A synthetic transcription platform for programmable gene expression in mammalian cells.

Precise, scalable, and sustainable control of genetic and cellular activities in mammalian cells is key to developing precision therapeutics and smart biomanufacturing. Here we create a highly tunable, modular, versatile CRISPR-based synthetic transcription system for the programmable control of gene expression and cellular phenotypes in mammalian cells. Genetic circuits consisting of well-characterized libraries of guide RNAs, binding motifs of synthetic operators, transcriptional activators, and additional genetic regulatory elements express mammalian genes in a highly predictable and tunable manner.

A versatile platform for generating engineered extracellular vesicles with defined therapeutic properties.

Extracellular vesicles (EVs) are an important intercellular communication system facilitating the transfer of macromolecules between cells. Delivery of exogenous cargo tethered to the EV surface or packaged inside the lumen are key strategies for generating therapeutic EVs. We identified two "scaffold" proteins, PTGFRN and BASP1, that are preferentially sorted into EVs and enable high-density surface display and luminal loading of a wide range of molecules, including cytokines, antibody fragments, RNA binding proteins, vaccine antigens, Cas9, and members of the TNF superfamily.

Exosome Surface Display of IL12 Results in Tumor-Retained Pharmacology with Superior Potency and Limited Systemic Exposure Compared with Recombinant IL12.

The promise of IL12 as a cancer treatment has yet to be fulfilled with multiple tested approaches being limited by unwanted systemic exposure and unpredictable pharmacology. To address these limitations, we generated exoIL12, a novel, engineered exosome therapeutic that displays functional IL12 on the surface of an exosome. IL12 exosomal surface expression was achieved via fusion to the abundant exosomal surface protein PTGFRN resulting in equivalent potency to recombinant IL12 (rIL12) as demonstrated by IFNγ production.

Small-molecule control of antibody N-glycosylation in engineered mammalian cells.

N-linked glycosylation in monoclonal antibodies (mAbs) is crucial for structural and functional properties of mAb therapeutics, including stability, pharmacokinetics, safety and clinical efficacy. The biopharmaceutical industry currently lacks tools to precisely control N-glycosylation levels during mAb production. In this study, we engineered Chinese hamster ovary cells with synthetic genetic circuits to tune N-glycosylation of a stably expressed IgG.

Comparison of Integrases Identifies Bxb1-GA Mutant as the Most Efficient Site-Specific Integrase System in Mammalian Cells.

Phage-derived integrases can catalyze irreversible, site-specific integration of transgenic payloads into a chromosomal locus, resulting in mammalian cells that stably express transgenes or circuits of interest. Previous studies have demonstrated high-efficiency integration by the Bxb1 integrase in mammalian cells. Here, we show that a point mutation (Bxb1-GA) in Bxb1 target sites significantly increases Bxb1-mediated integration efficiency at the Rosa26 locus in Chinese hamster ovary cells, resulting in the highest integration efficiency reported with a site-specific integrase in mammalian cells.

A multi-landing pad DNA integration platform for mammalian cell engineering.

Engineering mammalian cell lines that stably express many transgenes requires the precise insertion of large amounts of heterologous DNA into well-characterized genomic loci, but current methods are limited. To facilitate reliable large-scale engineering of CHO cells, we identified 21 novel genomic sites that supported stable long-term expression of transgenes, and then constructed cell lines containing one, two or three 'landing pad' recombination sites at selected loci. By using a highly efficient BxB1 recombinase along with different selection markers at each site, we directed recombinase-mediated insertion of heterologous DNA to selected sites, including targeting all three with a single transfection.

Glycine dimerization motif in the N-terminal transmembrane domain of the high density lipoprotein receptor SR-BI required for normal receptor oligomerization and lipid transport.

Scavenger receptor class B, type I (SR-BI), a CD36 superfamily member, is an oligomeric high density lipoprotein (HDL) receptor that mediates negatively cooperative HDL binding and selective lipid uptake. We identified in the N-terminal transmembrane (N-TM) domain of SR-BI a conserved glycine dimerization motif, G(15)X(2)G(18)X(3)AX(2)G(25), of which the submotif G(18)X(3)AX(2)G(25) significantly contributes to homodimerization and lipid uptake activity. SR-BI variants were generated by mutations (single or multiple Gly → Leu substitutions) or by replacing the N-TM domain with those from other CD36 superfamily members containing (croquemort) or lacking (lysosomal integral membrane protein (LIMP) II) this glycine motif (chimeras).

ApoE induces serum paraoxonase PON1 activity and stability similar to ApoA-I.

Serum paraoxonase (PON1) is an anti-atherogenic interfacially activated lipo-lactonase that was shown to selectively bind high-density lipoprotein (HDL) carrying apolipoprotein A-I (apoA-I). ApoA-I binding occurs with nanomolar affinity and induces a dramatic increase in enzyme stability and lactonase activity. This study examined the association of PON1 with reconstituted HDL (rHDL) carrying apolipoprotein E, and its consequences on the stability and enzymatic activity of PON1, and on its anti-atherogenic potential.

In vivo administration of BL-3050: highly stable engineered PON1-HDL complexes.

Background: Serum paraoxonase (PON1) is a high density lipoprotein (HDL)-associated enzyme involved in organophosphate (OP) degradation and prevention of atherosclerosis. PON1 comprises a potential candidate for in vivo therapeutics, as an anti-atherogenic agent, and for detoxification of pesticides and nerve agents. Because human PON1 exhibits limited stability, engineered, recombinant PON1 (rePON1) variants that were designed for higher reactivity, solubility, stability, and bacterial expression, are candidates for treatment.

The development of human sera tests for HDL-bound serum PON1 and its lipolactonase activity.

Serum paraoxonase (PON1) is a lipolactonase that associates with HDL-apolipoprotein A-I (HDL-apoA-I) and thereby plays a role in the prevention of atherosclerosis. Current sera tests make use of promiscuous substrates and provide no indications regarding HDL-PON1 complex formation. We developed new enzymatic tests that detect total PON1 levels, irrespective of HDL status and R/Q polymorphism, as well as the degree of catalytic stimulation and increased stability that follow PON1's tight binding to HDL-apoA-I.

The 192R/Q polymorphs of serum paraoxonase PON1 differ in HDL binding, lipolactonase stimulation, and cholesterol efflux.

Serum paraoxonase (PON1) is a HDL-associated enzyme exhibiting potentially antiatherogenic properties. Here, we examined the common PON1-192R/Q human polymorphism. Despite numerous studies, the effect of this polymorphism on the antiatherogenic potential of PON1 is yet unresolved.

Enhanced stereoselective hydrolysis of toxic organophosphates by directly evolved variants of mammalian serum paraoxonase.

We addressed the ability of various organophosphorus (OP) hydrolases to catalytically scavenge toxic OP nerve agents. Mammalian paraoxonase (PON1) was found to be more active than Pseudomonas diminuta OP hydrolase (OPH) and squid O,O-di-isopropyl fluorophosphatase (DFPase) in detoxifying cyclosarin (O-cyclohexyl methylphosphonofluoridate) and soman (O-pinacolyl methylphosphonofluoridate). Subsequently, nine directly evolved PON1 variants, selected for increased hydrolytic rates with a fluorogenic diethylphosphate ester, were tested for detoxification of cyclosarin, soman, O-isopropyl-O-(p-nitrophenyl) methyl phosphonate (IMP-pNP), DFP, and chlorpyrifos-oxon (ChPo).

The catalytic histidine dyad of high density lipoprotein-associated serum paraoxonase-1 (PON1) is essential for PON1-mediated inhibition of low density lipoprotein oxidation and stimulation of macrophage cholesterol efflux.

High density lipoprotein (HDL)-associated paraoxonase-1 (PON1) anti-atherogenic properties in macrophages, i.e. inhibition of cell-mediated oxidation of low density lipoprotein (LDL) and stimulation of cholesterol efflux, were studied using recombinant variants of PON1 and apoA-I expressed in Escherichia coli and reconstituted HDL (rHDL) particles composed of phosphatidylcholine/free cholesterol (PC/FC) and apoA-I.

High affinity, stability, and lactonase activity of serum paraoxonase PON1 anchored on HDL with ApoA-I.

Serum paraoxonase (PON1) is a high-density lipoprotein (HDL)-associated enzyme exhibiting antiatherogenic properties. This study examined the interaction of recombinant PON1 with reconstituted HDL comprised of PC, cholesterol, and various apolipoproteins (apoA-I, -II, and -IV). The affinity, stability, and lactonase activity were strongly correlated, with apoA-I exhibiting the strongest effects, apoA-IV exhibiting weaker yet significant effects, and apoA-II having a negative effect relative to protein-free particles.

Effects of acyl versus aminoacyl conjugation on the properties of antimicrobial peptides.

To investigate the importance of increased hydrophobicity at the amino end of antimicrobial peptides, a dermaseptin derivative was used as a template for a systematic acylation study. Through a gradual increase of the acyl moiety chain length, hydrophobicity was monitored and further modulated by acyl conversion to aminoacyl. The chain lengths of the acyl derivatives correlated with a gradual increase in the peptide's global hydrophobicity and stabilization of its helical structure.

The 'evolvability' of promiscuous protein functions.

How proteins with new functions (e.g., drug or antibiotic resistance or degradation of man-made chemicals) evolve in a matter of months or years is still unclear.

Structure and evolution of the serum paraoxonase family of detoxifying and anti-atherosclerotic enzymes.

Members of the serum paraoxonase (PON) family have been identified in mammals and other vertebrates, and in invertebrates. PONs exhibit a wide range of physiologically important hydrolytic activities, including drug metabolism and detoxification of nerve agents. PON1 and PON3 reside on high-density lipoprotein (HDL, 'good cholesterol') and are involved in the prevention of atherosclerosis.

Directed evolution of mammalian paraoxonases PON1 and PON3 for bacterial expression and catalytic specialization.

Serum paraoxonases (PONs) are a group of enzymes that play a key role in organophosphate (OP) detoxification and in prevention of atherosclerosis. However, their structure and mechanism of action are poorly understood. PONs seem like jacks-of-all-trades, acting on a very wide range of substrates, most of which are of no physiological relevance.

Analysis of membrane-binding properties of dermaseptin analogues: relationships between binding and cytotoxicity.

To understand relationships between membrane-binding properties of cytolytic peptides and resulting cytotoxicity, we investigated interactions of dermaseptin analogues with model bilayers by means of surface plasmon resonance. First, we tested the system by comparing two native dermaseptins, S1 and S4, whose binding properties were previously characterized in different experimental systems. Validation experiments revealed deviations from the one-to-one interaction model and indicated the binding to proceed by a two-stage mechanism.

Direct interaction of dermaseptin S4 aminoheptanoyl derivative with intraerythrocytic malaria parasite leading to increased specific antiparasitic activity in culture.

Antiplasmodial activity of the dermaseptin S4 derivative K(4)S4(1-13) (P) was shown to be mediated by lysis of the host cells. To identify antiplasmodial peptides with enhanced selectivity, we produced and screened new derivatives based on P and singled out the aminoheptanoylated peptide (NC7-P) for its improved antiplasmodial properties. Compared with P, NC7-P displayed both increased antiparasitic efficiency and reduced hemolysis, including against infected cells.

In vitro antiplasmodium effects of dermaseptin S4 derivatives.

The 13-residue dermaseptin S4 derivative K(4)S4(1-13)a (P) was previously shown to kill intraerythrocytic malaria parasites through the lysis of the host cells. In this study, we have sought peptides that will kill the parasite without lysing the erythrocyte. To produce such peptides, 26 compounds of variable structure and size were attached to the N terminus of P and screened for antiplasmodium and hemolytic activities in cultures of Plasmodium falciparum.

Antibacterial properties of dermaseptin S4 derivatives with in vivo activity.

Derivatives of the cytotoxic peptide dermaseptin S4 have recently emerged as potential antimicrobial agents. Here, we report on the antibacterial properties of three derivatives with improved toxicity profiles: a 28-residues K4K20-S4 and two shorter versions, K4-S4(1-16) and K4-S4(1-13). The range of MICs of K4K20-S4 against clinical isolates of Staphylococcus aureus, Pseudomonas aeruginosa, and Escherichia coli were, respectively, 1 to 4, 1 to 4, and 1 to 16 microg/ml.

Structural requirements for potent versus selective cytotoxicity for antimicrobial dermaseptin S4 derivatives.

To better understand the structural requirements for selective cytotoxicity of antimicrobial peptides, seven dermaseptin S4 analogs were produced and investigated with respect to molecular organization in solution, binding properties to model phospholipid membranes, and cytotoxic properties. Native dermaseptin S4 displayed high aggregation in solution and high binding affinity. These properties correlated with high cytotoxicity.