Dual Effect by Chemical Electron Transfer Enhanced siRNA Lipid Nanoparticles: Reactive Oxygen Species-Triggered Tumor Cell Killing Aggravated by Nrf2 Gene Silencing.

Insufficient endosomal escape presents a major hurdle for successful nucleic acid therapy. Here, for the first time, a chemical electron transfer (CET) system was integrated into small interfering RNA (siRNA) lipid nanoparticles (LNPs). The CET acceptor can be chemically excited using the generated energy between the donor and hydrogen peroxide, which triggers the generation of reactive oxygen species (ROS), promoting endosomal lipid membrane destabilization. Tetra-oleoyl tri-lysino succinoyl tetraethylene pentamine was included as an ionizable lipopeptide with a U-shaped topology for effective siRNA encapsulation and pH-induced endosomal escape. LNPs loaded with siRNA and CET components demonstrated a more efficient endosomal escape, as evidenced by a galectin-8-mRuby reporter; ROS significantly augmented galectin-8 recruitment by at least threefold compared with the control groups, with a p value of 0.03. Moreover, CET-enhanced LNPs achieved a 24% improvement in apoptosis level by knocking down the tumor-protective gene nuclear factor erythroid 2-related factor 2, boosting the CET-mediated ROS cell killing.

From structural design to delivery: mRNA therapeutics for cancer immunotherapy.

mRNA therapeutics have emerged as powerful tools for cancer immunotherapy in accordance with their superiority in expressing all sequence-known proteins in vivo. In particular, with a small dosage of delivered mRNA, antigen-presenting cells (APCs) can synthesize mutant neo-antigens and multi-antigens and present epitopes to T lymphocytes to elicit antitumor effects. In addition, expressing receptors like chimeric antigen receptor (CAR), T-cell receptor (TCR), CD134, and immune-modulating factors including cytokines, interferons, and antibodies in specific cells can enhance immunological response against tumors. With the maturation of in vitro transcription (IVT) technology, large-scale and pure mRNA encoding specific proteins can be synthesized quickly. However, the clinical translation of mRNA-based anticancer strategies is restricted by delivering mRNA into target organs or cells and the inadequate endosomal escape efficiency of mRNA. Recently, there have been some advances in mRNA-based cancer immunotherapy, which can be roughly classified as modifications of the mRNA structure and the development of delivery systems, especially the lipid nanoparticle platforms. In this review, the latest strategies for overcoming the limitations of mRNA-based cancer immunotherapies and the recent advances in delivering mRNA into specific organs and cells are summarized. Challenges and opportunities for clinical applications of mRNA-based cancer immunotherapy are also discussed.

In Vivo Endothelial Cell Gene Silencing by siRNA-LNPs Tuned with Lipoamino Bundle Chemical and Ligand Targeting.

Although small-interfering RNAs (siRNAs) are specific silencers for numerous disease-related genes, their clinical applications still require safe and effective means of delivery into target cells. Highly efficient lipid nanoparticles (LNPs) are developed for siRNA delivery, showcasing the advantages of novel pH-responsive lipoamino xenopeptide (XP) carriers. These sequence-defined XPs are assembled by branched lysine linkages between cationizable polar succinoyl tetraethylene pentamine (Stp) units and apolar lipoamino fatty acids (LAFs) at various ratios into bundle or U-shape topologies. Formulation of siRNA-LNPs using LAF4-Stp1 XPs as ionizable compounds led to robust cellular uptake, high endosomal escape, and successful in vitro gene silencing activity at an extremely low (150 picogram) siRNA dose. Of significance is the functional in vivo endothelium tropism of siRNA-LNPs with bundle LAF4-Stp1 XP after intravenous injection into mice, demonstrated by superior knockdown of liver sinusoidal endothelial cell (LSEC)-derived factor VIII (FVIII) and moderate silencing of hepatocyte-derived FVII compared to DLin-MC3-DMA-based LNPs. Optimizing lipid composition following click-modification of siRNA-LNPs with ligand c(RGDfK) efficiently silenced vascular endothelial growth factor receptor-2 (VEGFR-2) in tumor endothelial cells (TECs). The findings shed light on the role of ionizable XPs in the LNP in vivo cell-type functional targeting, laying the groundwork for future therapeutic applications.

Lipo-Xenopeptide Polyplexes for CRISPR/Cas9 based Gene editing at ultra-low dose.

Double pH-responsive xenopeptide carriers containing succinoyl tetraethylene pentamine (Stp) and lipo amino fatty acids (LAFs) were evaluated for CRISPR/Cas9 based genome editing. Different carrier topologies, variation of LAF/Stp ratios and LAF types as Cas9 mRNA/sgRNA polyplexes were screened in three different reporter cell lines using three different genomic targets (Pcsk9, eGFP, mdx exon 23). One U-shaped and three bundle (B2)-shaped lipo-xenopeptides exhibiting remarkable efficiencies were identified. Genome editing potency of top carriers were observed at sub-nanomolar EC50 concentrations of 0.4 nM sgRNA and 0.1 nM sgRNA for the top U-shape and top B2 carriers, respectively, even after incubation in full (≥ 90%) serum. Polyplexes co-delivering Cas9 mRNA/sgRNA with a single stranded DNA template for homology directed gene editing resulted in up to 38% conversion of eGFP to BFP in reporter cells. Top carriers were formulated as polyplexes or lipid nanoparticles (LNPs) for subsequent in vivo administration. Formulations displayed long-term physicochemical and functional stability upon storage at 4 °C. Importantly, intravenous administration of polyplexes or LNPs mediated in vivo editing of the dystrophin gene, triggering mRNA exon 23 splicing modulation in dystrophin-expressing cardiac muscle, skeletal muscle and brain tissue.

Tumor-targeted PROTAC prodrug nanoplatform enables precise protein degradation and combination cancer therapy.

Proteolysis targeting chimeras (PROTACs) have emerged as revolutionary anticancer therapeutics that degrade disease-causing proteins. However, the anticancer performance of PROTACs is often impaired by their insufficient bioavailability, unsatisfactory tumor specificity and ability to induce acquired drug resistance. Herein, we propose a polymer-conjugated PROTAC prodrug platform for the tumor-targeted delivery of the most prevalent von Hippel-Lindau (VHL)- and cereblon (CRBN)-based PROTACs, as well as for the precise codelivery of a degrader and conventional small-molecule drugs. The self-assembling PROTAC prodrug nanoparticles (NPs) can specifically target and be activated inside tumor cells to release the free PROTAC for precise protein degradation. The PROTAC prodrug NPs caused more efficient regression of MDA-MB-231 breast tumors in a mouse model by degrading bromodomain-containing protein 4 (BRD4) or cyclin-dependent kinase 9 (CDK9) with decreased systemic toxicity. In addition, we demonstrated that the PROTAC prodrug NPs can serve as a versatile platform for the codelivery of a PROTAC and chemotherapeutics for enhanced anticancer efficiency and combination benefits. This study paves the way for utilizing tumor-targeted protein degradation for precise anticancer therapy and the effective combination treatment of complex diseases.

Dynamic carriers for therapeutic RNA delivery.

Carriers for RNA delivery must be dynamic, first stabilizing and protecting therapeutic RNA during delivery to the target tissue and across cellular membrane barriers and then releasing the cargo in bioactive form. The chemical space of carriers ranges from small cationic lipids applied in lipoplexes and lipid nanoparticles, over medium-sized sequence-defined xenopeptides, to macromolecular polycations applied in polyplexes and polymer micelles. This perspective highlights the discovery of distinct virus-inspired dynamic processes that capitalize on mutual nanoparticle-host interactions to achieve potent RNA delivery. From the host side, subtle alterations of pH, ion concentration, redox potential, presence of specific proteins, receptors, or enzymes are cues, which must be recognized by the RNA nanocarrier via dynamic chemical designs including cleavable bonds, alterable physicochemical properties, and supramolecular assembly-disassembly processes to respond to changing biological microenvironment during delivery.

GalNAc- or Mannose-PEG-Functionalized Polyplexes Enable Effective Lectin-Mediated DNA Delivery.

A cationic, dendrimer-like oligo(aminoamide) carrier with four-arm topology based on succinoyl tetraethylene pentamine and histidines, cysteines, and N-terminal azido-lysines was screened for plasmid DNA delivery on various cell lines. The incorporated azides allow modification with various shielding agents of different polyethylene glycol (PEG) lengths and/or different ligands by copper-free click reaction, either before or after polyplex formation. Prefunctionalization was found to be advantageous over postfunctionalization in terms of nanoparticle formation, stability, and efficacy. A length of 24 ethylene oxide repetition units and prefunctionalization of ≥50% of azides per carrier promoted optimal polyplex shielding. PEG shielding resulted in drastically reduced DNA transfer, which could be successfully restored by active lectin targeting via novel GalNAc or mannose ligands, enabling enhanced receptor-mediated endocytosis of the carrier system. The involvement of the asialoglycoprotein receptor (ASGPR) in the uptake of GalNAc-functionalized polyplexes was confirmed in the ASGPR-positive hepatocarcinoma cell lines HepG2 and Huh7. Mannose-modified polyplexes showed superior cellular uptake and transfection efficacy compared to unmodified and shielded polyplexes in mannose-receptor-expressing dendritic cell-like DC2.4 cells.

Crosstalk Between NK Cell Receptors and Tumor Membrane Hsp70-Derived Peptide: A Combined Computational and Experimental Study.

Natural killer (NK) cells are central components of the innate immunity system against cancers. Since tumor cells have evolved a series of mechanisms to escape from NK cells, developing methods for increasing the NK cell antitumor activity is of utmost importance. It is previously shown that an ex vivo stimulation of patient-derived NK cells with interleukin (IL)-2 and Hsp70-derived peptide TKD (TKDNNLLGRFELSG, aa450-461) results in a significant upregulation of activating receptors including CD94 and CD69 which triggers exhausted NK cells to target and kill malignant solid tumors expressing membrane Hsp70 (mHsp70). Considering that TKD binding to an activating receptor is the initial step in the cytolytic signaling cascade of NK cells, herein this interaction is studied by molecular docking and molecular dynamics simulation computational modeling. The in silico results showed a crucial role of the heterodimeric receptor CD94/NKG2A and CD94/NKG2C in the TKD interaction with NK cells. Antibody blocking and CRISPR/Cas9-mediated knockout studies verified the key function of CD94 in the TKD stimulation and activation of NK cells which is characterized by an increased cytotoxic capacity against mHsp70 positive tumor cells via enhanced production and release of lytic granules and pro-inflammatory cytokines.

Lipoamino bundle LNPs for efficient mRNA transfection of dendritic cells and macrophages show high spleen selectivity.

Messenger RNA (mRNA) is a powerful tool for nucleic acid-based therapies and vaccination, but efficient and specific delivery to target tissues remains a significant challenge. In this study, we demonstrate lipoamino xenopeptide carriers as components of highly efficient mRNA LNPs. These lipo-xenopeptides are defined as 2D sequences in different 3D topologies (bundles or different U-shapes). The polar artificial amino acid tetraethylene pentamino succinic acid (Stp) and various lipophilic tertiary lipoamino fatty acids (LAFs) act as ionizable amphiphilic units, connected in different ratios via bisamidated lysines as branching units. A series of more lipophilic LAF4-Stp1 carriers with bundle topology is especially well suited for efficient encapsulation of mRNA into LNPs, facilitated cellular uptake and strongly enhanced endosomal escape. These LNPs display improved, faster transfection kinetics compared to standard LNP formulations, with high potency in a variety of tumor cell lines (including N2a neuroblastoma, HepG2 and Huh7 hepatocellular, and HeLa cervical carcinoma cells), J774A.1 macrophages, and DC2.4 dendritic cells. High transfection levels were obtained even in the presence of serum at very low sub-microgram mRNA doses. Upon intravenous application of only 3 µg mRNA per mouse, in vivo mRNA expression is found with a high selectivity for dendritic cells and macrophages, resulting in a particularly high overall preferred expression in the spleen.

Transcriptional Targeting of Dendritic Cells Using an Optimized Human Fascin1 Gene Promoter.

Deeper knowledge about the role of the tumor microenvironment (TME) in cancer development and progression has resulted in new strategies such as gene-based cancer immunotherapy. Whereas some approaches focus on the expression of tumoricidal genes within the TME, DNA-based vaccines are intended to be expressed in antigen-presenting cells (e.g., dendritic cells, DCs) in secondary lymphoid organs, which in turn induce anti-tumor T cell responses. Besides effective delivery systems and the requirement of appropriate adjuvants, DNA vaccines themselves need to be optimized regarding efficacy and selectivity. In this work, the concept of DC-focused transcriptional targeting was tested by applying a plasmid encoding for the luciferase reporter gene under the control of a derivative of the human fascin1 gene promoter (pFscnLuc), comprising the proximal core promoter fused to the normally more distantly located DC enhancer region. DC-focused activity of this reporter construct was confirmed in cell culture in comparison to a standard reporter vector encoding for luciferase under the control of the strong ubiquitously active cytomegalovirus promoter and enhancer (pCMVLuc). Both plasmids were also compared upon intravenous administration in mice. The organ- and cell type-specific expression profile of pFscnLuc versus pCMVLuc demonstrated favorable activity especially in the spleen as a central immune organ and within the spleen in DCs.

mCherry on Top: A Positive Read-Out Cellular Platform for Screening DMD Exon Skipping Xenopeptide-PMO Conjugates.

Phosphorodiamidate morpholino oligomers (PMOs) are a special type of antisense oligonucleotides (ASOs) that can be used as therapeutic modulators of pre-mRNA splicing. Application of nucleic-acid-based therapeutics generally requires suitable delivery systems to enable efficient transport to intended tissues and intracellular targets. To identify potent formulations of PMOs, we established a new in vitro-in vivo screening platform based on mdx exon 23 skipping. Here, a new in vitro positive read-out system (mCherry-DMDEx23) is presented that is sensitive toward the PMO(Ex23) sequence mediating DMD exon 23 skipping and, in this model, functional mCherry expression. After establishment of the reporter system in HeLa cells, a set of amphiphilic, ionizable xenopeptides (XPs) was screened in order to identify potent carriers for PMO delivery. The identified best-performing PMO formulation with high splice-switching activity at nanomolar concentrations in vitro was then translated to in vivo trials, where exon 23 skipping in different organs of healthy BALB/c mice was confirmed. The predesigned in vitro-in vivo workflow enables evaluation of PMO(Ex23) carriers without change of the PMO sequence and formulation composition. Furthermore, the identified PMO-XP conjugate formulation was found to induce highly potent exon skipping in vitro and redistributed PMO activity in different organs in vivo.

Receptor-Targeted Carbon Nanodot Delivery through Polymer Caging and Click Chemistry-Supported LRP1 Ligand Attachment.

Carbon nanodots present resistance to photobleaching, bright photoluminescence, and superior biocompatibility, making them highly promising for bioimaging applications. Herein, nanoprobes were caged with four-armed oligomers and subsequently modified with a novel DBCO-PEG-modified retro-enantio peptide ligand reL57, enhancing cellular uptake into U87MG glioma cells highly expressing low-density lipoprotein receptor-related protein 1 (LRP1). A key point in the development of the oligomers was the incorporation of ε-amino-linked lysines instead of standard α-amino-linked lysines, which considerably extended the contour length per monomer. The four-armed oligomer 1696 was identified as the best performer, spanning a contour length of ~8.42 nm for each arm, and was based on an altering motive of two cationic ε-amidated lysine tripeptides and two tyrosine tripeptides for electrostatic and aromatic stabilization of the resulting formulations, cysteines for disulfide-based caging, and N-terminal azidolysines for click-modification. This work highlights that well-designed four-armed oligomers can be used for noncovalent coating and covalent caging of nanoprobes, and click modification using a novel LRP1-directed peptide ligand facilitates delivery into receptor-expressing target cells.

Interleukin-6-controlled, mesenchymal stem cell-based sodium/iodide symporter gene therapy improves survival of glioblastoma-bearing mice.

New treatment strategies are urgently needed for glioblastoma (GBM)-a tumor resistant to standard-of-care treatment with a high risk of recurrence and extremely poor prognosis. Based on their intrinsic tumor tropism, adoptively applied mesenchymal stem cells (MSCs) can be harnessed to deliver the theranostic sodium/iodide symporter (NIS) deep into the tumor microenvironment. Interleukin-6 (IL-6) is a multifunctional, highly expressed cytokine in the GBM microenvironment including recruited MSCs. MSCs engineered to drive NIS expression in response to IL-6 promoter activation offer the possibility of a new tumor-targeted gene therapy approach of GBM. Therefore, MSCs were stably transfected with an NIS-expressing plasmid controlled by the human IL-6 promoter (IL-6-NIS-MSCs) and systemically applied in mice carrying orthotopic GBM. Enhanced radiotracer uptake by 18F-Tetrafluoroborate-PET/magnetic resonance imaging (MRI) was detected in tumors after IL-6-NIS-MSC application as compared with mice that received wild-type MSCs. Ex vivo analysis of tumors and non-target organs showed tumor-specific NIS protein expression. Subsequent 131I therapy after IL-6-NIS-MSC application resulted in significantly delayed tumor growth assessed by MRI and improved median survival up to 60% of GBM-bearing mice as compared with controls. In conclusion, the application of MSC-mediated NIS gene therapy focusing on IL-6 biology-induced NIS transgene expression represents a promising approach for GBM treatment.

High-resolution bioenergetics correlates the length of continuous protonatable diaminoethane motif of four-armed oligo(ethanamino)amide transfectants to cytotoxicity.

Recent clinical success with Onpattro and cationic ionizable lipid nanoparticle-based mRNA vaccines has rejuvenated research in the design and engineering of broader synthetic cationic vectors for nucleic acid compaction and transfection. However, perturbation of metabolic processes and cytotoxicity are still of concern with synthetic cationic vectors. Here, through an integrated bioenergetic and biomembrane integrity probing in three different human cell lines we reveal the dynamic effect of a library of sequence-defined four-arm oligo(ethanamino)amide transfectant on cell homeostasis, and identify metabolically safe building units over wide concentration ranges. The results show differential effects of the oligo(ethanamino)amide structure of comparable molecular weight on cell energetics. The severity of polycation effect on bioenergetic crisis follows with the length of continuous protonatable diaminoethane motif in the ascending order of glutaryl-triethylene tetramine, succinyl-tetraethylene pentamine and succinyl-pentaethylene hexamine. We further identify oligomeric structures that do not induce bioenergetic crisis even at high concentrations. Finally, transfection studies with a library of polyplexes carrying a reporter gene show no correlation between transfection efficiency and cytotoxicity. These observations demonstrate the usefulness of integrated high-resolution respirometry and plasma membrane integrity probing as a highly sensitive medium-throughput screening strategy for identification and selection of safe building units for transfectant engineering.

Peptide nucleic acid-zirconium coordination nanoparticles.

Ideal drug carriers feature a high loading capacity to minimize the exposure of patients with excessive, inactive carrier materials. The highest imaginable loading capacity could be achieved by nanocarriers, which are assembled from the therapeutic cargo molecules themselves. Here, we describe peptide nucleic acid (PNA)-based zirconium (Zr) coordination nanoparticles which exhibit very high PNA loading of [Formula: see text] w/w. This metal-organic hybrid nanomaterial class extends the enormous compound space of coordination polymers towards bioactive oligonucleotide linkers. The architecture of single- or double-stranded PNAs was systematically varied to identify design criteria for the coordination driven self-assembly with Zr(IV) nodes at room temperature. Aromatic carboxylic acid functions, serving as Lewis bases, and a two-step synthesis process with preformation of [Formula: see text] turned out to be decisive for successful nanoparticle assembly. Confocal laser scanning microscopy confirmed that the PNA-Zr nanoparticles are readily internalized by cells. PNA-Zr nanoparticles, coated with a cationic lipopeptide, successfully delivered an antisense PNA sequence for splicing correction of the [Formula: see text]-globin intron mutation IVS2-705 into a functional reporter cell line and mediated splice-switching via interaction with the endogenous mRNA splicing machinery. The presented PNA-Zr nanoparticles represent a bioactive platform with high design flexibility and extraordinary PNA loading capacity, where the nucleic acid constitutes an integral part of the material, instead of being loaded into passive delivery systems.

Chemical Evolution of Amphiphilic Xenopeptides for Potentiated Cas9 Ribonucleoprotein Delivery.

The introduction of the CRISPR/Cas9 system in the form of Cas9/sgRNA ribonucleoproteins (RNP) is an efficient, straightforward strategy for genome editing, and potent RNP carriers are in high demand. Here, we report a series of artificial peptides based on novel ionizable amino acids that are able to deliver Cas9 RNP into cells very efficiently. Systematic variation of hydrophobic properties revealed a relationship between the xenopeptide logD7.4 and genome editing potency. By correlating the physicochemical properties with biological activity, individual optima were found for different xenopeptide sequence architectures. The optimized amphiphilic carriers enable ∼88% eGFP knockout at an RNP dose of only 1 nM and up to 40% homology-directed repair (HDR) in eGFP/BFP switchable reporter cells by co-delivery with an ssDNA template. Mechanistic studies demonstrated that hydrophobically balanced xenopeptides are more resistant to ionic stress as well as concentration-dependent dissociation and promote endocytosis by both clathrin- and macropinocytosis-mediated pathways. The systematic study develops a versatile and adjustable carrier platform and highlights impactful structure-activity relationships, providing a new chemical guide for the design and optimization of nonviral Cas9 RNP nanocarriers.

Iron-Gallic Acid Peptide Nanoparticles as a Versatile Platform for Cellular Delivery with Synergistic ROS Enhancement Effect.

Cytosolic delivery of peptides is of great interest owing to their biological functions, which could be utilized for therapeutic applications. However, their susceptibility to enzymatic degradation and multiple cellular barriers generally hinders their clinical application. Integration into nanoparticles, which can enhance the stability and membrane permeability of bioactive peptides, is a promising strategy to overcome extracellular and intracellular obstacles. Herein, we present a versatile platform for the cellular delivery of various cargo peptides by integration into metallo-peptidic coordination nanoparticles. Both termini of cargo peptides were conjugated with gallic acid (GA) to assemble GA-modified peptides into nanostructures upon coordination of Fe(III). Initial pre-complexation of Fe(III) by poly-(vinylpolypyrrolidon) (PVP) as a template favored the formation of nanoparticles, which are able to deliver the peptides into cells efficiently. Iron-gallic acid peptide nanoparticles (IGPNs) are stable in water and are supposed to generate reactive oxygen species (ROS) from endogenous H2O2 in cells via the Fenton reaction. The strategy was successfully applied to an exemplary set of peptide sequences varying in length (1-7 amino acids) and charge (negative, neutral, positive). To confirm the capability of transporting bioactive cargos into cells, pro-apoptotic peptides were integrated into IGPNs, which demonstrated potent killing of human cervix carcinoma HeLa and murine neuroblastoma N2a cells at a 10 µM peptide concentration via the complementary mechanisms of peptide-triggered apoptosis and Fe(III)-mediated ROS generation. This study demonstrates the establishment of IGPNs as a novel and versatile platform for the assembly of peptides into nanoparticles, which can be used for cellular delivery of bioactive peptides combined with intrinsic ROS generation.

Molecular Chameleon Carriers for Nucleic Acid Delivery: The Sweet Spot between Lipoplexes and Polyplexes.

Taking advantage of effective intracellular delivery mechanisms of both cationizable lipids and polymers, highly potent double pH-responsive nucleic acid carriers are generated by combining at least two lipo amino fatty acids (LAFs) as hydrophobic cationizable motifs with hydrophilic cationizable aminoethylene units into novel sequence-defined molecules. The pH-dependent tunable polarity of the LAF is successfully implemented by inserting a central tertiary amine, which disrupts the hydrophobic character once protonated, resulting in pH-dependent structural and physical changes. This "molecular chameleon character" turns out to be advantageous for dynamic nucleic acid delivery via lipopolyplexes. By screening different topologies (blocks, bundles, T-shapes, U-shapes), LAF types, and LAF/aminoethylene ratios, highly potent pDNA, mRNA, and siRNA carriers are identified, which are up to several 100-fold more efficient than previous carrier generations and characterized by very fast transfection kinetics. mRNA lipopolyplexes maintain high transfection activity in cell culture even in the presence of ≥90% serum at an ultra-low mRNA dose of 3 picogram (≈2 nanoparticles/cell), and thus are comparable in potency to viral nanoparticles. Importantly, they show great in vivo performance with high expression levels especially in spleen, tumor, lungs, and liver upon intravenous administration of 1-3 µg luciferase-encoding mRNA in mice.

Mesenchymal Stem Cell-mediated Image-guided Sodium Iodide Symporter (NIS) Gene Therapy Improves Survival of Glioblastoma-bearing Mice.

PURPOSE: Mesenchymal stem cells (MSC) have emerged as cellular-based vehicles for the delivery of therapeutic genes in cancer therapy based on their inherent tumor-homing capability. As theranostic gene, the sodium iodide symporter (NIS) represents a successful target for noninvasive radionuclide-based imaging and therapy. In this study, we applied genetically engineered MSCs for tumor-targeted NIS gene transfer in experimental glioblastoma (GBM)-a tumor with an extremely poor prognosis. EXPERIMENTAL DESIGN: A syngeneic, immunocompetent GL261 GBM mouse model was established by subcutaneous and orthotopic implantation. Furthermore, a subcutaneous xenograft U87 model was used. Bone marrow-derived MSCs were stably transfected with a NIS-expressing plasmid driven by the constitutively active cytomegalovirus promoter (NIS-MSC). After multiple or single intravenous injection of NIS-MSCs, tumoral iodide uptake was monitored in vivo using 123I-scintigraphy or 124I-PET. Following validation of functional NIS expression, a therapy trial with 131I was performed on the basis of the most optimal application regime as seen by 124I-PET imaging in the orthotopic approach. RESULTS: A robust tumoral NIS-specific radionuclide accumulation was observed after NIS-MSC and radioiodide application by NIS-mediated in vivo imaging. NIS immunofluorescence staining of GBM and non-target tissues showed tumor-selective MSC homing along with NIS expression. Application of therapeutically effective 131I led to significantly delayed tumor growth and prolonged median survival after NIS-MSC treatment as compared with controls. CONCLUSIONS: A strong tumor-selective recruitment of systemically applied MSCs into GBM was found using NIS as reporter gene followed by successful therapeutic application of radioiodide demonstrating the potential use of NIS-based MSCs as therapy vehicles as a new GBM therapy approach.

Folate Receptor-Mediated Delivery of Cas9 RNP for Enhanced Immune Checkpoint Disruption in Cancer Cells.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system offers great opportunities for the treatment of numerous diseases by precise modification of the genome. The functional unit of the system is represented by Cas9/sgRNA ribonucleoproteins (RNP), which mediate sequence-specific cleavage of DNA. For therapeutic applications, efficient and cell-specific transport into target cells is essential. Here, Cas9 RNP nanocarriers are described, which are based on lipid-modified oligoamino amides and folic acid (FolA)-PEG to realize receptor-mediated uptake and gene editing in cancer cells. In vitro studies confirm strongly enhanced potency of receptor-mediated delivery, and the nanocarriers enable efficient knockout of GFP and two immune checkpoint genes, PD-L1 and PVR, at low nanomolar concentrations. Compared with non-targeted nanoparticles, FolA-modified nanocarriers achieve substantially higher gene editing including dual PD-L1/PVR gene disruption after injection into CT26 tumors in vivo. In the syngeneic mouse model, dual disruption of PD-L1 and PVR leads to CD8+ T cell recruitment and distinct CT26 tumor growth inhibition, clearly superior to the individual knockouts alone. The reported Cas9 RNP nanocarriers represent a versatile platform for potent and receptor-specific gene editing. In addition, the study demonstrates a promising strategy for cancer immunotherapy by permanent and combined immune checkpoint disruption.