Delivery Of Exogenous Proteins Research Articles

Delivery of exogenous proteins into eggs by injection into the mother's ovary (IMO) in zebrafish.

Genome editing has had profound effects on biological experimentation and can now be applied to many organisms, including non-conventional models. However, the introduction of genome editing components is time- and labor-consuming and sometimes requires special skills for microinjection. In this study, we developed a technique to deliver exogenous proteins into eggs by injection into the mother's ovary (IMO), which leads to the delivery of CRISPR/Cas9 into the eggs of oviparous animals, including fish. To test this technique, we examined whether exogenous proteins tagged with GFP or luciferase (Luc), and fluorescent-labeled RNP (Cas9 and sgRNA complex), can be delivered into eggs by IMO. When GFP-Luc or Cas9-Luc was delivered by IMO, their incorporation into fertilized eggs was confirmed by GFP fluorescence or luciferase activity; proteins were accumulated in the yolk. Cas9-RNP (targeting tyrosinase) was also incorporated into the eggs. However, genome editing of the target gene, tyrosinase, was not observed yet. This is presumably because the RNP delivered by IMO was packed in the yolk granules and did not reach into the embryonic nuclei. Thus, this report shows that exogenous molecules including Cas9-RNP were successfully delivered into fertilized eggs by IMO. Transferring the delivered RNP into nuclei will be critical for successful genome editing via the IMO delivery system.

Delivery of exogenous proteins by mesenchymal stem cells attenuates early memory deficits in a murine model of Alzheimer's disease

A promising intervention for Alzheimer's disease (AD) would ideally target key pathological factors that are involved in AD pathogenesis. Soluble factors produced by engrafted mesenchymal stem cells (MSCs) mediate potential therapeutic effects in AD. However, these therapeutic benefits are largely hampered by the limited paracrine capacity of MSCs. In this study, we used adenovirus-mediated gene transduction of bone marrow MSCs to deliver exogenous proteins into the brain of APPswe/PSEN1dE9 (APP/PS1) mice in the early stage of impairment. We observed that engrafted MSCs carrying exogenous (C-X3-C motif) ligand 1 (CX3CL1) alone reduced the production of the inflammatory cytokine TNF-ɑ and improved synapse-related protein expression but not cognitive function. Transplantation of MSCs carrying CX3CL1 and Wnt3a (CX3CL1-Wnt3a-MSC) significantly attenuated the learning and memory impairment when compared with a control group. The improvement of neurobehavioral functions in APP/PS1 mice treated with CX3CL1-Wnt3a-MSC was related to the inhibition of microglial neurotoxicity and promotion of hippocampal neurogenesis. Transplantation of CX3CL1-Wnt3a-MSC also regulated phosphoinositide 3-kinase/activated protein kinase B (PI3K/AKT) signaling to inhibit the activity of glycogen synthase kinase 3 beta (GSK3β). Taken together, these results indicate that the delivery of exogenous proteins via MSCs can modulate microglial function and enhance neurogenesis, thereby providing new insights into AD intervention.

Programmable RNA editing by recruiting endogenous ADAR using engineered RNAs.

Current tools for targeted RNA editing rely on the delivery of exogenous proteins or chemically modified guide RNAs, which may lead to aberrant effector activity, delivery barrier or immunogenicity. Here, we present an approach, called leveraging endogenous ADAR for programmable editing of RNA (LEAPER), that employs short engineered ADAR-recruiting RNAs (arRNAs) to recruit native ADAR1 or ADAR2 enzymes to change a specific adenosine to inosine. We show that arRNA, delivered by a plasmid or viral vector or as a synthetic oligonucleotide, achieves editing efficiencies of up to 80%. LEAPER is highly specific, with rare global off-targets and limited editing of non-target adenosines in the target region. It is active in a broad spectrum of cell types, including multiple human primary cell types, and can restore α-L-iduronidase catalytic activity in Hurler syndrome patient-derived primary fibroblasts without evoking innate immune responses. As a single-molecule system, LEAPER enables precise, efficient RNA editing with broad applicability for therapy and basic research.

The role of molecular adsorbent recirculating system dialysis for extracorporeal liver support in children

The majority of children with acute, acute-on-chronic, and progressive chronic liver failure require liver transplantation. Since organ availability is limited, extracorporeal liver support systems are increasingly applied to bridge the time until transplantation. At present, four different devices are available: the molecular adsorbent recirculating system (MARS), Prometheus dialysis, plasma exchange combined with hemodialysis (PE/HD), and single-pass albumin dialysis (SPAD). Randomized trials in adults have demonstrated efficient toxin removal, improved portal hypertension, hemodynamic stability, and improved hepatic encephalopathy compared with standard medical therapy. None of the liver support systems has yet been evaluated systematically in children. Knowledge of the specific indications and technical features of the different devices is essential if applied in children. MARS combines albumin dialysis with conventional hemodialysis and allows for efficient removal of water and protein-bound toxins without exogenous protein delivery and the associated infectious and allergic risks. It has successfully been applied in children with otherwise intractable cholestatic pruritus and with liver failure. The benefits, however, need to be balanced against the costs and the risk of volume and nitrogen overload if repeated plasma infusion is required. In cases of active bleeding, plasma exchange in combination with hemodialysis should be preferred.

Brain Involvement—The Ultimate Challenge

Lysosomal storage disorders (LSDs) typically present with a spectrum of phenotypes. More severe disease in terms of progression and complications is usually seen in patients with early neuronopathic manifestations. The primary central nervous system (CNS) pathology has traditionally been thought to be caused mainly by intracerebral storage of undegraded substrates of the deficient lysosomal enzymes. Visceral organs such as the liver, spleen, and kidney are easily reached by IV administration of enzyme replacement therapy. These therapies can overcome and prevent local substrate accumulation, whereas avascular tissues, such as cartilage and cornea, are not within reach for circulating enzymes. The brain presents a particular challenge, as the blood‐brain barrier (in health) prevents therapeutic enzymes from entering brain parenchyma. Despite this, some clinical indications of (partial) CNS progression/prevention effects of various “systemic-intended” enzyme replacement therapies in neuronopathic patients, especially with Gaucher disease, mucopolysaccharidosis (MPS) I, and MPS II, have been observed. As a result, additional adapted ideas regarding the possible etiopathology of CNS manifestations in LSDs have emerged. Among these are questioning whether the blood‐brain barrier is “leaky” in some LSDs, whether secondary damage to the brain can be a consequence of systemic disease (eg, hypoxia in MPS with sleep apneas), and whether aggregation of mutated proteins could cause disruption of neuronal cell function. New management approaches have also emerged. Among them is earlier and higher doses of IV enzyme replacement therapy. One concern with this treatment is that antigenicity may play a more important role in neuronopathic patients since they often have more nonsense mutations. The impact of antigenicity and immune response, especially in the CNS, to repeated delivery of exogenous proteins must be studied in more detail in animal models (MPS IIIA, MPS I). Another approach is the development of strategies to bypass the blood‐brain barrier, specifically targeting therapeutic enzymes to the CNS and peripheral nervous system. Lonser et al 1 recently published an approach with “direct convective delivery” of glucocerebrosidase in the CNS of 1 patient with chronic neuronopathic Gaucher disease. Other pharmacologic delivery techniques have been investigated, but they are limited in how they can be used clinically. Yet other therapeutic strategies—such as substrate reduction therapy, enzyme enhancement with chaperones, or gene therapy—have the theoretical potential to be effective in the brain but may have to be combined with enzyme replacement therapy. It is clear that specific advanced pathophysiologic changes (eg, neuronal cell loss, ectopic dendritogenesis) are not reversible; hence, early diagnosis, referral, intervention, and prevention are essential. The success of old and new therapeutic strategies depends on a small and early time window critical for intervention. In addition, timely and adequate use of supportive therapies and interventions is needed (eg, positive pressure ventilation in MPS, antiepileptic agents in Gaucher disease). Systematic and collaborative research in this field is warranted. Therefore, natural history studies, with a focus on manifestation and progression of neurologic signs and symptoms, as well as new techniques to examine and measure “neuronopathic disease,” are needed.

Making Proteins into Drugs: Assisted Delivery of Proteins and Peptides into Living Neurons

The introduction of exogenous proteins and peptides into cells is a valuable experimental approach. However, exogenous proteins are not internalized by living cells readily. This limitation has been overcome by the development of peptide-based methods that assist in the delivery of exogenous proteins into the cytoplasm and nucleus. These methods may facilitate the in vivo delivery of reagents in therapies aimed at neurodegenerative disorders and recovery from nervous system injury.