Ranit Kedmi

The systemic toxicity of positively charged lipid nanoparticles and the role of Toll-like receptor 4 in immune activation

Delivery of nucleic acids with positively charged lipid nanoparticles ((+)NPs) is widely used as research reagents and potentially for therapeutics due to their ability to deliver nucleic acids into the cell cytoplasm. However, in most reports little attention has been made to their toxic effects. In the present study, we performed comprehensive analyses of the potential toxicity associated with (+)NPs. Mice treated with (+)NPs showed increased liver enzyme release and body weight loss compared to mice treated with neutral or negatively charged NPs ((-)NPs), suggesting hepatotoxicity. Intravenous administration of (+)NPs induced interferon type I response and elevated mRNA levels of interferon responsive genes 15-25-fold higher than neutral and (-)NPs in different subsets of leukocytes. Moreover, treatment with (+)NPs provoked a dramatic pro-inflammatory response by inducing Th1 cytokines expression (IL-2, IFN gamma and TNF alpha) 10-75-fold higher than treatment with control particles. Finally, we showed that activation of TLR4 might serve as the underlying mechanism for induction of an immune response when (+)NPs are used. These results suggest that a careful attention must be made when different types of (+)NPs are being developed as nanotherapeutics.

RNAi nanoparticles in the service of personalized medicine

The understanding of cancer molecular origins and the different intracellular signaling pathways that are perturbed in the various types of malignancies have laid the foundation to realize why patients react diversely to therapy. For example, in colon cancer, an efficient treatment of EGF-receptor blockade with monoclonal antibody was found not to benefit patients with tumors positive for the K - ras mutation [1]. This slowly but steadily brought a shift from a ‘one size fits all’ therapy approach to a more person alized attitude, in which each patient is treated according to their tumor-specific genetic defects. To support this new approach, a substantial effort is invested in developing a new class of molecular diagnostic tools identifying patients’ tumor-personal gene-expression signatures, as well as mutation profiles. RNA interference (RNAi) is a is a ubiquitous, highly specific, endogenous mechanism of gene silencing that can be activated by incorporating small interfering (si)RNAs into the cell

Integrin-Targeted Nanoparticles for siRNA Delivery

Integrins are heterodimeric membrane glycoproteins composed of noncovalently associated α and β subunits. Integrins support cell attachment and migration on the surrounding extracellular matrix as well as mediate cell-cell interaction in physiological and pathological settings. Constant recycling of integrins from the plasma membrane to the endosome makes integrins ideal receptors for the delivery of drugs to the cell cytoplasm. RNA interference (RNAi) has evolved not only as a powerful tool for studying gene expression and validating new drug targets, but also as a potential therapeutic intervention. However, the major challenge facing the translation of RNAi into clinical practice is the lack of efficient systemic delivery to specific cell types. Utilizing integrins as delivery target, we have recently devised a strategy to target leukocytes termed Integrin-targeted and stabilized NanoParticles (I-tsNPs) that entrap high RNAi payloads and deliver them in a leukocyte-specific manner to induce robust gene silencing.

SNP Detection in mRNA in Living Cells Using Allele Specific FRET Probes

Live mRNA detection allows real time monitoring of specific transcripts and genetic alterations. The main challenge of live genetic detection is overcoming the high background generated by unbound probes and reaching high level of specificity with minimal off target effects. The use of Fluorescence Resonance Energy Transfer (FRET) probes allows differentiation between bound and unbound probes thus decreasing background. Probe specificity can be optimized by adjusting the length and through use of chemical modifications that alter binding affinity. Herein, we report the use of two oligonucleotide FRET probe system to detect a single nucleotide polymorphism (SNP) in murine Hras mRNA, which is associated with malignant transformations. The FRET oligonucleotides were modified with phosphorothioate (PS) bonds, 2′OMe RNA and LNA residues to enhance nuclease stability and improve SNP discrimination. Our results show that a point mutation in Hras can be detected in endogenous RNA of living cells. As determined by an Acceptor Photobleaching method, FRET levels were higher in cells transfected with perfect match FRET probes whereas a single mismatch showed decreased FRET signal. This approach promotes in vivo molecular imaging methods and could further be applied in cancer diagnosis and theranostic strategies.

A modular platform for targeted RNAi therapeutics

Previous studies have identified relevant genes and signalling pathways that are hampered in human disorders as potential candidates for therapeutics. Developing nucleic acid-based tools to manipulate gene expression, such as short interfering RNAs1–3 (siRNAs), opens up opportunities for personalized medicine. Yet, although major progress has been made in developing siRNA targeted delivery carriers, mainly by utilizing monoclonal antibodies (mAbs) for targeting4–8, their clinical translation has not occurred. This is in part because of the massive development and production requirements and the high batch-to-batch variability of current technologies, which rely on chemical conjugation. Here we present a self-assembled modular platform that enables the construction of a theoretically unlimited repertoire of siRNA targeted carriers. The self-assembly of the platform is based on a membrane-anchored lipoprotein that is incorporated into siRNA-loaded lipid nanoparticles that interact with the antibody crystallizable fragment (Fc) domain. We show that a simple switch of eight different mAbs redirects the specific uptake of siRNAs by diverse leukocyte subsets in vivo. The therapeutic potential of the platform is demonstrated in an inflammatory bowel disease model by targeting colon macrophages to reduce inflammatory symptoms, and in a Mantle Cell Lymphoma xenograft model by targeting cancer cells to induce cell death and improve survival. This modular delivery platform represents a milestone in the development of precision medicine.A self-assembled modular siRNA delivery platform enables the construction of a theoretically unlimited repertoire of carriers to target distinct cell surface receptors in the service of personalized medicine.

TAF4b and TAF4 differentially regulate mouse embryonic stem cells maintenance and proliferation.

TAF4b is a cell type‐specific subunit of the general transcription factor TFIID. Here, we show that TAF4b is highly expressed in embryonic stem cells (ESC) and is down‐regulated upon differentiation. To examine the role of TAF4b in ESC, we applied a knockdown (KD) approach. TAF4b depletion is associated with morphological changes and reduced expression of the self‐renewal marker alkaline phosphatase. In contrast, KD of TAF4, a ubiquitously expressed TAF4b paralog, retained and even stabilized ESC stemness. Retinoic acid‐induced differentiation was facilitated in the absence of TAF4b but was significantly delayed by TAF4 KD. Furthermore, TAF4b supports, whereas TAF4 inhibits, ESC proliferation and cell cycle progression. We identified a subset of TAF4b target genes preferentially expressed in ESC and controlling the cell cycle. Among them are the germ cell‐specific transcription factor Sohlh2 and the protein kinase Yes1, which was recently shown to regulate ESC self‐renewal. Interestingly, Sohlh2 and Yes1 are also targets of the pluripotency factor Oct4, and their regulation by Oct4 is TAF4b‐dependent. Consistent with that, TAF4b but not TAF4 interacts with Oct4. Our findings suggest that TAF4b cooperates with Oct4 to regulate a subset of genes in ESC, whereas TAF4 is required for later embryonic developmental stages.

Cell specific delivery of modified mRNA expressing therapeutic proteins to leukocytes

Therapeutic alteration of gene expression in vivo can be achieved by delivering nucleic acids (e.g., mRNA, siRNA) using nanoparticles. Recent progress in modified messenger RNA (mmRNA) synthesis facilitated the development of lipid nanoparticles (LNPs) loaded with mmRNA as a promising tool for in vivo protein expression. Although progress have been made with mmRNA-LNPs based protein expression in hepatocytes, cell specificity is still a major challenge. Moreover, selective protein expression is essential for an improved therapeutic effect, due to the heterogeneous nature of diseases. Here, we present a precision protein expression strategy in Ly6c+ inflammatory leukocytes in inflammatory bowel disease (IBD) induced mice. We demonstrate a therapeutic effect in an IBD model by targeted expression of the interleukin 10 in Ly6c+ inflammatory leukocytes. A selective mmRNA expression strategy has tremendous therapeutic potential in IBD and can ultimately become a novel therapeutic modality in many other diseases.Therapeutic alteration of protein expression using modified mRNA is limited by immunogenicity and instability in vivo. Here the authors use antibody-coated lipid nanoparticles to deliver mRNA to leukocytes and drive expression of anti-inflammatory cytokines in an inflammatory bowel disease mouse model.

Zooming in on selectins in cancer

Selectins are involved in leukocyte and cancer cell trafficking, which can be targeted with drugs and nanoparticles (Shamay et al., this issue). Selectins are involved in leukocyte and cancer cell trafficking, which can be targeted with drugs and nanoparticles (Shamay et al., this issue).

Correction: SNP Detection in mRNA in Living Cells Using Allele Specific FRET Probes

Figures 4 and 5 were incorrectly switched. The image currently appearing as Figure 4 belongs with the title and legend of Figure 5, and the image currently appearing as Figure 5 belongs with the title and legend of Figure 4. The titles and legends themselves are in correct order. In addition, there was an error in Figure 3. Please see the correct Figure 3 here: