In vivo selection of synthetic nucleocapsids for tissue targeting.

Controlling the biodistribution of protein- and nanoparticle-based therapeutic formulations remains challenging. In vivo library selection is an effective method for identifying constructs that exhibit desired distribution behavior; library variants can be selected based on their ability to localize to the tissue or compartment of interest despite complex physiological challenges. Here, we describe further development of an in vivo library selection platform based on self-assembling protein nanoparticles encapsulating their own mRNA genomes (synthetic nucleocapsids or synNCs). We tested two distinct libraries: a low-diversity library composed of synNC surface mutations (45 variants) and a high-diversity library composed of synNCs displaying miniproteins with binder-like properties (6.2 million variants). While we did not identify any variants from the low-diversity surface library that yielded therapeutically relevant changes in biodistribution, the high-diversity miniprotein display library yielded variants that shifted accumulation toward lungs or muscles in just two rounds of in vivo selection. Our approach should contribute to achieving specific tissue homing patterns and identifying targeting ligands for diseases of interest.

Efficacy and muscle safety assessment of fukutin-related protein gene therapy.

Limb-girdle muscular dystrophy type R9 (LGMDR9) is a muscle-wasting disease that begins in the hip and shoulder regions of the body. This disease is caused by mutations in fukutin-related protein (FKRP), a glycosyltransferase critical for maintaining muscle cell integrity. Here we investigated potential gene therapies for LGMDR9 containing an FKRP expression construct with untranslated region (UTR) modifications. Initial studies treated an aged dystrophic mouse model (FKRPP448L) with adeno-associated virus vector serotype 6 (AAV6). Grip strength improved in a dose- and time-dependent manner, injected mice exhibited fewer central nuclei and serum creatine kinase levels were 3- and 5-fold lower compared to those in non-injected FKRPP448L mice. Treatment also partially stabilized the respiratory pattern during exercise and improved treadmill running, partially protecting muscle from exercise-induced damage. Western blotting of C2C12 myotubes using a novel rabbit antibody confirmed heightened translation with the UTR modifications. We further explored the question of FKRP toxicity in wild-type mice using high doses of two additional muscle-tropic capsids: AAV9 and AAVMYO1. No toxic effects were detected with either therapeutic agent. These data further support the feasibility of gene therapy to treat LGMDR9.

Immunoglobulin deposition on biomolecule corona determines complement opsonization efficiency of preclinical and clinical nanoparticles.

Deposition of complement factors (opsonization) on nanoparticles may promote clearance from the blood by macrophages and trigger proinflammatory responses, but the mechanisms regulating the efficiency of complement activation are poorly understood. We previously demonstrated that opsonization of superparamagnetic iron oxide (SPIO) nanoworms with the third complement protein (C3) was dependent on the biomolecule corona of the nanoparticles. Here we show that natural antibodies play a critical role in C3 opsonization of SPIO nanoworms and a range of clinically approved nanopharmaceuticals. The dependency of C3 opsonization on immunoglobulin binding is almost universal and is observed regardless of the complement activation pathway. Only a few surface-bound immunoglobulin molecules are needed to trigger complement activation and opsonization. Although the total amount of plasma proteins adsorbed on nanoparticles does not determine C3 deposition efficiency, the biomolecule corona per se enhances immunoglobulin binding to all nanoparticle types. We therefore show that natural antibodies represent a link between biomolecule corona and C3 opsonization, and may determine individual complement responses to nanomedicines.

Publisher Correction: Immunoglobulin deposition on biomolecule corona determines complement opsonization efficiency of preclinical and clinical nanoparticles.

In the version of this Article originally published, a technical error led to Fig. 1a containing '!!!!!!!!' above the scale bar. This has now been corrected in all versions of the Article.

Isolation of Breast cancer CTCs with multitargeted buoyant immunomicrobubbles.

Circulating tumor cells (CTCs) are extremely rare cells found in blood of metastatic cancer patients. There is a need for inexpensive technologies for fast enrichment of CTCs from large blood volumes. Previous data showed that antibody-conjugated lipid shell immuno-microbubbles (MBs) bind and isolate cells from biological fluids by flotation. Here, blood-stable MBs targeted to several surface markers for isolation of breast tumor cells were developed. MBs coated with anti-human EpCAM antibodies showed efficient binding of EpCAM+ breast cancer cell lines SKBR-3, MCF-7, and MDA-MB-453, whereas anti-human EGFR MBs showed binding of EpCAMLOW/NEGATIVE cell lines MDA-MB-231 and BT-549. Multitargeted anti-human EpCAM/EGFR MBs bound all cell lines with over 95% efficiency. Highly concentrated MB-bound tumor cells were collected in a microliter volume via an inverted vacuum-assisted harvesting setup. Using anti-EpCAM and/or anti-EpCAM/EGFR MBs, an efficient (70-90%) recovery and fast (30min) isolation of the above-mentioned cells and cell clusters was achieved from 7.5mL of spiked human blood. Using anti-EpCAM MBs and anti-EpCAM/EGFR MBs, cytokeratin-positive, CD45-negative CTCs were detected in 62.5% (10/16) of patients with metastatic breast cancer and CTC clusters were detected in 41.7% (5/12) of CTC-positive samples. Moreover, in some samples MBs isolated cytokeratin positive, CD45 negative tumor-derived microparticles. None of these structures were detected in blood from non-epithelial malignancies. The fast and inexpensive multitargeted platform for batch isolation of CTCs can promote research and clinical applications involving primary tumors and metastases.

Variability of Complement Response toward Preclinical and Clinical Nanocarriers in the General Population.

Opsonization (coating) of nanoparticles with complement C3 component is an important mechanism that triggers immune clearance and downstream anaphylactic and proinflammatory responses. The variability of complement C3 binding to nanoparticles in the general population has not been studied. We examined complement C3 binding to dextran superparamagnetic iron oxide nanoparticles (superparamagnetic iron oxide nanoworms, SPIO NWs, 58 and 110 nm) and clinically approved nanoparticles (carboxymethyl dextran iron oxide ferumoxytol (Feraheme, 28 nm), highly PEGylated liposomal doxorubicin (LipoDox, 88 nm), and minimally PEGylated liposomal irinotecan (Onivyde, 120 nm)) in sera from healthy human individuals. SPIO NWs had the highest variation in C3 binding (n = 47) between subjects, with a 15-30 fold range in levels of C3. LipoDox (n = 12) and Feraheme (n = 18) had the lowest levels of variation between subjects (an approximately 1.5-fold range), whereas Onivyde (n = 18) had intermediate between-subject variation (2-fold range). There was no statistical difference between males and females and no correlation with age. There was a significant correlation in complement response between small and large SPIO NWs, which are similar structurally and chemically, but the correlations between SPIO NWs and other types of nanoparticles, and between LipoDox and Onivyde, were not significant. The calculated average number of C3 molecules bound per nanoparticle correlated with the hydrodynamic diameter but was decreased in LipoDox, likely due to the PEG coating. The conclusions of this study are (1) all nanoparticles show variability of C3 opsonization in the general population; (2) an individual's response toward one nanoparticle cannot be reliably predicted based on another nanoparticle; and (3) the average number of C3 molecules per nanoparticle depends on size and surface coating. These results provide new strategies to improve nanomedicine safety.

Interaction of extremophilic archaeal viruses with human and mouse complement system and viral biodistribution in mice.

Archaeal viruses offer exceptional biophysical properties for modification and exploration of their potential in bionanotechnology, bioengineering and nanotherapeutic developments. However, the interaction of archaeal viruses with elements of the innate immune system has not been explored, which is a necessary prerequisite if their potential for biomedical applications to be realized. Here we show complement activation through lectin (via direct binding of MBL/MASPs) and alternative pathways by two extremophilic archaeal viruses (Sulfolobus monocaudavirus 1 and Sulfolobus spindle-shaped virus 2) in human serum. We further show some differences in initiation of complement activation pathways between these viruses. Since, Sulfolobus monocaudavirus 1 was capable of directly triggering the alternative pathway, we also demonstrate that the complement regulator factor H has no affinity for the viral surface, but factor H deposition is purely C3-dependent. This suggests that unlike some virulent pathogens Sulfolobus monocaudavirus 1 does not acquire factor H for protection. Complement activation with Sulfolobus monocaudavirus 1 also proceeds in murine sera through MBL-A/C as well as factor D-dependent manner, but C3 deficiency has no overall effect on viral clearance by organs of the reticuloendothelial system on intravenous injection. However, splenic deposition was significantly higher in C3 knockout animals compared with the corresponding wild type mice. We discuss the potential application of these viruses in biomedicine in relation to their complement activating properties.