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.
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.
An effective strategy is developed to achieve both high mechanical strength and excellent nonfouling properties in saline environments, important for biomedical and engineering applications, by constructing a pure zwitterionic triple‐network hydrogel. Strong chain entanglement and electrostatic interactions provide effective energy dissipation to improve the mechanical strength and toughness of the hydrogel.
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.
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.
Enhanced biosynthesis of hyaluronic acid with engineered Corynebacterium glutamicum is achieved via metabolic regulation: Eight diverse operon combinations including HasA, HasAB, HasAC, HasAD, HasAE, HasABC, HasABD, and HasABE are compared and HasAB along with HasABC performed the best. LDH gene knockout is conducted by single crossover homologous recombination and the recombinant strain C. glutamicum/△ldh‐AB is constructed, meanwhile ATP supply is enhanced in the early stage of batch culture. Through glucose feeding, the maximum HA titer of C. glutamicum/△ldh‐AB in a 5 L fermenter highly reached 21.6 g L−1, more than threefolds of that of the wild Streptococcus.
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.
The hidden hydrophobicity of conventional polymers widely considered as hydrophilic is closely related to their immunogenicity when they are conjugated to immunogenic proteins.
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.
Structure breaking amino acids, P, S, and G, are incorporated into low immunogenic unstructured zwitterionic peptide fusion proteins. We find unique sequence motifs that exhibit charge balanced conformations and low immunogenicity.
Achieving therapeutic efficacy in protein replacement therapies requires sustaining pharmacokinetic (PK) profiles, while maintaining the bioactivity of circulating proteins. This is often achieved ...via PEGylation in protein-based therapies, but it remains challenging for proteins produced in vivo in mRNA-based therapies due to the lack of a suitable post-translational modification method. To address this issue, we integrated a genetically encoded zwitterionic polypeptide, EKP, into mRNA constructs to enhance the PK properties of product proteins. Composed of alternating glutamic acid (E), lysine (K), and proline (P), EKP exhibits unique superhydrophilic properties and low immunogenicity. Our results demonstrate that EKP fusion significantly extends the circulation half-life of proteins expressed from mRNA while preserving their bioactivity using human interferon alpha and Neoleukin-2/15 as examples. This EKP fusion technology offers a new approach to overcoming the current limitations in mRNA therapeutics and has the potential to significantly advance the development of mRNA-based protein replacement therapy.
Protein, nucleic acid, and small-molecule drugs frequently require chemical modification with polymers such as polyethylene glycol (PEG), to increase their in vivo circulation time and reduce their ...immunogenicity. Previously, we developed low-immunogenic zwitterionic polycarboxybetaine (PCB) to replace PEGylation. Herein, we delineate the design principles of zwitterionic polypeptide to mimic PCB. These studies establish an EKP polypeptide composed of glutamic acid (E), lysine (K), and proline (P) as a low-immunogenic, unstructured, zwitterionic peptide. Then, we demonstrate EKP peptide as a genetically encodable fusion protein platform. The unique zwitterionic design ensures high hydration, thereby increasing hydrodynamic size and improving stability. EKP fusion proteins achieve prolonged circulation, low immunogenicity, and maintained circulation time over multiple injections, thus avoiding accelerating blood clearance (ABC). Its performance substantiates EKP as a valuable platform technology for drugs and biologics.
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•A low-immunogenic zwitterionic EKP peptide is identified and confirmed in vivo.•Therapeutic proteins fused with genetically encoded EKP improve their biophysical properties and in vivo performance.•EKP is established as a platform technology for drugs and biologics.
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.
Cu everywhere: Mechanistic study of the tris(triazolylmethyl)amine ligand accelerated CuAAC reaction was performed in aqueous solutions. Reactive tri‐Cu and di‐Cu intermediates were identified by ESI‐MS and their CuAAC activity were quantitatively measured by LC‐MS.
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