Revolutionary delivery system enables precise protein transportation

Abstract

The capacity to transport customized proteins into certain cell types has enormous implications for life science research and disease therapy. However, challenges associated with cell targeting and protein transportation through cellular membranes still exist, necessitating the creation of complex systems that can continuously carry payload proteins into cells.1. In gene editing especially, achieving accurate targeted delivery is an intricate problem that warrants addressing. Endosymbiotic bacteria have developed complex delivery systems that enable them to interact with the host organism,2 wherein secreted contractile injection systems (CISs), which are analogous to bacteriophage tails, can be harnessed as nanodevices.3. These macromolecular complexes consist of a solid tubular structure encompassed in a compressible sheath that is attached to a baseplate and sharpened by a spike protein. It is hypothesized that payloads are packed into the lumen of the inner tube behind the spike, which upon recognition by the target cell are pushed into the target cell through the membrane via sheath contraction.4 Inspired by previous reports regarding CISs, recently, a team led by Professor Feng Zhang at the Broad Institute developed a redesigned protein delivery system; the corresponding results have been published in Nature.5 Therein, extracellular contractile injection systems (eCISs), syringe-like nanomachines mimicking bacteriophage tails that can transport payloads independently and extracellularly, served as a new tool to solve a long-standing problem, that is, how to deliver therapeutic molecules to specific types of human cells precisely and efficiently (Figure 1). The structural composition of eCISs originating from the Photorhabdus virulence cassette (PVC) is such that the tail fibers on the outside of one end recognize specific receptors on the cell surface and anchor to host cells; thus, in their study, the researchers speculated that modifying the structure of these tail fibers may enable them to recognize different cells. Given that the action and targeting mechanisms of eCISs in human cells remain a mystery, the team used AlphaFold, an artificial intelligence protein design platform that can predict protein structures from amino acid sequences, to redesign the injector of PVC to shift the targeting objective from insect cells to human cells. Cellular studies demonstrated that after modification, the syringe, carrying a variety of protein cargoes, could detect human and mouse cells. Further research on protein delivery to cultivated cells was carried out, and the modified PVC was proved to exhibit specific targeting toward epidermal growth factor receptor (EGFR) after genetic engineering. Schematic illustration of the extracellular contractile injection systems (eCISs) with re-engineered delivery performance in human cells and the mouse brain. The most promising application of the precise delivery based on such PVC-derived eCISs is the specific targeting of brain tissue and the nervous system after rationally redesigning PVC-derived eCISs via genetic engineering. The primary challenge in delivering proteins to the nervous system lies in the traversing of the blood-brain barrier (BBB) in the non-invasive and efficient manner. By pinpointing novel PVCs capable of targeting mouse cells with minimal systemic toxicity, the possibility of ideal intracranial delivery is expected. An extended tdTomato distribution representing is especially observed in the neurons of hippocampus region after intracranial injection of modified PVCs, while a negative control (Pvc10-free PVCs counterpart) fails to accumulate in the hippocampus region, which denotes that the modified PVCs have great potential for effectuating precise intracranial targeting without BBB restrictions. Particularly, the results regarding the in vivo delivery of the delicately designed eCISs, with high specificity and a lack of notable immune activity, along with their property of rapid purification from the brain, indicate their successful further application in humans. Typically, effective treatments for central nervous system (CNS) diseases involve three inter-related factors (a) delivering payloads (e.g., drugs, proteins, or other macromolecules) across the BBB, (b) achieving effective doses of the payloads at the target site, and (c) limiting the accumulation of drugs in non-target sites. The implementation of precise delivery by eCISs not only resolves a long-standing bottleneck of gene editing but also provides a viable solution to these key challenges. The tailored eCISs have paved the way for the creation of a novel protein delivery platform and provided a direction for future research in a wide range of application landscapes. Additionally, investigating potential delivery systems similar to PVCs for a range of payloads, including RNA or DNA, may become a priority for researchers henceforth. However, given the complex nature of the human immune