Ab-DeliverIN™ Transfection reagent

Effective therapies are urgently needed to safely target TDP-43 pathology as it is closely associated with the onset and development of devastating diseases such as frontotemporal lobar degeneration with TDP-43 pathology (FTLD-TDP) and amyotrophic lateral sclerosis (ALS). In addition, TDP-43 pathology is present as a co-pathology in other neurodegenerative diseases such as Alzheimer's disease and Parkinson's disease. Our approach is to develop a TDP-43-specific immunotherapy that exploits Fc gamma-mediated removal mechanisms to limit neuronal damage while maintaining physiological TDP-43 function. Thus, using both in vitro mechanistic studies in conjunction with the rNLS8 and CamKIIa inoculation mouse models of TDP-43 proteinopathy, we identified the key targeting domain in TDP-43 to accomplish these therapeutic objectives. Targeting the C-terminal domain of TDP-43 but not the RNA recognition motifs (RRM) reduces TDP-43 pathology and avoids neuronal loss in vivo. We demonstrate that this rescue is dependent on Fc receptor-mediated immune complex uptake by microglia. Furthermore, monoclonal antibody (mAb) treatment enhances phagocytic capacity of ALS patient-derived microglia, providing a mechanism to restore the compromised phagocytic function in ALS and FTD patients. Importantly, these beneficial effects are achieved while preserving physiological TDP-43 activity. Our findings demonstrate that a mAb targeting the C-terminal domain of TDP-43 limits pathology and neurotoxicity, enabling clearance of misfolded TDP-43 through microglia engagement, and supporting the clinical strategy to target TDP-43 by immunotherapy. SIGNIFICANCE STATEMENT: TDP-43 pathology is associated with various devastating neurodegenerative disorders with high unmet medical needs such as frontotemporal dementia (FTD), amyotrophic lateral sclerosis (ALS) and Alzheimer's disease. Thus, safely and effectively targeting pathological TDP-43 represents a key paradigm for biotechnical research as currently there is little in clinical development. After years of research, we have determined that targeting the C-terminal domain of TDP-43 rescues multiple patho-mechanisms involved in disease progression in two animal models of FTD/ALS. In parallel, importantly, our studies establish that this approach does not alter the physiological functions of this ubiquitously expressed and indispensable protein. Together, our findings substantially contribute to the understanding of TDP-43 pathobiology and support the prioritization for clinical testing of immunotherapy approaches targeting TDP-43.

Opioids targeting mu;μ (MOP) receptors produce analgesia in the peri-operative period and palliative care. They also produce side effects including respiratory depression, tolerance/dependence and addiction. The N/OFQ opioid receptor (NOP) also produces analgesia but is devoid of the major MOP side effects. Evidence exists for MOP-NOP interaction and mixed MOP-NOP ligands produce analgesia with reduced side effects. We have generated a HEKMOP/NOP human expression system and used bivalent MOP-NOP and fluorescent ligands to (i) probe for receptor interaction and (ii) consequences of that interaction. We used HEKMOP/NOP cells and two bivalent ligands; Dermorphin-N/OFQ (MOP agonist-NOP agonist; DeNO) and Dermorphin-UFP101 (MOP agonist-NOP antagonist; De101). We have determined receptor binding profiles, GTPγ[35S] binding, cAMP formation and ERK1/2 activation. We have also probed MOP and NOP receptor interactions in HEK cells and hippocampal neurones using the novel MOP fluorescent ligand, DermorphinATTO488 and the NOP fluorescent ligand N/OFQATTO594. In HEKMOP/NOP MOP ligands displaced NOP binding and NOP ligands displaced MOP binding. Using fluorescent probes in HEKMOP/NOP cells we demonstrated MOP-NOP probe overlap and a FRET signal indicating co-localisation. MOP-NOP were also co-localised in hippocampal tissue. In GTPγ[35S] and cAMP assays NOP stimulation shifted the response to MOP rightwards. At ERK1/2 the response to bivalent ligands generally peaked later. We provide evidence for MOP-NOP interaction in recombinant and native tissue. NOP activation reduces responsiveness of MOP activation; this was shown with conventional and bivalent ligands.

Recombinant immunoglobulin G (IgG) aggregates are formed during their production. However, the process underlying intracellular/extracellular aggregation in cell culture conditions is not well understood, and no effective method exists to assess IgG aggregates. Here, we establish an approach to detect intracellular aggregates using AF.2A1, a small artificial protein that binds to non-native IgG conformers and aggregates. Fluorescent-labeled AF.2A1 is prepared via conjugation and transfected into antibody-producing Chinese hamster ovary (CHO) cells. Micrographic images show intracellular IgG aggregates in CHO cells. The relative amount of intracellular aggregates (versus total intracellular IgG) differed depending on the type of additives used during cell culture. Interestingly, the relative amount of intracellular aggregates moderately correlates with that of in vitro extracellular IgG aggregates, suggesting they are secreted. This method will allow the investigation of antibody aggregation in cells, and may guide the production of therapeutic antibodies with high yield/quality.

Influenza A virus RNA genome exists as eight-segmented ribonucleoprotein complexes containing viral RNA polymerase and nucleoprotein (vRNPs). Packaging of vRNPs and virus budding take place at the apical plasma membrane (APM). However, little is known about the molecular mechanisms of apical transport of newly synthesized vRNP. Transfection of fluorescent-labeled antibody and subsequent live cell imaging revealed that punctate vRNP signals moved along microtubules rapidly but intermittently in both directions, suggestive of vesicle trafficking. Using a series of Rab family protein, we demonstrated that progeny vRNP localized to recycling endosome (RE) in an active/GTP-bound Rab11-dependent manner. The vRNP interacted with Rab11 through viral RNA polymerase. The localization of vRNP to RE and subsequent accumulation to the APM were impaired by overexpression of Rab binding domains (RBD) of Rab11 family interacting proteins (Rab11-FIPs). Similarly, no APM accumulation was observed by overexpression of class II Rab11-FIP mutants lacking RBD. These results suggest that the progeny vRNP makes use of Rab11-dependent RE machinery for APM trafficking.

Targeting cellular proteins with antibodies, to better understand cellular signaling pathways in the context of disease modulation, is a fast-growing area of investigation. Humanized antibodies are increasingly gaining attention for their therapeutic potential, but the collection of cellular targets is limited to those secreted from cells or expressed on the cell surface. This approach leaves a wealth of intracellular proteins unexplored as putative targets for antibody binding. Protein kinase Cθ (PKCθ) is essential to T cell activation, proliferation, and differentiation, and its phosphorylation at specific residues is required for its activity. Here we report on the design, synthesis, and characterization of a protein transduction domain mimic capable of efficiently delivering an antibody against phosphorylated PKCθ (Thr538) into human peripheral mononuclear blood cells and altering expression of downstream indicators of T cell activation and differentiation. We used a humanized, lymphocyte transfer model of graft-versus-host disease, to evaluate the durability of protein transduction domain mimic:Anti-pPKCθ modulation, when delivered into human peripheral mononuclear blood cells ex vivo. We demonstrate that protein transduction domain mimic:Antibody complexes can be readily introduced with high efficacy into hard-to-transfect human peripheral mononuclear blood cells, eliciting a biological response sufficient to alter disease progression. Thus, protein transduction domain mimic:Antibody delivery may represent an efficient ex vivo approach to manipulating cellular responses by targeting intracellular proteins.