RESUMEN
Polymer-protein bioconjugation offers a powerful strategy to alter the physical properties of proteins, and various synthetic polymer compositions and architectures have been investigated for this purpose. Nevertheless, conjugation of molecular bottlebrush polymers (BPs) to proteins remains an unsolved challenge due to the large size of BPs and a general lack of methods to transform the chain ends of BPs into functional groups suitable for bioconjugation. Here, we present a strategy to address this challenge in the context of BPs prepared by "graft-through" ring-opening metathesis polymerization (ROMP), one of the most powerful methods for BP synthesis. Quenching ROMP of PEGylated norbornene macromonomers with an activated enyne terminator facilitates the transformation of the BP Ru alkylidene chain ends into Pd oxidative addition complexes (OACs) for facile bioconjugation. This strategy is shown to be effective for the synthesis of two BP-protein conjugates (albumin and ERG), setting the stage for a new class of BP-protein conjugates for future therapeutic and imaging applications.
Asunto(s)
Polímeros , Proteínas , Polimerizacion , AlbúminasRESUMEN
Brain-derived neurotrophic factor (BDNF) acting through the tropomyosin-related receptor-B (TrkB) is an important signaling system for the maintenance and survival of neurons. Gene therapy using either recombinant adeno-associated virus (AAV) or lentiviral vectors can provide sustained delivery of BDNF to tissues where reduced BDNF signaling is hypothesized to contribute to disease pathophysiology. However, elevation in BDNF at target sites has been shown to lead to a downregulation of TrkB receptors, thereby reducing the effect of chronic BDNF delivery over time. A novel gene sequence has been designed coding both the ligand (BDNF) and the TrkB receptor in a single transgene separated by a short viral-2A sequence. The single transgene is efficiently processed intracellularly in vitro and in vivo to yield the two mature proteins, which are then independently transported to their final cellular locations: TrkB receptors to the cell surface, and BDNF contained within secretory vesicles. To accommodate the coding sequences of both BDNF and TrkB receptors within the narrow confines of the AAV vectors (4.7 kb pairs), the coding region for the pro-domain of BDNF was removed and the signal peptide sequence modified to improve production, intracellular transport, and secretion of mature BDNF (mBDNF). Intracellular processing and efficacy was shown in HEK293 cells and SH-SY5Y neuroblastoma cells using plasmid DNA and after incorporating the TrkB-2A-mBDNF into an AAV2 vector. Increased BDNF/TrkB-mediated intracellular signaling pathways were observed after AAV2 vector transfection while increased TrkB phosphorylation could be detected in combination with neuroprotection from hydrogen peroxide-induced oxidative stress. Correct processing was also shown in vivo in mouse retinal ganglion cells after AAV2 vector administration to the eye. This novel construct is currently being investigated for its efficacy in animal models to determine its potential to progress to human clinical studies in the future.