Tarmo Mölder

RIG-I and MDA-5 Detection of Viral RNA-dependent RNA Polymerase Activity Restricts Positive-Strand RNA Virus Replication

Type I interferons (IFN) are important for antiviral responses. Melanoma differentiation-associated gene 5 (MDA-5) and retinoic acid-induced gene I (RIG-I) proteins detect cytosolic double-stranded RNA (dsRNA) or 5′-triphosphate (5′-ppp) RNA and mediate IFN production. Cytosolic 5′-ppp RNA and dsRNA are generated during viral RNA replication and transcription by viral RNA replicases [RNA-dependent RNA polymerases (RdRp)]. Here, we show that the Semliki Forest virus (SFV) RNA replicase can induce IFN-β independently of viral RNA replication and transcription. The SFV replicase converts host cell RNA into 5′-ppp dsRNA and induces IFN-β through the RIG-I and MDA-5 pathways. Inactivation of the SFV replicase RdRp activity prevents IFN-β induction. These IFN-inducing modified host cell RNAs are abundantly produced during both wild-type SFV and its non-pathogenic mutant infection. Furthermore, in contrast to the wild-type SFV replicase a non-pathogenic mutant replicase triggers increased IFN-β production, which leads to a shutdown of virus replication. These results suggest that host cells can restrict RNA virus replication by detecting the products of unspecific viral replicase RdRp activity.

Elicitation of broad CTL response against HIV-1 by the DNA vaccine encoding artificial multi-component fusion protein MultiHIV—Study in domestic pigs

Broad CTL response against HIV-1 is one factor that helps to control the viral replication. We have constructed a DNA vaccine that encodes a large artificial fusion protein (MultiHIV) and shown it to be immunogenic in mice, swine and macaques. Inbred mice revealed CTL response only against two epitopes due to limited MHC class I variability. To assess the quality of the CTL response we addressed this question in domestic swine. Number of presented epitopes varied between 7 and 14 among the five selected animals. Epitopes detected in swine are localised in the same antigenic regions recognised in humans. This can be explained by the fact that swine MHC-I (SLA-I) complex is remarkably similar to human HLA-I. These results also indicate that immunogenicity profile of vaccines in domestic swine may predict the outcome of human immunisation.

Antibiotic-loaded nanoparticles targeted to the site of infection enhance antibacterial efficacy

Bacterial resistance to antibiotics has made it necessary to resort to using antibacterial drugs that have considerable toxicities. Here, we show that conjugation of vancomycin-loaded nanoparticles with the cyclic 9-amino-acid peptide CARGGLKSC (CARG), identified via phage display on Staphylococcus aureus (S. aureus) bacteria and through in vivo screening in mice with S. aureus-induced lung infections, increases the antibacterial activity of the nanoparticles in S. aureus-infected tissues and reduces the systemic dose needed, minimizing side effects. CARG binds specifically to S. aureus bacteria but not Pseudomonas bacteria in vitro, selectively accumulates in S. aureus-infected lungs and skin of mice but not in non-infected tissue and Pseudomonas-infected tissue, and significantly enhances the accumulation of intravenously injected vancomycin-loaded porous silicon nanoparticles bearing CARG in S. aureus-infected mouse lung tissue. The targeted nanoparticles more effectively suppress staphylococcal infections in vivo relative to equivalent doses of untargeted vancomycin nanoparticles or of free vancomycin. The therapeutic delivery of antibiotic-carrying nanoparticles bearing peptides targeting infected tissues may help combat difficult-to-treat infections.Nanoparticles carrying an antibiotic and conjugated with a peptide identified via phage display that binds specifically to Staphylococcus aureus effectively suppress staphylococcal infections in vivo.

Urokinase-controlled tumor penetrating peptide.

Tumor penetrating peptides contain a cryptic (R/K)XX(R/K) CendR element that must be C-terminally exposed to trigger neuropilin-1 (NRP-1) binding, cellular internalization and malignant tissue penetration. The specific proteases that are involved in processing of tumor penetrating peptides identified using phage display are not known. Here we design de novo a tumor-penetrating peptide based on consensus cleavage motif of urokinase-type plasminogen activator (uPA). We expressed the peptide, uCendR (RPARSGR↓SAGGSVA, ↓ shows cleavage site), on phage or coated it onto silver nanoparticles and showed that it is cleaved by uPA, and that the cleavage triggers binding to recombinant NRP-1 and to NPR-1-expressing cells. Upon systemic administration to mice bearing uPA-overexpressing breast tumors, FAM-labeled uCendR peptide and uCendR-coated nanoparticles preferentially accumulated in tumor tissue. We also show that uCendR phage internalization into cultured cancer cells and its penetration in explants of murine tumors and clinical tumor explants can be potentiated by combining the uCendR peptide with tumor-homing module, CRGDC. Our work demonstrates the feasibility of designing tumor-penetrating peptides that are activated by a specific tumor protease. As upregulation of protease expression is one of the hallmarks of cancer, and numerous tumor proteases have substrate specificities compatible with proteolytic unmasking of cryptic CendR motifs, the strategy described here may provide a generic approach for designing proteolytically-actuated peptides for tumor-penetrative payload delivery.

Identification of a peptide recognizing cerebrovascular changes in mouse models of Alzheimer’s disease

Cerebrovascular changes occur in Alzheimer’s disease (AD). Using in vivo phage display, we searched for molecular markers of the neurovascular unit, including endothelial cells and astrocytes, in mouse models of AD. We identified a cyclic peptide, CDAGRKQKC (DAG), that accumulates in the hippocampus of hAPP-J20 mice at different ages. Intravenously injected DAG peptide homes to neurovascular unit endothelial cells and to reactive astrocytes in mouse models of AD. We identified connective tissue growth factor (CTGF), a matricellular protein that is highly expressed in the brain of individuals with AD and in mouse models, as the target of the DAG peptide. We also showed that exogenously delivered DAG homes to the brain in mouse models of glioblastoma, traumatic brain injury, and Parkinson’s disease. DAG may potentially be used as a tool to enhance delivery of therapeutics and imaging agents to sites of vascular changes and astrogliosis in diseases associated with neuroinflammation.Cerebrovascular changes and astrogliosis occur in Alzheimer’s disease (AD). Using an in vivo phage display technique, the authors identified a peptide that upon systematic administration, can home to brain endothelial cells and astrocytes in mouse models of AD at the early stages of the disease.

Vascular changes in tumors resistant to a vascular disrupting nanoparticle treatment

&NA; Anti‐angiogenic and vascular disrupting therapies rely on the dependence of tumors on new blood vessels to sustain tumor growth. We previously reported a potent vascular disrupting agent, a theranostic nanosystem consisting of a tumor vasculature‐homing peptide (CGKRK) fused to a pro‐apoptotic peptide [D(KLAKLAK)2] coated on iron oxide nanoparticles. This nanosystem showed promising therapeutic efficacy in glioblastoma (GBM) and breast cancer models. However, complete control of the tumors was not achieved, and some tumors became non‐responsive to the treatment. Here we examined the non‐responder phenomenon in an aggressive MCF10‐CA1a breast tumor model. In the treatment‐resistant tumors we noted the emergence of CD31‐negative patent neovessels and a concomitant loss of tumor homing of the nanosystem. In vivo phage library screening in mice bearing non‐responder tumors showed that compared to untreated and treatment‐sensitive tumors, treatment sensitive tumors yield a distinct landscape of vascular homing peptides characterized by over‐representation of peptides that target &agr;v integrins. Our approach may be generally applicable to the development of targeted therapies for tumors that have failed treatment. Graphical abstract Figure. No caption available.

Publisher Correction: Identification of a peptide recognizing cerebrovascular changes in mouse models of Alzheimer’s disease

The original version of the Supplementary Information associated with this Article inadvertently omitted Supplementary Table 1. The HTML has now been updated to include a corrected version of the Supplementary Information.

Abstract 705: In vivo audition of tumor homing peptides using high-throughput sequencing and q-PCR

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA In vivo peptide phage display has been widely used to explore tumor vascular heterogeneity and to develop targeting ligands for the delivery of anticancer drugs and imaging agents. Numerous tumor homing peptides with various targeting specificities have been identified. To select a suitable targeting ligand for a particular tumor from an existing peptide panel, it is crucial to perform unbiased quantitative assessment of in vivo peptide homing. Here we describe an approach for parallel evaluation of in vivo tumor homing of peptide-displaying phage clones. Mice bearing orthotopic xenografts of gliomas, breast tumors, and prostate tumors were intravenously injected with equimolar mix of phage clones displaying 12 different tumor homing or control peptides. After 30 min circulation and removal of blood by perfusion, representation of each phage clone in tumors and control organs was determined by high throughput sequencing (HTS) and quantitative PCR (qPCR). The ratiometric analysis demonstrated a clear and reproducible homing signature for each tumor model. Calibrated copy number data obtained by qPCR correlated well with the HTS analysis. Selected phage clones were validated for tumor homing by conventional single-phage homing studies using immunofluorescence, phage titration, and qPCR. Our study shows that the ratiometric in vivo phage biodistribution analysis can be used to rapidly identify peptides that efficiently deliver payloads to particular tumor types. As the peptides are evaluated side-by-side in a same animal, inter-animal variability is eliminated and the number of animals required is dramatically decreased. The choice of analysis (HTS vs qPCR) depends on the complexity of phage pool to be audited. HTS enables the analysis of highly complex phage pools, but introduces a potential bias owing to multiple DNA amplification steps. On the other hand, qPCR allows precise quantification of the copy number of the target sequences, and is suitable for low complexity pools. Narrowing down a complex pool with HTS followed by precise evaluation with qPCR can be a potential combination of the two analytical methods. Citation Format: Kadri Toome, Tarmo Molder, Kuldar Koiv, Pille Saalik, Kazuki N. Sugahara, Erkki Ruoslahti, Tambet Teesalu. In vivo audition of tumor homing peptides using high-throughput sequencing and q-PCR. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 705. doi:10.1158/1538-7445.AM2014-705