Extracellular vesicles incorporating retrovirus-like capsids for the enhanced packaging and systemic delivery of mRNA into neurons.

The blood-brain barrier (BBB) restricts the systemic delivery of messenger RNAs (mRNAs) into diseased neurons. Although leucocyte-derived extracellular vesicles (EVs) can cross the BBB at inflammatory sites, it is difficult to efficiently load long mRNAs into the EVs and to enhance their neuronal uptake. Here we show that the packaging of mRNA into leucocyte-derived EVs and the endocytosis of the EVs by neurons can be enhanced by engineering leucocytes to produce EVs that incorporate retrovirus-like mRNA-packaging capsids. We transfected immortalized and primary bone-marrow-derived leucocytes with DNA or RNA encoding the capsid-forming activity-regulated cytoskeleton-associated (Arc) protein as well as capsid-stabilizing Arc 5'-untranslated-region RNA elements. These engineered EVs inherit endothelial adhesion molecules from donor leukocytes, recruit endogenous enveloping proteins to their surface, cross the BBB, and enter the neurons in neuro-inflammatory sites. Produced from self-derived donor leukocytes, the EVs are immunologically inert, and enhanced the neuronal uptake of the packaged mRNA in a mouse model of low-grade chronic neuro-inflammation.

Hidden hydrophobicity impacts polymer immunogenicity.

Antibodies against poly(ethylene glycol) (PEG) have been found to be the culprit of side reactions and efficacy loss of a number of PEGylated drugs. Fundamental mechanisms of PEG immunogenicity and design principles for PEG alternatives still have not been fully explored. By using hydrophobic interaction chromatography (HIC) under varied salt conditions, we reveal the "hidden" hydrophobicity of those polymers which are generally considered as hydrophilic. A correlation between the hidden hydrophobicity of a polymer and its polymer immunogenicity is observed when this polymer is conjugated with an immunogenic protein. Such a correlation of hidden hydrophobicity vs. immunogenicity for a polymer also applies to corresponding polymer-protein conjugates. Atomistic molecular dynamics (MD) simulation results show a similar trend. Based on polyzwitterion modification and with this HIC technique, we are able to produce extremely low-immunogenic protein conjugates as their hydrophilicity is pushed to the limit and their hydrophobicity is eliminated, breaking the current barriers of eliminating anti-drug and anti-polymer antibodies.

Motif-based zwitterionic peptides impact their structure and immunogenicity.

The linkage of zwitterionic peptides containing alternating glutamic acid (E) and lysine (K) amino acids exhibits protective effects on protein drugs due to their high hydration capacity. Previously, short EK peptides covering the surface of a protein drug showed significant protective effects and low immunogenicity. However, for high-molecular-weight single-chain (HMWSC) zwitterionic peptides, the incorporation of structure-disrupting amino acids such as proline (P), serine (S), and glycine (G) is necessary to improve their protective ability. Herein, we first probe the immunogenicity of eight EK-containing motif-based peptides, six of which incorporate structure-disrupting amino acids P, S, and G, linked to keyhole limpet hemocyanin (KLH). These studies uncover two sequence motifs, EKS and EKG, which show uniquely higher immunogenicity, while the other motifs, especially those containing P, exhibit lower immunogenicity. Additionally, the structure and dynamics of these sequence motifs are computationally modeled by Rosetta protein predictions and molecular dynamics (MD) simulations to predict properties of higher and lower immunogenicity peptides. These simulations revealed peptides with higher immunogenicity, namely EKS and EKG, exhibit regions of charge imbalance. Then, HMWSC zwitterionic sequences were linked to a typical protein drug, interferon-alpha 2a (IFN), which showed consistent immunogenic behaviors. Finally, epitope mapping and alanine scanning experiments using the serum collected from mice injected with HMWSC sequences also implicated a link between charge imbalance and peptide immunogenicity.

Phosphatidylserine Lipid Nanoparticles Promote Systemic RNA Delivery to Secondary Lymphoid Organs.

Secondary lymphoid organs (SLOs) are an important target for mRNA delivery in various applications. While the current delivery method relies on the drainage of nanoparticles to lymph nodes by intramuscular (IM) or subcutaneous (SC) injections, an efficient mRNA delivery carrier for SLOs-targeting delivery by systemic administration (IV) is highly desirable but yet to be available. In this study, we developed an efficient SLOs-targeting carrier using phosphatidylserine (PS), a well-known signaling molecule that promotes the endocytic activity of phagocytes and cellular entry of enveloped viruses. We adopted these biomimetic strategies and added PS into the standard four-component MC3-based LNP formulation (PS-LNP) to facilitate the cellular uptake of immune cells beyond the charge-driven targeting principle commonly used today. As a result, PS-LNP performed efficient protein expression in both lymph nodes and the spleen after IV administration. In vitro and in vivo characterizations on PS-LNP demonstrated a monocyte/macrophage-mediated SLOs-targeting delivery mechanism.

Mitigating the Immunogenicity of AAV-Mediated Gene Therapy with an Immunosuppressive Phosphoserine-Containing Zwitterionic Peptide.

Although recombinant adeno-associated viruses (AAVs) are considered low immunogenic and safe for gene delivery, the immunogenicity of capsids still represents a major obstacle to the readministration of AAV vectors. Here, we design an immunosuppressive zwitterionic phosphoserine (PS)-containing polypeptide to induce AAV-specific immune tolerance and eradicate the immunological response. AAVs modified with the zwitterionic PS polypeptide maintain their transduction activity and tissue tropism but suppress the induction of AAV-specific antibodies. In a hemophilia A mouse model (FVIII knockout mice), the readministration of zwitterionic PS polypeptide-modified AAV8-FVIII vectors successfully evades immunological response, corrects blood FVIII levels, and stops blood loss in tail-bleeding experiments. This potent and safe technology mimics the natural tolerance of apoptotic cells and controls the immunosuppressive, zwitterionic, and degradable polypeptide precisely, reducing the concern of toxicities upon readministrations. This work presents a new concept and a platform of engineered viral vectors by chemically linking immunosuppressive materials to AAV vectors, enabling the readministration of AAV vectors while maintaining their transduction efficiency to a considerable degree.

High-Strength and Nonfouling Zwitterionic Triple-Network Hydrogel in Saline Environments.

Zwitterionic hydrogels have received great attention due to their excellent nonfouling and biocompatible properties, but they suffer from weak mechanical strength in the saline environments important for biomedical and engineering applications due to the "anti-polyelectrolyte" effect. Conventional strategies to introduce hydrophobic or non-zwitterionic components to increase mechanical strength compromise their nonfouling properties. Here, a highly effective strategy is reported to achieve both high mechanical strength and excellent nonfouling properties by constructing a pure zwitterionic triple-network (ZTN) hydrogel. The strong electrostatic interaction and network entanglement within the triple-network structure can effectively dissipate energy to toughen the hydrogel and achieve high strength, toughness, and stiffness in saline environments (compressive fracture stress 18.2 ± 1.4 MPa, toughness 1.62 ± 0.03 MJ m-3 , and modulus 0.66 ± 0.03 MPa in seawater environments). Moreover, the ZTN hydrogel is shown to strongly resist the attachment of proteins, bacteria, and cells. The results provide a fundamental understanding to guide the design of tough nonfouling zwitterionic hydrogels for a broad range of applications.

Zwitterionic Polymer Conjugated Glucagon-like Peptide-1 for Prolonged Glycemic Control.

Glucagon-like peptide-1 (GLP-1) is of particular interest for treating type 2 diabetes mellitus (T2DM), as it induces insulin secretion in a glucose-dependent fashion and has the potential to facilitate weight control. However, native GLP-1 is a short incretin peptide that is susceptible to fast proteolytic inactivation and rapid clearance from the circulation. Various GLP-1 analogs and bioconjugation of GLP-1 analogs have been developed to counter these issues, but these modifications are frequently accompanied by the sacrifice of potency and the induction of immunogenicity. Here, we demonstrated that with the conjugation of a zwitterionic polymer, poly(carboxybetaine) (pCB), the pharmacokinetic properties of native GLP-1 were greatly enhanced without serious negative effects on its potency and secondary structure. The pCB conjugated GLP-1 further provided glycemic control for up to 6 days in a mouse study. These results illustrate that the conjugation of pCB could realize the potential of using native GLP-1 for prolonged glycemic control in treating T2DM.

Biosynthesis of Chondroitin in Engineered Corynebacterium glutamicum.

Chondroitin, the precursor of chondroitin sulfate, which is an important polysaccharide, has drawn significant attention due to its applications in many fields. In the present study, a heterologous biosynthesis pathway of chondroitin was designed in a GRAS (generally recognized as safe) strain C. glutamicum. CgkfoC and CgkfoA genes with host codon preference were synthesized and driven by promoter Ptac, which was confirmed as a strong promoter via GFPuv reporter assessment. In a lactate dehydrogenase (ldh) deficient host, intracellular chondroitin titer increased from 0.25 to 0.88 g/l compared with that in a wild-type host. Moreover, precursor enhancement via overexpressing precursor synthesizing gene ugdA further improved chondroitin titers to 1.09 g/l. Chondroitin production reached 1.91 g/l with the engineered strain C. glutamicum ΔL-CgCAU in a 5-L fed-batch fermentation with a single distribution Mw of 186 kDa. This work provides an alternative, safe and novel means of producing chondroitin for industrial applications.

Enhanced Biosynthesis of Hyaluronic Acid Using Engineered Corynebacterium glutamicum Via Metabolic Pathway Regulation.

Hyaluronic acid (HA) is a polysaccharide used in many industries such as medicine, surgery, cosmetics, and food. To avoid potential pathogenicity caused by its native producer, Streptococcus, efforts have been made to create a recombinant host for HA production. In this work, a GRAS (generally recognized as safe) strain, Corynebacterium glutamicum, is engineered for enhanced biosynthesis of HA via metabolic pathway regulation. Five enzymes (HasA-HasE) involved in the HA biosynthetic pathway are highlighted, and eight diverse operon combinations, including HasA, HasAB, HasAC, HasAD, HasAE, HasABC, HasABD, and HasABE, are compared. HasAB and HasABC are found to be optimal for HA biosynthesis in C. glutamicum. To meet the energy demand for HA synthesis, the metabolic pathway that produces lactate is blocked by knocking out the lactate dehydrogenase (LDH) gene using single crossover homologous recombination. Engineered C. glutamicum/Δldh-AB is superior and had a significantly higher HA titer than C. glutamicum/Δldh-ABC. Batch and fed-batch cultures of C. glutamicum/Δldh-AB are performed in a 5-L fermenter. Using glucose feeding, the maximum HA titer reached 21.6 g L-1 , more than threefolds of that of the wild-type Streptococcus. This work provides an efficient, safe, and novel recombinant HA producer, C. glutamicum/Δldh-AB, via metabolic pathway regulation.

Intermediates Stabilized by Tris(triazolylmethyl)amines in the CuAAC Reaction.

Tris(triazolylmethyl)amine ligands (TL) are widely used to accelerate the CuI -catalyzed azide-alkyne cycloaddition (CuAAC) reaction, but its mechanistic role remains unclear. Using electrospray ionization mass spectrometry, we detected for the first time the trinuclear TL-CuI3 -acetylide and the dinuclear TL-CuI2 -acetylide complexes in aqueous solution. The apparent second-order rate constants of their reaction with an azide were 27 and 783 m-1 ⋅s-1 when the alkyne was tethered to TL. In the catalytic system without the tether, the rate constant increased to >146 m-1 ⋅s-1 for the TL-CuI3 -acetylide, but dropped about 14-fold to approximately 55 m-1 ⋅s-1 for the TL-CuI2 -acetylide. The results indicated that TL accelerated the reaction by stabilizing the CuI2 - and CuI3 -acetylide and their azide-adduct intermediates, but this role is largely weakened by excess alkyne and other competing ligands under catalytic conditions.