Targeting the Inside of Cells with Biologicals: Toxin Routes in a Therapeutic Context.

Numerous toxins translocate to the cytosol in order to fulfil their function. This demonstrates the existence of routes for proteins from the extracellular space to the cytosol. Understanding these routes is relevant to multiple aspects related to therapeutic applications.

Targeting the Inside of Cells with Biologicals: Chemicals as a Delivery Strategy.

Delivering macromolecules into the cytosol or nucleus is possible in vitro for DNA, RNA and proteins, but translation for clinical use has been limited. Therapeutic delivery of macromolecules into cells requires overcoming substantially higher barriers compared to the use of small molecule drugs or proteins in the extracellular space. Breakthroughs like DNA delivery for approved gene therapies and RNA delivery for silencing of genes (patisiran, ONPATTRO, Alnylam Pharmaceuticals, Cambridge, MA, USA) or for vaccination such as the RNA-based coronavirus disease 2019 (COVID-19) vaccines demonstrated the feasibility of using macromolecules inside cells for therapy.

Applying Antibodies Inside Cells: Principles and Recent Advances in Neurobiology, Virology and Oncology.

To interfere with cell function, many scientists rely on methods that target DNA or RNA due to the ease with which they can be applied. Proteins are usually the final executors of function but are targeted only indirectly by these methods. Recent advances in targeted degradation of proteins based on proteolysis-targeting chimaeras (PROTACs), ubiquibodies, deGradFP (degrade Green Fluorescent Protein) and other approaches have demonstrated the potential of interfering directly at the protein level for research and therapy.

Cytosolic Delivery of Macromolecules in Live Human Cells Using the Combined Endosomal Escape Activities of a Small Molecule and Cell Penetrating Peptides.

Ineffective cellular delivery is a common problem in numerous biological applications. Developing delivery reagents that work robustly in a variety of experimental settings remains a challenge. Herein, we report how peptides derived from the prototypical cell penetrating peptide TAT can be used in combination with a small molecule, UNC7938, to deliver macromolecules into the cytosol of cells by a simple co-incubation protocol.

Radiometal-labeled anti-VCAM-1 nanobodies as molecular tracers for atherosclerosis - impact of radiochemistry on pharmacokinetics.

Radiolabeling of nanobodies with radiometals by chelation has the advantage of being simple, fast and easy to implement in clinical routine. In this study, we validated 68Ga/111In-labeled anti-VCAM-1 nanobodies as potential radiometal-based tracers for molecular imaging of atherosclerosis. Both showed specific targeting of atherosclerotic lesions in ApoE-/- mice.

Evaluating the Delivery of Proteins to the Cytosol of Mammalian Cells.

Delivery of proteins to the cytosol of living cells is a promising research tool. Delivery of antibodies in particular bears exciting applications such as in vivo tracking of proteins at endogenous expression levels or interference with cellular processes. In spite of the large number of methods published for protein delivery, successful applications so far are rare.

Antibodies inside of a cell can change its outside: Can intrabodies provide a new therapeutic paradigm?

Challenges posed by complex diseases such as cancer, chronic viral infections, neurodegenerative disorders and many others have forced researchers to think beyond classic small molecule drugs, exploring new therapeutic strategies such as therapy with RNAi, CRISPR/Cas9 or antibody therapies as single or as combination therapies with existing drugs. While classic antibody therapies based on parenteral application can only reach extracellular targets, intracellular application of antibodies could provide specific advantages but is so far little recognized in translational research. Intrabodies allow high specificity and targeting of splice variants or post translational modifications.

Recent Advances with ER Targeted Intrabodies.

ER intrabodies are recombinant antibody fragments produced and retained in the endoplasmatic reticulum (ER) of a cell or an organism with the purpose to induce phenotypes generated by interfering with the intracellular processing or by changing the location of the recognized antigen. The most common application is the generation of functional knockdowns of membrane proteins, which cannot reach their natural location on the cell surface when they are retained in the ER by the intrabody. Phenotypes generated by interfering with the secretion of extracellular or plasma proteins can be analyzed in a similar way.

Specific in vivo knockdown of protein function by intrabodies.

Intracellular antibodies (intrabodies) are recombinant antibody fragments that bind to target proteins expressed inside of the same living cell producing the antibodies. The molecules are commonly used to study the function of the target proteins (i.e.

Functional knock down of VCAM1 in mice mediated by endoplasmatic reticulum retained intrabodies.

Functional knockdowns mediated by endoplasmatic reticulum-retained antibodies (ER intrabodies) are a promising tool for research because they allow functional interference on the protein level. We demonstrate for the first time that ER intrabodies can induce a knock-down phenotype in mice. Surface VCAM1 was suppressed in bone marrow of heterozygous and homozygous ER intrabody mice (iER-VCAM1 mice).

Delivery of antibodies to the cytosol: debunking the myths.

The use of antibodies to target their antigens in living cells is a powerful analytical tool for cell biology research. Not only can molecules be localized and visualized in living cells, but interference with cellular processes by antibodies may allow functional analysis down to the level of individual post-translational modifications and splice variants, which is not possible with genetic or RNA-based methods. To utilize the vast resource of available antibodies, an efficient system to deliver them into the cytosol from the outside is needed.

Suppression of p75 neurotrophin receptor surface expression with intrabodies influences Bcl-xL mRNA expression and neurite outgrowth in PC12 cells.

Background: Although p75 neurotrophin receptor (p75NTR) is the first neurotrophin receptor isolated, its diverse physiological functions and signaling have remained elusive for many years. Loss-of-function phenotypic analyses for p75NTR were mainly focused at the genetic level; however these approaches were impacted by off-target effect, insufficient stability, unspecific stress response or alternative active splicing products. In this study, p75NTR surface expression was suppressed for the first time at the protein level by endoplasmic reticulum (ER) retained intrabodies.

Targeting antibodies to the cytoplasm.

A growing number of research consortia are now focused on generating antibodies and recombinant antibody fragments that target the human proteome. A particularly valuable application for these binding molecules would be their use inside a living cell, e.g.