High-performance renal imaging with a radiolabeled, non-excretable chimeric fusion protein.

Ideal nuclear imaging tracers should exhibit high target uptake and low background signal. Traditional renal scintigraphy and SPECT scans examine kidney function via static or dynamic tracing of radioactive probes in the kidneys. The lack of tracer affinity to specific biological processes and high background uptake from urinary excretion have added many difficulties to precision renal diagnosis. In this issue of Theranostics, Jin and colleagues innovatively devised a recombinant probe for preferential kidney imaging through targeting of tubular neonatal Fc receptor and proximal tubular basement membrane for sustained tubular reabsorption and accumulation. This work has broad implications regarding how an in depth understanding of physiology and pathology may be of service for tracer development, renal diagnosis, and disease theranostics.

Synthesis and pharmacokinetic characterisation of a fluorine-18 labelled brain shuttle peptide fusion dimeric affibody.

Brain positron emission tomography (PET) imaging with radiolabelled proteins is an emerging concept that potentially enables visualization of unique molecular targets in the brain. However, the pharmacokinetics and protein radiolabelling methods remain challenging. Here, we report the performance of an engineered, blood-brain barrier (BBB)-permeable affibody molecule that exhibits rapid clearance from the brain, which was radiolabelled using a unique fluorine-18 labelling method, a cell-free protein radiosynthesis (CFPRS) system. AS69, a small (14 kDa) dimeric affibody molecule that binds to the monomeric and oligomeric states of α-synuclein, was newly designed for brain delivery with an apolipoprotein E (ApoE)-derived brain shuttle peptide as AS69-ApoE (22 kDa). The radiolabelled products 18F-AS69 and 18F-AS69-ApoE were successfully synthesised using the CFPRS system. Notably, 18F-AS69-ApoE showed higher BBB permeability than 18F-AS69 in an ex vivo study at 10 and 30 min post injection and was partially cleared from the brain at 120 min post injection. These results suggest that small, a brain shuttle peptide-fused fluorine-18 labelled protein binders can potentially be utilised for brain molecular imaging.

An Improved Intracellular Synthetic Lipidation-Induced Plasma Membrane Anchoring System for SNAP-Tag Fusion Proteins.

The ability to chemically introduce lipid modifications to specific intracellular protein targets would enable the conditional control of protein localization and activity in living cells. We recently developed a chemical-genetic approach in which an engineered SNAP-tag fusion protein can be rapidly relocated and anchored from the cytoplasm to the plasma membrane (PM) upon post-translational covalent lipopeptide conjugation in cells. However, the first-generation system achieved only low to moderate protein anchoring (recruiting) efficiencies and lacked wide applicability. Herein, we describe the rational design of an improved system for intracellular synthetic lipidation-induced PM anchoring of SNAP-tag fusion proteins. In the new system, the SNAPf protein engineered to contain an N-terminal hexalysine (K6) sequence and a C-terminal 10-amino acid deletion, termed K6-SNAPΔ, is fused to a protein of interest. In addition, a SNAP-tag substrate containing a metabolic-resistant myristoyl-DCys lipopeptidomimetic, called mDcBCP, is used as a cell-permeable chemical probe for intracellular SNAP-tag lipidation. The use of this combination allows significantly improved conditional PM anchoring of SNAP-tag fusion proteins. This second-generation system was applied to activate various signaling proteins, including Tiam1, cRaf, PI3K, and Sos, upon synthetic lipidation-induced PM anchoring/recruitment, offering a new and useful research tool in chemical biology and synthetic biology.

Lysosome-targeting chimaeras for degradation of extracellular proteins.

The majority of therapies that target individual proteins rely on specific activity-modulating interactions with the target protein-for example, enzyme inhibition or ligand blocking. However, several major classes of therapeutically relevant proteins have unknown or inaccessible activity profiles and so cannot be targeted by such strategies. Protein-degradation platforms such as proteolysis-targeting chimaeras (PROTACs)1,2 and others (for example, dTAGs3, Trim-Away4, chaperone-mediated autophagy targeting5 and SNIPERs6) have been developed for proteins that are typically difficult to target; however, these methods involve the manipulation of intracellular protein degradation machinery and are therefore fundamentally limited to proteins that contain cytosolic domains to which ligands can bind and recruit the requisite cellular components. Extracellular and membrane-associated proteins-the products of 40% of all protein-encoding genes7-are key agents in cancer, ageing-related diseases and autoimmune disorders8, and so a general strategy to selectively degrade these proteins has the potential to improve human health. Here we establish the targeted degradation of extracellular and membrane-associated proteins using conjugates that bind both a cell-surface lysosome-shuttling receptor and the extracellular domain of a target protein. These initial lysosome-targeting chimaeras, which we term LYTACs, consist of a small molecule or antibody fused to chemically synthesized glycopeptide ligands that are agonists of the cation-independent mannose-6-phosphate receptor (CI-M6PR). We use LYTACs to develop a CRISPR interference screen that reveals the biochemical pathway for CI-M6PR-mediated cargo internalization in cell lines, and uncover the exocyst complex as a previously unidentified-but essential-component of this pathway. We demonstrate the scope of this platform through the degradation of therapeutically relevant proteins, including apolipoprotein E4, epidermal growth factor receptor, CD71 and programmed death-ligand 1. Our results establish a modular strategy for directing secreted and membrane proteins for lysosomal degradation, with broad implications for biochemical research and for therapeutics.

Beyond antibodies: ankyrins and DARPins. From basic research to drug approval.

This Pharmacological Perspective describes the pathway that, starting from the deep understanding of ankyrins - a family of proteins with high variability-binding and high specificity-binding characteristics - led to the development of a new class of recombinant-binding proteins, the DARPins (designed ankyrin repeat proteins). These are envisaged as alternatives to mAbs and related biologics, with the potential to overcome certain shortcomings of mAbs. DARPins have relatively low molecular weights (14-21kDas) and more favorable PK profiles than mAbs, are stable proteins that can be easily produced in Escherichia coli and can be used in their monovalent form or conjugated to other moieties, for example, polyethylene glycol (PEG) to enhance their half-life. DARPins can also be engineered to produce bi-specific or tri-specific compounds that bind different epitopes of the same target or two different targets. Abicipar, a first-in-class anti-VEGF-A DARPin had similar efficacy compared to anti-VEGF biologics (bevacizumab, ranibizumab) in preclinical studies and was not inferior to ranibizumab in the treatment of age-related macular degeneration (AMD) with a reduced number of intravitreal injections. Abicipar has recently been submitted for regulatory approval for use in AMD.

Highly Precise Protein Semisynthesis through Ligation-Desulfurization Chemistry in Combination with Phenacyl Protection of Native Cysteines.

Semisynthesis of proteins via expressed protein ligation is a powerful tool to furnish full-length proteins carrying site-specific (posttranslational) modifications. The development of various ß-mercapto amino acid building blocks coupled with ligation-desulfurization chemistry enabled further advances in this methodology by alleviating the need for cysteine residues at the desired ligation sites. However, this expansion in the availability of viable ligation sites is sometimes counterbalanced by the inadvertent desulfurization of unprotected native cysteines, which might be of structural and/or functional importance. Here, we provide a detailed protocol for using the cysteine-selective protecting group phenacyl (PAc) to achieve precise protein semisynthesis preserving native cysteine residues. The PAc group can be easily installed on cysteine(s) within recombinantly produced protein thioesters, withstands standard ligation, desulfurization and reversed phase HPLC conditions, and can be smoothly removed. We have previously demonstrated the utility of this protecting group through the semisynthesis of two model proteins, human small heat shock protein Hsp27 and Prion protein, in which one or two native cysteines, respectively, were maintained through the ligation-desulfurization sequence.

Use of the LC3B-fusion technique for biochemical and structural studies of proteins involved in the N-degron pathway.

The N-degron pathway, formerly the N-end rule pathway, is a protein degradation process that determines the half-life of proteins based on their N-terminal residues. In contrast to the well-established in vivo studies over decades, in vitro studies of this pathway, including biochemical characterization and high-resolution structures, are relatively limited. In this study, we have developed a unique fusion technique using microtubule-associated protein 1A/1B light chain 3B, a key marker protein of autophagy, to tag the N terminus of the proteins involved in the N-degron pathway, which enables high yield of homogeneous target proteins with variable N-terminal residues for diverse biochemical studies including enzymatic and binding assays and substrate identification. Intriguingly, crystallization showed a markedly enhanced probability, even for the N-degron complexes. To validate our results, we determined the structures of select proteins in the N-degron pathway and compared them with the Protein Data Bank-deposited proteins. Furthermore, several biochemical applications of this technique were introduced. Therefore, this technique can be used as a general tool for the in vitro study of the N-degron pathway.

PET imaging of a 68Ga labeled modified HER2 affibody in breast cancers: from xenografts to patients.

OBJECTIVE: Overexpression of human epidermal growth factor receptor-2 (HER2) in breast cancers provides promising opportunities for imaging and targeted therapy. Developing HER2 targeted positron emission tomography (PET) probes might be benefit for management of the disease. Small high-affinity scaffold proteins, affibodies, are ideal vectors for imaging HER2 overexpressed tumors. Despite of the initial success on development of 18F labeled ZHER2:342 affibody, the tedious synthesis producers, low yields and unfavorable pharmacokinetics may hinder the clinical use. 68Ga is an attractive positron emitter for PET imaging. A simple preparation of 68Ga labeled ZHER2:342 analog, 68Ga-NOTA-MAL-Cys-MZHER2:342, was reported in the study. The in vivo performances of the tracer for assessing HER2 status in breast cancers were also evaluated. METHODS: NOTA-MAL conjugated Cys-MZHER2:342 was radiolabeled with 68Ga. The probe was evaluated by in vitro tests including stability and cell binding studies in breast cancer cells with different HER2 levels. In vivo evaluation was performed in mice bearing tumors using microPET imaging and biodistribution experiments. A PET/CT imaging study was initially performed in patients with breast cancers. RESULTS: The tracer was synthesized in a straightforward chelation method with satisfactory non-decay corrected yield (81±5%) and radiochemical purity (>95%). In vivo micro-PET imaging showed that HER2 high levels expressed BT474 xenografts were more clear visualized than HER2 low levels expressed MCF-7 tumors (16.12 ± 2.69 ID%/g vs 1.32 ± 0.19 ID%/g at 1 h post-injection). The outcome was consistent with the immunohistochemical analysis. No significant radioactivity was accumulated in healthy tissues (less than 2% ID/g) except kidneys. In a preliminary clinical study, 68Ga-NOTA-MAL-Cys-MZHER2:342 PET imaging allowed more high-contrast detection of HER2 positive primary tumors (maximum standardized uptake value = 2.16±0.27) than those in HER2 negative primary focus (maximum standardized uptake value = 0.32±0.05). No detectable side-effects were found. CONCLUSION: In summary, this study indicates the significant efficiency of the 68Ga labeled HER2 affibody. Preclinical and clinical studies support the possibility of monitoring HER2 levels in breast cancers using 68Ga-NOTA-MAL-Cys-MZHER2:342. ADVANCES IN KNOWLEDGE: The research investigated the feasibility of a 68Ga labeled HER2 affibody modified with a hydrophilic linker for breast cancer PET imaging. Favorable outcomes showed that the probe might be valuable for determining HER2 status of the disease.

Facile and Efficient Chemoenzymatic Semisynthesis of Fc-Fusion Compounds for Half-Life Extension of Pharmaceutical Components.

The formation of Fc-fusions, in which biologically active molecules and the Fc fragment of antibodies are linked to each other, is one of the most efficient and successful half-life extension technologies to be developed and applied to peptide and protein pharmaceuticals thus far. Fc-fusion compounds are generally produced by recombinant methods. However, these cannot be applied to artificial middle molecules, such as peptides with non-natural amino acids, unnatural cyclic peptides, or pharmaceutical oligonucleotides. Here, we developed a simple, efficient, semisynthetic method for Fc-fusion production involving our previously developed enzymatic N-terminal extension reaction (i.e., NEXT-A reaction) and strain-promoted azide-alkyne cycloaddition, achieving quantitative conversion and high selectivity for the N-terminus of the Fc protein. An Fc-fusion compound prepared by this method showed comparable biological activity to that of the original peptide and a long-circulating plasma half-life. Thus, the proposed method is potentially applicable for the conjugation of a wide range of pharmaceutical components.

Transportan 10 improves the pharmacokinetics and pharmacodynamics of vancomycin.

In the presented study, transportan 10 (TP10), an amphipathic cell penetrating peptide (CPP) with high translocation activity, was conjugated with vancomycin (Van), which is known for poor access to the intracellular bacteria and the brain. The antibacterial activity of the conjugates was tested on selected clinical strains of methicillin-resistant Staphylococcus aureus (MRSA) and Enterococcus sp. It turned out that all of them had superior antimicrobial activity in comparison to that of free Van, which became visible particularly against clinical MRSA strains. Furthermore, one of the conjugates was tested against MRSA - infected human cells. With respect to them, this compound showed high bactericidal activity. Next, the same conjugate was screened for its capacity to cross the blood brain barrier (BBB). Therefore, qualitative and quantitative analyses of the conjugate's presence in the mouse brain slices were carried out after its iv administration. They indicated the conjugate's presence in the brain in amount >200 times bigger than that of Van. The conjugates were safe with respect to erythrocyte toxicity (erythrocyte lysis assay). Van in the form of a conjugate with TP10 acquires superior pharmacodynamic and pharmacokinetic.

Profile of asfotase alfa in the treatment of hypophosphatasia: design, development, and place in therapy.

Hypophosphatasia (HPP) is a multi-systemic metabolic disorder caused by loss-of-function mutations in the ALPL gene that encodes the mineralization-associated enzyme, tissue-nonspecific alkaline phosphatase (TNSALP). HPP is characterized by defective bone and dental mineralization, leading to skeletal abnormalities with complications resulting in significant morbidity and mortality. Management of HPP has been limited to supportive care until the introduction of a recently approved enzyme replacement therapy employing bone-targeted recombinant human TNSALP, asfotase alfa (AA). This new therapy has been transformative as it improves survival in severely affected infants, and overall quality of life in children and adults with HPP. This review provides an overview of HPP, focusing on important steps in the development of AA enzyme replacement therapy, including the drug design, preclinical studies in the HPP mouse model, and outcomes from clinical trials and case report publications to date, with special attention given to response to therapy of skeletal manifestations, biochemical features, and other clinical manifestations. The limitations, adverse effects, and outcomes of AA are outlined and the place in therapy for individuals with HPP is discussed.

Dynamic protein self-assembly driven by host-guest chemistry and the folding-unfolding feature of a mutually exclusive protein.

A novel exploration utilizing a well-designed fusion protein containing a redox stimuli-responsive domain was developed to construct dynamic protein self-assemblies induced by cucurbit[8]uril-based supramolecular interactions. The reversible interconversion of the morphology of the assemblies between nanowires and nanorings was regulated precisely by redox conditions.

Spotlight on romiplostim in the treatment of children with chronic immune thrombocytopenia: design, development, and potential place in therapy.

Primary immune thrombocytopenia (ITP) is an autoimmune disorder characterized by isolated thrombocytopenia. In approximately one-third of cases, the duration of thrombocytopenia will extend beyond 12 months consistent with a diagnosis of chronic ITP. Minor bleeding manifestations are common in chronic ITP while severe or life-threatening bleeding complications are uncommon. Moreover, spontaneous resolution occurs in the majority of children with chronic ITP necessitating treatment in only those children with ongoing bleeding manifestations or impairment in health-related quality of life (HRQOL). The characterization of thrombopoietin (TPO) and remarkable advancements in our understanding of the pathophysiology of ITP has led to the development of a new class of agents, the TPO-receptor agonists that have documented efficacy in the amelioration of thrombocytopenia and bleeding manifestations in chronic ITP. Romiplostim is a second-generation TPO-receptor agonist that has undergone limited evaluation in the treatment of chronic ITP in children. Evolving data suggest that romiplostim may be a safe and effective agent in the treatment of chronic ITP in children. Additional data are needed to confirm its ability to increase platelet counts, decrease bleeding manifestation, and improve the HRQOL of children and caregivers impacted by chronic ITP.

DRα1-MOG-35-55 treatment reduces lesion volumes and improves neurological deficits after traumatic brain injury.

Traumatic brain injury (TBI) results in severe neurological impairments without effective treatments. Inflammation appears to be an important contributor to key pathogenic events such as secondary brain injury following TBI and therefore serves as a promising target for novel therapies. We have recently demonstrated the ability of a molecular construct comprised of the human leukocyte antigen (HLA)-DRα1 domain linked covalently to mouse (m)MOG-35-55 peptide (DRα1-MOG-35-55 construct) to reduce CNS inflammation and tissue injury in animal models of multiple sclerosis and ischemic stroke. The aim of the current study was to determine if DRα1-MOG-35-55 treatment of a fluid percussion injury (FPI) mouse model of TBI could reduce the lesion size and improve disease outcome measures. Neurodeficits, lesion size, and immune responses were determined to evaluate the therapeutic potential and mechanisms of neuroprotection induced by DRα1-MOG-35-55 treatment. The results demonstrated that daily injections of DRα1-MOG-35-55 given after FPI significantly reduced numbers of infiltrating CD74+ and CD86+ macrophages and increased numbers of CD206+ microglia in the brain concomitant with smaller lesion sizes and improvement in neurodeficits. Conversely, DRα1-MOG-35-55 treatment of TBI increased numbers of circulating CD11b+ monocytes and their expression of CD74 but had no detectable effect on cell numbers or marker expression in the spleen. These results demonstrate that DRα1-MOG-35-55 therapy can reduce CNS inflammation and significantly improve histological and clinical outcomes after TBI. Future studies will further examine the potential of DRα1-MOG-35-55 for treatment of TBI.

Cell-Free Protein Synthesis Approach to Biosensing hTRß-Specific Endocrine Disruptors.

Here we introduce a Rapid Adaptable Portable In vitro Detection biosensor platform (RAPID) for detecting ligands that interact with nuclear hormone receptors (NHRs). The RAPID platform can be adapted for field use, allowing rapid evaluation of endocrine disrupting chemicals (EDCs) presence or absence in environmental samples, and can also be applied for drug screening. The biosensor is based on an engineered, allosterically activated fusion protein, which contains the ligand binding domain from a target NHR (human thyroid receptor ß in this work). In vitro expression of this protein using cell-free protein synthesis (CFPS) technology in the presence of an EDC leads to activation of a reporter enzyme, reported through a straightforward colorimetric assay output. In this work, we demonstrate the potential of this biosensor platform to be used in a portable "just-add-sample" format for near real-time detection. We also demonstrate the robust nature of the cell-free protein synthesis component in the presence of a variety of environmental and human samples, including sewage, blood, and urine. The presented RAPID biosensor platform is significantly faster and less labor intensive than commonly available technologies, making it a promising tool for detecting environmental EDC contamination and screening potential NHR-targeted pharmaceuticals.

Self-Assembled Materials Made from Functional Recombinant Proteins.

Proteins are potent molecules that can be used as therapeutics, sensors, and biocatalysts with many advantages over small-molecule counterparts due to the specificity of their activity based on their amino acid sequence and folded three-dimensional structure. However, they also have significant limitations in their stability, localization, and recovery when used in soluble form. These opportunities and challenges have motivated the creation of materials from such functional proteins in order to protect and present them in a way that enhances their function. We have designed functional recombinant fusion proteins capable of self-assembling into materials with unique structures that maintain or improve the functionality of the protein. Fusion of either a functional protein or an assembly domain to a leucine zipper domain makes the materials design strategy modular, based on the high affinity between leucine zippers. The self-assembly domains, including elastin-like polypeptides (ELPs) and defined-sequence random coil polypeptides, can be fused with a leucine zipper motif in order to promote assembly of the fusion proteins into larger structures upon specific stimuli such as temperature and ionic strength. Fusion of other functional domains with the counterpart leucine zipper motif endows the self-assembled materials with protein-specific functions such as fluorescence or catalytic activity. In this Account, we describe several examples of materials assembled from functional fusion proteins as well as the structural characterization, functionality, and understanding of the assembly mechanism. The first example is zipper fusion proteins containing ELPs that assemble into particles when introduced to a model extracellular matrix and subsequently disassemble over time to release the functional protein for drug delivery applications. Under different conditions, the same fusion proteins can self-assemble into hollow vesicles. The vesicles display a functional protein on the surface and can also carry protein, small-molecule, or nanoparticle cargo in the vesicle lumen. To create a material with a more complex hierarchical structure, we combined calcium phosphate with zipper fusion proteins containing random coil polypeptides to produce hybrid protein-inorganic supraparticles with high surface area and porous structure. The use of a functional enzyme created supraparticles with the ability to degrade inflammatory cytokines. Our characterization of these protein materials revealed that the molecular interactions are complex because of the large size of the protein building blocks, their folded structures, and the number of potential interactions including hydrophobic interactions, electrostatic interactions, van der Waals forces, and specific affinity-based interactions. It is difficult or even impossible to predict the structures a priori. However, once the basic assembly principles are understood, there is opportunity to tune the material properties, such as size, through control of the self-assembly conditions. Our future efforts on the fundamental side will focus on identifying the phase space of self-assembly of these fusion proteins and additional experimental levers with which to control and tune the resulting materials. On the application side, we are investigating an array of different functional proteins to expand the use of these structures in both therapeutic protein delivery and biocatalysis.

Two-Photon Tracer for Human Epidermal Growth Factor Receptor-2: Detection of Breast Cancer in a Live Tissue.

We have developed a two-photon fluorescent tracer (Pyr-affibody) that shows high selectivity for human epidermal growth factor receptor-2 (HER-2). Pyr-affibody showed absorption and emission maxima at 439 and 574 nm, respectively, with a two-photon absorption cross-section value of 40 × 10-50 cm4s/photon (GM) at 750 nm in aqueous buffer solution. The effective two-photon action cross-section value measured in HeLa cells was 600 GM at 730 nm, a value sufficient to obtain bright two-photon microscopy (TPM) images. Using Pyr-affibody, it was possible to detect HER-2 overexpressing cells and breast cancers at a depth of 90-130 µm in live mouse tissue by TPM.

A General Strategy for the Semisynthesis of Ratiometric Fluorescent Sensor Proteins with Increased Dynamic Range.

We demonstrate how a combination of self-labeling protein tags and unnatural amino acid technology permits the semisynthesis of ratiometric fluorescent sensor proteins with unprecedented dynamic range in vitro and on live cells. To generate such a sensor, a binding protein is labeled with a fluorescent competitor of the analyte using SNAP-tag in conjugation with a second fluorophore that is positioned in vicinity of the binding site of the binding protein using unnatural amino acid technology. Binding of the analyte by the sensor displaces the tethered fluorescent competitor from the binding protein and disrupts fluorescence resonance energy transfer between the two fluorophores. Using this design principle, we generate a ratiometric fluorescent sensor protein for methotrexate that exhibits large dynamic ranges both in vitro (ratio changes up to 32) and on cell surfaces (ratio change of 13). The performance of these semisynthetic sensor proteins makes them attractive for applications in basic research and diagnostics.

Molecular Self-Assembly Strategy for Generating Catalytic Hybrid Polypeptides.

Recently, catalytic peptides were introduced that mimicked protease activities and showed promising selectivity of products even in organic solvents where protease cannot perform well. However, their catalytic efficiency was extremely low compared to natural enzyme counterparts presumably due to the lack of stable tertiary fold. We hypothesized that assembling these peptides along with simple hydrophobic pockets, mimicking enzyme active sites, could enhance the catalytic activity. Here we fused the sequence of catalytic peptide CP4, capable of protease and esterase-like activities, into a short amyloidogenic peptide fragment of Aß. When the fused CP4-Aß construct assembled into antiparallel ß-sheets and amyloid fibrils, a 4.0-fold increase in the hydrolysis rate of p-nitrophenyl acetate (p-NPA) compared to neat CP4 peptide was observed. The enhanced catalytic activity of CP4-Aß assembly could be explained both by pre-organization of a catalytically competent Ser-His-acid triad and hydrophobic stabilization of a bound substrate between the triad and p-NPA, indicating that a design strategy for self-assembled peptides is important to accomplish the desired functionality.

Tackling amyloidogenesis in Alzheimer's disease with A2V variants of Amyloid-ß.

We developed a novel therapeutic strategy for Alzheimer's disease (AD) exploiting the properties of a natural variant of Amyloid-ß (Aß) carrying the A2V substitution, which protects heterozygous carriers from AD by its ability to interact with wild-type Aß, hindering conformational changes and assembly thereof. As prototypic compound we designed a six-mer mutated peptide (Aß1-6A2V), linked to the HIV-related TAT protein, which is widely used for brain delivery and cell membrane penetration of drugs. The resulting molecule [Aß1-6A2VTAT(D)] revealed strong anti-amyloidogenic effects in vitro and protected human neuroblastoma cells from Aß toxicity. Preclinical studies in AD mouse models showed that short-term treatment with Aß1-6A2VTAT(D) inhibits Aß aggregation and cerebral amyloid deposition, but a long treatment schedule unexpectedly increases amyloid burden, although preventing cognitive deterioration. Our data support the view that the AßA2V-based strategy can be successfully used for the development of treatments for AD, as suggested by the natural protection against the disease in human A2V heterozygous carriers. The undesirable outcome of the prolonged treatment with Aß1-6A2VTAT(D) was likely due to the TAT intrinsic attitude to increase Aß production, avidly bind amyloid and boost its seeding activity, warning against the use of the TAT carrier in the design of AD therapeutics.