellular reactive oxygen species and impaired chemotaxis. The disease phenotype was reversed with a noninhibitory small-molecule chaperone drug that enhanced glucocerebrosidase activity in the macrophages,reduced glycolipid storage,and normalized chemotaxis and production of reactive oxygen species. Macrophages differentiated from patient monocytes or patient-derived iPSCs provide cellular models that can be used to investigate disease pathogenesis and facilitate drug development. View Publication

Disentangling the complex interactions that govern stem cell fate choices of self-renewal,differentiation,or death presents a formidable challenge. Image-based phenotype-driven screening meets this challenge by providing means for rapid testing of many small molecules simultaneously. Pluripotent embryonal carcinoma (EC) cells offer a convenient substitute for embryonic stem (ES) cells in such screens because they are simpler to maintain and control. The authors developed an image-based screening assay to identify compounds that affect survival or differentiation of the human EC stem cell line NTERA2 by measuring the effect on cell number and the proportion of cells expressing a pluripotency-associated marker SSEA3. A pilot screen of 80 kinase inhibitors identified several compounds that improved cell survival or induced differentiation. The survival compounds Y-27632,HA-1077,and H-8 all strongly inhibit the kinases ROCK and PRK2,highlighting the important role of these kinases in EC cell survival. Two molecules,GF109203x and rottlerin,induced EC differentiation. The effects of rottlerin were also investigated in human ES cells. Rottlerin inhibited the self-renewal ability of ES cells,caused the cell cycle arrest,and repressed the expression of pluripotency-associated genes. View Publication

Intrasynovial flexor tendon lacerations of the hand are clinically problematic,typically requiring operative repair and extensive rehabilitation. The small-molecule connective tissue growth factor (CTGF) mimics,oxotremorine M (Oxo-M) and 4-PPBP maleate (4-PPBP),have been shown to improve tendon healing in small animal models by stimulating the expansion and differentiation of perivascular CD146+ cells. To enhance intrasynovial flexor tendon healing,small-molecule CTGF mimics were delivered to repaired canine flexor tendons via porous sutures. In vitro studies demonstrated that Oxo-M and 4-PPBP retained their bioactivity and could be released from porous sutures in a sustained manner. However,in vivo delivery of the CTGF mimics did not improve intrasynovial tendon healing. Histologic analyses and expression of tenogenic,extracellular matrix,inflammation,and remodeling genes showed similar outcomes in treated and untreated repairs across two time points. Although in vitro experiments revealed that CTGF mimics stimulated robust responses in extrasynovial tendon cells,there was no response in intrasynovial tendon cells,explaining the lack of in vivo effects. The results of the current study indicate that therapeutic strategies for tendon repair must carefully consider the environment and cellular makeup of the particular tendon for improving the healing response. View Publication

Chronic compressive cervical spinal cord injury (cCSCI),a debilitating condition,lacks effective treatment options. Addressing this gap,our study introduces a novel rat model of cCSCI developed through spinal cord compression via synthetic polyether sheet implantation,closely mimicking human pathology. We evaluated the model’s fidelity utilizing a comprehensive series of behavioral,electrophysiological,and histological assessments. Our research also explored the therapeutic potential of oligodendrogenic neural progenitor cells (oNPCs) derived from induced pluripotent stem cells. Transplanted oNPCs successfully integrated into the host spinal cord,differentiated into neurons,astrocytes,and oligodendrocytes,and demonstrated a remarkable capacity for enhancing neuroplasticity. Electrophysiological analyses revealed significant improvements in motor evoked potentials and a rectification of the excitability imbalance posttransplantation,indicating substantial recovery of motor circuits. Histological findings complemented these results,showing enhanced remyelination and a reduction in excitatory transmitter expression in the residual gray matter. Functionally,the transplantation of oNPCs led to marked improvements in grip strength,locomotor abilities,and sensory functions,surpassing those seen with standard treatments. This study not only provides a novel and reliable rat model of cCSCI for further research but also highlights the potential of oNPCs as a transformative approach for spinal cord injury therapy,suggesting their significant role in neural regeneration and repair. View Publication

The clinical success of CRISPR therapies hinges on the safety and efficacy of Cas proteins. The Cas9 from Francisella novicida (FnCas9) is highly precise,with a negligible affinity for mismatched substrates,but its low cellular targeting efficiency limits therapeutic use. Here,we rationally engineer the protein to develop enhanced FnCas9 (enFnCas9) variants and broaden their accessibility across human genomic sites by ~3.5-fold. The enFnCas9 proteins with single mismatch specificity expanded the target range of FnCas9-based CRISPR diagnostics to detect the pathogenic DNA signatures. They outperform Streptococcus pyogenes Cas9 (SpCas9) and its engineered derivatives in on-target editing efficiency,knock-in rates,and off-target specificity. enFnCas9 can be combined with extended gRNAs for robust base editing at sites which are inaccessible to PAM-constrained canonical base editors. Finally,we demonstrate an RPE65 mutation correction in a Leber congenital amaurosis 2 (LCA2) patient-specific iPSC line using enFnCas9 adenine base editor,highlighting its therapeutic utility. Subject terms: CRISPR-Cas9 genome editing,Molecular medicine,Genetic engineering,CRISPR-Cas9 genome editing View Publication

AbstractBackgroundThe pro-inflammatory cytokine,interleukin-18 (IL-18),plays an instrumental role in bolstering anti-tumor immunity. However,the therapeutic application of IL-18 has been limited due to its susceptibility to neutralization by IL-18 binding protein (IL-18BP),short in vivo half-life,and unfavorable physicochemical properties.MethodsIn order to overcome the poor drug-like properties of IL-18,we installed an artificial disulfide bond,removed the native,unpaired cysteines,and fused the stabilized cytokine to an IgG Fc domain. The stability,potency,pharmacokinetic and pharmacodynamic properties as well as efficacy of disulfide-stabilized IL-18 Fc-fusion (dsIL-18-Fc) were assessed via in vitro and in vivo studies.ResultsThe stability and mammalian host cell production yields of dsIL-18-Fc were improved,compared to the wild-type (WT) cytokine,while maintaining its biological potency and interactions with IL-18 receptor α (IL-18Rα) and IL-18BP. Recombinant fusion of the cytokine to an IgG Fc domain provided extended half-life. Notably,despite maintaining sensitivity to IL-18BP,dsIL-18-Fc was effective at activating both T and natural killer (NK) cells,and elicited a strong anti-tumor response,either as a single agent,or in conjunction with anti-programmed cell death-ligand 1 (anti-PD-L1) therapy.ConclusionsWe engineered IL-18 for reinforced stability,extended half-life,and improved manufacturability. The therapeutic benefit of dsIL-18-Fc,coupled with a more favorable manufacturability profile and enhanced drug-like properties,underscores the potential utility of this engineered cytokine in cancer immunotherapy. View Publication

IntroductionBispecific antibodies (BsAbs) can simultaneously target two epitopes of different antigenic targets,bringing possibilities for diversity in antibody drug design and are promising tools for the treatment of cancers and other diseases. T-cell engaging bsAb is an important application of the bispecific antibody,which could promote T cell-mediated tumor cell killing by targeting tumor-associated antigen (TAA) and CD3 at the same time.MethodsThis study comprised antibodies purification,Elisa assay for antigen binding,cytotoxicity assays,T cell activation by flow cytometry in vitro and xenogenic tumor model in vivo.ResultsWe present a novel bsAb platform named PHE-Ig technique to promote cognate heavy chain (HC)-light chain (LC) pairing by replacing the CH1/CL regions of different monoclonal antibodies (mAbs) with the natural A and B chains of PHE1 fragment of Integrin β2 based on the knob-in-hole (KIH) technology. We had also verified that PHE-Ig technology can be effectively used as a platform to synthesize different desired bsAbs for T-cell immunotherapy. Especially,BCMA×CD3 PHE-Ig bsAbs exhibited robust anti-multiple myeloma (MM) activity in vitro and in vivo.DiscussionMoreover,PHE1 domain was further shortened with D14G and R41S mutations,named PHE-S,and the PHE-S-based BCMA×CD3 bsAbs also showed anti BCMA+ tumor effect in vitro and in vivo,bringing more possibilities for the development and optimization of different bsAbs. To sum up,PHE1-based IgG-like antibody platform for bsAb construction provides a novel strategy for enhanced T-cell immunotherapy. View Publication

Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination (HR),with efficiency improved by CRISPR/Cas9 technology. Circular single-stranded DNA (cssDNA) has been used as a genome engineering catalyst (GATALYST) for efficient and safe gene knock-in. Here,we introduce enGager,an enhanced GATALYST associated genome editor system that increases transgene integration efficiency by tethering cssDNA donors to nuclear-localized Cas9 fused with single-stranded DNA binding peptide motifs. This approach further improves targeted integration and expression of reporter genes at multiple genomic loci in various cell types,showing up to 6-fold higher efficiency compared to unfused Cas9,especially for large transgenes in primary cells. Notably,enGager enables efficient integration of a chimeric antigen receptor (CAR) transgene in 33% of primary human T cells,enhancing anti-tumor functionality. This ‘tripartite editor with ssDNA optimized genome engineering (TESOGENASE) offers a safer,more efficient alternative to viral vectors for therapeutic gene modification. Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination. Here the authors present the TESOGENASE system which enhances CRISPR-based gene integration by tethering circular single-stranded DNA to Cas9. View Publication